WO2012074502A1

WO2012074502A1 - Secondary metabolite stimulation in photoautotrophic cultures

Info

Publication number: WO2012074502A1
Application number: PCT/US2010/058165
Authority: WO
Inventors: Daniel David Smolko
Original assignee: Chayil Technologies, Llc
Priority date: 2010-11-29
Filing date: 2010-11-29
Publication date: 2012-06-07

Abstract

Methods and systems are described herein for enhancing the growth, metabolite generation and extraction of an organism, tissues or cells by shifting the culture conditions by the addition of a stimulating agent from simple biomass accumulation to an enhanced condition that allows for the generation and recovery of specific classes of metabolites. This method can be used with plant, algal, diatom or cyanobacterial species and will enhance the generation of specific metabolites such as lipids, oils, fatty acids and other biomolecules.

Description

TITLE

Secondary Metabolite Stimulation in Photoautotrophic Cultures RELATED APPLICATION DATA

The application claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Application No. 61/275,072 filed on Aug. 25, 2009.

FIELD OF THE INVENTION

The present invention concerns methods for enhancing the growth, metabolite generation and metabolite removal in algae, diatom, cyanobacteria, photobacteria and plant tissues and cultures.

BACKGROUND

Plants, algae and other photosynthetic organisms have been used not only as our food supply and in our food supply chain but also as the source of extensive chemical substances including, pharmaceuticals, fragrances, oils, colors, dyes and agrochemicals etc. Algal, diatom, cyanobacterial, photobacterial and plant derived biomass and compounds can be classified as primary and secondary metabolites. Interest has risen for the production of lipids, oils and fatty acids classified as primary metabolites.

Biologically active compounds that are produced from plants are mostly secondary metabolites. There is a greater interest in secondary metabolites such as alkaloids, antiallergenics, quinones, antileukaemic agents, antimicrobials, antineoplastics, antivirals, flavonoids, insecticides, lignans, opiates, perfumes, pigments, sweeteners and

polysaccarides etc., because most of them exhibit physiological activity. There are more than 100,000 known plant secondary metabolites and around 25% of the medicines that are used in the U.S Pharmacopoeia contain plant-derived substances and every year more novel secondary metabolites are discovered.

Large scale cultures of both primary and secondary metabolites through in vitro techniques may have advantages of controlling external environmental conditions and producing these compounds on a scale necessary to generate the metabolites. In addition growth of organisms such as algal species require simple basic nutrients in a medium, such as nitrates, phosphates and a carbon source. Unfortunately in many instances the yields of these cultures by using ordinary culture techniques are too low to make these processes economically viable. Methods for improving the production could possibly be through gene expression specifically involved in regulation of metabolite production through genetic alterations.

Production of biologically active substances through plant cell culture could have distinct advantages. Cell, tissue and microbial culture are considered as an optimal method for continual production of biomass which are not influenced by environment and solve pending problems like the destruction of plant and organism habitats. Because of the slow growth rate and low productivity of plant and algal culture for the secondary metabolite production. To solve this problem optimization of the media, culture conditions and processes are required for higher productivity.

Production of secondary metabolites through large scale plant cell culture is

commercially possible only when there is a stable maintenance of rapid cell growth and high metabolite production during long term culture. The ability of the cell lines that could produce distinct metabolites are not stable which cause the cell lines to lose their initial productivity through subcultures. The success or failure of processes to enhance metabolite production are depended on the development of new methods and systems to overcome these problems.

SUMMARY OF THE INVENTION

Primary, secondary or novel metabolites are chemicals produced by algal, diatom, cyanobacterial, photobacterial or plant metabolism. Metabolites are molecules which can be extracted, released or separated from the biomass in the form of cellulose,

hemicellulose, pectin, lipopolysaccharides, protein, chlorophyll, fatty acids, lipids, oils, saccharides, glycerides, polyglycerides, terpenoids, quinones, lignans, flavonoids, alkaloids, polyions, chelators and other biomolecules.

Algal, cyanobacterial, photobacterial, diatom or plant culture based production systems offer an additional benefit in that they may be able to perform very specific

biotransformations in vivo. In addition to biodegradation, new compounds may be biosynthesized through the introduction of new or substituted precursors into the secondary metabolic pathways of the cells. By using plant, algal, diatom, photobacterial and cyanobacterial cultures quantities of these compounds will be obtained. In addition novel compounds not yet identified in algal, diatom, cyanobacterial or plant species may be induced.

Tremendous interest has risen for the production of lipids, oils and fatty acids. Growth of organisms such as algal species require basic nutrients in a medium such as nitrates, phosphates, a carbon source in the form of carbon dioxide, organic or inorganic carbon. There is a relationship between growth cycle and lipid generation for algae. Algal oil production appears to be enhanced during late exponential phase. Similar growth cycle and lipid generation relationships hold for plant, diatom, photobacterial and

cyanobacterial cultures. For instance the photoautotrophic microalga can be cultured in nitrate, nitrite or urea media and show a lipid content relationship based on growth cycle and illumination history. Gross biochemical composition of the lipid classes and fatty acids can be affected more by the growth phase than by the nitrogen source, carbon source or nutrient source. It does not matter what carbon, nitrogen, phosphorus or growth medium source is used as much as the culture growth phase and the addition of a stimulus to the culture to enhance the production of specific metabolites. Hence this invention comprises a metabolite generation process using algae, diatoms, cyanobacteria, photobacteria or plant biomass comprising: biomass generation followed by metabolite generation; wherein the addition of one, or more, stimulating agent(s) provide(s) for de novo or enhanced production of primary and/or secondary metabolites during the generation phase.

Agricultural wastewaters have very high BOD (biological oxygen demand), which is costly to treat aerobically. Anaerobic digestion is a very effective process for BOD removal, but is not an effective way to remove nutrients. Often further treatment of the effluent from anaerobic digesters is required before it can be discharged into the environment. Hence algae, diatoms, cyanobacteria, photobacteria, plants or a

combination of these organisms, including algae, may be used to convert waste to useful metabolites.

A chamber, reservoir, reactor, bioreactor, photbioreactor, tank or culture can be defined as but is not limited to a pond, covered pond, reactor, tank, flask, dish, tube, plastic bag, lake, ocean or other container. The term "medium" or "culture medium" refers to an aqueous liquid solution or suspension in the context of an algal, diatom, cyanobacterial or plant, plant tissue or plant cell growth medium. Medium also means water, pond water, lake water, steam water, river water, ocean water, saltwater and a nutrient containing aqueous liquid.

