WO2023081975A1

WO2023081975A1 - Production of biomass

Info

Publication number: WO2023081975A1
Application number: PCT/AU2022/051348
Authority: WO
Inventors: Duncan Royce Anderson; Cristian Coelho SILVA
Original assignee: Hydrobe Pty Ltd
Priority date: 2021-11-12
Filing date: 2022-11-11
Publication date: 2023-05-19

Abstract

The disclosure relates to a process for sequestering carbon from a gas stream that comprises carbon dioxide and a process for producing biomass. The process comprises: (i) producing an organic feedstock using a phototrophic source and a gas stream comprising carbon dioxide in a photosynthesis step; and (ii) converting the organic feedstock to biomass; wherein converting the organic feedstock to biomass comprises an aerobic bio-decomposition step that employs bacteria comprising at least one facultative anaerobic species. The present disclosure is also directed to biomass, and hydrogen, produced in accordance with the process and systems suitable for performing the process.

Description

Production of Biomass

Reference to Earlier Application

The present application claims priority from Australian Provisional Patent Application No. 2021903640, the entire contents of which is incorporated herein by reference.

Technical Field

This disclosure relates to a process and system for sequestering carbon from a gas stream that comprises carbon dioxide. The disclosure also relates to a process and system for producing biomass.

Background

A major contributor identified to the phenomenon of global warming is carbon dioxide (CO2) emissions. CO2 is mainly a by-product of combustion and it creates operational, economic, and environmental problems. The production of CO2 occurs in a variety of industrial applications such as the generation of electricity from coal at power plants and in the use of hydrocarbons that are typically the main components of fuels that are combusted in combustion devices, such as engines. Exhaust gas discharged from such combustion devices contains CO2 gas, which at present is simply released to the atmosphere.

Some methods have been proposed for sequestering the CO2 produced by coal fired and other power plants. Reacting the CO2 with large quantities of metal oxides, particularly with the oxide of calcium and oxide of magnesium, then burying the resulting carbonates is also one method for CO2 sequestration. This method has a disadvantage that it requires a large and continuous supply of minerals in order to sequester CO2. Another method involves removing the CO2 from the combustion gas at the power plant, compressing it, then shipping it by pipeline to a peridotite or serpentinite mine for conversion to a carbonate, before burying it at the mine site. However, for CO2, which is heavier than air, shipment of compressed CO2 presents material handling challenges because CO2 will stay close to the ground if it is accidentally released during transit and can thus be life threatening. Additionally, another problem may be the availability of mines sites for such burying.

Other techniques, which can be generally classified as geologic, terrestrial, or ocean systems have been attempted at sequestration of carbon (in the initial form of gaseous CO2). These techniques are primarily concerned with transporting generated carbon dioxide to physical sites and injecting the carbon dioxide into geologic, soil, or ocean repositories. Each of these sequestering techniques are expensive due to costs for preparing CO2 for transport, conducting transportation, and injecting the CO2 at the sequestration site. As such, these techniques may not be economically feasible and can, in some cases, consume more energy than the original carbon produced.

Many CO2 sequestering techniques focus on carbon storage, rather than converting carbon into a product that can then be utilised in the synthesis of other product. However, each of these technologies suffer due to the capital plant costs raised to uneconomic levels, and the effect of CO2 capture on the cost of electricity is prohibitive.

It is desirable to develop a CO2 sequestration technique that could convert carbon into a product that can then be utilised in the synthesis of other products. It is desirable to develop a CO2 sequestration technique which may assist in developing alternate sources of energy that may be economical, sustainable and environmentally friendly.

It is to be understood that, for any prior art publication or reference that is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Summary

A first aspect of the disclosure provides a process for sequestering carbon from a gas stream that comprises carbon dioxide. The process comprises: (i) converting the carbon dioxide in the gas stream to an organic feedstock using a phototrophic source in a photosynthesis step; (ii) converting the organic feedstock to biomass; wherein converting the organic feedstock to biomass comprises an aerobic bio-decomposition step that employs bacteria comprising at least one facultative anaerobic species. Also provided by this first aspect is a process for producing biomass, the process comprising: (i) producing an organic feedstock using a phototrophic source and a gas stream comprising carbon dioxide in a photosynthesis step; and (ii) converting the organic feedstock to biomass; wherein converting the organic feedstock to biomass comprises an aerobic bio-decomposition step that employs bacteria comprising at least one facultative anaerobic species.

In this first aspect, carbon is sequestered in the organic feedstock during the photosynthesis step. Aquatic plants, vegetative groundcover, particularly farms and forests can be considered carbon dioxide collectors. For example, microalgae have ability to use carbon in the form of CO2. Microalgae perform photosynthesis under a suitable light source (e.g. sunlight) and in the presence of CO2.

Microalgae are unicellular or multicellular organisms, and can be grown in marine, fresh and wastewater. The composition of microalgae differs with respect to species, cultivation environment and biochemical characteristics. They have about 50% carbon in dry weight, usually obtained from CO2. Generally, microalgae biomass consists mainly of proteins, carbohydrates and lipids. They are composed of 5-60% carbohydrates, 40-60% proteins, 5-10% nucleic acids and 8-30% lipids.

In the first aspect of the present disclosure, the term “phototrophic source” as used herein refers to one or more phototrophic organisms that are capable of photosynthesis. The phototrophic source may comprise an algal source. The term “algal source” as used herein is to mean one or more algal species (which may be one or more species of microalgae) capable of photosynthetically converting carbon dioxide into an organic feedstock. The algal source may include algae in the class Chlorophyceae and/or Trebouxiophyceae. The algal source may be a chiorophyte. The algal species may be part of the Chlorella genus. In an embodiment the algal species may be Chlorella vulgaris. Microalgae can include both eukaryotic and prokaryotic algae (e.g. cyanobacteria). In some embodiments, the phototrophic source may comprise a phototrophic bacteria source. The phototrophic bacteria source may be one or more species of phototrophic bacteria. Phototrophic bacteria may include cyanobacteria, green sulphur bacteria, purple sulphur bacteria, purple non-sulphur bacteria, and green non-sulphur bacteria. Phototrophic bacteria may include Rhodopseudomonas spp., Rhodospirillum spp., Rhodomicrobium spp. or Chloroflexus spp. The phototrophic source may comprise spirulina, such as one or more Arthrospira spp. (e.g. A. platensis, A. fusiformis, and/or A. maxima).

During the photosynthesis step, the phototrophic source (e.g. an algal source) converts the carbon dioxide into an organic feedstock. The term “organic feedstock” as used herein is to mean a feedstock having organic matter, such as biomass, that can include simple and complex carbohydrates, such as simple and complex sugars, biopolymers such as exopolysaccharides, algal debris and by-products from photosynthesis. The organic feedstock can also include material used during the photosynthesis step, such as materials and reagents present in a culture medium that is used for the photosynthetic conversion of carbon dioxide into the organic feedstock.

The organic feedstock is then converted into a biomass. As described in further detail below. This biomass may be a useful product. Alternatively or additionally, the conversion of the organic feedstock into the biomass may involve the production of valuable by-products.

In the first aspect, the conversion of the organic feedstock into biomass comprises an aerobic bio-decomposition step that employs bacteria comprising at least one facultative anaerobic species. Bio-decomposition refers to the conversion of the organic feedstock into other forms using one or more organisms in one or more biological processes. “Aerobic biodecomposition”, as used herein, is decomposition of organic materials in the presence of oxygen. In aerobic decomposition, living organisms, which use oxygen, feed upon the organic matter. They use the nitrogen, phosphorus, carbon, and other required nutrients.

Typically, aerobic bio-decomposition relies on aerobic bacteria (and other aerobic organisms). However, in the first aspect, the aerobic bio-decomposition employs bacteria comprising at least one facultative anaerobic species. Facultative anaerobic species, as used herein, are species of bacteria which preferably grow in anaerobic conditions but, in the presence of oxygen, can change their metabolic processes to grow in aerobic conditions. That is, facultative anaerobe are generally more efficient fermenters (i.e. an anaerobic process) than respirators (i.e. an aerobic process). Although facultative anaerobes are relatively more efficient fermenters, they can function and survive effectively in aerobic conditions.

The aerobic bio-decomposition step involves the decomposition (breakage) of cell walls of the phototrophic source (e.g. algal cell walls) and the production of a medium in which the sugars (e.g. algal sugars) are available. The sugars are then available in the medium for later use. These sugars may be in the final biomass produced in accordance with the first aspect. In some embodiments, these sugars may be utilised in later stages of the conversion process, as part of producing the final biomass. Without being bound by theory, it is believed that employing one or more facultative anaerobic species for the aerobic bio-decomposition step may increase the amount of sugars produced during the bio-decomposition. Also, without being bound by theory, it is believed that employing one or more facultative anaerobic species for the aerobic bio-decomposition step may increase the duration of sugar production.

In addition, without being bound by theory, it is believed that the aerobic bio-decomposition step may provide a more energy efficient method of lysing cells within the organic feedstock. Recovering any intracellular product may require less energy compared to the product embedded in the cell wall such as transmembrane proteins. Alternative cell disruption methods are known for lysing microalgae cells at industrial scale. Ultrasonication, high speed homogenisation, high pressure homogenisation, bead milling and thermal treatment are examples of physical processes. These physical processes are non-selective and energy intensive. Chemical cell lysis is another known method. However, it is also generally a non- selective process and may be toxic and/or affect product stability. Additional steps may be required to remove chemical(s) added for lysis in order to avoid or minimise chemical contamination of the final product. Enzymatic cell lysis may offer advantages over physical and chemical processes in terms of selectivity and non-toxicity. However, the use of purified enzymes alone to effect lysis can be an expensive and time-consuming alternative. The energy consumed to lyse the cells using physical, chemical and enzymatic approaches can be higher than the energy released by the sugars and complex carbohydrates from organic feedstock. Lysis via the aerobic bio-decomposition step may lyse cells within the organic feedstock and release the intracellular matrix into the surrounding media. The aerobic biodecomposition step may be less energy intensive than the known using physical, chemical and enzymatic approaches. In addition, the aerobic bio-decomposition step may be nontoxic compared to know methods of chemical cell lysis.

The one or more facultative anaerobic species may be selected from the group consisting of Escherichia spp. (such as E. coli); Klebsiella spp. (such as K. aerogenes); Staphylococcus spp.; Streptococcus spp.; Salmonella spp. (such as S. enterica and/or S. bongori); Listeria spp.; Corynebacterium spp.; Shewanella spp. (such as Shewanella oneidensis); and subspecies thereof; including combinations of the aforementioned bacteria. Pathogenic bacteria may not be suitable for certain applications of the process, such as when the biomass will be utilised in agricultural applications (e.g. as a biofertilizer) but may be suitable for other applications such as when the process generates valuable by-products in addition to the biomass.

In some embodiments, the aerobic bio-decomposition step employs a mixture of bacteria comprising the at least one facultative anaerobic species and one or more aerobic species. In these embodiments, the one or more aerobic species may be selected from obligate aerobic species and facultative aerobic species. Facultative aerobic species as used herein, are species of bacteria which preferably grow in aerobic conditions i.e. in the presence of oxygen, but in absence of oxygen can change their metabolic processes to grow in anaerobic conditions. That is, facultative aerobes are generally more efficient respirators (i.e. an aerobic process) than fermenters (i.e. an anaerobic process). Although facultative aerobes are relatively more efficient respirators, they may lay dormant, while retaining their morphological structure, in anaerobic conditions. In some cases, facultative aerobes may survive and metabolise gasses (perform fermentation) under anaerobic conditions. When used, the one or more aerobic species may be selected from the group consisting of: one or more Bacillus spp.; one or more Azospirillum spp. (such as Azospirillum brasilense); and/or one or more Lactobacillus spp. By way of example, Bacillus spp. can survive in an anaerobic regime and extreme environments, form spores and perform other absorption capacities. The multiplication capacity of Bacillus spp. is specific to (greater in) an aerobic regime. In an anaerobic regime, they can metabolize gases.

In some forms, the aerobic species comprise one or more species selected from one or more Gram-negative bacterial species and/or Gram-positive bacterial species.

Bacillus spp. have diverse antagonistic relationships. The one or more aerobic species may comprise B. subtilis. In some embodiments, the one or more Bacillus spp. comprise one or more selected from the group consisting of: B. subtilis; B. megaterium; B. pumilus; B. amyloliquefaciens; B. licheniformis; B. thuringiensis; and subspecies thereof. For example, the one or more aerobic bacteria species may comprise B. thuringiensis subsp. kurstaki. In some embodiments, the one or more aerobic species are a mixture of Bacillus spp. In some embodiments, the aerobic species include a combination of one or more Bacillus spp. in combination with other species. The one or more aerobic species may comprise B. subtilis, B. thuringiensis and B. megaterium.

Employing a mixture of bacteria comprising the at least one facultative anaerobic species and one or more aerobic species for the aerobic bio-decomposition step may be advantageous in some embodiments. At least one facultative anaerobic species and one or more aerobic species may live together and multiply together, such that the inoculation of these bacteria in the aerobic bio-decomposition step can be done concomitantly. Without being bound by theory, it is believed that there may be synergistic or symbiotic effects when such a mixture is used. When at least one facultative anaerobic species and one or more aerobic species are combined, there may be interactions between the bacteria and phototrophic source (e.g. algae) under aerobic conditions (i.e. when oxygen is present) and as sugars are released into the medium. These sugars may be more easily accessed by both the phototrophic source (e.g. algae) and bacteria, enhancing the peak cycle of favourable conditions for total sugar production.

Furthermore, without being bound by theory, competition between the one or more facultative anaerobic species and one or more aerobic species may result in greater bacterial activity. This in turn may promote bio-decomposition and greater release of sugars from the phototrophic source (e.g. algae).

Alternatively, or additionally, the presence of one or more facultative anaerobic species may promote maintenance or survivability of the one or more aerobic species. As noted above, without being bound by theory, it is believed that the combination of bacteria enhances the bio-decomposition process. This may result in an expansion of sugars available to the mixture of species of bacteria in the culture medium.

During bio-decomposition, some growth of the phototrophic source, such as an algal source, will continue, which can affect the pH of the organic source and oxygen levels. The use of a mixture of bacteria species, including one or more facultative anaerobic species, may provide conditions where there is an improved balance between ongoing phototrophic growth and the bio-decomposition processes effected by the bacteria during the aerobic biodecomposition step. For example, in some embodiments, the presence of oxygen and alkalinity-generating algal species is beneficial to the oxygen-consuming and aciditygenerating bacteria species. Accordingly, the combination of the organic feedstock and a balanced pH environment may result in a form of symbiosis which enhances maintenance and survivability of the bacteria. By promoting maintenance or survivability, there may be enhanced overall bacterial activity, increasing bio-decomposition performance.

By employing a mixture of bacteria comprising the at least one facultative anaerobic species and one or more aerobic species for the aerobic bio-decomposition step, the process of cell wall decomposition may occur without impacting significantly on the consumption of sugar, such that the decomposition process can occur simultaneously with the production of biomass. Furthermore, valuable by-products can also be produced during production of the biomass.

In some embodiments, the at least one facultative anaerobic species may comprise K. aerogenes and the one or more aerobic species may be one or more Bacillus spp. (such as B. subtilis). In some embodiments, the combination of one or more Bacillus spp. with at least one facultative anaerobic species bacteria such as K. aerogenes may increase the amount and duration of sugar production as compared to amount and duration of sugar produced by using only Bacillus spp. alone.

