WO2025240457A1

WO2025240457A1 - Conversion of biomass into industrial chemicals using engineered bacteria

Info

Publication number: WO2025240457A1
Application number: PCT/US2025/029115
Authority: WO
Inventors: Robert M. Kelly; Ryan G. BING; Daniel B. SULIS; Jack P. WANG; Daniel J. WILLARD; Kathryne C. FORD; Michael W. Adams
Original assignee: University of Georgia; North Carolina State University; University of Georgia Research Foundation Inc
Current assignee: University of Georgia; North Carolina State University; University of Georgia Research Foundation Inc
Priority date: 2024-05-13
Filing date: 2025-05-13
Publication date: 2025-11-20
Anticipated expiration: 2026-11-13

Abstract

Described herein are engineered extremely thermophilic bacterium including a heterologous gene encoding a bifunctional alcohol dehydrogenase. Also described herein are systems and methods for enhancing selective recovery of desirable products from plant biomass.

Description

Attorney Dkt.10620-156WO1 CONVERSION OF BIOMASS INTO INDUSTRIAL CHEMICALS USING ENGINEERED BACTERIA CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63/646,212, filed May 13, 2024, and to U.S. Provisional Application No.63/646,189, filed May 13, 2024, each of which is incorporated by reference herein in its entirety. STATEMENT ACKNOWLEDGING GOVERNMENT SUPPORT This invention was made with government support under DE-SC0022192 awarded by the U.S. Department of Energy, T32 GM008776 awarded by the National Institutes of Health, and 2018-67021-27716 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on May 13, 2025, as an .XML file entitled “10620- 156WO1_ST26” created on May 12, 2025, and having a file size of 201,611 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND Renewable alternatives for non-electrifiable fossil-derived chemicals are needed and plant matter, the most abundant biomass on earth, provide an ideal feedstock. However, the heterogeneous polymeric composition of lignocellulose makes conversion difficult. Lignin presents a formidable barrier to fermentation of non-pretreated biomass. Extensive chemical and enzymatic treatments can liberate fermentable carbohydrates from plant biomass, but microbial routes offer many advantages, including concomitant conversion to industrial chemicals. Current microbial fermentation pathways typically suffer from low yields and selectivity of desirable products, limiting their widespread industrial use. Thus, there is a need for fermentation methods and engineered bacteria that generate sufficient titers/yields to improve the selective conversion of biomass feedstocks to commercially valuable products. Attorney Dkt.10620-156WO1 SUMMARY Described herein are engineered extremely thermophilic bacteria and methods related to the conversion of a biomass to a carbonaceous product. In various aspects, described herein is a genetically modified extremely thermophilic bacterium, wherein the genetically modified extremely thermophilic bacterium comprises a heterologous gene encoding a bifunctional alcohol dehydrogenase. In some aspects, the engineered bacterium is a bacterium from the order Caldicellulosiruptorales. In some aspects, the genetically modified bacterium comprises Anaerocellum bescii. In some aspects, the heterologous gene comprises an AdhE gene from Thermoclostridium stercorarium. In some aspects, the AdhE gene from Thermoclostridium stercorarium comprises an Asp492Gly mutation. In some aspects, a gene encoding lactate dehydrogenase expression has been inactivated. In some aspects, a gene encoding lactate dehydrogenase has been inactivated by homologous recombination. In some aspects, the heterologous gene bifunctional AdhE is expressed from a plasmid. In some aspects, the heterologous gene encoding the bifunctional alcohol dehydrogenase is inserted into a genomic locus of the bacterium. In some aspects, the engineered bacterium further includes a promoter operably linked to the heterologous gene encoding the bifunctional alcohol dehydrogenase, wherein the promoter is configured to function at elevated temperatures. In some aspects, the heterologous bifunctional alcohol dehydrogenase gene is inserted downstream of Athe_0949 in the A. bescii genome. In some aspects, described herein are genetically modified A. bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C. In some aspects, described herein is a method for enhancing ethanol production. In some aspects, the method includes: obtaining a biomass sample; contacting the plant biomass sample with a genetically modified bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase under conditions effective to convert the plant biomass to ethanol. In some aspects, the conditions effective include one or more of: (i) maintaining pH between 5.8 and 7, and/or (ii) maintaining an operational temperature of 55-75°C. In some aspects, the genetically modified bacterium comprising A. bescii that has been genetically modified with the insertion of alcohol dehydrogenase gene. Attorney Dkt.10620-156WO1 In some aspects, described herein are genetically modified Anaerocellum bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C. In some aspects, described herein are genetically modified Anaerocellum bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C. Also described herein is a bioreactor system for enhancing ethanol production and selectivity relative to other fermentation byproducts from plant biomass, comprising: a temperature control system configured to maintain a fermentation temperature (e.g., from 55°C to 75°C) for a genetically modified A. bescii strain, the genetically modified A. bescii bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase; a pH control system configured to maintain a pH between 5.8-7.0 (e.g., using sodium bicarbonate and sodium hydroxide); a pressurization system configured to control carbon dioxide head pressure; and an agitation system configured to mitigate cell aggregation. In some aspects, the fermentation byproducts comprise acetate, acetone, lactate, hydrogen, carbon dioxide, acetoin, 2,3-butanediol, pyruvate, butanol, isopropanol, and/or higher chain alcohols. In some aspects, described is a method of producing ethanol from a plant biomass sample. The method can include: obtaining a plant biomass sample; contacting the plant biomass sample with any of the genetically modified thermophilic bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample; and fermenting the fermentable carbohydrate to produce ethanol. In some aspects, the plant biomass sample comprises a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. In some aspects, the poplar tree is a genetically modified poplar tree. In some aspects, the mixing of the plant biomass sample with the genetically modified bacterium occurs at about pH 5.8-7.0. In some aspects, the pH is maintained using NaOH or NaHCO₃. In some aspects, contacting the plant biomass sample with the genetically modified bacterium occurs at about 55-75˚C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample. In some aspects, described herein is a method of degrading lignin in a plant biomass sample, comprising: obtaining a plant biomass sample; mixing the plant biomass sample with Attorney Dkt.10620-156WO1 the genetically modified bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample. In some aspects, the method further includes: determining a methoxy content number in the plant biomass sample; wherein the methoxy content number is determined using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample. In some aspects, a value of less than or equal to 0.2 is indicative that the plant biomass can be solubilized without chemical pretreatment. In some aspects, the plant biomass sample is from a poplar tree. In some aspects, the poplar tree is a genetically modified poplar tree. In some aspects, mixing the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about pH 5.8-7.0. In some aspects, the pH is maintained using sodium hydroxide. In some aspects, the mixing of the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about 55-75˚C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample. In some aspects, the fermentable carbohydrate is converted to ethanol. In various aspects, described herein is a method of degrading lignocellulose in a plant biomass sample, comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; based on the methoxy content number being below a threshold value, contacting the plant biomass sample with a bacterium, wherein the bacterium degrades the lignocellulose; and forming a fermentable carbohydrate from the plant biomass sample. In some aspects, the threshold value is 0.17 or less. In some aspects, the bacterium is a thermophilic bacterium. In some aspects, the bacterium is Anaerocellum (e.g., Anaerocellum bescii), Acetivibrio (e.g., Acetivibrio thermocellus Caldicellulosiruptor, Attorney Dkt.10620-156WO1 Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium. In some aspects, the bacterium is a genetically modified strain. In some aspects, the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. In some