Different organisms can be used for primary and secondary metabolite production, including, for example, plants (plantae), algae, diatoms, photobacteria and cyanobacteria. Examples include but are not limited to the following organisms: Algae of the groups Archaeplastida, Rhizaria, Chromista, Cryptophyta, Dinoflagellates and Haptophyta (including Chlorophyceae and Charophyceae); Diatom algaes of the class

Bacillariophyceae (or Bacilliarophy); the heterokont, haptophyte, dinoflagellate, and euglenid phyla. The heterokonts which include both autotrophs (e.g. golden algae, kelp, diatoms) and heterotrophs (e.g. water moulds). Cyanophyta and Cyanobacteria are also known as blue-green algae and blue-green bacteria. Included are the classic orders of Gloeobacterales, Nostocales, Stigonematales, Pleurocapsales, Chroococcales,

Oscillatoriales and taxa related to or not yet validly published in the phylum

Cyanobacteria. Examples of the Cyanobacteria genera include but are not limited to Prochloron, Prochlorococcus, Prochlorothrix, Glaucophytes, Halospirulina and

Planktothricoides; Photobacteria including but not limited to the groups Purple Bacteria, Green Sulfur Bacteria, Heliobacteria including all Photoautotrophs and

Photoheterotrophs; Plants of the orders Embryphyta, Viridiphyta and Plastida; Algal examples include Neochloris oleoabundans a microalga belonging in the class Chlorophyceae; Scenedesmus dimorphus a unicellular algae in the class Chlorophyceae; Charophyceae Euglena gracilis; Phaeodactylum tricornutum a diatom; Pleurochrysis carterae a unicellular coccolithophorid alga of the class Haptophyta (Prymnesiophyceae; Prymnesium parvum; Tetraselmis chui a marine unicellular alga; Tetraselmis suecica; Isochrysis galbana a microalga; Nannochloropsis salina or other Nannochloropsis sp.; Botryococcus braunii or other Botryococcus sp. a green alga; Dunaliella tertiolecta; Spirulina species; strains of Chlorophyceae (green algae) such as Chlorella vulgaris; Rhodophyceae (red algae); Bacilliarophy (diatom algae).

Organisms herein used for the generation of biomass and metabolites include all organisms capable of photosynthetic growth, such as algae, diatoms, photosynthetic plants and cells and photosynthetic microbes in unicellular or multicellular form that are capable of growth in a medium or liquid phase. These terms may also include organisms modified by natural selection, selective breeding, directed evolution, synthetic assembly, or genetic manipulation.

It is understood to one skilled in the art that plants, algae, diatoms, photobacteria and cyanobacteria can be used interchangeably in most aspects of the present invention.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 hghgghghghghghgh

FIG. 2

DETAILED DESCRIPTION

Production Yield Issues

Yield issues associated with primary and secondary metabolites from plant, algal or cyanobacterial will be based on finding conditions for and promoting the growth environments, enhancing production and metabolite removal. Yields are improved through enhancement in various stages of growth and metabolite production. Methods include the addition of C02, growth regulators, exposure to light, extraction and/or removal of the final product. Metabolite synthesis is accompanied by transport and various degradation mechanisms.

High levels of metabolites interfere with the growth and viability of cultured cells. One type of selection pressure often ignored during cell line establishment is the autotoxicity that results when a metabolite is accumulated at high levels. If the product is accumulated within the cell or intracellularly those cells that acquire the highest content are debilitated. If the product is excreted those cells with the ability to sequester the product in their vacuoles or degrade it metabolically are selected. In either of these scenarios the productivities of newly established cultures can diminish with time. Problems with the accumulation of high product level arise from regulatory constraints within the cells such as feedback inhibition. The ability of the cell lines that could produce distinct metabolites become diminished and can cause the cell lines to lose their productivity through subcultures, contamination, culture conditions, genetic pressures or physical (epigenetic) changes. These problems can be reduced by employing the techniques described herein.

Bioreactor configurations conducive to low shear can be advantageous to preserve culture integrity, enhance biomass production and increase metabolite yields. A combination of strategies give sufficient yield improvements making the establishment of commercial algal, cyanobacterial or plant production processes economically feasible. It can be envisioned that multistage production systems can be used to separate or coordinate the nutritional requirements for cell growth and those for metabolite production. Production strategies would include stages for growth, enhanced metabolite yield and metabolite extraction. Operational configurations would include batch, semi-batch, fed-batch, perfusion, recycle or continuous processes. Reactor configurations could be in the form of pond, flat bed, flat plate, cylindrical, tubular or vertical reactors. In addition reactors, tanks, vessels or production systems can be switched out for other reactors, tanks, vessels or production systems in the system if they become contaminated with the wrong organisms or cell lines. These metabolites would include but are not limited to cellulose, hemicellulose, pectin, protein, chlorophyll, fatty acids, lipids, oils, saccharides, glycerides, triglycerides, polyglycerides, terpenoids, quinones, lignans, flavonoids, alkaloids, polychelating agents and other carbon based biomolecules.

Growth Phases

The nutritional environment of algae, diatoms, photobacteria and cyanobacteria has a significant bearing on their productivity in culture. Depending on the metabolite medium and culture conditions that favor growth do not always enhance metabolite biosynthesis. If low cell biomass or metabolite yields are obtained by using a single culture medium, then two different culture media are used sequentially. The first medium is optimized for growth, while the second is optimized for the biosynthesis of specific metabolites.

In other words if low cell or product yields are obtained by using a single culture medium then two different culture media can be used. The first medium is optimized for growth while the second is optimized for product biosynthesis. For example, a cell may produce more complex sugar molecules, carbohydrates or metabolites when switched to alternate conditions which may be useful in commercial products. As an example in some species protein is higher during exponential growth but the patterns change as specific nutrients are consumed and the cultures age, protein levels decrease, and lipids are one of the main constituents in algae with appropriate N (nitrogen) sources added in the late stationary phase.

