WO2013045931A1

WO2013045931A1 - Methods of ensiling algae, ensiled algae and uses of ensiled algae

Info

Publication number: WO2013045931A1
Application number: PCT/GB2012/052397
Authority: WO
Inventors: Roderick Wout; Hugh GREENWELL; David Davies; Michael Theodorou
Original assignee: University Of Durham; Silage Solutions Limited
Priority date: 2011-09-30
Filing date: 2012-09-27
Publication date: 2013-04-04
Also published as: GB201116841D0

Abstract

Methods of ensiling macroalgae, ensiled macroalgae and uses thereof are disclosed. One method involves determining whether a macroalgae species supports an epiphytic lactic acid bacteria (LAB)population sufficient to support a fermentation process under anoxic conditions that will lower the pH of the macroalgae effectively enough to ensile the macroalgae species. If it does so support such an epiphytic population of LAB, the method comprises storing harvested macroalgae of that species under conditions that are or will become anoxic to ensile the harvested macroalgae. Optionally, the method includes treating the harvested macroalgae prior to storage with an additive of an inoculant comprising at least one species of LAB capable of contributing to lowering the pH of the macroalgae under the storage conditions used and/or an additive of one or more enzymes capable of releasing soluble sugars from complex carbohydrates. Alternatively, the method involves treating harvested macroalgae with an additive capable of lowering the pH of the macroalgae effectively enough to ensile the harvested macroalgae under anoxic conditions and storing the treated macroalgae under conditions that are or will become anoxic to ensile the harvested macroalgae. In this instance, the additive may be an inoculant of at least one species of LAB capable of contributing to lowering the pH of the macroalgae under the storage conditions used, optionally in combination with an additive of one or more enzymes capable of releasing soluble sugars from complex carbohydrates,or the additive may be an acid or acid salt for effecting a substantially immediate pH reduction in the harvested material. Angiosperm plant material may be mixed with the harvested macroalgae as an additive when rich with epiphytic populations of LAB or as a co-silage component. Ensiled macroalgae and uses thereof as feedstock for human or livestock consumption or bioprocesses or as a soil conditioner or fertiliser or are also described.

Description

METHODS OF ENSILING ALGAE, ENSILED ALGAE AND USES OF ENSILED ALGAE

Field of the Invention

The invention relates to methods of ensiling algae, to ensiled algae silage and to uses of ensiled algae. In particular, the invention relates to utilising macroalgae in methods of making algae silage, to ensiled macroalgae products and to uses of ensiled macroalgae.

Background of the Invention

Owing to the seasonality of crop growing and harvesting in temperate regions of the world, it has long been necessary to store harvested crops for out-of-season use to sustain life during the winter. The globalisation of modern agricultural supply chains may have simplified the provision of crops between locations subject to different season but it has generated pressure to make many crop products available all year round, which availability may in many instances only be fulfilled using stored produce. The storage of produce may include grain storage (in silos, for example); fruit and vegetable stores, including in more recent times storage under inert gases and/or in refrigerated containers/buildings; and, to sustain sufficient livestock to meet the demand for meat and dairy products, preservation of grass and other agricultural fodder crops as hay and silage.

The storage of grass and other agricultural fodder crops for feeding livestock over the winter is particularly important and requires millions of tonnes of grass and other agricultural fodder crops to be preserved annually. The requirement for so much livestock feed may be exemplified by reference to DEFRA (the Department for the Environment, Food and Rural Affairs) statistics, which indicated that, in 2009 and 2010 in the UK alone, there were 10.1 million cattle and 31.0 million sheep. The preservation of fodder for livestock is therefore an essential part of livestock husbandry in temperate grassland regions and requires the grass and other agricultural fodder crops to be preserved in such a way that it provides a relatively low cost but nutritious feed for cattle and sheep when grazed grass is not available.

Hay making has been used for many thousands of years for conserving grass for feeding cattle and sheep through the winter. However, the vagaries of the weather tend to limit the effectiveness of making good quality hay and, owing to the intensification of livestock husbandry in recent decades increasing demand for winter feedstock, there has been a dramatic decline in hay making in favour of silage.

Silage consists of stored fodder crops, which may have a wide range of moisture contents and which may be fed to ruminant livestock; or which may be used as a biofuel, eg bioethanol, feedstock; or which may be used as feedstock for anaerobic digesters producing for example biogas. The ensiling process was developed in the 19th Century and is usually applied to land- based angiosperm plants such as grass or legume crops, including ryegrass, forage maize, sorghum or other cereals, using the entire green plant (not just the grain). Silage may also be made from many field crops, and special terms have been coined depending on type (eg, oatlage, haylage, baleage, etc). More recently, it has been proposed to apply an ensiling process to the aquatic angiosperm plant water hyacinth— see "Micro-organisms and Marine and Freshwater biomass", Micro-organisms as tools for rural processing of organic residues, Bioconversion of Organic Residues for Rural Communities (UNU, 1979) The United Nations University.

The silage making process involves storing the crop(s) in an anoxic environment and reducing the pH of the stored material to limit plant enzyme (particularly protease) activity and undesirable microbial activity during ensilage, thus retaining the nutritive value of the silage as close as possible to that of the original fodder crop(s).

Typically, the anoxic environment is achieved by placing and compacting freshly cut, green vegetation in a bunker silo and covering the top of the vegetation with earth or polythene sheeting to create a seal, or in a tower silo, or by wrapping large bales in plastic film. Polyethylene, sausage-like structures (such as Ag-Bags (trade mark) available from Ag-Bag Systems, Bow, Crediton, Devon, EX17 6EN) can also be used to store the preserved crop. Residual respiration in the freshly cut crop and some aerobic microbial activity contribute to utilise residual O2 and generate CO2 in the silo, bale or bag assisting in the production of anoxic conditions.

The reduction in the pH of the green vegetation may be achieved by permitting the indigenous, epiphytic population of lactic acid bacteria (LAB), dominated by species of Lactobacilli, Pediococci and Streptococci resident on the crop(s), to grow and feed off sugars in the sap of the crop(s) in a fermentation process. The LAB ferment grass sugars to organic acids, predominately lactic acid, thereby causing crop pH to decline (to pH 4.0 or below), effectively 'pickling' or preserving the crop.

