Anaerobic fermentation methods and systems

Abstract

The present invention relates to methods and systems for anaerobic fermentation, in particular methods and systems for enhancing anaerobic fermentation for the efficient production and accumulation of higher value byproducts including medium-chain carboxylic acids and alcohols.

Description

[0001] ANAEROBIC FERMENTATION METHODSAND SYSTEMS

[0002] FIELD

[0003] The present invention relates to methods and systems for anerobic fermentation. The invention has been developed primarily as methods and systems for enhancing anaerobic fermentation of algae for the efficient production and accumulation of higher value byproducts including medium-chain carboxylic acids and alcohols and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

[0004] BACKGROUND

[0005] The escalating global demand for renewable energy has prompted attention on the efficient transformation of low-value organic substances to value-added chemicals through biotechnology [1], Medium-chain fatty acids (MCFAs) are the straight-chain saturated monocarboxylates containing 6 to 12 carbon atoms, which have emerged as a prominent candidate among various conventional bioenergy chemicals due to their high energy density and hydrophobicity [2], So far, MCFAs have established themselves as crucial precursors for the production of food additives [3], biodiesels [4], antimicrobial agents [5] and bioplastics [6], Currently, the production of MCFAs mainly relies on the extraction from animal / plant oils and fossil fuels, imposing excessively high production costs that hinder their widespread applications [7], Thus, the generation of high-value MCFAs via upgrading low-value waste organic biomass is attractive for the general public interests.

[0006] To achieve economical and environmentally friendly MCFAs production at high productivity, the premise is to select the efficient and cost-effective organic feedstocks. Algae is a type of organism with high growth rate that can grow on sunlight and water. The application of algae as feedstocks for bioenergy production is promising attributed to their abundant content of non-cellulosic carbohydrates and proteins [8], On the other hand, uncontrolled growth of algae in water bodies can also induce serious environmental problems. Without prompt and effective treatment, the toxins released by algae can have considerable negative impacts on the health of organisms, water quality, and ecological balance [9], In order to prevent these hazards, algae are usually separated from the water systems by physical means, such as harvesting by ships

[0010] , This has contributed to the significant accumulation of organic waste, necessitating the urgent development of an efficient method to achieve its reduction. Taking into account both organic compositions of algae and environmental concerns, the conversion of algae as the substrate to produce MCFAs has emerged as a promising technology for efficient upgrading of low-value organic waste into high-value energy.

[0007] As a prevalent bioprocessing method for organic waste, anaerobic fermentation has been acknowledged as a platform for waste reduction and energy chemical recovery

[0011] , with shortchain fatty acids (SCFAs) serving as crucial precursors for MCFAs through chain elongation (CE) process

[0012] , In light of this, integrating anaerobic fermentation and CE allows for efficient upgrading of low-value algae biomass waste to MCFAs. Anaerobic functional microbes then utilize electron donors (ED) to generate acetyl-CoA, a vital component in bio-metabolic pathways. Subsequently, this coenzyme can couple with electron acceptors (EA, i.e., the primary fermentation products SCFAs), extending two carbons of fatty acids in a stepwise manner to form MCFAs ultimately [13, 14], It is evident that ED participation is indispensable forCE process, and thus, the selection of ED significantly affects the performances of MCFAs production. Till now, the most efficient and extensively studied ED for MCFAs production include ethanol and lactic acid

[0015] , However, using additional chemicals to produce medium-chain carboxylates is hindered by their high cost and resource consumption.

[0008] Although MCFAs with high carbon-to-oxygen ratio are recognized as the precursors of liquid biofuel, some additional products — alcohols such as butanol and hexanol — are better alternatives than MCFAs to directly replace gasoline as liquid fuel because it is compatible with current combustion engine designs [16, 17], Generally, the traditional routes for bioproduction of alcohols include two pathways [18, 19], (i) acetone-butanol-ethanol (ABE) fermentation from sugar-based sources and (ii) syngas fermentation. Regardless of these processes, a necessary step in alcohol production involves the bio-reduction of carboxylates

[0020] , resulting in the feasibility for co-producing MCFAs and alcohols via anerobic fermentation. However, the bioconversion of microalgae as a substrate for MCFA and alcohol production has not been thoroughly evaluated, despite its potential suitability due to its high sugar and protein content.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention broadly relates to novel methods and systems for anerobic fermentation which overcomes or ameliorates at least one of the disadvantages of the prior art, or to provide a useful alternative. In one or more aspects, the present invention provides methods and systems for anaerobic algae fermentation that are self-sufficient to achieve production of high-value bioenergy including medium chain fatty acid (MCFAs) and / or alcohols, advantageously without the need of supplementary electron donors.

[0011] In a first aspect, the present invention provides a method for anaerobic fermentation, comprising: (a) providing at least one substrate, wherein the at least one substrate comprises algae; (b) adding a first plurality of anaerobic microorganisms for carboxylate biosynthesis and chain elongation from the at least one substrate;

[0012] (c) adding one or more of a second microorganism for ethanol fermentation from the at least one substrate;

[0013] wherein the first plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol fermentation, and

[0014] wherein the method contributes to the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate.

[0015] In a second aspect, the present invention provides a system for anaerobic fermentation, comprising

[0016] (a) a first plurality of anaerobic microorganisms for carboxylate biosynthesis and chain elongation from at least one substrate, wherein the at least one substrate comprises algae; and

[0017] (b) one or more of a second microorganism for ethanol fermentation from the at least one substrate;

[0018] wherein the first plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol fermentation, and

[0019] wherein the system contributes to the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate.

[0020] Preferably, the one or more second microorganism comprises microorganisms capable of converting organic substrates into ethanol under anaerobic conditions.

[0021] In one embodiment, the one or more second microorganism is selected from yeast, thermophilic anaerobic bacterium, a-proteobacterium, or a combination thereof. In one embodiment, the one or more second microorganism is selected from Saccharomyces cerevisiae, Clostridium thermocellum, Candida shehatae, Zymomonas mobilis, or a combination thereof. In a preferred embodiment, the one or more second microorganism is yeast. In a particular preferred embodiment, the one or more second microorganism is Saccharomyces cerevisiae.

[0022] Preferably, the first plurality of anaerobic microorganisms and the second microorganism are added to the substrate in a single bioreactor. In one embodiment, the volatile solid content ratio for the at least one substrate and the first plurality of anaerobic microorganisms is 0.1: 1, preferably 0.5:1.

[0023] In one embodiment, the temperature for achieving chain elongation and ethanol production in a single anaerobic reaction system is within the range of mesophilic anaerobic fermentation, preferably between 30 °C and 40 °C.

[0024] In one embodiment, pH value of the single bioreactor is adjusted to 5.

[0025] Preferably, the second microorganism is added at a dosage of 0.2 to 0.5 g / g-VS.

[0026] In a third aspect, the present invention provides a method for anaerobic fermentation, comprising:

[0027] (a) combining at least one substrate and a plurality of anaerobic microorganisms, wherein the at least one substrate comprises algae;

[0028] (b) performing ethanol-type fermentation from the at least one substrate and the plurality of anaerobic microorganisms; and

[0029] (c) performing carboxylate biosynthesis and chain elongation from the at least one substrate and the plurality of anaerobic microorganisms

[0030] wherein the ethanol-type fermentation is performed at about pH 4-5, and wherein the carboxylate biosynthesis and chain elongation is performed at about pH 6-7,

[0031] wherein the plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol-type fermentation, and

[0032] wherein the method contributes to the production of medium-chain carboxylates and / or ethanol from the anaerobic fermentation of the at least one substrate.

[0033] In a fourth aspect, the present invention provides a system for anaerobic fermentation, comprising a plurality of anaerobic microorganisms for performing ethanol-type fermentation and carboxylate biosynthesis and chain elongation from at least one substrate, wherein the at least one substrate comprises algae,

[0034] wherein the ethanol fermentation is performed at about pH 4-5 or below and the carboxylate biosynthesis and chain elongation is performed at about pH 6-7,

[0035] wherein the plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol-type fermentation, and wherein the system contributes to the production of medium-chain carboxylates and / or ethanol from the anaerobic fermentation of the at least one substrate.

[0036] Preferably, the ethanol-type fermentation is performed at about pH 4.5.

[0037] Preferably, the carboxylate biosynthesis and chain elongation is performed at about pH 7. Preferably, the at least one bioproduct produced from the ethanol-type fermentation comprises ethanol and / or hydrogen.

[0038] Preferably, the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate comprises using at least one bioproduct produced from the ethanol-type fermentation as an electron donor to extend carbon chain length of short-chain carboxylates into medium-chain carboxylates.

[0039] In one embodiment, the at least one bioproduct produced from the ethanol-type fermentation as an electron donor is ethanol.

[0040] Preferably, the at least one bioproduct from the ethanol-type fermentation further contributes to controlling chain elongation of short-chain carboxylates into medium-chain carboxylates.

[0041] In an alternative embodiment, the at least one product produced from the ethanol-type fermentation that contributes to controlling chain elongation is hydrogen.

[0042] In one embodiment, no exogenous ethanol (and / or hydrogen) is added to the system or the method.

[0043] In one embodiment, additional exogenous ethanol (and / or hydrogen) is added to the system or the method for chain elongation of carboxylate.

[0044] Preferably, the algae comprises raw algae, pretreated algae or a combination thereof. In one embodiment, the pretreated algae is prepared by a pretreatment method selected from mechanical treatment, thermal treatment, chemical treatment, thermal hydrolysis or a combination thereof.

[0045] In one embodiment, the algae is pretreated with amylase.

[0046] Preferably, the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms further comprises microorganisms that are responsible for hydrolysis and acidification of the at least one substrate.

[0047] In one embodiment, the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms are combinations of functional anaerobic microorganisms responsible for hydrolysis of macromolecules in substrates, acidification and carbon chain extension of hydrolysis products.

[0048] In a preferred embodiment, the anaerobic microorganisms are combinations of functional anaerobic microorganisms extracted from existing anaerobic fermenters.

[0049] In one embodiment, the method according to the first or third aspect or the system according to the second or fourth aspect further comprising one or more reducing agent to drive reduction reactions that convert medium-chain fatty acid to long-chain alcohols.

[0050] In one embodiment, the one or more reducing agents is selected from reducing coenzymes, microcurrent, ascorbic acid, or a combination thereof.

[0051] In one embodiment, the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms further comprises microorganisms that are responsible for reduction reactions that convert medium-chain fatty acid to long-chain alcohols in the presence of the one or more reducing agent.

[0052] In one embodiment, the one or more reducing agents is selected from reducing coenzymes, microcurrent, ascorbic acid, or a combination thereof.

[0053] Preferably, the long-chain alcohols are selected from butanol, pentanol, hexanol, heptanol, octanol, ora combination thereof.

[0054] BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Schematic diagram of a system targeting high-value fatty acids production through bioaugmentation of anaerobic algae fermentation system.

[0055] FIG. 2. Schematic diagram of a system targeting high-value alcohols production through coupled bioaugmentation and reducing power supplementation.

[0056] FIG. 3. Schematic diagram of a system targeting ethanol production through the satisfaction of ethanol fermentation conditions.

[0057] FIG.4. Schematic diagram of a system targeting high-value fatty acids production through staged ethanol fermentation and carbon chain extension.

[0058] FIG. 5. Schematic diagram of a system targeting high-value alcohols production through coupled staged fermentation and reducing power supplementation.

[0059] FIG. 6. The carboxylate and alcohol profiles during anaerobic microalgae fermentation with sole ethanol (A), ethanol: lactic acid = 2:1 (B), 1:1 (C), and 2:1 (D), and sole lactic acid (E). (1) represents alcohol production performances, (2) indicates MCFAs production trends, with (3) demonstrating the accumulation of SCFAs with time.

[0060] FIG. 7. The final accumulated products (including carboxylates and alcohols) during algae fermentation process (A), the consumption of ED under different ED involvement conditions (B), and the proportions (C) and classifications (D) of various carboxylates and alcohols in the fermentation liquor.

[0061] FIG. 8. The microalgae degradation percentages within different anaerobic fermenters with different ED involvement.

[0062] FIG. 9. The details of carbon flux (COD basis) during algae anaerobic fermentation for MCFAs production when sole ethanol (A) or sole lactic acid (B) was considered as ED, as well as the efficiency of corresponding ED consumption in participating CE process. The square bars of different colours represent different species involved in the transformation of organic matters during the anaerobic fermentation process, and the width of the square bars represents the relative COD content of material transformation / generation.

[0063] FIG. 10. Simulation of cumulative MCFAs production profiles in different ED involved bioreactors (E1-E5) and the results of the fitted parameters.

[0064] FIG. 11. The main microalgae fermentation products and their corresponding theoretical biochemical reactions under different ED participation conditions.

[0065] FIG. 12. The thermodynamic feasibility comparisons of algae bioconversion into SCFAs (represented by acetate), MCFAs (represented by caproate) and alcohols (represented by butanol) under ED involvement of sole ethanol (A), Co-ED with ethanol to lactic acid COD ratio of 2:1 (B), 1:1 (C) and 1:2 (D), and sole lactic acid (E).

[0066] FIG. 13. Genome-based PCA analysis results of microbial populations from different microbial samples fermented by algae (A), and microbial community composition and corresponding gene abundance at the phylum level (B).

[0067] FIG. 14. Functional distribution of the non-redundant genes in ethanol, Co-ED and lactic acid samples. Functional categories were defined using KEGG database.

[0068] FIG. 15. The complex microbial metabolic pathway network diagram of algae microbial fermentation for MCFAs production and the comparison of the gene abundance of the target pathway in different microbial samples.

[0069] FIG. 16. The comparison of (A) SCFA production and (B) MCFA / alcohol production between control and yeast group. (C) The ethanol concentration of the yeast group within the initial nine days. (D) The product spectrum of algae fermentation with and without yeast. (E) The product distribution normalized with TCOD input of substrate. The statistical difference between control and yeast group is indicated as asterisk mark above the plots, (ns: P>0.05, *: 0.05> P>0.01, **: 0.01> P>0.001, ***: 0.001> P>0.0001, ****: p< 0.001)

[0070] FIG. 17. The microbial structure of control and yeast group at the end of fermentation under Phylum level. The abundance was obtained by using MetaPhlAn 4 from trimmed reads as input data.

[0071] FIG. 18. The heatmap of microbial structure in the control and yeast group under (A) Genus and (B) Species level. The abundance indicated in the heatmap was obtained by using MetaPhlAn 4 from trimmed reads as input data. All unassigned taxa were deleted but were still contained in the total abundance.

[0072] FIG. 19. The relative abundance of (A) glycoside hydrolase family (The bar plots indicate the proposition (%) of corresponding genes in the whole sample, while the differences between two group are showed in the scatter plots), (B) acyl chain elongation genes and (C) butanol production genes (exact Fisher’s test: * 0.01< P<0.05, **0.001 < P<0.01 ***: P<0.001).

[0073] FIG. 20. Proposed microbial pathway for MCFA and butanol production. Acetaldehyde dehydrogenase (ADA, EC 1.2.1.10), alcohol dehydrogenase (ADH, EC 1.1.1.1), phosphate acetyltransferase (PTA, EC 2.3.1.8), acetate kinase (ACK, EC 2.7.2.1), acetyl-CoA synthetase (ACS, EC 6.2.1.1), acetyl-CoA C-acyltransferase (ACAT, EC 2.3.1.16, EC 2.3.1.9), 3-hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.157, 1.1.1.35), enoyl-CoA hydratase (ECH, EC 4.2.1.55, EC 4.2.1.17), acyl-CoA dehydrogenase (ACD, EC 1.3.8.1, EC 1.3.8.7, EC 1.3.8.8), electron transfer flavoprotein (Etf, EC 1.5.5.1), thioesterase (TE, EC 3.1.2.20), 4-hydroxybutyrate CoA transferase (CoAT EC 2.8.3.1, EC 2.8.3.8, EC 2.8.3.9), ferredoxin-NAD+oxidoreductase-Na+ translocating (Rnf, EC 7.2.1.2), ferredoxin hydrogenase (H2ase, EC 1.12.7.2), bifurcating [Fe-Fe] hydorgenase (HydABC, EC 1.12.1.4), energy-conserving hydrogenase (EchABCDEF, EC 1.12.2.1), pyruvate ferredoxin oxidoreductase (PFOR, 1.2.7.1), aldehyde ferredoxin oxidoreductase (AOR, EC 1.2.7.5), aldehyde dehydrogenase (ALDH, EC 1.2.1.3), NADH-dependent reduced ferredoxin: NADP oxidoreductase (Nfn, EC 1.18.1.2, EC 1.19.1.1).

[0074] FIG. 21. The concentration of ethanol produced from Oedogoniumby yeast fermentation.

[0075] FIG. 22. The yield of ethanol and caproate produced from Oedogoniumby ethanol-type fermentation.

[0076] FIG. 23. The yield of ethanol, butanol and caproate produced from Oedogoniumby yeast fermentation with chain elongation.

[0077] FIG. 24 The yield of ethanol and caproate produced from Oedogonium by ethanol-type fermentation with chain elongation. DESCRIPTION OF EMBODIMENTS

[0078] In one or more aspects, the present invention generally correlates with the biotechnological conversion of algae into organic energy materials, particularly focusing on improving or enhancing anaerobic fermentation technology to achieve efficient production of high-value fatty acids and / or alcohols. In addition, the anaerobic fermentation methods and systems according to one or more aspects of the present invention provides a cost-effective technology and / or a self-sufficient biotechnology platform for algae energy recovery and upgrading that does not require external energy precursors. Specifically, the participation of electron donors (for example, ethanol) is essential to stimulate the corresponding biochemical reactions for the recovery of MCFAs from anaerobic fermentation. The anaerobic fermentation methods or systems according to one or more aspects of the present invention do not rely on the supplementation of exogenous ethanol, but the accumulation of self-sufficient ethanol through the modification of the anaerobic fermentation methods or systems.

[0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0080] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0081] The indefinite articles ‘a’ and ‘an’ are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining “one” or a “single” element or feature. For example, “a” cell includes one cell, one or more cells and a plurality of cells.

[0082] Unless the context requires otherwise, the terms “comprise”, “comprises” and “comprising”, or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

[0083] As used herein, unless otherwise specified the use of the ordinal adjectives “first”, “second”, “third”, etc., to describe a common object, merely indicate that different instances of like objects are being referred to and are not intended to imply that the objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner. As used herein, when referring to a measurable value such as an amount, a temporal duration, and the like, the term "about" is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0084] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0085] In a first aspect, the present invention provides a method for anaerobic fermentation, comprising:

[0086] (a) providing at least one substrate, wherein the at least one substrate comprises algae; (b) adding a first plurality of anaerobic microorganisms for carboxylate biosynthesis and chain elongation from the at least one substrate;

[0087] (c) adding one or more of a second microorganism for ethanol fermentation from the at least one substrate;

[0088] wherein the first plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol fermentation, and

[0089] wherein the method contributes to the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate.

[0090] In a second aspect, the present invention provides a system for anaerobic fermentation, comprising

[0091] (a) a first plurality of anaerobic microorganisms for carboxylate biosynthesis and chain elongation from at least one substrate, wherein the at least one substrate comprises algae; and

[0092] (b) one or more of a second microorganism for ethanol fermentation from the at least one substrate;

[0093] wherein the first plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol fermentation, and

[0094] wherein the system contributes to the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate. As use herein, “algae” are a type of single-cell or multi-cell microscopic algal organisms that usually live in water bodies. They can photosynthesize, using light energy to convert carbon dioxide and water into organic matter. Algae contain various organic substances, including carbohydrates, fatty acids, proteins, nucleic acids, vitamins and plant pigments. Among them, carbohydrates possess the highest content and are the main substrate for photosynthesis of algae, used for the synthesis of organic matter and energy storage.

[0095] In one embodiment, the algae may include, but are not limited to, species from prokaryotic algae (such as Cyanobacteria) and eukaryotic algae (such as green algae, red algae, brown algae, diatoms, and euglenoids). These may also include microalgae (e.g., Oedogonium, Chlorella, Spirulina) or macroalgae (e.g., Ulva, Porphyra, Laminaria), depending on the desired application.

[0096] “Anaerobic microorganisms” may include microbial populations extracted from existing anaerobic fermentation systems, which include the main functional microorganisms for traditional anaerobic fermentation. The anaerobic microorganisms may include those that perform various biochemical functions, including hydrolysis, acidification and carboxylates chain elongation. The anaerobic microorganisms disclosed herein may be composed of bacteria and archaea, with thermophilic species, mesophilic species, cellulolytic species, hemicellulolytic species, and glycolytic species being the main components. As such, the diversified organic compounds in algae can be bio-transformed into monomer molecule through hydrolysis. Subsequently, these simple organic chemicals can be fermented into carbon dioxide, hydrogen and short-chain fatty acids (notably acetate). The complex interactions of these individual biochemical reactions facilitate the basic function of traditional anaerobic fermentation.

[0097] Accordingly, the first plurality of anaerobic microorganisms preferably further comprises microorganisms that are responsible for hydrolysis and acidification of the at least one substrate.

[0098] In one embodiment, the first plurality of anaerobic microorganisms are combinations of functional anaerobic microorganisms responsible for hydrolysis of macromolecules in substrates, acidification and carbon chain extension of hydrolysis products.

[0099] In a preferred embodiment, the first plurality of anaerobic microorganisms are extracted from existing anaerobic fermenters.

[0100] Preferably, the one or more second microorganism comprises microorganisms capable of converting organic substrates into ethanol under anaerobic conditions.

[0101] In one embodiment, the one or more second microorganism is selected from yeast, thermophilic anaerobic bacterium, a-proteobacterium, or a combination thereof. In one embodiment, the one or more second microorganism is selected from Saccharomyces cerevisiae, Clostridium thermocellum, Candida shehatae, Zymomonas mobilis, or a combination thereof. In a preferred embodiment, the one or more second microorganism is yeast. In a particular preferred embodiment, the one or more second microorganism is Saccharomyces cerevisiae.

[0102] “Microorganisms capable of converting organic substrates into ethanol”, for example “yeast” (e.g., Saccharomyces cerevisiae), are single-celled fungi known for their capability to undergo alcoholic fermentation, a process converting carbohydrates into ethanol and carbon dioxide under anaerobic conditions. In one or more embodiments, this bioaugmentation technology is introduced to redirect the products of traditional anaerobic algae fermentation into ethanol production, an important precursor for high-value energy upgrading. The principal metabolism considering the conversion of carbohydrate into ethanol by yeast cells can be summarized as sugar metabolism and the acetaldehyde pathway. Yeast cells initially metabolize carbohydrates (typically glucose) via glycolysis, breaking them down into smaller molecules such as pyruvate or pyruvate analogs. These smaller molecules subsequently enter the glycolytic pathway, yielding pyruvate and acetaldehyde. Pyruvate and acetaldehyde undergo conversion into ethanol through the acetaldehyde pathway, with ethanol as the ultimate product.

[0103] “Ethanol-type fermentation” is one of the fermentation types in mixed cultures of acidogenesis aiming to achieve high-efficiency hydrogen production, as well as the accumulation of ethanol. Similar to traditional anaerobic algae fermentation, the organic components in algae form the substrate for ethanol-type fermentation.

[0104] Accordingly, in a preferred embodiment, bioproducts produced from the ethanol-type fermentation comprise ethanol and / or hydrogen.

[0105] As used herein, the term “fatty acid” or “carboxylates” refers to a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms. The chain-length range is from 2 to 80 but commonly from 12 up to 24. Short-chain fatty acids (SCFAs) are fatty acids with aliphatic tails of five or fewer carbons (e.g., butyric acid). Medium-chain fatty acids (MCFAs) are fatty acids with aliphatic tails of 6 to 12 carbons, which can form medium-chain triglycerides.

[0106] “Chain elongation” is an emerging biotechnological step to transform SCFAs into high-value MCFAs via the reverse β-oxidization pathway. The pathway of chain elongation is a cyclic process and utilizes an acetyl-CoA molecule each cycle, which comes from an electron donor (ED), coupling with short-chain fatty acids (as the electron acceptor (EA)) and extending its carbon chain length with two carbons (C2).

[0107] Accordingly, the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate preferably comprises using at least one bioproduct produced from the ethanol-type fermentation as an electron donor to extend the carbon chain length of the short-chain carboxylates into medium-chain carboxylates. In one embodiment, the at least one bioproduct produced from the ethanol-type fermentation as an electron donor is ethanol.

[0108] In addition to ethanol, hydrogen (H2) is also an important bioproduct of ethanol-type fermentation that can play a crucial role in the chain elongation (CE) process. Specifically, presence of H2can be beneficial for providing a reducing environment for efficient CE.