Fatty acid content in algal cultures is affected by the growth phase. Maximum polyunsaturated fatty acid values are observed at the early stationary phase and were found to decrease throughout the stationary phase. The highest fatty acid contents in the early stationary phase were observed in cultures with added urea; these cultures also had higher fatty acid content, with eicosapentaenoic acid and docosahexaenoic acid reaching levels of over 20% of total fatty acids, respectively. Our data indicate that algal lipid and fatty acid composition can be enhanced during or after the stages of biomass growth in a variety of reactor systems particularly by the addition of stimulating agents and particularly in perfusion, continuous and fed-batch reactor cultivation schemes. Our data indicate that algal lipid and fatty acid compositions can be enhanced during or after the stages of biomass growth in batch and fed-batch reactor cultivation schemes particularly in late -exponential cultures, stationary phase cultures and in continuous cultivation schemes.

Stimulating Agents of Metabolite Biosynthesis

Light & Modes of Growth

Light, or a lack thereof, stimulates the accumulation of metabolites of in phototrophic, heterotrophic and mixotropic conditions. The generation of metabolites herein pertains to cultures of plant, algae, diatoms, bacteria or cyanobacteria that can be grown under at least one or more of autotrophic, heterotrophic, or mixotrophic conditions. Species of organisms capable of being grown under autotrophic, heterotrophic, or mixotrophic conditions include green, red, brown and diatom algaes; cyanobacteria; and plant species.

In heterotrophic cultures from several different plant species, light is known to trigger anthocyanin and flavonoid synthesis. Flavonoids serve as natural U.V. protectants and the anthocyanins serve as natural pigments. However, neither flavonoids nor anthocyanins are produced without light irradiation. The spectral quality of the light is important as well, blue light stimulates anthocyanin synthesis while red light is ineffective. Hence conditions can be changed to induce plant and algal, diatom and cyanobacterial cultures to produce specific metabolites.

The invention demonstrates that organisms or tissues of organisms such as plants, algae, diatoms, photobacteria or cyanobacteria can be grown in at least two or more of autotrophic, heterotrophic, or mixotrophic conditions. Alternately organisms such as plants, algae, diatoms, photobacteria or cyanobacteria can be grown in at least two or more of autotrophic, heterotrophic, or mixotrophic conditions. Organisms such as plants, algae, diatoms, photobacteria or cyanobacteria can be grown in at least one or more of autotrophic, heterotrophic, or mixotrophic conditions in the absence or presence of a stimulus. The growth conditions for these organisms can enhance the production of metabolites such as lipids, oils, fatty acids and biomolecules. The organisms that can be used can be naturally occurring, naturally selected, cultured or modified. If modified the organisms can be hybridized, selectively bred, differentially selected, enhanced by directed evolution or genetically modified. "Modified" organisms are defined as those that are hybridized, selectively bred, differentially selected, enhanced by directed evolution or genetically altered. Algal, cyanobacterial, diatom, photobacteria or plant culture based production systems offer an additional benefit in that they may be able to perform very specific biotransformations in vivo. In one case the organism used in the biotransformation may be selected, cultured, modified or have the natural ability to create, modify or alter a biomolecule, metabolite or substance that is normally utilized by the algal, cyanobacterial, diatom or plant culture. In another example, the organism may be modified or have the natural ability to create, modify or alter a biomolecule, metabolite or substance that is not normally utilized or produced by that organism.

In yet another aspect, the invention involves methods and systems for preselecting, adapting, and conditioning one or more species of photosynthetic organisms to specific environmental and/or operating conditions to which the photosynthetic organisms will be exposed during utilization of a system of the invention. Because the cell walls of plant cells and some species of algae have natural tendency to adhere together, it is not possible to obtain a suspension which consists only of dispersed single cells. The proportion and the size of cell aggregates vary according to the plant and algal species and variety, and the medium in which the culture is grown. Cell aggregation leads to a difference in local environment between interior and exterior of the cell aggregrates, which can result in heterogeneity and differences in growth, gene expression, metabolism and susceptibility to the induction of metabolite biosynthesis. Reactors can be optimized to provide for low shear and growth conditions which maintain cell adhesion, aggregration and

susceptibility to metabolite biosynthesis.

This invention also includes preselecting, adapting and preconditioning the organisms to a stimulus, or a lack of stimulus, to which the photosynthetic organisms will be exposed during utilization of a system of the invention. In addition, this includes adapting the organisms to a light or illumination source, or a lack of light or illumination source, to which the photosynthetic organisms will subsequently be exposed during utilization of a system of the invention. The organisms include plant, algae, diatom, photobacteria or cyanobacteria species.

When the algal, cyanobacterial, photobacterial, diatom or plant is grown under autotrophic conditions, the generated cellular energy is derived from inorganic carbon or inorganic compounds 2, making the process very energy and cost efficient. Depending on selection pressures and whether the organism is naturally occurring, selected, cultured or modified, the resulting lipid content of the cells may be higher in total percentage and higher in specific desired lipid forms.

A photosynthetic organism 1,10,15,27 may be grown and maintained in an enhanced autotrophic environment by the addition of excess C02 2,11,16,28. The C02 2,11,16,28 is added from an industrial, combustion, synthetic or natural source. C02 2,11,16,28 transfer means to the culture medium include sparged, forced, sprayed, dissolved, liquid to gas or air contact or liquid on solid surface to gas or air contact method.

A heterotrophic medium 7,23,35 can increase total percentage of lipid produced and alter the ratio of lipids to favor those of a desired form(s). A heterotrophic medium can require an input of sugar 5,14,20,32, adding to the cost of production of these lipid products. As an alternative to the sugar in a heterotrophic medium other carbon sources, which include organic and inorganic waste 5,14,20,32, may be used. In a method of the invention, the above two production conditions can be combined in series, with biomass growth first in autotrophic conditions to optimize input efficiency, and then shifted to heterotrophic conditions 7,23,35 prior to lipid extraction 9,26,38 to optimize total lipid yields and desired lipid contents 9,26,38.

In an embodiment, a photosynthetic organism can be grown under autotrophic conditions utilizing a light source 3,12,17,29 to illuminate the growing system. After a certain period of time, an organic carbon source is added, thus starting heterotrophic growth and the lipid maturation phase. In an embodiment, when an organic carbon source is added to a reactor, light energy is provided to the organism, which creates mixotrophic growth conditions. Illumination energy can be minimized or eliminated 5,14,20,32 from the system, creating heterotrophic growth conditions 7,23,35.

