EA044251B1 - METHODS OF AGRICULTURAL PRODUCTION OF OILSEED CROPS BRASSICA CARINATA - Google Patents

Description

Cross references to related applications

This application claims priority to U.S. Provisional Patent Application No.

62/556,575, filed September 11, 2017, the contents of which are incorporated by reference in their entirety.

Field of technology to which the invention relates

The invention relates to the field of agriculture and discloses a new method involving growing the oilseed crop Brassica carinata as a replacement for fallow or existing cover crops used in crop rotations, using new farming techniques that retain the soil benefits of traditional cover crops or fallow rotations, but allow for the harvest of oil-rich grains that provide feedstock for low-carbon-intensive biofuel production, while achieving greater life-cycle greenhouse gas (GHG) emissions reductions as well as soil carbon sequestration.

Prior Art

Over-reliance on fossil fuels for transport, energy production, home heating, industrial power supply, etc. has led to an ever-increasing rate of emissions and accumulation of CO ₂ and GHGs in the atmosphere. This has led to the threat of global warming and its undesirable consequences. One strategy to reduce the rate of increase in emissions of CO2 and other greenhouse gases in the atmosphere is to reduce dependence on fossil fuels by replacing them with more environmentally friendly fuels, such as those derived from vegetable oils and biomass, which are less carbon-intensive over their life cycle. .

To control greenhouse gas emissions, governments have adopted regulations aimed at reducing the growth rate of carbon emissions to agreed upon target levels within their jurisdictions. To enable compliance with these regulations, mechanisms and methodologies have been developed to accurately audit the life cycle greenhouse gas emissions produced by specific fuels from well to reservoir. At the same time, measures are also being taken to incentivize emitters to meet these targets (carbon taxes, caps and trading systems). The end result of these measures was the creation of a system of payments for carbon dioxide emissions into the atmosphere, which must be borne by those that produce carbon dioxide.

This policy particularly targets industries such as those that produce and rely on fuels and energy sources. Thus, fuel producers, and by extension biofuel producers, have greater incentives to specify feedstocks, fuels, and production processes to meet their targets and minimize the impact of carbon pricing on their bottom line.

The need to reduce carbon emissions and strong incentives for industry to achieve carbon reductions in their sectors have been important factors leading to the development of new low-carbon-intensive pathways. However, the pricing of next-generation fuels relative to conventional fuels has acted as a barrier to their wider adoption. With the advent of carbon pricing, some of these disincentives have been removed, suggesting that the widening difference in the carbon intensity of biofuels relative to conventional fuels may be one factor that could have a significant impact on price differentials.

Carbon intensity (CI) is defined as a measure of the greenhouse gas (GHG) emissions of a fuel, determined using life cycle assessment (LCA). LCA identifies and estimates all GHG emissions from fuel production; from the cultivation or extraction of raw materials to the production of fuel, all the way to the final use of the fuel. Carbon intensity is defined as the mass of greenhouse gases emitted in carbon dioxide equivalent per unit of energy contained in the fuel, in units of grams of carbon dioxide equivalent per megajoule of energy (g CO ₂ -eq/MJ) (Regulatory requirements for determining the carbon intensity of renewable energy sources and low-carbon fuels (Fact Sheet RLCF-006), section 2, page 3, entitled What is Carbon Intensity?, published by the British Columbia Government's Department of Energy and Mines, published December 2010, revised July 2013). In 2017, the baseline CI values reported for fossil fuels under the EU Renewable Energy Directive (EU-RED) and for biodiesel under the US Renewable Fuel Standard (US RFS) were respectively 83.8 (EU-RED) g produced energy CO ₂ -eq./MJ and 91.8 g produced energy CO ₂ -eq./MJ (US RFS), as indicated in table. 3 from DeJong et al., 2017. Those skilled in the art will recognize that CI values for both fossil fuels and biofuels can change as LCA models and production methods evolve. The current CI value for various biofuel pathways can be found at (https://www.arb.ca.gov/fuels/lcfs/fuelpathways/current-pathways-01102017.xlsx).

Agricultural production provides the appropriate methodology for producing the next generation of biofuels. Modern agriculture produces food, feed and fiber on a huge scale and can be mobilized to provide raw materials for fuel production without the need to develop new production technologies or infrastructure.

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One of the attractive features of agricultural production is the use of plants' ability to utilize and fix atmospheric carbon dioxide through photosynthesis and thus act as an important carbon sink. The carbon stored in the biomass of the annual crop will eventually be remobilized, some of it as harvested material and the rest as plant debris (leaves, stems, trunks, roots), which are degraded by soil bacteria and fungi. Some of the carbon assimilated in the soil is used by soil microbes as an energy source and eventually converted to carbon dioxide gas, but some will also be stably retained in the soil, which is an important sink for carbon fixation and reduction of atmospheric emissions. Of all the carbon stocks in the environment, soil is second in size only to the oceans and has an estimated organic carbon content of more than 2.3 GT (Jobbagyand Jackson, 2000), which is 4 times the amount of carbon stored in total plant biomass. An additional benefit of restoring carbon to soils is the subsequent improvement in soil fertility and structure.

However, while annual crops fix carbon dioxide during their lifespan, and also return a significant portion of stored carbon dioxide to the soil for longer-term fixation, their cultivation can also directly and indirectly result in _CO2 and CO2-equivalent greenhouse gas emissions . These emissions occur during the cultivation of the crop, the subsequent conversion of the crop into raw materials, the conversion of raw materials into liquid fuels, the storage and transportation of raw materials and finished fuels, and finally the distribution and use of fuels. Greenhouse gas emissions associated with increased yield include the stages of seed development, field preparation, input crop production and application (fertilizers, pesticides/herbicides/seed treatments), crop planting, crop maintenance and harvesting, storage of harvested material, and storage and transportation to the processing plant.

Auditing methodologies such as the Greenhouse Gases, Regulated Emissions and Energy Use in Transport (GREET) model (Wang 1996), GHGenius ( S&T Squared. GHGenius, Model Version 4.03; www.ghgenius.ca; S&T Squared Consultants Inc. for Natural Resources Canada: Delta, British Columbia, 2017), based on a previously developed model (DeLucchi 1991), BioGrace (www.biograce.net ; Neeftet al., 2012) and others. This allows for more valid comparisons of the overall GHG impacts from biofuel production and use with those from fossil fuels, and also allows comparisons to be made between biofuels produced from different types of energy crops. The ability to accurately model and predict GHG emissions throughout the life cycle of biofuel production has made it possible to determine the value of carbon dioxide production. As a result of carbon pricing, national and international agreements have been concluded to reduce carbon dioxide/GHG emissions to specified targets. Examples of such policies are the Renewable Energy Directive (RED) in the EU, and the Renewable Fuel Standard (RFS) and California Low Carbon Fuel Standard (CA-LCFS) in the US.

Table 1 compares published carbon intensity (CI) values for selected biofuel pathways and compares them with values for conventional gasoline and/or diesel fuel. As can be seen, the FAME (fatty acid methyl ester) biodiesel pathways have CI values ranging from 67.32 to 51.35 g CO ₂ -eq/MJ compared to a CI of 102.4 g CO ₂ -eq/MJ for conventional diesel fuel, demonstrating the significant reduction in CI provided by biodiesel FAME pathways compared to their petroleum equivalents. In addition, at a CI of 44 g CO ₂ -eq/MJ, renewable or green diesel fuel produced by hydrotreating rapeseed oil provides an additional reduction in overall carbon intensity compared to the FAME process.

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Table 1

Carbon intensity of individual biofuel pathways

Raw materials Fuel path Carbon capacity (g COeq./MJ) Data source Model used Oil Petrol 93.3 DeJong et al., 2017 GREET BIOGRACE BIOGRACE Oil Diesel 91.8 Canola/ Rapeseed oil Canola/ Rapeseed oil Canola/ Rapeseed oil Canola/ Rapeseed oil Biodiesel (FAME) Biodiesel (FAME) Biodiesel (FAME) PPM*** 67.32 51.35 52 44 CAARB*

*https://www.arb.ca.gov/fuels/lcfs/fuelpathways/current-pathways-01102017.xlsx **http://eur-lex.europa.eu/legal-content/EN/ALL/?uri =CELEX%3A32009L0028 ***Hydrogenated vegetable oil

In a study focusing exclusively on greenhouse gas emissions from canola cultivation on the Canadian prairies between 1986 and 2006, the authors (Shrestha, et al., 2014) demonstrated that GHG emissions were reduced per unit area by 40% and on a dry matter basis by 65% in this period of time. The decline was due to a combination of factors, including reduced land-use change, increased grain yields, and increased soil organic carbon fixation through improved land management. In 2006, soil carbon fixation in this region averaged almost 500 kg CO ₂ /ha.

However, there is still a need for a separate parent crop whose production can be scaled up to meet the demand for high-quality liquid biofuel feedstocks such as biodiesel, clean diesel and jet fuel substitutes. However, other high-yielding and productive oilseed crops proposed as potential sources of feedstock, the most common species and varieties such as canola-type Brassica or soybean, produce edible oils that require additional costs compared to specialized biofuel feedstocks and which will also reduce supply edible oils.

For example, the cultivation of low-erucic acid canola or rapeseed to produce significant quantities of biofuels will almost certainly lead to land use changes to compensate for the decline in edible oil production. Moreover, the price premium for high-quality edible oils can be expected to push prices for canola biofuel feedstock to uncompetitive levels.

Soybeans, whose oil has been used as a feedstock for biofuel production, are cool-season legumes grown throughout much of North America, South America and Asia. As a source of edible oil, soybeans also currently account for more than 60% of edible oils consumed in the United States (data taken from Table 20: Local Market Reporting Year for Sales and Distribution of US Oilseeds and Products; and Table 21: Reporting Year year of the local market for the sale and distribution of US soybean crops and products; (https://apps.fas.usda.gov/psdonline/circulars/oilseeds.pdf)). Competition between its use as an edible oil and as a biofuel feedstock has resulted in price volatility, which likely reduces the economic justification for its need as a biofuel feedstock (Wisner 2010). Moreover, a significant shift from edible oils to biofuels will almost certainly result in indirect emissions from land use changes.

Palm oil, another major feedstock for biofuel production, is grown in Asia and South America. However, in many jurisdictions, palm oil faces significant barriers due to land use changes resulting from the establishment of palm plantations in sensitive ecosystems. The use of palm oil has been associated with high levels of greenhouse gas emissions due to massive deforestation resulting from the creation of monoculture palm plantations. So-called certified sustainable palm oil, or Roundtable on Sustainable Palm Oil (RSPO) palm oil, is distinguished from non-certified palm oil by the producer's commitment to preserving and protecting high-value natural forests. However, sustainable palm oil is significantly more expensive than non-certified palm oil, which discourages its use as a biofuel.

A member of the Brassicaceae (formerly Cruciferae) family, Brassica carinata is also known as Abyssinian cabbage, Ethiopian mustard, Abyssinian mustard, African sarson and Gomenzer. In addition to B. carinata, the genus Brassica includes several economically important oilseed species: B. juncea (L). Czern. (brown mustard), B. napus L. (rapeseed, Argentine canola), B. nigra (L.) W.D.J. Koch (black

- 3 044251 mustard) and B. rapa L. (field mustard, Polish canola), and also includes food crops B. oleracea L., including cabbage, broccoli, cauliflower, Brussels sprouts, kohlrabi and brown kale. The six species of Brassica are closely related genetically, as described in the Triangle of U (Nagaharu 1935). The natural range of Brassica carinata covers the central highlands of Ethiopia; however, recent efforts have been made to exploit the genetic differences inherent in Abyssinian mustard to develop varieties that are productive in more diverse agricultural environments, including semi-arid zones or regions where more marginal agricultural lands may predominate.

Brassica carinata produces abundant spherical seeds with a diameter of 1–1.5 mm (Mnzava and Schippers 2007), varying from yellow to tan or brown (Alemaw 1987, Rahman and Tahir 2010). The seeds are rich in oil, with an oil content of 37-44% based on the dry weight of the seed, depending on the variety and growing conditions. The protein content in the seeds is also high and amounts to 25-30% based on the dry weight of the seeds (Panet al., 2012). Unlike canola, Brassica carinata produces an inedible oil.

In Spain and Italy, Abyssinian mustard seed oil has been used for the production of biofuels (Cardoneet al., 2002, Cardoneet al., 2003, Bouaidet al., 2005, Gasolet al., 2007, Gasolet al., 2009) and as a bioindustrial feedstock for many purposes (eg in lubricants, paints, cosmetics, plastics). In North America, Abyssinian mustard has been evaluated as a biofuel feedstock (Drenthet al., 2014, Drenthet al., 2015), and the crude oil obtained from the seeds of B. carinata has been used to produce green or renewable diesel, biodiesel and biofuel for engines (Drenth et al. 2014). In October 2012, the National Research Council of Canada's experimental aviation flights using the world's first 100% biofuels for propulsion were successful (ReadiJet 100% biofuels flight - one of 2012's 25 most important scientific events, Popular Science Magazine, 2012(12).

Blackshaw and colleagues compared several oilseed species for suitability as FAME biodiesel sources in Western Canada (Blackshaw et al., 2011). Trials conducted at 5 sites in western Canada (during 2008-2009) tested a range of oilseed species and cultivars, including 3 canola cultivars (one each of Brassica napus, Brassica rapa and Brassica juncea). ), Brassica carinata, Camelina sativa, oriental mustard (B. juncea), yellow mustard (Sinapis alba), soybean and flax were tested for yield and quality of oil raw materials. Based on the results of these studies, Brassica carinata exceeded the yield of Brassica napus canola (control line) in only 1 of 9 sites for the year, accounting for the lowest combined yield ranking of all species tested in these trials, while for oil content, Brassica carinata ranked third place (higher than seasoning mustard and soy). However, it should be noted that the Abyssinian mustard variety, which is a heterogeneous common variety, was used in this study and not a commercial elite variety.

In contrast, in a comparison of Brassica oilseed cultivars conducted in Minnesota in 2012–2013, Gesch et al. (2015) wanted to demonstrate that new commercial Abyssinian mustard cultivars produced comparable grain yields to commercial canola cultivars such as Brassica napus, while producing nearly twice the above-ground biomass compared to Brassica napus cultivars. Gesch et al. indicate a lower seed to aboveground biomass ratio (harvest index) of Abyssinian mustard crops, and suggest that there is scope to improve grain yield through breeding. However, Gesch et al. What is not taught is that higher biomass of Brassica carinata may provide benefits in terms of the ability to return additional carbon to the soil.

Johnson and colleagues argue that Abyssinian mustard grain and biomass yields are positively correlated with increased nitrogen fertilizer application, and under conditions studied (up to 160-200 kg N/ha, depending on experiment), maximum straw and grain yields did not plateau (Johnsonet al., 2013). This may be taken to indicate that very high levels of nitrogen may be required to produce Abyssinian mustard grain; however, they also purportedly demonstrated that under conditions of high existing soil nitrogen mineralization, high grain yields could be obtained without the addition of nitrogen fertilizers. On the other hand, Johnson et al. have not reported the possible beneficial effect of including Brassica carinata in crop rotations with legumes such as lentils, peas or soybeans, which fix nitrogen and increase soil nitrogen mineralization, reducing the need for nitrogen fertilizer and the carbon intensity of Abyssinian mustard production.

As a first attempt to establish the carbon footprint of Brassica carinata cultivation, a greenhouse gas life cycle analysis was conducted on an Abyssinian mustard bioenergy system based on the use of all harvested above-ground biomass (including grain) as a lignocellulosic energy generation system (Gasol et al., 2007). Based on estimates of carbon associated with extensive root systems, Gasol et al. teach that up to 631 kg CO2/ha can be transferred to the soil, helping to reduce atmospheric CO2 emissions by up to 71% relative to a basic natural gas power generation system. However, Gasol et al. do not consider the possibility of additional return of nutrients to the soil due to the return of above-ground biomass

- 4 044251 after harvesting and harvesting of Abyssinian mustard grains, and do not consider the possibility of using the grain to extract raw materials for the production of biofuel and by-product in the form of flour for use as a high protein feed additive.

Although the above references suggest that Brassica carinata may be a suitable dedicated feed crop for biofuel production, it is unknown how this feedstock can be produced from Abyssinian mustard in a variety of regions, soil conditions and crop rotations to achieve the lowest possible the most beneficial carbon intensity for the biofuel route.

Summary of the invention

As a means to reduce dependence on fossil fuel use leading to subsequent increases in greenhouse gas emissions, and to promote sustainable agriculture, the invention described herein includes methods for growing Brassica carinata, a crop that produces oil from harvested seeds, which are feedstock to produce biofuels to replace fossil fuels, and to produce high-quality protein-rich flour as a by-product that can be used in commercial livestock feed. More specifically, the invention describes methods of cultivation to produce crops using optimal agronomic and land management practices applied in a variety of climate zones and regions, thereby significantly reducing atmospheric CO2 and GHG emissions compared to equivalent amounts of fossil fuels.

Brassica carinata can be sustainably cultivated in a variety of environments to produce high-quality biofuel feedstocks while achieving:

a) reducing greenhouse gas emissions associated with the production of raw materials, as well as the subsequent production of biofuels;

b) increasing the carbon content of the soil in which crops are grown;

c) providing conditions for increasing the yield of crops with which Brassica carinata is produced in crop rotation; And

d) achieve the above with little or no increase in land use change.

These characteristics allow credits to be awarded through schemes or programs designed to assign a value to carbon emissions, such as the RFS program in the US and the RED program in the EU. Such programs also monetize the cost of carbon generated during the production and use of fuels in a manner that reduces the price differential between fossil fuels and alternative biofuels. The end result is the recognition of carbon as the primary product of value. Similarly, the concept of Abyssinian mustard as a crop that is produced and valued as a source of a specific commodity (i.e. as a biofuel feedstock) is replaced by one whose value represents the necessary balance between carbon emissions and carbon reduction. In this light, Abyssinian mustard production represents a new category of agricultural production, namely one that can be characterized as carbon farming.

The present invention enables the cultivation of Abyssinian mustard in specific climate and soil zones and geographic regions using specific agricultural and land management practices to provide sustainable feedstocks for biofuel and feed while achieving measurable benefits in the form of reduced greenhouse gas emissions, improved soil structure and improved characteristics of the following crops grown with Abyssinian mustard in crop rotation.

Unlike canola, Brassica carinata produces an inedible oil, and its production can be done on marginal lands or as part of a crop rotation replacing summer or winter fallow, resulting in minimal displacement of food crops and little or no associated land use change.

Embodiments provide methods for cultivating Brassica carinata cultivars to provide winter crops with short day lengths in temperate or subtropical regions, and spring crops with long day lengths in regions with temperate cold dry climates.

In some embodiments, growing conditions are provided in which Abyssinian mustard is maintained on previously cultivated land, as a fallow replacement, and in a crop rotation accompanying or preceding legumes, pulses, or grains.

In still other embodiments, agronomic and land management practices are provided for growing and harvesting the oilseed crop Brassica carinata, including the use of fertilizers, herbicides and pesticides, seeding rates and seeding depth to achieve optimal grain and biomass.

Additional embodiments provide land management methods for Abyssinian mustard cultivation, such as returning above- and below-ground Abyssinian mustard plant biomass to the field to maximize soil carbon levels. The degree of carbon storage achievable with Abyssinian mustard is a surprising discovery. At that

- 5 044251 While other oilseeds such as canola are optimized for grain production by breeding for varieties that will channel plant energy into seed production at the expense of biomass production, Brassica carinata achieves high levels of grain production and biomass production simultaneously. The resulting increased biomass contains more carbon and can subsequently return more of this carbon to the soil after harvest.

In other embodiments, conditions are provided for growing Brassica carinata to produce grain, the oil of which is used as a feedstock for the production of biofuels, such as PBM, in the production of flour as a by-product of oil extraction, with protein, carbohydrate, fiber and caloric content as described for use as animal feed.

In other embodiments, methods for producing biofuel feedstocks with low carbon intensity are provided. The carbon intensity of biofuels produced from feedstocks produced in this way can be negative, resulting in enhanced reductions in greenhouse gas emissions.

In some environments where winter may be too harsh to support crop production, Brassica carinata can be planted immediately after winter, as soon as soil temperatures allow, as part of a crop rotation where Brassica carinata replaces the spring/summer fallow that typically follows harvest collected before the coming winter.

In one aspect of the present invention, a method is provided, including:

a) sowing the variety Brassica carinata as a second crop alternately with the first crop or to replace fallowing;

(b) implementing land management practices to reduce the use of fossil fuels and maximize atmospheric carbon capture by Brassica carinata biomass;

c) harvesting of the Brassica carinata variety for grain production;

d) returning approximately 70 to 90% of all plant material from the Brassica carinata variety, excluding grain, to the soil.

In some embodiments, the method includes planting the Brassica carinata variety immediately after harvest or at the same time as the first harvest to produce sequential crops without an intervening fallow period.

In some embodiments, the method further includes processing the grain to produce oil, where the oil is used as a feedstock to produce a low carbon-intensive biofuel.

In some embodiments, the method further comprises treating the grain such that, after extracting the oil fraction, a protein-rich, low-fiber flour fraction remains, which can be used as a protein-rich feed additive for livestock production.

In some embodiments, a new crop that is not Brassica carinata is planted immediately after or at the same time as Brassica carinata is harvested without an intervening fallow period, thereby increasing soil productivity while adding additional carbon to the soil. As a result, overall greenhouse gas emissions associated with agriculture are reduced.

Accordingly, in one embodiment of the present invention there is provided a method for cultivating Brassica carinata, comprising:

a) sowing the Brassica carinata variety immediately after harvest or simultaneously with the harvest of the first crop for successive cultivation of crops without an intermediate fallow period;

b) implementing land management practices to reduce the use of fossil fuels and maximize the capture of atmospheric carbon by Brassica carinata biomass;

c) harvesting of the Brassica carinata variety for grain production;

d) returning 70 to 90% of all Brassica carinata plant material, excluding grain, to the soil;

e) sowing a new crop, which may be the same as or different from the first crop, but is not Brassica carinata, immediately after or simultaneously with the harvest of Brassica carinata, without an intervening fallow period;

f) grain-to-oil processing, which uses oil as a feedstock to produce low-carbon-intensive biofuels; And

g) processing the grain so that after extraction of the oil fraction, a low-fiber, protein-rich flour fraction remains, which can be used as a protein-rich feed additive for livestock production.

Agricultural professionals will generally appreciate that fallow periods are a common practice in many regions. It will also be generally understood by those skilled in the art that a fallow period may often involve leaving the land uncultivated for a period of time equal to the typical period when the first crop is grown, or that a fallow period may also include planting a cover crop to control soil erosion or to

- 6 044251 preventing the growth of unwanted plants such as weeds. In each case, the term fallow is used to broadly describe a period of time when the land is not used to produce the first crop, but rather is managed either without a seeded crop or with a planted plant or crop that is simply used to provide vegetation cover above the ground. For each agricultural region, the timing and duration of fallowing will be different, since the climate may differ and practices vary from region to region, and this is usually obvious to agricultural specialists; however, fallow is a term to describe the portion of time when the soil is considered unproductive.

There are a number of cover crops that are used during the fallow period, ranging from wheat, rye, other grasses and even crops that produce seed oil such as Brassica napus, Brassica juncea, Camelina and Lesquerella. However, crops such as wheat, rye and other grasses do not produce oil that can be used for low carbon-intensive fuels, while crops such as Camelina and Lesquerella do not produce significant amounts of biomass to sequester sufficient carbon to provide greenhouse gas reductions observed for Brassica carinata. For example, it has been shown (Geschet al., 2015) that Brassica carinata can produce up to 2 times more biomass than Brassica napus and more than 4.5 times more biomass than Camelina under typical cropping conditions where fallowing is commonly used . In the present invention, we have demonstrated the unexpected and beneficial result that Brassica carinata can replace the fallow period and add more carbon to the soil, with the added benefit of producing grain that can be used to produce low carbon-intensive fuels.

It is an object of the present invention to provide a method in which fallow is prevented and replaced by Brassica carinata using techniques that maximize the capture of atmospheric carbon that is added to the soil at harvest. These techniques may include reducing the amount of fertilizer and reducing the use of additional water, such as for irrigation. The unique properties of Brassica carinata, which include increased tolerance to extreme climate changes such as frost or heat, allow Brassica carinata to be grown in regions where other oilseeds cannot grow or do not produce a productive product such as grain, which contains a high percentage of oils in grain.

In typical fallow protocols, the plant material that is grown during fallow is simply treated or killed with herbicides at the end of the fallow period for application to the soil. The present invention provides for more beneficial use of land, an increase in the amount of atmospheric carbon entering the soil through Brassica carinata biomass, and the added benefit of harvesting grains containing oil that can be used to produce low carbon-intensive fuels.

In some embodiments, the invention provides a method for producing grains for use in the production of low carbon-intensity vegetable oil feedstocks for biofuels; to add carbon to the soil; and/or to earn carbon credits.