[0109] Accordingly, the at least one bioproduct from the ethanol-type fermentation preferably further contributes to controlling chain elongation of the short-chain carboxylates into mediumchain carboxylates. In one embodiment, the at least one product produced from the ethanol-type fermentation that contributes to controlling chain elongation is hydrogen.

[0110] Preferably, the first plurality of anaerobic microorganisms and the second microorganism are added to the substrate in a single bioreactor.

[0111] In one embodiment, the volatile solid content ratio for the at least one substrate and the first plurality of anaerobic microorganisms is 0.1: 1, preferably 0.5:1.

[0112] In one embodiment, the temperature for achieving chain elongation and ethanol production in a single anaerobic reaction system is within the range of mesophilic anaerobic fermentation, preferably between 30 °C and 40 °C.

[0113] In one embodiment, the pH of the single bioreactor is maintained or adjusted between about pH 3 to pH 7, preferably between pH 4 to pH 6.

[0114] In a preferred embodiment, the pH of the single bioreactor is maintained or adjusted to about pH 5.

[0115] In one or more embodiment, the amount of the yeast added is between about 0.05 g to 10 g per 100 ml of the total volume of the anaerobic fermentation reaction. In one embodiment, the amount of the yeast added is about 0.1 g, 0.5 g, 1 g or 2 g per 100 ml of the total volume of the anaerobic fermentation reaction.

[0116] Preferably, the second microorganism is added at a dosage of 0.2 to 0.5 g / g-VS.

[0117] In a third aspect, the present invention provides a method for anaerobic fermentation, comprising:

[0118] (a) combining at least one substrate and a plurality of anaerobic microorganisms, wherein the at least one substrate comprises algae;

[0119] (b) performing ethanol-type fermentation from the at least one substrate and the plurality of anaerobic microorganisms; and (c) performing carboxylate biosynthesis and chain elongation from the at least one substrate and the plurality of anaerobic microorganisms

[0120] wherein the ethanol-type fermentation is performed at about pH 4-5, and wherein the carboxylate biosynthesis and chain elongation is performed at about pH 6-7,

[0121] wherein the plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol-type fermentation, and

[0122] wherein the method contributes to the production of medium-chain carboxylates and / or ethanol from the anaerobic fermentation of the at least one substrate.

[0123] In a fourth aspect, the present invention provides a system for anaerobic fermentation, comprising a plurality of anaerobic microorganisms for performing ethanol-type fermentation and carboxylate biosynthesis and chain elongation from at least one substrate, wherein the at least one substrate comprises algae,

[0124] wherein the ethanol fermentation is performed at about pH 4-5 or below and the carboxylate biosynthesis and chain elongation is performed at about pH 6-7,

[0125] wherein the plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol-type fermentation, and

[0126] wherein the system contributes to the production of medium-chain carboxylates and / or ethanol from the anaerobic fermentation of the at least one substrate.

[0127] During the fermentation process, pH is a primary control factor, which will pose significant effects on the physiological metabolism and transcriptomes of functional microorganisms related to this type of fermentation (i.e., Ethanoligenens harbinense).

[0128] As such, in one or more embodiment, the accumulated products of individual algae fermentation is redirected into ethanol by accurately controlling the pH of the fermentation system at about pH 4 to pH 5.

[0129] Preferably, the ethanol-type fermentation is performed at about pH 4.5.

[0130] Preferably, the carboxylate biosynthesis and chain elongation is performed at about pH 7. Preferably, the plurality of anaerobic microorganisms further comprises microorganisms that are responsible for hydrolysis and acidification of the at least one substrate. In one embodiment, the plurality of anaerobic microorganisms are combinations of functional anaerobic microorganisms responsible for hydrolysis of macromolecules in substrates, acidification and carbon chain extension of hydrolysis products.

[0131] In a preferred embodiment, the plurality of anaerobic microorganisms are extracted from existing anaerobic fermenters

[0132] Preferably, the at least one bioproduct produced from the ethanol-type fermentation comprises ethanol and / or hydrogen.

[0133] Preferably, the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate comprises using at least one bioproduct produced from the ethanol-type fermentation as an electron donor to extend the carbon chain length of the short-chain carboxylates into medium-chain carboxylates. In one embodiment, the at least one bioproduct produced from the ethanol-type fermentation as an electron donor is ethanol.

[0134] Preferably, the at least one bioproduct from the ethanol-type fermentation further contributes to controlling chain elongation of the short-chain carboxylates into medium-chain carboxylates. In one embodiment, the at least one product produced from the ethanol-type fermentation that contributes to controlling chain elongation is hydrogen.

[0135] In one embodiment, no exogenous ethanol (and / or hydrogen) is added to the system or the method.

[0136] In one embodiment, additional exogenous ethanol (and / or hydrogen) is added to the system or the method for chain elongation of carboxylate.

[0137] Preferably, the algae comprises raw algae, pretreated algae or a combination thereof. In one embodiment, the pretreated algae is prepared by a pretreatment method selected from mechanical treatment, thermal treatment, chemical treatment, thermal hydrolysis or a combination thereof.

[0138] As defined herein, “pre-treatment methods” for the reconstruction of algae streams (“pretreated algae”) may break down the feedstock to improve the bioavailability, increase the product yield, reduce the amount of raw substrate, and improve dewatering during the anaerobic treatment process. These processes may include conventional pretreatment methods, including biological hydrolysis (e.g., acid phase, enzymatic hydrolysis), mechanical hydrolysis, acid hydrolysis, thermal hydrolysis and chemical hydrolysis.

[0139] For example, thermal hydrolysis is a process used to prepare organic materials for subsequent biochemical or thermochemical conversion processes. During this pretreatment, organic materials are subjected to high temperature and pressure conditions in the presence of water. This treatment facilitates the breakdown of complex organic structures, such as lignocellulosic biomass, into simpler compounds, thereby enhancing their accessibility and susceptibility to further conversion processes.

[0140] In one embodiment, the algae is pretreated with amylase.

[0141] As would be understood by the skilled person, supplementing reducing power in fermentation processes aims to facilitate the conversion of fatty acids into alcohols. This process involves providing additional reducing agents to the fermentation system to drive the reduction reactions necessary for the conversion of fatty acids. In anaerobic fermentation pathways, fatty acids can be reduced to alcohols through enzymatic reactions involving specific reducing agents. However, in some cases, the endogenous reducing power within the fermentation system may be insufficient to drive these reduction reactions efficiently. Therefore, external reducing agents are added to enhance the overall reducing capacity of the system.

[0142] Accordingly, in one embodiment, the method according to the first or third aspect or the system according to the second or fourth aspect further comprising one or more reducing agent to drive reduction reactions that convert medium-chain fatty acid to long-chain alcohols.

[0143] In one embodiment, the one or more reducing agents is selected from reducing coenzymes, microcurrent, ascorbic acid, or a combination thereof.

[0144] The reducing agents can be utilized to achieve fatty acid reduction involve coenzymes supplementation, chemical reducing agents and microcurrent manipulation. For example, coenzymes such as NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) serve as electron carriers in biological redox reactions. Supplementing these coenzymes provides additional reducing power to facilitate the reduction of fatty acids to alcohols.

[0145] In one embodiment, the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms further comprises microorganisms that are responsible for reduction reactions that convert medium-chain fatty acid to long-chain alcohols in the presence of the one or more reducing agent.

[0146] As used herein, “long-chain alcohols” (or fatty alcohols) including propanol, butanol, pentanol, hexanol, and octanol, are usually high-molecular-weight, straight-chain primary alcohols, but can also range from as few as 4-6 carbons to as many as 22-26, derived from natural fats and oils. Long-chain alcohols usually have an even number of carbon atoms and a single alcohol group (-OH) attached to the terminal carbon. Some are unsaturated and some are branched. Preferably, the long-chain alcohols are selected from butanol, pentanol, hexanol, heptanol, octanol, ora combination thereof.

[0147] FIG. 1 discloses a microbial bioaugmentation drive system according to one particular aspect of the present invention, wherein yeast cells are integrated as bioaugmented microorganisms into an algae anaerobic fermentation system to facilitate the production of high-value medium-chain fatty acids. The anaerobic system utilizes algae as substrate to orient in different biochemical directions. The mixed microbial system extracted from the anaerobic digester undergoes traditional anaerobic fermentation process to accumulate short-chain fatty acids. Following another biochemical direction, yeast cells function to bio-convert carbohydrate in the algae into ethanol. Subsequently, the species responsible for carbon chain extension in the mixed microbial system utilize ethanol as electron donor and the short-chain fatty acids as electron acceptor to extend the carbon chain of fatty acids, thereby realizing the energy upgrading of algae to high-value fatty acids.

[0148] FIG. 2 discloses another microbial bioaugmentation drive system according to a particular aspect of the present invention, wherein alongside yeast bioaugmentation into the algae anaerobic fermentation system, additional reducing power is supplemented into the anaerobic system in a controlled manner to achieve the conversion of high-value fatty acids into alcohols. Microorganisms involved still utilize the organic compounds in algae as substrates for biochemical reactions, and they effectively produce high-value medium-chain fatty acids on the basis of accumulating simple ethanol and short-chain fatty acids. The difference is that the mixed microbial system reduces medium-chain fatty acids into valuable alcohols of corresponding chain length after receiving the reducing force.

[0149] FIG. 3 discloses an operating parameter control drive system for the production of ethanol according to a particular aspect of the present invention, wherein traditional algae anaerobic fermentation system is improved in operating parameters and converted into ethanol fermentation to achieve the accumulation of ethanol energy substances in the mixed fermentation system. This paradigm can be applied to existing algae fermentation facilities to convert traditional mixed fermentation products into ethanol through effective control of important operating conditions represented by pH.

[0150] FIG. 4 discloses an operating parameter control drive system for the production of MCFAs according to a particular aspect of the present invention, wherein the ethanol fermentation process discloses in FIG. 3 is used as the first stage of the entire technology, emerging as the basis for the anaerobic system to accumulate high-value energy products. The ethanol fermentation liquor is further transferred to a traditional algae anaerobic fermentation system, which stimulates the short-chain fatty acids generated through hydrolysis and acidification steps to further extend the carbon chain to produce extensive high-value medium-chain fatty acids. FIG. 5 discloses another operating parameter control drive system for the production of MCFAs according to a particular aspect of the present invention, controllable reducing power is supplemented into the anaerobic system that accumulates medium-chain fatty acids as disclosed in FIG.4. Accordingly, alcohols with extended carbon chains constitute the final target products of this system.

[0151] So that preferred embodiments of the invention may be fully understood and put into practical effect, reference is made to the following non-limiting examples.

[0152] EXAMPLES

[0153] Example 1 - Optimization of Electron Donors (ED)

[0154] To systematically evaluate which ED combination is the most beneficial for algae anaerobic fermentation targeting MCFAs production, five anaerobic bioreactor groups were set up and fed with sole ethanol, sole lactate, or the combination of ethanol: lactic acid controlled at ratios of 2:1, 1:1 and 1:2. The EDs fed into the system were controlled at the equivalent total chemical oxygen demand (COD). The efficiencies of ED utilization for CE process with different ED added were elucidated through detailing carbon flux calculations. Subsequent kinetics and thermodynamics analysis of different fermentative bioreactors confirmed distinctive contributions of various EDs to the compositions and concentrations of fermentation products from algae. Moreover, the exploration of genome facilitated the construction of complex microbial metabolic networks for algae fermentation, which is beneficial for comparing the mechanisms of MCFAs production under different ED stimulations. Therefore, this example would be beneficial for efficient management and energy recovery of blooming algae, through offering strategic directives for maximizing the accumulation of high-value energy resources.

[0155] Materials and Methods

[0156] Sources of algae and inoculum.

[0157] The algae used herewith were harvested from Taihu Lake, China by ship salvage, where the algal bloom is serious in summer. The main characteristics of algae were as follows: 17.4 ± 0.1 g / L total chemical oxygen demand (TCOD), 3.7 ± 0.1 mg / L soluble chemical oxygen demand (SCOD), 11.9 ± 0.2 g / L total solids (TS), and 9.6 ± 0.1 g / L volatile solids (VS).

[0158] The inoculum sludge was withdrawn from an anaerobic sludge digester, which was operated under mesophilic 35 ± 1 °C and semi-continuous mode with solids retention time (SRT) maintained around 20 day. The primary properties of the inoculated sludge were as follows: TS = 36.0 ± 0.2 g / L, VS = 16.9 ± 0.1 g / L, pH = 7.1 ± 0.1. MCFAs production from algae with different ED stimulations.

[0159] The experiments for evaluating the effects of ED on MCFAs production profiles during anaerobic algae fermentation were conducted in batch mode. Herein, five ED combinations with different ratios of ethanol to lactic acid were selected as the representative conditions for algae MCFAs fermentation, including sole ethanol (E1), ethanol: lactic acid= 2: 1 (E2), 1: 1 (E3), and 1:2 (E4), and sole lactic acid (E5). The ratio between ethanol and lactic acid was based on the associated COD value. Before commencing the experiment, the serum bottles were firstly fed with 10 mL algae mixture and 10 mL of inoculated sludge, followed by the addition of deionized water (DI) to ensure the final total working volumes was 100 mL. Afterwards, ED combinations with corresponding concentrations were introduced into different fermentation bioreactors. The ratio of total ED to available EA was maintained between 2 and 3, and the final TCOD in the serum bottles was controlled at 3 g / L. The calculation of ED concentrations was based on a previous study [3], with a focus on maximizing products accumulation efficiency of CE. pH of all the fermentation systems was adjusted to 5.5 ± 0.1 using hydrochloric acid and sodium hydroxide solution (3 M) to inhibit methanogenesis. Subsequently, the bottles were purged with N2 for 5 min and sealed to ensure the anaerobic condition. In addition, five sets of blank tests containing only DI (90 mL) and inoculated sludge (10 mL) were operated simultaneously to exclude the contributions of inoculum or ED to acid fermentation. The amount and the type of ED fed in the serum bottle were same to E1-E5 reactors. All anaerobic digestion experiments were conducted in triplicate, with the fermenters being cultivated in a constant-temperature incubator (35 ± 1 °C and 170 rpm). The anaerobic algae fermentation experiments lasted for around 30 days until the production profiles reached saturated levels. Liquid samples were collected periodically to quantify the concentrations of the organic acids and alcohols.

[0160] Calculation of ED utilization efficiencies for CE.

[0161] Apart from serving as the source of acetyl-CoA for CE pathway, the inoculated ethanol and lactic acid can also be consumed via excessive ethanol oxidation (EEC) and acrylate pathway. These two unwanted competitive pathways would affect the utilization efficiency of ethanol or lactic acid as ED for MCFAs biosynthesis. Specifically, ethanol is wasted and oxidized by ethanol-oxidizing microorganisms to form acetate with H2 released as the by-product via EEC reaction

[0021] , Lactic acid is misdirected to propionate formation via acrylate pathway rather than CE process

[0022] , A comprehensive understanding of the EDs utilization throughout the entire fermentation and the efficiency of the involvement of ED in CE is crucial to comprehend the mechanisms underlying distinctive MCFAs profiles under multiple ED situations. Herein, detailed stepwise calculations of carbon flux were calculated using sole ethanol and sole lactic acid groups as representatives to elucidate the details of carbon utilization in algae fermentation paradigms. It is worth noting that acetate and propionate were the main primary products generated without ED involvement during anaerobic fermentation

[0010] , As such, other abundant SCFAs, such as butyrate and valerate, are extensively accumulated though CE process. Based on this assumption, EEO and the acrylate pathways could contribute to the carbon fluxes of even-chain fatty acids and odd-chain fatty acids, respectively. The detailed process of carbon flux (COD based) calculation can be referred to Table 1 and Table 2. The final ED utilization efficiency for CE (fi, %) can be calculated using the following equation (Eq. 1):

[0162] EEO (acrylate pathway) consumed ED

[0163] - - - - - - - — - X 100 % ( 1 )

[0164]

[0165] total consumed ED / able 1. The calculation of detailed carbon utilization from microalqae and ED in the anaerobic algae paradigms with sole ethanol involved.

[0166] E1 (Sole Ethanol)

[0167] Ethanol C2 C3 C4 C5 Ca C7 Ca C4OH CsOH CaOH H2 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) 11586.7 %ngCCOD / L)n23527686 5 61 94398.9 455.6 2390.0 17.9 91.8 1325.2 18.4 89.7 130.6

[0168] (Final) g COD / mol 96 64 112 160 208 256 304 352 192 240 288 16

[0169] Calculation Reaction 1 6CH3CH2OH+5CH3(CH2)4COOH = 5CH3(CH2)6COOH+CH3COOH+4H2O+2H2

[0170] C2OH Ca Ca H2C2Concentration (13) = (15) / 352 / 5*6*96 (14) = (15) / 352*256 (15) = (8) (16) = (15) / 352 / 5*2*16 (17) = (15) / 352 / 5*64 (mg COD / L) 30.0 66.8 91.8 3.3 1.7 Reaction 2 2H2+CH3(CH2)4COOH = CH3(CH2)4CH2OH+H2O H2Ca CaOH Concentration (18) = (20) / 288*2*16 (19) = (20) / 288*256 (20) = (11) (mg COD / L) 10.0 79.8 89.7 Reaction 3 6CH3CH2OH+5CH3(CH2)3COOH = 5CH3(CH2)5COOH+CH3COOH+4H2O+2H2

[0171] C2OH C5C7 H2C2Concentration (21) = (15) / 304 / 5*6*96 (22) = (15) / 304*208 (23) = (7) (24) = (23) / 304 / 5*2*16 (25) = (15) / 304 / 5*64 (mg COD / L) 6.8 12.2 17.9 0.8 0.4 Reaction 4 2H2+CH3(CH2)3COOH = CH3(CH2)3CH2OH+H2O H2C5CsOH Concentration (26) = (28) / 240*2*16 (27) = (28) / 240*208 (28) = (10) (mg COD / L) 2.5 16.0 18.4 Reaction 5 6CH3CH2OH+5CH3(CH2)2COOH = 5CH3(CH2)4COOH+CH3COOH+4H2O+2H2

[0172] C2OH C4Ca H2C2Concentration (29) = (31)7256 / 5*6*96 (30) = (31) / 256*160 (31) = (6)+(14)+(19) (32) = (31) / 256 / 5*2*16 (33) = (31)7256 / 5*64 (mg COD / L) 1141.5 1585.4 2536.6 126.8 63.4 Reaction 6 6CH3CH2OH+5CH3CH2COOH = 5CH3(CH2)3COOH+CH3COOH+4H2O+2H2

[0173] C2OH C3C5H2C2Concentration (34) = (36)7208 / 5*6*96 (35) = (36) / 208*112 (36) = (5)+(22)+(27) (37) = (36) / 208 / 5*2*16 (38) = (36) / 208 / 5*64 (mg COD / L) 268.0 260.5 483.8 29.8 14.9 Reaction 7 2H2+CH3(CH2)2COOH = CH3(CH2)2CH2OH+H2O H2c4C4OH Concentration (39) = (41)7192*2*16 (40) = (41)7192*160 (41) = (9)

[0174]

[0175] (mg COD / L) 220.9 1104.3 1325.2 Reaction 8 6CH3CH2OH+4CH3COOH=5CH3(CH2)2COOH+4H2O+2H2

[0176] C2OH C2C4H2Concentration (42) = (44)7160 / 5*6*96 (43) = (44)7160 / 5*4*64 (44) = (4)+(30)+(40) (45) = (44)7160 / 5*2*16 (m COD / L) 5103.8 2268.4 7088.6 283.5 Microalgae Fermentation to C3 (3)+(35) 322.4 mg COD / L

[0177] Microalgae and Ethanol Conversion to C2 (2)+(43) 1954.9 mg COD / L

[0178] Total COD Conversion from Microalgae (COD Measurement) 248.3 mg COD / L

[0179] EEO Proportion in ED Utilization % 10.3 ED Utilization for CE % 89.7 able 2. The calculation of detailed carbon utilization from microalgae and ED in the anaerobic algae paradigms with sole lactic acid involved.

[0180] E5 (Sole Lactic Acid) Lactic Acid C2 C3 C4 C5 Ca C7 Ca (D (2) (3) (4) (5) (6) (7) (8) 11403.1 Concentration (Initial) „..._ _ „„ _ (mg COD / L) 32167 1736.2 773.0 3917.5 1031.3 1117.2 88.5 103.7

[0181] (Final) g COD / mol 96 64 112 160 208 256 304 352

[0182] Calculation Reaction 1 C3H6O3+CH3(CH2)4COOH = CH3(CH2)aCOOH+CO2+H2O LA Ca Ca Concentration (9) = (11 ) / 352*96 (10) = (11 ) / 352*256 (11) = (8) (mg COD / L) 28.3 75.5 103.7 Reaction 2 C3H6O3+CH3(CH2)3COOH = CH3(CH2)5COOH+CO2+H2O LA Cs C7 Concentration (12) = (14) / 304*96 (13) = (14) / 304*208 (14) = (7) (mg COD / L) 28.0 60.6 88.5 Reaction 3 C3H6O3+CH3(CH2)2COOH = CH3(CH2)4COOH+CO2+H2O LA C4 Ca Concentration (15) = (17) / 256*96 (16) = (17) / 256*160 (17) = (6)+(10)

[0183]

[0184] (mg COD / L) 447.2 745.4 1192.7 Reaction 4 C3H6O3+CH3CH2COOH = CH3(CH2)3COOH+CO2+H2O

[0185] LA C3C5

[0186] Concentration (18) = (20) / 208*96 (19) = (20) / 208*112 (20) = (5)+(13)

[0187] (mg COD / L) 503.9 587.9 1091.9

[0188] Reaction 5 C3H6O3+CH3COOH = CH3(CH2)2COOH+CO2+H2O

[0189] LA C2C4

[0190] Concentration (21) = (23) / 160*96 (22) = (23) / 160*64 (23) = (4)+(16)

[0191] (mg COD / L) 2797.7 1865.2 4662.9

[0192] Microalgae Fermentation to C2 (2)+(22) 3601.4 mg COD / L

[0193] Microalgae and Lactic Acid Conversion to C3 (3)+(19) 1360.9 mg COD / L

[0194] Total COD Conversion from Microalgae (COD Measurement) 1341.7 mg COD / L

[0195] Acrylate Pathway Proportion in ED Utilization % 44.2 ED Utilization Efficiency for CE % 55.8 Model fitting for MCFAs production curves with time.

[0196] Based on the trends of MCFAs accumulation during algae fermentation, a kinetic model was introduced to simulate the dynamic characteristics of anaerobic systems under diverse ED compositions. The kinetic formulation can be applied for the estimation of the potentials and production rates of MCFAs under different experimental conditions (Eq. 2), which was achieved based on the descriptions in previous publication

[0023] ,

[0197] Pt = Pmaxxexp {- exp (T - t) + 1]} (2)

[0198]

[0199] Where Ptis the cumulative MCFAs concentrations at the fermentation time (day), referred as t. T represents a constant with a value of 2.71828. The other parameters in the equation, including Pmax,Rm and Tcanbe computed through fitting the production profiles. In this context, Pmax(mg COD / L) represents the maximum production potential of total MCFAs, Rm(mg COD / L / day) implies the MCFAs production rates, while T (day) is the lag time.

[0200] Thermodynamic feasibility analysis of algae fermentation under different ED stimulations.

[0201] Thermodynamic analysis of the algae fermentation process can provide a theoretical foundation for understanding microbial energy metabolism. Insights concerning how different ED patterns affected the mechanisms was therefore provided via calculating Gibbs free energy. The exchange of actual Gibbs free energy at temperature T for any biochemical reaction can be calculated based on the standard reference free energy change (ArGT°), along with the activities (obtained from the concentrations or partial pressure of reactants and products) as indicated by Eq. 3.

[0202]

[0203] = &rGT° + RT In cti (3)

[0204] Herein, theoretical biochemical reaction stoichiometric equations under different ED stimulations were constructed based on the actual fermentation product compositions from experiments and the molecular formula of algae. C106H263O110N16P was deemed as the molecular formula of algae. Besides the desired compounds, the anaerobic fermentation products of this algae include ammonium derived from nitrogen (N) and phosphate derived from phosphorus (P). As such, based on these assumptions, the reference Gibbs free energy for each reaction at standard condition (ArG°, 25 °C and 1 atm) can be obtained using A^G0and Az / 7° of the individual chemicals (as listed in Table 3). After temperature was corrected to 35 °C and pH was adjusted to 5.5, this study would ultimately determine the thermodynamic characteristics of algae-derived MCFAs production within fermentation system containing various ED under real conditions

[0012] , Table 3. The Standard free Gibbs energy A^ G0,-Tjand standard enthalpy A^ H0iTof possible substances involved in the microalgae anaerobic fermentation process.