In another embodiment, photosynthetic organisms are grown in a plurality of ponds, chambers, or reactor under autotrophic conditions 3,12,17,29 and after a certain time, the organisms are then transferred to a second bioreactor that provides heterotrophic 7 or mixotrophic 6 growth conditions. Algae are grown in a plurality of modular PBRs under autotrophic conditions. Autotrophically grown algae can be transferred to a single larger chamber that provides heterotrophic growth conditions for the organisms. The transfer of the algae can be performed in series, semi-continuous, or continuous mode to the lipid maturation chamber 6,7,21,22,23,33,34,35.

In some embodiments a control system 39 and methodology are utilized in the operation of a system, which is configured to enable automatic, real-time optimization and adjustment of operating and growth parameters to achieve a shift from autotrophic 40 to heterotrophic (or mixotrophic) growth 41 conditions.

After another period of time, the algae can be harvested 42 from the lipid maturation chamber and the lipids can be collected and utilized for various processes including the production of biodiesel or other commercially useful products. A plurality of autotrophic chambers, such as ponds or photobioreactors, can be arranged to form a system for the growth and production of a photosynthetic biomass. As would be apparent to those skilled in the art, in some embodiments, a photobioreactor system can comprise one of a plurality of identical or similar photobioreactors interconnected in parallel, in series, or in a combination of parallel and series configurations, FIGS. A,B,C,D,E&F. For example, this could increase the capacity of the system (e.g., for a parallel configuration of multiple photobioreactors). The plurality of autotrophic chambers can also be coupled to a plurality of lipid maturation chambers FIGS. A,B,C&D. or a single lipid maturation chamber (FIG. E&F) that provide heterotrophic or mixotrophic growth conditions for improving the lipid content and/or characteristics of the biomass. In an embodiment, instead of transferring the biomass to a second bioreactor, an organic carbon source E is added to the plurality of reactors (FIGS. A,C&G) to create mixotrophic growth conditions 6. The PBRs can also be covered and provided with no light energy to create heterotrophic growth conditions for the photosynthetic biomass. All such configurations and arrangements of the inventive photobioreactor apparatus provided herein are within the scope of the invention.

Shifts between autotrophic, mixotrophic and hetrotrophic growth conditions may be desired. In this case one stage of the system is optimized for cell growth and a second stage is optimized for product biosynthesis, FIGS. A,C,D&F. In this configuration it would be advantageous for the second stage to be optimized for product recovery as well. If not sufficient a subsequent stage can be used for product recovery, FIGS. B&E. These stages can be interconnected in parallel, in series, or in a combination of parallel and series configurations, FIGS. A,C,D,E&F.

Each unit of a system of the invention can operate independently, FIGS.

A,B,C,D,E,F&G. The units can be modular and they can be easily swapped if desired. For example, if one unit becomes contaminated with another species of algae or other organism, it can be swapped for a different unit. The system of the invention can be intended to be modular and self-contained, harvest processes, medium recycling, water storage, power generation, and other processes may be grouped and distributed to individual units. Independent units can be connected in a network so that dispersal of medium and collection of biomass products can be coordinated.

In addition, operational configurations for each individual stage could include one of batch (FIGS. A3,C,D,E&F), semi-batch (FIGS. A,B,C,D,E&F), fed-batch (FIGS. A,B,C,D,E&F), perfusion (FIG. G), recycle (FIG. G) or continuous processes (FIG. H). Each stage could operate together, independently and in different modes.

It is understood as part of this invention, FIGS. 3&4, that instead of using a light or illumination source to induce the above changes in lipid generation and lipid composition a stimulus, or lack of a stimulus, may be used in the place of or in conjunction with a light or illumination source. To state more explicitly, in the place of a change in light or illumination source a stimulus, a lack of stimulus, FIGS. 3&4, is used to induce changes in lipid generation and lipid composition.

Stimulus then lack (or reduction) of stimulus (33,34,35)

No stimulus then stimulus (33,34,35)

This invention allows the use of a combination of stimulating agents in algal, diatom, cyanobacterial, photobacterial and plant cultures. To state more explicitly, in

combination with a light or illumination source a stimulus, or lack of stimulus, is used in combination with a light or illumination source to induce changes in lipid generation and lipid composition.

Light then light plus stimulus

Light plus stimulus then light (with no or reduced stimulus)

It is understood that as part of this invention that in the place of lipids other metabolites levels may be controlled by the use of a single or a combination of stimulating agents. To state more explicitly, in the place of, or in conjunction with a light or illumination source, a stimulus, or lack of stimulus, is used to induce changes in metabolite generation and metabolite composition. As an additional example, in combination with a light or illumination source a stimulus, or lack of stimulus, is used in combination with a light or illumination source to induce changes in metabolite generation and metabolite composition. As a further example, in the presence of, or in the absence of, light or illumination source, one or more stimulating agents is used to induce changes in metabolite generation and metabolite composition.

Based on our observations gross biochemical changes in the composition of the lipid classes, fatty acids, oils and metabolites are affected more by the growth phase than by the nitrogen source, carbon source or nutrient source. Hence, it does not matter what carbon, nitrogen, phosphorus or growth medium source is used as much as the growth phase, the presence or absence of a light or illumination source and the addition of a stimulus to the culture.

To state more explicitly, the addition of a stimulus causes the lipid composition to be altered and the lipid generation levels are changed by the application of a stimulus to a photoautotrophic, mixotrophic or heterotrophic culture. In addition to changing lipid composition, metabolite generation could be controlled in the same way. By example, the lipid, fatty acid and oil are generated in the presence or lack of a stimulus and then sent to a polishing reservoir to as a polishing step.

Bioconversion

The accumulation of secondary metabolites is part of a dynamic system which maintains the cell's internal chemical equilibrium. Product synthesis is always accompanied by product transport and various degradation mechanisms. There are four main routes for product turnover these include the interconversion of a transiently accumulating product into a more distantly related compound, the conjugation of the product with other compounds (i.e. sugars, amino acids, acyl groups) in the vacuole or the cytoplasm, the oxidative polymerization of the product into cell membranes or the cell wall, and the total degradation of the compound by hydrolysis, oxidation, reduction and normal enzymatic processes.

A bioconversion process can be used to produce or remove a variety of compounds & metabolites. One or more metabolites are produced by the addition of, or a lack of, a stimulus, a light or illumination source or a nutrient to the culture. The stimulus can be in the form of a polyolefin, plastic, starch, peptide, ionomer or polymer. The process generates metabolites by precursor feeding to effectively enhance or effect the

biodegradation, conversion or production of a compound or metabolite.