The ensilage process may be enhanced by the use of inoculants containing one or more strains of LAB, and the most common is Lactobaallus plantamm. Other bacteria used in inoculants include Lactobaallus buchneri, Enterococcus faedum and Vediococcus species. Microbial inoculants are selected strains of LAB, chosen for their rapid growth and ability to produce organic acid(s), predominantly lactic acid, and dominate and direct the silage fermentation. In many examples, they are rapidly growing, homofermentative LAB that produce just lactic acid from hexose sugar as their fermentation end product. Silage inoculants are distinct from many of the epiphytic LAB, which tend to be mixtures of both homo- and hetero- fermentative species. Collectively, the epiphytic population will produce mixed organic acids and CO2 and are therefore less effective at reducing crop pH. Alternatively, the pH of the green vegetation may be reduced by spraying the vegetation with acid or acid salts, to provide an instantaneous pH decline, as the vegetation is cut from the field and bailed or transported to the bunker silo. For example, the vegetation may be sprayed with formic acid or a formic acid salt in an appropriate amount at the time of ensiling resulting in an instantaneous decline in pH and causing crop preservation. Silages produced with chemical additives undergo either no or a restricted lactic acid fermentation (depending on the level of acid added at the time of ensiling) and as a consequence, tend to have higher residual soluble carbohydrate content than fermented crops.

Recently, to counteract greenhouse gas emissions from fossil fuels and remove our reliance on petro-chemicals, there has been significant scientific interest and economic investment in crops grown as dedicated energy crops for gaseous and liquid bio-fuel production and for the production of platform chemicals. Economic, environmental and energy security concerns arising from reliance on petroleum are forcing countries the world over to shift to alternative fuels such as bio-ethanol, bio-butanol, bio-diesel and biogas, and to chemical production methodologies (to produce succinic acid for example) based on sustainable, biogenic feedstocks. As with livestock production, these industries, as they develop, will require year-round supply of locally produced raw materials of consistent quality.

One outcome of such interest in bio-derived feedstocks for energy uses is that concerns have been raised that it could divert agricultural production away from food crops, especially in developing countries. The basic argument is that energy-crop programmes compete with food crops, thus causing food shortages and price increases.

More recently, and to counteract the food versus fuel debate, interest has been shown in the use of micro- and macro- algae as a raw material biomass feedstock for the production of biofuels and platform chemicals.

With respect to microalgae, existing microalgae businesses are based on a few high-value compounds such as astaxanthin and polyunsaturated fatty acids. However, the US Government has invested over US $250million in research grants over the last few years to look at the potential for making biodiesel from algal oils and the drive for algae-derived biofuels is now intense.

In a similar way, interest in macroalgae has also increased rapidly. The world market for seaweed is valued at roughly US $6billion, and splits into 80% for food consumption, with around 20% for hydrocolloids (mainly used as thickening agents in food, pharmaceutical and nutraceutical preparations etc). Other uses include as a fertilizer/soil conditioner for land crops, as a feed additive for livestock and as a proposed substrate for anaerobic digestion. Recent interest in evaluating macroalgae at a biofuel feedstock include the US Department of Energy (Macroalgae as a Biomass Feedstock, 2010), a European evaluation of micro- and macro- algae (EPOBIO report, 2007), and the setting up of initiatives such as Biomara (EU INTEREG project) involving the UK and Ireland and led by the Scottish Association for Marine Science (SAMS).

The production of macroalgae as feedstock for industrial biotechnology, anaerobic digestion or other uses is, in most cases, going to rely on cultivation rather than wild stock harvest. Some studies predict that yields of macroalgae will be better per hectare than those of terrestrial crops; however, the economies of such cultivation will be fragile. Yields may be significantly higher if fertilisers are used. In coastal or estuarine regions, sewage outfalls, sewage treatment works, and areas of excessive agricultural run-off, including pollution generated by fish farming, may be possible sources of the necessary nitrogen and phosphorous for macroalgae fertilisation. These sources would be beneficial to such cultivation because they are available at no cost. Additionally, seaweed farming in these areas will assist in the bio-remediation of the local environment without the need to use conventional water treatment procedures for removal of nitrate and phosphate pollutants from areas prone to eutrophication, which is both expensive and necessary.

Similarly to land-based crops, however, algae growth, at least in temperate climes, is seasonal, growth being primarily light dependent rather than temperature and light dependent as with land- based crops. Thus, harvesting periods for algae are similar to those for land-based crops and are selected for specific properties such as high weight or high sugar content, for example.

Year round availability of seaweed is therefore recognised as a significant constraint, hampering the development of algae farming. Progress towards reducing the seasonality effect for macroalgae has focused on the sequential harvesting of different varieties to 'extend' the harvest window and breeding programmes are ongoing, in Chile for example, to produce hybrid varieties with reduced seasonality, thereby offering lengthier harvest duration. Even with reduced seasonality, however, inclement weather conditions including the destructive forces of rough seas (which destroy the algal fronds) will conspire to limit the harvesting window for algal farms.

The seasonality of algae availability has required storage for out-of-season use. In common practise is the storage of both micro- and macro- algae by drying the algae; microalgae usually being in a powdered form and macroalgae being in a chopped form.

Perhaps not surprisingly owing to the rapidity with which algae deteriorates, the fact that algae can have a higher pH as compared to land-based crops, the generally different nature of carbohydrates in the algae as compared to land-based crops, eg the general absence of secondary wall thickening and the presence of distinctly different polysaccharide polymers, the presence of sugar alcohols rather than hexose sugars, and the (often significantly) lower or negligible epiphytic LAB populations on algae, the use of ensilage as a preservation methodology for freshly harvested macro- and micro- algae does not appear to have been considered as a viable storage solution for harvested algae despite the considerable attention that is now being directed towards aquaculture for the production of farmed algal biomass.

It is noted a paper entitled "The Uses of Seaweeds in Iceland", The State Electricity Authority, Geothermal Department, Reykjavik, Iceland (Fourth International Seaweed Symposium 1961 France) discloses that, in the year 1900, ensiling apparently successfully preserved fresh Laminanacae, which was wholesome and palatable fodder for sheep. However, that document does not disclose any details of the ensiled product, including the product age, or of the ensilage process used to achieve it.

It is also noted a paper entitled "The Preservation of Seaweed by Ensiling and Bactericides"— J. Sci. Food Agric, 6, January, 955 discloses the preservation of algae by ensiling for use as a possible feedstuff for farm animals or by killing off bacteria by treating the algae with biocides.