aspects, the poplar tree comprises a genetically modified poplar tree. In some aspects, contacting the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about pH 5.0-7.2. In some aspects, the pH is maintained with NaOH. In some aspects, the contacting of the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about 55˚C-85˚C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample.In some aspects, the fermentable carbohydrate is converted to ethanol. In some aspects, described herein is a method of determining an amount of a chemical pretreatment of a plant biomass sample, the method comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated _{using the formula: ,} wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; and determining, based on the methoxy content number, an amount of a chemical pretreatment needed for the plant biomass sample, wherein a higher methoxy content number indicates more chemical pretreatment and a lower methoxy content number indicates a less or no chemical pretreatment. In some aspects, the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. In some aspects, the poplar tree comprises a genetically modified poplar tree. DESCRIPTION OF DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the Attorney Dkt.10620-156WO1 description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures. Figure 1 - Correlation of A. bescii fermentation with lignin content and composition. Wild-type A. bescii 7-day fermentations of various plant biomasses. [Panel A] Linear regression of total fermentation products (acetate, lactate) generated by A. bescii as a function of total lignin mass fraction. [Panel B] Linear regression of total mass fraction of plant biomass solubilized during fermentation as a function of total lignin. [Panel C] Linear regression of total fermentation products as a function of plant biomass methoxy content number (Equation 1). [Panel D] Total mass fraction solubilized as a function of methoxy content number. For sugarcane bagasse, the effect of autoclave pretreatment is shown (indicated in Panels C, D); all other biomasses are not autoclaved. Plant biomasses included: sugarcane bagasse, spent coffee beans, Cave-in-Rock switchgrass, wheat straw, Frasier fir, corn stover, hemp fiber, wild-type poplar, and crystalline cellulose (Avicel). Figure 2 - Lignin monomers. Monolignols with methoxy group emphasized. Phenol ring carbon numbers of the phenol rings are indicated with small red numbers. Figure 3 - A. bescii fermentation of poplar lines. [Panel A] Total fermentation products (acetate, lactate, ethanol) generated by A. bescii over 7-days from 5 g/L milled poplar, plotted against relative tree growth (stem volume percent of wild-type poplar at 6- months) (367 fermentations across 134 poplar lines). [Panel B] Total fermentation products plotted against relative growth multiplied by total mass fraction of poplar solubilized by A. bescii during the 7-day fermentation (297 fermentations across 101 poplar lines). Error bars are ±1.5 standard error for biological replicate fermentation of poplar lines. [Panel C] Linear regression of total fermentation products as a function of lignin fraction of total poplar mass (n=64). [Panel D] Linear regression of total fermentation products as a function of poplar lignin S/G ratio (n = 32). WT indicates wild-type poplar. Two strains of A. bescii were employed, wild-type A. bescii DSM6725 and ethanol producing MACB1058, as indicated in Panel A. Figure 4 - Detailed Fermentative performance of H-4-1 poplar. [Panel A] Total mass fraction solubilized by A. bescii 7-day fermentation or relative tree growth (as percent stem volume of wild-type poplar) for 6 poplar lines. [Panel B] Fermentation products generated per gram poplar loaded into a 50 mL culture or relative growth multiplied by total mass fraction of poplar solubilized by A. bescii for 6 poplar lines. [Panel C] Mass fraction solubilized of H-4-1 poplar components from A. bescii fermentation, at 50 mL scale with no Attorney Dkt.10620-156WO1 pH control and 1 L scale with pH control. [Panel D] Mass fraction solubilized of H-4-1 poplar total lignin and total carbohydrate from A. bescii fermentation. All fermentations were in biological triplicate with the exception of wild-type poplar with wild-type A. bescii in panels A and B (n = 5), and 1 L, pH control in panels C and D (n = 2). Abbreviations: WT: wild- type poplar, SE: standard error, AIS: acid insoluble, AS: acid soluble. Figure 5 - Localization of A. bescii fermentative effects on poplar. Cross sections (250 