In some embodiments of the process of the first aspect, converting the organic feedstock to biomass comprises, after the aerobic bio-decomposition step: an anaerobic biodecomposition step. In some embodiments, the bacteria employed for the anaerobic biodecomposition may be grown to a production quantity during the aerobic bio-decomposition step. This may enhance the process, as it can avoid having to maintain a culture medium with a production quantity of the bacteria employed for the anaerobic bio-decomposition in a separate vessel of the system. Instead, the culture medium introduced for the aerobic biodecomposition step may only need a loading of the facultative anaerobic species that is sufficient to initiate growth to a production loading during the aerobic bio-decomposition step. In such embodiments, one or more aerobic bacteria may be used in combination with the facultative anaerobic species. These aerobic species will be provided at a production loading for the aerobic bio-decomposition step so that this step can proceed as the loading of the facultative anaerobic bacteria increases to the production loading for the anaerobic biodecomposition.

In some embodiments, the conditions in the anaerobic bio-decomposition step are such that one or more of the aerobic species used in the aerobic bio-decomposition will not persist in the anaerobic conditions. For example, when obligate aerobic species are used, these may be allowed not to survive the anaerobic bio-decomposition. Alternatively, it may be preferred that one or more aerobic species persist under anaerobic conditions. Persisting may include surviving (i.e. the bacteria are anaerotolerant) or growing and multiplying under anaerobic conditions.

As noted above, in some embodiments, the one or more aerobic species comprise one or more species selected from one or more facultative aerobic species. Facultative aerobic species may be advantageous in embodiments using an anaerobic bio-decomposition step, as facultative aerobic species can continue to grow and multiply under anaerobic conditions. The exemplary species mentioned above include facultative aerobic species. Some specific examples of facultative aerobic species are: B. subtilis; B. megaterium; B. pumilus; B. amyloliquefaciens; B. licheniformis; B. thuringiensis (incl. B. thuringiensis subsp. kurstaki); Azospirillum brasilense; and Lactobacillus spp.

In some embodiments, the aerobic bio-decomposition step will be performed until a desired sugar level is reached. For example, in some embodiments, the aerobic bio-decomposition step will be performed until peak sugar levels are reached. That is, the desired sugar level is the peak sugar level. In some other cases, the desired sugar level will be a level selected after achieving the peak sugar level. Sugar levels can be determined by monitoring the sugar levels in the culture medium. The peak sugar level can be determined by monitoring for the point at which the sugar available in the culture medium declines. At this point, the phototrophic source (e.g. the algae source) is no longer generating sufficient organic feedstock to maintain the bacteria. Peak sugar levels will occur when the rate of sugar available in the culture medium is no longer increasing. Once peak sugar levels have been obtained, the biomass may be collected (harvested). Alternatively, after reaching peak sugar levels, the process may progress from the aerobic bio-decomposition step to the anaerobic bio-decomposition step. Commencing the anaerobic bio-decomposition step after peak sugar levels have been obtained can be advantageous for the productions of valuable by- products, such as hydrogen gas. In some embodiments, the aerobic bio-decomposition step may continue beyond the peak sugar level point and until available sugars begin to decline. Such an arrangement may be selected if the process if being used for lower cost CO2 absorption.

In some embodiments, the desired sugar level for the aerobic bio-decomposition step many be reached after up to 30 days, such as up to about 15 to about 20 days. In some embodiments, the desired sugar level is reached after about 5 days.

In some embodiments, a further nutrient may be added before and/or during the aerobic biodecomposition step. The further nutrient may promote the growth of the one or more aerobic species, when the bacteria used comprises aerobic species. The further nutrient may comprise glycerine (glycerol). In some embodiments, the glycerine is added in the range of 1 to 10% w/V. Glycerol may provide fuel for the bacteria so that the bacteria decomposes the organic feedstock without consuming the released sugars, thus increasing the efficiency of the aerobic bio-decomposition step and the speed of consumption and growth.

In some embodiments, adding a further nutrient (e.g. glycerine) may improve the performance of the aerobic bio-decomposition step. In some embodiments, adding volumes of up to 3% of glycerine in the aerobic bio-decomposition may cause the one or more aerobic species (e.g. one or more Bacillus spp.) to grow exponentially, accelerating the decomposition of the phototroph’s cell walls (e.g. algae cell walls) in the organic feedstock. In some embodiments, the process may comprise monitoring bacteria growth during the aerobic bio-decomposition step and adjusting the amount of further nutrient that is added.

In some embodiments, the addition of a further nutrient before and/or during the aerobic biodecomposition step may be advantageous for any downstream anaerobic bio-decomposition step. For example, the addition of a nutrient, such as glycerine, may be useful for the production valuable by-products during the anaerobic bio-decomposition step. The addition of glycerine may favour more sugar in the medium. Glycerine may optimize the medium generating cellular osmotic balance, which may result in better anaerobic bio-decomposition. The addition of a nutrient, such as glycerine, may be beneficial for the production of valuable by-products such as hydrogen, alcohol, lipids etc. during the anaerobic bio-decomposition step. In some embodiments, alcohol may be methanol. In some embodiments, the amount of nutrient added, and the timing of that addition, may be selected to provide a balance of nutrient within a pre-determined range at the commencement of the anaerobic biodecomposition step. The nutrient balance may be monitored so as to adjust the amount and or timing of nutrient addition in order to arrive at a nutrient balance within the pre-determined range. Providing a nutrient (e.g. glycerine) balance within a pre-determined range may be advantageous for the generation of valuable by-products (e.g. hydrogen gas) during the anaerobic bio-decomposition step.

The process may comprise regulating or adjusting a temperature of step (i) and/or step (ii), such as with a heat source. For example, steps (i) and (ii) may both be maintained at about 35°C. The specific temperature of the photosynthesis step and/or the bio-decomposition step(s) may be determined by and regulated so as to favour the phototrophic source and/or bacteria used in these steps. In some embodiments, the bacteria used for the aerobic biodecomposition step may be mesophilic bacteria so as to minimise heating requirements. The bacteria used for the anaerobic bio-decomposition step (if performed) may be mesophilic bacteria so as to minimise heating requirements.

In some embodiments, the process comprises adjusting or providing light levels to within a pre-determined range during either or each of the aerobic bio-decomposition step and/or anaerobic bio-decomposition step (if the anaerobic bio-decomposition step is performed). Controlling light exposure levels can modulate the ongoing growth of the cells of the phototrophic source (e.g. algal cells) during the bio-decomposition step(s). Thus, the light exposure may be selected or adjusted to affect phototrophic source’s growth, which in turn can influence the activity of the bacteria. Selecting or adjusting the light exposure may assist in enabling the process of cell wall decomposition to occur without impacting significantly on the consumption of sugar.

In general, during the photosynthesis step, the more light (and COs gas) provided the faster the growth of the organism(s) in the phototrophic source. Typically, at the commencement of the aerobic bio-decomposition step the culture medium will be subjected to reduced or minimal light levels. Lower light levels promote the bio-decomposition process. In some embodiments, the light level may be increased for one or more periods during the aerobic bio-decomposition step. These one or more periods may be after the commencement of the bio-decomposition step, such as after a pre-selected level of bio-decomposition has occurred. Increasing the light level can stimulate carbon conversion by the phototrophic source and, consequently, new sugar generation activity. The light level may be adjusted to extend the duration of CO2 consumption. In some embodiments, light levels may be adjusted during process so that the biomass (and optionally other by-products) can be continuously produced.

Processes in accordance with the first aspect can be directed for sequestering carbon from a gas stream that comprises carbon dioxide, the process comprising: converting the carbon dioxide in the gas stream to an organic feedstock using a phototrophic source in a photosynthesis step. The carbon dioxide gas stream may be generated by combustion of hydrocarbons, such as in a coal- or gas-fired power station, or conversion of hydrocarbons into other gases that include carbon dioxide, such as occurs with steam reforming. An advantage of the disclosed process can be sequestering carbon from waste carbon dioxide, such as that generated by industrial processes, and in doing so converting it into product (the organics feedstock) that can then be utilised in the synthesis of another product, the biomass.

Embodiments of the process may often be conducted using gas streams that comprises carbon dioxide at concentrations greater than that found in atmospheric air, such as waste gas streams or flue gas. That is, the process may be used to prevent the release of carbon dioxide into the atmosphere by scrubbing waste gas streams. However, in some cases the process may be use for carbon capture from air. Thus, some embodiments may be used to sequester atmospheric carbon. Some embodiments may use direct air capture or alternatively use gas capture devices that produce a concentrated CO2 stream (i.e. a stream with CO2 in a concentration greater than the original air intake) that is fed to the process. Therefore, the process may be used to "scrub" or remove carbon dioxide from the atmosphere or from carbon dioxide producing activities. The disclosed process may be used in place of carbon dioxide sequestration such as where carbon dioxide is pumped and stored in geological formations.

As noted above, in some embodiments, valuable by-products can be generated when producing the biomass. In some embodiments, the by-product is hydrogen. Thus, in some embodiments, the process may be advantageous compared to existing carbon dioxide sequestration techniques as some embodiments of the present process may also produce hydrogen gas as a renewable gas source.

When converting the organic feedstock to biomass, the decomposition of the phototroph’s cells, e.g. algal cells, (into which the carbon has been sequestered) can result in the generation of carbon dioxide. The generation of this carbon dioxide will not outweigh the sequestration during the photosynthesis step so that there is effective sequestration of the carbon overall (in the resulting biomass). In some embodiments, the carbon dioxide produced during conversion of the organic feedstock to biomass will be collected and recycled. In some embodiments, the process may further comprise: collecting gaseous byproducts after the aerobic bio-decomposition step (or after converting the organic feedstock to biomass) and filtering the gaseous by-products to isolate a second carbon dioxide gas stream. The process may further comprise transferring the second carbon dioxide stream to step (i). The first and second carbon dioxide gas streams may be combined.

In some embodiments, the photosynthesis step may be performed in a microbial reactor that is fitted with a photon source. For the photosynthesis step, parameters such as media, pH, salinity, nutrient requirements, required light dosage rates, photosynthesis temperature, and so on will be adjusted according to requirements of the phototrophic source. The specific temperature and/or light dosage of the photosynthesis step may be determined by and regulated so as to favour the phototrophic source. Generally, the temperature of the photosynthesis conversion of carbon dioxide into the organic feedstock will range from about 30 °C to about 40 °C. The type of phototrophic source used, and the resulting organic feedstock produced, may be selected depending upon the requirements of the bacteria utilised to convert the organic feedstock into biomass. In some embodiments, more than one type of algal species may be used.

In a second aspect, there is provided a biomass produce by a process in accordance with the first aspect of the present disclosure. In some embodiments, the biomass may be the product of the aerobic bio-decomposition step. In some other embodiments, the biomass may be the product of the anaerobic bio-decomposition step. In some embodiments, the biomass may be a mixture comprising a first biomass fraction collected from the aerobic biodecomposition step and a second biomass fraction collected from the anaerobic biodecomposition step. In some embodiments, the biomass (or biomass fractions) may be collected (harvested) and then stored in one or more storage vessels.

During the aerobic bio-decomposition step, the cell walls of the organisms in the phototrophic source are broken. This process of cell breakage typically leaves very fine particles remain in the culture medium. Thus, much of the biomass produced in the process can be considered a liquid suspension. The biomass can contain metabolites, minerals, organic carbon, sugars and microbes.

The biomass may be harvested simply by collecting the biomass as produced. That is, in some embodiments, the biomass may be transferred into a storage vessel or packaging. The biomass as produced, with its original moisture content, may then be deployed in the desired application. In some other embodiments, the process may comprise extracting at least a portion of the water in the biomass before storing the biomass. That is, the process may comprise dewatering the biomass. Water removed from the biomass may be recovered and, in some cases, recycled. Various known dewatering methods can be used for dewatering the biomass, such as filtering. The dewatering method selected may depend on whether it is desirable to maintain any live bacteria or other organisms in the biomass. In such cases, the dewatering method selected may be one that avoids subjecting the biomass to shear forces that will cause significant cell disruption and losses in viability. Thus, the process may use suitably low shear dewatering. Suitably low shear dewatering is dewatering that is conducted below the shear rate at which significant cell disruption and losses in viability occur. The biomass obtained using the process of the first aspect may be suitable for used as a biofertilizer. Biofertilizers typically include living microbes that enhance plant nutrition by either by mobilizing or increasing nutrient availability in soils. The biomass may comprise bacteria species which may solubilize mineral and organic components in the soil; promote plant growth (e.g. act as growth promoters); and/or prevent or control diseases and pests. Thus, the step of converting the organic feedstock into biomass can be considered a step of growing bacteria for use in a biofertilizer.

In some embodiments, the biomass for use in as a biofertilizer may be harvested by emptying the contents of the reactor into a storage vessel. That is, the biomass may have a suitable composition and concentration for use as a biofertilizer, without requiring any removal of water to concentrate beneficial components of the biofertilizer.

In some embodiments, harvesting the biomass comprising transferring the biomass into a storage vessel or packaging. Harvesting the biomass may comprising draining and/or pumping into a storage vessel or packaging. The storage vessel may be a tank or bottle. The packaging may be a tote. Packaging the biomass into a tote can provide the biomass as a readily distributable product.

Once in the storage vessel or packaging, the biomass can be stored until distributed and/or deployed. In some cases, the biomass will be stored in a storage vessel before being packaged. After packaging, the biomass may be distributed and then stored for another period until being deployed.

As the biofertilizer contains live organisms (e.g. the bacteria), it can be desirable to minimise the period of storage before deployment in order to maintain viability of the organisms in the biofertilizer.

Bacteria is viable if it is alive and capable of reproduction or colonization. The concentration of viable bacteria in the biomass may need to exceed a certain threshold value or the beneficial effect of the bacteria may not be provided for certain applications of the biomass, such as when the biomass is used a biofertilizer. Quantities of bacteria are typically evaluated in terms of colony forming units (CPU). The biomass may have a high concentration of live bacteria. In some embodiments, the concentration of bacteria in the biomass may be more than 5.0 x10⁶ CFU/ml for one or more species in the biomass. In some embodiments, the concentration of bacteria in the biomass may be more than 7.0 x10⁸ CFU/ml, such as about 8.5 x10⁸ CFU/ml for one or more species in the biomass. In some embodiments, the concentration of bacteria in the biomass may be more than 1.0 x10⁹ CFU/ml, such as about 1.5 x10⁹ CFU/ml for one or more species in the biomass.

In order to maintain the viability of the bacteria during storage, the biomass may by cooled in storage. In some embodiments, the bacteria may be stored at a temperature of about - 20°C.

In some embodiments, an additive may be added to the biomass before storage. The additive may be used to enhance the survival of the bacteria in storage. For example, in some embodiments, the additive may have cryoprotective and/or lyoprotective properties for the bacteria. Thus, the additive may contribute to improvements in bacteria survival following storage of the biomass at low temperatures, for example following storage at -20°C.

The additive may be a glycerol-based cryoprotective additive, such as a glycerol-based medium (10%v/v), that is mixed with the biomass before storage. The additive may comprise one or more disaccharides, oligosaccharides and/or polysaccharides, such as trehalose.