[0205] Name hfGoi Ts(KJ / mol) (KJ / mol) C106H263O110N16P

[0206]

[0207] -260.31*106 -190.27*106 H2O -237.2 -285.8 Ethanol -181.8 -288.3 Lactate -517.1 -687 n-butanol -171.8 -274.9 NH3-79.4 -133.3 H3PO4 -1130.3 -1296.3 acetate -369.4 -486 propionate -367.3 -453.9

[0208] n-butyrate -358.7 -474.5

[0209] n-valerate -350.2 -495.1 n-caproate -341.8 -515.8

[0210] n-heptanoate -333.2 -536.3

[0211] n-caprylate -324.6 -557

[0212] H+0 0 H20 0 CO2-394.4 -393.5

[0213] Nucleic acid extraction, library construction, and metagenome analysis.

[0214] Prior to metagenomic analysis, microbial samples at the end of the fermentation experiments were withdrawn from bioreactors with sole ethanol (E1), Co-ED (E3 with the highest MCFAs yields) and sole lactic acid (E5) involved. The genomic DNA of the microbial sample was extracted using a DNA extraction kit, followed by the guality control of the obtained DNA by measuring its concentration and purity. Subseguently, Illumina shotgun metagenomic seguencing library was constructed on Illumina Hiseg4000 platform (Illumina Inc., San Diego, CA, USA). After removing the low-quality reads by Sickle (https: / / github.com / najoshi / sickle), raw reads of 51284544 (E1), 49613378 (E3) and 45750568 (E5) were generated in this study.

[0215] The contigs derived from clean read assembly were subjected to open reading frames (ORF) prediction using Prodigal v2.6.3 (https: / / github.com / hyattpd / Prodigal), with the

[0216]

[0217] corresponding detailed procedures being referenced to previous study. Afterwards, CD-HIT (http: / / www.bioinformatics.org / cd-hit / ) was introduced to cluster the predicted genes, during which the longest sequences of each cluster were considered as the representative for constructing non-redundant gene set. The gene abundances in each sample were then quantified by comparing the high-quality reads with non-redundant gene set by SOAPaligner. To annotate sequences in non-redundant gene sets, BLASTP was applied with an E-value of 1 E-5 as cutoff. Specially, taxonomic and functional were annotated against NCBI NR database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively.

[0218] Other analytical techniques. In this study, the components of alcohols and organic acids (i.e., fatty acids and lactic acid) in the algae fermentation liquor were quantified. The concentrations of lactic acid were measured using a high-performance liquid chromatography (HPLC, Agilent-1260) equipped with a UV detector. Prior to the subsequent analyses of the product yields under different ED situations, the concentrations of alcohols and fatty acids were obtained via a gas chromatography (GC) equipped with a flame ionization detector (FID), with the detailed information being referred to existing literature

[0024] , In addition, the model fitting and plotting of the experimental data were performed using Origin 2020 software. The basic parameters representing the concentration and degradation of the fermentation algae, including TS, VS and COD, were measured following the standard methods

[0012] ,

[0219] Results

[0220] Algae fermentation bioreactor performances under different ED stimulations.

[0221] During the algae fermentation targeting MCFAs as the desired products, the accumulation concentrations of the total products (including SCFAs, MCFAs and alcohols) reached steady levels from Day 21 to Day 30 (FIG. 6). The fermentation group containing sole ethanol proved to be the best for accumulating total fermentation products (FIG. 7A). This group yielded the highest total carboxylate and alcohol yield, reaching 9356.0 ± 222.1 mg COD / L) simultaneously. Moreover, the high-value products of MCFAs in this fermentation group with only ethanol serving as ED also attained the highest concentration (i.e., 2499.7 ± 90.2 mg COD / L). The bioreactor with pure lactic acid introduction was the second optimal group in terms of MCFAs production, with 1309.5 ± 33.2 mg COD / L MCFAs yield attained (FIG. 7A). The Co-ED group that exhibited comparable MCFAs production to that of sole lactic acid-added group involved initial ethanol and lactic acid with the COD ratio of 1:1 (FIG. 7A, 7C & 7D). Based on these comparisons of product concentrations, the type and relative content of ED significantly affected the MCFAs accumulation in the algae fermentation liquor. However, the trends of MCFAs were not linearly correlated with the relative concentrations of ethanol and lactic acid in the system.

[0222] Considering that SCFAs and alcohols were also important products in addition to the desirable MCFAs, the variations in their accumulation trends under different ED were also disclosed in this study. Primarily, it was evident that the accumulated alcohol concentrations (including butanol, pentanol and hexanol) progressively decreased as the relative content of ethanol in ED decreased (from E1 to E5) (FIG. 7A & 7C). Therefore, the existence of ethanol as ED was critical for upgrading algae into alcohols. For the bioreactors where lactic acid was applied as the sole ED, no alcohol was formed. This might be attributed to the absence of H2 in the CE byproducts when lactic acid acted as the acetyl-CoA precursor [3], Consequently, lactic acid-involved algae fermentation system would be unable to produce alcohols because of insufficient reducing power. However, it was noteworthy that E5 fermenters (with only lactic acid) exhibited the highest content of odd-chain fatty acids in the final liquor (FIG. 7A & 7C), confirming the contribution of acrylate pathway in this anaerobic system.

[0223] Overall ED consumption performances and their utilization efficiencies for CE.

[0224] Theoretically, in the paradigm for algae treatment to produce MCFAs, it was desirable to couple the inherent ability of anaerobic microorganisms to utilize algae as the substrate for anaerobic fermentation with their ability to perform CE under ED stimulation. The algae and ED can then be upgraded into high-value organic chemicals. However, efficient transformation of algae and ED were inevitably impeded by the bioavailability of organic substrates and the microbial toxicity of accumulated products

[0025] , On the other hand, the competitive pathways, such as EEO and acrylate pathway, have been identified as the hindering factors for efficient utilization of ED in this study based on the product profiles shown in FIG. 7. Thus, it was crucial to evaluate the efficiency of the substrate utilization in the conjunction with ED during the fermentation processes. FIG. 8 displayed the degradation percentages of algae based on the measured COD compositions before and after fermentation. The highest algae COD conversion was observed in E1, which was corresponding to the total fermentation product yield in FIG. 7A.

[0225] As EDs acted as the precursors for MCFAs production, their consumption and structure were linked with the compositions and concentrations of the final fermentation products. FIG. 7B showed the variations of ethanol and lactic acid concentrations before and after fermentation in the 5 fermentation groups. The total ED consumptions in each experimental group were 9234.0 ± 829.3 mg COD / L (E1), 5547.2 ± 117.7 mg COD / L (E2), 7321.1 ± 181.6 mg COD / L (E3), 6600.7 ± 152.2 mg COD / L (E4), and 8186.4 ± 151.6 mg COD / L (E5), respectively. Notably, the potential conversion of ethanol and lactic acid to acetate and propionate would pose effects not only on CE efficiency of ED, but also on the acid fermentation of algae. This is because algae were the additional source of EA. To quantify the potential conversions of algae to simple fatty acids, the carbon fluxes of the fermentation process with sole ethanol and sole lactic acid participation were comprehensively analysed, as depicted in FIG. 9. Acetate and propionate were the initial products of fatty acids, which would participate in CE and be gradually upgraded to MCFAs at the expense of ED. The corresponding calculation details were listed in Table 1 and Table 2. The computation results show that 10.3% of total ethanol and 44.2% of total lactic acid were utilized in the EEO and acrylate pathways, respectively. This contributed to an overall ethanol utilization efficiency of 89.7% and a lactic acid utilization efficiency of 55.8%. Therefore, lactic acid was more prone to be converted to simple fatty acids by the competitive pathway compared to ethanol. This is one of the key reasons for the lower MCFAs yields in the fermentation bioreactors containing lactic acid.

[0226] Fitting of an empirical kinetic model for MCFAs production profiles. To explore the kinetic characteristics of MCFAs accumulation in the fermentation systems under different ED introduction, a dynamic model was introduced to simulate the trends of actual MCFAs profiles in this study, with the fitting parameter of R2>0.9 in all groups. E1 exhibited the highest fitting potential (2516.8 ± 67.3 mg COD / L) and production rate of total MCFAs (259.2 ± 24.6 mg COD / L / day), verifying the excellent stimulation effects of ethanol for CE in anaerobic algae fermentation systems (FIG. 10). The order of the fitted total MCFAs potentials and the associated production rates for the fermenters incorporating lactic acid as ED was as follows: sole lactic acid > E: L=1:1 > E: L=2:1 > E: L=1:2. The results of the kinetic simulations were consistent with the trends of overall MCFAs accumulation profiles, demonstrating that single type of ED was more effective in algae fermentation to produce MCFAs than a mixture of ED.

[0227] Thermodynamic evidence for the algae fermentation performances with different ED involvement.

[0228] According to the second law of thermodynamics, microbial growth can only be guaranteed if the biochemical reactions are exergonic. The investigation of thermodynamic properties of different fermentation paradigms can then reflect the feasibility of bioreactions in this study. Based on the accumulated product distribution under different ED conditions, theoretical biochemical reaction equations were constructed using the molecular formulas of algae and the molecular ratios of algae to ethanol or lactic acid in different fermentation systems. Notably, fermentation products with COD contribution greater than 5% were considered as major products, being the components of the stoichiometric reactions. Under the anaerobic condition of 35 °C and pH 5.5, the theoretical biochemical reactions in the algae fermentation systems with different ED introductions all exhibited negative Gibbs free energy (FIG. 11), thereby confirming the rationality of the major product compositions in the actual fermentation experiments.

[0229] To further assess how stoichiometric relationships between algae and ED affected product selectivity in different fermentation systems, chemical reaction equations were constructed separately for the conversion of substrates and ED into only SCFAs, only MCFAs, and only alcohols. Acetate, caproate, and butanol were the representative SCFAs, MCFAs, and alcohols for this stoichiometric analysis, respectively. Afterwards, the theoretical Gibbs free energies associated with these three pathways were compared. Overall, the product compositions can be reflected by the variations in Gibbs free energies of these model chemical reactions (FIG. 12A-E). The highest free energy release was achieved when algae were exclusively converted into alcohols in the ethanol-fed fermentation system, and the second highest is the conversion of algae to caproate in the same situation (FIG. 12A). This result aligned with the high yield of MCFAs and alcohols in the corresponding experiment group. For the mixed-ED-fed case where the proportion of ethanol was higher than lactate, more acetate and butanol instead of caproate were produced from algae (FIG. 12B), which verified the previous observed insufficient MCFAs production regardless of the considerable alcohol yield. Under the experimental conditions where the COD ratio of ethanol to lactate were 1:1 and 1:2, the alcohol pathway was calculated to possess the highest absolute value of Gibbs free energy (FIG. 12C & 12D), consistent with the obvious accumulation of butanol in the final products. For the sole lactic acid involvement group, the substrate of algae was more inclined to SCFAs rather than MCFAs (FIG. 12E). Thus, compared to lactic acid, sole ethanol emerged as more efficient ED for CE process targeting MCFAs. Herein, the methodology proposed for thermodynamic analysis allowed for the mechanistic investigation of product selectivity in complex biochemical reaction networks, thus providing a theoretical foundation for the optimization of efficient and reliable biochemical production.

[0230] Key microbiota in algae fermenters under different ED conditions.

[0231] To evaluate the genomic variations of the algae fermentation systems with different ED participation, microbial samples were extracted from E1, E3, and E5 respectively. E3 was the fermentation group with the most effective MCFAs accumulation among Co-ED groups. The relative percentages of sequences assigned to taxa or functions can be compared to further elucidate the sources of distinctive MCFAs production performances in different microbial samples. After taxonomy annotation, PCA analysis was performed based on beta-diversity to quantify the differences in microbial community compositions among the microbial samples. Specifically, the dissimilarity was visualized in FIG. 13A through the distances between the scatter points, where 2 principal coordinates (PC1 and PC2) with the overall species variation of 84.9% + 15.1% were utilized. The scatter points were dispersed along the PC1 and PC2 directions, indicating the obvious differences in the species annotation of the genomes.

[0232] The top 10 phyla identified herewith were commonly observed in the anaerobic fermentation systems (FIG. 13B). Among them, the phylum Firmicutes accounted for the highest sequence percentage in all the samples, which was reported as a consortium of acidification and CE-related microorganisms

[0024] , The corresponding gene abundance in Ethanol (E1) was 30.6%, while 25.5% and 23.9% of the genome in Co-ED (E3) and Lactic Acid (E5) samples were assigned to be phylum Firmicutes, respectively. This variation might explain the highest MCFAs yield in E1 group in the actual experimental results. The similar final MCFAs concentrations in E3 and E5 groups may also be explained by the associated microbial structure. Other abundant phyla, including Proteobacteria

[0026] , Chloroflexi, Actinobacteria, Acidobacteria

[0027] , Candidatus_Aminicenantes and Bacteroidetes

[0028] , were associated with the production of SCFAs in anaerobic systems. The total gene abundances of these SCFAs producers in the three samples were 52.8%, 58.9%, and 56.7%, respectively. Planctomycetes, the annamox microorganism, also existed in the microbial samples (with the gene abundances in the range of 3.7% to 3.8%), which implied the potential for converting N element to ammonium and the subsequent annamox within algae fermentation. The taxonomy annotation results can only provide preliminary evidence for genome analysis, a detailed analysis of function annotation in the microbial networks was therefore performed to better understand the mechanisms underlying the differential MCFAs profiles in different algae fermentation paradigms.

[0233] Functional traits of microbiota in algae fermenters under different ED conditions. The non-redundant gene sets extracted from various microbial samples in algae fermentation systems could exhibit a diverse array of functional characteristics, as depicted in FIG. 14. In general, the functions were categorized into metabolism, cellular respiration, genetic information processes, signalling and others. The high proportions of relevant sequences for these functions confirmed the basic cellular growth and metabolic capabilities of all algae fermentation systems under anaerobic conditions.

[0234] According to the functional annotated against KEGG database, the metabolic pathways detected in the anerobic algae fermentation system included acidification pathway, acrylate pathway, ethanol oxidation, MCFAs pathway, and alcohol pathway (FIG. 15). Notably, fatty acids biosynthesis (FAB) pathway, one representative metabolism related to CE process, exhibited the highest gene abundance, confirming the ability of the anaerobic systems to produce MCFAs. The corresponding total gene abundances in ethanol, Co-ED, and lactic acid samples were 0.17%, 0.17%, and 0.12%, respectively. Reverse beta oxidation (RBO) pathway, constituting the other significant pathway for CE

[0012] , was identified in the three microbial samples by the sequences annotation, with the highest abundance of the gene sequences being observed in the Lactic acid sample. The relative abundance of RBO-related genes in the E5 sample was 166.4% and 152.6% of those in E1 and E3 samples, respectively. This finding revealed that the variations in MCFAs production under different ED conditions might be attributed to the distinct functional pathways associated with CE. The dominant genes also included those responsible for the conversion of carbohydrate and amino acid to pyruvate, forming the essential step for fatty acids formation during algae fermentation. The order of gene abundance associated with ethanol oxidation and alcohol pathways (Ethanol > Co-ED > Lactic acid) was aligned with the observed fermentation profiles, as described previously. Also, this observed order was also in line with the associated microbial community observed in this study. Furthermore, although the proportion of gene sequences related to acrylate pathway remained at low levels, these genes may be highly expressed, as gene abundance was not necessarily linked to gene expression. Based on the carbon flux calculation, a significant percentage of lactic acid consumed by E5 group was confirmed to be transformed into propionate. The above analysis concerning microbial metabolic network provided detailed information for the distinctive algae-derived MCFAs production under different ED conditions from the perspective of microbial functions.

[0235] Discussion

[0236] A systematic analysis was conducted to optimize the ED components and their relative contents on the performances of algae fermentation systems specifically focusing on MCFAs recovery. This analysis not only examined the performances of these systems but also delved into the underlying mechanisms at play. Basically, sole-ethanol, sole-lactate and hybrid-ED composed of ethanol and lactate were the main components. Considering the saturated MCFAs yields, sole-ED exhibited better stimulation performances for MCFAs accumulation. However, in the anaerobic systems for fermenting liquor-making wastewater [3] and lactose

[0029] , Co-ED was suggested to be superior in participating in MCFAs production. This was mainly because that CO2 derived from lactic acid can be used for the growth of ethanol-based CE microorganisms. The reutilization of gas byproducts (i.e., H2 and CO2) via homoacetogenesis was the additional reason for the high MCFAs production under Co-ED situation. The differential effects of ED on the MCFAs accumulation from liquor-making wastewater and algae originated from the distinctions of organic composition in the two substrates. In contrast to liquor-making wastewater, algae serves as a more complex reservoir containing various organic compounds such as proteins, carbohydrates and lipids. In the proposed paradigm of algae anaerobic fermentation for the production of MCFAs observed herewith, fundamental anaerobic fermentation of algae was coupled with CE process. Thus, the beneficial effects of Co-ED for CE cannot be effectively addressed if the acidification at the front ends failed to provide sufficient EA during the early sage of bioreactor operation. Furthermore, with excessive ED and inadequate EA, ethanol and lactic acid would undergo EEC and acrylate pathway to firstly ensure SCFAs provision. The hybrid ED system, originally possessing a stronger CE potential, would not be capable of enhancing MCFAs production because of this negative feedback regulation.

[0237] In different single ED systems, ethanol was observed to be more conducive for MCFAs production than lactic acid. This distinction can be supported by the difference in substrate utilization, ED utilization, and thermodynamic feasibility within the overall anaerobic systems. In addition to the high yields of MCFAs, longer-chain alcohols also comprised an important component in the total liquid fermentation products. As important industrial chemical platform and fuels

[0030] , these alcohols can greatly enhance the environmental sustainability of product recovery during algae fermentation. The MCFAs accumulation performances in the Co-ED systems also exhibited obvious variations, among which the equivalent COD of ethanol and lactic acid contributed to the highest MCFAs yields. Hence, it can be inferred that there was a balance among the utilization of EA, the selection of CE process, and the regulatory role in the front-end acidification step for ethanol and lactic acid bioconversion. The balance and feedback regulation of hybrid ED proposed by this study can also provide an optimization basis for high-value liquid biofuel production from the perspective of ED utilization.

[0238] When ethanol participated in the CE process, the bioconversion of fatty acids with n carbon atoms to fatty acids with 2 more carbon atoms requires net equimolar amounts of ethanol to provide the carbon source. The same goes to lactate-based CE process

[0031] , Therefore, to gradually upgrade simple fatty acids such as acetate and propionate to MCFAs, increasing the participation of ethanol or lactate is required under stepwise accumulation. From the carbon flux calculation (FIG. 9), the overall material conversion indicated that the algae providing EA through acidification contributed less to the product COD compared to ED. Considering this aspect, the economic concerns should be focused on the exogenously addition of ED for energy upgrading. Moreover, the environmental burdens that commercialize ethanol production process could be incorporated with the algae fermentation for MCFAs production through its involvement as ED

[0032] , The dual considerations of environmental and economic sustainability have propelled the emergence of future research focal points, aiming to explore whether it is feasible to engineer the in-situ utilization of organic compounds within substrates as ED to achieve the maximum COD high-value utilization from waste streams such as algae.

[0239] Conclusions

[0240] This example emphasized the importance of the compositions of ED, the essential precursor of the CE process, for maximizing the MCFAs production from anaerobic algae fermentation. In the microbial systems, both sole-ED (with only ethanol or lactic acid involvement) and hybrid-ED (with both ethanol and lactic acid involvement) were investigated. The products profiles indicated that sole ED, especially pure ethanol addition, was more conducive to high MCFAs yields. Subsequent calculations of ED utilization efficiency for CE process, dynamic analysis, and thermodynamic characterizations all aligned with the varying trends of MCFAs accumulation, as well as the final byproducts (i.e., SCFAs and alcohols) concentrations. According to the taxa and function annotation of genomes, the complex microbial network regarding the pathways during algae anaerobic fermentation, establishing a foundation for gaining insights into the functional differences among microbial samples affected by different ED. Overall, this example represents a fundamental exploration in the resource reutilization of algae, thus providing a solid theoretical cornerstone for the enhancement and widespread application of this biotechnology while addressing the environmental burdens induce by the algae partially.

[0241] Example 2 - Bioaugmentation of algae fermentation with yeast to improve microbial chain elongation performance

[0242] In order to improve the performance of algae fermentation and decreasing the cost of electron donor in biorefining microalgal biomass within a circular loop, the inventor has developed a novel integrated process that involves in-situ production of both electron donor and acceptor from algae through bioaugmentation of algae fermentation with yeast. The feasibility of this innovative system for the production of medium-chain carboxylic acids was assessed. Subsequently, metagenomic analysis was utilized to elucidate the microbial community structure and metabolic potentials within this novel process.

[0243] Materials and Methods Source of algae, inoculum and yeast

[0244] Algae were obtained from Huachu Trading Co., Ltd. (Henan, China) and were stored in a refrigerator at -20 °C before use. The main species of algae was Chlorella. The total suspended solids (TSS) of algae were 52.43 ± 0.06 g / L and its volatile suspended solids (VSS) were 48.82 ± 0.04 g / L. Prior to the tests, the algae were diluted to a VSS concentration of 15 g / L using tap water. The total chemical oxygen demand (COD) of diluted algae was 22.56 ± 10.64 g / L, which had a composition of 55.1% ± 5.2% protein, 17.5% ± 3.1% total carbohydrates.

[0245] The inoculum used in the study was anaerobic digestion sludge (ADS) and obtained from our lab-scaled mesophilic reactor, which was used to treat excess activated sludge. To remove residual organics, the sludge was self-digested for a minimum of a month without any external organic supply. The final inoculum had TSS and VSS concentrations of 46.26 ± 0.29 g / L and 20.01± 0.16 g / L, respectively. The commercial dry baker’s yeast used in the study was purchased from Angel Company (China) and consisted primarily of Saccharomyces cerevisiae.

[0246] Anaerobic algae fermentation protocol

[0247] Anaerobic algae fermentation was conducted using a short-term batch experiment in 150 mL serum flasks. Specifically, two yeast groups were prepared by combining 36 mL of algae, 64 mL of ADS, and 2 g dry yeast, resulting in a total volume of 100 mL. This achieved a substrate-to-inoculum volatile solids (VS) ratio of 0.5. Two control groups were also included, identical to the experimental groups but without yeast. To assess the fermentation performance of the inoculum and saccharomyces cerevisiae, four blank groups were set up, replacing algae with tap water. The NaOH and HCI solutions (3 M) was used to adjust pH of fermentation broth to 5.0 ± 0.1. After each sampling, the pH was also adjusted manually to maintain 5.0. The pH was relatively stable throughout the fermentation (<0.5 units change), indicating limited fluctuations during the experiment. The pH of 5.0 was chosen based on its demonstrated optimal chain elongation performance in the pre-experiment. To maintain anaerobic conditions, pure nitrogen was purged to the headspace of reactor for 15 minutes after sampling. Subsequently, all bottles were placed in an incubator shaker at 35.0 ± 0.1°C and 130 rpm for the fermentation process.

[0248] Chemical analytical methods

[0249] The head gas sample (1 mL) was collected by syringe with seal valve and then was injected into a gas chromatograph (GC-112A, China) equipped with a thermal conductivity detector (TCD) to analyses the concentration of H2, CO2 and CH4. Simultaneously, the liquid sample (2 mL) was collected, centrifuged and filtered. A 5 pL / mL-sample of HCOOH was added to ensure the carboxylic acids as free molecules. A gas chromatograph (GC-2010 Plus, SHIMMADZU, Japan) was used to determine the carboxylic acids and butanol. This gas chromatograph was equipped with a flame ionization detector (FID) and a capillary column (SH-Stabllwax-DA, 30 m x 0.32 mm x 0.25 pm).