µm) of milled poplar (wild-type, E-3-1, or H-4-1) before and after fermentation by A. bescii were imaged with scanning electron microscopy (SEM), widefield microscopy, and confocal microscopy. Fluorescence in widefield and confocal microscopy was the result of Calcofluor White staining of cellulose (blue) and lignin autofluorescence (red). “Before” and “After” indicate poplar particles from before and after fermentation. Figure 6 - Relationship between microbial fermentative outcomes and lignocellulose methoxy content. A. bescii and Ac. thermocellus fermentation of various plant biomasses. [Panels A, B] Includes non-genetically modified biomasses (excluding autoclaved sugarcane) from Figure 1 (green points) and additional data from 32 genetically modified poplar lines (black points). [Panel A] Linear regression of total fermentation products (acetate, lactate, ethanol) from 7-day A. bescii fermentations of plant biomass as a function of methoxy correlation (Equation 1). [Panel B] Linear regression of total biomass mass fraction solubilized by A. bescii as a function of methoxy correlation. [Panel C] Linear regression of total fermentation products and methoxy content for Ac. thermocellus from the non- genetically modified biomasses, including wild-type poplar. [Panel D] Linear regression of mass solubilization and methoxy content for the non-genetically modified plant biomasses fermented with Ac. thermocellus. Additionally, for Panels C, D, autoclaved sugarcane bagasse is compared against not autoclaved sugarcane bagasse, as indicated. Spent coffee and hemp fiber are not included for Ac. thermocellus in [Panels C, D], due to reported toxicity of spent coffee and no data for hemp fiber. Figure 7 – A. bescii central metabolism. Central metabolism of carbohydrates, particularly hexoses and the associated electron carriers and carbon outlets. Engineered strains of A. bescii have deleted the lactate dehydrogenase (ldh) responsible for lactate production, and inserted ethanol formation genes (namely the bifunctional alcohol dehydrogenase, adhE). Strain RKCB92 has a NAD(P)H dependent AdhE; all other AdhE strains are NADH dependent. Bf-Nfn = NADH-dependent reduced ferredoxin:NADP+ oxidoreductase; Bf-H2ase = bifurcating [FeFe] hydrogenase, MBH = ferredoxin-dependent Attorney Dkt.10620-156WO1 membrane-bound hydrogenase; Pta = phosphate acetyltransferase; Ack = acetate kinase; Fd_ox/red = oxidized or reduced ferredoxin. Figure 8 - Protein alignment of bifunctional alcohol dehydrogenases. AdhE protein sequences from Acetivibrio thermocellus and Thermoclostridium stercorarium subsp. strain RKWS1 Asp492Gly used to create A. bescii strain RKCB92 with the indicated Asp492Gly mutation. Shown are SEQ ID NOs:2 and 3. Figure 9 - Effect of temperature on bottle cultures of RKCB92. Cultures (50 ml) of RKCB92 were grown on 20 g/L cellobiose with 30 mM MOPS, 30 mM MES initial pH of 7.2, with 36 mM NH₄Cl, 2x vitamins, 2x phosphate, 0.3 mM uracil, with otherwise standard D516 medium. Cultures done in quadruplicate. Error bars represent ± 1 standard deviation. [Panel A] 65°C, [Panel B] 70°C, [Panel C] 75°C culture temperatures. [Panels A, C, E] are 680 nm optical density of cultures, with minimum doubling times (T_d,min) indicated. [Panels B, D, F] are major water-soluble fermentation products and sugar consumption (glucose equivalents). Figure 10 - Ethanol strain reoccurring aggregation phenotype. A. bescii strains cultured for 36 h without agitation. Ethanol strains were grown at temperatures for maximum ethanol:acetate selectivity (60°C for MACB1058 and RKCB89, and 68°C for RKCB92). Figure 11 - Effect of bicarbonate on RKCB92 ethanol production from cellulose. [Panels A, B] Major water-soluble fermentation products. [Panels C, D] Cell density of planktonic cells and mole fraction of ethanol of all water-soluble products measured (ethanol, acetate, pyruvate, acetoin, amino acids). [Panels E, F] Ethanol/acetate (mol/mol) selectivity. [Panels A, C, E] Bioreactors with 2.6 g/L (31mM) sodium bicarbonate initial, then pH controlled with ~9% sodium bicarbonate (saturated at 22°C). [Panels B, D, F] Bioreactors with 0.1 g/L (1.2 mM) sodium bicarbonate, pH controlled with 2 M sodium hydroxide. All Initial pHs were 7.1 ± 0.05, allowed to acidify to and then held at 6.0 ± 0.05. Cultures were 1.5 L bioreactors with 50 g/L Avicel (microcrystalline cellulose) in D516 medium with 2x vitamins, 2 mM phosphate, 36 mM NH4Cl (initial, further fed batch to 60 mM in bicarbonate and 48 mM in hydroxide case). Figure 12 - Cell Morphology