In some embodiments, a method of producing grain for use in the production of low carbon-intensity fuel feedstock is provided, comprising:

a) sowing the Brassica carinata variety immediately after harvest or simultaneously with the harvest of the first crop for sequential cultivation without an intermediate fallow period;

(b) implementing land management practices to reduce the use of fossil fuel inputs and maximize atmospheric carbon sequestration by Brassica carinata biomass;

c) harvesting of the Brassica carinata variety for grain production;

d) returning approximately 70 to 90% of all plant material from the variety Brassica carinata, excluding grain, to the soil;

e) grain processing to extract oil and flour fractions; And

f) converting oil into a low carbon-intensive fuel and flour into a high protein feed additive for livestock.

In other embodiments, the following is provided.

1. A method of growing Brassica carinata, including:

a) sowing the variety Brassica carinata as a second crop alternately with the first crop or to replace fallowing;

(b) implement land management practices to reduce the use of fossil fuels and maximize atmospheric carbon sequestration by Brassica carinata plant material;

c) harvesting of the Brassica carinata variety for grain production;

d) returning approximately 70 to 90% of all plant material from the Brassica carinata variety, excluding grain, to the soil.

2. The method of embodiment 1, further comprising sowing the variety Brassica carinata

- 7 044251 immediately after harvest or simultaneously with the harvest of the first crop for sequential cultivation of crops without an intermediate fallow period.

3. The method of embodiment 1 or 2, further comprising processing the harvested grain to extract oil and obtain a flour fraction.

4. The method of embodiment 3, further comprising using the oil as a feedstock to produce low carbon-intensive biofuels.

5. The method of embodiment 4, wherein the low carbon-intensity biofuel has a carbon-intensity value that is reduced by about 50-200 g CO ₂ -eq/MJ relative to the carbon-intensity value of the corresponding fossil-derived fuel.

6. The method of embodiment 4, wherein the GHG emissions resulting from the production of the low carbon-intensive biofuel during its life cycle are reduced by approximately 60,400% compared to the GHG emissions resulting from the production of the corresponding fossil fuel.

7. The method of embodiment 3, further comprising producing a protein-enriched livestock feed additive from the flour fraction.

8. The method of any one of embodiments 1-7, further comprising planting a new crop, which may be the same as the first crop or different from the first crop, but which is not Brassica carinata, immediately after or simultaneously with harvesting the Brassica carinata without an intermediate fallow period.

9. The method of any one of embodiments 1-8, further comprising fixing carbon in the soil.

10. The method of any one of embodiments 1-9, providing fixation of about 0.5 to 5 tons of CO2 per hectare per year in the soil.

11. The method as in any one of embodiments 1-10, wherein the land management practice includes no-till, shallow-till, or medium-till cultivation.

12. The method as in any one of embodiments 1-11, wherein the land management practice includes eliminating irrigation or reducing irrigation compared to the normal amount of irrigation required for another oilseed crop for the same growing environment.

13. The method as in any one of embodiments 1-12, wherein the land management practice includes reducing the use of inorganic nitrogen fertilizer compared to the recommended amount of nitrogen fertilizer for Brassica carinata for the growing environment.

14. The method of embodiment 13, comprising reducing the use of inorganic nitrogen fertilizer to about 40-100% of the recommended amount of nitrogen fertilizer for Brassica carinata in the growing environment.

15. The method of embodiment 13, comprising reducing the use of inorganic nitrogen fertilizer to about 40-90% of the recommended amount of nitrogen fertilizer for Brassica carinata in the growing environment.

16. The method of embodiment 13, comprising reducing the use of inorganic nitrogen fertilizer to about 50-70% of the recommended amount of nitrogen fertilizer for Brassica carinata in the growing environment.

17. The method as in any one of embodiments 1-12, wherein the land management practice includes using manure to provide from about 20 to 100% of the nitrogen fertilizer needed to grow Brassica carinata.

18. The method as in any one of embodiments 1-12, wherein the land management practice includes using manure to provide about 30 to 90% of the nitrogen fertilizer needed to grow Brassica carinata.

19. The method as in any one of embodiments 1-12, wherein the land management practice includes using manure to provide about 40 to 80% of the nitrogen fertilizer needed to grow Brassica carinata.

20. The method as in any one of embodiments 1-12, wherein the land management practice includes using manure to provide about 50 to 75% of the nitrogen fertilizer needed to grow Brassica carinata.

21. The method as in any one of embodiments 17-20, wherein the manure is chicken manure, cattle manure, or sheep manure.

22. The method as in any one of embodiments 1-21, wherein the Brassica carinata variety is grown on the land during a period of time when the land would normally lie fallow.

23. The method as in any one of embodiments 1-22, wherein there is minimal or no land use change.

24. The method as in any one of embodiments 1-23, wherein the first crop is a legume.

25. The method of embodiment 24, wherein the legume is beans, peas, lentils, soybeans, peanuts or alfalfa.

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26. The method of embodiment 24, wherein the legume is peanut, lentil or soybean.

27. The method as in any one of embodiments 1-23, wherein the first crop is a cereal crop.

28. The method of embodiment 27, wherein the grain crop is wheat, barley, rye, oats or corn.

29. The method of embodiment 27, wherein the grain crop is wheat or corn.

30. The method as in any one of embodiments 1-23, wherein the first crop is cotton or sesame.

31. The method as in any one of embodiments 1-30, wherein the growing environment is in a region with a tropical rainy climate, and wherein the land management practices include planting Brassica carinata in the fall or winter for harvest in the spring or summer.

32. The method as in any one of embodiments 1-30, wherein the growing environment is in a region with a tropical rainy climate, and wherein the land management practices include planting Brassica carinata in the spring for harvest in the summer or fall.

33. The method of embodiment 31, wherein the first crop is cotton or sesame.

34. The method of embodiment 30 or 31, wherein the first crop is a legume or grain crop.

35. The method of embodiment 34, wherein the legume is peanut, lentil or soybean.

36. The method of embodiment 34, wherein the grain crop is corn or wheat.

37. The method as in any one of embodiments 1-23, wherein the first crop is a pulse crop, the growing environment is in a warm temperate, humid climate region, and the land management practice includes planting Brassica carinata in the fall or winter for harvest in the spring or summer.

38. The method of embodiment 37, wherein the legume is peanut, lentil or soybean.

39. The method as in any one of embodiments 1-23, wherein the first crop is a cereal crop, the growing environment is in a region with a warm temperate humid climate, and the land management practice includes planting Brassica carinata in the spring or summer for harvest in the fall or winter.

40. The method as in any one of embodiments 1-23, wherein the first crop is a cereal crop, the growing environment is in a region with a temperate warm dry climate, and the land management practice includes planting Brassica carinata in the fall or winter for harvest in the spring or summer.

41. The method as in any one of embodiments 1-23, wherein the first crop is a cereal crop, the growing environment is in a region with a temperate cold dry climate, and the land management practice includes planting Brassica carinata in the spring for harvest in the summer or fall.

42. The method as in any one of embodiments 1-23, wherein the first crop is a cereal crop, the growing environment is in a region with a temperate cold wet climate, and the land management practice includes planting Brassica carinata in the spring for harvest in the summer or fall.

43. The method as in any one of embodiments 1-23, wherein the first crop is a cereal crop, the growing environment is in a tropical arid climate region, and the land management practice includes planting Brassica carinata in the fall or winter for harvest in the spring or summer.

44. The method as in any one of embodiments 39-43, wherein the grain crop is corn or wheat.

45. The method of any one of embodiments 1-44, wherein the method results in the fixation of about 0.5 to 5 tons of CO2/ha/year in the soil.

46. The method as in any one of embodiments 1-45, wherein harvesting is carried out using a combine harvester.

47. The method of embodiment 46, wherein harvesting is carried out by direct harvesting.

Description of the drawing

The drawing shows the stages of production of HVO (hydrotreated vegetable oil) from cultivated Brassica carinata grains, which are within the system boundary from The distribution coefficient for the first three stages is 0.613. The output of GRM is 0.58 MJ/MJ of Abyssinian mustard seeds.

- 9 044251

Definitions

A number of terms are used in the descriptions, examples, and tables that follow. To assist in a clear and consistent understanding of the invention, the following definitions are provided.

Biofuels are fuels produced from feedstocks derived from a biological (plant, animal or bacterial) hydrocarbon source, as opposed to fuels derived from a fossil source.

Types of biofuels include fuels that are classified as the following.

a) First generation: Generally, first generation biofuels are those fuels derived from biomass isolated from food crops, such as ethanol obtained from the fermentation of starches and sugars, or biodiesel obtained from the transesterification of edible vegetable oils.

b) Second generation: Second generation biofuels are produced from feedstocks derived from the biomass of target non-food energy crops or from crop residues of food crops. The former category includes feedstock oils from specialty energy crops such as jatropha, while the latter category includes lignin, cellulose and hemicellulose from corn stover, sugarcane bagasse, etc.

c) Advanced biofuels or third generation biofuels: biofuels obtained from algal feedstock.

Biomass is broadly defined as material of organic origin that includes a living or recently living organism. Aboveground plant biomass includes all biomass associated with that part of the plant that is above the soil surface at the time of harvest. Likewise, belowground plant biomass includes all biomass associated with the part of the plant that is below the soil surface at the time of harvest. It follows that total plant biomass is defined as the sum of above-ground biomass and all below-ground biomass at the time of harvest.

Carbon intensity (CI) refers to the amount of greenhouse gas (GHG) emissions produced by a unit amount of fuel over its entire life cycle, compared to the energy released when that unit of fuel is burned. Greenhouse gas production is determined by a thorough life cycle analysis (LCA), which lists all greenhouse gas emissions emitted during the production and use of a unit of fuel. For biofuels, this includes all emissions resulting from the cultivation of the crop and the subsequent transportation of the harvested material, the subsequent processing of the harvested material into fuel feedstock, the storage and transportation of feedstocks, the production of fuel from feedstocks, the storage and distribution of fuels, through to the final use of the fuel. CI is recorded as the mass of carbon dioxide equivalent greenhouse gases emitted per unit of energy contained in the fuel, in units of grams of carbon dioxide equivalent per megajoule of energy produced (g CO ₂ -eq/MJ).

Abyssinian mustard refers to the seeds or plants of the species Brassica carinata, containing both the B genome from Brassica nigra (black mustard) and the C genome from Brassica oleracea (cabbage) (Nagahuru, 1935).

Cereals, or grain crops, are the term applied to grasses that are grown for their grain, including, but not limited to, barley, corn, oats, rice, rye, and wheat.

Climate zones, climatic regions, or climate as used herein, are terms referring to geographic divisions of the earth's surface that delineate regions based on similarities in historical average temperatures, precipitation, and seasonal variations. The climate zones used in this invention are based on the zones that are described in the EU document entitled Commission Decision of 10 June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC, which in turn , are based on climate zones established by the IPCC (IPCC 2006, 2006 IPCC, National Greenhouse Gas Inventory Program Guidelines for National Greenhouse Gas Inventories, Eggleston H.S., Buendia L., Miwa K., Ngara T., and Tanabe K. (eds. .).Published: IGES, Japan). Zones are defined based on common altitude, mean annual temperature (MAT), mean annual precipitation (MAP), mean annual precipitation to potential evapotranspiration ratio (MAP:PET), and frost frequency. There are 12 climatic regions: tropical mountain; tropical rainy; tropical humid; tropical dry; moderate warm, humid; moderate warm, dry; moderate cold, damp; moderate cold, dry; boreal humid; boreal dry; polar wet; and polar dry (described in Table 2 below).

- 10 044251

table 2

IPCC (Intergovernmental Panel on Climate Change) climate zone definitions

Climate zone Defining Characteristics Tropical mountain - Average annual temperature above 18°C - Up to seven days with negative temperatures per year - Altitude above sea level more than 1000 m tropical rainy - Average annual temperature above 18°C - Up to 7 days with negative temperatures per year - Average annual precipitation more than 200 mm - Less than 3 months of dry weather in winter tropical wet - Average annual temperature above 18°C - Up to 7 days with negative temperatures per year - Average annual precipitation from 100 to 200 cm - Less than 3-5 months of dry weather in winter Tropical dry Average annual temperature above 18°C - Up to 7 days of frost per year - 5-8 months of dry weather in winter Moderate warm, wet - Average annual temperature above 10°C - Damp weather; no dry season - Ratio of average annual precipitation to potential evapotranspiration more than 1 Moderate warm, dry - Average annual temperature above 10°C - Seasonally dry weather; winter rains - Ratio of potential evapotranspiration to average annual precipitation more than 1 Moderate cold, wet - Average annual temperature above 10°C (4-8 months with temperature above 10°C) - The ratio of average annual precipitation to potential evapotranspiration is more than 1 Moderate cold, dry - Average annual temperature above 10°C (4-8 months with temperature above 10°C - The ratio of potential evapotranspiration to average annual precipitation is more than 1 Boreal wet - 3 months or less with a temperature of 10°C or higher - The ratio of average annual precipitation to potential evapotranspiration is more than 1 Boreal dry - 3 months or less with a temperature of 10°C or higher - The ratio of potential evapotranspiration to average annual precipitation is more than 1 polar wet - All months with temperatures below 10°C - The ratio of average annual precipitation to potential evapotranspiration is more than 1 Polar dry - All months with temperatures below 10°C - Ratio of potential evapotranspiration to average annual precipitation more than 1

Harvesting refers to the process of reaping and collecting the seed pods from a ripe crop, threshing the pods to release the seeds (grain), and winnowing to separate and extract the grain from the empty pods, stems, and branches (collectively called chaff). These once separate operations are now often combined using a multifunctional mechanized device known as a combine harvester.

Cover crops are annual plants grown primarily to enrich or improve soil. They grow by preventing soil degradation due to erosion and leaching of nutrients from the root zone of root crops. They can also help conserve soil moisture, act as reservoirs to maintain soil nutrients, improve soil structure by increasing soil carbon, and counteract disease by preventing the persistence of plant pathogens. By nature, cover crops are planted during one season to maintain or improve the soil's potential to support the growth of the next season's crop. Generally, cover crops are not grown in anticipation of harvesting material that has its own economic value, such as suitability for food, fuel, or fiber production. Rather, the plant material produced by a cover crop is usually incorporated into the soil during or toward the end of its life cycle. In contrast, Abyssinian mustard, which provides similar benefits to both the soil and subsequent crops, is harvested to produce an oil-rich grain that can be processed into biofuel feedstocks as well as protein-rich flour. which can be used in animal feed, which provides a direct economic benefit to the farmer.

Growing. Cultivation refers to the conditions under which crops are sown, maintained, and harvested.

For Abyssinian mustard, growing factors include the following.

a) Sowing time: Abyssinian mustard is a medium to long season crop requiring a slightly longer growing season than other mustards. Therefore, early sowing provides better results. The ideal planting date depends largely on geography and weather. However, as a general rule, soils should be at least 40 F or higher before planting.

i) Canadian Prairies and US Boreal: Typical planting occurs in the spring from early April to late May.

ii) Southeastern United States: Typical planting occurs in the fall from October to December.

iii) South America (Uruguay): The optimal planting time is autumn or winter (ie usually between the beginning of May and the end of June).

b) Planting: Soil type and previous planting history will influence the type of plowing required to prepare the soil for planting. Reduced or minimal tillage can improve water conservation, soil organic matter, fuel efficiency and erosion control. Abyssinian mustard can be sown in soil subjected to conventional or minimal tillage, or it can be sown without tillage in standing stubble. Sowing is carried out at a rate designed to achieve a plant density in the range of 80 to 180 plants per square meter. B. carinata can be sown at a constant depth of 0.5 to 5.0 cm, for example at a depth of 1.35 to 2.5 cm.

c) Fertility: The soil fertility requirements for Abyssinian mustard are similar to those for other mustards and canola. Adequate amounts of nitrogen, phosphorus, potassium and sulfur are required to achieve true yield potential. Manure can be used as a source of organic nitrogen to replace some or all of the recommended inorganic nitrogen for Brassica carinata growth in a particular soil composition. Fertilizer rates vary depending on the growing zone and soil fertility.

d) Tilling: Abyssinian mustard can be planted in soil subjected to conventional plowing, where conventional plowing or deep plowing involves substantial loosening of the soil, repeated several times a year, so that little crop residue remains on the soil surface at the time of sowing. Alternatively, Abyssinian mustard may be planted in soil that is maintained under conservation plowing practices in which the extent and frequency of plowing is significantly reduced compared to conventional plowing (called medium- or shallow-till soil management), or it may not be subjected to plowing when planted in standing stubble. A more detailed description of each plowing practice is provided below in the definition of land management practices.

e) Moisture: Abyssinian mustard requires adequate soil moisture at sowing and emergence to establish a good stand, but can tolerate lower humidity thereafter and withstands the semi-arid mid-summer conditions of the Southern Canadian prairies.

f) Temperature: Abyssinian mustard is a temperate crop, but it has been adapted to more extreme conditions in the prairies of Southern Canada and the northernmost regions of the United States. During the initial stand establishment, Abyssinian mustard can recover from short periods of frost, and tolerates higher temperatures during flowering and seeding better than other Brassica oilseeds.

g) Herbicides: Abyssinian mustard is an aggressive crop and will outcompete many weeds if it is well established. However, some weed species, if allowed to grow early and maintained, can affect the quality and yield of all crops, including Abyssinian mustard. Examples of weeds that can negatively affect the yield and quality of Abyssinian mustard include kochia, wild mustard, and wild radish. Thus, weed control is an important aspect of modern agricultural practice and involves several different but complementary approaches, including physical methods of removing weeds before seed sowing, such as cultivation, plowing and weeding, and the use of chemical agents or herbicides to suppress or killing weeds before they take root and/or can form and release their seeds. Herbicides are a class of pesticides and include a large group of chemical compounds that interfere with specific biological processes of plants in a way that blocks their growth and survival. Herbicides are grouped into classes defined by the biological process with which they interact. These may include inhibition of lipid biosynthesis, inhibition of amino acid biosynthesis, hormonal regulation of plant growth, inhibition of photosynthesis, inhibition of nitrogen metabolism, inhibition of biosynthesis or function of plant pigments; agents that can damage cell membranes

- 12 044251 branes, and agents that inhibit seedling growth (Sherwani et al., 2015). In general, different compounds and classes of herbicides may demonstrate varying degrees of effectiveness against particular weed species. Moreover, some crop species may be more resistant to certain classes of herbicides than others. Thus, in a particular geographic region, the use of a particular herbicide for weed control may be dictated by the nature of the crop being grown and the native weeds found in that region. Thus, a particular herbicide may be determined to be registered for use on a crop based on its efficacy record and proven ability to control the relevant weeds without significantly affecting crop yield. Registered use also defines the specific methods of application of the herbicide, including the recommended concentration of the herbicide, use of suitable diluents, adjuvants, surfactants, etc., method of delivery (i.e., spray or granule), timing of application at the appropriate stage of cultivation for to ensure minimal crop damage, timing of application and number of applications to ensure optimal weed control, location of application (leaf or soil), recommended weather conditions for optimal weed control. Some examples of herbicides recommended for use with Abyssinian mustard grown in the southeastern United States are listed in:

(https://agrisoma.com/ckfinder/userfiles/files/2017_18_SE_Handbook.pdf).

The above is provided by way of example and is in no way intended to limit the scope of the invention.

h) Fungicides are a class of pesticides that include a diverse range of chemical agents that can prevent or reduce the severity of plant infection by pathogenic fungi. As with herbicides, there are many classes of fungicides. FRAC (Fungicide Resistance Action Committee; http://www.frac.info/home) lists 12 classes based on the different biochemical pathways targeted by the fungicides in the class, as well as a 13th class that includes fungicides with unknown modes of action. Fungicides also vary in delivery method and site of action: some fungicides are sprayed onto the surface of plants, while others are applied to the soil surface as granules or as a liquid poured onto the soil surface. Fungicides applied to the soil are typically absorbed through the roots and transported to all plant tissues through the xylem. Fungicides that are foliar can be topical, i.e. protect only those surfaces with which they come into contact, systemically, i.e. absorbed by the upper parts of plants, but then transferred by xylem to all above-ground tissues, or partially systemic, i.e. they may be locally absorbed, but may only be transported short distances to protect a slightly larger surface area than the fungicide's initial point of contact. Additionally, as with herbicides, there is a fungicide registration system that limits the use of specific fungicides to specific crops and fungal diseases where their use has been found to be most effective and safe. Fungal diseases of Brassica oilseeds can reduce the yield and quality of harvested grain. Depending on the nature and severity of the fungal pathogen infection, the impact can range from minor to complete crop loss. Fungicides can help reduce the risk of losses caused by fungal infection, but the costs of spraying fungicides are significant enough to require a benefit-risk type analysis before deciding to apply. Examples of economically significant fungal diseases of Brassica and mustard oilseeds include:

i) Sclerotinia, caused by a fungus whose spores infect Brassica primarily during flowering, and the incidence is associated with periods of high humidity. The stems develop lesions that can eventually kill the plant. Fungicides are available that can control the severity of the infection, but must be applied at specific times in the plant's life cycle (i.e., early and mid-bloom) to achieve the best effect. Often several applications are required within this period of time.

ii) Alternaria blight is a fungal disease of Brassicas that affects plants at all stages of growth from early seedling to maturity, although mature plants are more susceptible. The greatest economic impact is noted on the yield and quality of grain. Application of a foliar fungicide late in the flowering stage is an effective way to mitigate the more detrimental effects of the disease on yield and grain quality.

ii i) Blackleg, a fungal disease of Brassica oilseeds, infects plants at all stages, but early stage infections have the most severe consequences, often resulting in plants with necrotic lesions on lower stems that can actually sever the plants at the base. Fungicides are only partially effective, having little protective effect when applied at an early stage of plant growth.

iv) Clubroot is a soil fungus that attacks the roots of Brassica oilseed crops. The spores can persist in the soil for a long time, and there is currently no effective fungicidal treatment. Control can

- 13 044251 require the use of crop rotations that limit the frequency of mustard planting.

i) Insecticides are a third group of pesticide compounds designed to reduce or eliminate crop loss due to pests. Like herbicides and fungicides, insecticides are classified according to their mode of action and the biochemical pathways they target. One classification scheme (IRAC MoA) proposed by the Insecticide Resistance Action Committee (IRAC; http://www.irac-online.org) lists 29 classes of insecticides grouped by common biochemical processes and pathways targeted by insecticidal compounds. As with herbicides and fungicides, the function and persistence of insecticides can also be influenced by their sites of action, that is, whether they are active only on the plant surface when applied, or act as systemic agents. Further differentiation between certain groups of insecticides may be apparent based on whether they exhibit selectivity for certain types of insects, due to distinctive aspects of the biology of these insects. Given that some insects perform beneficial roles such as controlling plant pests, serving as plant pollinators, and improving soil nutrient ratios, it is important that insecticides are not applied indiscriminately, but rather are used in a manner that limits their effects to those needed as much as possible. species. Thus, all conditions such as timing of application, amount and method of application, as well as restrictions on the types of insecticides used and the crops on which they can be used, are included in the registered criteria for the use of insecticides as a means of ensuring their safety and effectiveness. The following are examples of insect species that can have a significant negative economic impact on the yield and quality of Brassica grain and mustard oilseeds:

j) Cruciferous flea beetle is a very common pest of oilseed Brassica and mustard crops, feeding on both leaves and stems. When flea beetle is present in the early stages of crop development, it can cause severe thinning of the crop canopy, which will ultimately have a significant impact on grain yield. There are insecticidal products that can be applied as a foliar spray that are effective in controlling the early stages of an infestation.

k) The adult cabbage moth lays eggs on the surface of the leaves of Brassica and mustard oilseeds, and when hatched, the resulting larva attacks the leaves and stems of the crop. The larvae from the eggs, hatched at later stages, also feed on the seed pods. The most severe infestations are those that begin early in plant growth, as the moth's life cycle can support up to four generations during a typical Brassica oilseed growing season, resulting in ever-increasing cycles of crop damage. In such cases, insecticide treatments are most effective when applied early in the season when larvae are first observed.

l) Both the adult seed moth and its larvae can cause significant damage to Brassica oilseed crops, mainly by disrupting flowering and seed pod development. Adults feed on flower buds, severely weakening them and making them more susceptible to heat-induced damage and loss. The adults lay eggs in seed pods, and the larva feeds on the developing seeds. When the larvae reach adulthood, they emerge from the pod and continue to feed on seeds through the pod wall. Application of insecticides early in the flowering stage is necessary to control the primary infestation to stop progression to a secondary infestation.

m) Seed treatment. Often, specific fungicides and insecticides can be formulated with chemical agents and binders to form a composition that can be applied to the surface of the seeds. This seed treatment forms a stable coating on the surface of the seeds. The resulting processed seed can then be packaged and sold to the farmer. When the seeds are subsequently planted, the fungicide and insecticide will be present at the optimum dose to enable the developing plant to resist early season insect and fungal infestation when the plant is most vulnerable and to allow the seedling to develop a more vigorous stem. Benefits include increased yield potential and reduced need for early season spraying.

Emission rate is the average level of emissions of a given pollutant from a given source relative to the intensity of a particular activity. As a specific example, carbon intensity is the amount of carbon (often expressed in grams of _CO2 ) released during energy production (in megajoules).