The biomass may have high amounts of one or more of N, P, K⁺, Ca²⁺, Mg²⁺, Fe, Mn, Cu, Zn, B, C, organic matter etc. Table 1 A below outlines the amount of N, P, K⁺, Ca²⁺, Mg²⁺, Fe, Mn, Cu, Zn, B, C, Organic Matter that may be present in some embodiments. It should be understood that each amount may be present in an embodiment, with differing amounts of the remaining nutrients. Thus, each entry in Table 1 A should may be individual or in combination with any one or more of the other entries in the table for some embodiments.

Table 1A

The biomass is made up of the organic matter and inorganic residue. That is, the inorganic residue percentage balances the organic matter percentage. The inorganic residue is the fixed mineral residue from the biomass (e.g. sodium, potassium, magnesium, calcium, iron, phosphorous, copper, chloride, aluminium, zinc, manganese and other mineral compounds).

The organic matter and inorganic residue percentages are calculated based on the loss on ignition mass. Loss on ignition mass loss is determined by burning a dried sample of the biomass in an oven at a high temperature (typically, 500-600°C). The mass remaining after burning is the inorganic residue, with the mass lost on ignition being the organic matter. The CaCh pH range for ideal plant growth is between 5-8. The biomass may have a CaCh pH within range for ideal plant growth. In some embodiments, the CaCh pH of the biomass may be about 6.7-7.2. Different plants prefer different soil pH conditions (e.g. some plants prefer slightly acidic, or slightly alkaline). The phototrophic source and/or the bacteria used to convert the organic feedstock into the biomass can affect pH. In some embodiments, the phototrophic source and/or the bacteria, as well as the growth conditions, may be selected to provide a biomass at a desirable pH for use as a biofertilizer for one or more plants. Thus, in some embodiments, the pH of the biomass may be within a pre-determined range based on the intended use of the biomass. In some embodiments, a combination of oxygen- and alkalinity-generating phototrophic species and oxygen-consuming and acidity-generating bacteria species is used to achieve a desired biomass pH, with the relative growth of the alkalinity-generating species and acidity-generating species being controlled to adjust biomass pH.

In biological farming systems, the ideal "balanced" ratio of carbomnitrogen is 24:1. In general, to provide a ratio approaching the desired level it is necessary to add nitrogen to soils. In some embodiments, the biomass may have a carbon mitrogen ratio of about 200 to about 250. By having a higher carbomnitrogen ratio, the biomass, when used as a biofertilizer, may provide fuel to support adding more microbes to the soil to generate nitrogen. Alternatively or additionally, the biomass can provide direct nitrogen when deployed as a biofertilizer.

As noted above, the biomass can be stored until distributed and/or deployed. In some embodiments, such as when the biomass is used for an agricultural treatment (e.g. a soil and plant treatment or a biofertilizer), the biomass may be deployed using spray equipment, such as spray equipment typically utilised in agricultural applications. Alternatively, the biomass may be deployed using equipment used for irrigation, such as drip lines.

The biomass produced in accordance with the first aspect may be compatible with commercial fertilizers. Alternatively or additionally, the biomass may have diverse applications in agriculture, soil and plant treatment. In some embodiments, the biomass may be used in soil remediation for mining sites. In some other embodiments, the biomass may be applied as a treatment to mining deposits, such as deposits which depend on biogeochemical cycles. Indirectly, by increasing soil biomass, the use of the biomass as a biofertilizer or soil treatment may enhance drainage control and/or absorption.

In some embodiments, the biofertilizer may include the biomass and one or more other components. In some embodiments, the biomass may be combined with one or more bacteria species (one or more supplementary bacteria species) and/or one or more fungi species. For example, in embodiments in which an anaerobic bio-decomposition step is used, the biomass may be mixed with one or more bacteria species (and/or fungi species) that would not have tolerated the anaerobic bio-decomposition step in order to produce the biofertilizer. Such bacteria may be obligate aerobic bacteria that are desirable for use in biofertilizers. The one or more bacteria species and/or fungi species added to the biomass may be species that would not have be beneficial to the process of producing the biomass. That is, the one or more bacteria and/or fungi species may not have been desirable for use in the aerobic bio-decomposition step and/or anaerobic bio-decomposition step (if the anaerobic bio-decomposition step is performed). The one or more supplementary bacteria may be selected from the group consisting of: Azospirillum brasilense; Bacillus subtilis; Trichoderma harzianum; Bacillus aryabhattai; Saccharopolyspora spinose; Chromobacterium subtsugae; Bacillus thuringiensis (BT); Beauveria bassiana; Bacillus pumilus; and any combinations thereof. The one or more fungi species may be selected from the group consisting of: Isaria fumosorosea; Metarhizium anisopliae; and any combinations thereof. It will be appreciated that the one or more supplementary bacteria species may be selected from a species that is otherwise suitable for use as bacteria employed in the conversion of the organic feedstock into a biomass. In some embodiments, the bacteria species may not have been utilised for the production of the biomass in that embodiment of the process and, accordingly, is added as a supplementary species. Alternatively, or additionally, the addition of supplementary bacteria may increase the amount of a desired bacteria species already in the biomass to a level desired for the biofertilizer.

The biomass may be combined with the one or more supplementary bacteria species at the time of deployment or shortly before deployment. For example, the supplementary bacteria species may be mixed with the biomass at the time of spraying or may be mixed with the biomass immediately prior commencing spraying (e.g. the supplementary bacteria species may be mixed with the biomass in the storage vessel of the spraying equipment). Alternatively, the supplementary bacteria species may be combined with the biomass immediately or shortly before the biomass is loaded into the deployment equipment (e.g. the spraying or irrigation system). In some embodiments, the supplementary bacteria species may be combined with the biomass while it is in storage. The supplementary bacteria species may be combined with the biomass shortly after or during harvesting. In some embodiments, the mixture of the supplementary bacteria species and biomass is transferred to the storage vessel or packaging. In some embodiments, the supplementary bacteria species may be combined (e.g. mixed) with the biomass at the time of packaging so that the mixture is packaged.

In embodiments in which the biomass is collect for use as, or as part or, a biofertilizer, the bacteria selected for the bio-decomposition step(s) may be selected to provide a biofertilizer with a desired bacterial composition. For example, the at least one facultative anaerobic species may comprise a nitrogen-metabolising anaerobe(s). In some embodiments, the at least one facultative anaerobic species may comprise a urease-producing anaerobe(s). For example K. aerogenes produces urease by metabolism. Urease catalyses the hydrolysis of urea into carbon dioxide and ammonia. Thus, the inclusion of urease-producing anaerobe(s) can facilitate the effective utilisation of urea-containing fertilizers that can be used in combination with the biofertilizer. Urease may also enhance soil biomineralization and biocementation conditions.

In some embodiments, a biofertilizer comprising urease-producing anaerobe(s) may be advantageous as it catalyses the hydrolysis of urea into carbon dioxide and ammonia. The ammonia may then be directly absorbed by plants. Alternatively, or additionally, the ammonia may be converted by one or more microbes in the biofertilizer into nitrate. The biofertilizer may comprise one or more ammonia-oxidizing bacteria and/or nitrifying bacteria that convert the ammonia into nitrate. Nitrate can have a higher uptake rate by plants than ammonia. In some embodiments, the ammonia-oxidizing bacteria and/or nitrifying bacteria may be one or more species selected from Nitrosomonas spp., Nitrosospira spp., Nitrosococcus spp., and NitrosoIobus spp. In some embodiments, the ammonia-oxidizing bacteria may be a supplementary bacteria species. In some other forms, an aerobic species utilised in the process, and present in the resulting biomass, is ammonia-oxidizing bacteria ammonia- oxidizing bacteria and/or nitrifying bacteria. In some of those embodiments, the ammonia- oxidizing bacteria and/or nitrifying bacteria may be one or more species selected from Nitrosomonas spp., Nitrosospira spp., Nitrosococcus spp., and NitrosoIobus spp.

In some forms, the least one facultative anaerobic species utilised in the process, and present in the resulting biomass, is K. aerogenes. K. aerogenes may generate various metabolites, including urease.

In some embodiments, the bacteria may comprise one or more phosphorus-solubilizing bacteria. Such bacteria may mobilize poorly available phosphorus via solubilization and mineralization. Phosphorus-solubilizing bacteria may include Bacillus spp., Pseudomonas spp., and Agrobacterium spp.. Thus, the one or more aerobic bacteria used in some embodiments may comprise one or more phosphorus-solubilizing bacteria, such as Bacillus spp. Alternatively, the at least one facultative anaerobic species may comprise one or more species selected from Pseudomonas spp., and Agrobacterium spp.

In some embodiments, bacteria utilised in the process and present in the resulting biomass may provide protection against diseases, protection against pests, and/or may control growth of crops. One or more embodiments of the biomass may provide one or more of soil pest control, reduction of water stress, solubilization, leaf disease protection, root disease protection and nematode protection, nutrition, caterpillar control, and bedbug control.

In some embodiments, the aerobic bacteria utilised in the process and present in the resulting biomass may comprise a mixture of Bacillus spp. In a biofertilizer, B. megatherium may enable mineral solubilization, B. subtilis may provide protection against diseases and B. thuringiensis, may provide protection against pests. Accordingly, in some embodiments, it can be advantageous to use a mixture comprising B. subtilis, B. thuringiensis and B. megaterium. Such a combination of bacteria may provide a biomass suitable for use in the production of a biofertilizer for soybean and/or corn crops.

In some embodiments, it is desirable for the least one facultative anaerobic species utilised in the process, and present in the resulting biomass, to be K. aerogenes and for the one or more aerobic species utilised in the process, and present in the resulting biomass, to be B. subtilis, B. thuringiensis and B. megaterium.

In some embodiments, the biomass produced by the process of the first aspect may be used as a feedstock into other biological processes. For example, the biomass may be a suitable feedstock for generating other products such as alcohol(s), polyunsaturated fatty acids and other fatty acids. In some embodiments, the bacteria may be selected such that the anaerobic bio-decomposition step generates by-products such as alcohol(s), polyunsaturated fatty acids and other fatty acids.

In some embodiments, a by-product-producing bacteria may be used in, or added to, the aerobic bio-decomposition step and/or anaerobic bio-decomposition step in order to produce a valuable by-product at the same time as converting the organic feedstock into biomass. In some embodiments, a by-product-producing bacteria may be added to either or each of the aerobic bio-decomposition step and anaerobic bio-decomposition step. For example, a by- product-producing that is unable to tolerate the aerobic bio-decomposition step may be added when the anaerobic bio-decomposition step commences so that the by-product can be generated during this step. The by-product-producing bacteria may comprise methanogenic bacteria, methanobacteria, aceto bacteria, acetogenic bacteria, liquefaction bacteria, Clostridium spp. (methane), Bacillus spp., Escherichia spp., Staphylococcus spp., Methanobacter spp., Methanococcus spp., Methanosarcina spp., Saccharomyces spp.. For example, the by-product-producing bacteria may be Methanobacterium (Mb.) omlianskii (methane), Mb. formicicum (methane), Mb. soehngenii (methane), Mb. thermoautotrophicum (methane), Mb. ruminantium (methane), Mb. mobile (methane), Mb. methanica (methane), Mb. suboxydans (methane), Mb. propionicum (methane), Methanococcus (Me.) mazei (methane Me. vannielii (methane), Methanosarcina (Ms.) bovekeri (methane), Ms. methanica (methane), Ms. alcaliphilum (methane), Ms. acetivorans (methane), Ms. thermophilia (methane), Ms. barker/ (methane), Ms. vacuolata (methane), Propionibacterium acidi-propionici (methane), Saccharomyces cerevisiae (ethanol), S. ellipsoideus (ethanol), Clostridium propionicum (propanol), Clostridium acetobutylicum (butanol), Clostridium saccharoperbutylacetonicum (butanol), Clostridium butyricum (hydrogen), wherein the chemical in parentheses indicates a useful material which that by-product-producing bacteria produces.

In some embodiments of the present invention, hydrogen gas may be generated during the aerobic bio-decomposition step and/or anaerobic bio-decomposition step. This hydrogen may be collected, separately from the biomass, and then stored in one or more storage vessels. Hydrogen generation may be performed by conducting the photosynthesis step and one or both of the bio-decomposition steps in accordance with the hydrogen generation process described in International Patent Application No. PCT/AU2020/050285 (published as International Publication No. WO/2020/191442) the contents of which are incorporated by reference, except that in the process of the present disclosure the aerobic biodecomposition step that employs bacteria comprising at least one facultative anaerobic species.

The bacteria selected for use in the aerobic bio-decomposition step and/or anaerobic biodecomposition step may comprise hydrogen-producing bacteria. One or more bacteria species may be selected so that hydrogen is generated during either or each of the aerobic bio-decomposition step and anaerobic bio-decomposition step. In some embodiments, one or more hydrogen-producing bacteria may be added to either or each of the aerobic biodecomposition step and anaerobic bio-decomposition step. For example, a hydrogenproducing bacterium that is unable to tolerate the aerobic bio-decomposition step may be added when the anaerobic bio-decomposition step commences so that hydrogen can be generated during this step.

In some embodiments, the hydrogen-producing bacteria may comprise one or more of Bacillus spp. and K. aerogenes.

Thus, in some embodiments, there is a provided a process in which hydrogen is generated during one or both of the aerobic bio-decomposition step and the anaerobic biodecomposition step, and the biomass produced after hydrogen production is suitable for use as, or as part of, a biofertilizer. The biomass may be abundant in organic matter, minerals and proteins remaining from the microorganisms utilised during the bio-decomposition step(s), as well as living bacteria. Thus, the biomass may comprise nutrients and bacteria that are beneficial for plant cultivation. In a third aspect, there is provided hydrogen that is produced by a process in accordance with the first aspect of the present disclosure.

A further aspect of the present disclosure provides a system suitable for performing the process of the first aspect. There is provided a system for sequestering carbon from a gas stream that comprises carbon dioxide, the system comprising: one or more reactors; said reactors comprising: at least one reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source, said reactor having an inlet for receiving a carbon dioxide gas stream; and at least one reactor configured for subjecting the organic feedstock to biodecomposition, said reactor being configured to alternate between operating under aerobic and anaerobic conditions.

In accordance with this aspect, there is also provided a system for producing biomass, the system comprising: one or more reactors; said reactors comprising: at least one reactor configured to produce an organic feedstock using a phototrophic source and a gas stream comprising carbon dioxide, said reactor having an inlet for receiving a carbon dioxide gas stream; and at least one reactor configured for subjecting the organic feedstock to biodecomposition, said reactor being configured to alternate between operating under aerobic and anaerobic conditions. The first carbon dioxide gas stream may be a first waste carbon dioxide gas stream.

Thus, the system for performing the process of the present disclosure may have a single reactor that is configured for both conducting the photosynthesis step and the conversion of the organic feedstock to biomass. This reactor can be considered both a bio-decomposition- capable reactor and a photosynthesis-capable reactor. The system may have two or more reactors that are configured for both conducting the photosynthesis step and the conversion of the organic feedstock to biomass. In some embodiments of the system, at least one of said one or more reactors is configured both to convert a carbon dioxide gas stream into the organic feedstock using the phototrophic source and for subjecting the organic feedstock to bio-decomposition. As described above, in some embodiments, the biomass may be the product of the aerobic bio-decomposition step. In some other embodiments, the biomass may be the product of the anaerobic bio-decomposition step. In some embodiments, the biomass may be a mixture comprising a first biomass fraction collected from the aerobic biodecomposition step and a second biomass fraction collected from the anaerobic biodecomposition step. It will be appreciated that the system may adopt different modes of operation so as to produce and harvest biomass from the aerobic bio-decomposition step and/or anaerobic bio-decomposition step.