With regard to the ensilage of seaweed, classic grassland ensilage methodology was employed, namely packing chopped seaweed into pipe silos and sealing the top with bitumen paper and soil.

The ensilage process appeared to rely on natural bacteria present on the seaweed. Although the paper suggests the process was successful, this may be questionable as quality assessments

(particularly the measurement of lactic acid concentrations) were not made and the ensiled seaweed was apparently not palatable to sheep suggesting the quality of the ensiled seaweed was poor.

It is also noted in papers "Combinations of lactic acid bacteria and yeast suitable for preparation of marine silage", Uchida, M., Amakasu, H., Satoh, Y. and Murata, M. (2004) Fisheries Science 70, 507-5 7 and "Development of a new dietary material from unutilized algal resources using fermentation skills", Uchida, M. and Miyoshi, T. (2010), Bull. Fish Res Agen. 31, 25-29 that dried, powdered seaweed was subjected to decomposition using cellulase and then fermented using LAB and yeast.

Summary of the Invention

It is desirable therefore to provide methodology for ensiling freshly harvested macroalgae and to provide ensiled macroalgae products that are useful. In accordance with the invention, this may be achieved by ensiling macroalgae as described below to produce an ensiled product.

According to the invention, a method of ensiling macroalgae comprises either:

a) determining whether a sample of macroalgae species to be harvested has an epiphytic population of lactic acid bacteria (LAB) sufficient to support a fermentation process under anoxic conditions that will lower the pH of the macroalgae effectively enough to ensile the macroalgae species; and

b) if a positive result is obtained in step a), storing harvested macroalgae of that species under conditions that are or will become anoxic to ensile the harvested macroalgae, optionally having treated the harvested macroalgae prior to storage with an additive comprising an inoculant comprising at least one species of LAB capable of contributing to lowering the pH of the macroalgae under the storage conditions used and/or one or more enzymes capable of releasing soluble sugars from complex carbohydrates;

or

c) treating harvested macroalgae with an additive capable of lowering the pH of the macroalgae effectively enough to ensile the harvested macroalgae under anoxic conditions; and

d) storing the treated macroalgae from step c) under conditions that are or will become anoxic to ensile the harvested macroalgae.

In a particularly preferred embodiment of the invention as herein described, the method comprises treating harvested macroalgae with an additive capable of lowering the pH of the macroalgae effectively enough to ensile the harvested macroalgae under anoxic conditions and storing the treated macroalgae under conditions that are or will become anoxic to ensile the harvested macroalgae.

The macroalgae are Eukaryotes and include all species belonging to the red, green and brown algae. These are all of the multicellular species within the Rhodophyta, Chlorophyta and Phaeophyta.

Common examples of each are as follows:

Rhodophyta: Valmana palmata, A/raciophora hypnoides, Mastocarpus stellat s, Chondrus crispus. These are mostly multicellular marine macroalgae, although some are fresh water species are known (within the freshwater genus, Fem ned), and estimates indicate as many as 10,000 species.

Chlorophyta: Ulva lobata, Caulerpa racemosa, Cladophora r pestns, Monostroma grevillei. These are mostly unicellular, but include some multicellular, macroalgae estimates indicate there are about 7000 species. Many species live in fresh water and, like land plants, they all contain chlorophylls a, b and store true starch in their plastids. True land plants evolved from the Chlorophyta.

Phaeophyta; Laminana hyperborea, Fucus serrat s, Ascophylluym nodosum, Sargassum muticum. These are mainly multicellular marine macroalgae and worldwide there are about 1500-2000 species of brown algae.

The harvested macroalgae may be used in the invention in the freshly collected and substantially whole condition, or, more preferably, the freshly collected macroalgae may be subjected to a chopping or comminution process to reduce the macroalgae to relatively small pieces, for example typically roughly 2 cm to 20 cm long/wide. By "freshly collected" is meant the macroalgae is subjected to the ensiling process typically within 72 hours, more preferably within 48 hours, especially within 24 hours, as the method of the invention includes allowing the microalgae to wilt, ie to dry out, somewhat to increase the dry matter content thereof prior to ensilage.

In one embodiment of the invention, angiosperm plants, whole or, more preferably, chopped or comminuted, may be mixed with the harvested algae. The angiosperm plants may be any suitable land-based or aquatic plants and such plants, as previously described, may include grass or legume crops, including ryegrass, forage maize, sorghum or other cereals, field crops and water hyacinth. Preferably, (and, on a fresh matter weight (FW) basis), at least 10% by weight, more especially at least 20% by weight, based on the total weight of macroalgae and angiosperm plants, of angiosperm plants may be added to the harvested macroalgae. Preferably, not more than 90% by weight, more especially not more than 80% by weight, based on the total weight of macroalgae and angiosperm plants, of angiosperm plants may be added to the harvested macroalgae.

The determination as to whether the sample of macroalgae species to be harvested has an epiphytic population of lactic acid bacteria (LAB) sufficient to support a fermentation process under anoxic conditions that will lower the pH of the macroalgae effectively enough to ensile the macroalgae species may be performed by any suitable method. A simple means of making such a determination is to try and ensile the sample under anoxic conditions, for example using the laboratory ensilage method as described in Example 1 below. If the sample is successfully ensiled and lactic acid is detected in sufficient quantity to reduce the pH of the macroalgae, the epiphytic population of LAB is sufficient; conversely, if the sample is not successfully ensiled and lactic acid is not detected in sufficient quantity to reduce the pH of the macroalgae, the epiphytic population of LAB is not sufficient.

In determining whether the macroalgae sample has been successfully ensiled, it is noted the exact concentration of lactic acid and pH required to ensile the macroalgae is dependant on a number of variables such as the dry matter (DM) content of the macroalgae to be ensiled, the chemical characteristics (in particular the concentration of the various nitrogen containing fractions) and the amount and type of pre-ensilage processing. Taking all these variables into account, a successfully ensiled macroalgae biomass would contain between 10 and 180 g kg DM of lactic acid with a pH decline from the original macroalgae of less than 1 log unit to greater than 3 log units.

If the macroalgae has an epiphytic population of bacteria sufficient to ensile it under anoxic conditions, the macroalgae may be processed using step (b) of the method of the invention. Alternatively, irrespective of whether step (a) is performed or in the event of a negative outcome of step (a), ie the macroalgae does not have an epiphytic population of bacteria sufficient to ensile it under anoxic conditions, the macroalgae may be ensiled using steps (c) and (d) of the method of the invention.