Fallowing refers to the agricultural practice of leaving farmland bare of crops or vegetation for one or more growing seasons after a period in which the land is intensively cultivated. The purpose of fallowing is to increase the likelihood of higher yields the following season. The fallow season gives the farmer the opportunity to deal with weed problems in the fields. Weeds can grow in fields that are fallow and then can be

- 14 044251 destroyed either by physical means or by treatment with herbicides. This can be repeated several times during one fallow season and thus depletion of the weed seed bank can be achieved more effectively than using pre-planting, growing and post-harvest measures. Periods of fallow can also benefit the soil by allowing it to store moisture to replace what was depleted by the previous crop. During fallowing, organic material from crop residues and roots of previous crops can be more thoroughly broken down, benefiting the soil even more by improving its structure and nutrient content. Fallow periods also allow the soil microbiome and other soil organisms to replenish their numbers, while a break in crop production can also deplete certain plant pathogens that use crop species as hosts. Those skilled in the art will appreciate that fallow periods built into a crop rotation can provide significant benefits to the health and yield of subsequent and future crops, but it will also be understood that during the fallow period, a field fallow does not provide the farmer with the opportunity for economic returns from cash crops. In contrast, growing Brassica carinata as a cover crop instead of fallowing can provide many of the soil and crop rotation benefits described above, but with the added benefit of allowing the farmer the opportunity for significant economic returns from harvesting valuable oilseed grains. Growing Brassica carinata as a cover crop instead of fallowing also benefits the environment by providing feedstock for low-carbon-intensive biofuel production, as well as providing the opportunity to offset life-cycle GHG emissions by sequestering and transferring significant amounts of carbon to the soil.

The term feedstock as used herein refers to oils obtained from crushing oilseeds and subjected to surface refinement to make them suitable and sufficient primary feedstocks for the production of biofuels using the specified methods.

Fertilizers (inorganic fertilizers, chemical fertilizers, mineral fertilizers) are manufactured nutrients added to the soil by the farmer/producer to supplement existing soil-based nutrients to optimize the growth, yield and productivity of the plants and crops being grown. Nitrogen (N), phosphorus (P) and potassium (K) are the main macronutrients of fertilizers.

Chemical components commonly used as chemical fertilizers include, but are not limited to, the following.

Nitrogen fertilizers: ammonia (NH ₃ ), sodium nitrate (NaNO ₃ ), ammonium nitrate (NH ₄ NO ₃ ), calcium ammonium nitrate (Ca(NO ₃ ) ₂ NH ₄ NO ₃ ), monoammonium phosphate or MAF (NH ₄ H ₂ PO ₄ ), diammonium phosphate or DAP ((NH ₄ ) ₂ HPO ₄ ) and urea (CO(NH2)2).

Phosphorus fertilizers: phosphorus pentoxide (P ₂ O ₅ ), superphosphate or OSP (monocalcium phosphate or Ca (H ₂ PO ₄ ) ₂ ), phosphorites, MAP and DAP.

Potash fertilizers: potassium, potassium oxide (K2O), potassium chloride (KCl), potassium nitrate ( _KNO3 ), potassium sulfate ( _K2SO4 ), potassium phosphate ( _KH2PO4 ) and potassium phosphate (K2HPO4).

While the chemical forms of N, P and K in fertilizers may vary, to allow comparisons between fertilizers containing different forms of the three elements, the proportions are standardized as follows: nitrogen content is expressed as elemental nitrogen, phosphorus content is expressed as _P2 equivalent O ₅ , and the potassium content is expressed in K2O5 equivalent. Conversion factors allow the masses of different mineral forms to be converted into the corresponding standard mass. Testing the soil for pre-existing nutrients before planting is the most reliable way to determine optimal fertilizer application levels. Excessive application of fertilizers at levels higher than necessary based on crop needs and soil nutrient content is not recommended for several reasons. Often the additional costs associated with applying excess fertilizer do not result in increased yields to cover the costs. In addition, excess nutrients can have a detrimental effect on crop growth. For example, excess nitrogen following the establishment of Brassica oilseeds can lead to excessive foliage growth at the expense of flowering and seed nutrition. In addition, excess nitrogen fertilizer may be released from the soil either as material leaching into groundwater and then into water bodies, or through direct evaporation. This leached and/or vaporized material can be converted through an indirect process to N2O (see below) and thus contribute to GHG emissions. Excess nitrogen and phosphate fertilizers leaching from the soil into groundwater can leach into freshwater bodies (lakes and rivers) and reach levels that are sufficient or cause eutrophication and deoxygenation, resulting in damage to the aquatic environment.

The term grain in relation to Brassica carinata refers to the seeds collected at maturity and supplied as a source of oil and flour products.

Greenhouse gases (GHGs) are a subset of gaseous by-products emitted

- 15 044251 from anthropogenic sources, such as from the combustion of hydrocarbon fuels or the release of volatile components of hydrocarbon-containing products, which increase global warming by facilitating the capture of radiant solar energy in the atmosphere. The main greenhouse gases are CO2 (carbon dioxide), CH ₄ (methane), N2O (nitrous oxide) and CFCs (chlorofluorocarbons). Emissions of CFCs, a class of compounds used in aerosol propellants and refrigerants, typically result from their direct release. _N2O emissions can occur during the combustion of hydrocarbon fuels, as well as when released from applied fertilizer. The most important GHGs are CO2, CH ₄ and N2O. GHGs are ranked in terms of their global warming potential (GWP), or ability to promote global warming, based on concentration. When compared in terms of their relative contribution to global warming (global warming potential) on a mass basis, N2O is 265 times stronger than CO2 and CH ₄ is 28 times stronger than CO2 (values taken from the IPCC Fifth Assessment Report: IPCC , 2014: Climate Change 2014: Synthesis Report Contributions of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (Core Writing Group, RK Pachauri and LA Meyer (eds.)). IPCC, Geneva, Switzerland, 151 pp.) Based on the above relative capacities, GHG emissions are often expressed as CO2-equivalent emissions (combining and normalizing the effects of the three greenhouse gases emitted during energy production relative to _CO2 ). GHG emissions and their impact on global warming are typically associated with the combustion of fossil fuels, but GHGs can also be released from the combustion of biomass-based fuels. In the latter case, greenhouse gas emissions are offset by CO ₂ , which is absorbed by plants and crops through photosynthesis.

The term harvest or harvest as used herein refers to the collection of a part of a plant that has matured sufficiently during the growing season to have value as a source of food, feed, fiber, raw material, structural material, or as shoots of the plant itself. Abyssinian mustard is harvested, for example, mechanically, ideally when seed maturity is reached (seeds, pods and stems change color from green to yellow, seed moisture content is 9.5% or less). Abyssinian mustard can be harvested by direct mowing or, if necessary, can be processed at an early stage, dried naturally or using a desiccant, then the dried strip can be harvested by a combine harvester. Canola, which has a higher tendency to droop when mature, is often windrowed before harvesting. Windrowing refers to cutting the canola near the base of the plant and leaving the plant horizontal in the field for several days to allow the grain to reach proper dryness. After drying, the windrow is collected by combining. Another option is called popping, which is similar to swathing except the plant is physically pushed onto its side and allowed to dry out for several days before harvesting with a combine. For all of these changes in harvesting, the common final stage is harvesting with a combine. However, because Abyssinian mustard has a much stronger stem than canola, the preferred method of harvesting Abyssinian mustard is direct harvesting at maturity rather than swathing or popping and then combining. Direct combining allows crops to be harvested in one pass along the rows in the field. Single-pass harvesting produces less CO ₂ than harvesting that involves swathing or pushing in addition to combining, due to reduced fuel consumption.

Harvest index (Hay, 1995) refers to the ratio between the weight of grain harvested from a plant at maturity and the weight of remaining above-ground plant material, which may include stems and branches, remaining associated leaves and empty seed pods (chaff).

Land Use Change (LUC). The term land-use change, as used in the context of environmental sciences, refers to changes in land use that result in significant changes in stored carbon and associated changes in levels of atmospheric CO2 and other greenhouse gas emissions. Examples where LUC results in increased _CO2 and GHG emissions include forest clearing to increase arable land available for agricultural production and grassland clearing to increase arable land for agricultural production. Examples of LUCs resulting in reductions in atmospheric CO ₂ and GHG levels include returning previously cultivated lands to their natural state.

Land management methods. For purposes of the present invention, the term land management practices refers, for a given land use, to those practices or changes in those practices that affect soil carbon, nutrient, and water levels and may also alter levels of atmospheric CO2 and other GHGs. These may include: types of plowing and crop residue management, types and amounts of fertilizer (or other inputs) used, and use of specific crop rotations or fallow seasons. Types of land use and resource use are given in Table. 3 EU document entitled Commission Decision of 10 June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC, including:

i) deep plowing: significant soil disturbance with complete loosening and/or frequent

- 16 044251 year) plowing operations. At planting, a small amount (eg, <30%) of the surface is covered with residue;

ii) reduced plowing: primary and/or secondary plowing, but with reduced soil disturbance (usually shallow and without completely loosening the soil) and usually leaving a surface with >30% residue coverage at planting;

iii) no-till (or no-till): direct seeding without major tillage, with minimal soil disturbance in the seeding area. Herbicides are commonly used to control weeds;

iv) Shallow plowing (or shallow tillage): Low residue return occurs when it is due to residue removal (through collection or burning), frequent fallowing without vegetation, production of low residue crops (e.g. vegetables, tobacco, cotton), without mineral fertilizer or nitrogen-fixing crop;

v) medium plowing (or medium tillage): is an example of annual sowing of grain crops where all crop residues are returned to the field. If residues are removed, then additional organic matter (such as manure) is added. Also requires mineral fertilizer or nitrogen-fixing crops in crop rotation;

vi) high level of manure management: significantly higher carbon input compared to cropping systems with medium carbon input due to the additional practice of regularly adding animal manure; and vii) high level of manure-free tillage: represents a significantly higher input of crop residues compared to medium carbon input cropping systems due to additional practices such as high residual yield crops, green manures, cover crops, improved fallow with vegetation, irrigation, frequent use of perennial grasses in annual crop rotations, but without applying manure (see line above).

Beans (or legumes) are plants of the family Fabaceae (or Leguminosae) that are grown primarily for their seeds; the dried forms are referred to as beans. Legumes are also grown as feed. An important characteristic of legumes is their roots, which have uniquely developed structures known as root nodules that can be colonized by symbiotic nitrogen-fixing rhizobacteria. These symbiotic bacteria give the bean the ability to fix atmospheric nitrogen in the form of ammonia, which is subsequently used by the plant for the biosynthesis of amino acids and proteins. When a plant dies, nitrogen stored as protein returns to the soil and is eventually converted to NO ₃ , which then becomes available to other plants.

Life cycle assessment (LCA) is a systematic set of procedures for compiling and studying the inputs and outputs of materials and energy and their associated environmental impacts directly resulting from the operation of a product or service system throughout its life cycle (ISO 14040.2 Project: Life Cycle Assessment cycle - Principles and Guidelines). LCA is a method for assessing the potential environmental aspects and potential aspects associated with a product (or service) by listing relevant inputs and outputs, assessing the potential environmental impacts associated with those inputs and outputs, and interpreting the results of the inventory and impact phases according to in relation to the objectives of the study (draft ISO 14040.2: Life cycle assessment - Principles and guidelines).

Aspects of LCA include the following.

A functional unit that defines and delineates what is being studied, the relevant process steps, defines the relevant inputs and outputs, and provides a basis for comparison between alternative fuels, fuel production processes, or raw materials.

System boundaries that define which process(es) should be included in the analysis of a particular system: for transport fuels, the most commonly used system boundary is called the complete fuel production cycle and includes all stages from raw material extraction, processing, transportation, storage, distribution network to final combustion in the vehicle engine. An option for a full fuel production cycle is from well to reservoir, which includes all stages of the fuel production system, with the exception of fuel combustion in the vehicle engine.

Allocation methods that are used to separate process emissions when one or more by-products, along with the main product, are produced during the process. A specific example of this is the distribution that occurs when oilseed-derived biofuels are analyzed from well to reservoir until the oil is extracted from the oilseed. The BioGrace model used here takes into account the fact that grinding oilseeds produces not only oil (the biofuel feedstock) but also a protein-rich flour by-product. Before processing oilseeds into separate flour and oil fractions, a partition coefficient is applied to all process emissions, which reflects the proportion of energy associated with the oil portion of the oilseeds, based on the oil's LHV (lower heating value). After separating the oil and flour fractions, all energy consumption and emissions are attributed to the oil, while flour no longer contributes.

Low carbon intensity biofuels, or low CI biofuels, as used herein, are biofuels whose production results in a lower carbon intensity than the corresponding petroleum-based fuel under one or more renewable fuel regulations. For example, using the values indicated in table. 1 from DeJong et al., a low CI biofuel will have a CI value of less than 83.8 g CO ₂ eq/MJ according to the EU-RED standard, and a low CI biodiesel will have a CI value less than 91.8 g CO ₂ -eq./MJ in accordance with US RFS requirements.

A low-GHG biofuel, as used herein, is a biofuel whose production results in lower GHG emissions than the corresponding petroleum-based fuel, as determined using one or more LCA models, in accordance with one or more rules use of renewable fuels.

Macronutrients used in the present invention relate to nitrogen (N), phosphorus (P), potassium (K) and sulfur (S). Nitrogen, as the main component of amino acids, proteins and chlorophyll, primarily promotes the growth of leaves and foliage. Phosphorus, contained in DNA/RNA polymers, nucleoside precursors and coenzymes, membrane phospholipids, etc., is necessary for the formation of roots and flowers, as well as for the development and maturation of seeds and fruits. Potassium is an important regulator of water movement, turgor, flowering and fruiting.

Manure, as used in the present invention, refers to organic matter, mainly derived from animal feces, which can be used as an organic fertilizer in agriculture. Manure contributes to soil fertility by adding organic matter and nutrients such as nitrogen, which are used by bacteria, fungi and other organisms in the soil. Most animal manure consists of feces. A common form of animal manure is farmyard manure (FYM), which may also contain plant material (often straw) that was used as animal bedding and absorbed feces and urine. Manure from different animals has different qualities and requires different application rates when used as fertilizer. For example, sheep manure is high in nitrogen and potassium, and cattle manure is a good source of nitrogen as well as organic carbon. Chicken manure has a concentrated content of both nitrogen and phosphate.

Maturity is defined as the stage at which the filling of the seed pods is complete, the pods and seeds have lost all green color, and the seed moisture content is less than 9%. At this point, most, if not all, leaves have fallen off, the stems and trunks have turned yellow, and the plant is considered dead.

Micronutrients: In addition to the major macronutrients (N, P and K), smaller amounts of secondary macronutrients including calcium (Ca), magnesium (Mg) and sulfur (S), and trace amounts of micronutrients (such as boron, copper, iron, manganese , zinc) can also promote optimal plant growth and productivity.

_N2O emissions: Cultivated agricultural soils can emit nitrous oxide ( _N2O ), a potent greenhouse gas 265 times more potent than CO2. Nitrous oxide can be released directly or indirectly. Direct emissions of nitrous oxide can result from nitrification and denitrification of soil nitrogen by microorganisms. Soil nitrogen can arise from the use of synthetic nitrogen fertilizers (urea, ammonia or nitrate based), organic fertilizers (mulch, manure), natural animal or poultry waste (manure/urine), decomposition of crop/agricultural residues, ongoing mineralization/demineralization organic matter in the soil. Indirect nitrous oxide emissions result from a multi-step process. The first stage involves the release of ammonia or nitrate/nitrite (NOx)-based gases into the atmosphere. These emissions can arise from a number of sources: direct volatilization of nitrogen-containing compounds found in synthetic fertilizers, organic fertilizers or animal waste; burning of plant waste/crop residues; and combustion of fuels for agricultural machinery. The second stage involves the deposition of these atmospheric nitrogen compounds (ammonium, NOx) through precipitation, for example on the surface of soil or water bodies, and the last stage involves subsequent conversion through denitrification/nitrification to N2O and release to the atmosphere. A secondary source of ammonium and NOx for indirect nitrous oxide emissions involves the leaching of nitrogen fertilizers, organic fertilizers or animal waste from the soil into groundwater levels and then into water bodies, after which they can be converted by denitrification/nitrification to N2O. Formulas for calculating direct and indirect N2O emissions from agricultural lands based on mass amounts of synthetic fertilizers, organic fertilizers, above- and below-ground biomass at harvest, agricultural fuel use, and soil saturation during the growing season are provided by the BioGrace model (and are based on described in IPCC 2006, 2006, IPCC Guidelines for National Greenhouse Gas Inventories, prepared by the National Greenhouse Gas Inventory Program, Eggleston H.S., Buendia L., Miwa K.,

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Ngara T. and Tanabe K. (eds). Published by: IGES, Japan.)

Seed pods are specialized structures that contain seeds as they develop and mature into grains. Seed pods protect the seeds from the external environment and provide energy and nutrients for seed development. When the seeds reach full maturity, the seed capsule becomes dry and brittle, and, having lost all chlorophyll, becomes yellow in color. At the same time, the seed capsule becomes susceptible to cracking, physical opening of the structure, which allows the release of the mature seed (grain).

Sequence cropping is the practice of growing two or more crops in succession on the same piece of land in a crop year and allows farmers to expand their use of the land during the season, that is, during the winter when crops are not usually cultivated. This allows the farmer to earn additional income. Consistent crop production does not entail changes in land use since the land has already been cleared and used for agricultural production. Additionally, using a cover crop such as Brassica carinata as a succession crop allows the farmer to benefit from the soil during the fallow period and generate income from the sale of Abyssinian mustard grains.

Soil is made up of minerals, organic matter, gases, liquids and various animal and plant organisms. Soil is formed over time by the interaction of climatic, geological, hydrological and atmospheric factors on the minerals that make up the earth's crust. Given enough time, soil develops layers or horizons that vary in structure and composition, determined by the relative proportions of sand, silt, and clay.

Soil carbon: Soil contains organic and inorganic (mineral) forms of carbon. The organic carbon fraction may consist of dead and decaying matter or contain living plants, insects, fungi, or microbial matter. The soil organic carbon stock reference value (SOCST) can be estimated from the known carbon content of a region's reference soil type, modified by the effects of the region's climate (based on the climate classification scheme described earlier). In the table below. 3, adapted from table. 1 of the EU document entitled Commission Decision of 10 June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC, summarizes the estimated values of SOC _ST (in tons of carbon per hectare in the soil layer at a depth of 0- 0.3 m) for topsoil classes in a given climate region.

Table 3

Topsoil SOCST values for mineral soil types in specific climate zones

Climate type Soil organic carbon content for different soil types (T/ha) Clay soil with high activity Clay soil with low activity Sandy soils Alkaline soils Moderate cold dry 50 33 14 - Moderate cold wet 95 85 71 115 Moderate warm dry 38 24 19 - Moderate warm humid 88 63 34 - tropical dry 38 35 31 - tropical wet 65 47 39 -

The value of soil organic carbon (SOC), taking into account factors such as land use, land management and agricultural inputs, can then be calculated for the cultivated land (SOC=SOC _ST xF _LU xF _MG xFI, where SOC = soil organic carbon measured as mass of carbon per hectare ; SOCST = standard soil organic carbon in the top 0-30 cm of soil, measured as mass of carbon per hectare and determined as described above, FLU = land use coefficient, reflecting the difference in soil organic carbon associated with land use type compared to standard soil organic carbon; F _MG = management factor, reflecting the difference in soil organic carbon associated with a management practice compared to standard soil organic carbon; FI = input factor, reflecting the difference in soil organic carbon associated with different levels of carbon input into the soil, compared to standard soil organic carbon. Table 4, adapted from table. 2 EU document entitled: COMMISSION DECISION of 10 June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC, provides values for FLU, F _MG , FI for crops grown in different climatic zones under certain types of land use, land management practices and levels of resource input.

Table 4

Values for FLU, F _MG and FI

Climate zone Land use Land management Land use costs Flu Fmg Fi Moderate cold/warm dry Cultivation Deep plowing Low 0.80 1.00 0.95 Average 0.80 1.00 1.00 Tall, with manure 0.80 1.00 1.37 Tall, no manure 0.80 1.00 1.04 Medium plowing Low 0.80 1.02 0.95 Average 0.80 1.02 1.00 Tall, with manure 0.80 1.02 1.37 Tall, no manure 0.80 1.02 1.04 No plowing Low 0.80 1.10 0.95 Average 0.80 1.10 1.00 Tall, with manure 0.80 1.10 1.37 Tall, no dung 0.80 1.10 1.04 Moderate cold/warm humid Cultivation Deep plowing Low 0.69 1.00 0.92 Average 0.69 1.00 1.00 Tall, with manure 0.69 1.00 1.44 Tall, no dung 0.69 1.00 1.11 Medium plowing Low 0.69 1.02 0.92 Average 0.69 1.02 1.00 Tall, with manure 0.69 1.02 1.44 Tall, no manure 0.69 1.02 1.11 No plowing Low 0.69 1.10 0.92 Average 0.69 1.10 1.00 Tall, with manure 0.69 1.10 1.44 Tall, no dung 0.69 1.10 1.11 tropical dry Cultivation Deep plowing Low 0.58 1.00 0.95 Average 0.58 1.00 1.00 Tall, with manure 0.58 1.00 1.37 Tall, no dung 0.58 1.00 1.04 Medium plowing Low 0.58 1.02 0.95 Average 0.58 1.02 1.00 Tall, with manure 0.58 1.02 1.37 Tall, no dung 0.58 1.02 1.04 No plowing Low 0.58 1.10 0.95 Average 0.58 1.10 1.00 Tall, with manure 0.58 1.10 1.37 Tall, no dung 0.58 1.10 1.04 tropical wet Cultivation Deep plowing Low 0.48 1.00 0.92 Average 0.48 1.00 1.00 Tall, with manure 0.48 1.00 1.44 Tall, no manure 0.48 1.00 1.11 Medium plowing Low 0.48 1.02 0.92 Average 0.48 1.02 1.00 Tall, with manure 0.48 1.02 1.44 Tall, no manure 0.48 1.02 1.11 No plowing Low 0.48 1.10 0.92 Average 0.48 1.10 1.00 Tall, with manure 0.48 1.10 1.44 Tall, no manure 0.48 1.10 1.11

If cropping practices, land management practices, or costs associated with a particular agricultural land base have changed and the consequences of the change(s) in carbon stocks need to be known, then SOC values for the reference scenario (SOCR) as well as the actual scenario can be calculated in a similar manner (SOCA) and use the values to calculate soil carbon storage (Esca) using the formula E _sca =-(SOC _R -SOC _A )*3.664/20 years, or in tons of carbon (as CO ₂ /year over a period of 20 years If the E _sca value is negative, it represents a loss of soil carbon, whereas if it is positive, it represents a net accumulation of soil carbon.

Soil classification. Soils are classified based on the World Soil Resources Reference Base (WRB), which proposes 30 soil reference groups. These 30 reference soil groups are divided into 10 classes as described below.

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Class No. 1 includes all organic soils. Organic soils (histosols) are soils that are unusually rich in organic matter in various stages of decomposition, and where the rate of decomposition is hampered by prolonged exposure to low temperatures and/or wet conditions. The remaining mineral soil groups are classified into nine classes based on their most specific identifying factors that are key to their formation and differentiation.

Class No. 2 includes all types of mineral soils that are especially caused by human influence. This class consists of one abstract soil group: anthrosols.

Class No. 3 includes mineral soils, the formation of which is determined by the properties of their source material. The class includes three abstract soil groups:

Andosols of volcanic origin and from volcanic areas;

arenosols, which include sandy soils of desert areas, beaches, inland sand dunes, etc.; and Vertisols - heavy clay soils found in marshy areas, river banks and basins.

Class No. 4 includes mineral soils, the formation and characteristics of which are influenced by their topographic/physiographic or hydrological conditions. This class consists of four abstract soil groups:

stratified fluvisols found in lowlands and marshy areas;

unstratified gleysols, usually found in marshy areas;

Leptosols, shallow soils found in higher areas, usually above rocky subsoils; and regosols, deeper soils of upland areas found above gravelly subsoils.

Class No. 5 includes soils that are only moderately developed due to their relatively young age and are therefore representatives of a fairly diverse reference soil group: Cambisols.

Class No. 6 includes soils determined by the climate of arid tropical regions. The six reference soil groups in this class share a long history of dissolution and transport of weathering products, which led to the formation of deep and genetically mature soils:

plinth salts, consisting of a mixture of clay and quartz (plinthite);

Ferralsols with very low cation exchange capacity and the absence of any weathering components;

aluminum-rich alisols with high cation exchange capacity;

Nitisols, deep soils with a red tint, high in iron;

acrisols - soils with low fertility, high clay content and high concentrations of aluminum; and lixisols, soils with low fertility, low cation exchange capacity, but high base saturation.

Class No. 7 includes soils determined by climate in arid and semi-arid regions. Five abstract soil groups are collected in class No. 7:

solonchaks, with a high content of soluble salts, solonetz, with a high percentage of adsorbed sodium ions, gypsisols with a horizon of secondary enrichment in gypsum, durisols with a layer or nodules of soil material that is cemented by silica, and calcisols with secondary carbonate enrichment.