In some embodiments, in use, the reactor(s) of the system will first receive a medium comprising the phototrophic source. During the photosynthesis step, the phototrophic source will be provided with light and carbon dioxide. Thus, the reactor may comprise one or more light sources (photon sources). After the phototrophic source has reached a suitable loading for the organic feedstock, a medium containing the bacteria comprising at least one facultative anaerobic species is added to the reactor and the light levels are reduced or minimised so that the aerobic bio-decomposition step may commence. As discussed above, in some embodiments, the light levels may be adjusted during bio-composition of the organic feedstock. The medium containing the bacteria may be transferred from one or more bacterial medium vessels in the system. The bacterial medium vessel(s) may be used to maintain a culture of the bacteria for the aerobic bio-decomposition step.

In some embodiments, the system comprises two or more reactors configured both to convert a carbon dioxide gas stream into the organic feedstock using the phototrophic source and for subjecting the organic feedstock to bio-decomposition. The two or more reactors may be fluidly connected.

In use, one or some of the reactors may receive a medium comprising the phototrophic source and be used to perform the photosynthesis step. Once a suitable organic feedstock has been generated, a portion of the organic feedstock can be provided to the other reactor(s) in the system (i.e. those not used for the photosynthesis step). This redistribution of the organic feedstock may provide space in each reactor to receive a medium containing the bacteria comprising at least one facultative anaerobic species. As noted above, this medium may be transferred from a bacterial medium vessel(s).

In some embodiments, only one or some of the reactors are configured for performing the photosynthesis step, as only one or some of the reactors may be used to prepare the organic feedstock before it is distributed between the reactors of the system. Thus, in some other embodiments, the system comprises: one or more reactors configured both to convert a carbon dioxide gas stream into the organic feedstock using the phototrophic source and for subjecting the organic feedstock to bio-decomposition; and one or more bio-decomposition reactors, each of said bio-decomposition reactors being a reactor configured for subjecting the organic feedstock to bio-decomposition, said reactor being configured to alternate between operating under aerobic and anaerobic conditions. The or each reactor configured for subjecting the organic feedstock to bio-decomposition may comprise a first gas inlet connected to a source of oxygen-containing gas and a second gas inlet connected to a source of oxygen-free gas. Thus, in some embodiments, the reactor (the bio-decomposition-capable reactor) may be configured to alternate between operating under aerobic and anaerobic conditions by having a first gas inlet connected to a source of oxygen-containing gas (e.g. air) and a second gas inlet connected to a source of oxygen- free gas (e.g. nitrogen or hydrogen gas). To switch between operating under aerobic and anaerobic conditions, the supply of oxygen-containing gas can be switched off and the supply of oxygen-free gas can commence or vice versa. In some other embodiments, the or each reactor may have a single gas supply line may be connected to both the source of oxygen-containing gas and the source of oxygen-free gas. A valve arrangement may be used to switch between the gases provided using the line.

The bio-decomposition-capable reactor can be configured to alternate between generating an oxygen-containing and an oxygen-free atmosphere. In some embodiments, the biodecomposition-capable reactor is configured to generate anaerobic conditions by injecting nitrogen into the reactor, while ceasing air ingress and regulating temperature parameters. During the switching process, the injection of the oxygen-free gas can be used to purge the oxygen-containing gas from the reactor.

One or more of the reactors may be configured for pressurisation. Thus, in some embodiments, during use the reactor may be pressurised. Pressurisation may increase the solubilisation of carbon dioxide. Pressurisation may also enable pressurised gaseous byproducts to be collected from the system. Alternatively or additionally, a reactor configured for performing the photosynthesis step may also be configured to receive an excess flowrate of carbon dioxide gas. The excess flowrate may be used to agitate the phototrophic source, enhancing contact with the carbon dioxide. In embodiments using an excess flowrate, the system may be configured to recycle excess carbon dioxide back through the reactor. The excess flowrate may be a flow rate of up to 5 VVM.

In some embodiments, each reactor will be configured for only one of conducting the photosynthesis step and the conversion of the organic feedstock to biomass. Thus, the present disclosure provides a system for sequestering carbon from a gas stream that comprises carbon dioxide, the system comprising: a photosynthesis reactor configured to convert a first carbon dioxide gas stream into an organic feedstock using a phototrophic source, the photosynthesis reactor having an inlet for receiving a carbon dioxide gas stream and an organic feedstock outlet; and a bio-decomposition reactor comprising an inlet in communication with the organic feedstock outlet for receiving the organic feedstock, the bio-decomposition reactor being configured to alternate between operating under aerobic and anaerobic conditions. Also provided is a provides a system for producing biomass, the system comprising: a photosynthesis reactor configured to produce an organic feedstock using a phototrophic source and a gas stream comprising carbon dioxide, the photosynthesis reactor having an inlet for receiving a carbon dioxide gas stream and an organic feedstock outlet; and a bio-decomposition reactor comprising an inlet in communication with the organic feedstock outlet for receiving the organic feedstock, the bio-decomposition reactor being configured to alternate between operating under aerobic and anaerobic conditions. The first carbon dioxide gas stream may be a first waste carbon dioxide gas stream.

In some embodiments, the bio-decomposition reactor may be configured to alternate between operating under aerobic and anaerobic conditions by having a first gas inlet connected to a source of oxygen-containing gas (e.g. air) and a second gas inlet connected to a source of oxygen-free gas (e.g. nitrogen gas). To switch between operating under aerobic and anaerobic conditions, the supply of oxygen-containing gas can be switched off and the supply of oxygen-free gas can commence or vice versa. The bio-decomposition reactor can be configured to alternate between generating an oxygen-containing and an oxygen-free atmosphere. In some embodiments, the bio-decomposition reactor is configured to generate anaerobic conditions injecting nitrogen into the reactor, while ceasing air ingress and regulating temperature parameters.

The system may further comprise one or more storage vessels for collecting and storing biomass and any by-products (e.g. hydrogen). The biomass and any by-product(s) are typically stored in separate storage vessels. The one or more storage vessels may be in fluid communication with the reactor (such as a bio-decomposition-capable reactor or a dedicated bio-decomposition reactor) for receiving and storing the selected material (i.e. biomass or by-product) produced or generated in the bio-decomposition step. In some embodiments, the system may comprise a biomass collection line and a hydrogen gas stream line, each connected to a respective storage vessel, each line be configured to transport the select material to the relevant storage vessel.

The system may further comprise an auxiliary carbon dioxide supply line for transferring carbon dioxide generated in a reactor for performing the bio-decomposition step(s) (such as a bio-decomposition-capable reactor or a dedicated bio-decomposition reactor) to a reactor for performing the photosynthesis step (such as a photosynthesis-capable reactor or a dedicated photosynthesis reactor). A filter or scrubber may be provided along the auxiliary carbon dioxide supply line may comprise for removing gases other than carbon dioxide.

In some embodiments, the system may comprise one or more heat exchangers configured to heat either or each of the one or more reactors. The system may comprise one or more heat exchangers to heat either or each of the photosynthesis reactor and bio-decomposition reactor.

The system may further comprise a controller for controlling the reactor(s). Controlling one or more of the reactors may comprise alternating operation between aerobic to anaerobic conditions. The system may further comprise a controller for controlling the photosynthesis reactor and/or the bio-decomposition reactor.

The system may further comprise a combustion chamber in fluid communication with and upstream of a reactor for performing the photosynthesis step (such as a photosynthesis- capable reactor or a dedicated photosynthesis reactor). The combustion chamber may be configured to combust a fuel source to generate the first carbon dioxide gas stream.

In some embodiments, the system, further comprises a water supply for supplying water to one, some of all of the reactors. For example, the water supply may be for supplying water to the photosynthesis reactor and/or the bio-decomposition reactor. Water from a water source may be supplied to the photosynthetic reactor.

Brief Description of the Drawings

Various embodiments will now be described with reference to the following accompanying non-limiting drawings, provided by way of example only.

Figure 1A shows a schematic of a system used to generate biomass and hydrogen accordance with an embodiment of the disclosure.

Figure 1 B shows a schematic of a system used to generate biomass and hydrogen accordance with an embodiment of the disclosure.

Figure 2A shows a schematic of a system used to generate biomass and hydrogen in accordance with another embodiment of the disclosure.

Figure 2B shows a schematic of a system used to generate biomass and hydrogen in accordance with another embodiment of the disclosure. Figure 3A shows a schematic of a system used to generate biomass and hydrogen in accordance with another embodiment of the disclosure.

Figure 3B shows a schematic of a system used to generate biomass and hydrogen in accordance with another embodiment of the disclosure.

Figure 4 shows a schematic of a system used to generate biomass and hydrogen in accordance with another embodiment of the disclosure.

Figure 5 shows an embodiment of a photosynthesis reactor.

Figure 6 shows a schematic of a system used to generate hydrogen in accordance with another embodiment of the disclosure.

Figure 7 shows a schematic of a system used to generate biomass and hydrogen in accordance with an embodiment of the disclosure.

Figure 8 illustrates the average C. vulgaris biomass (i.e. organic feedstock) biomass growth curve of Test ID TH20/21 samples and shows a comparison of growth with and without CO2 injection.

Figure 9 illustrates the average C. vulgaris biomass (i.e. organic feedstock) growth curve for the second part of Test ID TH20/21 .

Figure 10 illustrates the average biomass growth curve of Test ID TH22/21 samples and shows a comparison of growth with and without CO2 injection.

Figure 11 illustrates sugar levels measured for days 0, 4 and 11 during Test ID TH17/21 .

Figure 12 illustrates Bacillus spp. concentration measured for days 0 and 11 during Test ID TH17/21.

Figure 13 shows medium pH levels measured for days 0, 4 and 1 1 during Test ID TH17/21 .

Figure 14 illustrates sugar levels measured for days 0, 1 , 2, 5 and 7 during Test ID TH19/21.

Figure 15 shows Bacillus spp. concentration measured for days 0 and 7 during Test ID TH19/21.

Figure 16 shows medium pH levels measured for days 0, 1 , 2, 5 and 7 during Test ID TH19/21.

Figure 17 illustrates sugar levels measured for days 0, 1 , 6 and 9 during Test ID TH21/21. Figure 18 shows Bacillus spp. and K. aerogenes concentration measured for days 0 and 13 during Test ID TH21/21 .

Figure 19 shows medium pH levels measured for days 0, 1 , 6 and 9 during Test ID TH21/21 .

Figure 20 illustrates sugar levels measured for days 0, 3 and 6 during Test ID TH24/21 .

Figure 21 shows medium pH levels measured for days 0, 3 and 6 during Test ID TH24/21 .

Figure 22 shows Bacillus spp. and K. aerogenes concentration measured for days 0 and 6 during Test ID TH24/21.

Figure 23 illustrates sugar levels measured for days 0 and 5 during Test ID TH25/21 .

Figure 24 shows medium pH levels measured for days 0 and 5 during Test ID TH25/21 .

Figure 25 shows Bacillus spp. and K. aerogenes concentration measured for days 0 and 5 during Test ID TH25/21.

Figure 26 shows final carbon dioxide and hydrogen gas percentages measured for Test ID TH25/21.

Figure 27A shows photomicrographs (1 OOOx magnification) of C. vulgaris cells in: A) a growth assay (organic feedstock) and B) a bacterial bio-decomposition assay, with Figure 27B being a drawing reflecting Figure 27A.

Figure 28A shows a photomicrograph (1 OOOx magnification) of C. vulgaris cells in a bacterial bio-decomposition assay, with Figure 28B being a drawing reflecting Figure 28A.

Figure 29A shows a photomicrograph of C. vulgaris bio-decomposition by Bacillus spp. and K. aerogenes, with Figure 29B being a drawing reflecting Figure 29A.

Figure 30 shows a soybean phonological scale.

Detailed description

In the following, reference is made to accompanying drawings which form a part of the detailed description. The illustrative embodiments described in the detailed description, depicted in the drawings and defined in the claims, are not intended to be limiting. Other embodiments may be utilised, and other changes may be made, without departing from the spirit or scope of the subject matter presented. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings can be arranged, substituted, combined, separated and designed in a wide variety of different configurations, all of which are contemplated in this disclosure. In one aspect, the present disclosure relates to a system for sequestering carbon from a gas stream that comprises carbon dioxide, the system comprising: one or more reactors; said reactors comprising: at least one reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source, said reactor having an inlet for receiving a carbon dioxide gas stream; and at least one reactor configured for subjecting the organic feedstock to bio-decomposition, said reactor being configured to alternate between operating under aerobic and anaerobic conditions. In accordance with this aspect, there is also provided a system for producing biomass, the system comprising: one or more reactors; said reactors comprising: at least one reactor configured to produce an organic feedstock using a phototrophic source and a gas stream comprising carbon dioxide, said reactor having an inlet for receiving a carbon dioxide gas stream; and at least one reactor configured for subjecting the organic feedstock to bio-decomposition, said reactor being configured to alternate between operating under aerobic and anaerobic conditions.

An embodiment of a system 100 used for sequestering carbon and producing a biomass is shown in Figure 1A. System 100 has a reactor 124. In this embodiment, the reactor 124 is a dual-purpose bioreactor which is configured to work as photobioreactor and biodecomposition reactor. That is, the reactor 124 is a bio-decomposition-capable reactor and a photosynthesis-capable reactor. In photobioreactor mode, reactor 124 can convert carbon dioxide into an organic feedstock using photosynthesis. The system 100 has a carbon dioxide supply line 129 that feeds carbon dioxide from a carbon dioxide source 121 into the reactor 124 for use in the photosynthesis step. The carbon dioxide supply line 129 may include a filter to filter off gases other than carbon dioxide.

The carbon dioxide delivered to the dual-purpose bioreactor 124 may be mixed with other gases, such as air. In an embodiment, a concentration of the carbon dioxide delivered to the dual-purpose bioreactor 124 ranges up to about 50%. In an embodiment, a concentration of the carbon dioxide delivered to the dual-purpose bioreactor 124 ranges from about 8% to about 20%. Carbon dioxide may be supplied to the reactor 124 at a rate of about 0.2 to about 0.8 VVM. In some embodiments, the carbon dioxide may be supplied to the reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source at a rate of about 0.2 to about 5 VVM, such as from about 0.5 to about 3 VVM. In an embodiment, a mixing manifold is provided (not shown) to allow a concentration of carbon dioxide in the waste carbon dioxide gas stream to be adjusted.