The process can consist of the biodegradation, bioconversion or production of a compound or metabolite wherein the stimulus consists of a polyolefin, plastic, starch, peptide, ionomer or polymer product. In a further illustration, the bioconversion process for a compound or metabolite wherein exposure to U.V. or visible light of an aqueous solution containing the dissolved or partially dissolved compound aids in the photolysis of the compound in order to aid in the biodegradation or conversion of the compound. In greater scope a bioconversion process for a compound, metabolite or stimulus, wherein exposure to a light or illumination source, such as artificial, fluorescence, incandescent, solar, full spectrum, UV, red, blue, far-red; or a combination thereof, of an aqueous solution containing the dissolved or partially dissolved compound, metabolite or stimulus aids in the photolysis of the compound in order to aid in the biodegradation or conversion of the compound.

Growth regulators

Plant growth regulators can be divided functionally into two main groups, the growth promoters and the growth inhibitors. Auxins, cytokinins and giberellins tend to be plant growth promoters. Abscisic acid and ethylene are plant growth inhibitors. Growth regulator type and growth regulator level influence their relative effect. The absolute auxin level, auxin type and auxin ratio to other growth promoters in culture determines whether an auxin acts as a developmental growth promoter or as a secondary metabolism inducer. Most culture media contain only auxins and cytokinins. Specific bioregulators, such as the aminoethylphenylethers, which effect carotenoid and isoprenoid biosynthesis in plants have come into use as secondary product inducers.

Other growth regulators appear to stimulate production through the generation of ethylene. Ethylene generation follows spermidine addition. Gibberellins can be used to reduce starch formation, increase organogenesis and specific metabolite yields.

Stimulating and Stress-Inducing Agents

Stress is normally defined as the condition which has a negative effect on the increase of the dry matter of a plant. When dealing with plant tissue culture stress can be defined as the application of an external constraint which influences the primary and secondary metabolism of the cultivated plant tissues. Thus, a positive effect of stress is an increase in yield of particular secondary metabolites even though the growth of the cells may be compromised. A variety of chemical and biochemical stress-inducing agents can be used to increase the biosynthesis of important secondary products and to shorten fermentation times. We will use the terms "stress", "stress-inducing", "stimulus" and "stimulating agents" interchangeably to ease the illustration of the principal in this text.

Following exposure to stimulating agents metabolite production in plant tissue cultures can occur by two means. Production can occur anew (de novo) or production can be enhanced after treatment. In both of these cases the stimulus is called a "stimulus- inducing" agent, and the secondary metabolite can be called a stress or "stimulus- induced" metabolite. In some case the metabolite is undetectable in one cell line but is present at an easily detectable level in another. The stimulus induced metabolite. Stimulus inducing agents can be classified as biotic if they are obtained from other organisms or as abiotic if they are inorganic or produced by other means. A few examples of biotic and abiotic agents include:

Biotic antibiotics - actinomycin-D, nigeran, nystatin; glycoproteins - microbial culture filtrates, microbial culture homogenates; yeast preparations; hydrolytic enzymes - amylases, cellulases, hemicellulases, pectinases, pectate lyases, proteases, lipases; lipids - arachidonic acid, eicosapentaenoic acid, lysolecithin; glucans - chitosan, glutathione, microbial homogenates, oligosaccharins, oligosaccharides; proteins - cytochrome C, peptides, polylysine, protamines; or a combination thereof

Abiotic detergents - triton, tween, nonionic, lysolecithin, phosphate based; chelators - EDTA, EGTA; heavy metal ions - copper sulfate, mercuric chloride, vanadyl sulfate, organic solvents - acetone, DMSO, ether, chloroform; thermal shock & stress (between 0°C and 50°C) osmotic stress & shock by varying concentrations from 0 to 15% w/v mannitol (preferably 6 to 10 % w/v), 0 to 10% w/v sucrose (preferably 6 to 10%), saccharides, polysacharrides, 0.01% to 36% w/v NaCl (preferably between 0% to 10% w/v); and 0.01% to 37% w/v KC1 (preferably between 0% to 10% w/v); pesticides - carbamate, organophosphorus; light - artificial, fluorescence, incandescent, solar, full spectrum, UV, Red, Blue, Far-red; or a combination thereof

Heavy metal stimulus inducing agents can be used to enhance oil biomass and secondary product accumulation in microalgae, macroalgae and cyanobacteria. Stimulus inducing agents that are metal based include, but are not limited to an ionic solution, salt, hydrate, hydride, hydroxide, oxide, nitrate, chlorate, sulfate, phosphate, fluoride, chloride, bromide, iodide, molybdate, sulfide, selenide, carbide, chelate or organo-metallic compound of zinc, vanadium, copper, manganese, titanium, cobalt, iron, calcium, magnesium, selenium, molybdenum, zirconium, bismuth, barium or other metals or a combination thereof. For example, a stimulating agent consisting of a metal or compound thereof would be in the concentration range of 0 to 100 millimolar. Perferably the stimulating agent would be in a concentration range of 0.010 to 0.100 millimolar.

For example stimulating agents based on zinc are in the form of and include but are not limited to ZnO, ZnS, ZnSe, ZnO?, ZnFb, ZnC₂, ZnF? ,ZnCR ZnBr?, Zn , Zn²⁺ ions, the hydroxide Ζη(ΟΗ In stronger alkaline solutions, this hydroxide is dissolved to form zincates ([Zn(OH)₄]^2"). The nitrate Zn(NCh)₂, chlorate Zn(ClChK sulfate Z11SO4, phosphate ZmfPO^ and molybdate ZnMoQ₄ are also applicable forms of inorganic compounds of zinc. One of the simplest examples of an organic compound of zinc is the acetate

Other forms of zinc will work as well. Other metals in addition to zinc will work as well. Other metals with low toxicity may work as well.

In addition a variety of viability stabilizers may be used to prolong high secondary biosynthetic enzyme activities. These stabilizers include calcium, polyamines, osmolytes and other nutritional factors. Stimulating Product Secretion:

Detriments to Optimal Growth and Product Formation

One type of selection pressure often ignored during cell line establishment is the autotoxicity that results when a secondary product is accumulated at high levels by the cultured cell lines. High levels of metabolites often interfere with the growth and viability of cell cultures. If the product is accumulated intracellularly those cells that acquire the highest content are debilitated. If the product is excreted those cells with the ability to sequester the product in their vacuoles or degrade it metabolically are selected. In either of these two scenarios the productivities of newly established cultures diminish with time. Another problem with the accumulation of high product levels arises from regulatory constraints within the cells such as feedback inhibition.