Class No. 8 includes soils that occur in the steppe zone between a dry climate and a temperate zone with uniform moisture, and includes three reference soil groups:

chernozems with deep, very dark surface soils and carbonate enrichment in the subsoil, chestnut soils with shallower brownish surface soils and accumulation of carbonate and/or gypsum at some depth (these soils are found in the driest parts of the steppe zone), and phayozems, dark red soils prairies with high base saturation, but without visible signs of secondary carbonate accumulation.

Class No. 9 contains brownish and grayish soils of regions with a temperate climate with uniform moisture and includes five abstract soil groups:

acidic podzols with a bleached elution horizon above an accumulation horizon of organic matter with aluminum and/or iron, planosols with a bleached topsoil above a dense, slowly permeable soil, low-base albeluvisols with a bleached elution horizon grading into a clay-rich subsurface horizon, rich the bases are Luvisols with a distinct clay accumulation horizon, and Umbrisols with a thick, dark, acidic surface horizon rich in organic matter.

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Class No. 10 includes soils from permafrost regions and is included in one reference soil group: cryosols.

In the EU Commission decision of 10 June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC (http://eurlex.europa.eu/legalcontent/EN/TXT/?uri=uriserv% 3AOJ.L_.2010.151.01.0019.01.ENG) 30 abstract soil groups were further grouped into 6 main soil categories, including sandy (arenosols), wetland soils (gleysols), volcanic soils (andosols), alkaline soils (podzols), low-active soils clay soils (with low cation exchange capacity or CEC) and highly active clay soils (with high CEC).

Straw is defined as all the above-ground parts of a plant (excluding grain) that are collected during the harvesting and combining process and then separated from the grain and later deposited back into the field.

Stubble is the post-harvest residue left in the field that contains material below the point of harvesting by the combine and that which is not collected for subsequent grain threshing and winnowing operations. In shallow-till or no-till models, most or all of the stubble remains in the field when the next crop is planted. This requires special seeding equipment that can open an unobstructed path to the soil, ensuring good seed-to-soil contact in the middle of the stubble. This is especially important in situations where the stubble may be particularly dense, such as stubble from a previous corn crop.

Variety refers to a botanical taxonomic designation by which a variety is classified below a species or subspecies, and a legal definition by which the term variety refers to a commercial variety of a plant that is protected in accordance with the terms set out in the International Convention for the Protection of New Varieties of Plants, the international agreement administered by UPOV (Union for the Protection of New Varieties of Plants). The term cultivar (under UPOV) describes a new, physically distinct, uniform and stable cultivar of a plant developed by a breeder. The latest definition provides precise rights of protection and ownership between treaty countries for a plant breeder of a legal variety if the above four criteria are met.

Detailed Description of the Invention

Carbon dioxide emissions from agricultural practices and land use changes may contribute to the overall carbon intensity of the biofuels pathway. The present invention describes the use of Brassica carinata as a distinct agricultural feedstock for biofuels, and also lists the associated climate zones and crop rotations used in cultivation, as well as associated agronomic practices to maximize the reduction of carbon dioxide emissions during its rise, even before such to the extent that the net flow of carbon during cultivation and harvesting contributes to a net reduction in atmospheric _CO2 levels (i.e., negative carbon intensity).

The present invention describes the production of Brassica carinata, a crop whose cultivation has not previously been demonstrated to be useful for the production of feedstocks that can be used to produce biofuels with low carbon intensity. Brassica carinata, due to its unique growth habit and ability to withstand frost, drought and heat, provides these benefits that were previously unknown. In this invention, as well as in the examples and description provided herein, the practical utility of Brassica carinata as a crop rotation option in a number of growing scenarios in a variety of production practices has been demonstrated as worked examples. Examples are provided where, under optimal conditions, negative overall carbon intensity for crude oil and meal production can be achieved, which can offset the carbon intensity encountered during biofuel production and distribution and results in a significant amount of carbon per hectare per year being returned to the soil.

Brassica carinata has a unique growth habit among the oilseed Brassicaceae in producing a mature plant that is more branched than other oilseed Brassica species (Geschet al., 2015). When comparing biomass between existing commercial Brassica napus and Brassica carinata cultivars, aboveground biomass accumulation was found to be 1.8-2 times higher per unit area for Brassica carinata cultivars compared to modern commercial Brassica napus cultivars. In the case of Brassica carinata, seed yields approach those of the most modern rapeseed varieties such as Brassica napus, with the volume of aboveground biomass almost doubling (Geschet al., 2015).

The deep and extensive taproot system of Brassica carinata can extend 60–90 cm below the soil surface with more than 50% of the root mass in the top 30 cm (e.g., see Seepaulet al., 2016). Taproots can penetrate compacted layers of soil, improving soil structure in the process. They can absorb minerals and nutrients that would normally leach into groundwater and make those nutrients available to subsequent crops in the rotation. Roots also make up a significant proportion of the plant's total biomass—approximately 20–25% of the plant's aboveground biomass measured at maturity (Ganet al., 2009a)—and provide additional carbon sequestration that will be returned to the soil after harvest. Roots are not only

- 22 044251 are a carbon sink, but also serve as a channel through which carbon-containing molecules can also be released into the environment at the root-soil interface. The release of carbon by living root tissue, also known as rhizodeposition, occurs during plant growth and maturation and involves three sources of carbon deposition into the surrounding soil: carbon originating from detached root cells; carbon derived from secreted mucus; and carbon from small molecules that leak out from root cells, the latter representing an important source of root-sequestered carbon (Nguyen, 2003). The carbon sequestered by Brassica napus roots was estimated to be about 350 kg/ha of carbon per growing season (Ganet al., 2009b).

The carbon content of Brassica carinata is estimated to be between 45 and 47% of the dry biomass (Gasolet al., 2007, Ducaet al., 2015) and thus represents a significant pool of carbon stored both aboveground and belowground. in parts during the growing season. At maturity, Abyssinian mustard grains are usually harvested by harvesting, which mows and collects the above-ground plant material, consisting of stems and branches where the seed pods are located. The seed pods are threshed and the grains collected, and any remaining material, including the now empty pods, stems, branches and petioles (collectively referred to as crop straw), is returned to the field, which, together with the remaining plant stubble, can now potentially contribute to increasing soil carbon levels by breakdown of residues by soil bacteria, fungi and mold.

When examining pathways for crop-based biofuel production, most opportunities to reduce carbon intensity lie in feedstock production and, in particular, at the crop production stage. Given that crops absorb more CO ₂ than they emit during their life cycle, it is possible, with some modification of cropping practices, to introduce negative carbon intensity at this stage of the pathway, resulting in a reduction in the overall carbon intensity of the pathway.

There are significant opportunities to reduce CO2 and GHG emissions from growing, harvesting, storing, shipping and processing crops. For example, reducing costs, especially of inorganic nitrogen fertilizers, can have a significant impact on carbon-based emissions, both by reducing emissions associated with fertilizer production and by reducing soil nitrogen, which, if present in excess compared with the needs of agricultural crops, can be released into the atmosphere in the form of nitrous oxide, which is 265 times more active than CO2. Although nitrogen is an essential nutrient for most annual crops, its application can be finely adjusted based on known crop requirements and determination of pre-existing nitrogen levels in the soil. In addition, annual leguminous crops, which are known to fix atmospheric nitrogen in the soil, can be used in crop rotations with other non-nitrogen-fixing crops to reduce the latter's need for added nitrogen fertilizers.

GHG emissions can also occur due to indirect land use change (ILUC). ILUC is a consequence of the increasing demand for land to grow new energy and feed crops, displacing food crops. To continue to meet the demand for displaced food crops, new land must be found to replace the land currently used for commodity production. This may involve clearing forest or grassland, releasing large amounts of previously stable, fixed CO2 and other GHGs in the process. A crop that can be successfully grown on underutilized, marginal cropland, as a cover crop, or as a fallow replacement in crop rotations will have a major advantage as an energy or feed crop due to its reduced potential for ILUC.

Crop rotation is an important means of reducing greenhouse gas emissions from ILUC by increasing the efficiency of existing land use and reducing the need for new agricultural land. Crop rotation also takes advantage of favorable relationships between complementary crop species to increase yield and productivity. For example, a subsequent crop that is different from the preceding crop may prevent the long-term establishment or persistence of a disease specific and/or endemic to the preceding crop (ie, the succeeding crop serves as a green manure crop). The following crop can also act as an alternative to the fallow cycle and provide the benefits of a cover crop, e.g. preventing soil erosion, helping to conserve moisture and recirculating essential minerals and nutrients and improving soil structure. Some crops, such as legumes, can fix atmospheric nitrogen in the soil and reduce the need for exogenously added nitrogen fertilizers for subsequent crops.

Crop crops such as Brassica species can release compounds with antimicrobial properties (i.e. glucosinolates) into the soil, which can result in protection against plant pathogens for the next crop. Glucosinolates are a unique class of sulfur-containing compounds

- 23 044251 synthesized by Brassicaceae, which, along with their catabolites, have powerful antimycotic and antimicrobial activity. Synthesis of glucosinolates occurs in a number of plant compartments, including the roots, and the release of glucosinolates and their catabolites in root exudates is believed to contribute to the ability of Brassica oilseeds to provide effective disease control when included in cereal rotations. Thus, yields from crops grown in crop rotation can often exceed yields from crops grown as a monoculture, and crop rotations that include cover crops that replace winter fallow are more productive and sustainable than those that rely on fallow. For example, in reviews of data related to wheat cultivation in Australia, Europe and North America, Angus and colleagues (Angus et al., 2011, Angus et al., 2015) teach that cultivating wheat after growing Brassica napus or Brassica juncea consistently leads to to increase the yield of the next wheat crop compared to wheat after wheat.

Those skilled in the art will understand that the references are provided by way of example and are not intended to limit the scope of the invention.

In one aspect, a means for producing biofuel feedstocks with low carbon intensity is provided. In particular, the invention describes methods of using agricultural practices, including land management practices, to provide feedstocks for the production of biofuels with low carbon intensity as a consequence of growing the oilseed crop Brassica carinata in a crop rotation sequence. A specified crop rotation sequence that covers periods of time when conventional cash crops are not grown has been found to provide significant benefits when the carbon intensity and GHG emissions associated with that cultivation are assessed according to various established carbon intensity and GHG assessment schemes.

For example, growing Brassica carinata during winter in tropical and temperate climates has produced the unexpected result of good economic yields of Brassica carinata grain. In addition, dryland cultivation of Brassica carinata further provides the unexpected result of successful harvesting of Brassica carinata grains, which provide feedstocks suitable for the production of advanced low-carbon-intensive biofuels such as hydrogenated vegetable oils (HVEs), for the production of renewable diesel and jet fuels .

The present invention also provides agricultural methods, which include crop rotation strategies and land management techniques to reduce fossil fuel inputs and maximize atmospheric carbon fixation during cultivation, for producing Brassica carinata seeds to provide feedstock that can be used to produce biofuels and other products with low carbon intensity. These production methods and crop rotation strategies had not been previously described, and the low carbon intensity and low GHG profile of the resulting crop were neither obvious nor predictable.

The unique characteristics of the Brassica carinata cultivars described in the present invention, when combined with specific land management practices, seasonal timing of crop rotations, and upstream crops in said rotation, enable the production of feedstocks for low carbon-intensive biofuels and other renewable products.

The use of Brassica carinata oilseeds to produce low-carbon biofuel feedstocks also provides a plant meal or protein source as a by-product after oil extraction. It is noted that the same GHG reduction associated with the oil component of the grain is also associated with the flour portion or byproduct of oil extraction. Accordingly, the present invention relates to a new protein-rich feed additive with low greenhouse gas emissions, a product that is useful for raising livestock in a more environmentally friendly manner. Therefore, the present invention describes a flour product with a low PG value for use as an animal feed additive.

In some embodiments, Brassica carinata is shown to be grown in tropical and warm climates as a winter cover crop interspersed with summer crops such as beans, cotton, peanuts or sesame where conventional fallow cultivation practices have been followed (Seepaulet al. , 2015). This is the first example of Brassica oilseeds providing a consistent yield in this geographic area when planted in early to late November, and is made possible by the unique ability of the established Abyssinian mustard to survive and recover from heavy frost when other Brassica oilseeds such as canola, could not recover adequately (Seepaul et al., 2015). Oilseed crops such as soybean are highly susceptible to frost kill (Hume and Jackson, 1981) and therefore would not be considered as a possible winter cover crop in this environment. Benefits of using Brassica carinata as a winter crop in this environment include the ability to retain winter moisture and nutrients in the soil, reduce the leaching of nitrogen, phosphate and other residual nutrients into local waterways, and provide a means to increase soil organic carbon (Newmanet al ., 2010 (corrected)). This introduces a new and viable variety

- 24 044251 bringing a winter oilseed crop to a region where it did not previously exist offers benefits in terms of improved soil structure and additional moisture for crops planted after the Abyssinian mustard harvest and provides conditions for increased yields of subsequent crops. From a sustainability perspective, growing Brassica carinata as winter cover may not necessarily displace food crop production; since the land was previously agricultural, there are no direct impacts of land use change.

In other embodiments, Brassica carinata can be grown in semi-arid regions as a summer crop as part of a crop rotation with summer and winter grain crops (eg, winter and spring wheat). Similarly, Brassica carinata can be grown in perennial crop rotations in combination with legumes (such as peas, lentils, peanuts and soybeans) and grain crops (such as corn, wheat, barley, rye, oats or spelt) in areas with high summer temperatures (average July temperature 18-24°C) and limited total precipitation (less than 200-500 mm per year). In the southern hemisphere, the crop can be sown in late autumn or early winter in moist soil. In areas with higher rainfall it can be sown as early as early spring.

Brassica crops have long been shown to be beneficial when grown in crop rotations with cereal crops such as wheat, an important food crop suitable for production in semi-arid regions due to its shorter growing season and tolerance to extreme climatic conditions. Crop rotations with oilseeds, as well as with Brassica forages, have consistently shown a positive impact on the yield of the next crop due to the effect of improving soil structure and moisture conservation, as well as the ability to provide a break in the disease cycle that affects cereal performance (Angus, et al. , 2011). The ability to break disease cycles in cereals is due to the lack of susceptibility of Brassica to many cereal diseases, but may also be due to the ability to actively resist the persistence of pathogens in the soil through the biofumigant activity of root exudates and residues (Kirkegaard and Sarwar, 1998). Brassica carinata also lends itself to conservation or no-till models that further conserve soil moisture as well as reduce the release of stable organic carbon deposits from disturbed soil layers. Again, in a semi-arid environment, the application of the invention would enable sustainable production of biofuel feedstock from a non-food crop, either as part of a crop rotation where its cultivation replaces fallowing, or on marginal lands. In any case, direct or indirect land use change will be minimal due to the cultivation of Abyssinian mustard in this environment. Fixing atmospheric _CO2 as soil organic carbon will further reduce life-cycle emissions of GHGs with the added benefit of providing conditions for increased yields of food crops grown in crop rotation.

In other embodiments of the invention, Brassica carinata can also be grown by planting in the spring, with harvest in the fall in northern temperate regions as part of a crop rotation with summer and winter grain crops, whereby Brassica carinata follows the harvest of the previous winter cereal, replacing the summer one. fallow, and after harvesting winter grains are sown.

Suitable grains include wheat, barley, rye or oats. As well as the benefits gained from replacing fallow, the additional benefits of increased overall productivity and reduced direct and indirect land use changes mean that biofuels produced from second generation (non-fuel) oil seed feedstocks such as Abyssinian mustard oil can comply with the EU directive, which approves second generation raw materials, allowing double counting of mandatory emission limits. Brassica carinata's greater resistance to early-season frost and its ability to better cope with higher temperatures and lower humidity during flowering and seeding, as well as its resistance to lodging, allows it to better withstand early- and late-season weather extremes (Seepaul et al. , 2015), making it a more reliable oilseed crop option for growers in semi-arid regions.

Similarly, Brassica carinata can be grown in perennial crop rotations in combination with legumes (such as peas, lentils, peanuts and soybeans) and grain crops (such as corn, wheat, barley, rye, oats or spelt) in areas with high summer temperatures (average July temperatures 18-24°C) and limited total precipitation (less than 200-500 mm per year). In the southern hemisphere, the crop can be sown in late autumn or early winter in moist soil. In areas of high rainfall, it can be sown as early as early spring.

Thus, Brassica carinata can be cultivated in a range of climates in crop rotation with various summer or winter grains, legumes or other crops to produce oilseeds that provide both oil feedstock for biofuel production and livestock feed. Raw materials obtained from grain make up almost the entire mass of seeds with little or no waste. Significant plant residues left after grain harvesting are returned

- 25 044251 in the field and make a major contribution to increasing the organic carbon content in the soil and reducing the amount of carbon released into the atmosphere in the form of CO2. Increased soil carbon results in improved soil structure, moisture retention and nutrient balance, which improves growing conditions for subsequent crops. In addition, when rotated with other crops, Abyssinian mustard can provide an interruption of infection, increasing the productivity of subsequent crops. Brassica carinata can also be sown directly into stubble left over from previous crops. This practice, known as conservation tillage or no-till agricultural practices, conserves soil moisture in semi-arid regions, maintains soil structure, and reduces GHG emissions from fuel use during tillage equipment operation. Overall, Abyssinian mustard cultivation provides feedstock for biofuel production while providing measurable GHG emissions reductions (as assessed using various GHG audit models) across a range of production scenarios and geographic regions.

Based on its oilseed crop, Brassica carinata not only provides raw materials for the production of a potential alternative to fossil fuels, but by increasing biomass production, it can also provide an effective mechanism for fixing and returning carbon to the soil. Soils are also a potential sink for fixing carbon and reducing air emissions. Of all the carbon stocks in the environment, soil is second in size only to the oceans, and contains an estimated organic carbon content of more than 2.3 GT (Jobbagy and Jackson, 2000), more than 4 times the amount of carbon stored in all plant biomass. Moreover, due to factors such as intensive farming, deforestation, erosion, etc., actual soil carbon stocks are depleted relative to their maximum storage capacity. It is estimated that the additional carbon sequestration capacity in soils may exceed 50-100 GT (Lal 2008a, Lal 2008b).

In one aspect of the invention, Brassica carinata is sown into the stubble of the harvested crop, with or without intermediate fallowing, such that the preceding crop, which is not Brassica carinata, is the last crop to be harvested before sowing the Abyssinian mustard.

In one embodiment, the precursor crop is a legume crop, which may include the following annual crops: beans, peas, lentils, soybeans, peanuts, or alfalfa. Legumes are a useful crop choice in crop rotations due to their ability to fix atmospheric nitrogen, increasing the nitrogen content of the soil. Oilseed crops such as Brassica carinata are characterized by requiring significant amounts of nitrogen to achieve maximum yield. As a follow-on crop to legumes, Brassica carinata can utilize stored nitrogen in the soil, which in turn reduces its need for nitrogen-containing fertilizers. It is well known that the production of ammonia-based fertilizers using methods such as the Haber process results in significant emissions of CO2, which is a major by-product of the reaction. In addition, reducing the amount of exogenously added inorganic nitrogen fertilizers can also reduce soil emissions of nitrous oxide produced by soil bacteria and microflora. Nitrous oxide, a potent greenhouse gas whose one gram is equivalent to 265 grams of CO2, also contributes significantly to the overall carbon intensity of plant-based biofuel pathways. As an ultimate benefit of Abyssinian mustard following legumes in crop rotation, the residue left after legume harvesting has a consistency that does not affect good contact of Abyssinian mustard seeds with the soil, which leads to better germination and establishment of the Abyssinian mustard crop, and also allows use the advantages of agricultural production without plowing or with shallow plowing.

To reduce GHG production resulting from the application of excess amounts of inorganic nitrogen fertilizer, in one embodiment of the invention, land management techniques include reducing the use of inorganic nitrogen fertilizer compared to the recommended amount of nitrogen fertilizer for Brassica carinata for the growing environment. In some embodiments, the land management methods include reducing the use of inorganic nitrogen fertilizer to about 40-100% of the recommended amount of nitrogen fertilizer for Brassica carinata in the growing environment. In some embodiments, the land management methods include reducing the use of inorganic nitrogen fertilizer to about 40-90% of the recommended amount of nitrogen fertilizer for Brassica carinata in the growing environment. In other embodiments, land management methods include reducing the use of inorganic nitrogen fertilizer to about 50-70% of the recommended amount of nitrogen fertilizer for Brassica carinata in the growing environment. This practice of reducing the use of inorganic nitrogen fertilizer would be beneficial, for example, when soil nitrogen levels before Brassica carinata are planted are high, such as when Brassica carinata is planted after a legume harvest or after the first crop to which a large amount of nitrogen fertilizer has been applied.

In one embodiment of the invention, the Brassica carinata variety is planted in stubble and collected

- 26 044251 crops with or without intermediate fallowing in regions with a climate classified as tropical rainy in accordance with the guidelines established by Directive 2009/28/EC, so that all months can be frost-free, with temperatures above 18°C in maritime areas, and while the climate is mostly humid, 3-5 months of winter can be drier. In some embodiments, Brassica carinata is planted in the fall or winter for harvest in the spring or summer. In other embodiments, Brassica carinata is sowed in the spring or summer for harvest in the fall or winter. In some embodiments, the Brassica carinata cultivar is selected from regionally adapted cultivars selected for one or more traits from the group consisting of superior oil yield per planted area, shorter ripening time, drought tolerance, increased disease resistance, or crack resistance seeds

In another embodiment of the invention, Brassica carinata is planted in the stubble of a harvested crop, with or without intermediate fallowing, in regions with a climate classified by warm temperature and rainfall, as defined in Directive 2009/28/EC as a climate with moderate to high humidity during throughout the year, with no particular dry season and more than 8 months with temperatures of 10°C or higher. In some embodiments, Brassica carinata is planted in the fall or winter for harvest in the spring or summer. In other embodiments, Brassica carinata is planted in the spring or summer for harvest in the fall or winter. In some embodiments, the Brassica carinata cultivar is selected from regionally adapted cultivars selected for one or more traits from the group consisting of superior oil yield per planted area, shorter ripening time, drought tolerance, increased disease resistance, or crack resistance seeds

Crop Rotation Scenarios: The present invention can be implemented in a number of different climate zones in which Brassica carinata, when planted in rotation with the first crop, is planted in the stubble of the harvested first crop. The planting and harvesting season for Brassica carinata can vary depending on geography and crop rotation practices in each region. As described above, crop rotations that include cereals and Brassica oilseeds, such as Brassica carinata, can be beneficial for grain yield and quality because the Brassica oilseed, which is unable to infect or serve as a host for pathogens, allows temporary and a physical break in the disease cycle that affects cereals, preventing these diseases from becoming resistant. The roots and crop residues of Brassica carinata contain toxic substances such as glucosinolates, which can also actively inhibit the spread of pathogens in the soil.

Scenario A. In one embodiment of the present invention, Brassica carinata is sown into the stubble of a harvested crop with or without intermediate fallow in regions with a climate classified as tropical humid, with Brassica carinata planted in the fall or winter for harvest in the spring or early summer. In some embodiments, the harvested crop is a leguminous crop, including, but not limited to, beans, peas, peanuts, lentils, and soybeans. In other embodiments, the harvested crop is a grain crop, including, but not limited to, wheat, barley, rye, oats, or corn. In other embodiments, the harvested crop is cotton or sesame.

Scenario B: In one embodiment of the present invention, Brassica carinata is sown into the stubble of a harvested crop with or without intermediate fallowing in regions with a climate classified as tropical humid, where Brassica carinata is planted in the spring for harvest in the summer or fall. In some embodiments, the harvested crop is a legume crop, including, but not limited to, peas, lentils, and soybeans. In other embodiments, the harvested crop is a grain crop, including, but not limited to, wheat, barley, rye, oats, or corn.

Scenario C. In one embodiment of the present invention, Brassica carinata is sown into the stubble of a harvested crop with or without intermediate fallowing in regions with a climate classified as warm-temperate humid, resulting in Brassica carinata being planted in the fall or winter for harvest in the spring or summer. . In some embodiments, the harvested crop is a legume crop, including, but not limited to, peas, lentils, and soybeans.

Scenario D: In one embodiment of the present invention, Brassica carinata is seeded into the stubble of a harvested crop with or without intermediate fallowing in regions with a climate classified as warm-temperate humid, with Brassica carinata planted in the spring or summer for harvest in the fall. In some embodiments, the harvested crop is a grain crop, including, but not limited to, wheat, barley, rye, oats, or corn.

Scenario E. In one embodiment of the present invention, Brassica carinata is sown into the stubble of a harvested grain crop, with or without intermediate fallow, in regions with a climate classified as temperate warm dry, with Brassica carinata planted in the fall or

- 27 044251 in winter for harvesting in spring or summer. In some embodiments, the harvested grain crop is corn. In other embodiments, the harvested grain crop is wheat.