In some embodiments, the flowrate of carbon dioxide may be supplied to the reactor, configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source, at an excess flowrate. When the carbon dioxide is dispensed at an excess flowrate, there is a greater flow of carbon dioxide into the phototrophic source than can be converted. Supplying an excess flow rate can be advantageous as the excess flow rate may ensure that there is no undersupply of carbon dioxide to the phototrophic source. In addition, the excess flowrate may cause agitation of the phototrophic source, enhancing the contact between the phototrophic source and the carbon dioxide, further promoting efficient conversion of the carbon dioxide into the organic feedstock. In embodiments where the carbon dioxide is provided at an excess flowrate, it may be preferred to avoid loss of the excess carbon dioxide into the atmosphere. In some embodiments, the system for sequestering carbon from a gas stream may be configured to recycle any excess carbon dioxide through the reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source. An embodiment of a system 100 suitable for sequestering carbon from a carbon dioxide stream supplied at an excess flowrate is shown in Figure 1 B. System 100 of Figure 1 B is similar to Figure 1 A, with the same reference numerals used to denote like features. The system of Figure 1 B has a reactor 124. In this embodiment, like that of Figure 1A, the reactor 124 is a dual-purpose bioreactor which is configured to work as photobioreactor and bio-decomposition reactor. The system 100 has a carbon dioxide supply line 129 that feeds carbon dioxide from a carbon dioxide source 121 into the reactor 124 for use in the photosynthesis step. This embodiment of the system also has a carbon dioxide return line 129’ to return excess gas to the source 121 , where the returned carbon dioxide can be combined with fresh carbon dioxide to continue supplying carbon dioxide at the excess flowrate. In some embodiments, including the one illustrated by Figure 1 B, carbon dioxide may be supplied to the reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source at a rate of about 0.2 to about 5 VVM, such as from about 0.5 to about 3 VVM. In an embodiment, a mixing manifold is provided (not shown) to allow a concentration of carbon dioxide in the waste carbon dioxide gas stream to be adjusted.

In embodiments such as those illustrated in Figures 1 A and 1 B, the reactor when used to process the photosynthesis step is partially emptied to create room for the adding microorganisms e.g. bacteria. When a suitable organic feedstock has been produced, bacteria can then be added from bacteria storage vessel 131 to the dual-purpose bioreactor 124 via conduit 132 to begin the aerobic bio-decomposition step. In bio-decomposition reactor mode, the reactor 124 can to switch between aerobic and anaerobic biodecomposition conditions (if necessary). The reactor 124 is for subjecting the organic feedstock to bio-decomposition and comprises a first gas inlet (not shown) connected to a source of oxygen-containing gas (e.g. a source of air) 123 and a second gas inlet (not shown) connected to a source of oxygen-free gas (e.g. a source of nitrogen gas) 128. In use, to switch between operating under aerobic and anaerobic conditions, the supply of oxygencontaining gas can be switched off and the supply of oxygen-free gas can commence or vice versa.

The biomass produced during bio-decomposition using the reactor 124 is transferred via a conduit 141 to a biomass storage vessel in the form of a storage vessel (e.g. tank) 140. It will be appreciated that in some modes of use, conversion of the organic feedstock into biomass may comprise an aerobic bio-decomposition step, without the need for a subsequent anaerobic bio-decomposition step. Alternatively, the reactor 124 may be operated so as to subject the medium therein to anaerobic conditions. Such a mode of operation may be preferred when the production of hydrogen gas is particularly desired.

As noted above, during the bio-decomposition, hydrogen gas can be generated using system 100. The hydrogen generated is transferred via a conduit 130 to a hydrogen storage vessel in the form of storage vessel (e.g. tank) 126. Conduit 130 includes a pump 125 to pump the generated hydrogen to the storage vessel 126. The pump 125 can allow the storage vessel 126 to be pressurised. However, the pump 125 is not required in all embodiments. Also, in some embodiments, a pump is provided along line 141 to pump the biomass to the storage vessel 140. It should be appreciated that for, this and other embodiments, the term “storage vessel” is to be interpreted broadly to include any form of closed/closable vessel that is capable of storing the product in question. Thus, “storage vessel” can includes materials that can adsorb (i.e. reversibly adsorb) hydrogen such as carbonaceous materials, metal-organic frameworks and molecular sieves.

In the embodiments of Figures 1 A and 1 B, a heat exchanger 122 is in thermal communication with the microbial dual-purpose bioreactor 124. The heat exchanger 122 is connected to a heat source 127 to supply heat to the microbial dual-purpose bioreactor 124. In some embodiments, one or more heat exchangers may be in thermal communication with the reactor of the system. Alternatively, the system may not include a heat exchanger.

The microbial dual-purpose bioreactor 124 may include numerous sensors including pH sensors, temperature sensors, reactor level sensors, and sensors to monitor feedstock generation from the photosynthesis step and biomass and/or gas generation from the biodecomposition step(s). In an embodiment, the microbial dual-purpose bioreactor 124 is fitted with rotameters to monitor the gas inflow into the reactor. The system 100 also includes a control system (not shown) that receives information from the various sensors. The control system can adjust parameters such as, for example, reactor temperature, phototrophic source (e.g. algal) and bacteria loading rates and pH to optimise the reaction conditions to allow the most efficient generation of biomass and/or hydrogen. Generally, each of the supply lines, are fitted with valves that are actionable and controllable by the control system to control the flow of the various components around the system 100. The control system can also include a datalogger.

Another embodiment of a system 100 used for sequestering carbon and producing a biomass is shown in Figure 7. System 100 has a two reactors 124a, 124b. Each reactor 124a, 124b can be configured in the same way as reactor 124 described above with reference to Figure 1 A. In a further variation not shown, each reactor may be configured so that an excess flow rate of carbon dioxide can be supplied, with each reactor being configured to recycle the excess carbon dioxide through the reactor. For example, each reactor may be configured with a carbon dioxide return line 129’ such as that illustrated in Figure 1 B. Returning to Figure 7, in this embodiment, there are two reactors 124a, 124b which are each a dual-purpose bioreactor configured to work as photobioreactor and biodecomposition reactor. The system 100 has a carbon dioxide supply line 129 that can feed carbon dioxide from a carbon dioxide source 121 into either or each reactor 124a, 124b for use in the photosynthesis step. The carbon dioxide supply line 129 may include a filter to filter off gases other than carbon dioxide.

The carbon dioxide delivered to either or each reactor 124a, 124b may be mixed with other gases, such as air. In an embodiment, a concentration of the carbon dioxide delivered ranges up to about 50%. In an embodiment, a concentration of the carbon dioxide delivered to either or each reactor 124a, 124b ranges from about 8% to about 20%. Carbon dioxide may be supplied to either or each reactor 124a, 124b at a rate of about 0.2 to about 0.8 VVM. In some embodiments, the carbon dioxide may be supplied to the reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source at a rate of about 0.2 to about 5 VVM, such as from about 0.5 to about 3 VVM. As noted above, in a variation of the embodiment illustrated in Figure 7, the system may be configured to supply the carbon dioxide from the source 121 at an excess flowrate. In an embodiment, a mixing manifold is provided (not shown) to allow a concentration of carbon dioxide in the waste carbon dioxide gas stream to be adjusted.

In some modes of use, only one of the reactors 124a, 124b is used to perform the photosynthesis step. When a suitable organic feedstock has been produced, a portion of the organic feedstock produced in the reactor 124a, 124b used for the photosynthesis step is transferred to the other reactor 124a, 124b via transfer line 150. Thus, the organic feedstock is divided between the reactors 124a, 124b. This can provide space in each reactor 124a, 124b for adding bacteria from bacteria storage vessel 131. In some other embodiments four or more reactors may be used in this way, with half the reactors used to form the organic feedstock which is then distributed between the reactors in the system before introduction of the bacteria.

In bio-decomposition reactor mode, each reactor 124a, 124b can to switch between aerobic and anaerobic bio-decomposition conditions. Each reactor 124a, 124b is for subjecting the organic feedstock to bio-decomposition and comprises a first gas inlet (not shown) connected to a source of oxygen-containing gas (e.g. a source of air) 123 and a second gas inlet (not shown) connected to a source of oxygen-free gas (e.g. a source of nitrogen gas) 128. In use, to switch between operating under aerobic and anaerobic conditions, the supply of oxygen-containing gas can be switched off and the supply of oxygen-free gas can commence or vice versa.

The biomass produced during bio-decomposition using each reactor 124a, 124b is transferred to a biomass storage vessel in the form of a storage vessel (e.g. tank) 140. During the bio-decomposition, hydrogen gas can be generated using system 100. The hydrogen generated is transferred to a hydrogen storage vessel in the form of storage vessel (e.g. tank) 126.

An embodiment of a system 200 used for the production of biomass and hydrogen is shown in Figure 2A. System 200 has a photobioreactor 232 and a bio-decomposition reactor 233. Photobioreactor 232 is configured to convert carbon dioxide into an organic feedstock using photosynthesis. The system 200 also has a carbon dioxide supply line 229 that feeds carbon dioxide from a carbon dioxide source 221 into the photobioreactor reactor 232. The carbon dioxide supply line may include a filter to filter off gases other than carbon dioxide. The photobioreactor chamber 232 and bio-decomposition reactor chamber 233 are connected to one another via a conduit 234. The photobioreactor chamber 232 may be fitted with a photon source (not shown). The bio-decomposition reactor 233 is configured to switch (alternate) between aerobic and anaerobic bio-decomposition. Bacteria may be added from bacteria storage vessel 231 to the bio-decomposition reactor chamber 233 via conduit 235 to begin the bio-decomposition step. The bio-decomposition reactor 233 has a first gas inlet (not shown) connected to a source of oxygen-containing gas (e.g. a source of air) 223 and a second gas inlet (not shown) connected to a source of oxygen-free gas (e.g. a source of nitrogen gas) 228. When bio-decomposition reactor is used for aerobic bio-decomposition, the air supply from the source 223 can be initiated to provide a supply of oxygen-containing gas. Anaerobic bio-decomposition in bio-decomposition reactor 233 can be initiated by injecting nitrogen from supply 228 to purge the air. In some other embodiments, a single gas supply line may be connected to both the source of oxygen-containing gas and the source of oxygen-free gas. A valve arrangement may be used to switch between the gases provided using the line.

In an embodiment, only a portion of the organic feedstock generated in the photobioreactor chamber 232 is transferred to the bio-decomposition chamber 233. For example, a portion of the organic feedstock is kept as an inoculum. In an embodiment, 60% of the organic feedstock produced in the photobioreactor chamber 232 is transferred to the biodecomposition reactor 233 and 40% of the organic feedstock is retained as an inoculum for further use in the photobioreactor chamber 233. The reactors 232 and 233 can be operated as batch, semi-batch or continuous processes.

In some embodiments of the present invention, the system may be periodically or continuously operated so that biomass or an intermediate product of the bio-decomposition can be provided to a reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source (e.g. a photobioreactor). That is, in some embodiments, biomass or an intermediate product of a bio-decomposition step are provided as a nutrient source to the phototrophic source. In some embodiments, at least a portion of the products of the aerobic biological decomposition step may be mixed, e.g. recycled, with the phototrophic source. In some embodiments, the bio-decomposition step performed to produce the product provided to the phototrophic source as a nutrient source is a feed biodecomposition step. In embodiments using a feed bio-decomposition step, the process conditions and/or bacteria species may be selected so as to provide a desirable nutrient source for the phototrophic source. The feed bio-decomposition step may use a different ratio and/or combination of bacteria in order to provide a desired nutrient source. In some embodiments, the feed bio-decomposition step may comprise an aerobic bio-decomposition step, an anaerobic bio-decomposition step, or a combination thereof (such as an aerobic bio-decomposition step followed by an anaerobic bio-decomposition step) to provide the desired nutrient source. In some embodiments, the nutrient is the biomass into which the organic feedstock was converted.

Without being bound by theory, it is believed that mixing at least a portion of the biodecomposition products with the phototrophic source may increase glucose production, which can be advantageous for subsequent bio-decomposition of the organic feedstock. In addition, using a bio-decomposition product as a nutrient source may adjust the parameters of the organic feedstock, including pH, so that more efficient biological treatment in the biodecomposition reactor is promoted.

In embodiments such as those illustrated in Figures 2A and 2B, the mode of operation may be adjusted so that periodically biomass or an intermediate product from the biodecomposition reactor chamber 233 can be provided to the photobioreactor chamber 232. The biomass or an intermediate product from the bio-decomposition reactor chamber 233 can be provided to the photobioreactor chamber 232 via conduit 234 or, in a variation, a separate conduit (not shown). Other embodiments with plural reactors, such as those shown in Figures 3A, 3B, 4, 6 and 7, may be similarly operated periodically so that biomass or an intermediate product from the bio-decomposition step can be provided to the reactor with the phototrophic source using the illustrated conduits between the reactors. In variations of those embodiments shown in Figures 3A, 3B, 4, 6 and 7, a separate conduit (not shown) may be provided between reactors so that the biomass or intermediate product can be provided to the reactor(s) with the phototrophic source. In embodiments such as that shown in Figure 7, in which the reactors 124a, 124b can be used to perform the photosynthesis step and biodecomposition, the product from the bio-decomposition can be provided to the phototrophic source by retaining at least a portion of the bio-decomposition product in the reactor 124a, 124b so that the phototrophic source can be added to it before the reactor 124a, 124b is used to perform the photosynthesis step.

Returning to Figure 2, the biomass produced in the bio-decomposition reactor 233 is transferred via a conduit to a biomass storage vessel in the form of a storage vessel (e.g. tank) 240.

The hydrogen generated in the bio-decomposition reactor chamber 233 is transferred via a conduit 230 to a hydrogen storage vessel in the form of storage vessel (e.g. tank) 226. Conduit 230 includes a pump 225 to pump generated hydrogen to the storage vessel 226. The pump 225 can allow the storage vessel 226 to be pressurised. However, the pump 225 is not required in all embodiments.

In an alternative embodiment shown in Figure 2B, the bio-decomposition reactor chamber 233 can be fitted with an auxiliary carbon dioxide supply line 236 that transfers any carbon dioxide generated by the bio-decomposition reactor chamber 233 to the photobioreactor chamber 232 (i.e. a carbon dioxide recycle line). This means that carbon dioxide generated by the bio-decomposition reactor chamber can be used as a feedstock for the photobioreactor chamber 232. The auxiliary carbon dioxide supply line 236 may help improve the efficiency of the system 200 as a greater biomass and hydrogen yield can be achieved per unit of carbon dioxide delivered to the system by carbon dioxide supply line 229. The auxiliary carbon dioxide supply line 236 is fitted with a filter 237, such as a membrane filter, for filtering the carbon dioxide gas from other gases e.g. hydrogen and hydrocarbons.

The carbon dioxide delivered to the photobioreactor 232 may be mixed with other gases, such as air. In an embodiment, a concentration of the carbon dioxide delivered to the bioreactor 232 ranges up to about 50%. In an embodiment, a concentration of the carbon dioxide delivered to the photobioreactor 232 ranges from about 8% to about 20%. Carbon dioxide may be supplied to the photobioreactor 232 at a rate of about 0.2 to about 0.8 VVM. In some embodiments, the carbon dioxide may be supplied to the reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source at a rate of about 0.2 to about 5 VVM, such as from about 0.5 to about 3 VVM. The system may be configured to supply the carbon dioxide from the source 221 at an excess flowrate. In a further variation not shown, the photobioreactor 232 may be configured so that an excess flow rate of carbon dioxide can be supplied, with the reactor being configured to recycle the excess carbon dioxide through the reactor. For example, the photobioreactor 232 may be configured with a carbon dioxide return line 129’ such as that illustrated in Figure 1 B. In an embodiment, a mixing manifold is provided (not shown) to allow a concentration of carbon dioxide in the waste carbon dioxide gas stream to be adjusted.