The additive used in the method of the invention may comprise an inoculant and/ or an enzyme or an acid or acid salt as hereinafter described. In a preferred embodiment, the additive used in the method of the invention consists essentially of an inoculant, optionally in combination with an enzyme, or an acid or acid salt as hereinafter described.

In one embodiment of the invention, the additive comprises an inoculant and, as previously described, it may optionally be used in step b) of the method of the invention or it is used in step c) of the method of the invention and it is at least one species of bacterium that will react with the carbohydrate energy source in the macroalgae and, optionally, with the carbohydrate energy source in any added angiosperm plants in a fermentation reaction to produce organic acids, preferably predominately lactic acid, to lower the pH of the macroalgae to ensile it.

The inoculant may consist of only a single species of bacterium, for example Lactobacillus plantarum, which is active over the full range of pH values from 7 to 4 found in the macroalgae during the ensiling process. Alternatively, the inoculant may comprise more than one, preferably several, species of bacteria each of which function most efficiently over different parts of the pH range generated in the macroalgae during the ensiling process.

Useful species of bacteria include Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus cremoris, Lactobacillus curvatus, Lactobacillus xylosus, and Lactobacillus salivarius and mixtures thereof. Such bacterial species are all used to improve (enhance) the pH decline; however, some species/strains are only effective once the initial pH is below 5. Some other species may be introduced into the inoculant to initiate pH decline when the starting pH is closer to 7. Such species include Enterococcusfaecium (pseudonyms, Lactococcus faecium or Streptococcus faecium) and Lactococcus lactis, which function most efficiently at the higher pH range of between 6.5 and 5.5. The inoculant may also include species that function most efficiently at lower pH values, eg between 6 and 4.5, for example Pediococci (Pediococcus pentocaseous, Vediococcus a dilactici, Vediococcus cerevisiae).

Other useful species of bacteria, which produce lactic acid, acetic acid and propionic acid fermentation products, include Lactobacillus buchnen, Lactobacillus brevis, Vropionibactenum jensenii, Vropionibactenum propionicum, and Vropionibactenum shermanii and even some Bacillus species such as Bacillus pumilis, Bacillus Licheniformis and Bacillus subtilis.

Preferably, the inoculant comprises about 1 x 10^s colony forming units (cfu) per g of crop dry matter of bacteria, more preferably at a minimum of about 1 x 10⁵ cfu per g of crop dry matter of bacteria and more especially at least about 1 x 10⁶ cfu per g of crop dry matter of bacteria. In an alternative version of this embodiment of the invention, the inoculant may comprise angiosperm plant matter that is sufficiently rich in epiphytic populations of suitable species of lactic acid producing bacteria (such as the hetero- and homo- fermentative lactobacilli) to ensure fermentation occurs during the ensiling process. In this embodiment, an inoculant of at least one additional species of bacterium as described above may also be included in the additive.

In another embodiment of the invention, the additive may comprise one or more enzymes capable of releasing soluble sugars from complex carbohydrates, for example storage carbohydrates such as starch or structural carbohydrates such as cellulose and hemicelluloses.

Thus, the additive may comprise enzyme complexes such as cellulase complexes comprising of endoglucanase, exoglucanase and cellobiohydrolase, the enzymes of which act synergistically to hydrolyse cellulose and cellulose in cellulose-containing substrates; and hemicellulose complexes comprising endo-1 ,4-xylanases, including both arabinose- and non-arabinose- liberating endo- xylanases, and exo-xylosidases, the enzymes of which act synergistically to hydrolyse hemicellulose and hemicellulose in hemicellulose-containing complexes.

Other useful enzymes for use as the additive comprise enzymes such as esterases and amylases, preferably additionally to the cellulolase and hemicellulase complexes described above.

In preferred embodiments of the invention, the additive may comprise an inoculant in combination with an enzyme

In an alternative version of this embodiment of the invention, when the epiphytic population of LAB on the macroalgae is sufficient to support a fermentation process under anoxic conditions that will lower the pH of the macroalgae effectively enough to ensile the macroalgae species, the additive may comprise one or more enzymes as described above without additional inoculants of LAB species being present in the additive.

The additive may comprise an acid or an acid salt for effecting a substantially immediate pH reduction in the harvested material, for example a significant pH reduction within 1 to 5 days. The acid or acid salt may be an organic acid or acid salt or an inorganic acid or acid salt or mixtures of organic acids or acid salts or inorganic acids or acid salts. The organic acid or acid salt is preferably a carboxylic acid or carboxylic acid salt and may be an aliphatic acid or acid salt or an aromatic acid or acid salt. Preferably, the acid or acid salt is a straight chain fatty acid or acid salt, more preferably a O to C12 straight chain fatty acid or acid salt. In particular, the straight chain fatty acid or acid salt is selected from the group consisting of formic acid, acetic acid and propionic acid, salts of such acids and mixtures thereof. A useful aromatic acid is benzoic acid and, more preferably, a benzoic acid salt. Inorganic acids may also be used notwithstanding their corrosive nature and health and safety considerations that may arise from such use. The use of inorganic acids, particularly such as sulphuric acid, in the method of the invention may be particularly economically attractive when the ensiled macroalgae is intended for use in bioprocesses rather than as an animal feedstock or for human consumption.

Preferably, the anoxic storage conditions under which the macroalgae is placed to ensile it may be achieved by packing the macroalgae into a bunker, long polythene 'sausage-shaped silo' of the Ag Bag type or tower silo, or by wrapping bales of varying shapes and sizes either with or without pre-ensiled mechanical chopping/ processing in plastic film.

In an alternative embodiment, the anoxic storage conditions under which the macroalgae is placed to ensile it may be achieved by packing the macroalgae into a vacuum pack bag or similar container, extracting air from the bag or similar container and sealing the bag or similar container. As will be appreciated, if the macroalgae is being processed under a gas, eg nitrogen, other than air, then the reference to extracting air is intended to include the removal of any other such gas. This process is particularly advantageous in that, as macroalgae lacks significant structural components owing to the absence of lignin in almost all macroalgae species, this makes the harvested macroalgae exceptionally amenable to packing under vacuum. One of the advantages of vacuum packing for ensiling macroalgae and ensiled macroalgae is a significant increase in bulk density, assisting in the compaction, storage and transportation of the biomass.