Scenario F: In one embodiment of the present invention, Brassica carinata is sown into the stubble of a harvested cereal crop with or without fallow in regions with a climate classified as temperate cool dry, with Brassica carinata planted in the spring for harvest in the summer or fall. In some embodiments, the harvested grain crop is corn. In other embodiments, the harvested grain crop is wheat.

Scenario G In one embodiment of the present invention, Brassica carinata is seeded into the stubble of a harvested grain crop with or without intermediate fallowing in regions with a climate classified as cool-humid temperate, with Brassica carinata planted in the spring for harvest in the fall. In some embodiments, the harvested grain crop is corn. In other embodiments, the harvested grain crop is wheat.

Scenario H: In one embodiment of the present invention, Brassica carinata is sown into the stubble of a harvested grain crop, with or without intermediate fallowing, in regions with a climate classified as tropical dry, where Brassica carinata is planted in the fall or winter for harvest in the spring or summer. In some embodiments, the harvested grain crop is corn. In other embodiments, the harvested grain crop is wheat.

In any of the above embodiments and crop rotation scenarios, the field may be subject to reduced (medium) plowing, shallow plowing, or no plowing prior to planting. As will be known to those skilled in the art, seeding Abyssinian mustard into stubble, particularly cereal stubble, under circumstances where no-till or shallow-till cultivation methods are used, will entail the use of seeding methods and machinery designed to ensure constant contact at the appropriate depth between the seeds and the soil surface in the specified stubble. It is also known to those skilled in the art that snow can further compact the soil, and as described previously, where shallow or no-till tillage practices do not allow heavy crop stubble to be removed or compacted topsoil to be loosened, due consideration should be given to planting Abyssinian mustard using appropriate methods and mechanisms to ensure constant seed-to-soil contact at the appropriate soil depth.

In any of the above embodiments and crop rotation scenarios, sowing of Brassica carinata is carried out using a seeder or similar tool set at a depth of 0.50 cm; 0.63 cm; 1.25 cm; 1.9 cm; 2.5 cm; 3.75 cm or 5 cm, or any depth in the specified range, at a seeding rate of 3.0 kg seeds/ha, 4.0 kg seeds/ha, 5.0 kg seeds/ha, 5.6 kg seeds/ha, 6.7 kg seeds/ha, 7.8 kg seeds/ha, 9.0 kg seeds/ha, 10.1 kg seeds/ha, 11.2 kg seeds/ha or any other rate in the specified range. The spacing between rows can be set to 10 cm, 20 cm, 30 cm, 40 cm, 50 cm or any distance within the specified range. As will be known to those skilled in the art, as previously described, when shallow or no-till land management practices are unable to remove heavy crop stubble or loosen compacted topsoil, proper care should be given to planting Abyssinian mustard using appropriate techniques and machinery to ensure consistent contact of seeds with soil at the appropriate soil depth.

In any of the above-described embodiments and crop rotation scenarios, inorganic (mineral) fertilizer is applied by surface feeding, inter-row feeding, broadcast application, or foliar application. In some embodiments, the inorganic (mineral) fertilizer includes one or more of an inorganic nitrogen (N) fertilizer, a phosphate fertilizer, a potassium fertilizer, and a sulfur fertilizer. In some embodiments of the invention: inorganic nitrogen (N) fertilizer is applied at the rate of 30 kg/ha, 45 kg/ha, 56 kg/ha, 67 kg/ha, 78 kg/ha, 90 kg/ha, 101 kg/ha, 112 kg/ha, 123 kg/ha, 135 kg/ha, 150 kg/ha, 165 kg/ha or any quantity within the specified range; phosphorus (P) fertilizer is added at the rate of 22, 34, 45 or 56 kg or any amount in the specified range in P ₂ O ₅ equivalents per hectare; potassium (K) fertilizer is added in the amount of 30, 45, 56, 67, 78, 90, 101 or any amount in the specified range, equivalent to K2O per hectare; and sulfur (S) fertilizer is added at the rate of 11 kg/ha, 17 kg/ha, 22 kg/ha, 28 kg/ha, 34 kg/ha, 40 kg/ha or any amount within the specified range. In some embodiments, inorganic nitrogen fertilizer and sulfur fertilizer are applied in a split dose, one half at planting and the other half before flowering, while P and K fertilizers are applied in a single dose at planting. In loamy soils where inorganic N-fertilizer and S-fertilizer are applied in separate doses, one-quarter to one-third of the inorganic N-fertilizer and one-third to one-half of the S-fertilizer are added at planting, and the remainder is added at stemming, while While fertilizers P and K are used in a single dose during sowing. In deep sandy soils, fertilizer can be applied in three doses: at sowing or the appearance of the first sprout, a third of the inorganic fertilizer N, half of the S fertilizer, half of the K fertilizer and all P fertilizer is applied at sowing or the appearance of the first sprout; at

- 28 044251 during stemming, add a third of inorganic nitrogen fertilizer and the remaining S and K fertilizers; and finally, at early flowering, add the remaining N fertilizer.

In any of the above embodiments and crop rotation scenarios, manure and/or organic fertilizer can be used to provide part or all of the nitrogen fertilizer needed during Abyssinian mustard cultivation. Manure can be applied by broadcasting, banding, incorporation, or other methods known to one skilled in the art, using a manure spreader, clod spreader, tanker, or other suitable equipment known to one skilled in the art. The manure may be one or more types of poultry manure, cattle feces, pig feces, or other agricultural wastes rich in nitrogen and other nutrients. As will be known to one skilled in the art, the amount of manure applied to a field will depend on the composition of the manure, in particular the nitrogen content. Typical manure application rates range from 0.5 to 10 t/ha or any rate within that range. For example, manure can be applied at a rate of about 0.5; 1; 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 7; 8; 9 or 10 t/ha. When applied at this rate, manure can provide approximately 20 to 100%, or any percentage of the specified range, of nitrogen fertilizer required during Brassica carinata cultivation. For example, manure can provide about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95% or 100% of the nitrogen fertilizers needed to grow Brassica carinata. In some embodiments, the manure may provide from about 30 to 90% or any percentage of the specified range of nitrogen fertilizer needed to grow Brassica carinata. In other embodiments, the manure may provide from about 40 to 80%, or any percentage within the specified range, of the nitrogen fertilizer required to grow Brassica carinata. In other embodiments, the manure may provide from about 50 to 75%, or any percentage within the specified range, of the nitrogen fertilizer required to grow Brassica carinata.

Based on a conservative yield estimate for Abyssinian mustard in semi-arid regions of the United States of 2090 kg grain per hectare (equivalent to 899 kg oil per hectare assuming 43% oil by weight), by 2022, at a nutrient input of 45-90 kg/ha inorganic N fertilizers, 17-34 kg/ha P-fertilizer, 0-11 kg/ha K-fertilizer, 3.1 kg/ha pesticide and 32.7 l/ha diesel fuel, and estimated GHG emissions associated with crushing, storage and oil transportation, biofuel production, and biofuel distribution that were roughly equivalent to those for soybeans and camelina, the EPA's estimated total GHG emissions reductions in a hypothetical Abyssinian mustard pathway to produce biodiesel from biomass or advanced fuels such as GBM would allow the producer to receive RIN credits Type 4 or Type 5 (EPA-HQ-OAR2015-0093-; FRL-9926-80-OAR; Notice of Opportunity to Comment on the analysis of greenhouse gas emissions associated with the production and transportation of Brassica carinata oil for use in biofuel products. Federal Register, Vol. 80, No. 79, Friday April 24, 2015, p 22996-23003; https://www.gpo.gov/fdsys/pkg/FR-2015-04-24/pdf/2015-09618.pdf). Thus, an aspect of this invention is that Abyssinian mustard is a non-edible oilseed crop that can be grown in semi-arid environments to provide optimal biofuel feedstock and achieve significant reductions in GHG emissions while improving soil quality, which can help improve subsequent yields. food crops.

When grown in accordance with any of the above embodiments or crop rotation scenarios, B. carinata will fix approximately 0.5 to 5.0 tons of CO ₂ per hectare per year, or any amount of CO 2 in that range, in the soil. For example, the plant B. carinata in any of the above embodiments or crop rotation scenarios will record 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5 or 5.0 t CO2 per hectare per year, or any amount of CO2 in the specified range, in the soil.

Feedstock derived from B. carinata grain harvested from B. carinata in accordance with any of the embodiments or crop rotation scenarios described above can be used to produce low carbon intensity (low CI) biofuels such as biodiesel or jet fuel. In some embodiments, the low CI biofuel has a carbon intensity value that is reduced by at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, or more g CO ₂ -eq/J, by relative to the carbon intensity of the corresponding conventional fossil fuel. In other embodiments, the low carbon-intensity biofuel has a carbon-intensity value that is reduced by about 50-200 g CO2e/MJ of energy produced, or any amount in that range, relative to the carbon-intensity value of the corresponding fossil-derived fuel. In other embodiments, the low carbon-intensity biofuel has a carbon-intensity value that is reduced by about 75-200 g CO ₂ -eq/MJ of energy produced or any amount in that range relative to the carbon-intensity value of the corresponding fossil-derived fuel. In other embodiments, the low carbon intensity biofuel has a carbon intensity value that is reduced by 100-200 g CO ₂ eq/MJ of energy produced or by

- 29 044251 any amount within a specified range relative to the carbon intensity of the relevant fossil fuel.

Similarly, growing B. carinata in any of the above embodiments or crop rotation scenarios will result in a reduction in life cycle GHG emissions of approximately 60-400% or any percentage in that range when used to produce green (renewable) diesel fuel and when measured relative to GHG production during refining and production of conventional diesel fuel from fossil fuels. For example, growing B. carinata in any of the embodiments or crop rotation scenarios described above will result in life cycle GHG emissions reductions of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%. 350%, 375% or 400% when used to produce green (renewable) diesel and when measured against GHG output from refining and production of conventional diesel from fossil fuels. In some embodiments or crop rotation scenarios, growing B. carinata will result in a reduction in life cycle GHG emissions of approximately 75-300%, or any percentage within that range, when used to produce green (renewable) diesel fuel and when measured relative to GHG production during the time of refining and production of conventional diesel from fossil fuels. In some embodiments or crop rotation scenarios, the growth of B. carinata will result in a reduction in life cycle GHG emissions of approximately 90-250%, or any percentage within that range, when used to produce green (renewable) diesel fuel and when measured relative to production GHGs during refining and production of conventional diesel fuel from fossil fuels.

Examples

Example 1: Successive cultivation of Brassica carinata as winter cover after peanuts in a crop rotation. This example demonstrates the cultivation of Brassica carinata as a cover crop in a tropical humid climate to produce low carbon biofuel feedstock and protein-rich meal for use as livestock feed. As an example of cultivation in this area, Brassica carinata was grown sequentially during the winter of 2015-2016 as a fallow-replacing winter cover crop on two farms in the southeastern United States (Northern Florida). Farm 1 was located near Jay, Florida and Farm 2 was located near Alta, Florida. The previous crop grown on both farms was peanuts, a type of legume.

In table Figure 5 shows how the cultivation of Abyssinian mustard was carried out on each farm. Both farms were located in northern Florida, an area classified as tropical humid as described above and in Table 1. 2. The soil in the North Florida region where both farms are located was classified as acrisol, which belongs to Soil Classification Class #6 in the above definitions.

The fields were prepared for planting using a no-till approach. Sowing was carried out using Brassica carinata AAC-A120 with the sowing rate and depth within the optimal ranges suggested in the Agrisoma 2015 Growing Guide for the region (https://agrisoma.com/ckfinder/userfiles/files/2017_18_SE_Handbook.pdf).

The inputs used in both trusses (listed in Table 5) are also within the suggested ranges given in the manufacturer's manual. To more accurately assess the totality of GHG emissions associated with Abyssinian mustard cultivation, farm managers recorded the fuel consumption of all agricultural equipment used during the cultivation and harvest of Abyssinian mustard (shown in Table 5). All irrigation was done through natural rainfall, so no additional irrigation was required or used. Upon reaching maturity, the seeds were collected by direct mowing with a combine, and virtually all plant materials except harvested grain were returned to the field. The net yield and yield per hectare of harvested Abyssinian mustard grain (at 10% moisture content) from both farms are given in Table. 5.

Table 5

Details of growing Abyssinian mustard (farm 1 and farm 2)

Units Farm 1 (FL) Farm 2 (FL) Cultivation details Location North Florida North Florida Previous culture Peanut Peanut Abyssinian mustard planting area ha 23.87 8.09 Seeding rate kg/ha 5.60 4.48 Total seeds used kg 133.68 36.25

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Harvest kg 46,576.98 13,957.69 Yield/Area kg/ha 1.951 1.725 Energy sources used in cultivation Diesel fuel consumption MJ ha' ¹ year' ¹ 928.14 3,704.37 Agrochemicals N-fertilizer(kg N) kg N ha' ¹ year' ¹ 78.4 129.9 Manure kg N ha' ¹ year' ¹ - - CaO fertilizer (kg CaO) kg CaO ha' ¹ year 1 - - KgO-fertilizer (kg KgO) kg KgO ha' ¹ year ¹ 78.4 89.6 Р2О5- fertilizer (kg Р2О5) kg Р2О5 ha' ¹ year ¹ 22.4 44.8 Pesticides kg ha' ¹ year' ¹ - -

Example 2: Brassica carinata grown successively as winter cover behind cereals (corn) in a tropical humid climate (FL/SE USA). This example demonstrates the cultivation of Brassica carinata as a cover crop in a tropical humid climate to provide feedstock for biofuel production and protein-rich meal for use as livestock feed.

Brassica carinata was grown successively during the winter of 2015-2016 as a fallow replacement winter cover crop on farms in the SE United States. The previous crop grown was corn, an example of a type of grain crop, and in contrast to previous practice, the residues of the previous grain crops were not introduced into the soil by plowing. In table Table 6 summarizes the details of Abyssinian mustard cultivation carried out in the tropical humid climate zone, as described above and in table. 2. The soils in this region of North Florida are of the sandy arenosol type (see Soil Classification Class #3 in Definitions above).

Table 6

Details of growing Abyssinian mustard

Units Data Cultivation details Location Florida Previous culture Corn Seeding rate kg/ha 4 Harvest kg/ha 2 114 Harvest ⁽¹⁾ MJ/ha 50 229 Energy sources used in cultivation Application of diesel fuel MJ ha' ¹ year' ¹ 1 385 Agrochemicals N-fertilizer (kg N) kg N ha' ¹ year' ¹ 141.1 Manure kg N ha' ¹ year' ¹ - CaO fertilizer (kg CaO) kg CaO ha' ¹ year' ¹ 340.0 KgO-fertilizer (kg KgO) kg KgO ha' ¹ year' ¹ 89.6 Р2О5- fertilizer (kg Р2О5) kg Р2О5 ha' ¹ year' ¹ 88.5 Pesticides kg ha' ¹ year' ¹ - Grain transport Truck to grain loader km 33.8

⁽¹⁾ Based on Net Heating Value (HCH) for rapeseed at 0% moisture content of 26.4 MJ/kg, taken from: JEC E3 Database (version 31-7-2008).

⁽²⁾ Based on density 832 kg/ ^m3 , NTS 43.1 kg/MJ, taken from: JEC E3 database (version 31-7-2008)

In the above environment, Brassica carinata was sown in mid to late November into the stubble of the previous corn crop, usually at a depth of 1.25-2.5 cm. Two inbred open-pollinated Abyssinian mustard cultivars are currently recommended for this growing environment: Resonance AACA120 (currently time previously protected by Plant Breeders Rights of Canada, Application No. 15-8718) or Avanza 641 (WO 2017/181276A), the latter variety selected based on regional

- 31 044251 adaptation, high yield, reduced glucosinolate content and increased frost resistance. The seeding rate was increased to 4 kg/ha to achieve an optimal plant density in the range of 80-180 plants per ^m2 . The resources used were as described in Table. 6, and included inorganic nitrogen, potassium and phosphorus fertilizers in the proposed quantities. Arenosol soils are moderately acidic, which requires the addition of dolomitic lime (CaO). Inorganic nitrogen fertilizer was applied at a rate of 141.1 kg/ha, which, although higher than typically recommended for Abyssinian mustard, may be justified for sandy soil types in regions with humid tropical climates where nitrogen, which may leach from root zones, tends to be washed out from the root zone.

To most accurately estimate total GHG emissions during cultivation, the consumption of all motorized agricultural equipment fuel consumption across the entire farm used during cultivation and harvesting of Abyssinian mustard was recorded. All irrigation was provided by rainfall, so additional irrigation was not required or used. Grown as a winter cover crop under short daylight conditions, Brassica carinata took just over 5 months to reach maturity, at which point Abyssinian mustard was harvested by direct harvesting with a combine. The grain was collected, and virtually all plant materials except the harvested grain were returned to the field. Grain yield per hectare (at 10% moisture content) and total fuel consumption are given in Table. 6.

Example 3: Brassica carinata as summer cover after legumes (lentils) in cool dry temperate (NT) climates. This example demonstrates the cultivation of Abyssinian mustard as a summer cover crop to produce biofuel feedstock and protein-rich flour for livestock feed. Brassica carinata was grown as a summer cover crop sown into the stubble of a previous lentil crop in a cool, dry temperate climate zone in the northern United States and the southern prairies of Canada. In table Table 7 provides details of cultivation carried out in a climate region classified as a temperate cold dry region, as described above and in Table. 2. Examples of such regions include the northern states of the United States, as well as the southern prairies of western Canada. The soils in these regions are classified as highly active clayey soils (Commission Decision of June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC). Lentils, a legume species, are an increasingly important crop in these regions and are often grown in crop rotations that include cereals such as wheat and/or Brassica oilseeds.

In the environment described above, Brassica carinata was sown from mid-April to early May, typically when soil temperatures were above 4-5°C, into the stubble of the previous lentil crop and at a depth of 1.25-2.5 cm. Two inbred open-pollinated varieties of Abyssinian mustard, Resonance AACA120 (Plant Breeder's Rights Canada, filed April 9, 2015, Application No. 158718) and 3A22 (U.S. Provisional Patent Application No. 62/326111, filed April 22, 2016 and currently International Patent Application No. PCT/CA 2017 /050474, filed April 18, 2017) are currently recommended for this growing environment, with the latter variety selected based on regional adaptation, high yield, lower glucosinolate content, and earlier maturity. The seeding rate was adjusted to achieve plant densities in the range of 80-180 plants per ^m2 , which corresponds to a seeding rate of 5 to 9 kg/ha. Resources correspond to the description in the table. 7 and include inorganic nitrogen, potassium and phosphorus fertilizers in the indicated quantities. The soil pH in these regions is typically 7.0 or higher and therefore dolomite lime is not required. Although 90kg/ha of inorganic nitrogen is the recommended rate, as lentils do increase soil nitrogen levels through the ability of their roots to fix atmospheric nitrogen, the amount of nitrogen added can be reduced accordingly for subsequent crops. Thus, in table. 7 lists two cultivation scenarios that differ only in the amount of inorganic nitrogen added: one (scenario 1) at the usual recommended rate and the other (scenario 2) using inorganic nitrogen halved to take advantage of the nitrogen provided by the previous lentil harvest.

- 32 044251

Table 7

Details of growing Abyssinian mustard

Units Scenario 1 Scenario 2 Cultivation details Location Northern states Northern states Previous culture Lentils Lentils Seeding rate kg/ha 5 5 Harvest kg/ha 1 800 1 800 Harvest ⁽¹⁾ MJ/ha 42,768 42,768 Energy sources used in cultivation Application of diesel fuel ⁽²⁾ MJ ha' ¹ year' ¹ 1000 1000 Agrochemicals N-fertilizer (kg N) kg N ha' ¹ year' ¹ 90 45 Manure kg N ha ⁴ year' ¹ - - CaO fertilizer (kg CaO) kg CaO ha ⁴ year ⁴ - - KgO-fertilizer (kg KgO) kg KgO ha ⁴ year ⁴ 80 80 Р2О5- fertilizer (kg Р2О5) kg Р2О5 ha ⁴ year ⁴ 40 40 Pesticides kg ha ⁴ year ⁴ 10 10 Grain transportation Truck to grain loader km 100 100

⁽¹⁾ Based on Net Heating Value (HCH) for rapeseed at 0% moisture content of 26.4 MJ/kg, taken from: JEC E3 Database (version 31-7-2008).

⁽²⁾ Based on density 832 kg/ ^m3 , NTS 43.1 kg/MJ, taken from: JEC E3 database (version 317-2008)

In order to more accurately estimate the totality of GHG emissions associated with cultivation using all agricultural implements and machinery used at all stages of growing and harvesting Abyssinian mustard, the amount of diesel fuel used in all operations was recorded. For this purpose, a default value of 1000 MJ/ha diesel use was used, which represents a moderately high level of motorized agricultural implement use. All watering was achieved from natural precipitation, so additional irrigation was not required or used. When grown as a summer cover crop, Brassica carinata usually reaches maturity within 4 months, after which it is harvested by direct mowing with a combine. The grain was collected, and virtually all plant materials except the harvested grain were returned to the field. The net yield and yield per hectare of harvested Abyssinian mustard grain (at a moisture content of 10%) for both nitrogen use scenarios are shown in Table. 7.

Example 4: Brassica carinata grown successively as winter cover after legumes (soybeans) in a warm temperate, humid climate (Uruguay). This example demonstrates the successive cultivation of Brassica carinata as a fallow replacement winter cover crop to produce biofuel feedstock and protein-rich meal for use as livestock feed. Brassica carinata was grown as a winter cover crop sown into the stubble of the preceding soybean crop in the temperate, warm, humid climate of Uruguay. In table Table 8 summarizes the details of the cultivation carried out in the winter of 2015, in a climate region classified as warm-temperate humid, as described above and in Table. 2. Examples of such regions include most of Uruguay's arable land. The soils in these regions are classified as high activity clayey soils (Commission Decision of June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to Directive 2009/28/EC). Lentils, a legume species, are an increasingly important crop in these regions and are often grown in crop rotations that include cereals such as wheat and/or Brassica oilseeds.

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Table 8

Details of growing Abyssinian mustard

Units Data Cultivation details Location Uruguay Previous culture Soybeans Seeding rate kg/ha 7 Harvest kg/ha 2 100 Output ⁽¹⁾ MJ/ha 49,896 Energy sources used in cultivation Application of diesel fuel ⁽²⁾ MJ ha' ¹ year' ¹ 277 Agrochemicals N-fertilizer (kg N) kg N ha' ¹ year' ¹ 59.7 Manure kg N ha' ¹ year' ¹ - CaO fertilizer (kg CaO) kg CaO ha' ¹ year' ¹ 18.3 KgO-fertilizer (kg K ₂ O) kg KgO ha' ¹ year' ¹ 67.4 Р2О5- fertilizer (kg Р2О5) kg Р2О5 ha' ¹ year' ¹ 61.0 Pesticides kg ha' ¹ year' ¹ 13 Grain transportation Truck to grain loader km 376

⁽¹⁾ Based on Net Heating Value (HCH) for rapeseed at 0% moisture content of 26.4 MJ/kg, taken from: JEC E3 database (version 31-7-2008).

⁽²⁾ Based on density 832 kg/ ^m3 , NTS 43.1 kg/MJ, taken from: JEC E3 database (version 31-7-2008)

Between mid and late May, seventeen farms in Uruguay covering more than 2,400 hectares were sequentially planted with Abyssinian mustard in the stubble of the previous soybean crop and at a depth of 1.25-2.5 cm. Two inbred open-pollinated plants were recommended for this growing environment. Abyssinian mustard cultivars, Resonance AAC-A120 (currently pre-protected by PBR in Canada, Application No. 15-8718) and Avanza 641 (US plant variety patent pending), the latter cultivar selected based on regional adaptation, high yield, lower glucosinolate content, frost resistance and earlier maturity. In table Table 8 shows the average values of seeding rates, resources used and yields for all farms. The seeding rate was adjusted to achieve optimal plant density corresponding to an average seeding rate of 7 kg/ha. The resources used correspond to those given in table. 8 and include the average value of inorganic nitrogen, potassium, phosphorus and calcium (lime) fertilizers used on all farms. The soil pH value in these regions is often moderately acidic, up to pH 5.7, and thus dolomitic lime was used to reduce soil acidity. Based on soil nitrogen analysis results, an average of 59.7 kg/ha of nitrogen was applied. This is below the recommended level of applied nitrogen (90 kg/ha) but reflects levels of pre-existing nitrogen in the soil, likely from the previous legume crop. In table Figure 8 also shows average levels of pesticide use (including pesticides, herbicides and fungicides) across all farms, since the production of these products consumes energy and therefore contributes to GHG emissions; Using the BioGrace model, the _CO2e emissions from these products were determined and combined into total emissions during the growing phase.

As described previously, to most accurately estimate the total GHG emissions associated with Abyssinian mustard cultivation, all agricultural implements and machinery used during the cultivation and harvest of Abyssinian mustard and the amount of diesel fuel used in their operation were recorded. The average consumption of diesel fuel in all farms was 277 MJ of diesel fuel per hectare. All irrigation was provided by rainfall, so additional irrigation was not required or used. When grown as a winter cover under short-day conditions, Brassica carinata reached maturity within 5–6 months (one to two months longer than required under summer growing conditions), at which point Abyssinian mustard was harvested by direct mowing with a combine harvester. The grain was harvested and virtually all plant materials other than harvested grain were returned to the field. The net yield and yield per hectare of harvested Abyssinian mustard grain (at a moisture content of 10%) are given in table. 8.