The reactors 232 and 233 can each includes numerous sensors including pH sensors, temperature sensors, reactor level sensors, and sensors to monitor feedstock generation from the photobioreactor and gas generation from the bio-decomposition reactor. In an embodiment the both reactors 232 and 233 are fitted with rotameters to monitor the gas inflow into the reactors. The system 200 also includes a control system (not shown) that receives information from the various sensors. The control system can adjust parameters such as, for example, reactor temperature, phototrophic source (e.g. algal) and bacteria loading rates and pH to optimise the reaction conditions to allow the most efficient generation of hydrogen. Generally, each of the supply lines, are fitted with valves that are actionable and controllable by the control system to control the flow of the various components around the system 200. The control system can also include a datalogger.

An embodiment of a system 300 used for the production of biomass and hydrogen is shown in Figure 3A. System 300 has a microbial reactor in the form of photobioreactor 312 that is configured to convert carbon dioxide into an organic feedstock using photosynthesis. The system 300 also has a carbon dioxide supply line 329 that feeds carbon dioxide from a carbon dioxide source 311 into the photobioreactor reactor 312. The carbon dioxide supply line 329 may include a filter to filter off gases other than carbon dioxide. The system 300 also includes a bio-decomposition reactor 314. The bio-decomposition reactor 314 is configured to switch between aerobic and anaerobic bio-decomposition. The bio-decomposition reactor 314 has a first gas inlet (not shown) connected to a source of oxygen-containing gas (e.g. a source of air) 323 and a second gas inlet (not shown) connected to a source of oxygen-free gas (e.g. a source of nitrogen gas) 328. When bio-decomposition reactor is used for aerobic bio-decomposition, the air supply from the source 323 can be initiated to provide a supply of oxygen-containing gas. Anaerobic bio-decomposition in bio-decomposition reactor 314 can be initiated by injecting nitrogen from supply 328 to purge the air. In some other embodiments, a single gas supply line may be connected to both the source of oxygencontaining gas and the source of oxygen-free gas. A valve arrangement may be used to switch between the gases provided using the line.

The photobioreactor 312 focuses on the photosynthesis step. This reactor has built in LED lighting to provide a suitable light source of the photosynthesis. In use, once peak sugar is reached, the liquid biomass can be transferred to a bio-decomposition reactor 314, where it is mixed with bacteria. The bio-decomposition reactor 314 of this embodiment does not have a light source. Instead, light levels are minimised for the bio-decomposition steps in the biodecomposition reactor 314.

The photobioreactor 312 and bio-decomposition reactor 314 are connected to one another via a conduit 330. The conduit 330 passes the organic feedstock from an organic feedstock outlet of the photobioreactor 312 to an inlet of the bio-decomposition reactor 314. The organic feedstock may be provided as a slurry and/or liquid. In an embodiment, the organic feedstock is provided as a solution that is fed to the bio-decomposition reactor 314. In an embodiment, the conduit 330 has a pump or auger for pumping or conveying the organic feedstock from the photobioreactor 312 to the bio-decomposition reactor 314. The biodecomposition reactor 314 is set up to convert the organic feedstock into biomass and hydrogen. In an embodiment a filter is provided at the photobioreactor 312 so that only the organic feedstock is passed from the photobioreactor 312 to the bio-decomposition reactor 314. In an embodiment, only a portion of the organic feedstock generated in the photobioreactor 312 is transferred to the bio-decomposition reactor 314. For example, a portion of the organic feedstock is kept as an inoculum. In an embodiment, 60% of the organic feedstock produced in the photobioreactor 312 is transferred to the biodecomposition reactor 314 and 40% of the organic feedstock is retained as an inoculum for further use in the photobioreactor 312. The reactors 312 and 314 can be operated as batch, semi-batch or continuous processes.

The biomass produced in the bio-decomposition reactor 314 is transferred via a conduit to a biomass storage vessel in the form of a storage vessel (e.g. tank) 340.

The hydrogen generated in the bio-decomposition reactor chamber 314 is transferred via a conduit 324 to a hydrogen storage vessel in the form of storage vessel (e.g. tank) 316. Conduit 324 includes a pump 325 to pump generated hydrogen to the storage vessel 316. The pump 325 can allow the storage vessel 316 to be pressurised. However, the pump 325 is not required in all embodiments.

In use, the bio-decomposition reactor 314 generates biomass, hydrogen and waste carbon dioxide and/or waste hydrocarbons. The relative amounts of biomass, hydrogen, carbon dioxide and hydrocarbons generated in the bio-decomposition reactor 314 generally depends on the bio-decomposition reactor conditions. Because the photobioreactor 312 uses carbon dioxide as a feedstock, the bio-decomposition reactor 314 can be fitted with an auxiliary carbon dioxide supply line 332 that transfers any carbon dioxide generated by the bio-decomposition reactor 314 to the photobioreactor 312 (i.e. a carbon dioxide recycle line). Carbon dioxide generated by the bio-decomposition reactor 314 can then be used as a feedstock for the photobioreactor 312. The auxiliary carbon dioxide supply line 332 can help improve the efficiency of the system 310 as a greater biomass and hydrogen yield can be achieved per unit of carbon dioxide delivered to the system by carbon dioxide supply line 329.

The auxiliary carbon dioxide supply line 332 can be connected to the bio-decomposition reactor 314 or alternatively the auxiliary carbon dioxide supply line 332 can branch off conduit 324. In either configuration, the auxiliary carbon dioxide supply line 332 is fitted with a filter 333, such as a membrane filter, for filtering the carbon dioxide gas from other gases e.g. hydrogen and hydrocarbons.

A photosynthesis heat exchanger 318 is in thermal communication with the photobioreactor 312 and a bio-decomposition heat exchanger 320 is in thermal communication with the biodecomposition reactor 314. The heat exchangers 318 and 320 may be connected to heat source 317 to supply heat to the reactors 312 and 314. The heat exchangers may be connected in parallel to the heat source 317, as illustrated. In some other embodiments, the heat exchangers may be connected in series. In another embodiment, shown in Figure 3B, a water supply 321 is in fluid communication with photobioreactor 312 and photosynthesis heat exchanger 318 is in thermal communication with the water supply 321. With this arrangement, heat provided to the photobioreactor 312 is passed to the bio-decomposition reactor 314 through the transfer of the organic feedstock from the photobioreactor 312 to the biodegradation reactor 314. However, other embodiments may also include a bio-decomposition heat exchanger. In the embodiment shown in Figure 3B, the water supply 321 can include a mist generator for generating a mist of water from the water supply. The photosynthesis heat exchanger 318 can be in thermal communication with the mist generator.

In a variation of the embodiment of Figure 3A, a water supply 321 is not in thermal communication with the heat exchanger 318 and instead the heat exchanger 318 is in direct thermal communication with the photobioreactor 312.

The water supply 321 can have two water supply channels, one leading directly to the photobioreactor 312 and another leading to a carbon dioxide mixing chamber 340 (see Figure 3B). The carbon dioxide mixing chamber 340 receives carbon dioxide e.g. from carbon dioxide supply line 329 to form a carbon dioxide-enriched solution that is then delivered to the photobioreactor 312. In an embodiment, the mixing chamber 340 forms an emulsion of carbon dioxide and water.

Generally, the heat exchangers 318 and 320 will heat their respective reactors to maintain the reactors at required temperatures. Typically, the reactors 312 and 314 are maintained at a temperature ranging from about 28-30 ^eC to about 40 ^eC. However, if reactor 312 and/or 314 includes extremophiles, the operational temperature may be in excess of 40 ^eC, such as greater than 80 ^eC. It should also be appreciated that the heat exchangers 318 and 320 may also be operated to cool their respective reactors. Alternatively, or additionally, photobioreactor 312 may be in thermal communication with bio-decomposition reactor 314 to transfer heat between the reactors 312 and 314, for example if one reactor requires constant cooling and the other reactor requires constant heating.

The photobioreactor 312 and the bio-decomposition reactor 314 can each includes numerous sensors including pH sensors, temperature sensors, reactor level sensors, and sensors to monitor feedstock generation from the photobioreactor 312 and gas generation from the bio-decomposition reactor 314. In an embodiment, the reactors 312 and 314 are fitted with rotameters to monitor the gas inflow into the reactors. The system 300 also includes a control system (not shown in Figures) that receives information from the various sensors. The control system can adjust parameters such as, for example, reactor temperature, phototrophic source (e.g. algal) and bacteria loading rates and pH to optimise the reaction conditions to allow the most efficient generation of hydrogen. Generally, each of the supply lines, such as auxiliary carbon dioxide supply line 332 and conduits 329, 330 and 324, are fitted with valves that are actionable and controllable by the control system to control the flow of the various components around the system 300. The control system can also include a datalogger.

The reactors in the illustrated embodiments described above are each depicted as a single reactor. However, in some embodiments, the or each reactor can include a plurality of reactors, sub-reactors or reaction chambers. For example, Figure 4 shows an embodiment of the photobioreactor 412 having six reactors 412a-412f. The reactors 412a-412f are connected in parallel. The photobioreactor 412 is configured for use with an algal source but can be adapted to other phototrophic sources. A gas manifold 439 connects the carbon dioxide supply line 428 to the reactors 412a-412f. An algal manifold 441 connects an algal supply line 429 to the reactors 412a-412f. The reactors 412a-412f are arranged for counter current flow of carbon dioxide and algal material. In a variation to the embodiment of Fig. 4, the reactors 412a-412f are connected in series.

An outlet gas line 431 is provided to allow excess gas(es) to be removed from the reactors 412a-412f. If the excess gases include carbon dioxide, the excess gases can be reintroduced into carbon dioxide supply line 428. When the reactors 412a-412f are connected in series, the carbon dioxide and algal flow may be co-current or counter-current. Figure 4 is exemplary only and the person skilled in the art will appreciate that reactors 124, 232, 233, 312, 314 in the embodiments described above can each be configured to include a plurality of reactors, sub-reactors or reaction chambers. In an embodiment, each of the plurality of reactors, subreactors or reaction chambers are modular units. An example of a modular photoreactor (i.e. a photosynthesis reactor) is shown in Figure 5. The modular photoreactor 500 is a hollow tube 502 fitted with a light source in the form of lamp 504 in an internal space of the tube 502. The reactor 500 has a capacity of about 1200L. A power source 506 is connected to the lamp 504. The reactor 500 can have a plurality of lamps 504. The lamp 504 may emit visible and/or UV light. The hollow tube 502 in use is filled with reaction media 512 that includes a phototrophic source. The reactor 500 has a gas inlet 508 fitted near an in-use bottom end of the hollow tube 502. The gas inlet 508 is used to pass carbon dioxide into the hollow tube 502. Input line 510 is positioned near an in-use top end of the hollow tube 502. Input line 510 is used to add phototrophic sources (e.g. algal sources), reaction media, buffers, pH adjusters and so on to the hollow tube 502. The reactor 500 also has an outlet (not shown) for extracting the organic feedstock generated by the photosynthetic conversion of carbon dioxide. The lamp 504 can be powered using renewable energy.

Another embodiment of a system 600 is shown in Figure 6. System 600 is similar to system 300 except that the carbon dioxide source 31 1 is a waste carbon dioxide gas stream generated from a gas reformer 622. Gas reformer 622 converts a hydrocarbon source 626, such as methane or plumbed natural gas, into hydrogen via steam forming. A by-product of steam reforming is carbon dioxide. In the embodiment of Figure 6, the carbon dioxide byproduct is collected and passed from the gas reformer 622 through carbon dioxide supply line 628 to the photobioreactor 612. To separate the carbon dioxide in supply 628 from other gases generated by the gas reformer 622, such as carbon monoxide, steam and hydrogen, gas filter 629 may be provided on carbon dioxide supply line 628.

The hydrogen produced by the gas reformer 622 is collected and passed into storage vessel 616 via conduit 636. Conduit 636 may be provided with filter 637 to remove any contaminants from the hydrogen gas stream. In an embodiment, the bio-decomposition reactor 614 also produces hydrocarbons when the organic feedstock from the photobioreactor 612 is converted into hydrogen. An auxiliary hydrocarbon feed line 634 connects the biodecomposition reactor 614, via the manifold 602, with the gas reformer 622 for passing hydrocarbon generated by the bio-decomposition reactor 614 to the gas reformer 622. In an embodiment, the auxiliary hydrocarbon supply line 634 is fitted with a filter 635 for purifying the hydrocarbons generated by the bio-decomposition reactor 614 prior to delivery to the reformer 622.

Supplying the gas reformer 622 with hydrocarbons generated from the bio-decomposition reactor 614, and also supplying the photobioreactor 612 with carbon dioxide generated from the bio-decomposition reactor 614, may help to increase the amount of hydrogen generated per unit of hydrocarbon (e.g. source 626) from about 40% to about 65%, representing about a 63% increase in the amount of hydrogen generated.

In an embodiment, supply lines 632 and 634, and conduit 624, are connected to a manifold 602 as shown in Figure 6. Manifold 602 is connected to a gas outlet of bio-decomposition reactor 614. Manifold 602 is also fitted with a filter so that the hydrogen, carbon dioxide and any hydrocarbons generated by the bio-decomposition reactor 614 are filtered and passed in respective lines 624, 632 and 634. In an alternative to the embodiment depicted in Figure 6, auxiliary hydrocarbon feed line 634 may alternatively join feed line 627 to form a single supply of hydrocarbons rather than having two hydrocarbon input lines into the reformer 622. The gas reformer 622 is in thermal communication with the heat exchangers 618 and 620 so that the heat generated by the gas reformer 622 is used to heat the reactors 612 and/or 614. Utilising the heat generated from the reformer 622 to heat the reactors 612 and 614 helps to reduce the energy requirements of reactors 612 and 614.

As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the invention. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

In addition, where dimensions are described herein, it will be appreciated that plus or minus (±) typical manufacturing tolerances are applicable to those values. As appreciated by those in the art, manufacturing tolerances may be determined to achieve a desired mean and standard deviation of manufactured components in relation to the ideal component profile.

Examples

Various embodiments will be described herein with reference to the following non-limiting examples and comparative examples.

Photosynthesis Step Examples

To assess the autotrophic growth of microalgae Chlorella vulgaris and its CO2 absorption, the experiments described below were performed.

To start the experiments, pre-established volumes of algae, with known concentration, were added in closed vessels containing sterile Bold's Basal Medium (BBM) medium and the assays were performed in triplicate. The specific parameters for each experiment are presented in Table A. Table 1 below contains the details of methodology and parameters for each experiment.

Table 1 - Parameters of the experiments performed for algae growth and CO2 absorption.

TEST ID Experimental Summary Experimental Parameters

Table A - Parameters used in the Photosynthesis Step Experiments.

The parameters evaluated in the experiments were: pH, cell concentration, biomass weight, visual staining and CO2 absorption. Table 2 describes the methodologies for evaluating each parameter. As used for these experiments, biomass refers to the C. vulgaris generated. Accordingly, biomass in these examples illustrates the generation of an organic feedstock for the process.

Table 2 - Methodology used to evaluate all parameters analysed in co growth and absorption testss.

Test ID TH20/21 - Results

The product of the treatments for Test ID TH20/21 was divided into two parts. One part was used for further experiments with the Test ID TH24/21. Test ID TH24/21 is described in further detail below.