When the additive comprises an inoculant of LAB and/ or one or more enzymes as hereinbefore described, it will be necessary for the bag or similar container to have a valve mechanism for venting C02 generated during the fermentation processes occurring within the biomass.

When the additive comprises an acid or an acid salt that only partially inhibits the epiphytic population from participating in microbial fermentation, the bag or similar container will also require a valve mechanism for venting gas.

When the additive comprises an acid or an acid salt that completely inhibits the epiphytic population from participating in microbial fermentation, the bag or similar container will not require a valve mechanism for venting gas.

It is also within the scope of the present invention to vacuum pack ensiled macroalgae extracted from storage in for example a silo to aid transport and/or distribution to the point of use of the ensiled macroalgae. Clearly, this would be particularly beneficial if the point of use of the ensiled macroalgae is not adjacent the silo location as the ensiled macroalgae would retain its condition during such transport and/ or distribution to the point of use. This may be particularly important if the ensiled macroalgae is intended for use as a livestock feedstock.

The bag or similar container is conveniently made of a gas impermeable polymer, for example polyethylene or polypropylene. Alternatively, and especially when the ensiled macroalgae is to be used in aqueous-based bioprocesses and ambient levels of moisture during storage are insufficient to degrade it, the gas impermeable polymer may be a water-soluble polymer such as polyvinyl alcohol.

Also, according to the invention, an ensiled macroalgae comprising preserved macroalgae in which residual amounts of epiphytic and/or inoculant lactic acid bacteria (LAB) and/or enzyme(s) are present, or preserved macroalgae in which residual amounts of additive acid and/or acid salts are present, the epiphytic LAB and the additive comprising inoculant LAB and/or enzyme(s) or acid and/or acid salts being as herein before described and defined.

Ensiled macroalgae in accordance with the invention exhibits a pH decline from the original macroalgae of less than 1 log unit to greater than 3 log units. Preferably, the ensiled macroalgae has a pH of less then 7, more especially less than 6.5. Typically, the ensiled macroalgae has a pH of less than 6. The ensiled macroalgae typically has a pH of not less than 3. When the ensiled macroalgae has been generated using an additive comprising an inoculant of LAB, the total lactic acid concentration (L+D lactic acid) after 90 days of storage under the anoxic conditions is between 10 and 200g kg ¹ DM.

The ensiled macroalgae of the invention is useful in a number of applications and the invention includes the use of the ensiled macroalgae in such applications. For example, the ensiled macroalgae may be used as a feedstock for human or livestock consumption; or may be used as a soil conditioner and/or fertiliser; or may be used as a feedstock in a bioprocess, whether the process be for generating energy or commodity or specialty monomeric and polymeric chemicals (e.g., for pharmaceutical and/ or nutraceutical use).

The invention also includes a method of bioprocessing organic feedstock material comprising feeding to a bioprocessing unit at least one feedstock comprising ensiled macroalgae of the invention.

The use of ensiled macroalgae of the invention in such a method is particularly useful if there is available to the bioprocessing plant two or more ensiled macroalgae feedstocks, or, indeed, other ensiled or fresh organic feedstocks that may be being used, that vary in composition of dry matter, chemical constituents etc, for example from being harvested at different times of the year. In this instance, two or more of the feedstocks may be mixed to produce a feed stream into the bioprocessing plant that is of relatively consistent composition.

Such a combination of at least two ensiled macroalgae feedstocks to generate a feedstock of relatively consistent composition may also be useful for other chemical plant or for livestock feeding.

Brief Description of the Drawings

The present invention will now be described with reference to Examples 1 and 2 below and with reference to the accompanying drawings in which: Figure 1 shows photographs of macroalgae Faminana hyperborean that has been held under ensiling conditions as described in Example 1 below, the photographs being of a control sample, a sample treated with an additive consisting of an inoculant lactic acid bacteria (LAB) and a sample treated with an additive consisting of an acid;

Figure 2 shows photographs of macroalgae Fucus serratus held under ensiling conditions as described in Example 1 below, the photographs being of a sample treated with an additive consisting of an inoculant lactic acid bacteria (LAB) and a sample treated with an additive consisting of an acid;

Figures 3 and 4 show photographs of macroalgae Fucus serratus and Laminana hyperborean, respectively, held under ensiling conditions as described for samples (e) and (c), respectively, in Example 2 for 434 days in vacuum pack bags; and

Figure 5 shows a photograph of a vacuum pack of macroalgae Faminana hyperborean held under ensiling conditions as described for samples (b) in Example 2 for 434 days.

Example 1

The following methodology was used in this Example.

Determination of Macroalgae Silage Dry Matter (DM) Content

One aliquot (100 g fresh matter (FM)) of the thoroughly mixed contents from each ensilage jar (see below) was placed in polythene sampling bags and kept frozen at -20°C for several weeks prior to dry matter (DM) content determination. DM content was determined by placing the thawed 100 g aliquots in weighed aluminium trays and drying to constant weight in a fan-assisted drying oven (approximately 48 h at 60°C).

Determination of pH and Chemical Analyses

Two aliquots (10 g and 20 g FM, respectively) of the thoroughly mixed contents from each ensilage jar (see below) were placed in polythene sampling bags.

Distilled water (90 ml) was added to the 10 g FM aliquots which were then 'stomached' for 2 minutes (Stomacher 400 Circulator (trade mark) available from Seward Ltd., Dominion House, Easting Close, Worthing, West Sussex, BN 4 8HWQ, UK) to liberate cell contents prior to pH determination using a pH electrode.

The 20 g FM aliquots were frozen at -20°C until needed for chemical analyses. Following thawing, distilled water (80 ml) was added to each of the aliquots and the resultant mixture lightly massaged in the bag to ensure adequate mixing. The bags and their contents were left at 4°C overnight and, on the following day, the supernatant liquid from each aliquot was collected by filtration under vacuum through glass fibre paper. The collected supernatant liquid from each aliquot was used immediately for chemical analysis. A YS1 2700 Select Biochemistry Analyser (YS1 (UK) Ltd., 119 Fleet Road, Fleet Hampshire GU51 3PD) was used according to the manufacturers instructions to determine the L-lactic acid and glucose (dextrose) content of the filtered supernatant liquid samples.

Results for L-lactic acid and glucose were expresses as g kg⁴ DM of ensiled seaweed. The aqueous and DM content of the extracted samples were taken into account in making these calculations.