Compared to other oilseed varieties grown in Uruguay, Brassica carinata produces both high yield and high biomass. In a 2016 study in Uruguay, the Brassica carinata variety Avanza 641 was planted under identical conditions in three sites, along with several modern open-pollinated and hybrid spring commercial canola-type varieties of Brassica napus. During cultivation, graphs were generated for plant density, pod density, aboveground biomass at harvest, grain yield at harvest, and yield index.

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The data is given in table. 9.

Table 9

Grain and biomass yield for Brassica carinata Avanza 641 grown in parallel with hybrid canola varieties

Material Repetition Plants/ ^m2 Pods/ ^m2 Biomass (kg/ha) Grain yield (kg/ha) V. carinata Avanza 641 1 41.18 9932.35 16544.12 4369.12 V. carinata Avanza 641 2 66.18 13902.94 21691.18 5866.18 V. carinata Avanza 641 3 51.47 10105.88 16617.65 4586.03 V. napus HYOLA 50 1 27.94 5591.18 10000.00 2510.29 V. napus HYOLA 50 2 26.47 5222.06 9117.65 2277.21 B. napus HYOLA 50 3 20.59 7841.18 13676.47 3238.97 B. napus HYOLA 575 CL 1 54.41 7614.71 14044.12 2833.82 B. napus HYOLA 575 CL 2 45.59 5847.06 9411.76 1780.15 B. napus HYOLA 575 CL 3 44.12 5335.29 9044.12 1422.79 B. napus RIVETTE 1 27.94 5182.35 10558.82 2645.59 B. napus RIVETTE 2 25.00 3979.41 7455.88 1732.35 B. napus RIVETTE 3 25.00 4483.82 10529.41 2889.71

Least square means (LSM) were calculated across replicates and means were compared using Tukey's test to determine if any significant differences were observed among the varieties tested (see Table 10). LSM values using the same notation for each dimension are not significantly different.

Table 10

Grain yield and biomass accumulation for Brassica carinata Avanza 641 compared to canola hybrids

Variety Yield (kg/ha) Biomass (kg/ha) Pods/ ^m2 Plants/ ^m2 LSM Designations LSM Designations LSM Designations LSM Designations Avanza 641 4940 A 18284 A 11314 A 53 A HYOLA 50 2675 IN 10931 IN 6218 IN 48 A HYOLA 575 CL 2012 IN 10833 IN 6266 IN 26 IN RIVETTE 2422 IN 9515 IN 4549 IN 25 IN

As can be seen, under the cultivation conditions used in these Uruguayan studies, Brassica carinata significantly outperformed even the most modern hybrid spring canola varieties. This yield advantage was contributed by traits such as pod density and plant density, which were significantly higher for Brassica carinata AVANZA 641. Previous work has demonstrated higher above-ground biomass production of spring-grown Brassica carinata cultivars compared to other oilseed Brassica species in the northern United States (Gesch, et al. 2015). The results presented here demonstrate that Brassica carinata cultivars selected for short-day winter cultivation also produce significantly higher levels of aboveground biomass than other commercial Brassica oilseed crops while maintaining high yield potentials. Abundant biomass production, if managed in conjunction with land management practices such as returning crop residues to the field, smart tillage, and stubble maintenance, can contribute to significant returns of plant nutrients and carbon to the soil (see below).

Example 5: Brassica carinata grown successively as a winter cover crop after cereals (wheat) in New South Wales. This example demonstrates the cultivation of Abyssinian mustard as a cover crop in temperate warm dry and tropical dry climate zones, as seen in the New South Wales wheat belt of eastern Australia, for the production of biofuel feedstock and protein-rich flour as livestock feed. Here, to take advantage of the increased moisture that the winter season provides, Brassica oilseeds (primarily canola varieties) are sown in the fall, grown in winter, and harvested in spring or early summer after a growing season of up to 5-7 months. Similarly, Brassica carinata has been consistently grown as a winter cover crop to replace winter fallow on farms in a subregion known to receive relatively high winter rainfall. The predecessor crop is wheat, which serves as an example of a type of grain crop, and in contrast to previous practice, residues from the predecessor crops are not introduced into the soil by plowing. In table 11 summarizes the details of the cultivation of Abyssinian mustard, carried out in a zone of moderate warm dry climate, as described above and in table. 2. Most soil in this region is classified as Luvisol, Vertisol or Calcisol, which are described respectively in Class No. 9, Class No. 3 and Class No. 7 in the soil classification definitions above.

In the environment described above, Brassica carinata was sown sequentially from mid to late April to late May into the stubble of the previous wheat crop, usually at a depth of 1.25-2.5 cm. The seeding rate was adjusted to 5 kg/ha to achieve an optimal plant density in the range of 80 -180 plants per ^m2 . The resources used were as described in Table. 11, and included inorganic nitrogen, potassium and phosphorus fertilizers in the proposed quantities. 110 kg/ha of inorganic nitrogen fertilizer was used, which, although higher than the generally recommended level for Abyssinian mustard, may be justified for sandy soils in humid tropical environments where nitrogen tends to leach from the root zone.

Table 11

Details of growing Abyssinian mustard

Units Data Cultivation details Location Australia (NSW) Previous culture Wheat Seeding rate kg/ha 5 Harvest kg/ha 2000 Harvest ⁽¹⁾ MJ/ha 47520 Energy sources used in cultivation Diesel applications ⁽²⁾ MJ ha' ¹ year' ¹ 1000 Agrochemicals N-fertilizer (kg N) kg N ha' ¹ year' ¹ 110 (high)/55 (low) Manure kg N ha' ¹ year' ¹ - CaO fertilizer (kg CaO) kg CaO ha' ¹ year' ¹¹ - K ₂ O-fertilizer (kg K ₂ O) kg K2O ha' ¹ year' ¹ thirty Р2О5- fertilizer (kg Р2О5) kg Р2О5 ha' ¹ year' ¹ thirty Pesticides kg ha' ¹ year' ¹ 10 Grain transport Truck to grain collector km 376

⁽¹⁾ Based on Net Heating Value (HCH) for rapeseed at 0% moisture content of 26.4 MJ/kg, taken from: JEC E3 Database (version 31-7-2008).

⁽²⁾ Based on density 832 kg/ ^m3 , NTS 43.1 kg/MJ, taken from: JEC E3 database (version 31-7-2008)

Case Study 6: GHG Emission Reductions for Low Carbon Intensity Biofuels Using Feedstocks Derived from Brassica carinata sequentially grown as a winter cover crop following peanuts in a crop rotation. The BioGrace v1.4 model (http://www.Biograce.net) was used to calculate greenhouse gas emissions from Abyssinian mustard cultivation in the Abyssinian mustard growing example described in Example 1. The model complies with the sustainability criteria of the Renewable Energy Directive (2009/28/EC, RED), which are also specified in the Fuel Quality Directive (2009/30/EC). BioGrace Excel calculations are based on life cycle assessment (LCA) to estimate GHG emissions per MJ of fuel.

This means the following.

The functional unit is the production and use of one MJ of fuel.

All stages of the life cycle from biomass production to fuel distribution are taken into account (see Table 12) and are presented in the calculation sheet in a separate module representing one stage in the biofuel journey. For biofuels, the use phase has no greenhouse gas emissions because the CO2 released is biogenic (and the CH4 emissions generated when the fuel is burned are negligible).

The module collects consumption data and calculates emissions of the three main gases contributing to climate change ( _CO2 , CH4 and N2O). Detailed information about the contribution of each gas is presented at the last stage of the calculation. The sum of all three gases is expressed in the equivalent amount of CO ₂ (CO ₂ -eq.) required to obtain the same greenhouse gas effect (g CO ₂ -eq./MJ of timing fuel produced).

The GHG emissions of each module were then summed to obtain the GHG emissions of the entire path.

However, for the purposes of this example, although the resulting feedstock will be used primarily for the production of fuel oil for use as a fuel replacement in transportation and aviation fuels, the BioGrace model is only used to account for GHG emissions from the cultivation phase of the Abyssinian mustard biofuel pathway, including harvest , drying and transporting grain to storage in order to establish the potential of Abyssinian mustard cultivation to reduce carbon intensity in the associated fuel pathway under cultivation conditions where Abyssinian mustard is grown as a winter cover, replacing fallow and following previous legumes (peanuts) in re- 36 044251 gion in the tropical humid climate zone. However, in order to estimate GHG emissions consistently and in a manner that is consistent with the functional unit, the estimated GBM yield for the shortcut was assumed to be 0.58 MJ GBM/MJ Abyssinian mustard seeds. The production steps for GRM from cultivated Brainica carinata, which are within the well-to-reservoir system boundary of the BioGrace GHG Emissions Calculator, are shown in the drawing. The distribution coefficient for the first three stages of using Abyssinian mustard oil is 0.613.

GHG emissions for input resources and fuels used during cultivation can be estimated based on the amount of input or fuel consumed by multiplying by appropriate emission factors similar to those presented in the BioGrace spreadsheet. Emissions derived from fuels used to transport seeds, oils or fuels can be calculated based on similarly provided emission factors for the relevant fuel type, multiplied by the distance traveled and the fuel use efficiency of the specific mode of transport (e.g. rail, road or marine) .

Lower values of calorific value, expressed in MJ/kg and obtained from a table of such values provided in the BioGrace analytical table, were used to determine the energy content of grains, oils, flour at various stages of the path, and were converted into MJ per MJ timing, taking into account functional unit.

Diesel used to fuel tractors and agricultural equipment used in Abyssinian mustard cultivation (for field preparation, sowing, application of agrochemicals and harvesting), as well as electrical energy used to dry harvested Abyssinian mustard grains, also contribute to greenhouse gas emissions. and they are also counted as part of the growing phase.

GHG emissions for fuels used during cultivation can be estimated based on the amount of fuel used by multiplying by appropriate emission factors similar to those presented in the BioGrace spreadsheet.

The production of inputs used in growing crops, such as fertilizers and pesticides, has associated emissions that should be included as part of the life cycle GHG emissions of the biofuel production pathway; they are calculated based on the amount of inputs used to grow crops and the default emission factor (g P/G/kg feedstock produced) available for the production process of the relevant feedstock (JEC E3 database; version 31-7-2008). There is also an additional source of emissions from cultivation that needs to be accounted for, resulting from field emissions of nitrous oxide (N2O), a greenhouse gas 265 times more potent than CO2. Such field emissions fall into three additional categories: direct N2O emissions from fields, indirect N2O emissions from leaching and runoff, and indirect N2O emissions from volatilization of NH3 and NOx. Field emissions are a consequence of the decomposition or combustion of organic matter derived from crop residues, as well as a consequence of the use of nitrogen fertilizers itself, and are the estimated N2O emissions module in the BioGrace spreadsheet (as described in the Definitions section of this invention).

Transport of grain to collection and storage points supported by commercial grain transporters was also a potential source of GHG emissions. The nature of transport, fuel used during transport and distance traveled were recorded and used to determine net GHG emissions (Table 12). For the purposes of this example, only the transportation of grain to a local storage facility is considered.

Table 12

Transportation of grain, oil and fuel

Vehicles Fuel Distance from Farm 1 (km) Distance from Farm 2 (km) Grain to local storage Truck Diesel 45.05 80.5 Grain from storage to port 1 Truck Diesel 800.17 505.44 Grain from port 1 to port 2 Cargo ship Diesel 8423 Grain to the processing plant Truck Diesel thirty Oil for refining Truck Diesel 1178 GRM to the distribution center Truck Diesel 150 Timing station to gas station Truck Diesel 150

All emissions tabulated during the cultivation, drying and transport phases were added to determine the total emissions for the growing phase (see Table 13 for growing phase emissions for Farm 1 and Table 14 for growing phase emissions for Farm 2 ). For the cultivation of Abyssinian mustard and the subsequent transportation of grain for storage in a local grain storage facility, a partition coefficient is applied to emissions that takes into account the fact that the oil fraction of Abyssinian mustard makes up 63% of the energy of the seed and is the fraction of the seed that is uniquely processed in the GRM. Thus, up to the point where the oil is processed into the timing belt, the emissions produced are multiplied by the distribution coefficient.

Table 13

Emissions from Abyssinian mustard cultivation (Farm 1 from Example 1)

Distribution coefficient Not- distributed results Distributed Results Total All results in CO ₂ -eq./MJ GRM Growing Abyssinian mustard 61.3% 50.47 30.91 Drying Abyssinian mustard 61.3% 0.99 0.61 E _EU cultivation 31.5 Grain transportation (truck) 61.3% 0.28 0.17 Transportation eta 0.17

Table 14

Emissions associated with Abyssinian mustard cultivation (Farm 2 from Example 1)

Distribution coefficient Not- distributed results Distributed Results Total All results in CO ₂ -eq./MJ GRM Growing Abyssinian mustard 61.3% 93.66 57.37 Drying Abyssinian mustard 61.3% 0.99 0.61 E _EU cultivation 58.0 Grain transportation (truck) 61.3% 0.48 0.29 Transportation eta 0.29

The BioGrace model takes into account another factor when calculating net greenhouse gas emissions - that is, the expected reduction in greenhouse gas emissions that could result from improved land management practices if a bioenergy crop is grown, compared to conventional practice. The emissions reduction, called E _sca , suggests that improved land management practices lead to increased carbon sequestration on managed lands, offsetting some of the emissions produced during the growing, processing and transport stages of the journey. In the specific case of growing Abyssinian mustard as described in the present invention, further reductions in emissions are expected due to the transition from deep tillage to no-tillage and the replacement of fallow with a ground cover crop that returns most of its biomass back to the soil at harvest. The BioGrace model quantifies and assigns an E _sca value in terms of tonnes of CO2 returned to soil/ha/year based on these improvements (see E _sca values, Table 15). This is subsequently converted into tons of CO2 returned to soil/MJ of GRM biofuel produced, which is then used to reduce net emissions along the way (see Tables 16 and 17).

As can be seen for the cultivation of Abyssinian mustard on farm 1, emissions of CO ₂ -eq. per MJ of GRM produced are negative (-35.6 tCO ₂ -eq./MJ of GRM produced) if only considering the cultivation phase along the GRM production path, indicating the net reduction in atmospheric GHG levels per unit of fuel produced as a result of growing Abyssinian mustard under conditions growing on farm 1. On farm 2 emissions of CO ₂ -eq. per MJ of the produced timing belt are also negative: -17.6 t CO ₂ -eq/MJ of timing gear is produced. Factors contributing to greater emissions reductions at Farm 1 included: (a) lower use of inorganic nitrogen fertilizer, which contributed to lower field GHG emissions as well as life cycle emissions associated with nitrogen fertilizer production; (b) lower fuel consumption for agricultural machinery used during cultivation, although the number of hectares cultivated was actually higher.

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Table 15

Improved land management (farms 1 and 2)

Actual land use Reference land use Link Climatic region Tropical Tropical Section 6.1 wet wet Solution Soil type Low activity clay Low activity commissions Section 6.2 Tillage No plowing clay Deep Commission decision Table 3, Level of resources used Tall, without plowing Short Commission decision Table 3, SOCst manure 47 tons C/ha 47 tons C/ha Commission decision Table 1, Flu 0.48 tons C/ha 0.48 tons C/ha Commission decision, using climatic region and soil type Table 2, Fmg 1.22 tons C/ha 1.0 tons C/ha Commission decision Table 2, Fi 1.11 tons C/ha 0.92 tons C/ha Commission decision Table 2, soa* 30.6 tons C/ha 20.8 tons C/ha Commission decision p** Mca 1.79 tons CO2/ha/year

*SOCi=SOCsT X Flu X Fmg X Fi **Esca=(SOCA-SOCR)*3.664/20

Table 16

Emissions associated with Abyssinian mustard cultivation (farm 1 of case study 1)

Distribution coefficient Not- distributed results Distributed Results Total All results in COg-Eq./MJ GRM Growing Abyssinian mustard 61.3% 50.47 30.91 Drying Abyssinian mustard 61.3% 0.99 0.61 E _EU cultivation 31.5 Grain transportation (truck) 61.3% 0.28 0.17 Transportation eta 0.17 Bonus OR Esca 100.0% (67.3)* (67.3)* (67.3)* Total (15.6)* (35.63)*

*numbers in brackets are negative

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Table 17

Emissions from growing Abyssinian mustard (farm 2 from case study 1)

Distribution coefficient Not- distributed results Distributed Results Total All results in COg-Eq./MJ GRM Growing Abyssinian mustard 61.3% 93.66 57.37 Drying Abyssinian mustard 61.3% 0.99 0.61 E _EU cultivation 58.0 Grain transportation (truck) 61.3% 0.48 0.29 Transportation eta 0.29 Bonus OR Esca 100.0% (76.2)* (76.2)* (76.2)* Total (52.9)* (17.64)*

*numbers in brackets are negative

Example 7: GHG emission reductions associated with the entire GRM production pathway using Abyssinian mustard as a feedstock when grown sequentially as a winter cover after peanuts in a crop rotation. This example demonstrates the GHG reductions achieved throughout the entire production chain of a PMG using Abyssinian mustard feedstock grown sequentially as a cover crop in a tropical humid climate after legumes (peanuts). As described in the previous example, Brassica carinata was grown during the winter of 2015-2016 as a fallow replacement winter cover crop on two farms in the southeastern United States. The previous crop grown on both farms was peanuts, a type of legume. Contrary to common practice, peanut residues were not incorporated into the soil by plowing after harvest, but instead a no-till treatment was used to keep the peanut residues in the field.

To calculate the greenhouse gas impacts of Abyssinian mustard cultivation in these examples, the BioGrace v.1.4 model (http://www. Biograce.net) was used as previously described. In table Figure 18 summarizes the relevant modules from the BioGrace V1.4 model, which take into account all relevant emissions produced along the GRM biofuel production path (from well to reservoir). The example described here, unlike the previous example which only looked at emissions from cultivation, considers all the emission sources listed above.

During the growing phase, emissions associated with the preparation of seeds and fields, direct and indirect emissions associated with the use of resources, emissions associated with the use of agricultural equipment for sowing, application of inputs, harvesting, etc., emissions associated with the use of energy for drying of grain, and emissions resulting from the release of N2O under field conditions are as described in the previous example (Example 6) and are summarized in Table. 13 and 14.

Table 18

Sources of GHG emissions accounted for by the BioGrace 1.4 model

Emissions during cultivation (E _ec ) Growing Abyssinian mustard Drying Abyssinian mustard Processing Emissions (E _p ) Oil extraction Oil refining Hydrogenation of Abyssinian oil mustard Transport emissions (E _t d) Transportation of Abyssinian mustard grain Transportation of Abyssinian mustard oil Transportation of timing belts to storage Transportation to a gas station Emissions due to change land use (Ei) Emission reduction (Bonus or E _sca )

For processing steps that include oil extraction and biofuel processing, hydrotreated vegetable oil (HVO) production has been selected as the most likely end use of Abyssinian mustard feedstock. During the crushing and oil recovery phase, electricity for operating the press and crushing line equipment, and a natural gas-fired steam generator for heating, are the main sources of GHG emissions considered in the LCA model. Chemicals used in extracting oil from flour (eg hexane) and in degumming and refining the extracted oil (eg NaOH and phosphoric acid) also contribute to life cycle GHG emissions and are also taken into account. For recycling

- 40 044251 Timing Oil Electricity and steam generator powered by natural gas are the main energy source contributing to greenhouse gas emissions, as well as hydrogen used in the hydrotreating process itself. Typically, default emission values are used for these processing steps because they are well-established processes that do not vary much. The existing default values for rapeseed oil extraction and hydrotreating were used for the Abyssinian mustard pathway as they are not expected to be significantly different whether applied to rapeseed or Abyssinian mustard. Emissions at the processing stage are given in table. 19. Although different total quantities of butter are produced and processed due to differences in grain yield, since these emissions are normalized by the total amount of oil produced from each farm's contribution, the normalized processing emissions are equal for each farm's grain production.

Table 19

Emissions from processing Abyssinian mustard grains into biofuels with low carbon intensity

Distribution coefficient Not- distributed results Distributed Results Total All results in COg-Eq./MJ GRM Oil extraction 61.3% 6.41 3.92 Hydrogenation of vegetable oil 100.0% 9.34 9.34 E _r cultivation 13.3

For transportation-related emissions, distances traveled between the farm gate, the nearest grain elevator, crushing plant, timing oil refinery, and gas station are used to estimate transportation fuel requirements. The cost of producing electricity in storage facilities is based on the amount of raw materials and biofuel, which, in turn, is calculated based on grain yield. In this particular example, grain grown on farms in North Florida and South Georgia was transported to one of three collection points and then trucked to the Port of Tampa, where the grain was collected and loaded into the holds of an ocean freight carrier. The grain was transported by sea to Rouen, France, and then by road to Grand Crones for crushing. The vegetable oil was then transported by truck to Antwerp, Belgium, for storage, and then by truck to an oil refinery in Donges, France, to be converted into fuel by VRM refining. Distances traveled and fuel used during transportation (summarized in Table 20) were used to determine emissions produced during transportation phases for both Farm 1 (Table 21) and Farm 2 (Table 22).

For the growing stage, transporting Abyssinian mustard grains to the crusher, oilseed crushing and oil extraction stages, a partition factor was applied to emissions to account for the fact that the oil fraction of Abyssinian mustard constitutes 63% of the seed energy, and it is the seed fraction that is uniquely processed in the GRM. Thus, up to the point where the oil is processed into the timing belt, emissions are multiplied by the distribution coefficient (0.63), while in subsequent stages emissions are considered equal to 100% of the calculated values.

Table 20

Transportation of grain, oil and fuel

Vehicles Fuel Distance from Farm 1 (km) Distance from Farm 2 (km) Grain to local storage Truck Diesel 45.05 80.5 Grain from storage to port 1 Truck Diesel 800.17 505.44 Grain from port 1 to port 2 Cargo ship Diesel 8423 Grain to the processing plant Truck Diesel 15 Oil for refining Truck Diesel 1193 GRM to the distribution center Truck Diesel 150 Timing station to gas station Truck Diesel 150

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Table 21

Emissions from transportation (farm 1)

Distribution coefficient Not- distributed results Distributed Results Total All results in COeq/MJ GRM Grain transportation (truck) 61.3% 0.27 0.17 Grain transportation (truck) 61.3% 4.72 2.89 Grain transportation (sea) 61.3% 6.50 3.98 Transportation of Abyssinian mustard oil 100.0% 0.07 0.07 Transportation of Abyssinian mustard oil 100.0% 2.91 2.91 Transportation of timing belts 100.0% 0.41 0.41 in storage Transportation to a gas station 100.0% 0.74 0.74 Transportation eta 8.3

Table 22

Transportation Emissions (Farm 2)

Distribution coefficient Not- distributed results Distributed Results Total All results in CO3-eq/MJ GRM Grain transportation (truck) 0.48 0.29 Grain transportation (truck) 61.3% 2.98 1.83 Grain transportation (sea) 61.3% 6.50 3.98 Transportation of Abyssinian mustard oil 61.3% 0.07 0.07 Transportation of Abyssinian mustard oil 100.0% 2.91 2.91 Transporting the timing belt to storage 100.0% 0.41 0.41 Transportation to a gas station 100.0% 0.74 0.74 Transportation etd 8.4

As described previously, indirect land use changes can contribute significantly to GHG emissions in the biofuel pathway and are included in the BioGrace model as a potential source of greenhouse gas (GHG) emissions that can be added to the above-mentioned phases. However, in the method of growing Abyssinian mustard according to the present invention, no indirect land use change occurs because the cultivation of Abyssinian mustard replaces the fallow period in the crop rotation and does not displace any other crop.

The BioGrace model takes into account another factor when calculating net GHG emissions - that is, the expected reduction in GHG emissions that could result from improved land management practices for growing a bioenergy crop compared to current practices. The emission reduction, called E _sca , suggests that improved land management practices lead to increased carbon sequestration, thereby offsetting some of the emissions produced during the growing, processing and transport stages. In the specific case of Abyssinian mustard, as described here, additional emissions reductions are expected due to a shift from deep tillage to no-tillage and replacing fallow with a cover crop that returns most of its biomass back to the soil. The BioGrace model quantifies and assigns an E _sca value based on these improvements, which is then subtracted from the net emissions of the entire pathway (Table 23).