[0250] The chemical oxygen demand (COD) was measured using the fast digestion-spectrophotometric method. The VS and TS were analyzed using the same methods as previous research

[0033] , The concentration of protein and reducing sugar in the algae was determined by reformative Lowry method and the 3,5-dinitrosalicylic acid colorimetry

[0034] ,

[0251] DNA extraction and metagenomic sequencing

[0252] Twenty milliliters anaerobic sludge samples collected from two duplicate fermentation bottles of both the control and experiment groups were evenly mixed for genomic DNA extraction. Following the completion of experiment, the liquid sample underwent harvesting through centrifugation. The E. Z. N. A. Soil DNA Extraction Kit Genomic DNA extraction kit was used to extract genomic DNA in accordance with the manufacturer's guidelines. To assess the purity of the extracted DNA, absorbance measurements (A=260 nm, A=280 nm) were conducted using a NanoDrop spectrophotometer (USA). Subsequently, the Covaris M220 (Covaris, USA) was utilized to fragment the DNA to achieve a target size of approximately 400 bp. Subsequently, the NEXTFLEX™ Rapid DNA-Seq Kit was used to metagenomic library preparation. The resulting DNA library was subjected to paired-end sequencing (2 x 150 bp) on the Illumina Hiseq4000 platform (Illumina, USA).

[0253] Metagenomic analysis

[0254] Metagenomic sequencing reads underwent meticulous processing. Fastp (v0.20.0) was employed for trimming and adaptor removal. To ensure the accuracy of metagenomic analysis by preventing interference from the yeast genome, Bowtie 2 (v.2.3.5.1) was used to remove the genome of Saccharomyces cerevisiae, obtained from NCBI (GCF_000146045.2). Subsequently, the profiling of the microbial community composition was analysed by MetaPhlAn 4.0 (v4.0.6). For an in-depth exploration of metabolic potentials, the trimmed reads were assembled with MEGAHIT (v1.1.2). Prodigal (v2.6.3) was employed for predicting protein-coding genes. All predicted genes were merged and clustered with CD-HIT (v4.6.1, -aS 0.9, -c 0.9) to construct non-redundant gene sets. Reads after quality control were mapped back to the non-redundant gene sets with 95 % identity using salmon (1.10.2), and the reads count were used to calculate the gene abundance, expressed as transcripts per kilobase per million mapped reads (TPM) in each sample. All of the genes were taxonomically aligned to EggNOG and dbCAN2 by Diamond vO.8.35.

[0255] Results

[0256] Yeast bioaugmentation effectively prolonged the carbon chain of fermentation products into medium-chain carboxylic acids The final concentrations of the main products in the control and yeast groups are shown in FIG. 16A and 16B. Algae showed a feasible fermentation performance for carboxylate production. In the single algae fermentation, the total concentration of carboxylate was 54.0 ± 9.9 mM-C during 25 day- fermentation. The addition of yeast significantly increased the concentration to 184.6 ± 16.2 mM-C. Among them, acetate, butyrate, and valerate concentrations in the yeast group were 3.9-fold, 2.1 -fold, and 2.4-fold higher, respectively, compared to the control group. This enhancement would be attributed to the in-situ production of ethanol by the yeast fermentation. FIG. 16C illustrates the ethanol concentration in the yeast group during the first nine days, peaking at 28.2 ± 0.4 mM-C. The concentration of reducing sugar in algae was 3.95 g / L. In the yeast group, the ethanol yield was 0.45 g / (g reducing sugar) near the theoretical ethanol yield of saccharomyces cerevisiae which indicated the yeast plays a crucial role in the in-situ production of ethanol.

[0257] The consumption of ethanol during subsequent fermentation was linked to the accumulation of butyrate and caproate, signifying the involvement of the rBOX in the chain elongation of carboxylic acids. However, the accumulated acetate (93.93 ± 14.80 mM-C, accounting -37% of the COD fraction in the end products) was observed in the yeast group. Acetate accumulation would be attributed to the inhibition of CE in the acidic conditions (pH 5.0) and the toxicity of products

[0035] , A transient acetate accumulation was observed in our research, possibly from ethanol oxidation

[0036] , The produced ethanol serving as the electron donor to elongate the carboxylic acids into the longer carbon chain carboxylic acids, particularly caproate (from 0 to 21.9 ± 2.7 mM-C) and caprylate (from 0 to 4.6 ± 0.4 mM-C). Besides, the butanol production was also observed in the yeast group, with 1.9 ± 0.2 mM-C. The butanol production would also be attributed to the ethanol consumption. In this process, ethanol can serve as the reducing power to transform butyrate into butanol via butanol dehydrogenase (bdh) or alcohol dehydrogenase (adh) under reduced environments [37, 38],

[0258] In the control groups (n = 2) without added yeast, the fermentation broth exhibited the presence of SCCA metabolites, while no medium-chain carboxylic acid (MCFA) was detected throughout the 25-day fermentation period. The absence of significant metabolites in the absence of added yeast, along with the carbon and electron balances, supports MCFA and butanol produced through CE facilitated by added yeast, instead of the oxidation process from the algae biomass.

[0259] The addition of yeast significantly increased the fermentation performance, the total concentration of carboxylic acids increased by 4-fold compared to control group (FIG. 16D). In addition, the accumulative hydrogen production was also increased from 0.08 ± 0.01 mmol to 0.58 ± 0.02 mmol, indicating the better chain elongation performance in the yeast group. Notably, the enhanced carbon dioxide production was also detected in the yeast group, indicating the ethanol production was mainly due to the yeast fermentation. Whatever, the production of butyrate and valerate were still produced via chain elongation, however the electron donor was some amino acid hydrolyzed from proteins in the algae and the lower reduced power hindered the carbon chain further elongating

[0039] ,

[0260] FIG.16E demonstrated the COD fraction of the main fermentation products. During the period of the batch experiments, the control groups converted 24.21% ± 0.55% of total substrate COD to carboxylic acids. In comparison, yeast group utilized 83.03 ± 0.80% of algae COD to ferment. Specifically, the average conversion of COD in algae to MCFAs was 14.79 ± 0.22%, and MCFAs accounted for 18.43 ± 1.83% of the total carboxylic acids produced. The results indicated that the addition of yeast increased the substrate utilization rate and direct the metabolic potential towards chain elongation.

[0261] Yeast bioaugmentation shaped microbial community structure for chain elongation The community composition at the phylum level in the control and yeast groups is presented in Fig. 17. Specifically, the microbial community structures were primarily influenced by the proliferation of bacteria belonging to the Firmicutes phylum, attributed to ethanol production, resulting in a rise in relative abundance from 3.0% to 17.2%. Firmicutes has been previously documented to exhibit high prevalence in chain elongation systems fueled by ethanol and carboxylic acids

[0040] , The enrichment of Firmicutes has consistently been observed in labscale bioreactors engaged in chain elongation, utilizing both simple substrates and complex feedstocks. In addition to Firmicutes, other phyla displayed varying degrees of increase, except for Bacteroidetes (decreased from 62.4% to 26.5%) and Ignavibacteriae (decreased from 2.3% to 1.8%).

[0262] Given the community composition with the addition of yeast, the differences of microbial structure between control and yeast groups at Genus level was further examined (FIG. 18A). The most of genus have been enriched at different extent especially the acidogenic microbes including Clostridium, Lactococcus, Enterococcus etc. Notably, Clostridium belongs to Firmicutes have significant increased from 1.7% to 8.8% due to its chain elongation ability

[0041] , After investigating the specific species, one identified species (FIG. 18B), C. butyricum was significantly increased in relative abundance under yeast-existence CE. C. butyricum, is a strictly butyrate-producing bacterium

[0042] , The increased acidogenic microbes explained the enhanced performance for carboxylic acid production. In addition, some reported gas-independent Genus have also enriched in the yeast groups such as Thermomonas which can utilize hydrogen to stabilize pH level of fermentation borth

[0043] , Methanothrix was also increase from 1.0% to 1.7% which was reported to use carbon dioxide

[0044] , Considering the higher head concentration of hydrogen and carbon dioxide in the yeast groups, these microorganisms can utilize them to growth indicating the shaped microbial structure can utilize multiple substrates to increase the algae utilization rate. Also, some genus which can directly utilized algae or yeast cells were increased such as Anaerolinea (0 to 0.02%) [45, 46], The increase of this Genus was attributed to the increased algae utilization rate in the yeast group.

[0263] The metabolic potentials in this bioaugmentation process

[0264] o Metabolic potentials to utilize complex carbohydrates

[0265] Carbohydrates were the most attractive substances in our study, as they accounted for a large proportion of the algae COD (-17.5%) and was verified to sufficiently convert to MCFA in the previous research. Therefore, to investigate the substrate utilization performance, the CAZyme database was used to analyze the microbial metabolic potentials to degrade complex carbohydrates. The glycoside hydrolases (GHs) class was of particular interest in our study because they can release sugars that can be easily metabolized by microorganisms as well as yeast that do not express complex carbohydrate-degrading enzymes. The results showed that the yeast group has a better carbohydrates hydrolysis potential due to a higher abundance of GHs except for GH13 (FIG. 19A, Table 4). This result indicated that the added yeast increased the carbohydrates hydrolysis potential, thereby enhancing the acid production performance. Table 4. Abundance difference in glycoside hydrolases (GH) class between yeast and control group.

[0266] Control Yeast (%) p-values Effect 95.0% 95.0% CAZy

[0267] (%) size lower Cl upper Cl 6.66315287496e- GH13 17.001 19.977 -2.975 -4.300 -1.650 06

[0268] GH5 10.891 9.391 0.004 1.500 0.465 2.534 GH9 10.099 8.598 0.003 1.501 0.502 2.500 GH3 8.039 8.144 0.827 -0.105 -1.044 0.834 GH31 7.465 7.625 0.723 -0.160 -1.071 0.750 GH29 3.873 2.941 0.003 0.932 0.299 1.564 GH18 3.377 3.001 0.208 0.376 -0.239 0.990 GH20 3.275 2.587 0.018 0.688 0.097 1.278 GH23 3.232 4.076 0.010 -0.844 -1.500 -0.188 GH77 3.175 3.935 0.016 -0.759 -1.407 -0.112 GH33 2.729 2.210 0.049 0.520 -0.026 1.065 GH38 2.262 3.116 0.002 -0.854 -1.424 -0.284 GH51 2.188 1.991 0.440 0.197 -0.309 0.702 GH26 2.032 1.687 0.131 0.346 -0.133 0.824 GH95 1.994 1.481 0.027 0.512 0.049 0.976 GH65 1.960 1.475 0.026 0.485 0.024 0.946 GH43 1.890 1.639 0.272 0.250 -0.217 0.718 GH94 1.734 1.960 0.343 -0.227 -0.705 0.252 GH57 1.631 1.815 0.432 -0.184 -0.647 0.280 GH30 1.305 0.922 0.035 0.383 0.006 0.761 GH19 0.919 1.174 0.180 -0.255 -0.623 0.114

[0269]

[0270] GH15 0.905 0.760 0.353 0.145 -0.186 0.477 GH32 0.836 1.272 0.015 -0.436 -0.806 -0.065 GH101 0.831 1.211 0.027 -0.380 -0.745 -0.015 GH105 0.779 0.643 0.364 0.136 -0.172 0.445 GH103 0.724 1.276 0.001 -0.552 -0.914 -0.190 GH102 0.588 0.934 0.023 -0.347 -0.666 -0.027 GH116 0.532 0.480 0.720 0.052 -0.214 0.317 GH8 0.510 0.327 0.116 0.183 -0.060 0.426 GH66 0.504 0.290 0.043 0.214 -0.024 0.451 GH97 0.474 0.330 0.183 0.144 -0.095 0.383 GH39 0.417 0.592 0.151 -0.175 -0.441 0.090 GH130 0.381 0.281 0.377 0.101 -0.119 0.320 GH37 0.328 0.247 0.423 0.081 -0.125 0.288 GH6 0.268 0.267 1.000 0.001 -0.200 0.202 GH88 0.227 0.284 0.503 -0.057 -0.254 0.140 GH63 0.155 0.181 0.835 -0.026 -0.192 0.139 GH68 0.129 0.180 0.514 -0.051 -0.211 0.109 GH47 0.054 0.059 1.000 -0.004 -0.112 0.103 GH16 0.046 0.080 0.495 -0.034 -0.147 0.078 GH4 0.040 0.179 0.018 -0.139 -0.278 0.001 GH73 0.036 0.049 1.000 -0.014 -0.111 0.084 GH36 0.032 0.034 1.000 -0.003 -0.092 0.086 GH28 0.027 0.049 0.678 -0.022 -0.116 0.072 GH99 0.021 0.023 0.613 -0.003 -0.081 0.075 GH127 0.019 0.044 0.359 -0.025 -0.113 0.063 GH14 0.011 0.024 0.613 -0.013 -0.086 0.059 GH121 0.010 0.013 1.000 -0.003 -0.068 0.061 GH54 0.009 0.018 1.000 -0.009 -0.076 0.058 GH74 0.008 0.012 1.000 -0.004 -0.066 0.058 GH27 0.006 0.007 1.000 -0.001 -0.057 0.055 GH17 0.006 0.009 0.483 -0.003 -0.060 0.054 GH1 0.006 0.028 0.233 -0.022 -0.095 0.050 GH52 0.005 0.010 0.483 -0.004 -0.062 0.053 GH59 0.005 0.032 0.233 -0.027 -0.102 0.048 GH84 0.000 0.013 0.483 -0.013 -0.069 0.043

[0271]

[0272] GH79 0.000 0.016 0.483 -0.016 -0.076 0.043

[0273] o Metabolic potentials for chain elongation

[0274] In addition, the microbiome gene abundance data was analyzed to predict the chain elongation potential between control and yeast groups. FIG. 19B and Table 5 show the whole acyl-related gene for chain elongation. The results indicated that the addition of yeast significantly increased some of the key genes including cetyl-CoA C-acyltransferase (A CAT), acyl-CoA dehydrogenase (ACD) and alcohol dehydrogenase (ADH). These genes were responsible for the conversion of SCCA and ethanol to the corresponding acyl-CoA (FIG. 20). Acetyl-CoA is an essential intermediate product in the rBOX process of acetate and ethanol. The conversion of acetate to acetyl-CoA can through two pathways, 1) a one-step pathway involving ATP-dependent acetyl-CoA synthase (ACS) or 2) a three-step pathway involving acetate kinase (ACK), phosphate acetyltransferase (PTA) and CoA transferase (CoAT)

[0047] , Our study revealed a higher abundance of these genes in the yeast group compared to the control group. The lack of statistical significance in the obtained results could be ascribed to the relatively brief cultivation period (29 days) in the batch experiments. This limited timeframe may not have allowed for substantial substrate utilization, resulting in an insufficient shaping of the microbial structure, especially when compared to continuous reactor conditions. To holistically explore the influence of yeast addition on the chain elongation process, it is imperative to conduct metagenomic and metatranscriptome analyses within a continuous reactor setup. This approach will provide a more in-depth understanding, as the continuous reactor model offers a more stable environment for microbial interactions and metabolic processes.

[0275] Table 5. Abundance difference in key gene for MCCA and butanol production between control and yeast group.

[0276] Pathway Enzyme Control Yeast p-values Effect 95.0% 95.0% Enzyme (%) (%) size lower upper Cl descriptio Cl n 2.3.1.16 0.112 0.127 0.110146 -0.015 -0.033 0.003 ACTA 2.3.1.9 0.335 0.386 0.001335 -0.052 -0.083 -0.020 ACTA 4.2.1.17 0.302 0.326 0.118167 -0.024 -0.053 0.006 ECH 4.2.1.55 0.013 0.012 0.807558 0.001 -0.005 0.007 ECH 6.2.1.1 0.192 0.208 0.185745 -0.016 -0.039 0.008 ACS 1.1.1.157 0.115 0.099 0.084022 0.016 -0.002 0.033 HAD 1.1.1.35 0.178 0.191 0.234185 -0.014 -0.036 0.009 HAD 1.3.8.1 0.071 0.082 0.188923 -0.010 -0.025 0.005 ACD 1.3.8.7 0.239 0.308 7.66561687411e-07 -0.070 -0.097 -0.042 ACD 1.3.8.8 0.010 0.014 0.225593 -0.004 -0.010 0.002 ACD rBOX

[0277] genes 1.1.1.1 0.174 0.204 0.010159 -0.030 -0.053 -0.007 ADH 2.7.2.1 0.052 0.056 0.525362 -0.004 -0.017 0.008 ACK 2.3.1.8 0.058 0.059 0.824607 -0.001 -0.014 0.011 PTA 1.2.1.10 0.028 0.034 0.286199 -0.005 -0.015 0.004 ADA 2.8.3.1 0.025 0.027 0.801954 -0.001 -0.010 0.007 CoAT 2.8.3.8 0.024 0.012 0.001175 0.012 0.005 0.019 CoAT 2.8.3.9 0.023 0.012 0.002579 0.011 0.004 0.018 CoAT 3.1.2.20 0.011 0.010 0.896136 0.001 -0.005 0.006 TE 1.5.5.1 0.044 0.052 0.177445 -0.008 -0.020 0.004 ETF 1.12.2.1 0.003 0.004 0.815287 -0.001 -0.004 0.002 Ech 1.12.7.2 0.013 0.016 0.318827 -0.003 -0.010 0.003 H2ase 1.1.1.1 0.174 0.204 0.010159 -0.030 -0.053 -0.007 ADH 1.12.1.4 0.009 0.006 0.262117 0.003 -0.002 0.007 Hyd Butnaol

[0278] production 1.18.1.2 0.079 0.083 0.602820 -0.004 -0.019 0.011 Nfn 1.19.1.1 0.077 0.080 0.631089 -0.004 -0.018 0.011 Nfn

[0279]

[0280] 1.2.1.3 0.221 0.197 0.063404 0.023 -0.001 0.047 ALDH 1.2.7.1 0.225 0.231 0.611893 -0.006 -0.032 0.019 PFOR 1.2.7.5 0.121 0.129 0.361054 -0.008 -0.027 0.010 AOR 2.3.1.8 0.058 0.059 0.824607 -0.001 -0.014 0.011 PTA

[0281]

[0282] 2.7.2.1 0.052 0.056 0.525362 -0.004 -0.017 0.008 ACK

[0283] In both the control and yeast groups, a small number of genes involved in ferredoxin hydrogenase production were detected, providing support for the hypothesis that H2 production contributes to the MCFA producers. Additionally, we investigated the presence of two other hydrogenases, EchABCDEF and HydABC, which are known to generate energy through proton translocation or electron confurcation using nicotinamide adenine dinucleotide hydrogen (NADH) and reduced ferredoxin, respectively

[0048] , These genes encoding the known components of these enzyme complexes, indicating that these systems likely play a significant role in H2 production in these microbiomes (FIG. 19B and 19C, Table 5).

[0284] o Metabolic potentials for butanol production

[0285] Two distinct pathways are responsible for the butanol production from butyryl-CoA as illustrated in FIG. 20 and Table 5 [38, 49], In the direct pathway, butyryl-CoA is enzymatically converted to butyraldehyde and subsequently reduced to butanol by a bifunctional alcohol dehydrogenase (ADH)

[0050] , On the other hand, the indirect pathway involves the initial conversion of butyryl-CoA to butyrate, with the intermediate butyryl phosphate (butyryl-P) formed through the action of PTA. The subsequent step involves the transformation from butyryl-P to butyrate by ACK, leading to the production of ATP via substrate-level phosphorylation

[0051] , Undissociated butyric acid is reduced by an aldehyde:ferredoxin oxidoreductase (AOR) using electrons from reduced ferredoxin (Fdred), leading to the formation of butyraldehyde

[0052] , The changes in the relative abundance of these genes are depicted in FIG. 19C. The results demonstrate that the addition of yeast significantly increased the abundance of ADH genes from 0.17% to 0.20%. The yeast group showed a decrease in ALDH abundance and a slight increase in AOR genes, suggesting that the addition of yeast altered the metabolic potential of the microbiome towards the indirect pathway for butanol production. Furthermore, the higher concentrations of acetate and butyrate in the yeast group indicate a potential increase in AOR activity compared to the control group. In prior studies, it has been documented that the introduction of exogenous acetate can induce a further shift in the product composition towards alcohol in specific acetogens [51, 53], Richter et al. (2016) provided evidence indicating that the production of alcohol is governed by thermodynamic principles rather than transcriptional or translational regulation. Although their model was initially proposed to elucidate the factors influencing ethanol production, its applicability extends to our findings of enhanced butanol production, considering the involvement of similar enzyme systems

[0054] ,

[0286] Discussion This study presents pioneering evidence of yeast significantly enhancing the production of carboxylates and butanol from algae biomass. The addition of yeast resulted in a fourfold increase in total carboxylate concentration, reaching 184.6 ± 16.2 mM-C compared to the control group. Given the absence of ethanol production in the control group, the enhanced performance can be entirely attributed to the in-situ production of ethanol by yeast. This ethanol not only acted as an electron donor for chain elongation but also served as a reducing force for butyrate reduction, elucidating the observed enhanced performance in the experimental group. This synergistic action effectively prolonged the carbon chain of fermentation products into MCFAs with a total concentration of 28.0 ± 3.4 mM-C and butanol with a concentration of 1.9 ± 0.2 mM-C.

[0287] Previous research has delved deeply into the influence of ethanol on chain elongation and alcohol synthesis. For example, Wu etal. achieved an MCFA yield of approximately 19.8 mM-C / g VS by utilizing waste activated sludge alkaline fermentation liquid as a substrate with the addition of 226.6 mM ethanol

[0055] , Similarly, Wang et al. obtained a caproic acid yield of 53.6 mM-C / g VS by using 651.0 mM ethanol and anaerobic fermentation liquid of sewage sludge as substrates

[0056] , In our study, we achieved an MCFA yield of 46.3 mM-C / g VS without the addition of any extra ethanol, demonstrating the feasibility of this novel process for MCFA production. It is imperative to note that equivalent MCFA yields do not necessarily indicate identical fermentation efficiencies. Elevated ethanol concentrations could potentially instigate excessive ethanol oxidation, subsequently providing an abundance of electron acceptors for chain elongation

[0057] , Consequently, the high MCFA yield might predominantly originate from ethanol rather than biowastes. In our methodology, external ethanol supplementation was circumvented, with the requisite ethanol for chain elongation being intrinsically generated from algae by the yeast. This resulted in an augmented substrate utilization efficiency, bolstering the economic viability of the yeast-mediated chain elongation system.

[0288] In addition, the high accumulation of acetate suggests a significant inhibition in chain elongation, likely attributable to the weak acidic conditions present. The COD fraction of SCCAs among total fermentation derivatives, representing approximately 70% (FIG. 16E), points to suboptimal chain elongation efficacy. This diminished efficacy can be ascribed to the pH conditions, specifically those below pH 5, which predominantly favor ethanol synthesis through yeast fermentation

[0058] , The pH plays a pivotal role in determining the primary products in the chain elongation system. Studies have demonstrated that neutral pH conditions are conducive to MCFA synthesis [59, 60], Nonetheless, a mildly acidic environment is advocated to suppress the methanogenic pathway, thereby minimizing the need for methanogenic inhibitors. A salient limitation of acidic environments in the chain elongation system is the pronounced product toxicity to the chain elongator, even at minimal MCFA concentrations. Furthermore, research confirmed that upon achieving a certain concentration of MCFA, the methanogenic pathway could also be inhibited due to the inherent toxicity of MCFA, even in a neutral pH setting

[0061] , While certain studies reported elevated MCFA yields under acidic conditions, these were primarily attributed to specific acidic-tolerant species [62, 63], Our investigation, however, did not identify such species in our reactor, potentially due to an incompatible substrate. Consequently, considering the methanogenic inhibition and the pH discrepancies between yeast and chain elongation, subsequent research could enhance MCFA production rates by segregating the yeast fermentation and chain elongation processes, operating them under distinct pH regimes.

[0289] The enhanced performance can be attributed to the growth of chain elongators (Clostridium). The utilization of gases by some species also increased the substrate utilization rate. The addition of yeast directed the conversion of reducing sugars into ethanol, facilitating MCFA production through chain elongation. Moreover, the production of carbon dioxide from yeast metabolism increased the proportion of Thermomonas and Methanothrix which is known to reduce carbon dioxide through hydrogen [43, 44], This enhanced gas utilization further increased the carbon utilization rate in the yeast-induced chain elongation system. Metagenomic analysis supported these observations, showing increased abundance of genes related to acetyl-CoA production in the yeast group, while genes associated with the utilization of complex substrates were more abundant in the control group. This metabolic shift redirected the carbon and electron flow towards the production of MCFAs and butanol rather than biomass increase. In addition, the taxonomic origins analysis indicated that the most common species involved in the rBOX pathway belonged to the phyla Firmicutes, Proteobacteria, and Actinobacteria (Table 6-32). Although previous research identified various Firmicutes capable of chain elongation, few were associated with Proteobacteria and Actinobacteria

[0064] , Our study suggested that these phyla might also produce MCFAs through the rBOX pathway due to the presence of relevant genes. However, metatranscriptomic analysis should be conducted to provide further evidence.