Ensilage of Macroalgae in Accordance with the Invention

The macroalgae chosen for this Example were the benthic marine brown macroalgal species, L minaria hyperbore and Fucus serratus. Both species are indigenous and plentiful in the coastal waters surrounding the UK. Sufficient quantities of the macroalgae were collected from below MLWS (mean low water springs) at Berwick on Tweed. The collection method involved identification and removal of the chosen species, using a sharp knife to cut them from the rocks. The macroalgae were harvested in 100 kg capacity permeable nylon bags and the water drained off in the bags. Approximately 5 h after collection, the macroalgae samples were removed from their respective bags, were laid out in a shed on a dry concrete floor and left overnight to wilt to achieve a target dry matter of between 15 and 35 % by weight.

Each species of macroalgae was then chopped separately using a Bosch 2200Hi industrial garden shredder with a splined cylinder cutter. Once chopped the macroalgae was spread thinly on a polythene sheet to prepare three samples of each species: (a) a control sample, (b) a sample which was treated with an additive consisting of an inoculant of freshly cultured Lactobaallus plantamm applied at 0⁶ cfu g^"1 FM, and (c) a sample which was treated with an additive consisting of a formic acid based additive (Additive F NC available from Kelvin Cave Ltd, Bridgewater, Somerset) at a dosage rate of 4 litres tonne⁴ FM.

The additives were applied through an airbrush sprayer directly onto the chopped macroalgae samples. Each macroalgae sample to which additive was added was thoroughly mixed and three replicates of each of the samples (a) to (c) were packed into Week 1.5 litre glass preservation jars fitted with gas-tight rubber seals, glass lids and lid-fastening, spring retaining clips to retain anaerobic conditions during storage. The samples were pressed by hand into the jars until they were completely full (approximately 1.3kg of macroalgae per jar) and the lids fastened above the compacted macroalgae samples, taking care to exclude air spaces insofar as it was practicable to do so to enable anoxic conditions to prevail. Following a storage period of 40 days at a constant temperature of 18-20°C, the jars were opened and the contents of each jar was removed and thoroughly mixed prior to being sub-sampled for subsequent pH, dry matter determinations and chemical analyses (which included L-lactic acid and dextrose (glucose) analyses).

The results of pH measurements on fresh macroalgae and on the samples (a) to (c) after 40 days are shown in Table 1. As can be seen, the pH of all of the samples reduced during the 40-day period under anoxic conditions. For the Laminana hyperborea samples, the pH decreased by approximately 1.7 units in the untreated (a) sample to approximately 2.9 units in the treated (b) and (c) samples. With regard to the Fucus serratus samples, the pH declined by approximately 2.1 — 2.4 for each of the three samples (a) to (c).

Visual inspection of the ensiled samples (photographed after thawing 100 g FM aliquots for DM determination) showed quite distinct differences relating to treatment— see Figures 1 and 2.

In the case of Laminana hyperborea, Figure 1 , sample (a), the control, appeared contaminated and degraded (rotting) and it was difficult to observe the typical shape or texture of the original macroalgae. In contrast, samples (b) and (c), the samples treated with additives consisting of inoculant LAB or acid, respectively, retained substantially the appearance of the original chopped macroalgae samples placed into the preservation jars 40-days before (Figure 1).

Table 1

Treatment L. hyperborea pH* F. serratus pH*

Fresh (no ensilage) 7.43 8.28

Sample (a) (untreated) 5.77 (0.078) 6.22 (0.224)

Sample (b) (additive - inoculant LAB) 4.51 (0.097) 6.11 (0.111)

Sample (c) (additive - acid) 4.55 (0.113) 5.92 (0.312)

*Mean (SD); 3 replicates

With regard to the Fucus serratus, Figure 2, there was no apparent deterioration of the control material, sample (a), and all three samples of ensiled macroalgae were preserved and looked remarkably similar. As sample (a) looked essentially like samples (b) and (c), only samples (b) and (c) are shown in Figure 2.

For both Laminana hyperborea and Fucus serratus, samples (c), the samples treated with additives consisting of acid, were a slightly darker brown (Laminana hyperborea), less green (Fucus serratus) colour than the respective samples (a), the control samples, and (b), the samples treated with additives consisting of inoculant LAB.

For Laminana hyperborea, the reduced decline in pH value of the control sample (a) (1.7 pH units), relative to the decline in pH values of the samples (b) and (c) treated with additives (2.9 pH units), was consistent with its different visual appearance and likely caused by acidic fermentation products from the spoilage micro-organisms involved in rotting the macroalgae.

The low pH value for the control sample (a) of Fucus serratus macroalgae, which is comparable to the pH values of samples (b) and (c) suggest the epiphytic LAB population on the Fucus serratus macroalgae was sufficient to support the fermentation process under anoxic conditions effectively enough to ensile the Fucus serratus macroalgae species without the need for an additive of an inoculant LAB.

The dry matter contents (% DM) of the samples post the 40-day period is shown in Table 2.

For the Faminana hyperborea macroalgae, values ranged from 15.61 % for the untreated control sample (a) to 18-19 % for the samples (b) and (c) treated with additives. In the case of the Fucus serratus macroalgae, the post ensilage percentage DM values were significantly higher, at 29-30%, than the values recorded for the Faminana hyperborea macroalgae. The lower DM value recorded for the Faminana hyperborea macroalgae sample (a), relative to the higher values for samples (b) and (c) suggested losses arising from the emission of gaseous fermentation products, which is consistent with the rotted appearance of sample (a).

The values for L-lactic acid production (g kg⁴ DM), a key indicator of the ensilage process, are shown in Table 3.

Table 2

Treatment F. hyperborea (% DM)* F. serratus (% DM)*

Sample (a) (untreated) 15.61 (0.095) 29.44 (0.023)

Sample (b) (additive - inoculant 8.56 (1. 33) 29.85 (0.433)

LAB)

Sample (c) (additive - acid) 9.04 (1.624) 30. 0 (0.289)

*Mean (SD); 3 replicates

Table 3

*Mean (SD); 3 replicates In sample (b) of the Laminana hyperborea macroalgae, and in samples (a) and (b) of the Fucus serratus macroalgae, L-lactic acid was detected in quantities equivalent to those found in poor to average grass silages and was significantly in excess of the amounts found in the other samples. In the (b) samples of the macroalgae, it can be concluded that the inoculant Factobacillus plantarum was responsible for most {Fucus serratus) or all (Laminaria hyperborean) of the lactic acid production. In the control sample (a) of the Fucus serratus macroalgae, as discussed above in relation to the pH value and appearance of this sample, it seems likely that an epiphytic population of LAB was responsible for the production of lactic acid found in this sample which, although just 36% of that detected in sample (b), was sufficient to cause effective ensilage of the sample.