of RKCB92. Epifluorescence micrographs of acridine orange stained cells from the sodium bicarbonate and sodium hydroxide pH controlled bioreactors grown on 50 g/L Avicel. Images from are from 170 and 215 h for the bicarbonate stationary and late stationary cases, and from 36 and 103 h for the hydroxide stationary and late stationary cases. Attorney Dkt.10620-156WO1 Figure 13 - Evaluation of engineered A. bescii genomes. Nanopore long read assemblies for wildtype (DSM6725), MACB1018, MACB1034, MACB1058, MACB1062, RKCB89, and RKCB92. [Panel A] Circos plot of assemblies for all strains, outer bar shows the wildtype genome G/C content, blue bars indicate presence of ISCbe4 Lre2 family transposon that occurs in all strains (including wild type), green bars indicate insertion sequences (>50 bp) scaled proportional to the insertion size, orange bars indicate deleted regions (>50 bp), grey bars and regions denote mutations that were inherited from the parent strain. [Panel B] Table summarizing insertion and deletions >50 bp, Region number matches the red numbers in [Panel A]. [Panel C] Lineage tree indicating the intended genomic edits for each strain. Figure 14 - Transcriptomic analysis of RKCB92 sodium bicarbonate versus hydroxide Avicel bioreactors. RNA sequencing results from late-log phase cells. Heat mapped log₂ fold change of the sodium bicarbonate (NaHCO₃) condition compared to sodium hydroxide (NaOH) condition. Log2 counts per million (CPM) are also reported for both conditions. Individual genes are mapped clockwise in order of RKCB92 Gene ID. Specific areas of interest (A-Q) and the 8 most expressed genes with significant fold changes in the bicarbonate condition (1-8) are indicated. Details of these regions and genes are shown in Figure 17. Figure 15 shows a table providing a summary of reported fermentation performance of ethanol producing strains. Figure 16 shows a table providing a summary of RKCB89 and MACB1058 cultures. Figure 17 shows a summary of RKCB92 Fermentations. Figure 18 - Plasmids Maps. Plasmid maps for the five plasmids used in this study. pRGB025 and pRGB026 are E. coli expression vectors for Thermoclostridium stercorarium AdhE with and without the Asp492Gly mutation. pRGB008 and pRGB011 are A. bescii acetate gene (pta, ack) knockout non-replicating vectors. pRGB032 is A. bescii non- replicating vector for integration of T. stercorarium RKWS1 AdhE with Asp492Gly mutation into the Athe_0949 locus. DETAILED DESCRIPTION The present invention can be understood more readily by reference to the following detailed description, examples, drawings, and claims, and their previous and following Attorney Dkt.10620-156WO1 description. However, before the present articles, systems, and/or methods are disclosed and described, it is to be understood that this invention is not limited to the specific or exemplary aspects of articles, systems, and/or methods disclosed unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. The following description of the invention is provided as an enabling teaching of the invention in its best, currently known aspect. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various aspects of the invention described herein, while still obtaining the beneficial results of the present invention. It will also be apparent that some of the desired benefits of the present invention can be obtained by selecting some of the features of the present invention without utilizing other features. Accordingly, those of ordinary skill in the pertinent art will recognize that many modifications and adaptations to the present invention are possible and may even be desirable in certain circumstances and are a part of the present invention. Thus, the following description is again provided as illustrative of the principles of the present invention and not in limitation thereof. General Definitions It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination in a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of” and “consisting essentially of.” 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 this invention belongs. In this specification and in the claims, which follow, reference will be made to a number of terms that shall be defined herein. Attorney Dkt.10620-156WO1 For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used. Further, ranges can be expressed herein as from “about” one particular value and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2%, or 1%) of the particular value modified by the term “about.” 