The treatments for Test ID TH20/21 used five Roux vials as reactors. Two reactors (F1 and F2) had no CO2 injection and only utilized atmospheric gas. Three reactors (F3, F4 and F5) had injection of 10% CO2, i.e. an enriched atmosphere. Figure 8 shows the growth curve (mean) of the samples from Test ID TH20/21 , while Table 3 shows the raw data of each reactor, along with the amount of CO2 absorbed.

Figure 8 shows that the growth of the microalgae was significantly higher in the samples that had injection enriched with 10% CO2. It was also observed that a higher initial concentration of microalgae (F5 - 1 %) presented higher yield and higher CO2 absorption (see T able 3) than the samples with lower concentration (F3 and F4 - 0.1 %). What differed among the samples was their growth rate, the assays with 0.1% algae had a faster growth than the sample with 1 % microalgae. It was also observed that the samples without CO2 injection had already reached the stability phase and started the phase of growth decline at the end of the third day of the experiment. Meanwhile, samples with CO2 injection were still in their exponential growth phase at the end of the same period. For this reason, the experiment was continued for a second part, after the removal of the aliquot required for Test ID TH24/21 , with CO2 injection in all remaining samples.

Table 3 - Data on biomass growth and CO2 absorption of the first part of Test ID TH20/21

After 6 days from the beginning of the experiment, the entire content of reactors F3 and F5 was collected for use in Test ID TH24/21 . Also, about 200 mL of the contents of reactor F4 was collected for use in Test ID TH24/21 . To the remaining content of the reactor F4, a further 200 mL of sterile BBM medium was added. At this stage, 10% COs was injected in all three remaining reactors (F1 , F2 and F4). This continued testing was directed to assessing the maximum cell growth that the cultures could achieve, as well as in how many days (retention time) before the cultures would enter the stationary and growth decline phase. Based on the results presented in Figure 9 and Table 4, it was observed that from the seventh day to the twelfth day there was a production of approximately 1 .5 g/L of biomass, while in the remaining 10 days the biomass increased by about 1.0 g/L, showing that the exponential phase of growth happened in the first week of production. However, after 17 days of production the microalgae had not yet reached its growth decline phase. Exponential growth was measured until the 23rd day.

The maximum biomass growth occurred in reactor F2, with 10% COs used in this exclusively autotrophic process, which was 2.98 g/L, and a total of 5.459g of CO2 was absorbed.

Table 4 - Data on biomass growth and CO2 absorption of the second part of Test ID TH20/21

Test ID TH22/21 - Results Table 5 shows the biomass growth and CO2 absorption data from Test ID TH22/21 , together with an outline of the treatments used. Figure 10 illustrates the average biomass growth curve of Test ID TH22/21 samples and shows a comparison of growth with and without CO2 injection (i.e. a comparison of Treatment 1 and Treatment 2 of Test ID TH22/21 ).

It was observed that the results of Test ID TH22/21 reinforce the observations from Test ID TH20/21 : (I) the presence of CO2 is essential for greater biomass growth (I.e. organic feedstock); (II) the highest growth rate, as well as the most pronounced exponential growth takes place in the first week and; (III) even with 16 days of production, a rate of decline was not achieved for the microalgae growth, with the microalgae continuing to generate biomass and absorb CO2. Table 5 - Biomass growth and CO2 absorption data from the test, with the description of the treatments

Test ID TH22/21

The photosynthetic process of growth of C. vulgaris and CO2 absorption was satisfactory, with cellular concentrations in the range of 10⁷ to 10⁸ cells per mL and reaching 2.8g/L of biomass in 22 days of retention with autotrophic growth and medium enriched with 10% CO2. In this condition, a maximum daily growth rate of 400mg of biomass was obtained and average daily growth of 152mg was obtained. A final absorption of 5.2g of CC^ per litre of culture medium was obtained at the end of 23 days.

Examples of the Conversion of an Organic Feedstock into Biomass

Described herein are experiments assessing the conversion an organic feedstock comprising C. vulgaris. The conversion uses aerobic and anaerobic bio-decomposition stages. The bacteria employed are Bacillus spp. which may or may not be combined with K. aerogenes (a facultative anaerobic species). Photomicrographs (1000x magnification) of C. vulgaris cells are shown in Figures 27A and 27B. Figures 27A and 27B show: A) growth assay (organic feedstock) and B) a bacterial biodecomposition assay. The numbered arrows in the figure show distinct patterns: 1 - cell with the cell wall intact; 2 - cell in reproduction by binary division; 3 - cell with cell wall damage and loss of intracellular content; and 4 - heap of bacteria attacking a set of algae. Figure 28A is a photomicrograph (1000x magnification) of C. vulgaris cells which shows a bacterial bio-decomposition assay with arrows indicating bacteria decomposing algae cells, with Figure 28B being a drawing reflecting Figure 28A.

Figure 29A shows a photomicrograph of C. vulgaris bio-decomposition by Bacillus spp. and K. aerogenes, with Figure 29B being a drawing reflecting Figure 29A. As indicated by the circle, there are cell walls thicker than normal, indicating cell protection mechanism. Arrows in Figures 29A and 29B indicating bacteria decomposing algae cells.

Table 6 sets out the details of the experiments. The specific parameters for each experiment are presented in Table 2A. These experiments use a mixture of Bacillus spp.: B. subtilis, B. megaterium and B. thuringiensis.

Table 6 - Description of the tests carried out for the bio-decomposition of microalgae.

Table 2A - Parameters used in each bio-decomposition experiment

For Test IDs TH17/21 , TH19/21 and TH21/21 , sterile BBM medium was used with the C. vulgaris microalgae added at the beginning of the assays at known concentrations. For Test IDs TH23/21 , TH24/21 and TH25/21 , the BBM medium with the C. vulgaris microalgae previously grown was used. The other parameters used in each of these experiments are presented in Table 2A.

The parameters evaluated in these experiments were: pH, sugar concentration, biomass weight, bacterial concentration, staining and gas quantification. Table 7 describes the methodologies for evaluating each parameter.

Table 7 - Methodology used to evaluate all parameters analysed in sugar decomposition and gas production tests.

Test ID TH17/21 - Results

Test ID TH17/21 , the decomposition of the cell wall of C. vulgaris algae employed bacteria of Bacillus spp. Test ID 17/21 was used to evaluated whether the presence of lighting would influence bio-decomposition by Bacillus spp.

At the beginning of Test ID TH17/21 (day 0), as shown in Figure 11 , the samples that had the addition of bacteria showed a significantly higher amount of sugars compared to the control, this is due to the Tryptic Soy Broth (TSB) which is rich in sugars and is used for preparation of the inoculum together with bacteria. The composition of the TSB and the BBM are set out in Tables 8 and 9 below.

Table 8 - Composition of BBM synthetic medium for microalgae cultivation.

Formulation Concentration (g/L)

K2HPO4 (Dipotassium Phosphate) 0,075000

KH2PO4 (Monopotassium Phosphate) 0,014000

MgSOu^,'7Hu^,O(Magnesium Sulfate Heptahydrate) 0,075000 NaNOs (Sodium Nitrate) 0,090000

CaCl2.2H2O (Dihydrated Calcium Chloride) 0,025000

NaCI (Sodium Chloride) 0,025000

EDTA-Na₄ 0,050000

FeSCk. 7H2O (Heptahydrate Ferrous Sulfate) 0,004980

H3BO3 (Boric acid) 0,011420

MnCl2.4H2O (Tetrahydrate Manganese Chloride) 0,000232

ZnSC>4.7H2O (Heptahydrate Zinc Sulfate) 0,001410

CuSCk .5H2O (Pentahydrate Copper Sulfate) 0,000252

NaMoC>4.2H2O (Dihydrate Sodium Molybdate) 0,000192

Co CI2.6H2O (Hexahydrate Cobalt Chloride) 0,000008

Yeast extract 0,300000

Table 9 - Composition of synthetic medium TSB. r'L.i Concentration

Sodium Chloride

Pancreatic digest of casein 17,00

Papic soy digest 3.00 dextrose 2,50

Sodium Chloride ,00

Dipotassium phosphate 2,50

On the fourth day Test ID TH17/21 , there was a significant increase in the concentration of dissolved sugars in the medium, showing that the bacteria degraded the microalgae cells, with subsequent dissolution in the medium.

On the 1 1th day of the trial there was a sharp drop in sugar concentration, suggesting that the time of exposure of the microalgae to bacteria may have been high, causing them to have consumed the sugar that was released from the microalgae. This demonstrates the need to control the retention time inside the reactor, causing this parameter to be effectively monitored and adjusted, so that aerobic bacteria do not overlap in the biota, inhibiting the reaction of the anaerobic process.

The influence of the presence of light and light intensity on algal decomposition was assessed by observing the results in Figures 1 1 and 12. Treatments 1 and 2, with a light intensity of 500 and 950 lux, respectively, were proportionally the most effective for algae cell wall decomposition (See Figure 11 ). These treatments had produced, between days 0 and 4, of 0.0228 g/L and 0.0217 g/L of dissolved sugars (Treatment 1 and 2 respectively). The bacteria in Treatment 3 have multiplied more (see Figure 12), having a higher consumption of sugars consequently.

The influence of the presence of light and light intensity on the algal decomposition process may be illustrated by the pH of the medium (see Figure 13). It was observed that the Control (without the presence of Bacillus spp. showed a significant increase in pH, while the Treatments with the addition of bacteria maintained their pH values throughout the time of the assay. These data suggest that the presence of light causes the cells of the microalgae to continue to increase, which increases pH values. However, bacteria of the genus Bacillus are well-known for promoting a large acidification of the culture medium. Thus, the acid produced by the bacteria was neutralized by the alkalinity of microalgal growth. This behaviour may not be favourable. As Bacillus spp. have optimal growth in pH in the neutral range, there may be a higher consumption of sugar from the decomposition of microalgae.

Test ID TH19/21 - Results

Test ID TH19/21 was used to assess the decomposition of the cell wall of the algae in the absence of light. Therefore, for Test ID TH19/21 , employment of the mixture of Bacillus spp. mix for decomposition of the algal cell wall was evaluated, with the bio-decomposition conducted in the absence of light (i.e. no photoperiod).

Figure 14 illustrates sugar levels measured for days 0, 1 , 2, 5 and 7 during Test ID TH19/21. Figure 14 shows a behaviour similar to that observed in Test ID TH17/21 : there is a peak of sugars at the beginning of the assay (derived from TSB broth) with subsequent reduction (TSB intake), followed by a new peak (degradation of the algal cell wall). However, in Test ID TH19/21 , it was not possible to observe a new fall in sugar indices, since Test ID TH19/21 was completed before a consumption of the released sugars occurred.

The results for Test ID TH19/21 indicate lighting control is effective for the bio-decomposition of algae cells, making the algae sugars available in the medium for later use in the anaerobic bio-decomposition process.

It was observed that the concentration of Bacillus spp. remained around 10⁶ (see Figure 15), while in Test ID TH17/21 it reached 10⁸. This suggests that an average bacterial concentration would be ideal to promote efficient degradation of the algal cell wall, but without providing a consumption of released sugars. Figure 16 shows medium pH levels measured for days 0, 1 , 2, 5 and 7 during Test ID TH19/21. The performance of the test in the absence of light was also more efficient, since it reached significant sugar values, promoted an acidification of the medium (see Figure 16), thus avoiding an uncontrolled proliferation of Bacillus spp., and avoided the use of a source of energy expenditure for the process.

Effect of lighting

In view of the results of Test IDs TH17/21 and TH19/21 , it is considered that lighting control is effective for the bio-decomposition of algae cells, making the algae sugars available in the medium for later use in the anaerobic bio-decomposition process. Performance in the absence of light was also more efficient, since it reached significant sugar values, promoted an acidification of the medium (thus avoiding an uncontrolled proliferation of Bacillus spp.), and avoided the use of a source of energy expenditure for the process. C. vulgaris is very sensitive to light, making indexes of up to 300 - 400 lux sufficient for cells to remain resistant (these cells are usually isolated for maintenance of the medium). Variation in light levels may lead to the following: dark - total bio-decomposition; 400 lux - symbiosis with Bacillus spp.; 900 lux - symbiosis growth and medium consumption; 2000 lux - medium alkalinisation and algae growth.

In the process of the first aspect, controlling light intensity during the bio-decomposition step can allow the phototrophic source to continue photosynthesizing, and thus, the amount of biomass in the medium is increased, making more biomass available to be decomposed by the bacteria. In this example, controlling light intensity during the bio-decomposition process allows the microalgae to continue photosynthesizing, and thus, the amount of biomass in the medium is increased, making more biomass available to be decomposed by Bacillus spp. Thus, there is an increase in general biomass, both from Bacillus spp. (via an increase in cell size, due to the consumption of the medium), and C. vulgaris (due to cellular multiplication by photosynthesis).

During bio-decomposition with light exposure, compared to bio-decomposition without light exposure, the phototrophic source (the microalgae) continues to grow during the biodecomposition period, which can increase the levels of sugar available in the medium. Without light exposure, the bio-decomposition process can occur quickly. In some embodiments, after alight exposure time during bio-decomposition (e.g. during the aerobic bio-decomposition step), it is possible to “switch off” the light to control the bio-decomposition of the organic feedstock. For example, after an initial period of bio-decomposition with light exposure, it may be possible to conduct further bio-decomposition without light exposure or with reduced light exposure, by inoculating the medium (e.g. one containing a mixture of Bacillus spp. and Chlorella spp.) with a facultative anaerobic species (e.g. K. aerogenes). The facultative anaerobic species (e.g. K. aerogenes) may then produce H₂ with the remaining nutrients in the medium. The final biomass, after the production of H₂, can be harvested for use as an agricultural fertilizer.

In some embodiments, controlling light intensity and/or light exposure may be used to reduce the energy expenditure of the process. For example, controlling light intensity and/or light exposure may facilitate or enable symbiosis between the main target microorganisms.

Test ID TH21/21 - Results

Test ID TH21/21 was conducted to evaluate whether there is symbiotic effect of the Bacillus spp. mixture in combination with K. aerogenes on cell decomposition.

Figure 17 shows that from the sixth day of the test there began to be a greater decomposition of the algal cell wall, with consequent greater release of sugars in the culture medium. It appears K. aerogenes had a significant influence on this process, since the treatments employing K. aerogenes alone or in combination with the Bacillus spp. mixture had the best results.

Figure 18 shows Bacillus spp. and K. aerogenes concentration measured for days 0 and 13 during Test ID TH21/21 . It was observed that the Bacillus spp. had a slight increase in cell concentration during the course of the experiment (about 1 log CFU/mL), while K. aerogenes increased a little more, about 2 log CFU/ mL. Without being bound by theory, this behaviour may be due to the generation rate of each microorganism, in which K. aerogenes may present a shorter generation time than Bacillus spp. Also, without being bound by theory, this behaviour may be related the use of K. aerogenes displaying a greater decomposition of the algal cell wall, with a consequent higher concentration of dissolved sugars.

Figure 19 shows medium pH levels measured for days 0, 1 , 6 and 9 during Test ID TH21/21 . From Figure 19 is can be observed that there were slight increases in these values. This was expected, since, due to the low concentration of dissolved sugars, the bacteria present probably did not have enough substrate to carry out their normal metabolism (carbon consumption with acid production). Furthermore, the microorganisms were dissolved in BBM, which has a vast saline composition, which may have provided buffering effect and, therefore, made extreme changes in pH difficult. Test ID TH24/21 - Results

Test ID TH24/21 included the addition of glycerine to the culture medium to assess the behaviour of bacteria in the decomposition of the cell wall of C. vulgaris.