With regard to the (c) samples of the macroalgae species treated with the acid additive, from the absence of lactic acid, and appearance of the ensiled macroalgae, it can be concluded that the additive inhibited any epiphytic LAB populations that may have been present on the samples and rapidly lowered the pH of the samples and, under the anoxic conditions, the samples were ensiled as is apparent from Figures 1 and 2. Thus, as expected, in the samples (c), lactic acid would be expected to be present in relatively small amounts, which it is as shown in Table 3.

As previously described, ensiled macroalgae made in accordance with the invention is useful in a number of applications; for example, the ensiled macroalgae may be used as a feedstock for human or livestock consumption; or may be used as a soil conditioner and/or fertiliser; or may be used as a feedstock in a bioprocess, whether the process be for generating energy or commodity or specialty monomeric or polymeric chemicals (e.g., for pharmaceutical and/or nutraceutical use).

Example 2

The following methodology was used in this Example.

The determinations of macroalgae silage DM content, pH and chemical analyses were conducted as described for Example 1. However, in this Example 2, glucose was not measured but the volatile fatty acids (VFA), acetic, propionic and butyric acids were measured and total lactic acid was also measured.

Determination of VFA

Aqueous samples (as used and described for L-lactic acid analysis in Example 1) were analysed for acetic acid, propionic acid, n-butyric acid and iso-butyric acid concentration by GC-FID using internal standard quantification. Prior to analysis, samples were centrifuged and the supernatant removed for analysis. Samples were diluted 2:1 with internal standard (2 -methyl valeric acid) prior to analysis and quantified against a 5-point calibration. Samples and standards were prepared by volume using a positive displacement pipette, and analyte concentration calculated in mg L⁴. Results have been reported in concentration units of mg L⁴ for the samples as received. All samples were analysed in triplicate.

VFA Analytical Conditions

Perkin Elmer Clarus 500 GC-FID. ZB-FFAP column (30 m x 0.32mm x 0.25μτη), 2 mL min⁴ He carrier gas. Oven temperature programme: hold @ 110°C for 0.5 min, ramp @ 10°C min⁴ to 165°C, ramp @ 45°C min ¹ to 260°C, hold 2 min. 300°C detector temperature, 0.5 μί_^ injection, 250°C inlet temperature.

All VFA and lactic acid results were expressed as g kg⁴ DM of ensiled seaweed as in example 1. Ensiling of Macroalgae in Accordance with the Invention

As in Example 1 , the macroalgae chosen for this Example 2 were the benthic marine brown macroalgal species, Faminana hyperborea and Fucus serratus. Sufficient quantities of the macroalgae were collected from the area known as Marsden bay, near South Shields, UK, at a depth of 12 m below the surface by divers. The collection method involved identification and removal of the chosen species, using a sharp knife, from rocks. The macroalgae were harvested in 100 kg capacity permeable nylon bags and the water drained off in the bags once they were removed from the sea. Approximately 5 hours after collection, the macroalgae samples were removed from their respective bags, were laid out in a shed on a dry concrete floor and were left to wilt either over night or over two nights.

Immediately prior to ensiling on each consecutive day, each species of macroalgae was chopped as described in Example 1. Once chopped the macroalgae was spread thinly on a polythene sheet to prepare five samples of each species as follows:

(a) a control untreated sample;

(b) a sample which was treated with an additive consisting of an inoculant of Factobaallus buchneri (a heterofermentative lactic acid bacterium) applied at 106 cfu g⁴ FM;

(c) a sample which was treated with an additive consisting of an inoculant of Factobacillus plantarum (a homofermentative lactic acid bacterium) applied at 106 cfu g⁴ FM;

(d) a sample which was treated with an additive consisting of a mixture of sodium benzoate, sodium nitrite and potassium sorbate (Safesil, available from Kelvin Cave Ltd, Bridgewater, Somerset) at a dosage rate of 4 litres tonne⁴ FM; and

(e) a sample which was treated with an additive consisting of a formic acid based additive

(Additive F NC available from Kelvin Cave Ltd, Bridgewater, Somerset) at a dosage rate of 6 litres tonne⁴ FM, ie at a higher application rate for this additive than used in Example 1. The additives were applied through an airbrush sprayer directly onto the chopped macroalgae samples. Each macroalgae sample to which additive was added was thoroughly mixed.

For each macroalgae species, three replicates of each of the samples (a) to (e) were packed into Week 1.5 litre glass preservation jars as in Example 1 and two further sets of three replicates of each of the samples (a) to (e) were packed into polythene bags and sealed with a vacuum packer. The vacuum packs were made up by placing 100 g FM of chopped seaweed was packed into polythene bags (dimensions 300x150 mm; Kalle Nalo, Witham, UK), which are designed for vacuum packing food produce. A vacuum packer, model MVS35 (Minipack Torre Ltd, Corby, UK) was used to remove 95% of the air from the bags, which were then heat sealed by the machine to provide a perfect seal to inhibit ingress of air after sealing. The heat seal was positioned 150 mm from the base of the bag and, following sealing, surplus plastic was automatically cut 5 mm above the seal.

The sets of samples were held at a constant temperature of 8-20°C. The set of samples in the jars and one set of samples in the bags were held for a first ensiling period of 105 days and the second set of samples in the bags was held for a second ensiling period of 434 days.

Following the respective storage periods, the jars and bags were opened and the contents of each jar or bag was removed and thoroughly mixed prior to being sub-sampled for subsequent pH, dry matter determinations and chemical analyses (which included total lactic acid and VFA).

Figures 3 and 4 show macroalgae structures are still intact and well preserved even after 434 days of storage, indicating visually that the process described herein will successfully preserve macroalgae.

The pHs of the wilted, pre-treatment macroalgae samples in this Example 2 are shown in Table 4. As will be apparent, the pH of the pre-treatment macroalgae of Example 2 was lower than the pH of the pre-treatment macroalgae of Example . This is likely to be due to the fact that the macroalgae used in Example 2 were harvested from under the water in contrast to the macroalgae used in Example 1 which were harvested from the shore line and were not permanently covered by water and so possible more likely to be contaminated with sand. The results from both Examples 1 and 2 therefore show that the methodologies described herein are suitable for the storage of both freshly cultivated and harvested macroalgae, plus beach cast macroalgae that can cause problems to councils in areas where tourism is important.