[0290] The addition of yeast also induced the production of butanol. Metagenomic evidence suggested the significant role of theAOR pathway in butanol production. This pathway utilizes an Fd-dependent reaction to reduce organic acids to aldehydes, which are then further reduced to alcohols by NAD(P)H-dependent alcohol dehydrogenase (ADH). Several studies have indicated that the AOR pathway is the primary route for ethanol and butanol production in autotrophic or sugar-based fermentation by acetogens such as C. Ijungdahlii and C. ragsdalei. Species like C. saccharoperbutylacetonicum and C. thermoanaerobacter species with AOR and heterologous alcohol dehydrogenase (adh) genes can produce ethanol from glucose and convert exogenous carboxylic acids into their corresponding alcohol [65, 66], For example, Richter et al. achieved a butanol yield of 0.4 g / (L h) by reducing supplemented butyrate in continuous cultures of a C. saccharoperbutylacetonicum reactor

[0067] , Our metagenomic data indicated that most AOR genes were found in Proteobacteria and Chloroflexi, except for Clostridium (Table 12), suggesting more metabolic diversity than previously reported. The strong correlation between butyrate concentration and AOR abundance indicated the significant role of the AOR pathway in butanol production (Fig. 16A and Fig. 19C). The high concentration of organic acids could accelerate the regeneration of reducing equivalents, leading to faster metabolism and increased growth rates

[0068] , Additionally, higher hydrogen supply in the yeast group resulted in a higher nicotinamide adenine dinucleotide (NADH / NAD+) ratio, thereby increasing the rate of organic acid reduction. However, it remains to be determined to what extent AOR, ALDH, and alcohol dehydrogenases contribute to the reduction of organic acids to their corresponding alcohols.

[0291] This work aimed to verify the feasibility of yeast in promoting MCFA production. The integration of yeast fermentation and algae fermentation demonstrates the feasibility in resource recovery from waste algae biomass. able 6. Gene annotation (EC 1.1.1.1)

[0292] query evalue score COG.category Description Preferred.name EC yeast_k141_99806_1 8.77e-70 221 C alcohol dehydrogenase mdh1 1.1.1.1,1.1.1.61 yeast_k141 100493_ hydroxyacid-oxoacid

[0293] 2.66e-47 163 C CT0951 1.1.1.1,4.3.3.7

[0294] 1 transhydrogenase activity

[0295] Alcohol dehydrogenase GroES- yeast_k141_10237_1 5.19e-53 176 c - 1.1.1.1

[0296] like domain

[0297] yeast_k141_103797_ Iron-containing alcohol

[0298] 1.02e-228 652 c adhE 1.1.1.1,1.2.1.10 1 dehydrogenase

[0299] yeast_k141_105881_

[0300] 2.24e-52 174 c alcohol dehydrogenase - 1.1.1.1

[0301] 1

[0302] yeast_k141_111794_

[0303] 2.27e-47 162 c alcohol dehydrogenase adhA 1.1.1.1

[0304] 1

[0305] yeast_k141_112536_ 1.1.1.1,1.1.1.284,1.2.1.4

[0306] 4.46e-99 297 E Dehydrogenase adhB

[0307] 1 6

[0308] yeast_k141_114348_ Alcohol dehydrogenase GroES- 1.57e-68 219 - 1.1.1.1

[0309] 1 c like domain

[0310] yeast_k141 114840_

[0311] 2.57e-62 202 c alcohol dehydrogenase - 1.1.1.1,1.1.1.284 1

[0312] Belongs to the zinc-containing

[0313] yeast_k141_115418_

[0314] 4.23e-40 142 c alcohol dehydrogenase family. frmA 1.1.1.1,1.1.1.284 1

[0315] Class-Ill subfamily

[0316] c Iron-containing alcohol

[0317] yeast_k141_11657 1 1.46e-37 142 dhaT1 1.1.1.1,1.1.1.202

[0318] dehydrogenase

[0319] yeast_k141 _118881 _ belongs to the iron- containing

[0320] 2.98e-23 100 c adhE 1.1.1.1,1.2.1.10 1 alcohol dehydrogenase family

[0321] yeast_k141_120229_ Alcohol dehydrogenase GroES- 1.08e-105 315 c ccrA 1.1.1.1,1.3.1.85 1 like domain

[0322] yeast_k141_121963_

[0323] 4.38e-44 154 E alcohol dehydrogenase - 1.1.1.1

[0324] 4

[0325] yeast_k141_122388.

[0326] 8.49e-54 179 E alcohol dehydrogenase - 1.1.1.1

[0327] 1

[0328] yeast_k141_126145. Alcohol dehydrogenase GroES- 1.19e-67 216 - 1.1.1.1

[0329]

[0330] 1 s like domain Belongs to the zinc-containing

[0331] yeast_k141_12785_2 4.18e-230 637 C alcohol dehydrogenase family. - 1.1.1.1,1.1.1.284

[0332] Class-Ill subfamily

[0333] yeast_k141_128421_ COG1064 Zn-dependent alcohol

[0334] 1.52e-74 231 S adhP 1.1.1.1

[0335]

[0336] 1 dehydrogenases

[0337] able 7. Gene annotation (EC 1.1.1.35)

[0338] query evalue score COG_category Description Preferred_name EC

[0339] 3-hydroxyacyl-CoA

[0340] yeast_k141_641_1 7.98e-91 289 I fadB 1.1.1.35

[0341] dehydrogenase

[0342] Belongs to the enoyl-CoA

[0343] yeast_k141_479061_1 2.19e-96 301 I ech-8 1.1.1.35,4.2.1.17,5.3.3.8 hydratase isomerase family

[0344] Belongs to the enoyl-CoA

[0345] yeast_k141_630338_1 2.44e-33 129 I ech-8 1.1.1.35,4.2.1.17,5.3.3.8 hydratase isomerase family

[0346] Belongs to the enoyl-CoA

[0347] yeast_k141_732607_1 1.83e-66 220 I ech-8 1.1.1.35,4.2.1.17,5.3.3.8 hydratase isomerase family

[0348] 3-hydroxyacyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_19946_1 2.07e-30 120 I - dehydrogenase 5.3.3.8

[0349] 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_33216_1 5.95e-42 156 I enoyl-CoA hydratase isomerase MA20_44845

[0350] 5.3.3.8

[0351] 3-hydroxyacyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_46411_1 1.12e-07 56.2 I - dehydrogenase 5.3.3.8

[0352] PFAM Enoyl-CoA hydratase 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_58226_1 1.23e-105 331 I - isomerase 5.3.3.8

[0353] Belongs to the enoyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_74854_1 2.23e-35 135 I MA20_44845

[0354] hydratase isomerase family 5.3.3.8

[0355] Belongs to the enoyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_113939_1 8.44e-63 211 I MA20_44845

[0356] hydratase isomerase family 5.3.3.8

[0357] 3-hydroxyacyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_251397_1 7.11e-53 185 I - dehydrogenase 5.3.3.8

[0358] Involved in the aerobic and

[0359] anaerobic degradation of long- 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_367381_1 2.01e-10 63.9 I chain fatty acids via betafadB

[0360] 5.3.3.8

[0361] oxidation cycle. Catalyzes the

[0362]

[0363] formation of 3-oxoacyl-CoA from enoyl-CoA via L-3-hydroxyacyl- CoA. It can also use D-3- hydroxyacyl-CoA and cis-3-enoyl- CoA as substrate

[0364] 3-hydroxyacyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_371556_1 6.75e-35 135 I - dehydrogenase 5.3.3.8

[0365] 3-hydroxyacyl-CoA

[0366] 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_379858_1 5.16e-53 185 I dehydrogenase, NAD binding - 5.3.3.8

[0367] domain

[0368] 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_389269_2 3.5e-23 97.8 I enoyl-CoA hydratase isomerase MA20_44845

[0369] 5.3.3.8

[0370] Belongs to the enoyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_393144_1 2.58e-62 214 I - hydratase isomerase family 5.3.3.8

[0371] Belongs to the enoyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_394436_1 4.11e-73 237 I MA20_44845

[0372] hydratase isomerase family 5.3.3.8

[0373] Belongs to the enoyl-CoA 1.1.1.35,4.2.1.17,5.1.2.3, yeast_k141_415712_3 1.08e-200 590 I -

[0374]

[0375] hydratase isomerase family 5.3.3.8

[0376] able 8. Gene annotation (EC 1.1.1.157)

[0377] query evalue score COG_category Description Preferred_name EC

[0378] 3-hydroxyacyl-CoA

[0379] yeast_k141_966733_

[0380] 7.17e-80 251 C dehydrogenase, NAD binding paaH 1.1.1.157

[0381] 1

[0382] domain

[0383] 3-hydroxyacyl-CoA

[0384] yeast_k141_964348_

[0385] 5.68e-73 226 I dehydrogenase, C-terminal hbd 1.1.1.157

[0386] 1

[0387] domain

[0388] yeast_k141_963215_ 3-hydroxyacyl-coa

[0389] 3.82e-09 57.4 I paaH 1.1.1.157

[0390] 2 dehydrogenase

[0391] yeast_k141_958823_ PFAM 3-hydroxyacyl-CoA 1.1.1.157,1.1.1.35,4.2.1.

[0392] 6.85e-76 236 I - 1 dehydrogenase 17

[0393] 3-hydroxyacyl-CoA

[0394] yeast_k141_957776_

[0395] 6.81e-39 139 I dehydrogenase, C-terminal - 1.1.1.157

[0396] 1

[0397] domain

[0398] yeast_k141_952179_ 3-hydroxyacyl-CoA

[0399] 9.25e-85 256 I hbd 1.1.1.157

[0400]

[0401] 2 dehydrogenase yeast_k141_948296_

[0402] 5.34e-136 392 I Dehydrogenase paaH1 1.1.1.157

[0403] 2

[0404] yeast_k141_947505_ 3-hydroxyacyl-CoA

[0405] 7.63e-36 139 I - 1.1.1.157

[0406] 1 dehydrogenase

[0407] yeast_k141_943644_ 3-hydroxyacyl-CoA

[0408] 1.7e-48 168 C paaH 1.1.1.157

[0409] 1 dehydrogenase

[0410] 3-hydroxyacyl-CoA

[0411] yeast_k141_933015_

[0412] 1.76e-147 429 I dehydrogenase, C-terminal paaH 1.1.1.157

[0413] 1

[0414] domain

[0415] 3-hydroxyacyl-CoA

[0416] yeast_k141_931664_

[0417] 1.18e-101 307 I dehydrogenase, C-terminal paaH 1.1.1.157

[0418] 1

[0419] domain

[0420] 3-hydroxyacyl-CoA

[0421] yeast_k141_928381_ 1.1.1.157,1.1.1.35,4.2.1.

[0422] 7.56e-36 134 C dehydrogenase, NAD binding - 1 17,5.1.2.3

[0423] domain

[0424] yeast_k141_926515_ 3-hydroxyacyl-CoA

[0425] 5.17e-16 77.4 I hbd 1.1.1.157

[0426] 1 dehydrogenase

[0427] 3-hydroxyacyl-CoA

[0428] yeast_k141_92566_2 0.000514 41.6 I - 1.1.1.157

[0429] dehydrogenase

[0430] 3-hydroxyacyl-CoA

[0431] yeast_k141_923853_

[0432] 3.56e-40 146 I dehydrogenase, NAD binding - 1.1.1.157

[0433] 1

[0434] domain

[0435] 3-hydroxyacyl-CoA

[0436] yeast_k141_923780_

[0437] 2.93e-32 123 I dehydrogenase, C-terminal - 1.1.1.157

[0438] 1

[0439] domain

[0440] 3-hydroxyacyl-CoA

[0441] yeast_k141 _919866_

[0442] 1.93e-16 77.4 I dehydrogenase, C-terminal hbdA 1.1.1.157

[0443] 2

[0444] domain

[0445]

[0446] yeast_k141_916211_2 9.79e-59 189 I dehydrogenase hbd 1.1.1.157

[0447] able 9. Gene annotation (EC 1.2.1.3)

[0448] query evalue score COG_category Description Preferred_name EC yeast_k141_2692_1 6.98e-85 268 C Aldehyde dehydrogenase family calB 1.2.1.3,1.2.99.10

[0449] belongs to the aldehyde

[0450] yeast_k141_99896_1 2.85e-81 255 C - 1.2.1.3

[0451]

[0452] dehydrogenase family yeast_k141_968156_ Belongs to the aldehyde

[0453] 5.67e-20 88.6 C aldB 1.2.1.3

[0454] 1 dehydrogenase family

[0455] yeast_k141_966476_ Belongs to the aldehyde 1.2.1.3,1.2.1.32,1.2.1.39,

[0456] 1.57e-128 380 C - 1 dehydrogenase family 1.2.1.8,1.2.1.85 yeast_k141_966290_

[0457] 6.68e-196 558 C Psort location Cytoplasmic, score ywdH 1.2.1.3,1.2.99.10

[0458] 1

[0459] yeast_k141_963447_ Belongs to the aldehyde

[0460] 1.22e-26 108 C - 1.2.1.3,1.2.1.32,1.2.1.85 1 dehydrogenase family

[0461] yeast_k141_961509_ Belongs to the aldehyde

[0462] 8.95e-26 108 C - 1.2.1.3

[0463] 1 dehydrogenase family

[0464] yeast_k141 _960139_ belongs to the aldehyde

[0465] 1.24e-43 163 C - 1.2.1.3,1.2.1.68

[0466] 23 dehydrogenase family

[0467] yeast_k141_958651_ Belongs to the aldehyde

[0468] 6.17e-251 699 C - 1.2.1.3

[0469] 2 dehydrogenase family

[0470] yeast_k141_954976_

[0471] 7.24e-25 106 C Aldehyde dehydrogenase family calB 1.2.1.3,1.2.99.10

[0472] 1

[0473] yeast_k141_952143_ belongs to the aldehyde

[0474] 4.88e-43 154 C - 1.2.1.3

[0475] 1 dehydrogenase family

[0476] yeast_k141_951336_ belongs to the aldehyde

[0477] 2.85e-25 106 C - 1.2.1.3

[0478] 1 dehydrogenase family

[0479] yeast_k141_949313_

[0480] 2.54e-82 258 C Aldehyde dehydrogenase family - 1.2.1.3

[0481] 1

[0482] yeast_k141_946933_ Belongs to the aldehyde

[0483] 4.74e-33 128 C ywdH 1.2.1.3,1.2.99.10

[0484] 1 dehydrogenase family

[0485] yeast_k141_946048_

[0486] 1.18e-67 222 C Aldehyde dehydrogenase family calB 1.2.1.3,1.2.99.10

[0487] 1

[0488] yeast_k141 _945952_ belongs to the aldehyde

[0489] 2.95e-62 205 C - 1.2.1.3

[0490] 1 dehydrogenase family

[0491] yeast_k141 _944922_ belongs to the aldehyde

[0492] 2.63e-23 98.2 C - 1.2.1.3

[0493] 1 dehydrogenase family

[0494] yeast_k141_943451_ belongs to the aldehyde

[0495] 3.63e-17 80.1 C - 1.2.1.3

[0496]

[0497] 1 dehydrogenase family able 10. Gene annotation (EC 1.2.1.10)

[0498] query evalue score COG_category Description Preferred_name EC

[0499] Catalyzes the conversion of

[0500] acetaldehyde to acetyl-CoA,

[0501] using NAD(H) and coenzyme A.

[0502] yeast_k141_961563_1 4.96e-49 165 Q Is the final enzyme in the meta- 1.2.1.10

[0503] cleavage pathway for the

[0504] degradation of aromatic

[0505] compounds

[0506] belongs to the iron- containing

[0507] yeast_k141_954280_1 3.47e-09 58.9 C adhE 1.1.1.1,1.2.1.10

[0508] alcohol dehydrogenase family

[0509] belongs to the aldehyde

[0510] yeast_k141_949014_1 4.14e-45 159 C - 1.1.1.1,1.2.1.10,1.2.1.81 dehydrogenase family

[0511] belongs to the iron- containing

[0512] yeast_k141_948035_1 3.37e-67 223 C adh 1.1.1.1,1.2.1.10

[0513] alcohol dehydrogenase family

[0514] Iron-containing alcohol

[0515] yeast_k141_926374_1 6.38e-46 164 C adhE 1.1.1.1,1.2.1.10

[0516] dehydrogenase

[0517] belongs to the iron- containing

[0518] yeast_k141_909823_1 1.87e-159 470 C adh 1.1.1.1,1.2.1.10

[0519] alcohol dehydrogenase family

[0520] Catalyzes the conversion of

[0521] acetaldehyde to acetyl-CoA,

[0522] using NAD(H) and coenzyme A.

[0523] yeast_k141_909032_1 5.17e-69 216 Q Is the final enzyme in the metamhpF 1.2.1.10

[0524] cleavage pathway for the

[0525] degradation of aromatic

[0526] compounds

[0527] Catalyzes the conversion of

[0528] acetaldehyde to acetyl-CoA,

[0529] using NAD(H) and coenzyme A.

[0530] yeast_k141_904116_2 3e-121 357 Q Is the final enzyme in the metahsaG 1.2.1.10

[0531] cleavage pathway for the

[0532] degradation of aromatic

[0533] compounds

[0534] belongs to the iron- containing

[0535] yeast_k141_902470_1 1.88e-91 290 C adh 1.1.1.1,1.2.1.10

[0536]

[0537] alcohol dehydrogenase family yeast_k141_900405_1 4.24e-46 162 C Aldehyde dehydrogenase family - 1.2.1.10

[0538] belongs to the iron- containing

[0539] yeast_k141_895177_1 9.9e-133 400 C adh 1.1.1.1,1.2.1.10 alcohol dehydrogenase family

[0540] Catalyzes the conversion of

[0541] acetaldehyde to acetyl-CoA,

[0542] using NAD(H) and coenzyme A.

[0543] yeast_k141_862891_1 5.97e-65 205 Q Is the final enzyme in the metahsaG 1.2.1.10

[0544] cleavage pathway for the

[0545] degradation of aromatic

[0546] compounds

[0547] Catalyzes the conversion of

[0548] acetaldehyde to acetyl-CoA,

[0549] using NAD(H) and coenzyme A.

[0550] yeast_k141_857769_1 1.45e-14 71.6 Q Is the final enzyme in the meta- 1.2.1.10

[0551] cleavage pathway for the

[0552] degradation of aromatic

[0553] compounds

[0554] Catalyzes the conversion of

[0555] acetaldehyde to acetyl-CoA,

[0556] using NAD(H) and coenzyme A.

[0557] yeast_k141_849750_2 2.67e-44 153 Q Is the final enzyme in the metahsaG 1.2.1.10

[0558] cleavage pathway for the

[0559] degradation of aromatic

[0560] compounds

[0561] Catalyzes the conversion of

[0562] acetaldehyde to acetyl-CoA,

[0563] using NAD(H) and coenzyme A.

[0564] yeast_k141_842384_2 1.65e-18 82.8 H Is the final enzyme in the meta- 1.2.1.10

[0565] cleavage pathway for the

[0566] degradation of aromatic

[0567] compounds

[0568] Catalyzes the conversion of

[0569] acetaldehyde to acetyl-CoA,

[0570] yeast_k141_824735_1 5.29e-70 220 Q using NAD(H) and coenzyme A. mhpF 1.2.1.10

[0571] Is the final enzyme in the meta

[0572]

[0573] cleavage pathway for the degradation of aromatic

[0574] compounds

[0575] yeast_k141_820303_2 0 1691 C alcohol dehydrogenase adhE 1.1.1.1,1.2.1.10

[0576] Catalyzes the conversion of

[0577] acetaldehyde to acetyl-CoA,

[0578] using NAD(H) and coenzyme A.

[0579] yeast_k141_815920_1 1.3e-45 156 Q Is the final enzyme in the metahsaG 1.2.1.10

[0580] cleavage pathway for the

[0581] degradation of aromatic

[0582]

[0583] compounds

[0584] able 11. Gene annotation (EC 1.2.7.1)

[0585] query evalue score COG_category Description Preferred_name EC yeast_k141_9839_1 4.04e-56 194 C Domain of unknown function nifJ 1.2.7.1

[0586] Oxidoreductase required for the

[0587] yeast_k141_97295_1 1.08e-113 360 C transfer of electrons from - 1.2.7.1

[0588] pyruvate to flavodoxin

[0589] Pyruvate:ferredoxin

[0590] yeast_k141_97271_2 3.28e-26 105 c - 1.2.7.1

[0591] oxidoreductase core domain II

[0592] 2-oxoacid acceptor

[0593] yeast_k141_97271_1 4e-29 113 c oxidoreductase, gamma subunit, - 1.2.7.1

[0594] pyruvate 2-ketoisovalerate

[0595] 2-oxoacid ferredoxin

[0596] yeast_k141_9721_1 1.37e-71 227 c porA 1.2.7.1,1.2.7.10 oxidoreductase, alpha subunit

[0597] Oxidoreductase required for the

[0598] yeast_k141_964525_1 2.53e-74 248 c transfer of electrons from nifJ 1.2.7.1

[0599] pyruvate to flavodoxin

[0600] Oxidoreductase required for the

[0601] yeast_k141_961554_2 1.72e-41 152 c transfer of electrons from nifJ 1.2.7.1

[0602] pyruvate to flavodoxin

[0603] TIGRFAM pyruvate

[0604] yeast_k141_961553_3 7.23e-81 244 c ketoisovalerate oxidoreductase, porC-1 1.2.7.1

[0605] gamma subunit

[0606]

[0607] yeast_k141_961553_2 4.62e-19 81.3 c 4Fe-4S dicluster domain - 1.2.7.1 Pyruvate ferredoxin / flavodoxin

[0608] yeast_k141_958556_1 3.87e-62 194 C porG 1.2.7.1,1.2.7.3 oxidoreductase

[0609] yeast_k141_951957_1 2.11e-11 63.2 C PFAM Thiamine pyrophosphate porB 1.2.1.58,1.2.7.1

[0610] Oxidoreductase required for the

[0611] yeast_k141_951353_1 7.06e-140 431 C transfer of electrons from porA 1.2.7.1

[0612] pyruvate to flavodoxin

[0613] Oxidoreductase required for the

[0614] yeast_k141_95079_1 1.05e-65 224 C transfer of electrons from - 1.2.7.1

[0615] pyruvate to flavodoxin

[0616] Oxidoreductase required for the

[0617] yeast_k141_946701_1 1,24e-45 167 C transfer of electrons from - 1.2.7.1

[0618] pyruvate to flavodoxin

[0619] Oxidoreductase required for the

[0620] yeast_k141_946571_1 9.78e-76 254 C transfer of electrons from nifJ 1.2.7.1

[0621] pyruvate to flavodoxin

[0622] 2-oxoacid acceptor

[0623] yeast_k141_945624_2 7.78e-66 212 C oxidoreductase, gamma subunit, - 1.2.7.1

[0624] pyruvate 2-ketoisovalerate

[0625] Oxidoreductase required for the

[0626] yeast_k141_945096_1 2.41e-105 333 C transfer of electrons from nifJ 1.2.7.1

[0627] pyruvate to flavodoxin

[0628] Thiamine pyrophosphate

[0629] yeast_k141 _944981 _ 1 1,29e-57 187 C enzyme, C-terminal TPP binding porB 1.2.1.58,1.2.7.1

[0630]

[0631] domain

[0632] able 12. Gene annotation (EC 1.2.7.5)

[0633] query evalue score COG_category Description Preferred_name EC Aldehyde ferredoxin

[0634] yeast_k141_186_1 7.69e-44 157 C - 1.2.7.5

[0635] oxidoreductase

[0636] Aldehyde ferredoxin

[0637] yeast_k141_483_1 6.07e-26 108 C oxidoreductase, N-terminal - 1.2.7.5

[0638] domain

[0639] Aldehyde ferredoxin

[0640] yeast_k141_1546_3 4.68e-120 367 c oxidoreductase, N-terminal - 1.2.7.5

[0641]

[0642] domain Aldehyde ferredoxin

[0643] yeast_k141_2947_2 5.79e-252 716 C oxidoreductase, N-terminal - 1.2.7.5

[0644] domain

[0645] Aldehyde ferredoxin

[0646] yeast_k141_5820_1 3.03e-93 289 C oxidoreductase, N-terminal - 1.2.7.5

[0647] domain

[0648] PFAM Aldehyde ferredoxin

[0649] yeast_k141_8877_1 6.12e-22 97.1 C oxidoreductase, N-terminal - 1.2.7.5

[0650] domain

[0651] aldehyde ferredoxin

[0652] yeast_k141_12285_1 4.72e-57 196 C - 1.2.7.5, 1.2.7.6 oxidoreductase activity