In Test IDs TH17/21 , TH19/21 and TH21/21 , centrifuged microalgae from growths in a previous isolated medium was added to a sterile BBM medium. In Test ID TH24/21 , the culture medium from the Test ID TH20/21 was used. Thus, Test ID TH24/21 used an alga already adapted to the culture medium and that grew in the presence of high amounts of CO2, which may favour a greater accumulation of sugars inside.

A significant difference was observed when using adapted microalgae (an organic feedstock), as there was a significant increase in the contents of dissolved sugars in the medium (see Figure 20), in relation to the previous trials (Test IDs TH17/21 , TH19/21 and TH21/21 ). The measured sugars are due to the decomposition of the algal cell wall. Glycerine, also known as glycerine, glycerol or propanetriol, is a polyol. Accordingly, it is not detected by the methodology used for sugar concentration analysis.

From the results shown in Figures 20, 21 and 22, it was noticed that the addition of glycerine had a positive influence on the release of sugars. It may be that glycerol provides fuel for bacteria so that the bacteria decompose the algal cell wall without consuming the released sugars, thus increasing the efficiency of bio-decomposition and the speed of consumption and growth.

Test ID TH25/21 - Results

In Test ID TH25/21 , a C. vulgaris organic feedstock was subjected to bio-decomposition with the subsequent production of hydrogen gas (H₂). As Test IDs TH21/21 and TH24/21 suggest that employing K. aerogenes in combination with the Bacillus spp. may be advantageous for the bio-decomposition of the cell wall of the C. vulgaris organic feedstock. Test ID TH25/21 was conducted to test the hypothesis that the bio-decomposition process can occur simultaneously with the production of H₂. Evaluation parameters (pH, sugar concentration and bacterial concentration) were analysed at the beginning and at the end of Test ID TH25/21.

Based on the results shown in Figure 23, it was observed that the treatments with the addition of 10% glycerine presented the highest sugar contents. However, in this case, this is not a good result, as it indicates that the bacteria did not consume the sugars present and, as a consequence, there was little hydrogen gas production. This is illustrated by the gas analysis shown in Figure 26, which show that these two treatments were the ones with the lowest H₂ production. It is known that glycerol is a good energy nutrient for bacteria and that they can produce good rates of H₂ using this compound. In Test ID TH24/21 good results were obtained using 2.5% glycerol. In some embodiments, it may be that the addition of glycerine only once at the beginning of the anaerobic bio-decomposition step (during which hydrogen gas may be generated) would result in good production of H₂ or could act as antagonists for bacterial cells.

Based on the result shown in Figures 24 and 25, it can be concluded that this high concentration of glycerine (10%) had a negative influence on Test ID TH25/21. Treatments 3 and 4 showed a significant reduction in cell concentration of both Bacillus spp. and K. aerogenes, which may have led to the low production of H₂ in these treatments. The excess of glycerine in the TSB and BBM medium did not allow a direct consumption of glycerol and gas generation, causing the inhibition of the gas formation process.

Final generation of CC and H? gases in Test ID TH25/21

As shown in Figure 26, treatments that had only microalgae as an energy source for bacteria showed a satisfactory production of H₂, indicating that a process using an aerobic biodecomposition of the organic feedstock may be a promising technique for the production of H₂.

The addition of glycerine is a promising and a cheaper resource, as a source of energy sugars and subsequent formation of gases, when complementing the medium with substances that simulate the TSB medium (proteins and minerals), used for the isolation and growth of K. Aerogenes. This is because it optimizes the medium generating cellular osmotic balance and better bio-decomposition. Rates of up to 20% of H₂ may be generated in the system.

Addition of a nutrient (glycerine)

As described above, the results in Figures 20, 21 and 22 show that the addition of glycerine had a positive influence on the release of sugars. Figure 23 indicates that the treatments with the addition of 10% glycerine presented the highest sugar contents. However, in this case, this is not a good result. Figures 25 and 26 show that a high concentration of glycerine (10%) may have a negative influence on bio-decomposition efficiency.

In the aerobic bio-decomposition process there were satisfactory results, with rapid decomposition of the algae and subsequent consumption of sugar derived from the breaking of the cell wall and the dispersion of its intracellular content in the environment. This was observed by the gradual presence of sugars in the medium over time with the bacteria. As K. aerogenes and Bacillus spp. can live together and multiply together, the inoculation of these bacteria in the aerobic bio-decomposition process was done concomitantly. Thus, the bio-decomposition of algae occurs in a retention period with atmospheric air, rich in O2. Soon after completion of the aerobic bio-decomposition period (5 days) (i.e. when peak sugar available in the medium is reached), the anaerobic process can be put into motion, by making the atmosphere and the environment anaerobic using an inert gas (N₂).

By controlling that glycerine dosage, it is possible to achieve rates of up to 20% hydrogen gas production with glycerine rates in the range of 10%.

For every 1 kg of algae biomass (organic feedstock), 1.83 kg of CO₂ may be absorbed, requiring 800 to 1000 litres of liquid nutrition medium for this, considering an approximate algae concentration of 2.5 g/L. To achieve the maximum conversion rate in H₂ it is necessary to perform the nutritional replacement of sugars (glucose, glycerine), allowing the production of 3.3 L of H₂ per later of medium with algae every 24 hours. Therefore, 1000 litters of algal medium may produce 3,300 L/day of hydrogen gas.

Using microalgae as the substrate (organic feedstock) for the production of H₂ by K. aerogenes, the maximum conversion reached 5.3% of H₂. Therefore, to increase productivity, it may be necessary to dose energy sources at an intermediate point in the process. The addition of glycerine had a positive influence on the release of sugars. Its addition made it possible to achieve rates of up to 20% hydrogen production with glycerine rates in the range of 10%. Using 2.5% glycerol good results were obtained.

Analysis of fertilizer of the final biomass for Test ID TH25/21

The final biomass of the processes can be suitable for use as a biofertilizer. Its chemical and physical characteristics may be compatible with other commercial fertilizers and the biomass may have a high concentration of bacteria (above 4.0 x 10⁸ CFU/ml). Thus, the biomass may have diverse applications in agriculture, soil and plant treatment.

A sample of the final biomass from Reactor F2 (Treatment 1 ) of Test ID TH25/21 was analysed. The composition of the biomass sample is shown in Table 10 below.

Bacteria of the genus Bacillus spp. have diverse antagonistic relationships, being growth promoters, solubilizing mineral and organic components in the soil, promoting plant growth, balance of diseases and pests. K. aerogenes is urease-producing bacteria. Urease is produced as a metabolite in its natural bio-decomposition process. Urease has applications in soil biocementation (also known as Microbially Induced Calcite Precipitation MICP), is capable of composing the biomineralization of the land, and possesses a capacity to improve soils in, for example: • agricultural areas that require better drainage conditions; or

• mining deposits which depend on biogeochemical cycles, to provide drainage control and absorption.

Table 10 - Analysis of the composition of organic nutrients and minerals from sample of the final biomass from Reactor F2 (Treatment 1 ) of Test ID TH25/21 (i.e. the biomass after hydrogen gas production).

Implementation plan for soybean crop tests using a biomass as a biofertilizer

It is proposed to obtain a biomass in accordance with the process of the present disclosure.

The embodiment of the process: • used C. vulgaris as the phototrophic source; and • employed K. aerogenes, B. subtilis, B. megaterium and B. thuringiensis for the aerobic bio-decomposition step.

The biomass can be collected after the aerobic bio-decomposition step. As a cryoprotectant addition, 10% glycerine can be added, before storing the biomass at -20°C.

It is proposed to use the biomass as a biofertilizer for soybeans. A testing area of ten (10) hectares of soybeans is proposed for the application of biofertilizer from planting to harvesting of soybeans. As set out in Table 11 , it is proposed to make ten (10) applications of the biomass from planting to harvesting. The proposed dosage regime is an initial does of 80 ml/hectare in the planting groove, followed by doses of 50ml/hectare that are applied using a spray bar. Thus, after planting, the total application will be 500ml of biofertilizer that can be taken in storage vessel (bottle) for application during spraying.

It is proposed to use the following equipment and materials for the biomass trial:

• Tractor: Massey Ferguson 7219;

• Planter: John Deere 11 13 - 1 1 lines - with fluid application tank 650 L;

• Untreated Seed (White Soybean Seed);

• Spray tank: 2500 L with 15-meter bar; and

• Harvester: John Deere S440.

As set out in Table 1 1 , one or more supplementary bacteria species are proposed to be used with the biomass. It is proposed to mix the supplementary bacteria species into the biomass when it is in the tank of the spray equipment.

Tables 1 1 and 12 each refer to the plant (soybean) phase of growth. These phases are illustrated in Figure 30, which shows the soybean phonological scale.

Table 11 - Proposed Application of a biomass-containing biofertilizer

After the final application of the biomass-containing biofertilizer, it is proposed to subject the crop to a desiccation phase prior to harvest. This is a step used in legume crops (pulses) such as soybean to remove moisture to promote an even (or faster) ripening of the crop. As stated in Table 12 it is proposed to apply a bacteria species to the crop (without use of the biomass) for the desiccation phase. Table 12 - Supplementary bacteria species proposed for the implementation plan

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Embodiments have been described herein with reference to the accompanying drawings. However, some modifications to the described embodiments may be made without departing from the spirit and scope of the described embodiments, as described in the appended claims.

Claims

Claims:

1 . A process for sequestering carbon from a gas stream that comprises carbon dioxide, the process comprising:

(i) converting the carbon dioxide in the gas stream to an organic feedstock using a phototrophic source in a photosynthesis step;

(ii) converting the organic feedstock to biomass; wherein converting the organic feedstock to biomass comprises an aerobic biodecomposition step that employs bacteria comprising at least one facultative anaerobic species.

2. The process as claimed in claim 1 , wherein the aerobic bio-decomposition step employs a mixture of bacteria comprising the at least one facultative anaerobic species and one or more aerobic species.

3. The process as claimed in claim 2, wherein said one or more aerobic species is selected from: one or more Bacillus spp.; one or more Azospirillum spp.; and/or one or more Lactobacillus spp..

4. The process as claimed in claim 3, wherein the one or more Bacillus spp. comprise one or more selected from the group consisting of: B. subtilis; B. megaterium; B. pumilus; B. amyloliquefaciens', B. licheniformis; B. thuringiensis; and subspecies thereof.

5. The process as claimed in claim 4, wherein the one or more Bacillus spp. comprise B. thuringiensis subsp. kurstaki.

6. The process as claimed in claim 3, 4 or 5, wherein the one or more aerobic species comprises B. subtilis, B. thuringiensis and B. megaterium.

7. The process as claimed in claim 3, 4 or 5, wherein the one or more aerobic species comprises B. subtilis.

8. The process as claimed in any one of claims 3 to 7, wherein the one or more Azospirillum spp. comprise Azospirillum brasilense.

9. The process as claimed in any one of claims 2 to 8, wherein the one or more aerobic species comprises one or more facultative aerobic species.

10. The process as claimed in any one of claims 2 to 9, further comprising: adding, before and/or during the aerobic bio-decomposition step, a further nutrient for promoting the growth of said one or more aerobic species of bacteria.

11. The process as claimed in claim 10, wherein the further nutrient comprises glycerine.

12. The process as claimed in claim 1 1 , wherein the glycerine is added in the range of 1 to 10% w/V.

13. The process as claimed in any one of the preceding claims, wherein converting the organic feedstock to biomass comprises, after the aerobic bio-decomposition step: an anaerobic bio-decomposition step.

14. The process as claimed in claim 13, further comprising: collecting a gas generated during the anaerobic bio-decomposition step.

15. The process as claimed in claim 14, wherein the gas generated is hydrogen.

16. The process as claimed in any one of the preceding claims, wherein the at least one facultative anaerobic species comprises one or more species selected from: one or more Klebsiella spp..

17. The process as claimed in claim 16, wherein the at least one facultative anaerobic species comprises K. aerogenes.

18. The process as claimed in any one of the preceding claims wherein the biomass is suitable for use as a fertilizer.

19. A biomass produced by a process according to any one of the preceding claims.

20. A system for sequestering carbon from a gas stream that comprises carbon dioxide, the system comprising: a photosynthesis reactor configured to convert a first carbon dioxide gas stream into an organic feedstock using a phototrophic source, the photosynthesis reactor having an inlet for receiving a carbon dioxide gas stream and an organic feedstock outlet; and a bio-decomposition reactor comprising an inlet in communication with the organic feedstock outlet for receiving the organic feedstock, the biodecomposition reactor being configured to alternate between operating under aerobic and anaerobic conditions.

21. The system as claimed in claim 20, wherein the bio-decomposition reactor comprises a first gas inlet connected to a source of oxygen-containing gas and a second gas inlet connected to a source of oxygen-free gas.

22. The system as claimed in claim 21 , wherein the source of oxygen-containing gas is a source of air.

23. The system as claimed in claim 21 or 22, wherein the source of oxygen-free gas is a source of nitrogen gas.

24. The system as claimed in any one of claims 20 to 23, further comprising one or more product storage vessels in communication with the bio-decomposition reactor for receiving and storing the products generated in the bio-decomposition reactor.

25. The system as claimed in any one of claims 20 to 24, further comprising one or more heat exchangers configured to heat each of the photosynthesis reactor and bio-decomposition reactor.

26. The system as claimed in any one of claims 20 to 25, further comprising a water supply for supplying water to the photosynthesis reactor and/or the biodecomposition reactor.

27. A system for sequestering carbon from a gas stream that comprises carbon dioxide, the system comprising: one or more reactors; said reactors comprising: at least one reactor configured to convert a carbon dioxide gas stream into an organic feedstock using a phototrophic source, said reactor having an inlet for receiving a carbon dioxide gas stream; and at least one reactor configured for subjecting the organic feedstock to biodecomposition, said reactor being configured to alternate between operating under aerobic and anaerobic conditions.

28. The system according to claim 27, wherein at least one of said one or more reactors is configured both to convert a carbon dioxide gas stream into the organic feedstock using the phototrophic source and for subjecting the organic feedstock to bio-decomposition.

29. The system according to claim 28, wherein the system comprises two or more reactors configured both to convert a carbon dioxide gas stream into the organic feedstock using the phototrophic source and for subjecting the organic feedstock to bio-decomposition.

30. The system according to claim 29, wherein said two or more reactors are fluidly connected.

31 . The system as claimed in any one of claims 27 to 30, wherein the or each reactor configured for subjecting the organic feedstock to bio-decomposition comprises a first gas inlet connected to a source of oxygen-containing gas and a second gas inlet connected to a source of oxygen-free gas.

32. The system as claimed in claim 31 , wherein the source of oxygen-containing gas is a source of air.

33. The system as claimed in claim 31 or 32, wherein the source of oxygen-free gas is a source of nitrogen gas.

34. The system as claimed in any one of claims 27 to 33, further comprising one or more product storage vessels in communication with one or more of said at least one reactor configured for subjecting the organic feedstock to bio- decomposition, said one or more product storage vessels for receiving and storing the products generated in said one or more reactors.

The system as claimed in any one of claims 27 to 34, further comprising one or more heat exchangers configured to heat either or each of the one or more reactors.

The system as claimed in any one of claims 27 to 35, further comprising a water supply for supplying water to either or each of the one or more reactors.