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Table 23

Amount of emissions associated with the route (production of timing biofuel from Abyssinian mustard oil)

Total All results in g CO _2-eq./MJ grm Farm 1 Farm 2 E _EU cultivation 31.5 58.0 E _r processing 13.3 13.3 Transportation etd 8.3 8.4 Bonus OR Esca (67.3)* (76.2)* Total (14.2)* 3.5

*numbers in brackets are negative

In table 23 also summarizes the greenhouse gas emissions calculated for all paths from Abyssinian mustard oil to the timing belt when Abyssinian mustard was produced on farm 1 and farm 2. Since both farms, located on the same soil and in the same climatic zones, are in geographical proximity to each other and the crop is processed identically to the same end point, it follows that the processing and transport steps will be very similar in terms of emissions. As can be seen, the only phase that shows a difference in emissions between them is the growing phase and reflects differences in the practices used on each farm. These include differences in planted area, seeding rates, levels of input use (especially nitrogen-based), energy used in cultivation, and ultimately yield. However, as shown in table. 24, the resulting life cycle GHG emissions for the path from Abyssinian mustard to GRM are 14.2 g CO ₂ eq./MJ (Farm 1) and 3.5 g CO ₂ eq./MJ (Farm 2) are significantly lower than the life cycle emissions associated with the production of petroleum-derived diesel fuel at 83.8 g CO ₂ eq/MJ (WTT Annex 1, v.3, paragraphs 2.1 and 3; Z1), ensuring a reduction in GHG emissions compared to diesel fuel by 96-117%.

Table 24

Reduced carbon intensity (CI) and GHG emissions compared to fossil fuel benchmark

Fossil fuel (diesel) Biodiesel from raw materials produced from Abyssinian mustard grains Farm 1 Farm 2 CI (g CO2-eq/MJ) 83.8* -14.2 3.5 Reducing GHG emissions — 117% 96%

*DeJong et al., 2017

Example 8: Brassica carinata grown successively after soybean as winter cover; the impact of manure use on greenhouse gas emissions from farming. Brassica carinata was grown sequentially during the winter of 2015-2016 as a winter cover crop to replace fallowing on two farms in the southeastern United States (Farm A, located near Fort Valley, Georgia, and Farm B, located near Dublin, Georgia). . This region belongs to the climate zone classified as a warm-humid temperate climate zone, as described above and in Table. 2. The soils found in this region of Georgia fall within the general classification of low activity clay soil types (see 2010/335/EU; Commission Decision of 10 June 2010 on guidelines for the calculation of land carbon stocks for the purposes of Annex V to the Directive 2009/28/EC). Both farms' fields were planted with Brassica carinata Avanza 641 according to the procedures described in the Agrisoma Growing Guide for the Region (https://agrisoma.com/ckfmder/userfiles/files/2017_18_SE_Handbook.pdf);

For specific cultivation details, see table. 25. The amounts of applied fertilizers used in both farms are listed in table. 25 and are based on soil analysis results to determine the amount of nutrients added to achieve the recommended ranges suggested in the manufacturer's manual. For Farm A, all nitrogen was applied in the form of inorganic N fertilizer, while Farm B used a mixture of inorganic N fertilizer and manure.

To ensure an accurate estimate of all GHG emissions associated with Abyssinian mustard cultivation, farm managers recorded the fuel consumption of all agricultural equipment used during the cultivation and harvest of Abyssinian mustard (Table 26). Watering at both locations was provided by a combination of natural rainfall as well as supplemental irrigation. Once mature, the seeds were collected by direct harvesting with a combine, and virtually all plant materials except harvested grain were returned to the field. The yield per unit area of harvested Abyssinian mustard grain (at a given moisture content) from both farms is given in Table. 25.

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Table 25

Details of growing Abyssinian mustard (farm A and farm B)

Cultivation details Location Fort Valley, GA Dublin, GA Previous culture Soybeans Soybeans Abyssinian mustard planting area ha 35.64 17.82 Seeding rate kg/ha 4.0 6.0 Harvest kg/ha' ¹ year' ¹ 1912.14 1754.81 Grain moisture 8.2% 9.3% Sources of energy used during cultivation Units Farm A Farm B Application of diesel fuel MJ ha' ¹ year' ¹ 858.93 862.24 Agrochemicals N-fertilizer(kg N) kg N ha' ¹ year' ¹ 110.63 135.39 Manure kg N ha' ¹ year' ¹ - 87.63 CaO fertilizer (kg CaO) kg CaO ha' ¹ year' ¹ - - KgO-fertilizer (kg KgO) kg KgO ha' ¹ year' ¹ - - Р2О5- fertilizer (kg Р2О5) kg Р2О5 ha' ¹ year' ¹ - - Pesticides kg ha' ¹ year' ¹ 3.54 4.14

To calculate greenhouse gas emissions from the cultivation of Abyssinian mustard with and without manure, the BioGrace v.1.4 model (http://www.Biograce.net) was used as described in Example 6, using the same principles for accounting for greenhouse gas emissions for the use of resources and fuel during cultivation and transportation. However, for the purposes of this example, although the resulting feedstock will be used primarily to produce fuel oil for fuel replacement applications in transportation and aviation fuels, the BioGrace model is only used to account for GHG emissions during the growing phase of the Abyssinian mustard biofuel pathway, including harvest , drying and transporting grain to storage to demonstrate the potential of Abyssinian mustard cultivation to reduce carbon intensity in the associated biofuel pathway in a growing environment where Abyssinian mustard is grown as a fallow replacement winter cover crop on a farm in a warm temperate humid climate, and to evaluate the influence of manure used at the cultivation stage on the carbon intensity of the resulting GRM biofuel. However, in order to estimate GHG emissions consistently and in a manner that is consistent with the functional unit, the estimated GBM output for the shortened path was assumed to be 0.58 MJ GBM/MJ harvested Abyssinian mustard grain. The partition coefficient for the first three stages of Abyssinian mustard oil production (growing, drying and transporting the grain) is 0.613, as described previously.

All emissions tabulated during the growing, drying and transport phases were added to obtain the total emissions (expressed as carbon intensity) for the growing phase (see Table 26 for growing phase emissions for Farm A and Farm B). As described previously, for the grain growing, drying and transport stages, a partition factor is applied to emissions to account for the fact that the Abyssinian mustard oil fraction represents 63% of the seed energy and is the fraction that is uniquely processed in the GRM. Thus, up to the point where the Abyssinian mustard oil is extracted from the grain, the emissions produced are multiplied by the mentioned distribution factor to obtain the so-called allocated emissions for each of the stages of grain growing, drying and transport (see Table 27).

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Table 26

Emissions from growing Abyssinian mustard ______without manure (farm A) or with manure (farm B)__________________

Farm A, emissions in COg-eq Farm B, emissions in COg-eq All values in g COg-eq/MJ timing belt Growing Diesel fuel consumption 2.77 3.07 N fertilizer (inorganic) 23.93 32.31 Manure* np 0 Pesticides 1.59 1.84 Seed material 0.11 0.17 N2O field emissions 34.13 66.48 Poditot cultivation 62.52 103.87 Drying grain 0.70 0.71 Grain transportation 1.26 1.69

The use of manure will not result in direct emissions due to production, but will result in indirect emissions (considered when calculating field N2O emissions) Table 27

Emissions from growing Abyssinian mustard without manure (farm A) or with manure (farm B)

Growing Drying Distribution coefficient Farm A, Unallocated Emissions Farm A distributed emissions Farm B, Unallocated Emissions Farm B distributed emissions All values in g CO2-eq/MJ GRM 61.3% 61.3% 62.52 0.70 38.29 0.43 103.87 0.71 63.62 0.43 Transportation 61.3% 1.26 0.77 1.69 1.03 Total 39.49 65.08

The BioGrace model takes into account another factor when calculating net GHG emissions, that is, the expected reduction in GHG emissions that could result from improved land management practices during bioenergy crop production compared to a baseline situation where management practices were not changed. The emissions reduction, called E _sca , suggests that improved land management practices result in increased carbon sequestration on managed lands, offsetting some of the emissions produced during the growing, processing and transport stages of the journey. In the specific case of Abyssinian mustard cultivation, as described here, additional emissions reductions are expected due to a shift from medium tillage to shallow tillage, replacing fallow with a ground cover crop that returns most of its biomass back to the soil at harvest, and also by using manure, which also helps store carbon in the soil. The BioGrace model quantifies and assigns an E _sca value in terms of tons of CO2 returned to the soil/ha/year based on these improvements (see E _sca values in Table 28 for Farm A and Table 29 for Farm B). This is subsequently converted into tonnes of CO2 returned to soil/MJ of GRM biofuel produced, serving to reduce net emissions along the way.

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Table 28

Changes in soil organic carbon resulting from improved land management (farm A - no manure application)

Actual land use Reference land use Link Climatic region Moderate warm humid Moderate warm humid Paragraph 6.1, Commission decision Soil type Low activity clay Low activity clay Paragraph 6.2, Commission decision Tillage Shallow plowing Deep plowing Table 3, Commission decision Resource utilization level Tall, no dung Average Table 3, Commission decision SOCst 63 tons C/ha 63 tons C/ha Table 1, Commission decision, using climate region and soil type Flu 0.69 tons C/ha 0.69 tons C/ha Table 2, Commission decision Fmg 1.08 tons C/ha 1.0 tons C/ha Table 2, Commission decision Fi 1.11 tons C/ha 1.0 tons C/ha Table 2, Commission decision SOCi* 52.1 tons C/ha 43.5 tons C/ha p** J-'Sca 1.583 tons CO2/ha/year p***r^sca 58.23 g COeq/MJ grm

*SOG= SOCst X Flu X Fmg X Fi **E _S ca = (SOCa- SOCr)*3.664/20 *** g CO _2-eq./MJ of produced hydrocarbons = tons of CO _{2-eq./ha/year} X (10 ⁶ g/t)/MJ of GR produced/ha/year, where MJ of GR produced/ha/year = MJ of rapeseed produced/ha/year X 55.5% (calculated path conversion efficiency)

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Table 29

Changes in soil organic carbon resulting from improved ______land management (Farm B using manure)______

Actual land use Reference land use Link Climatic region Soil type Tillage Resource utilization level SOCst Moderate warm humid Low activity clay Shallow plowing High, without manure 63 tons C/ha Moderate warm humid Low activity clay Deep plowing Average 63 tons C/ha Paragraph 6.1, Panel Decision Paragraph 6.2, Panel Decision Table 3, Panel Decision Table 3, Panel Decision Table 1, Panel Decision, using climate region and soil type Flu 0.69 tons C/ha 0.69 tons C/ha Table 2, Commission decision Fmg 1.44 tons C/ha 1.0 tons C/ha Table 2, Commission decision Fi 1.11 tons C/ha 1.0 tons C/ha Table 2, Commission decision SOCi* 67.6 tons C/ha 43.5 tons C/ha p** J-'Sca 4.421 tons CO2/ha/year P J-sca 179.43 g COgeq/MJ grm

*SOG= SOCst X Flu X Fmg X Fi **E _S ca = (SOCa- SOCr)*3.664/20 *** g CO _2-eq./MJ of produced hydrocarbons = tons of CO _{2-eq./ha/year} X (10 ⁶ g/t)/MJ of GR produced/ha/year, where MJ of GR produced/ha/year = MJ of rapeseed produced/ha/year X 55.5% (calculated path conversion efficiency)

As can be seen from table. 30, CO ₂ eq. per MJ of produced GR at the stages of growing, drying and shipping grain for both farms are negative (i.e. -18.7 t CO ₂ -eq/MJ of produced GR for Farm A, and -114.35 t СО ₂ -eq. /MJ GRM produced for Farm B), indicating that growing Abyssinian mustard under the conditions and methods described herein resulted in a net reduction in CO ₂ eq. atmosphere, respectively, essentially by increasing soil organic carbon levels due to the net incorporation of carbon-containing crop residues, leaf litter and root material, reducing soil carbon losses through the use of reduced tillage and, in the case of Farm B, by improving soil structure and carbon fixation through the use of manure.

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Table 30

Amount of emissions associated with the production route (GRM products from Abyssinian mustard raw materials) without manure (Farm A) or with manure (Farm B)

Farm A emissions Distributed results Emissions on farm B Distributed results All results are in g CO ₂ . _E sq./MJ timing Growing 38.29 63.62 Plus drying 0.43 0.43 Plus transportation 0.77 1.03 Decrease in E _sca (58.23)* (179.43)* Total (18.74)* (114.35)*

*Numbers in parentheses are negative.

As can be seen from table. 26, the use of manure on farm B entails a significant increase in CO ₂ -eq emissions associated with direct and indirect N ₂ O emissions. Indeed, considering only the contribution of the stages of growing, drying and transporting grain, farm 2 shows a 1.65 times higher _CO2e emissions than Farm A. However, this is more than offset by the 2.8 times increase in annual soil carbon fixation observed for Farm B compared to Farm A (see Tables 28 and 29) . Thus, in cases where a farmer may wish to grow Abyssinian mustard in soils with relatively low fertility (especially in terms of nitrogen levels), it is clearly beneficial for the farmer to use manure as a replacement for inorganic fertilizers (especially inorganic nitrogen) to achieve required fertility levels and maximum yield of Abyssinian mustard, since greenhouse gas emission levels can be offset and further reduced due to the positive impact of manure application on soil carbon storage.

Although the current analysis only considers part of the GRM biofuel pathway, it will be apparent to one skilled in the art that the application of the optimal practices described herein for growing Abyssinian mustard to produce GRM feedstock and low carbon-intensive biofuels will allow for significant accrual reductions in the growing, drying stages and transportation of Abyssinian mustard grains to emissions produced at later stages of the journey (for example, oil extraction, transportation and storage of the original oil, conversion of the feedstock to the GRM, transportation of the GMR for storage and distribution). Indeed, if the techniques described for Farm B were applied to the CBM production pathway described in Example 7, a net negative carbon intensity for the entire CBM production pathway would be easily achieved. The greater the negative carbon intensity that can be achieved during the growing, drying and transport stages of grain, the greater the range of transportation options for feedstocks and GBMs that can be considered while minimizing overall greenhouse gas emissions along the biofuel route.

Example 9: GHG mitigation potential for low carbon-intensity biofuels produced from feedstocks derived from sequential cultivation of Brassica carinata as a winter cover crop following a cereal (corn) in a tropical humid climate (Florida/SE US). This example demonstrates the GHG reductions achieved by Abyssinian mustard grown successively as a cover crop in a tropical humid climate, as exemplified by post-grain Abyssinian mustard production in North Florida (described previously in Example 2). As in the previous examples, emissions caused by growing Abyssinian mustard as a winter cover crop after maize in a tropical humid climate were calculated using the BioGrace model, assuming the GRM as the final product, and are summarized in Table 1. 31 as g CO ₂ -eq./MJ of produced timing belt. Emissions associated with growing and harvesting, drying and transporting grains were tabulated before and after applying a partition factor, which is used to account for the fact that only the oil portion of the grain contributes to GHG emissions from that part of the biofuel pathway. As can be seen, after applying the partition coefficient, it was found that the total emissions from growing, drying and transporting grain are 47.9 g CO ₂ -eq./MJ of produced GR.

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Table 31

Emissions from Abyssinian mustard cultivation

Distribution coefficient Not- distributed results Distributed Results Total All results in CO ₂ -eq./MJ GRM Growing Abyssinian mustard 61.3% 77.18 43.72 Drying Abyssinian mustard 61.3% 0.97 0.59 E _EU cultivation 47.9 Grain transportation (truck) 61.3% 0.20 0.12 Transportation eta 0.12

Table 32 summarizes the benefits that can be achieved through maize and Abyssinian mustard crop rotation and associated improved land management practices. The BioGrace model compares soil carbon storage before and after new agricultural practices are introduced. In the base situation, the soil is tilled with shallow tillage and then maintained under fallow conditions while using low input levels, whereas in the modified situation, the Abyssinian mustard cover crop is grown under no-till conditions and high input levels. The net result of this change in practice is a significant annual net contribution of carbon to existing soil stocks due to the return of stored carbon from crop residues and root material remaining after harvest. The BioGrace model predicts a net increase in soil carbon of 1.02 _tCO2 /ha/year from growing Abyssinian mustard under improved land management practices compared to baseline practices. Since carbon is primarily derived from plants through photosynthetic fixation of atmospheric CO2, this represents a net removal of CO2 from the atmosphere and fixation in the soil. The net GHG emission reduction can also be expressed relative to the amount of GBM produced, 36.59 g CO ₂ -eq/MJ GBM, and this bonus or E _sca value can be used to offset the GHG emissions that are generated throughout the biofuel journey. This is shown in table. 33, where E _sca values are subtracted from the net emissions resulting from growing, drying and transporting grain. As can be seen, the net GHG emissions of 11.42 g CO ₂ -eq/J of the engine are obtained after subtracting the E _sca factor. In contrast to some of the above examples of Abyssinian mustard cultivation, the carbon intensity of the pathway involving growing Abyssinian mustard as a fallow after maize in a tropical humid climate, drying and transporting the harvested grain to collection points remains positive even after subtracting the E _sca bonus, indicating that net GHG emissions are released. This is partly due to the high levels of nitrogen used in the cultivation of Abyssinian mustard in this study and the associated contribution to greenhouse gas emissions in the field during the cultivation stage.

However, if nitrogen input could be reduced by 50% (i.e. from 141 kg/ha to 70 kg/ha) without significantly affecting Abyssinian mustard yield (low nitrogen use), GHG emissions during the growing phase could be reduced from 47 .9 g CO ₂ -eq./MJ GRM to 30.1 g CO ₂ -eq./MJ GRM (Table 33) due to a reduction in life cycle emissions associated with the production of nitrogen fertilizers, as well as a reduction in emissions in field conditions. When transport and E _sca are taken into account, emissions become negative (-6.4 g _CO2e /MJ GRM), indicating a net reduction in atmospheric CO2 levels from Abyssinian mustard cultivation that can be used to offset emissions for other phases of the journey biofuels. This example illustrates how maximizing nitrogen use efficiency in Abyssinian mustard cultivation can have a significant impact on reducing greenhouse gas emissions associated with low-carbon-intensity biofuel production.

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Table 32

Reduced emissions from improved land management

Actual land use Reference land use Link Climatic region tropical wet tropical wet Paragraph 6.1, Commission decision Soil type Sandy Sandy Paragraph 6.2, Commission decision Tillage No plowing Shallow plowing Table 3, Commission decision Resource utilization level Tall, no dung Short Table 3, Commission decision SOCst 39 tons C/ha 39 tons C/ha Table 1, Commission decision, using climatic region and soil type Flu 0.48 tons C/ha 0.48 tons C/ha Table 2, Commission decision Fmg 1.22 tons C/ha 1.15 tons C/ha Table 2, Commission decision Fi 1.11 tons C/ha 0.92 tons C/ha Table 2, Commission decision SOCi* 25.4 tons C/ha 19.8 tons C/ha p** J-'Sca 1.02 tons CO2/ha/year p***c-sca 36.59 g COeq/MJ grm

SOCi= SOCst X Flu X Fmg X Fi **Esca=(SOCA-SOCR)*3.664/20 *** g CO _2-eq./MJ of produced hydrocarbons = tons of CO _{2-eq./ha/year} X (10 ⁶ g/t)/MJ of GR produced/ha/year, where MJ of GR produced/ha/year = MJ of rapeseed produced/ha/year X 55.5% (calculated path conversion efficiency)

Table 33

Emissions from Abyssinian mustard cultivation

Total All results in g COi-^b/MJ grm 110 kg/ha inorganic N 55 kg/ha inorganic N E _EU cultivation 47.9 30.1 Transportation eta 0.12 0.12 Bonus OR E _SC a (36.6)* (36.6)* Total 11.42 (6.4)*

*numbers in brackets are negative

Example 10: Greenhouse gas emissions from growing Brassica carinata as a summer cover crop following legumes (lentils) in a temperate, cold, dry climate. This example demonstrates the reduction in greenhouse gas emissions achieved by using Abyssinian mustard as a summer cover crop grown for biofuel feedstock. As in the previous examples, emissions from growing Abyssinian mustard as a summer cover crop after lentils in a cool-dry temperate climate zone are calculated using the BioGrace model, taking the GRM as the final product as described previously, and the data are summarized in Table 1. 34 (Inorganic nitrogen use scenario 1 with

110 kg/ha) and table. 35 (Scenario 2 use of inorganic nitrogen with 55 kg/ha) as g CO ₂ -eq./MJ of produced GRM. Emissions associated with grain growing and harvesting, grain drying, and grain transportation are tabulated before and after applying a partition factor, which is used to account for the fact that only the oil portion of the grain contributes to GHG emissions from that portion of the biofuel pathway. As can be seen from comparing the data in table. 34 and 35, GHG emissions from grain drying and transport are identical for both nitrogen use scenarios, but differ significantly for the growing phase, with scenario 2 showing significantly lower emissions predicted by the BioGrace model. This reflects lower field emissions due to less nitrogen fertilizer applied to the crop. Thus, lower nitrogen fertilizer requirements coupled with the ability to maintain yields is an expected benefit of using lentils and other pulse crops in crop rotations with Abyssinian mustard and has the added benefit of significant reductions in greenhouse gas emissions during cultivation.

Table 34

Emissions from Abyssinian mustard cultivation (scenario 1)

Growing Abyssinian mustard Drying Abyssinian mustard E _EU cultivation Transportation of grain (truck) Distribution coefficient 61.3% 61.3% 61.3% Not- distributed results All results in ( 61.1 1.02 0.61 Distributed results: o ₂ -eq./ MJ GRM 37.4 0.62 0.37 Total 38.1 Transportation eta 0.37 Table 35 Emissions associated with Abyssinian mustard cultivation (scenario 2) Growing Abyssinian mustard Drying Abyssinian mustard Distribution coefficient 61.3% 61.3% Un-Allocated Total distributed results results All results in CO2-eq/MJ GRM 40.4 1.02 24.8 0.62 E _EU cultivation 25.4 Grain transportation (truck) 61.3% 0.61 0.37 Transportation eta 0.37

In table 36 summarizes the benefits that can be achieved by adopting a lentil/Abyssinian mustard crop rotation and associated improved land management practices. The BioGrace model compares soil carbon storage before and after new agricultural practices are introduced. In the original situation the land is left fallow and low levels of inputs are used, while in the modified situation the cover crop Abyssinian mustard is grown. Although this entails the use of more resources, the net result of growing Abyssinian mustard is a significant annual net contribution of carbon to existing soil stocks due to the return of stored carbon from crop residues and root material returned after harvest. The BioGrace model predicts a net increase in soil carbon of 0.73 _tCO2 /ha/year from Abyssinian mustard cultivation compared to baseline. Since carbon is predominantly derived from plants through photosynthetic fixation of atmospheric CO ₂ , this represents a net removal of CO ₂ from the atmosphere and fixation in the soil. The net GHG emission reduction can also be expressed relative to the amount of GBM produced, 30.32 g CO ₂ -eq/MJ GBM, and this bonus E _sca value can be used to offset the GHG emissions that are generated along the way. This is shown in table. 37, where E _sca is subtracted from the net emissions accumulated from growing, drying and transporting grain. As can be seen from scenario 1 (high nitrogen use), a net GHG emission of 8.2 g CO ₂ -eq./MJ of the engine is obtained after adding the E _sca factor; however, in scenario 2 (low nitrogen use) a net GHG emission reduction of 4.5 g CO ₂ -eq/MJ GBM is achieved. This negative carbon intensity can be used to offset emissions that may occur at other stages of the oil production pathway, such as processing, refining and hydrotreating of Abyssinian mustard oil, helping to reduce the overall emissions of the pathway. Thus, increasing nitrogen use efficiency during the cultivation phase and improving land management practices associated with cultivation could result in a more negative carbon intensity for this phase of the pathway, and could significantly reduce overall GHG emissions along the entire biofuel pathway.

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Table 36

Improved land management (scenario 1 and 2 for inorganic nitrogen)

Actual land use Reference land use Link Climatic region Moderate cold dry Moderate cold dry Paragraph 6.1, Commission decision Soil type Highly active clayey Highly active clayey Paragraph 6.2, Commission decision Tillage No plowing No plowing Table 3, Commission decision Resource utilization level Tall, no dung Short Table 3, Commission decision SOCst 50 tons C/ha 50 tons C/ha Table 1, Commission decision, using climate region and soil type Flu 0.8 tons C/ha 0.8 tons C/ha Table 2, Commission decision Fmg 1.1 tons C/ha 1.1 tons C/ha Table 2, Commission decision Fi 1.1 tons C/ha 0.95 tons C/ha Table 2, Commission decision SOCi* 45.8 tons C/ha 41.2 tons C/ha P** J-sea 0.73 tons CO ₂ /ha/year p*** J-sea 30.32 g CO ₂ . _E sq./MJ timing belt

*SOQ=SOCst X Flu X Fmg X Fi **E _sca =(SOCA-SOCR) X 3.664/20 *** g CO _2-eq./MJ of produced hydrocarbons = tons of CO _{2-eq./ha/year} X ( 10 ⁶ g/t)/MJ of GR produced/ha/year, where MJ of GR produced/ha/year = MJ of rapeseed produced/ha/year X 55.5% (calculated path conversion efficiency)

Table 37

Emissions associated with Abyssinian mustard cultivation with high (110 kg/ha) and low (55 kg/ha) inorganic N use

Total All results are in g CO ₂ . _E sq. / MJgrm High N level Low N level E _EU cultivation 38.1 25.4 E _t d transportation 0.37 0.37 Bonus OR Esca (30.32)* (30.32)* Total 8.2 (4.5)*

*numbers in brackets are negative.