The accumulation of secondary products is only part of a dynamic system which maintains the cell's internal chemical equilibrium. Product synthesis is always accompanied by product transport and various degradation mechanisms. There are four main routes for product turnover. These include the interconversion of a transiently accumulating product into a more distantly related compound, the conjugation of the product with other compounds (i.e. sugars, amino acids, acyl groups) in the vacuole or the cytoplasm, the oxidative polymerization of the product into cell membranes or the cell wall, and the total degradation of the compound by hydrolysis, oxidation, reduction and normal enzymatic processes.

Degradation could be due to heat-shock observed in plants and cell suspension cultures which leads to the activation of peroxidases and thus to non-specific degradation of intermediates.

Enhancing Bio-Product Formation along with Optimal Growth

The nutritional environment of plant, algal, diatom and cyanobacterial tissues will have a significant bearing on productivity in cell culture. There is a relationship between culture medium composition and culture growth and productivity. In most cases medium conditions that favor cell growth do not favor secondary product biosynthesis. Therefore, if low cell or product yields are obtained by using a single culture medium then two or more different culture media can be used sequentially. The first medium is optimized for cell growth while the second is optimized for product biosynthesis. Addition of stimulating agents to cells may lead to increased excretion of some secondary products together with the stimulation of biosynthesis.

Because the cell walls of plant cells and some species of algae have natural tendency to adhere together, it is not possible to obtain a suspension which consists only of dispersed single cells. The proportion and the size of cell aggregates vary according to the plant and algal species and variety, and the medium in which the culture is grown. Cell aggregation leads to a difference in local environment between interior and exterior of the cell aggregates, which can result in heterogeneity and differences in growth, gene expression, metabolism and susceptibility to the induction of metabolite biosynthesis. Reactors can be optimized to provide for low shear and growth conditions which maintain cell adhesion, aggregation and susceptibility to metabolite biosynthesis.

A culture operation is termed batch when all the required nutrients are supplied initially. The only exceptions are for carbon dioxide, oxygen and light. Batch culture

configurations are commonly used for experimental studies because of their simplicity. The production of growth- as well as nongrowth-associated products can be achieved in batch bioreactors by the optimization of different nutrient levels. In batch operating mode at least one component in the medium becomes growth-limiting during cultivation. As this component is metabolized by the cells there is a cessation of rapid growth. As a rule, nongrowth-associated product biosynthesis is dependent on the depletion of the correct nutrient. For microbial systems the growth limiting nutrient is usually the carbon, nitrogen or phosphorus source. In plant, algal, diatom and cyanobacterial species the main limiting factor may also be a growth regulator.

A two-stage culture process is appropriate, if a single bioreactor stage physically or chemically limits the productivity of a nongrowth-associated product. Even chemostats are appropriate for nongrowth-associated products when they are operated in tandem, because the conditions in the first bioreactor can be optimized for biomass production while those in the second can be optimized for secondary product synthesis. A two stage system can also be applied to batch reactors where nutritional conditions for growth and production are sequentially separated. A batch process using such a system with microalgae would take 12-48 hours.

Even though there are some potential problems with two-stage operating systems, these configurations are often better suited for large-scale plant, algal, diatom and

cyanobacterial cell culture. Large-scale production must guard against spontaneous variations in genomes of plant, algal, diatom and cyanobacteria cells. Length of time in culture, exposure to high phytohormones concentrations or sudden changes in ploidy can render semi-continuous or continuous culture systems non-productive. However, in immobilized or two-stage batch production systems this problem is not so critical since fresh cell lines are introduced into these reactors during each operating cycle.

One type of selection pressure often ignored during cell line establishment is the autotoxicity that results when a secondary metabolite is accumulated at high levels by the cultured cell lines. High levels of products often interfere with the growth and viability of cell cultures. In large quantities lipophilic compounds can damage plant cell membranes. In cultured cells, feedback inhibition due to lipophilic toxicity seems to hinder the release and biosynthesis of terpenes. If the product is accumulated intracellularly those cells that acquire the highest content are debilitated. If the product is excreted those cells with the ability to sequester the product in their vacuoles or degrade it metabolically are selected. In either of these two scenarios the productivities of newly established cultures diminish with time. Another problem with the accumulation of high product levels arises from regulatory constraints within the cells such as feedback inhibition. In order to remove accumulated metabolites permeabilizing agents could be used which include: Antibiotics - nystatin; calcium chelators - EDTA, EGTA; detergents - lysolecithin, Triton-XlOO; electroporation; freeze-thaw cycles; organic solvents - toluene, ether and DMSO; osmotic shock; polycations - polylysine, chitosan

In plant, algal and cyanobacterial cultures product traps could be used and would include a variety of solid and liquid phase adsorbents and extractants. These solid and liquid phase adsorbents would be insoluble in the liquid culture medium and provide reasonably high product partition coefficients (K>1). Examples of solid adsorbents would include activated carbon, ionic resins, reverse phase resins, celluloses, polysaccharides, polymers, zeolites, other solids or a combination thereof. Liquid phase absorbents would include alcohols, silicon oil, mineral oil, toluene, organic oils, organic solvents, polymers, ion exchange resins, various other organic phases or a combination thereof. In addition it may be possible to optimize product removal by integrating bioreactor or culture operation with a two-phase counter-current membrane extraction system. As an example, the two phase extraction system is used with or without the membrane, with a different separator (partition) other than the membrane and in counter-current, cocurrent or mixed mode. In such a system it would be envisioned that an extractant phase with a high partition coefficient would be separated from the culture by a membrane, sieve, mesh, surface contact, liquid/gas contact, liquid/liquid contact or liquid/solid contact. This system would remove feedback inhibition in the cultured cells, enhance metabolite production and promote product excretion. A reverse phase resin can consist of a C8, CI 2, C18 resin, a resin of chain length C(X), where C(X) varies between between C2 to C500. In addition the reverse phase resin can be a solid or liquid resin and be used as an oil, lipid, wax, fatty acid or fatty acid ester trap. This would allow for the preferential extraction of oil, lipid, wax, fatty acid or fatty acid esters.