[0653] Aldehyde ferredoxin

[0654] yeast_k141_13045_1 1,66e-36 137 C - 1.2.7.5

[0655] oxidoreductase

[0656] PFAM Aldehyde ferredoxin

[0657] yeast_k141_13251_1 3.43e-28 116 C - 1.2.7.5

[0658] oxidoreductase

[0659] aldehyde ferredoxin

[0660] yeast_k141 _14824_1 8.42e-38 146 C - 1.2.7.5

[0661] oxidoreductase

[0662] PFAM Aldehyde ferredoxin

[0663] yeast_k141_21147_1 7.82e-81 258 C - 1.2.7.5

[0664] oxidoreductase

[0665] Aldehyde ferredoxin

[0666] yeast_k141_22171_1 8.28e-57 194 C oxidoreductase, N-terminal - 1.2.7.5

[0667] domain

[0668] PFAM Aldehyde ferredoxin

[0669] yeast_k141_25395_1 7.27e-29 115 C aor 1.2.7.5

[0670] oxidoreductase

[0671] Aldehyde ferredoxin

[0672] yeast_k141_26069_1 1.05e-44 161 C oxidoreductase, N-terminal - 1.2.7.5

[0673] domain

[0674] Aldehyde ferredoxin

[0675] yeast_k141_32847_1 6.41e-167 481 C aorA 1.2.7.5

[0676] oxidoreductase

[0677] Aldehyde ferredoxin

[0678] yeast_k141_40290_1 0 972 C oxidoreductase, N-terminal - 1.2.7.5

[0679] domain

[0680] Aldehyde ferredoxin

[0681] yeast_k141_41776_1 6.25e-16 79.3 C oxidoreductase, N-terminal - 1.2.7.5

[0682]

[0683] domain Aldehyde ferredoxin

[0684] yeast_k141_46471_1 7.15e-21 92.8 C - 1.2.7.5

[0685] oxidoreductase

[0686] able 13. Gene annotation (EC 1.3.8.1)

[0687] query evalue score COG_category Description Preferred_name EC

[0688] Acyl-CoA dehydrogenase, C- yeast_k141_6074_5 2.23e-223 622 I bed 1.3.8.1

[0689] terminal domain

[0690] yeast_k141_99301_1 8.73e-05 43.9 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12

[0691] Acyl-CoA dehydrogenase, C- yeast_k141_136500_1 1,22e-46 163 I - 1.3.8.1,1.3.99.12 terminal domain

[0692] Acyl-CoA dehydrogenase, C- yeast_k141_171417_1 9.52e-55 182 I fadE19 1.3.8.1,1.3.99.12 terminal domain

[0693] yeast_k141_183909_1 2.42e-70 222 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12

[0694] Acyl-CoA dehydrogenase, C- yeast_k141_190463_5 3.03e-223 622 I fadE19 1.3.8.1,1.3.99.12 terminal domain

[0695] yeast_k141_217421_1 2.75e-97 293 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12 yeast_k141_281298_1 0.000326 42 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12 yeast_k141_300012_1 1.11e-35 131 I acyl-CoA dehydrogenase activity yngJ 1.3.8.1,1.3.99.12 yeast_k141_310488_1 8.54e-42 148 I Dehydrogenase yngJ 1.3.8.1,1.3.99.12 yeast_k141_313132_1 1,65e-32 127 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12

[0696] Acyl-CoA dehydrogenase, C- yeast_k141_315604_2 3e-45 157 I fadE19 1.3.8.1,1.3.99.12 terminal domain

[0697] yeast_k141_353340_1 6.41 e-63 204 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12

[0698] Acyl-CoA dehydrogenase, C- yeast_k141_447282_2 1.61e-05 47.8 I fadE19 1.3.8.1,1.3.99.12 terminal domain

[0699] yeast_k141_539048_3 6.04e-10 58.9 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12

[0700] Acyl-CoA dehydrogenase, C- yeast_k141_541591_1 7.06e-10 62.4 I fadE19 1.3.8.1,1.3.99.12 terminal domain

[0701] yeast_k141_592931_1 4.39e-126 369 I acyl-CoA dehydrogenase fadE19 1.3.8.1,1.3.99.12

[0702]

[0703] yeast_k141_697253_1 2.62e-73 232 I acyl-CoA dehydrogenase activity yngJ 1.3.8.1,1.3.99.12 able 14. Gene annotation (EC 1.3.8.7)

[0704] query evalue score COG_category Description Preferred_name EC

[0705] Acyl-CoA dehydrogenase, C- yeast_k141_740_2 2.18e-51 176 I - 1.3.8.7

[0706] terminal domain

[0707] yeast_k141_773_1 3.32e-66 214 C acyl-CoA dehydrogenase 1.3.8.7 yeast_k141_778_1 9.44e-129 375 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141 _14092_1 2.78e-175 496 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141_20153_2 1.73e-11 63.2 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141_84175_1 1.05e-72 228 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141_136275_4 5.78e-182 516 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8

[0708] Acyl-CoA dehydrogenase, C- yeast_k141_180363_1 7.67e-08 58.5 I - 1.3.8.7, 1.3.8.8 terminal domain

[0709] yeast_k141_189273_2 5.72e-24 99 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8

[0710] Acyl-CoA dehydrogenase, C- yeast_k141_280600_2 5.75e-11 62.8 I fadE 1.3.8.7, 1.3.8.8 terminal domain

[0711] Acyl-CoA dehydrogenase, C- yeast_k141_315407_1 5.37e-05 48.9 I - 1.3.8.7, 1.3.8.8 terminal domain

[0712] yeast_k141_339736_2 2.21e-21 91.7 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8

[0713] PFAM acyl-CoA dehydrogenase

[0714] yeast_k141_388052_1 1,46e-73 231 C - 1.3.8.7, 1.3.8.8 domain protein

[0715] yeast_k141_409396_1 8.37e-72 227 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8 yeast_k141_419944_1 1,24e-73 230 I acyl-CoA dehydrogenase fadE20 1.3.8.7, 1.3.8.8 yeast_k141_479057_1 3.99e-44 154 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8

[0716] Acyl-CoA dehydrogenase, C- yeast_k141_493251_1 4.66e-94 284 I - 1.3.8.7, 1.3.8.8 terminal domain

[0717] Acyl-CoA dehydrogenase, C- yeast_k141_498808_1 4.05e-13 68.6 c hcaD 1.3.8.7, 1.3.8.8

[0718]

[0719] terminal domain

[0720] able 15. Gene annotation (EC 1.3.8.8)

[0721] query evalue score COG_category Description Preferred_name EC yeast_k141_740_2 9.44e-129 375 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8

[0722]

[0723] yeast_k141_773_1 1.58e-151 434 I acyl-CoA dehydrogenase - 1.3.8.8 yeast_k141_778_1 2.78e-175 496 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141 _14092_1 1.73e-11 63.2 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141_20153_2 8.61e-144 412 I acyl-CoA dehydrogenase 1.3.8.8 yeast_k141_84175_1 1.05e-72 228 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8 yeast_k141_136275_4 4.08e-38 136 I acyl-CoA dehydrogenase 1.3.8.8 yeast_k141_180363_1 5.78e-182 516 I acyl-CoA dehydrogenase 1.3.8.7, 1.3.8.8

[0724] PFAM acyl-CoA dehydrogenase

[0725] yeast_k141_189273_2 7.16e-76 237 C - 1.3.8.8

[0726] domain protein

[0727] Acyl-CoA dehydrogenase, C- yeast_k141_280600_2 7.67e-08 58.5 I - 1.3.8.7, 1.3.8.8

[0728] terminal domain

[0729] yeast_k141_315407_1 5.72e-24 99 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8

[0730] PFAM acyl-CoA dehydrogenase

[0731] yeast_k141_339736_2 5.34e-103 308 C - 1.3.8.8

[0732] domain protein

[0733] yeast_k141_388052_1 5.22e-54 179 I acyl-CoA dehydrogenase - 1.3.8.8 yeast_k141_409396_1 1.58e-83 256 I acyl-CoA dehydrogenase - 1.3.8.8

[0734] Acyl-CoA dehydrogenase, C- yeast_k141_419944_1 5.75e-11 62.8 I fadE 1.3.8.7, 1.3.8.8

[0735] terminal domain

[0736] Acyl-CoA dehydrogenase, C- yeast_k141_479057_1 5.37e-05 48.9 I - 1.3.8.7, 1.3.8.8

[0737] terminal domain

[0738] PFAM acyl-CoA dehydrogenase

[0739] yeast_k141_493251_1 7.42e-52 177 C - 1.3.8.8

[0740] domain protein

[0741]

[0742] yeast_k141_498808_1 2.21e-21 91.7 I acyl-CoA dehydrogenase - 1.3.8.7, 1.3.8.8

[0743] able 16. Gene annotation (EC 1.5.5.1)

[0744] query evalue score COG_category Description Preferred_name EC

[0745] Is involved in the reduction of 2,3- digeranylgeranylglycerophospholi

[0746] pids (unsaturated archaeols) into

[0747] 2,3- yeast_k141_3489_1 8.52e-29 113 C diphytanylglycerophospholipids - 1.3.1.101,1.3.7.11,1.5.5.1

[0748] (saturated archaeols) in the

[0749] biosynthesis of archaeal

[0750] membrane lipids. Catalyzes the

[0751]

[0752] formation of archaetidic acid (2,3- di-O-phytanyl-sn-glyceryl

[0753] phosphate) from 2,3-di-O- geranylgeranylglyceryl phosphate

[0754] (DGGGP) via the hydrogenation

[0755] of each double bond of the

[0756] isoprenoid chains

[0757] Electron transfer flavoprotein- yeast_k141 _11402_1 2.8e-76 242 C - 1.5.5.1 ubiquinone oxidoreductase

[0758] Electron transfer flavoprotein- yeast_k141_20437_2 2.33e-34 129 C - 1.5.5.1 ubiquinone oxidoreductase

[0759] Electron transfer flavoprotein- yeast_k141_43648_1 4.85e-94 289 C - 1.5.5.1 ubiquinone oxidoreductase

[0760] Electron transfer flavoprotein- yeast_k141_80901_1 1.08e-91 288 C ubiquinone oxidoreductase, 4Fe- - 1.5.5.1

[0761] 4S

[0762] Electron transfer flavoprotein- yeast_k141_83728_1 1,48e-70 227 C etf 1.5.5.1 ubiquinone oxidoreductase

[0763] Electron transfer flavoprotein- yeast_k141_87002_1 1,43e-88 276 C etf 1.5.5.1 ubiquinone oxidoreductase

[0764] Electron transfer flavoprotein- yeast_k141_99483_1 1.07e-51 180 C - 1.5.5.1 ubiquinone

[0765] Electron transfer flavoprotein- yeast_k141_104871_1 9.82e-79 250 C - 1.5.5.1 ubiquinone oxidoreductase

[0766] Electron transfer flavoprotein- yeast_k141_118423_2 1,46e-42 152 C - 1.5.5.1 ubiquinone oxidoreductase

[0767] Electron transfer flavoprotein- yeast_k141_124331_1 4.87e-134 395 C etf 1.5.5.1 ubiquinone oxidoreductase

[0768] Electron transfer flavoprotein- yeast_k141_138946_1 1.31e-74 239 C - 1.5.5.1 ubiquinone oxidoreductase

[0769] Electron transfer flavoprotein- yeast_k141_142096_1 4.59e-59 196 C ubiquinone oxidoreductase, 4Fe- etf 1.5.5.1

[0770] 4S

[0771] Electron transfer flavoprotein- yeast_k141_142857_1 1.48e-81 256 C - 1.5.5.1 ubiquinone oxidoreductase

[0772] Electron transfer flavoprotein- yeast_k141_150019_2 1.61e-12 66.6 C etf-QO 1.5.5.1

[0773]

[0774] ubiquinone oxidoreductase Electron transfer flavoprotein- yeast_k141_152571_1 8.82e-129 380 C - 1.5.5.1

[0775] ubiquinone oxidoreductase

[0776] Electron transfer flavoprotein- yeast_k141_153364_1 5.09e-67 218 C etf 1.5.5.1

[0777] ubiquinone oxidoreductase

[0778] Electron transfer flavoprotein- yeast_k141_155135_1 3.4e-85 266 C etf 1.5.5.1

[0779]

[0780] ubiquinone oxidoreductase

[0781] able 17. Gene annotation (EC 1.12.1.4)

[0782] query evalue score COG_category Description Preferred_name EC

[0783] Thioredoxin-like [2Fe-2S]

[0784] yeast_k141_340216_1 1,66e-42 144 C - 1.12.1.4,1.6.5.3

[0785] ferredoxin

[0786] Thioredoxin-like [2Fe-2S]

[0787] yeast_k141_480834_2 8.37e-41 141 C - 1.12.1.4,1.6.5.3

[0788] ferredoxin

[0789] yeast_k141_873690_2 1,68e-65 202 c 2 iron, 2 sulfur cluster binding - 1.12.1.4,1.6.5.3 yeast_k141_940687_1 3.49e-20 85.5 c 2 iron, 2 sulfur cluster binding - 1.12.1.4,1.6.5.3

[0790] NDH-1 shuttles electrons from

[0791] NADH, via FMN and iron- sulfur

[0792] yeast_k141_208327_1 8.69e-200 570 c - 1.12.1.3,1.12.1.4,1.6.5.3

[0793] (Fe-S) centers, to quinones in the

[0794] respiratory chain

[0795] NDH-1 shuttles electrons from

[0796] NADH, via FMN and iron- sulfur

[0797] yeast_k141_267875_1 2.14e-67 221 c - 1.12.1.3,1.12.1.4,1.6.5.3

[0798] (Fe-S) centers, to quinones in the

[0799] respiratory chain

[0800] yeast_k141_275677_2 1.42e-51 181 c iron-sulfur cluster assembly - 1.12.1.3,1.12.1.4,1.6.5.3

[0801] NDH-1 shuttles electrons from

[0802] c NADH, via FMN and iron- sulfur

[0803] yeast_k141_705840_1 1.08e-78 250 - 1.12.1.3,1.12.1.4,1.6.5.3

[0804] (Fe-S) centers, to quinones in the

[0805] respiratory chain

[0806] NDH-1 shuttles electrons from

[0807] c NADH, via FMN and iron- sulfur

[0808] yeast_k141_719122_1 4.68e-68 223 - 1.12.1.3,1.12.1.4,1.6.5.3

[0809] (Fe-S) centers, to quinones in the

[0810] respiratory chain

[0811]

[0812] yeast_k141_808416_1 1.88e-79 254 c iron-sulfur cluster assembly - 1.12.1.3,1.12.1.4,1.6.5.3 NDH-1 shuttles electrons from

[0813] NADH, via FMN and iron- sulfur

[0814] yeast_k141_827491_1 4.62e-72 233 C - 1.12.1.3,1.12.1.4,1.6.5.3

[0815] (Fe-S) centers, to quinones in the

[0816] respiratory chain

[0817] NDH-1 shuttles electrons from

[0818] NADH, via FMN and iron- sulfur

[0819] yeast_k141_964857_1 9.57e-155 451 C - 1.12.1.3,1.12.1.4,1.6.5.3

[0820] (Fe-S) centers, to quinones in the

[0821] respiratory chain

[0822] 1.12.1.3,1.12.1.4,1.17.1. yeast_k141_164712_1 6.03e-59 196 C Iron hydrogenase small subunit - 9, 1.6.5.3 1.12.1.3,1.12.1.4,1.17.1. yeast_k141_722445_1 5.38e-160 464 C Iron hydrogenase small subunit - 9, 1.6.5.3 1.12.1.3,1.12.1.4,1.17.1. yeast_k141_957361_1 3.52e-16 77 C Iron hydrogenase small subunit - 9, 1.6.5.3 1.12.1.2,1.12.1.3,1.12.1. yeast_k141_564955_1 4.87e-41 154 C iron-sulfur cluster assembly -

[0823]

[0824] 4, 1.6.5.3

[0825] able 18. Gene annotation (EC 1.12.2.1)

[0826] query evalue score COG_category Description Preferred_name EC

[0827] Belongs to the NiFe NiFeSe 1.12.2.1,1.12.5.1,1.12.99 yeast_k141_67356_1 9.13e-105 320 C hynA

[0828] hydrogenase large subunit family.6

[0829] Belongs to the NiFe NiFeSe 1.12.2.1,1.12.5.1,1.12.99 yeast_k141_75274_1 1.37e-47 166 C hynA

[0830] hydrogenase large subunit family.6

[0831] TIGRFAM hydrogenase (NiFe)

[0832] yeast_k141_184216_1 4.1e-45 158 c hyaS 1.12.2.1,1.12.99.6

[0833] small subunit (hydA)

[0834] c Hydrogenase (NiFe) small 1.12.2.1,1.12.5.1,1.12.99 yeast_k141_215776_2 1.02e-08 57.4 hynB

[0835] subunit HydA.6

[0836] c TIGRFAM hydrogenase (NiFe)

[0837] yeast_k141_676836_2 4.03e-19 84.7 - 1.12.2.1

[0838] small subunit (hydA)

[0839] NiFe / NiFeSe hydrogenase small

[0840] yeast_k141_723021_1 3.94e-104 314 c - 1.12.2.1,1.12.5.1

[0841] subunit C-terminal

[0842] c NiFe / NiFeSe hydrogenase small

[0843] yeast_k141_760948_1 1.33e-154 449 - 1.12.2.1,1.12.5.1

[0844] subunit C-terminal

[0845] c Belongs to the NiFe NiFeSe 1.12.2.1,1.12.5.1,1.12.99 yeast_k141_784519_1 9.37e-30 116 hynA

[0846]

[0847] hydrogenase large subunit family.6 TIGRFAM hydrogenase (NiFe)

[0848] yeast_k141_851713_2 2.32 e- 174 496 C hybS 1.12.2.1,1.12.99.6 small subunit (hydA)

[0849] TIGRFAM hydrogenase (NiFe)

[0850] yeast_k141_947014_1 1.7e-40 146 C - 1.12.2.1,1.12.99.6

[0851]

[0852] small subunit (hydA)

[0853] able 19. Gene annotation (EC 1.12.7.2)

[0854] query evalue score COG_category Description Preferred_name EC NiFe / NiFeSe hydrogenase small

[0855] yeast_k141_2253_1 9.45e-59 189 C hoxB 1.12.7.2

[0856] subunit C-terminal

[0857] Respiratory-chain NADH

[0858] yeast_k141_5628_1 8.97e-15 74.7 C - 1.12.7.2

[0859] dehydrogenase, 30 Kd subunit

[0860] yeast_k141_23973_1 1.76e-39 144 c #NAME? - 1.12.7.2

[0861] c Iron only hydrogenase large

[0862] yeast_k141_53846_2 1.06e-45 160 - 1.12.7.2

[0863] subunit, C-terminal domain

[0864] yeast_k141_84786_2 5.18e-52 165 s Iron hydrogenase small subunit - 1.12.7.2

[0865] c Iron only hydrogenase large

[0866] yeast_k141_84786_3 9.89e-204 572 - 1.12.7.2

[0867] subunit, C-terminal domain

[0868] PFAM NADH ubiquinone

[0869] yeast_k141_119806_2 1,63e-70 220 c hoxB 1.12.7.2

[0870] oxidoreductase, 20

[0871] c Membrane bound hydrogenase

[0872] yeast_k141_125932_1 7.32e-57 187 - 1.12.7.2

[0873] subunit

[0874] c NiFe / NiFeSe hydrogenase small

[0875] yeast_k141_132002_1 1.16e-76 238 hoxB 1.12.7.2

[0876] subunit C-terminal

[0877] yeast_k141_165014_1 9.78e-06 50.8 c hydrogenase large subunit - 1.12.7.2

[0878] c Iron only hydrogenase large

[0879] yeast_k141_183340_2 1.47e-17 81.3 - 1.12.7.2

[0880] subunit, C-terminal domain

[0881] c Iron only hydrogenase large

[0882] yeast_k141_204204_5 1.01e-31 124 - 1.12.7.2

[0883] subunit, C-terminal domain

[0884] c NiFe / NiFeSe hydrogenase small

[0885] yeast_k141_216833_2 2.23e-24 99 hoxB 1.12.7.2

[0886] subunit C-terminal

[0887] yeast_k141_252335_1 3.11e-257 712 c #NAME? - 1.12.7.2

[0888] NiFe / NiFeSe hydrogenase small

[0889] yeast_k141_274865_1 9.65e-38 136 c hoxB 1.12.7.2

[0890]

[0891] subunit C-terminal 4Fe-4S ferredoxin iron-sulfur

[0892] yeast_k141_285892_2 1,22e-07 51.6 C - 1.12.1.2,1.12.7.2

[0893] binding domain protein

[0894] NiFe / NiFeSe hydrogenase small

[0895] yeast_k141_290293_1 1.72e-181 508 C hoxB 1.12.7.2

[0896]

[0897] subunit C-terminal

[0898] able 20. Gene annotation (EC 1.18.1.2)

[0899] query evalue score COG_category Description Preferred_name EC

[0900] PFAM Oxidoreductase FAD

[0901] yeast_k141_341949_1 2.53e-53 175 P - 1.18.1.2,1.8.1.2

[0902] NAD(P)-binding

[0903] yeast_k141_759250_3 PFAM Oxidoreductase FAD

[0904] 1.58e-204 566 P - 1.18.1.2,1.8.1.2

[0905] 1 NAD(P)-binding

[0906] 1.18.1.2,1.19.1.1,1.8.7.3, yeast_k141_428317_1 7.65e-26 110 C 'glutamate synthase preT 1.8.98.4,1.8.98.5,1.8.98.

[0907] 6

[0908] C 4fe-4S ferredoxin, iron-sulfur

[0909] yeast_k141_42031_2 1.83e-32 124 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0910] 4fe-4S ferredoxin, iron-sulfur

[0911] yeast_k141_172353_1 3.38e-88 274 c trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0912] 0 Pyridine nucleotide-disulphide

[0913] yeast_k141_211306_1 1.74e-21 95.9 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0914] c 4fe-4S ferredoxin, iron-sulfur

[0915] yeast_k141_242571_1 1.3e-167 485 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0916] c 4fe-4S ferredoxin, iron-sulfur

[0917] yeast_k141_338265_1 3.31e-63 206 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0918] c 4fe-4S ferredoxin, iron-sulfur

[0919] yeast_k141_373402_1 4.01e-22 96.7 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0920] Pyridine nucleotide-disulphide

[0921] yeast_k141_429303_1 2.9e-30 118 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0922] 0 Pyridine nucleotide-disulphide

[0923] yeast_k141_449199_1 2.87e-45 157 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0924] c 4fe-4S ferredoxin, iron-sulfur

[0925] yeast_k141_468431_1 2.05e-99 306 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0926] c ferredoxin-NADP+ reductase

[0927] yeast_k141_565417_1 5.02e-48 163 ypdA 1.18.1.2,1.19.1.1,1.8.1.9

[0928]

[0929] activity ferredoxin-NADP+ reductase

[0930] yeast_k141_854443_1 3.76e-51 173 C ypdA 1.18.1.2,1.19.1.1,1.8.1.9 activity

[0931] Pyridine nucleotide-disulphide

[0932] yeast_k141_883754_1 5.73e-32 122 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0933] Pyridine nucleotide-disulphide

[0934] yeast_k141_961070_1 1.21e-28 112 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0935] Iron-sulfur cluster binding domain

[0936] 1.18.1.2,1.19.1.1,1.4.1.1 yeast_k141_18469_1 3.25e-47 160 CH of dihydroorotate dehydrogenase gitA

[0937] 3,1.4.1.14

[0938]

[0939] B

[0940] able 21. Gene annotation (EC 1.19.1.1)

[0941] query evalue score COG_category Description Preferred_name EC 1.18.1.2,1.19.1.1,1.8.7.3, yeast_k141_428317_1 7.65e-26 110 C 'glutamate synthase preT 1.8.98.4,1.8.98.5,1.8.98.