Example 11: Reducing GHG emissions by successively growing Brassica carinata as a winter cover after a legume (soybean) in a warm temperate, humid climate (Uruguay). This example demonstrates the GHG reductions achieved by using Abyssinian mustard as a fallow replacement winter cover crop grown for biofuel feedstock. Emissions associated with growing Abyssinian mustard as a successive winter cover crop after soybean in a warm-humid temperate climate were then calculated using the BioGrace model, taking the GRM as the end product as described previously, and summarized in Table 1. 38 in the form of g CO ₂ -eq/MJ produced by the timing belt. As described above, emissions associated with growing and harvesting, drying and transporting grain were tabulated before and after applying a partition factor, which is used to account for the fact that only the oil portion of the grain contributes to GHG emissions in that portion of the journey biofuels. As can be seen, after applying the distribution coefficient, it was found that the total emissions from growing, drying and transporting grain are 27.1 g CO ₂ -eq./MJ GRM.

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Table 38 Abyssinian related

Distribution coefficient Not- distributed results Distributed Results Total All results in CO ₂ -eq./MJ GRM Growing Abyssinian mustard 61.3% 41.49 25.44 Drying Abyssinian mustard 61.3% 0.55 0.34 E _EU cultivation 25.8 Grain transportation (truck) 61.3% 2.19 1.34 Transportation eta 1.3

In table 39 summarizes the benefits that can be achieved by adopting a soybean/Abyssinian mustard crop rotation and associated improvements in land management practices. The BioGrace model compares soil carbon storage before and after new agricultural practices are introduced. In the original situation, the land was left fallow and low levels of inputs were used, while in the modified situation a cover crop was grown and higher levels of inputs were applied. Although this entails the use of more resources, the net result of growing Abyssinian mustard is a significant annual net contribution of carbon to existing soil stocks due to the return of stored carbon from crop residues and root material after harvest. The BioGrace model predicted a net increase in soil carbon of 1.41 _tCO2 /ha/year from Abyssinian mustard cultivation compared to the base case. Since carbon is predominantly a plant product produced by photosynthetic fixation of atmospheric CO2, this would represent a net removal of atmospheric CO2 fixed in the soil. The net GHG emission reduction was also expressed relative to the amount of GBM produced, 50.91 g CO ₂ -eq/MJ GBM, and this bonus or E _sca value was then used in the BioGrace model to offset the GHG emissions generated along the way. This is shown in table. 39, where the E _sca value was subtracted from the net emissions accumulated during grain growing, drying and transportation, resulting in a negative GHG emission of 23.8 g CO ₂ -eq/MJ GR. In other words, under the growing conditions used in this study, cultivating Brassica carinata for oilseed production can reduce atmospheric CO ₂ levels. This negative carbon intensity can be used to offset emissions that may occur at other stages of the oil production pathway, such as processing, refining and hydrotreating of Abyssinian mustard oil, helping to reduce the overall emissions of this pathway. Negative carbon intensity at the growing stage is influenced by factors such as nitrogen use efficiency and improved land management practices associated with Abyssinian mustard cultivation.

Table 39

Improved land management

Actual land use Reference land use Link Climatic region Moderate warm humid Moderate warm humid Paragraph 6.1, Commission decision Soil type Highly active clayey Highly active clayey Paragraph 6.2, Commission decision Tillage No plowing No plowing Table 3, Commission decision Resource utilization level Tall, no dung Average Table 3, Commission decision SOCst 88 tons C/ha 88 tons C/ha Table 1, Commission decision, using climate region and soil type Flu 0.69 tons C/ha 0.69 tons C/ha Table 2, Commission decision Fmg 1.15 tons C/ha 1.15 tons C/ha Table 2, Commission decision Fi 1.11 tons C/ha 1.0 tons C/ha Table 2, Solution

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commissions SOCi* 77.5 tons C/ha 69.8 tons C/ha P ** -bsca 1.41 tons CO2/ha/year R * * * -bsca 50.91 g CO ₂ eq./MJ timing belt

*SOQ=SOCst X Flu X Fmg X Fi **Esca=(SOCA-SOCR) X 3.664/20 *** g CO _2-eq./MJ of produced hydrocarbons = tons of CO _{2-eq./ha/year} X (10 ⁶ g/t)/MJ of GR produced/ha/year, where MJ of GR produced/ha/year = MJ of rapeseed produced/ha/year X 55.5% (calculated path conversion efficiency)

Table 40

Emissions associated with Abyssinian mustard cultivation

All results in g CO ₂ -eq. / MJgrm Total E _EU cultivation 25.8 E _t d transportation 1.3 Bonus or E _sca (50.91)* Total (23.81)*

*numbers in brackets are negative

Case Study 12: Greenhouse gas emission reductions resulting from sequential cultivation of Brassica carinata as a winter cover crop following cereals (wheat) in New South Wales. This example demonstrates the GHG reductions achieved by using Abyssinian mustard as a cover crop in warm-temperate and tropical-dry climates such as the New South Wales wheat belt in eastern Australia. As in the previous examples, emissions caused by sequentially growing Abyssinian mustard as a winter crop after wheat in a warm temperate climate were calculated using the BioGrace model, taking the GRM as the final product, and the data are summarized in Table 1. 41 (with a high level of inorganic nitrogen use) and in table. 42 (with a low level of inorganic nitrogen use) in the form of g CO ₂ -eq./MJ GRM. Emissions associated with grain growing and harvesting, grain drying, and grain transportation are tabulated before and after applying a partition factor, which is used to account for the fact that only the oil portion of the grain contributes to GHG emissions from that portion of the biofuel pathway. As can be seen, after applying the partition coefficient, the total emissions from growing, drying and transporting grain are 38.8 g CO ₂ -eq/MJ GRM, obtained in a scenario where a large amount of inorganic nitrogen fertilizer is used when growing Abyssinian mustard, and 25. 5 g CO2eq/MJ GRM obtained in a scenario where a low amount of inorganic nitrogen fertilizer is used.

Table 41

Emissions from Abyssinian mustard cultivation (high levels of inorganic nitrogen)

Not- distributed results Distribution coefficient Distributed Results Total All results in CO ₂ -eq / MJ GRM Growing Abyssinian mustard 59.71 61.3% 36.57 Drying Abyssinian mustard 1.57 61.3% 0.96 E _EU cultivation 37.5 Grain transportation (truck) 2.19 61.3% 1.34 Transportation eta 1.3

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Table 42

Emissions from Abyssinian mustard cultivation (low levels of inorganic nitrogen)

Not- distributed results Distribution coefficient Distributed Results Total All results in COg-Eq./MJ GRM Growing Abyssinian mustard 38.01 61.3% 23.28 Drying Abyssinian mustard 1.57 61.3% 0.96 E _EU cultivation 24.2 Grain transportation (truck) 2.19 61.3% 1.34 Transportation eta 1.3

In table 43 summarizes the benefits that can be achieved by implementing a wheat/Abyssinian mustard crop rotation and associated improvements in land management practices in specified climate zones and soil types. The BioGrace model compares soil carbon storage before and after new agricultural practices are introduced. In the base situation, the soil is shallow-tilled and then maintained under fallow conditions while using low input levels, whereas in the modified situation, the Abyssinian mustard cover crop is grown under no-till conditions and high input levels. The net result of this change in practice is a significant annual net contribution of carbon to existing soil stocks due to the return of stored carbon from crop residues and root material remaining after harvest.

The BioGrace model predicts a net increase in soil carbon of 0.97 _tCO2 /ha/year due to Abyssinian mustard grown under improved land management practices compared to baseline. Since carbon is predominantly a plant product produced by photosynthetic fixation of atmospheric CO2, this represents a net removal of CO2 from the atmosphere and fixation in the soil. The net reduction in GHG emissions can also be expressed relative to the amount of GBM produced, 35 g CO2eq/MJ of GBM, and this bonus or E _sca value can be used to offset the GHG emissions that are generated along the biofuel's entire journey. This is shown in table. 44, where E _sca values are subtracted from the net emissions accumulated during grain growing, drying and transportation. As can be seen in the case of high inorganic nitrogen cultivation, the net GHG emission of 3.8 g CO ₂ -eq/MJ GRM is obtained after subtracting the E _sca factor. In contrast to other examples of Abyssinian mustard cultivation, the carbon intensity of the pathway involving the cultivation of Abyssinian mustard as a winter fallow after wheat in the warm-temperate warm-dry/tropical-dry climate zone covering New South Wales, followed by drying and transporting the harvested grain to collection points remains positive even after subtracting the E _sca bonus, indicating that net GHG emissions are released. This is partly due to the high level of nitrogen used in Abyssinian mustard cultivation, which in this study contributes to field GHG emissions during the cultivation stage.

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Table 43

Reducing emissions through improved land management

Actual land use Reference land use Link Climatic Temperate Warm Dry/Tropical Paragraph 6.1, Decision region dry commissions Soil type Highly active clayey Paragraph 6.2, Commission decision Tillage No plowing Shallow plowing Table 3, Commission decision Resource utilization level Tall, no dung Short Table 3, Commission decision SOCst 38 tons C/ha 38 tons C/ha Table 1, Commission decision, using climate region and soil type Flu 0.8 tons C/ha 0.8 tons C/ha Table 2, Commission decision Fmg 1.1 tons C/ha 1.1 tons C/ha Table 2, Commission decision Fi 1.04 tons C/ha 0.95 tons C/ha Table 2, Commission decision SOCi* 34.8 tons C/ha 29.5 tons C/ha -C'sca 0.97 tons CO ₂ /ha/year p*** -bsca 35 g CO2-eq/MJ GRM

*SOQ=SOCst X Flu X Fmg X Fi **Esca=(SOCA-SOCR) X 3.664/20 *** g CO _2-eq./MJ of produced hydrocarbons = tons of CO _{2-eq./ha/year} X (106 g/t)/MJ of GR produced/ha/year, where MJ of GR produced/ha/year = MJ of rapeseed produced/ha/year X 55.5% (calculated path conversion efficiency)

If nitrogen input can be reduced by 50% (i.e. from 110 kg/ha to 55 kg/ha) without significantly affecting Abyssinian mustard yield (low nitrogen use), GHG emissions during the cultivation phase can be reduced from 37.5 g CO ₂ -eq./MJ GRM up to 24.2 g CO ₂ -eq./MJ GRM (Table 44) due to reductions in life cycle emissions associated with the production of nitrogen fertilizers, as well as reductions in field emissions. When transport and E _sca are taken into account, total emissions in the low-nitrogen scenario are reduced to -9.5 CO ₂ -eq/MJ GBM, which represents the net reduction in atmospheric CO2 levels resulting from growing Abyssinian mustard under these conditions. This example and the preceding example serve to illustrate the impact that these differences in soil type and climate region can have on the ability to grow Abyssinian mustard and associated best practices for reducing greenhouse gas emissions.

Table 44

Emissions associated with Abyssinian mustard cultivation

Total All results in g CO2-eq. / MJgrm High inorganic N levels Low inorganic N levels E _EU cultivation 37.5 24.2 Transportation eta 1.3 1.3 Bonus or E _sca (35)* (35)* Total 3.8 (9.5)*

*numbers in brackets are negative.

Example 13: Impact of manure management on greenhouse gas emissions and fixation during Brassica carinata cultivation. During the winter of 2016-2017, Brassica carinata was grown on 13 independent farms located in central Georgia, USA. To assess commitment to environmentally friendly practices, Abyssinian mustard production on these farms was carefully monitored. Data obtained on energy use and GHG emissions for all stages of the growing process were analyzed using the BioGrace Biofuels GHG Emissions Calculator spreadsheet, version 4d, as described in the previous Examples. Of particular interest was assessing the impact of manure use (in this case, chicken manure) on GHG emission levels from Abyssinian mustard when used as a partial or complete replacement for inorganic nitrogen. Six of the 13 farms used manure as a fertilizer in combination with inorganic nitrogen or, in one case, as a complete replacement for inorganic nitrogen, while

- 56 044251 the rest used inorganic nitrogen exclusively in their fertilizer mixtures. Cultivation on all farms included the use of improved land management practices described in the present invention, including shallow tillage and the use of Abyssinian mustard as a cover crop in rotation with cereals, legumes, cotton or sesame.

Table 45 summarizes the data obtained from these farms. For the purposes of this study, it was assumed that the resulting Abyssinian mustard grain would provide the feedstock for the production of GRM biodiesel, and therefore intermediate GHG calculations for this pathway were normalized to the energy content of GRM biodiesel as previously described. CO2 equivalent emissions were calculated from cultivation data and included emissions from the following steps: production of raw materials, use of fuel for agricultural machinery, production of commercial seed used to start cultivation, seed drying and seed transport. Direct and indirect emissions from organic and inorganic nitrogen applied to the field were also quantified and included. Improved land management and farming practices prevented some CO ₂ emissions from entering the atmosphere and instead included them in the soil organic carbon pool, reducing net emissions. This latter effect, known as E _sca , can be quantified as described previously and then subtracted from the CO ₂ -eq produced by the above sources to obtain the net crop emission for each farm. As can be seen from table. 45, all farms produced negative emissions for the growing phase of the pathway, indicating that growing Abyssinian mustard using the methods described in the present invention resulted in a net removal of atmospheric CO2. Farms using manure as a nutrient source achieved greater reductions in atmospheric CO2 than those using only inorganic nutrients. One reason for this can be seen in the impact of manure use on soil carbon storage, where farms that used manure showed several times higher levels of soil carbon storage than farms using only inorganic nitrogen.

In the study described in this paper, data were not directly obtained from the final stages of the timing belt production path. However, after the grain has been combined, the subsequent stages of the path can be considered to be common to all grain sources. Emissions associated with the energy used in extracting oil and converting oil feedstocks into timing biodiesel are well studied and largely depend on the amount of feedstock used. Although distances and conditions for transporting, distributing and storing raw materials and finished fuels can be highly variable, for the purposes of this example, default distances and transportation fuel type were used to provide data for calculating net emissions for such a sample path, and these were added to the above actuals the growing phase emissions data previously described to obtain the total carbon intensity for the GBM resulting from agricultural production of Abyssinian mustard and Abyssinian mustard feedstock. As can be seen from table. 45, in most cases, the carbon intensity of the GEM produced by this route is negative by default, indicating a net reduction in atmospheric GHG levels compared to diesel production from fossil fuels. The greatest GHG reduction benefits come from feedstocks produced on farms using manure during the growing phase.

It is clear that the more one is able to reduce CO ₂ -eq emissions during the growing phase through the improved practices described here, including the use of shallow or no-till practices, reduced irrigation, and the use of manure, the more one can offset emissions arising during subsequent phases. stages of the journey not related to cultivation, which are more dependent on variable factors of distance and conditions of transportation, distribution and storage of raw materials and finished fuel.

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Table 45

Impact of manure use on _CO2e emissions. as a result of growing Abyssinian mustard and the carbon intensity of diesel fuel produced using Abyssinian mustard feedstock

Resources used Growing CI for converting raw materials into timing belts Trusses ¹ Truss D Truss Μ Truss A Truss K Manure nitrogen (kg/ha) 79 70 70 70 Inorganic nitrogen (kg/ha) 118 134 0 134 %Ν from manure 40 34 100 34 Net emissions (g with _2.eq./MJ GRM) ² -219.9 -164 -151.4 -124 Soil carbon accumulation (tons CO ₂ /ha/g) 4.421 3.784 3.784 3.784 CI ³ (g CO _2eq / MJ) -205.5 -149.5 -136.9 -109.5 GHG reduction ^{3 4} 345% 278% 263% 231% Farm E 88 135 39 -114.4 4.421 -99.9 219% Farm L 70 90 44 -103.8 4.421 -89.5 207% Farm F 0 101 0 -57 1,583 -42.5 151% Farm J 0 125 0 -56.1 1,583 -41.7 150% Farm N 0 118 0 -38.4 1,583 -24 129% Farm G 0 137 0 -29.3 1,583 -14.8 118% Farm C 0 111 0 -18.8 1,583 -4.3 105% Farm B 0 179 0 -14.2 1,583 0.4 100% Farm I 0 125 0 -10.8 1,583 3.6 96%

¹ All farms listed grew Brassica carinata in central Georgia during the winter of 2016-2017.

² Includes emissions in CO ₂ -eq. from growing, drying grain and transporting grain is less than the value of E _sca , as described in example 8.

³ Based on a path containing actual growing data for each farm, supplemented by default emissions data from oil extraction and processing, and modeled emissions data from oil and fuel transportation, storage and distribution.

⁴ Based on standard petroleum diesel CI value of 83.8 CO ₂ -eq/MJ, according to BioGrace v emissions calculator. 1.4d.

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All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually designated for inclusion by reference. Reference to any publication is intended to disclose it prior to its filing date and should not be construed as an admission that the present invention is not entitled to precede such publication by virtue of a prior invention.

Although the foregoing invention has been described in some detail for purposes of illustration and example for clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made in light of the teachings of this invention without departing from the spirit and scope of the appended claims.

It should be noted that, as used in this specification and the accompanying claims, the singular forms include a reference to the plural unless the context clearly dictates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates.

The expression and/or, as used herein in the description and claims, is to be understood to mean one or both of the elements so combined, that is, elements which in some cases are present together and in other cases separately.

Multiple elements listed by the expression and/or are to be construed in the same way, that is, one or more elements so combined. If desired, other elements may be present other than those specifically identified in the proposal and/or whether or not related to those elements specifically identified. Thus, as a non-limiting example, a reference to A and/or B, when used in conjunction with an open expression such as containing, may refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, only on B (if necessary, including elements other than A); in yet another embodiment, to A and B (including other elements if necessary); etc.

As used herein in the specification and claims, the term or should be understood to cover the same meaning as and/or as defined above. For example, when separating elements in a list, either or and/or should be interpreted as inclusive, that is, including at least one, but also including more than one, of a number or list of elements, and optionally,

-

Claims (24)

additional unregistered elements. As used in the present invention, in the description or in the accompanying claims, the transitional terms containing, including, bearing, having, containing, involving and the like are to be understood as including or open-ended (i.e. to mean including but not limiting), and they do not exclude unaccounted for items, materials, or process steps. Only transition phrases consisting of and essentially consisting of, respectively, are closed or semi-closed transition phrases with respect to the claims and exemplary embodiments of the present invention. The transitional phrase consisting of excludes any element, step or ingredient that is not specifically stated. A transition phrase consisting essentially of limits the scope of application to specified elements, materials or steps, and those that do not significantly affect the essential characteristic(s) of the invention disclosed and/or claimed herein. CLAIM 1. A method of growing Brassica carinata to obtain oilseed raw materials for the production of biofuel with low carbon intensity, including: a) planting Brassica carinata as a second crop in a rotation with the first crop or to replace fallow land, and implementing land management practices to reduce the use of fossil fuels and maximize atmospheric carbon sequestration by Brassica carinata plant material; b) harvesting of the Brassica carinata variety for grain production; c) returning approximately 70 to 90% of all plant material from the Brassica carinata variety, excluding grain, to the soil; d) processing the harvested grain to extract oil; e) the use of oil as a feedstock for the production of low-carbon-intensity biofuels that have a carbon-intensity value reduced by at least 20 g CO ₂ eq./MJ of energy produced compared to the carbon-intensity value of the corresponding conventional fossil-derived fuel. 2. A method of growing Brassica carinata to obtain oilseed raw materials for the production of biofuel with low carbon intensity, including: a) planting Brassica carinata as a second crop in a rotation with the first crop or to replace fallow land, and implementing land management practices to reduce the use of fossil fuels and maximize atmospheric carbon sequestration by Brassica carinata plant material; b) harvesting of the Brassica carinata variety for grain production; c) returning approximately 70 to 90% of all plant material from the Brassica carinata variety, excluding grain, to the soil; wherein the oil extracted from the harvested grain is intended to be used as a feedstock for the production of a low carbon-intensity biofuel that has a carbon-intensity value reduced by at least 20 g CO2e/MJ of energy produced compared to the carbon-intensity value of the corresponding conventional fuel produced from fossil raw materials. 3. A method for producing oilseed raw materials for biofuel with low carbon intensity, including: a) obtaining grain produced by a method including: i) planting Brassica carinata as a second crop in rotation with the first crop or to replace fallow land, and implementing land management practices to reduce the use of fossil fuels and maximize atmospheric carbon sequestration by Brassica carinata plant material; ii) harvesting the Brassica carinata variety for grain production; iii) returning approximately 70 to 90% of all Brassica carinata plant material, excluding grain, to the soil; And b) extracting oil from the harvested grain to obtain oilseed raw materials; wherein the oilseed-derived biofuel has a carbon intensity value that is reduced by at least 20 g CO2e/MJ of energy produced compared to the carbon intensity of the corresponding conventional fossil-derived fuel. 4. A method for producing biofuels with low carbon intensity, including: a) obtaining oilseed raw materials prepared by a method including: i) planting Brassica carinata as a second crop in rotation with the first crop or to replace fallow land, and implementing land management practices to reduce the use of fossil fuels and maximize atmospheric carbon sequestration by Brassica carinata plant material; ii) harvesting the Brassica carinata variety for grain production; - 62 044251 iii) returning approximately 70 to 90% of all plant material from the variety Brassica carinata, excluding grain, to the soil; iv) extracting oil from harvested grains to obtain oilseed raw materials; And b) production of biofuels with low carbon intensity from oilseeds; wherein the oilseed-derived biofuel has a carbon intensity value that is reduced by at least 20 g CO ₂ eq./MJ of energy produced compared to the carbon intensity value of the corresponding conventional fossil-derived fuel. 5. A method according to any one of claims 1 to 4, wherein said land management techniques include one or more of: i) no-till or reduced/reduced tillage with at least 30% of crop residue remaining on the soil surface during sowing of Brassica carinata, ii) use of less than 165 kg/ha of inorganic nitrogen fertilizer for Brassica carinata, iii) ) the use of manure to provide approximately 20 to 100% of the total nitrogen fertilizer applied to Brassica carinata. 6. The method according to claim 1 or 2, further comprising sowing the Brassica carinata variety immediately after harvest or simultaneously with the harvest of the first crop for sequential cultivation of crops without an intervening period of fallow land. 7. The method according to claim 3 or 4, where the method for obtaining said grain or said oilseed raw material further comprises sowing the Brassica carinata variety immediately after harvesting or simultaneously with harvesting the first crop to grow the crop sequentially without an intervening period of fallow land. 8. The method according to any one of claims 1 to 4, wherein the low carbon-intensity biofuel has a carbon-intensity value that is reduced by about 50-200 g CO2e/MJ compared to the carbon-intensity value of the corresponding fossil-derived fuel. 9. The method according to any one of claims 1-4, where the biofuel has a carbon intensity value that is reduced by at least 40, 60, 80, 100, 120, 140, 160, 180, 200 g CO ₂ eq./MJ of energy produced compared to the carbon intensity of the corresponding conventional fossil-derived fuel. 10. The method according to any one of claims 1 to 9, wherein the greenhouse gas emissions resulting from the production of low carbon-intensive biofuels during its production cycle are reduced by approximately 60-400% compared to the greenhouse gas emissions resulting from the production of the corresponding fossil fuels. 11. The method according to any one of claims 1 to 4, wherein the flour fraction remaining after extracting the oil from the harvested grain is used to obtain a protein-enriched feed additive for livestock. 12. The method according to claim 1 or 2, further comprising sowing a new crop, which may be the same as the first crop or different from the first crop, but which is not Brassica carinata, immediately after or simultaneously with harvesting the Brassica carinata without intervening period of fallow land. 13. A method according to any one of claims 1 to 12, which fixes approximately 0.5 to 5 tons of CO ₂ per hectare per year in the soil. 14. The method of any one of claims 1 to 13, comprising reducing the use of exogenously added nitrogen fertilizer to less than about 60% of the total amount of nitrogen fertilizer used to grow the Brassica carinata variety. 15. Method according to any one of claims 1 to 14, wherein the manure is chicken manure, cattle manure or sheep manure. 16. Method according to any one of claims 1 to 15, where the first crop is a legume. 17. The method of claim 16, wherein the legume is peanuts, soybeans, lentils, beans or peas. 18. Method according to any one of claims 1 to 17, wherein the first crop is a cereal crop. 19. The method according to claim 18, where the grain crop is wheat, barley, rye, oats or corn. 20. The method according to any one of claims 1 to 15, where the first crop is cotton or sesame. 21. The method according to any one of claims 1 to 20, wherein the growing environment is in a region with a tropical humid climate and wherein the land management techniques include sowing Brassica carinata in the fall or winter for harvest in the spring or summer, or sowing Brassica carinata in the spring for harvest. in the fall. 22. The method according to any one of claims 1 to 20, wherein the growing environment is in a region with a tropical, dry climate and where land management practices include planting Brassica carinata in the fall or winter for harvest in the spring or summer. 23. The method of any one of claims 1 to 20, wherein the growing environment is in a region with a temperate, cold, dry climate and wherein land management practices include planting Brassica carinata in the spring for harvest in the summer or fall. 24. The method according to any one of claims 1 to 20, where the growing medium is located in a region with moderate cold -