This product trapping mechanism would prevent autotoxicity, remove feedback inhibition and prevent product degradation. It can be envisioned that select lipophilic or

hydrophilic metabolites and compounds generated in a two-phase culture system would not only preserve the excreted products, but in some cases it may stimulate the synthesis of individual compounds or a specific biosynthetic pathway. In other cases the excreted product is safely removed from the culture medium before it is degraded.

Plant, algal and cyanobacterial biomass can be used in an alternate fashion as

biocatalysts. Metal biocatalysts or chelates can be manufactured through the use of a metal-biopolymer complex consisting of the combination of metal ions or complexes with plant fibers, cellulose, hemicellulose, chitin, chitosan, polysaccharides,

disaccharides, monosaccharides, pectin, protein, peptides, amino acids, other

biopolymers, synthetic polymers or combinations thereof.

Biocatalysts could be formed through the stimulated production of specific enzymes or enzymes enhanced by the addition of select metal ions. These biocatalysts could be formed directly in culture. The hydroxyl, amide, amine and/or carboxyl in the polymer or monomer can form a complex with the metal salts and then combined with carboxyl and amino groups to produce a catalyst, condensation solution, an oxidizing solution, an oxidizing condensation solution, degradation solution or other reacting solutions. The resulting solutions of metal-polymer chelate can then be used for various applications such as catalysis, ion-exchange, analyte detection, ionic polymer solutions, disinfectants, aides in biochemical reactions during fermentation, interaction moieties, matrices and aides in protein or metabolite purification, for the activation of enzymes, cells and metabolites, as metabolite sequestration and preservation systems for cells, bacteria, organisms, protein, enzyme, culture medium, medical treatments, oil products, plants and metal enzyme catalysis. Special functional groups can be added and developed into metal polymer chelates to provide high-efficiency, high-density, low-activation for catalytic reactions. Gas, liquid or solid phase reactions can be catalyzed without high temperature or high pressure and occur at room temperature.

The inventions have been disclosed in the context of several embodiments, processes, descriptions and methods. It is understood to those of average and advanced skill in the art in light of the teachings of these inventions that certain changes and modifications may be made thereto without departing from the spirit or scope of the inventions and the claims. It is also understood to those of average and advanced skill in the art in light of the teachings of these inventions that the inventions in terms of processes, descriptions and methods claimed herein extend beyond the examples, embodiments, processes, descriptions and methods herein and we claim all similar embodiments, processes, descriptions and methods.

Claims

We claim:

1. A metabolite generation process using algae, diatoms, cyanobacteria,

photobacteria or plant biomass comprising: biomass generation and metabolite generation; wherein the addition a stimulating agent provides for de novo or enhanced production of primary or secondary metabolites during the generation phase.

2. A metabolite generation process of Claim 1 where a single stimulating agent is used to provide for de novo or enhanced production of primary or secondary metabolites during the generation phase.

3. A metabolite generation process of Claim 1 where two or more stimulating agents are used to provide for de novo or enhanced production of primary and/or secondary metabolites during the generation phase.

4. A metabolite generation process of Claim 1 where in the place of a light or

illumination source, a stimulus, or lack of stimulus, is used to induce changes in lipid generation and lipid composition.

5. A metabolite generation process of Claim 1 where in combination with a light or illumination source, a stimulus, or lack of stimulus, is used in combination with a light or illumination source to enhance changes in lipid generation and lipid composition.

6. A metabolite generation process of Claim 1 where in the place of a light or

illumination source, a stimulus, or lack of stimulus, is used to induce changes in metabolite generation and metabolite composition.

7. A metabolite generation process of Claim 1 wherein combination with a light or illumination source, a stimulus, or lack of stimulus, is used in combination with a light or illumination source to induce changes in metabolite generation and metabolite composition.

8. A metabolite generation process of Claim 1 wherein the presence of, or in the absence of, light or an illumination source, one or more stimulating agents can be used to induce changes in metabolite generation and metabolite composition.

9. The metabolite generation process of Claims 1 to 8 wherein the biomass consists of members of algae, diatom, cyanobacteria, photobacteria or a plant species.

10. The biomass as in Claims 2 and 3 can be, but is not limited to the following

groups Algae of the groups Archaeplastida, Rhizaria, Chromista, Cryptophyta, Dinoflagellates and Haptophyta (including Chlorophyceae and Charophyceae); Diatom algaes of the class Bacillariophyceae (or Bacilliarophy); Organisms of the heterokont, haptophyte, dinoflagellate, and euglenid phyla. The heterokonts which include both autotrophs (e.g. golden algae, kelp, diatoms) and heterotrophs (e.g. water moulds). Cyanophyta and Cyanobacteria which are also known as blue-green algaeand blue-green bacteria. Included are the classic orders of Gloeobacterales, Nostocales, Stigonematales, Pleurocapsales, Chroococcales, Oscillatoriales and taxa related to or not yet validly published in the phylum Cyanobacteria. Examples of the Cyanobacteria genera include but are not limited to Prochloron, Prochlorococcus, Prochlorothrix, Glaucophytes,

Halospirulina and Planktothricoides; Photobacteria including but not limited to the groups Purple Bacteria, Green Sulfur Bacteria, Heliobacteria including all Photoautotrophs and Photoheterotrophs; Plants of the orders Embryphyta, Viridiphyta and Plastida; Neochloris oleoabundans a microalga belonging in the class Chlorophyceae; Scenedesmus dimorphus a unicellular algae in the class Chlorophyceae; Charophyceae Euglena gracilis; Phaeodactylum tricornutum a diatom; Pleurochrysis carterae a unicellular coccolithophorid alga of the class Haptophyta (Prymnesiophyceae; Prymnesium parvum; Tetraselmis chui a marine unicellular alga; Tetraselmis suecica; Isochrysis galbana a microalga;

Nannochloropsis salina or other Nannochloropsis sp.; Botryococcus braunii or other Botryococcus sp. a green alga; Dunaliella tertiolecta; Spirulina species; strains of Chlorophyceae (green algae) such as Chlorella vulgaris; Rhodophyceae (red algae); Bacilliarophy (diatom algae).

1 1. The metabolite of claims 1 to 10 wherein the primary metabolites consists of cellulose, hemicellulose, pectin, protein, terpenoids, quinones, lignans, flavonoids chlorophyll, fatty acids, lipids, oils.

12. The metabolite of claims 1 to 10 wherein the secondary metabolites consist of fatty acids, lipids, oils, saccharides, glycerides, polyglycerides, terpenoids, quinones, lignans, flavonoids, alkaloids, polychelating agents (PCA) and other carbon based biomolecules.