[0942] 6

[0943] 4fe-4S ferredoxin, iron-sulfur

[0944] yeast_k141_42031_2 1.83e-32 124 C trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0945] 4fe-4S ferredoxin, iron-sulfur

[0946] yeast_k141_172353_1 3.38e-88 274 c trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0947] Pyridine nucleotide-disulphide

[0948] yeast_k141_211306_1 1.74e-21 95.9 0 trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0949] 4fe-4S ferredoxin, iron-sulfur

[0950] yeast_k141_242571_1 1.3e-167 485 c trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0951] 4fe-4S ferredoxin, iron-sulfur

[0952] yeast_k141_338265_1 3.31 e-63 206 c trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0953] 4fe-4S ferredoxin, iron-sulfur

[0954] yeast_k141_373402_1 4.01 e-22 96.7 c trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0955] Pyridine nucleotide-disulphide

[0956] yeast_k141_429303_1 2.9e-30 118 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0957] Pyridine nucleotide-disulphide

[0958] yeast_k141_449199_1 2.87e-45 157 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0959] 4fe-4S ferredoxin, iron-sulfur

[0960] yeast_k141_468431_1 2.05e-99 306 c trxB_2 1.18.1.2,1.19.1.1,1.8.1.9 binding domain protein

[0961] ferredoxin-NADP+ reductase

[0962] yeast_k141_565417_1 5.02e-48 163 c ypdA 1.18.1.2,1.19.1.1,1.8.1.9

[0963]

[0964] activity ferredoxin-NADP+ reductase

[0965] yeast_k141_854443_1 3.76e-51 173 C ypdA 1.18.1.2,1.19.1.1,1.8.1.9 activity

[0966] Pyridine nucleotide-disulphide

[0967] yeast_k141_883754_1 5.73e-32 122 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0968] Pyridine nucleotide-disulphide

[0969] yeast_k141_961070_1 1.21e-28 112 0 ypdA 1.18.1.2,1.19.1.1,1.8.1.9 oxidoreductase

[0970] Iron-sulfur cluster binding domain

[0971] 1.18.1.2,1.19.1.1,1.4.1.1 yeast_k141_18469_1 3.25e-47 160 CH of dihydroorotate dehydrogenase gitA

[0972] 3,1.4.1.14

[0973] B

[0974] Iron-sulfur cluster binding domain

[0975] 1.18.1.2,1.19.1.1,1.4.1.1 yeast_k141_27378_1 4.76e-75 231 CH of dihydroorotate dehydrogenase nfnA

[0976] 3,1.4.1.14

[0977] B

[0978] 1.18.1.2,1.19.1.1,1.4.1.1 yeast_k141_171504_2 5.77e-57 186 C 2 iron, 2 sulfur cluster binding gitA

[0979]

[0980] 3,1.4.1.14

[0981] able 22. Gene annotation (EC 2.3.1.8)

[0982] query evalue score COG_category Description Preferred_name EC

[0983] N-terminal half of MaoC

[0984] yeast_k141_41943_1 1.38e-48 167 I - 2.3.1.8,4.2.1.119

[0985] dehydratase

[0986] Phosphate acetyl / butaryl

[0987] yeast_k141_314437_1 1.5e-07 51.6 C pta 2.3.1.8,3.6.3.21

[0988] transferase

[0989] Phosphate acetyl / butaryl

[0990] yeast_k141_648741_1 2.78e-111 326 C pta 2.3.1.8,3.6.3.21

[0991] transferase

[0992] Phosphate acetyl / butaryl

[0993] yeast_k141_695763_1 1.43e-17 80.1 c pta 2.3.1.8,3.6.3.21

[0994] transferase

[0995] c Phosphate acetyl / butaryl

[0996] yeast_k141_769729_2 1,62e-228 630 pta 2.3.1.8,3.6.3.21

[0997] transferase

[0998] c Phosphate acetyl / butaryl

[0999] yeast_k141_925491_1 3.22e-32 122 pta 2.3.1.8,3.6.3.21

[1000] transferase

[1001] c Phosphate acetyl / butaryl

[1002] yeast_k141_1143_2 4.15e-65 208 pta 2.3.1.8

[1003] transferase

[1004] c Phosphate acetyl / butaryl

[1005] yeast_k141_8836_1 2.2e-117 346 pta 2.3.1.8

[1006] transferase

[1007] c Phosphate acetyl / butaryl

[1008] yeast_k141_11392_3 3.02e-164 468 pta 2.3.1.8

[1009]

[1010] transferase Bifunctional enoyl-CoA hydratase

[1011] yeast_k141_20049_1 8.74e-64 209 Cl - 2.3.1.8

[1012] phosphate acetyltransferase

[1013] PFAM phosphate acetyl butaryl

[1014] yeast_k141_21209_1 3.42e-42 147 C pta 2.3.1.8

[1015] transferase

[1016] Phosphate acetyl / butaryl

[1017] yeast_k141_23671_2 4.61 e-39 138 C pta 2.3.1.8

[1018] transferase

[1019] yeast_k141_39167_5 4.69e-179 506 C Psort location Cytoplasmic, score pta 2.3.1.8

[1020] Phosphate acetyl / butaryl

[1021] yeast_k141_40368_1 1,44e-99 298 C pta 2.3.1.8

[1022] transferase

[1023] Phosphate acetyl / butaryl

[1024] yeast_k141_73205_1 1.34e-43 152 C pta 2.3.1.8

[1025] transferase

[1026] Bifunctional enoyl-CoA hydratase

[1027] yeast_k141_97212_2 1.35e-22 96.3 Cl pta 2.3.1.8

[1028] phosphate acetyltransferase

[1029]

[1030] yeast_k141_102845_1 1.87e-47 167 C belongs to the CobB CobQ family pta 2.3.1.8 able 23. Gene annotation (EC 2.3.1.9)

[1031] query evalue score COG_category Description Preferred_name EC yeast_k141_717_2 7.18e-61 198 I Belongs to the thiolase family fadA4 2.3.1.9 yeast_k141_337182_2 2.36e-37 145 I Enoyl-CoA hydratase / isomerase - 2.3.1.9,4.2.1.149 yeast_k141_909422_1 1.83e-54 190 I Enoyl-CoA hydratase / isomerase - 2.3.1.9,4.2.1.149

[1032] Belongs to the enoyl-CoA

[1033] yeast_k141_935036_1 1,25e-99 320 I - 2.3.1.9,4.2.1.149 hydratase isomerase family

[1034] Catalyzes the synthesis of

[1035] acetoacetyl coenzyme A from two

[1036] molecules of acetyl coenzyme A.

[1037] It can also act as a thiolase,

[1038] yeast_k141_1418_2 1.5e-109 325 I - 2.3.1.9

[1039] catalyzing the reverse reaction

[1040] and generating two-carbon units

[1041] from the four-carbon product of

[1042] fatty acid oxidation

[1043] yeast_k141_2380_1 1,65e-05 45.8 I Belongs to the thiolase family fadA 2.3.1.9 yeast_k141_3710_2 3.07e-82 255 I Belongs to the thiolase family - 2.3.1.9 yeast_k141_6074_8 2.55e-208 585 I Thiolase, C-terminal domain thlA 2.3.1.9

[1044]

[1045] yeast_k141_6735_1 2.2e-81 255 I Thiolase, C-terminal domain - 2.3.1.9 yeast_k141_6925_1 3.96e-84 259 I Belongs to the thiolase family phbA 2.3.1.9 yeast_k141_9726_1 1.34e-43 154 I Belongs to the thiolase family - 2.3.1.9 yeast_k141_10658_1 2.01e-224 624 I Belongs to the thiolase family bktB 2.3.1.9 yeast_k141 _11893_1 8.86e-221 616 I Belongs to the thiolase family - 2.3.1.9

[1046] Catalyzes the synthesis of

[1047] acetoacetyl coenzyme A from two

[1048] molecules of acetyl coenzyme A.

[1049] It can also act as a thiolase,

[1050] yeast_k141_11896_3 3.12e-260 717 I - 2.3.1.9

[1051] catalyzing the reverse reaction

[1052] and generating two-carbon units

[1053] from the four-carbon product of

[1054] fatty acid oxidation

[1055] Catalyzes the synthesis of

[1056] acetoacetyl coenzyme A from two

[1057] molecules of acetyl coenzyme A.

[1058] It can also act as a thiolase,

[1059] yeast_k141 _12484_1 2.44e-42 153 I yfcY 2.3.1.9

[1060] catalyzing the reverse reaction

[1061] and generating two-carbon units

[1062] from the four-carbon product of

[1063] fatty acid oxidation

[1064] yeast_k141_12674_1 1.61e-213 595 I Belongs to the thiolase family - 2.3.1.9

[1065]

[1066] yeast_k141_15256_1 4.13e-111 330 I Belongs to the thiolase family - 2.3.1.9 able 24. Gene annotation (EC 2.3.1.16)

[1067] query evalue score COG_category Description Preferred_name EC yeast_k141_1095_1 3.97e-92 280 I Belongs to the thiolase family - 2.3.1.16 yeast_k141_2456_1 2.36e-43 151 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_17683_1 2.06e-67 217 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_22875_1 8.17e-62 202 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_27071_1 4.63e-90 275 I Thiolase, C-terminal domain fadA6 2.3.1.16,2.3.1.9 yeast_k141_47690_1 1.27e-135 394 I Belongs to the thiolase family pcaF 2.3.1.16,2.3.1.9 yeast_k141_63920_1 5.85e-90 275 I Belongs to the thiolase family fadA 2.3.1.16,2.3.1.9

[1068]

[1069] yeast_k141_68111_1 4.53e-47 162 I Belongs to the thiolase family vraB 2.3.1.16,2.3.1.9 yeast_k141_72352_1 3.61 e-53 179 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_77172_1 5.08e-91 276 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_93104_2 6.99e-31 120 I Thiolase, C-terminal domain - 2.3.1.16,2.3.1.9 yeast_k141_94831_2 1.24e-19 86.7 I Belongs to the thiolase family pcaF 2.3.1.16,2.3.1.9 yeast_k141_109979_1 6.97e-144 417 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_114863_1 1.23e-104 312 I Belongs to the thiolase family pcaF 2.3.1.16,2.3.1.9 yeast_k141_126540_2 3.57e-117 346 I Belongs to the thiolase family - 2.3.1.16,2.3.1.9 yeast_k141_131209_2 7.58e-07 49.7 I Belongs to the thiolase family pcaF 2.3.1.16,2.3.1.9

[1070]

[1071] yeast_k141_131354_1 3.7e-17 81.3 I Belongs to the thiolase family bktB 2.3.1.16,2.3.1.9 able 25. Gene annotation (EC 2.7.2.1)

[1072] query evalue score COG_category Description Preferred_name EC Catalyzes the formation of acetyl

[1073] phosphate from acetate and ATP.

[1074] yeast_k141_3471_1 3.91 e-68 218 C ackA 2.7.2.1

[1075] Can also catalyze the reverse

[1076] reaction

[1077] Catalyzes the formation of acetyl

[1078] phosphate from acetate and ATP.

[1079] yeast_k141_4688_1 5.87e-125 366 C ackA 2.7.2.1

[1080] Can also catalyze the reverse

[1081] reaction

[1082] Catalyzes the formation of acetyl

[1083] phosphate from acetate and ATP.

[1084] yeast_k141 _11828_1 1.92e-63 204 H ackA 2.7.2.1

[1085] Can also catalyze the reverse

[1086] reaction

[1087] Catalyzes the formation of acetyl

[1088] phosphate from acetate and ATP.

[1089] yeast_k141_12564_1 1.06e-19 86.7 F ackA 2.7.2.1

[1090] Can also catalyze the reverse

[1091] reaction

[1092] Catalyzes the formation of acetyl

[1093] phosphate from acetate and ATP.

[1094] yeast_k141_12564_2 2.34e-110 326 F ackA 2.7.2.1

[1095] Can also catalyze the reverse

[1096] reaction

[1097] Catalyzes the formation of acetyl

[1098] yeast_k141_17424_1 4.95e-100 301 C ackA 2.7.2.1

[1099]

[1100] phosphate from acetate and ATP. Can also catalyze the reverse

[1101] reaction

[1102] Catalyzes the formation of acetyl

[1103] phosphate from acetate and ATP.

[1104] yeast_k141_20165_1 1.36e-29 115 F ackA 2.7.2.1

[1105] Can also catalyze the reverse

[1106] reaction

[1107] Catalyzes the formation of acetyl

[1108] phosphate from acetate and ATP.

[1109] yeast_k141_22932_1 2.53e-28 112 C ackA 2.7.2.1

[1110] Can also catalyze the reverse

[1111] reaction

[1112] yeast_k141_23599_2 2.65e-92 289 F Acetokinase family - 2.7.2.1

[1113] Catalyzes the formation of acetyl

[1114] phosphate from acetate and ATP.

[1115] yeast_k141_23671_1 3.15e-20 89.7 F ackA 2.7.2.1

[1116] Can also catalyze the reverse

[1117] reaction

[1118] Catalyzes the formation of acetyl

[1119] phosphate from acetate and ATP.

[1120] yeast_k141_32838_1 2.39e-89 275 H ackA 2.7.2.1

[1121] Can also catalyze the reverse

[1122] reaction

[1123] Catalyzes the formation of acetyl

[1124] phosphate from acetate and ATP.

[1125] yeast_k141_35550_3 1.72e-212 597 C ackA 2.7.2.1

[1126] Can also catalyze the reverse

[1127] reaction

[1128] Catalyzes the formation of acetyl

[1129] phosphate from acetate and ATP.

[1130] yeast_k141_36427_1 1,66e-69 220 H ackA 2.7.2.1

[1131] Can also catalyze the reverse

[1132] reaction

[1133] Catalyzes the formation of acetyl

[1134] phosphate from acetate and ATP.

[1135] yeast_k141_38427_1 1.35e-72 230 F ackA 2.7.2.1

[1136] Can also catalyze the reverse

[1137] reaction

[1138] Catalyzes the formation of acetyl

[1139] phosphate from acetate and ATP.

[1140] yeast_k141_44361_1 6.42e-20 90.9 F ackA 2.7.2.1

[1141] Can also catalyze the reverse

[1142]

[1143] reaction Catalyzes the formation of acetyl

[1144] phosphate from acetate and ATP.

[1145] yeast_k141_46167_1 9.74e-13 68.2 H ackA 2.7.2.1

[1146] Can also catalyze the reverse

[1147] reaction

[1148]

[1149] yeast_k141 _51656_1 7.2e-83 255 F acetate kinase activity ackA 2.7.2.1 able 26. Gene annotation (EC 2.8.3.1)

[1150] query evalue score COG_category Description Preferred_name EC yeast_k141_44887_1 5.84e-79 252 I ketone body catabolic process ydiF 2.8.3.1,2.8.3.8

[1151] CoA transferase having broad

[1152] substrate specificity for shortchain acyl-CoA thioesters with the

[1153] yeast_k141_9924_2 2.38e-54 186 I - 2.8.3.1

[1154] activity decreasing when the

[1155] length of the carboxylic acid chain

[1156] exceeds four carbons

[1157] yeast_k141_13371_1 1.88e-16 78.6 I Coenzyme A transferase - 2.8.3.1

[1158] Malonate decarboxylase, alpha

[1159] yeast_k141_19209_1 2.48e-187 536 I - 2.8.3.1

[1160] subunit, transporter

[1161] CoA transferase having broad

[1162] substrate specificity for shortchain acyl-CoA thioesters with the

[1163] yeast_k141_28764_2 6.98e-15 73.6 I - 2.8.3.1

[1164] activity decreasing when the

[1165] length of the carboxylic acid chain

[1166] exceeds four carbons

[1167] Belongs to the 3-oxoacid CoA- yeast_k141_29550_1 1.08e-61 203 I - 2.8.3.1

[1168] transferase family

[1169] CoA transferase having broad

[1170] substrate specificity for shortchain acyl-CoA thioesters with the

[1171] yeast_k141_57272_2 1.61e-09 57.8 I - 2.8.3.1

[1172] activity decreasing when the

[1173] length of the carboxylic acid chain

[1174] exceeds four carbons

[1175] CoA transferase having broad

[1176] yeast_k141_58531_1 5.56e-10 59.3 I substrate specificity for short- 2.8.3.1

[1177]

[1178] chain acyl-CoA thioesters with the activity decreasing when the

[1179] length of the carboxylic acid chain

[1180] exceeds four carbons

[1181] Malonate decarboxylase, alpha

[1182] yeast_k141_59956_1 2.88e-57 194 I - 2.8.3.1 subunit, transporter

[1183] Malonate decarboxylase, alpha

[1184] yeast_k141_60080_1 5.06e-49 172 I - 2.8.3.1 subunit, transporter

[1185] CoA transferase having broad

[1186] substrate specificity for shortchain acyl-CoA thioesters with the

[1187] yeast_k141_69852_1 1.01e-22 97.4 I - 2.8.3.1 activity decreasing when the

[1188] length of the carboxylic acid chain

[1189] exceeds four carbons

[1190] Malonate decarboxylase, alpha

[1191] yeast_k141_73287_2 1,49e-25 105 I - 2.8.3.1 subunit, transporter

[1192] CoA transferase having broad

[1193] substrate specificity for shortchain acyl-CoA thioesters with the

[1194] yeast_k141_89617_1 4.73e-69 223 I - 2.8.3.1 activity decreasing when the

[1195] length of the carboxylic acid chain

[1196] exceeds four carbons

[1197] CoA transferase having broad

[1198] substrate specificity for shortchain acyl-CoA thioesters with the

[1199] yeast_k141_97773_1 1.49e-61 204 I ydiF 2.8.3.1 activity decreasing when the

[1200] length of the carboxylic acid chain

[1201] exceeds four carbons

[1202] yeast_k141_114123_1 6.24e-41 147 I Coenzyme A transferase - 2.8.3.1

[1203] CoA transferase having broad

[1204] substrate specificity for shortchain acyl-CoA thioesters with the yeast_k141_140051_1 1.75e-67 221 I - 2.8.3.1 activity decreasing when the

[1205] length of the carboxylic acid chain

[1206] exceeds four carbons

[1207] Malonate decarboxylase, alpha

[1208] yeast_k141_140584_1 1.38e-262 733 I - 2.8.3.1

[1209]

[1210] subunit, transporter able 27. Gene annotation (EC 2.8.3.8)

[1211] query evalue score COG_category Description Preferred_name EC

[1212] TIGRFAM 3-oxoacid CoA- yeast_k141_19465_1 9e-47 157 I - 2.8.3.8, 2.8.3.9

[1213] transferase, A subunit

[1214] 3-oxoacid CoA-transferase, B

[1215] yeast_k141_19465_2 1.95e-101 300 I ctfB 2.8.3.8, 2.8.3.9

[1216] subunit

[1217] yeast_k141_30987_1 2.88e-47 157 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9 yeast_k141_44887_1 5.84e-79 252 I ketone body catabolic process ydiF 2.8.3.1,2.8.3.8

[1218] 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_145700_1 1.25e-146 415 I PFAM Coenzyme A transferase scoA

[1219] 8.3.9

[1220] TIGRFAM 3-oxoacid CoA- yeast_k141_152011_1 6.6e-62 204 I - 2.8.3.5, 2.8.3.8, 2.8.3.9 transferase, B subunit

[1221] yeast_k141_225861_1 2.93e-64 209 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9 yeast_k141_241837_1 5.51e-14 69.3 I Coenzyme A transferase atoD 2.8.3.8, 2.8.3.9

[1222] Acyl CoA acetate 3-ketoacid CoA

[1223] yeast_k141_264504_1 5.72e-51 167 I - 2.8.3.5, 2.8.3.8, 2.8.3.9 transferase beta subunit

[1224] yeast_k141_290718_1 3.46e-45 152 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9 yeast_k141_316986_1 5.23e-29 109 I Coenzyme A transferase - 2.8.3.8, 2.8.3.9

[1225] 3-oxoacid CoA-transferase, a 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_383982_1 5.11e-76 232 I scoA

[1226] subunit 8.3.9

[1227] COG 1788 Acyl CoA acetate 3- yeast_k141_431072_1 1e-30 115 I ketoacid CoA transferase, alpha - 2.8.3.8, 2.8.3.9

[1228] subunit

[1229] 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_507559_2 7.05e-39 135 I PFAM Coenzyme A transferase scoA

[1230] 8.3.9 yeast_k141_520609_1 4.94e-39 135 I Coenzyme A transferase - 2.8.3.8, 2.8.3.9 yeast_k141_520609_2 9.75e-24 97.1 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9

[1231]

[1232] yeast_k141_575934_5 2.23e-133 380 I Coenzyme A transferase - 2.8.3.8, 2.8.3.9

[1233] able 28. Gene annotation (EC 2.8.3.9)

[1234] query evalue score COG_category Description Preferred_name EC

[1235] TIGRFAM 3-oxoacid CoA- yeast_k141_19465_1 9e-47 157 I - 2.8.3.8, 2.8.3.9

[1236]

[1237] transferase, A subunit 3-oxoacid CoA-transferase, B

[1238] yeast_k141_19465_2 1.95e-101 300 I ctfB 2.8.3.8, 2.8.3.9

[1239] subunit

[1240] yeast_k141_30987_1 2.88e-47 157 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9

[1241] 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_145700_1 1.25e-146 415 I PFAM Coenzyme A transferase scoA

[1242] 8.3.9

[1243] TIGRFAM 3-oxoacid CoA- yeast_k141_152011_1 6.6e-62 204 I - 2.8.3.5, 2.8.3.8, 2.8.3.9 transferase, B subunit

[1244] yeast_k141_225861_1 2.93e-64 209 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9 yeast_k141_241837_1 5.51e-14 69.3 I Coenzyme A transferase atoD 2.8.3.8, 2.8.3.9

[1245] Acyl CoA acetate 3-ketoacid CoA

[1246] yeast_k141_264504_1 5.72e-51 167 I - 2.8.3.5, 2.8.3.8, 2.8.3.9 transferase beta subunit

[1247] yeast_k141_290718_1 3.46e-45 152 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9 yeast_k141_316986_1 5.23e-29 109 I Coenzyme A transferase - 2.8.3.8, 2.8.3.9

[1248] 3-oxoacid CoA-transferase, a 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_383982_1 5.11e-76 232 I scoA

[1249] subunit 8.3.9

[1250] COG 1788 Acyl CoA acetate 3- yeast_k141_431072_1 1e-30 115 I ketoacid CoA transferase, alpha - 2.8.3.8, 2.8.3.9

[1251] subunit

[1252] 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_507559_2 7.05e-39 135 I PFAM Coenzyme A transferase scoA

[1253] 8.3.9 yeast_k141_520609_1 4.94e-39 135 I Coenzyme A transferase - 2.8.3.8, 2.8.3.9 yeast_k141_520609_2 9.75e-24 97.1 I Coenzyme A transferase - 2.8.3.5, 2.8.3.8, 2.8.3.9 yeast_k141_575934_5 2.23e-133 380 I Coenzyme A transferase - 2.8.3.8, 2.8.3.9

[1254] 2.8.3.5, 2.8.3.6, 2.8.3.8, 2. yeast_k141_576548_2 4.05e-100 294 I CoA-transferase scoB

[1255]

[1256] 8.3.9

[1257] able 29. Gene annotation (EC 3.1.2.20)

[1258] query evalue score COG_category Description Preferred_name EC yeast_k141_59735_1 1.47e-41 141 I acyl-coa hydrolase HA62_15520 3.1.2.20

[1259] Lysophospholipase L1 and

[1260] yeast_k141_143477_1 1.81e-42 146 E - 3.1.2.20

[1261] related esterases

[1262] yeast_k141_144246_2 7.53e-52 167 I acyl-coa hydrolase HA62_15520 3.1.2.20

[1263]

[1264] yeast_k141_229361_1 2.33e-41 140 I acyl-coa hydrolase HA62_15520 3.1.2.20 yeast_k141_235536_1 6.76e-34 121 I acyl-coa hydrolase ykhA 3.1.2.20

[1265] GDSL-like Lipase / Acylhydrolase

[1266] yeast_k141_287946_2 6.04e-80 244 E - 3.1.2.20

[1267] family

[1268] yeast_k141_316303_3 7.59e-69 212 I acyl-coa hydrolase ykhA 3.1.2.20 yeast_k141_321853_1 1,27e-50 163 I acyl-coa hydrolase HA62_15520 3.1.2.20 yeast_k141_367929_2 1.23e-11 62.4 I acyl-coa hydrolase HA62_15520 3.1.2.20

[1269] PFAM lipolytic protein G-D-S-L

[1270] yeast_k141_383356_1 6.85e-25 99.4 E - 3.1.2.20

[1271] family

[1272] yeast_k141_405365_1 2.27e-37 131 I acyl-coa hydrolase vdID 3.1.2.20 yeast_k141_440652_1 3.37e-48 158 I acyl-coa hydrolase HA62_15520 3.1.2.20 yeast_k141_544573_2 1,44e-46 154 I acyl-coa hydrolase HA62_15520 3.1.2.20