The results of the macroalgae silage analyses after opening are shown in Tables 5, 6 and 7, respectively, for the set of macroalgae samples stored in jars and stored for 105 days, the first set of macroalgae samples stored in vacuum packs and stored for 05 days and the second set of macroalgae samples stored in vacuum packs and stored for 434 days. Each Table shows the mean and standard deviation (shown in brackets) for the parameters indicated. Table 4

Table 5

ND=Not Determined

*Mean (SD); 3 replicates

The results for the chemical analyses for macroalgae ensiled in jars opened after 105 days (shown in Table 5) show that all silages were well preserved with the exception of the Fucus serratus 1 day wilt untreated silage. The Fucus serratus 1 day wilted untreated silage had a higher pH (4.70) and low levels of lactic acid accompanied by high levels of VFA, namely acetic, propionic and butyric acids. The untreated macroalgae silage samples (a) were generally less well preserved than any of the other treated macroalgae samples (b) to (e). The samples (a) had higher pH values with generally lower levels of the beneficial acid, lactic acid, and relatively higher levels of the VFA's. The results indicate that whilst some fermentation may be achieved without an additive, in most cases better preservation will be achieved using an additive. All additive-treated silages were well preserved without exception. The use of formic acid at 6 1 t FM application inhibited most of the fermentation as can be seen from the low levels of all acids both beneficial (lactic) and detrimental VFA. All inoculated silages had a Lactic:VFA ratio of greater than 5:1 which indicates an excellent preservation.

The results for the chemical analyses for macroalgae ensiled in vacuum packed bags opened after 105 days (shown in Table 6) show that all silages were well preserved. Similarly to the macroalgae ensiled in the jars, the untreated silage samples (a) (Table 6) were less well preserved compared to the samples (b) to (e) ensiled with an additive. The sample (a) silages also had a greater degree of variability between replicates as indicated by the standard deviations, indicating that there is a greater risk of poorer fermentation when not using an additive.

Table 6

*Mean (SD); 3 replicates

Generally macroalgae ensiled in the vacuum packed bags were better preserved than the same macroalgae ensiled in the jars. The ratio of lactic:VFA in all vacuum packed bag silage samples (b) to (e) was greater than 5:1 which indicated excellent preservation as in the silage samples (b) to (e) in the jars. Whilst both the jar and vacuum packed bag ensiling methodologies are successful in the preservation of macroalgae, the pH values indicate a lower pH and the proportion of lactic to VFA in the vacuum packed bag samples indicates that this method was, in this example, superior to the jar method. The jar methodology is used throughout Europe (Eire, Germany, Sweden, UK, Hungary, Czech Republic) as an experimental method to give a good simulation of large-scale pit, bunker and 'Ag bag' or 'Sausage' silos.

The results for the chemical analyses for macroalgae ensiled in vacuum packed bags opened after 434 days (shown in Table 7) show that all silages were well preserved. The results for the 434- day preserved macroalgae silages followed the same trends as for the 105-day silages. These results indicate that the methodologies highlighted in this process are able to maintain macroalgae in a preserved state for a time period in excess of 1 year.

Table 7

ND = Not determined

*Mean (SD); 3 replicates The results shown in this example also indicate that macroalgae can be successfully ensiled after differing lengths of wilting with the data showing that both the 1 day and 2 day wilted silages could be preserved by the process outlined and produce seaweed silage with a low pH and high levels of lactic acid and correspondingly low levels of acetic, propionic and butyric acids.

The results also show that different species of macroalgae can be successfully preserved by the techniques indicated for periods in excess of 1 year.

Claims

A method of ensiling macroalgae comprising either:

b) if a positive result is obtained in step a), storing harvested macroalgae of that species under conditions that are or will become anoxic to ensile the harvested macroalgae, optionally having treated the harvested macroalgae prior to storage with an additive comprising an inoculant comprising at least one species of LAB capable of contributing to lowering the pH of the macroalgae under the storage conditions used and/ or one or more enzymes capable of releasing soluble sugars from complex carbohydrates;

or

The method according to claim 1, which comprises mixing angiosperm plants with the harvested algae.

The method according to claim 1 or claim 2 in which the additive that may optionally be used in step b) or is used in step c) comprises an inoculant comprising at least one species of bacterium that will react with carbohydrate energy source in the macroalgae and, when dependent on claim 2, with sugars in any of the added angiosperm plants in a fermentation reaction to produce organic acids, preferably predominately lactic acid, to lower the pH of the macroalgae to ensile it.

The method according to claim 3 when dependent on claim 2 in which the inoculant comprises an epiphytic population of LAB present on said angiosperm plants that is sufficiently rich in suitable species of bacterium to ensure fermentation occurs during the ensiling process.

The method according to any one of the preceding claims in which the inoculant that may optionally be used in step b) or is used in step c) comprises one or more enzymes capable of releasing soluble sugars from complex carbohydrates.

6. The method according to claim 1 or claim 2 in which the additive that is used in step c) comprises an acid or an acid salt for effecting a substantially immediate pH reduction in the harvested material.

7. The method according to any one of the preceding claims comprises packing the harvested macroalgae into a bunker, plastic bag or tower silo, or wrapping large bales of the harvested macroalgae in plastic film to enclose it whereby to create anoxic conditions or permit anoxic conditions to be created.

8. The method according to claim 6 comprises packing the harvested macroalgae into a vacuum pack bag or similar container, extracting air from the bag or similar container and sealing the bag or similar container.

9. An ensiled macroalgae comprising preserved macroalgae in which residual amounts of epiphytic and/or inoculant lactic acid bacteria (LAB) and/or enzyme(s) are present, or preserved macroalgae in which residual amounts of additive acid and/or acid salts are present.

10. Use of ensiled macroalgae according to claim 9 as a feedstock for human or livestock consumption.

11. Use of ensiled macroalgae according to claim 9 as a soil conditioner and/ or fertiliser.

12. Use of ensiled macroalgae according to claim 9 as a feedstock in a bioprocess.

13. A method of bioprocessing organic feedstock material comprising feeding to a bioprocessing unit at least one feedstock comprising ensiled macroalgae according to claim 9.