13. The metabolites as in claim 11 whose levels can be enhanced by the addition of a stimulating agent.

14. The metabolites as in claim 12 whose levels can be enhanced by the addition of a stimulating agent.

15. The metabolite production process of claims 1 to 10 wherein the stimulating agent consists of an abiotic stimulating agent.

16. The metabolite production process of claim 1 to 10 wherein the stimulating agent consists of a biotic stimulating agent.

17. The metabolite production process of claims 1 to 10 wherein the stimulating agent consists of ethylene, ethaphon, ethylene generators, auxin, cytokinin, giberrelin, absissic acid, aminoethylphenylethers, norflurazon, 2,4-D, natural growth regulators or synthetic growth regulators.

18. The metabolite production process of claim 15 wherein the abiotic stimulating agent consists of a metal preferably in the form of a water soluble ionic compound or a water soluble organo-metallic compound.

19. A stimulating agent as in claim 15 in the form of, but not limited to, an ionic

solution, salt, hydrate, hydride, hydroxide, oxide, nitrate, chlorate, sulfate, phosphate, fluoride, chloride, bromide, iodide, molybdate, sulfide, selenide, carbide, chelate or organo-metallic compound of zinc, vanadium, copper, manganese, titanium, cobalt, iron, calcium, magnesium, selenium, molybdenum, zirconium, bismuth, barium or other metal or a combination thereof.

20. A metal stimulating agent as in claim 15 wherein the stimulating agent is in the form of ZnO, ZnS, ZnSe, Zn02, ZnH2, ZnC2, ZnF2 ,ZnC12, ZnBr2, ZnI2, Zn2+ ions, zinc hydroxide Zn(OH)2, a zincate ([Zn(OH)4]2-), zinc nitrate Zn(N03)2, zinc chlorate Zn(C103)2, zinc sulfate ZnS04, zinc phosphate Zn3(P04)2, zinc molybdate ZnMo04 or forms of organic zinc such as zinc acetate

(Zn(02CCH3)2).

21. A stimulating agent consisting of a metal or compound thereof as in claims 19 and 20 in the concentration range of 0 to 100 millimolar, and preferably in the 0.010 to 0.100 millimolar range.

22. The metabolite production process of claims 1 to 21 where the addition of the stimulating agent enhances the release of the primary or secondary metabolite.

23. The metabolite production process of claims 1 to 21 where the stimulating agent is added to the cells in exponential growth, late exponential growth or stationary phase to enhance metabolite production.

24. The metabolite production process of claims 1 to 21 wherein the stimulating agent is added to the cells during exponential, late-exponential or stationary phase in a batch production process to enhance metabolite production.

25. The metabolite production process of claims 1 to 21 wherein the stimulating agent is added directly to the cells in a batch, semi-batch, fed-batch, perfusion or continuous production process to enhance metabolite production.

26. A batch, semi-batch, fed-batch, perfusion or continuous process as in claims 1 to 12 wherein the biomass is grown in a reservoir and part of the biomass is removed or flowed into a secondary or subsequent reservoir wherein a stimulating agent as in claims 13 to 25 is added to the cells to enhance metabolite production.

27. The metabolite production process of claims 1 to 10 wherein the addition of a permeabilizing agent enhances the release of the primary or secondary metabolite.

28. The metabolite production process of claims 1 to 10 wherein the extraction of the product can be effected through a membrane, sieve, mesh, surface contact, liquid/gas, liquid/liquid contact or liquid/solid contact by flotation, flocculation, foam fractionation, absorption, adsorption or extraction.

29. An extraction process as in claim 28 wherein a reduction of primary or secondary metabolite concentration is achieved in the growth or production phase.

30. A nonmembrane based extraction process wherein the fluid contacting the

biomass is a permeabilizing agent as in claim 27.

31. Permeabilizing agents as in claims 27 and 30 consisting of alcohols, dimethyl sulfoxide, silicon oil, mineral oil, organic oils, organic solvents, polymers, ionomers, ion exchange resins, or a combination thereof.

32. Plant, algal and cyanobacterial culture metabolite product traps which include a variety of solid and liquid phase adsorbents and extractants.

33. Metabolite product traps as in claim 32, which would be mostly or partially

insoluble in the culture liquid or medium and provide reasonably high product partition coefficients (K>1). Examples of solid adsorbents would include but are not limited to activated carbon, ionic resins, reverse phase resins, celluloses, polysaccharides, polymers, zeolites or a combination thereof. Liquid phase absorbents would include alcohols, silicon oil, mineral oil, toluene, organic oils, organic solvents, polymers, ion exchange resins, various other organic phases or a combination thereof.

34. A reverse phase resin as in Claim 32 and 33 consisting of C8, CI 2, C18 resin, a resin of chain length C(X), where C(X) varies between between C2 to CIOO.

35. A reverse phase resin as in Claims 32, 33 and 34 where the solid or liquid resin can be used as an oil, lipid, wax, fatty acid or fatty acid ester trap.

36. A metabolite product trapping mechanism or removal as in any of claims 32 to 35 which prevents autotoxicity, removes feedback inhibition and prevents product degradation.

37. The metabolite production process wherein precursor feeding is used to

effectively enhance or effect the biodegradation, conversion or production of a compound.

38. The biodegradation, byconversion or production of a metabolite as in claim 37 wherein the compound consists of, but is not limited to, a polyolefin, plastic, starch, peptide, other polymer or waste product.

39. A compound as in claims 37 and 38 wherein exposure to U.V. or visible light of an aqueous solution containing the dissolved or partially dissolved compound aids in the photolysis of the compound in order to aid in the biodegradation or conversion of the compound.

40. A process as in claims 1 to 10, 37, 38 and 39 whereby one or more metabolites are produced by the addition of, or a lack of, a stimulus, a light or illumination source or a nutrient to the culture.

41. A process as in Claims 15, 16, 38, 39 and 40 whereby the stimulus can be in the form of a polyolefin, plastic, starch, peptide or other polymer.

42. The use of plant, algal, diatom, photobacterial and cyanobacterial biomass in metal biocatalysts or chelates can be manufactured through the use of a metal- biopolymer complex or metal ions or compounds with plant fibers, cellulose, hemicellulose, chitin, chitosan, polysaccharides, disaccharides, monosaccharides, pectin, protein, peptides, amino acids, other biopolymers, synthetic polymers or combinations thereof.