[1273] PFAM lipolytic protein G-D-S-L

[1274] yeast_k141_571737_1 2.06e-07 51.6 E - 3.1.2.20

[1275] family

[1276] PFAM lipolytic protein G-D-S-L

[1277] yeast_k141_609003_1 1.13e-37 134 E - 3.1.2.20

[1278] family

[1279] yeast_k141_660251_1 4.83e-72 220 I acyl-coa hydrolase ykhA 3.1.2.20

[1280]

[1281] yeast_k141_692903_5 1.7e-52 169 I acyl-coa hydrolase HA62_15520 3.1.2.20

[1282] able 30. Gene annotation (EC 4.2.1.17)

[1283] query evalue score COG_category Description Preferred_name EC

[1284] Belongs to the enoyl-CoA

[1285] yeast_k141_790647_1 3.3e-13 71.6 I - 4.2.1.17,5.3.3.8

[1286] hydratase isomerase family

[1287] yeast_k141_472380_2 2.46e-86 261 I Enoyl-CoA hydratase / isomerase - 4.2.1.17,5.3.3.18 yeast_k141_600682_1 3.95e-05 46.6 I Enoyl-CoA hydratase / isomerase - 4.2.1.17,5.3.3.18

[1288] Belongs to the enoyl-CoA

[1289] yeast_k141_724245_2 9.69e-09 58.9 I - 4.2.1.17,5.3.3.18

[1290] hydratase isomerase family

[1291] yeast_k141_776849_2 5.54e-29 110 I Enoyl-CoA hydratase / isomerase - 4.2.1.17,5.3.3.18

[1292] Belongs to the enoyl-CoA

[1293] yeast_k141_778899_2 5.8e-75 236 I - 4.2.1.17,5.3.3.18

[1294] hydratase isomerase family

[1295] yeast_k141_842926_2 2.7e-80 248 I Enoyl-CoA hydratase / isomerase - 4.2.1.17,5.3.3.18

[1296] 4.2.1.17,4.2.1.18,4.2.1.5 yeast_k141_130099_1 3.21 e-65 209 I Enoyl-CoA hydratase liuC

[1297]

[1298] 7 Belongs to the enoyl-CoA

[1299] yeast_k141_23048_2 8.63e-66 212 I - 4.2.1.17,4.2.1.18 hydratase isomerase family

[1300] yeast_k141_79746_1 9.16e-22 92.4 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18 yeast_k141_147340_1 1.27e-14 74.3 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18 yeast_k141_160284_2 1.37e-83 257 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18 yeast_k141_229083_1 1.04e-26 105 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18 yeast_k141_240872_1 4.87e-93 281 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18 yeast_k141_244483_1 3.86e-69 215 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18 yeast_k141_417207_1 4.58e-19 86.3 I Enoyl-CoA hydratase / isomerase 4.2.1.17,4.2.1.18

[1301] Belongs to the enoyl-CoA

[1302] yeast_k141_425100_1 1.04e-59 191 I crt 4.2.1.17,4.2.1.18

[1303]

[1304] hydratase isomerase family

[1305] able 31. Gene annotation (EC 4.2.1.55)

[1306] query evalue score COG_category Description Preferred_name EC

[1307] PFAM MaoC domain protein

[1308] yeast_k141_25125_1 6.59e-15 70.1 I - 4.2.1.55

[1309] dehydratase

[1310] yeast_k141_217847_1 1.34e-11 62.4 I dehydratase - 4.2.1.55

[1311] N-terminal half of MaoC

[1312] yeast_k141_252916_2 6.02e-34 122 I - 4.2.1.55

[1313] dehydratase

[1314] N-terminal half of MaoC

[1315] yeast_k141_252935_1 9.13e-70 213 I - 4.2.1.55

[1316] dehydratase

[1317] N-terminal half of MaoC

[1318] yeast_k141_260204_2 3.31 e-63 196 I - 4.2.1.55

[1319] dehydratase

[1320] yeast_k141_261330_1 1.05e-23 92.8 I COG2030 Acyl dehydratase phaJ 4.2.1.55 yeast_k141_261626_1 0.000841 41.6 I MaoC like domain - 4.2.1.55

[1321] PFAM MaoC domain protein

[1322] yeast_k141_266714_1 1.04e-42 144 I - 4.2.1.55

[1323] dehydratase

[1324] yeast_k141_273064_2 2.62e-23 92.8 I dehydratase - 4.2.1.55

[1325] PFAM MaoC domain protein

[1326] yeast_k141_301458_1 1.55e-24 95.5 I - 4.2.1.55

[1327] dehydratase

[1328] yeast_k141_459706_2 2.86e-73 222 I MaoC like domain - 4.2.1.55

[1329] N-terminal half of MaoC

[1330] yeast_k141_463614_1 7.48e-47 153 I - 4.2.1.55

[1331]

[1332] dehydratase yeast_k141_476468_1 9.35e-10 60.1 I dehydratase maoC 4.2.1.119,4.2.1.55

[1333] PFAM MaoC domain protein

[1334] yeast_k141_507911_2 1.16e-21 87.8 I phaJ 4.2.1.55

[1335] dehydratase

[1336] N-terminal half of MaoC

[1337] yeast_k141_514013_1 2.17e-27 103 I - 4.2.1.55

[1338] dehydratase

[1339] yeast_k141_558814_2 7.03e-17 77 I COG2030 Acyl dehydratase - 4.2.1.55

[1340] Hydroxyacyl-thioester

[1341] yeast_k141_580405_1 4.25e-08 54.7 I - 2.7.4.3,4.2.1.55

[1342]

[1343] dehydratase type 2

[1344] able 32. Gene annotation (EC 6.2.1.1)

[1345] query evalue score COG_category Description Preferred_name EC Catalyzes the conversion of

[1346] acetate into acetyl-CoA (AcCoA),

[1347] an essential intermediate at the

[1348] junction of anabolic and catabolic

[1349] pathways. AcsA undergoes a two- step reaction. In the first half

[1350] yeast_k141_1428_1 1.39e-34 132 I reaction, AcsA combines acetate acsA 6.2.1.1

[1351] with ATP to form acetyl-adenylate

[1352] (AcAMP) intermediate. In the

[1353] second half reaction, it can then

[1354] transfer the acetyl group from

[1355] AcAMP to the sulfhydryl group of

[1356] CoA, forming the product AcCoA

[1357] yeast_k141 _111040_4 0 952 I AMP-binding enzyme acsA 6.2.1.1,6.2.1.32 yeast_k141_207348_1 1.31e-42 152 I AMP-binding enzyme acsA 6.2.1.1,6.2.1.32 yeast_k141_209463_1 0 974 I Psort location Cytoplasmic, score acsA 6.2.1.1,6.2.1.32

[1358] AMP-binding enzyme C-terminal

[1359] yeast_k141_653970_1 3.01 e-44 158 I MA20_27440 6.2.1.1,6.2.1.32 domain

[1360] AMP-binding enzyme C-terminal

[1361] yeast_k141_660249_1 2.95e-52 179 I acsA 6.2.1.1,6.2.1.32 domain

[1362] AMP-binding enzyme C-terminal

[1363] yeast_k141_899283_1 1.32e-51 179 I acsA 6.2.1.1,6.2.1.32

[1364]

[1365] domain AMP-binding enzyme C-terminal

[1366] yeast_k141_856959_1 3.44e-19 89.4 I MA20_27440 6.2.1.1,6.2.1.2,6.2.1.32 domain

[1367] PFAM AMP-dependent

[1368] yeast_k141_230781_1 3.41e-246 689 I - 6.2.1.1.6.2.1.2

[1369] synthetase and ligase

[1370] AMP-binding enzyme C-terminal

[1371] yeast_k141_495173_1 1.3e-18 84.7 I - 6.2.1.1.6.2.1.2

[1372] domain

[1373] AMP-binding enzyme C-terminal

[1374] yeast_k141_625078_1 1.19e-88 275 I - 6.2.1.1.6.2.1.2

[1375] domain

[1376] AMP-binding enzyme C-terminal

[1377] yeast_k141_740454_1 1.95e-66 216 I - 6.2.1.1.6.2.1.2

[1378] domain

[1379] AMP-binding enzyme C-terminal

[1380] yeast_k141_932915_1 9.44e-170 487 I - 6.2.1.1.6.2.1.2

[1381] domain

[1382] COG0365 Acyl-coenzyme A

[1383] yeast_k141 _14124_1 0.000911 40.8 I synthetases AM P-(fatty) acid PrpE 6.2.1.1,6.2.1.17

[1384] ligases

[1385] PrpE from Ralstonia

[1386] solanacearum can produce

[1387] acetyl-, propionyl-, butyryl- and

[1388] yeast_k141_25610_1 1.07e-119 360 I acrylyl-coenzyme A, and PrpE 6.2.1.1,6.2.1.17

[1389] Salmonella enterica produces

[1390] propionyl- and butyryl-coenzyme

[1391] A

[1392] Acetyl-coenzyme A synthetase N- yeast_k141_72198_1 1.33e-92 288 I PrpE 6.2.1.1,6.2.1.17

[1393] terminus

[1394] PrpE from Ralstonia

[1395] solanacearum can produce

[1396] acetyl-, propionyl-, butyryl- and

[1397] yeast_k141_107312_1 8.48e-47 165 I acrylyl-coenzyme A, and PrpE 6.2.1.1,6.2.1.17

[1398] Salmonella enterica produces

[1399] propionyl- and butyryl-coenzyme

[1400]

[1401] A Conclusion

[1402] This example demonstrated a novel integrated process that involves in-situ production of both electron donor and acceptor from algae through bioaugmentation of algae fermentation with yeast. The results revealed that the addition of yeast led to a fourfold increase in carboxylate yield. This novel process elongates the carbon chain of fermentation products, resulting in the synthesis of medium-chain carboxylic acids with a yield of 46.3 mM-C / g VS. This enhancement was not solely due to the in-situ production of ethanol but also to the high substrate utilization rate in the yeast group. The microbial structure unveiled the pivotal role of heightened chain elongator presence in this enhancement. Moreover, the organized microbial guild emerged as a key factor in enhancing substrate utilization rates. Metabolic potential analysis underscored the increased abundance of genes linked to acetyl-CoA production as the primary pathway for MCFA synthesis, while the AOR-related pathway orchestrated butanol production. In essence, this research pioneers a revolutionary chain elongation process for the production of MCFAs and butanol, achieving this milestone without reliance on external electron donors.

[1403] Example 3 - Biologically transforming Oedogonium into high-value liquid biofuels Oedogonium is a green algae specially generated by RegenAqua that was grown in wastewater. It typically represents a substantial but largely untapped renewable energy resource as it carries a substantial amount of chemical energy, just like other organic wastes we mentioned above. The aim of this example is to use various strategies to produce biofuels from Oedogonium harvested from wastewater treatment and evaluate the feasibility of biotransforming Oedogonium into high-value liquid biofuels.

[1404] Methods and Materials

[1405] Source of algae and inoculum

[1406] Oedogonium was collected from real wastewater, which were stored in a refrigerator at -20 °C before use. Prior to the tests, the algae were diluted to a VSS concentration of 15 g / L using tap water.

[1407] The inoculum used in the study was anaerobic digestion sludge (ADS) and obtained from our lab-scaled mesophilic reactor, which was used to treat excess activated sludge. To remove residual organics, the sludge was self-digested for a minimum of a month without any external organic supply. The final inoculum had TSS and VSS concentrations of 46.26 ± 0.29 g / L and 20.01± 0.16 g / L, respectively.

[1408] Anaerobic algae fermentation reactor setup The anaerobic algae fermentation tests were conducted using long-term continuous experiments. Specifically, four groups of anaerobic fermenters were prepared by adding algae as substrate and introducing ADS and / or yeast as inoculum, aiming to achieve substrate-to-inoculum volatile solids (VS) ratios of 0.5. Strategy 1 focused on yeast fermentation using varying amounts of dry yeast (0.1 g, 0.5 g, and 1 g) with algae as the substrate. The pH was adjusted to 5.0 using NaOH or HCI (3 M) and maintained throughout the experiment. Strategy 2 explored ethanol-type fermentation with algae and ADS at three pH levels: 4.0, 4.5, and 5.0. The pH was manually maintained using 3 M NaOH or HCI as needed. Strategy 3 evaluated the production of bio-MCFAs by combining ADS, algae, and yeast under a neutral pH (7.0). This aimed to optimize chain elongation and fatty acid synthesis. Strategy 4 investigated a two-step approach, starting with acidic fermentation (pH 4.5) of algae and ADS, followed by a second step where the supernatant liquor was transferred to ADS at pH 7.0 for further elongation and product formation. For all strategies, the fermentation bioreactors were purged with nitrogen for 15 minutes to maintain anaerobic conditions, and sealed to maintain anaerobic conditions. Reactors were placed under the temperature of 35 ± 0.1°C with agitation at 130 rpm. Sampling was conducted periodically to monitor pH, gas, and liquid composition. The pH was adjusted manually after each sampling to maintain the desired levels.

[1409] Chemical analytical methods

[1410] The headspace gas sample (1 mL) was extracted using a syringe equipped with a sealing valve and analyzed using a gas chromatograph (GC-112A, China) with a thermal conductivity detector (TCD) to measure the concentrations of H2, CO2, and CH4. Simultaneously, a liquid sample (2 mL) was taken, centrifuged, and filtered. To stabilize carboxylic acids in their free molecular form, a 5 pL / mL solution of HCOOH was added. Carboxylic acids and butanol were quantified using a gas chromatograph (GC-2010 Plus, SHIMADZU, Japan) equipped with a flame ionization detector (FID) and a capillary column (SH-Stabilwax-DA, 30 m x 0.32 mm x 0.25 pm).

[1411] Results

[1412] Bioethanol production from Oedogonium via anaerobic fermentation

[1413] FIG. 21 showed the concentration of ethanol produced from Oedogonium by yeast fermentation as strategy 1. Amylase pretreatment is significantly more effective in ethanol production than cellulase pretreatment, because amylase pretreatment can effectively destroy the structure of biomass, resulting in more fermentable sugars. During yeast fermentation of pretreated green algae, the higher the yeast dosage, the higher the ethanol concentration (15.7g / L). The ethanol yield can reach up to 0.31g / g-VS Oedogonium at the highest yeast dosage (0.2g / g-VS) in this study. FIG. 22 showed the yield of ethanol produced from Oedogonium by Ethanol-type fermentation as strategy 2. Some species (e.g., Ethanoligenens) in ADS can utilize carbohydrates to produce ethanol, known as ethanol-type fermentation. The pH is a critical parameter to maintain the activity of ethanol producers because they can tolerant the low pH levels, but other microorganisms cannot. Acidic conditions (pH=4) under amylase pretreatment are more conducive to ethanol production (0.17g / g-VS green algae), while the changes of ethanol production under different pH conditions with cellulase pretreatment are not obvious and significantly lower than the former. Apart from ethanol, a certain amount of MCFAs (caproate, 0.01-0.09 g / g-VS green algae) were also observed at acidic conditions, although neutral conditions are more conducive to chain elongation.

[1414] Bio-MCFAs production from Oedogonium via anaerobic fermentation In strategy 3, yeast is inoculated in the system to produce ethanol from Oedogonium as electron donor of chain elongation. Fermentative bacterium in ADS firstly converts the carbohydrates in the Oedogonium to short chain fatty acids (SCFAs) as electron acceptor of chain elongation. Then chain elongator in ADS can elongate the carbon chains of SCFAs by adding 2 carbon atoms from ED (ethanol) per cycle, thereby producing MCFAs. FIG. 23 showed the yield of MCFAs produced from Oedogonium by yeast fermentation with chain elongation as strategy 3. During 9 days’ anaerobic fermentation, ethanol is continuously produced by yeast from Oedogonium and gradually consumed for chain elongation. The 0.27 g / g-VS-algae MCFAs (i.e., caproate) was produced from Oedogonium with amylase pretreatment, which is higher than that (0.09 g / g-VS algae) with cellulase pretreatment. A small amount of butanol (0.01 g / g-VS algae) is also gained from Oedogonium with amylase pretreatment.

[1415] In strategy 4, ethanol is in suit produced from Oedogonium by ADS through ethanol-type fermentation at pH 4.5 in the first reactor. Then ethanol is served as electron donor for chain elongation of SCFAs produced from Oedogonium in the second reactor at pH 7.0, which is more conducive to chain elongation than acid pH. FIG. 24 showed the yield of MCFAs produced from Oedogonium by ethanol-type fermentation with chain elongation as strategy 4. The 0.21 g / g-VS-algae MCFAs (i.e., caproate) was produced from Oedogonium with amylase pretreatment, which is higher than that (0.05 g / g-VS algae) with cellulase pretreatment.

[1416] Oedogonium versus microalgae as feedstock

[1417] As shown in Table 33, Oedogonium is a high-quality feedstock for the microbial communities to produce biofuels based our preliminary experiments, comparable or slightly better than microalgae (Chlorella) previously tested. This should be due to the high carbohydrate content in Oedogonium harvested from wastewater treatment. It is expected that the production rate can be further improved / optimised if the microbial communities can be enriched that would be particularly suitable for Oedogonium bioconversion in next stage experiments. Table 33. Oedogonium vs. Chlorella as feedstock for bioethanol and bio-MCFA production Feedstock Bioethanol production Bio-MCFA production

[1418] Oedogonium Strategy 1 - 0.31 g / g-VS algae Strategy 1 - 0 g / g-VS algae Strategy 2 - 0.17 g / g-VS algae Strategy 2 - 0.09 g / g-VS algae Strategy 3 - 0.14 g / g-VS algae Strategy 3 - 0.27 g / g-VS algae Strategy 4 - 0.04 g / g-VS algae Strategy 4 - 0.21 g / g-VS algae Chlorella Strategy 1 - 0.23 g / g-VS algae Strategy 1 - 0 g / g-VS algae Strategy 2 - 0.13 g / g-VS algae Strategy 2 - 0.08 g / g-VS algae Strategy 3 - 0.11 g / g-VS algae Strategy 3 - 0.27 g / g-VS algae Strategy 4 - 0.02 g / g-VS algae Strategy 4 - 0.18 g / g-VS algae Conclusions

[1419] These four strategies developed by the inventor are promising and feasible to produce bioethanol and bio- MCFAs from Oedogonium harvested from wastewater treatment.

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[1497] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

[1498] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[1499] Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in the field.

Claims

CLAIMS1. A method for anaerobic fermentation, comprising:(a) providing at least one substrate, wherein the at least one substrate comprises algae; (b) adding a first plurality of anaerobic microorganisms for carboxylate biosynthesis and chain elongation from the at least one substrate;(c) adding one or more of a second microorganism for ethanol fermentation from the at least one substrate;wherein the first plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol fermentation, andwherein the method contributes to the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate.

2. A system for anaerobic fermentation, comprising(a) a first plurality of anaerobic microorganisms for carboxylate biosynthesis and chain elongation from at least one substrate, wherein the at least one substrate comprises algae; and(b) one or more of a second microorganism for ethanol fermentation from the at least one substrate;wherein the first plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol fermentation, andwherein the system contributes to the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate.

3. The method of claim 1 or the system of claim 2, wherein the one or more second microorganism comprises microorganisms capable of converting organic substrates into ethanol under anaerobic conditions.

4. The method or the system according to any one of claims 1 to 3, wherein the one or more second microorganism is selected from yeast, thermophilic anaerobic bacterium, a- proteobacterium, ora combination thereof.

5. The method or the system according to any one of claims 1 to 4, wherein the one or more second microorganism is yeast.

6. The method or the system according to any one of claims 1 to 5, wherein the one or more second microorganism is Saccharomyces cerevisiae.

7. The method or the system according to any one of claims 1 to 6, wherein the first plurality of anaerobic microorganisms and the second microorganism are added to the substrate in a single bioreactor.

8. The method or the system of claim 7, wherein the volatile solid content ratio for the at least one substrate and the first plurality of anaerobic microorganisms is 0.1:1, preferably 0.5:1.

9. The method or the system of claim 7 or 8, wherein the temperature for achieving chain elongation and ethanol production in a single anaerobic reaction system is within the range of mesophilic anaerobic fermentation, preferably between 30 °C and 40 °C.

10. The method or the system of any one of claims 7-9, wherein pH value of the single bioreactor is adjusted to 5.

11. The method or the system according to any one of claims 1 to 10, wherein the second microorganism is added at a dosage of 0.2 to 0.5 g / g-VS.

12. A method for anaerobic fermentation, comprising:(a) combining at least one substrate and a plurality of anaerobic microorganisms, wherein the at least one substrate comprises algae;(b) performing ethanol-type fermentation from the at least one substrate and the plurality of anaerobic microorganisms; and(c) performing carboxylate biosynthesis and chain elongation from the at least one substrate and the plurality of anaerobic microorganismswherein the ethanol-type fermentation is performed at about pH 4-5, and wherein the carboxylate biosynthesis and chain elongation is performed at about pH 6-7,wherein the plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol-type fermentation, andwherein the method contributes to the production of medium-chain carboxylates and / or ethanol from the anaerobic fermentation of the at least one substrate.

13. A system for anaerobic fermentation, comprising a plurality of anaerobic microorganisms for performing ethanol-type fermentation and carboxylate biosynthesis and chain elongation from at least one substrate, wherein the at least one substrate comprises algae,wherein the ethanol fermentation is performed at about pH 4-5 or below and the carboxylate biosynthesis and chain elongation is performed at about pH 6-7,wherein the plurality of anaerobic microorganisms comprises microorganisms that are responsible for chain elongation of carboxylate using at least one bioproduct produced from the ethanol-type fermentation, andwherein the system contributes to the production of medium-chain carboxylates and / or ethanol from the anaerobic fermentation of the at least one substrate.

14. The method of claim 12 or the system of claim 13, wherein the ethanol-type fermentation is performed at about pH 4.5.15 The method or the system according to any one of claims 12 to 14, wherein the carboxylate biosynthesis and chain elongation is performed at about pH 7.

16. The method or the system according to any one of claims 1 to 15, wherein the at least one bioproduct produced from the ethanol-type fermentation comprises ethanol and / or hydrogen.

17. The method or the system according to any one of claims 1 to 16, wherein the production of medium-chain carboxylates from the anaerobic fermentation of the at least one substrate comprises using at least one bioproduct produced from the ethanol-type fermentation as an electron donor to extend carbon chain length of short-chain carboxylates into medium¬ chain carboxylates.

18. The method or the system according to claim 17, wherein the at least one bioproduct produced from the ethanol-type fermentation as an electron donor is ethanol.

19. The method or the system according to any one of claims 1 to 18, wherein the at least one bioproduct from the ethanol-type fermentation further contributes to controlling chain elongation of short-chain carboxylates into medium-chain carboxylates.

20. The method or the system according to claim 19, wherein the at least one product produced from the ethanol-type fermentation that contributes to controlling chain elongation is hydrogen.

21. The method or the system according to any one of claims 1 to 20, wherein no exogenous ethanol (and / or hydrogen) is added to the system or the method.

22. The method of the system according to any one of claims 1 to 20, wherein additional exogenous ethanol (and / or hydrogen) is added to the system or the method for chain elongation of carboxylate.

23. The method or system according to any one of claims 1 to 22, wherein the algae comprises raw algae, pretreated algae or a combination thereof.

24. The method or system according to claim 23, wherein the pretreated algae is prepared by a pretreatment method selected from mechanical treatment, thermal treatment, chemical treatment, thermal hydrolysis or a combination thereof.

25. The method or system according to claim 23 or 24, wherein the algae is pretreated with amylase.

26. The method or system according to any one of claims 1 to 25, wherein the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms further comprises microorganisms that are responsible for hydrolysis and acidification of the at least one substrate.

27. The method or system according to any one of claims 1 to 26, wherein the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms are combinations of functional anaerobic microorganisms responsible for hydrolysis of macromolecules in substrates, acidification and carbon chain extension of hydrolysis products.

28. The method or system according to any one of claims 1 to 27, wherein the anaerobic microorganisms are combinations of functional anaerobic microorganisms extracted from existing anaerobic fermenters.

29. The method or system according to any one of claims 1-28, further comprising one or more reducing agent to drive reduction reactions that convert medium-chain fatty acid to long-chain alcohols.

30. The method or system according to claim 29, wherein the one or more reducing agents is selected from reducing coenzymes, microcurrent, ascorbic acid, ora combination thereof.

31. The method or system according to claim 29 or 30, wherein the first plurality of anaerobic microorganisms or the plurality of anaerobic microorganisms further comprisesmicroorganisms that are responsible for reduction reactions that convert medium-chain fatty acid to long-chain alcohols in the presence of the one or more reducing agent.

32. The method or system according to any one of claims 29 to 31, wherein the long-chain alcohols are selected from butanol, pentanol, hexanol, heptanol, octanol, or a combination thereof.

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