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, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range. As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance generally, typically, or approximately occurs. As used herein, the term “substantially,” in, for example, the context “substantially identical” or “substantially similar” refers to a method or a system, or a component that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about Attorney Dkt.10620-156WO1 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% by similar to the method, system, or the component it is compared to. As used herein, “amino acid” refers to a compound containing both amino (—NH2) and carboxyl (—COOH) groups generally separated by one carbon atom. The central carbon atom may contain a substituent which can be either charged, ionisable, hydrophilic or hydrophobic. Any of 22 basic building blocks of proteins having the formula NH2—CHR— COOH, where R is different for each specific amino acid, and the stereochemistry is in the ‘L’ configuration. Additionally, the term “amino acid” can optionally include those with an unnatural ‘D’ stereochemistry and modified forms of the ‘D’ and ‘L’ amino acids. As used herein, the terms “polypeptide,” “peptide,” or “protein” generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally-occurring amino acids. The term “protein”, as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and/or quaternary structure. The term “protein” excludes small peptides by definition, the small peptides lacking the requisite higher-order structure necessary to be considered a protein. Different modifications of, and/or additions to, the polypeptides constituting the population according to the invention may be performed in order to tailor the polypeptides to the specific use intended. Such modifications and additions are described in more detail below, and may comprise additional amino acids comprised in the same polypeptide chain, or labels and/or therapeutic agents that are chemically conjugated or otherwise bound to the polypeptides constituting the population. In some embodiments additional amino acid residues on the C-terminal end may be preferred. These additional amino acid residues may play a role in the binding of the polypeptide, but may equally well serve other purposes, related for example to one or more of the production, purification, stabilization, coupling or detection of the polypeptide. Such additional amino acid residues may comprise one or more amino acid residues added for purposes of chemical coupling. An example of this is the addition of a cysteine residue at the very first or very last position in the polypeptide chain, i.e. at the N- or C-terminus. A cysteine residue to be used for chemical coupling may also be introduced by replacement of another amino acid on the surface of the protein domain, preferably on a portion of the surface that is not involved in target binding. Such additional amino acid residues may also comprise a “tag” for purification or detection of the polypeptide, Attorney Dkt.10620-156WO1 such as a hexahistidyl (His6) tag, or a “myc” tag or a “FLAG” tag for interaction with antibodies specific to the tag. The skilled person is aware of other alternatives. The “additional amino acid residues” discussed above may also constitute one or more polypeptide domain(s) with any desired function, such as another binding function, or an enzymatic function, or a metal ion chelating function, or a fluorescent function, or mixtures thereof. The “percentage of sequences identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The term “identical” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides in length. The terms “similarity” or “percent similarity” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of Attorney Dkt.10620-156WO1 the following sequence comparison algorithms or by manual alignment and visual inspection. Sequences are “substantially similar” to each other if they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similar to each other. Optionally, this similarly exists over a region that is at least about 25 amino acids in length (e.g., at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75), or more typically over a region that is at least about 100 to 500 or 1000 or more amino acids in length. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters. As described herein, “engineered” or “modified” organisms are produced via the introduction of genetic material into a host or parental microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material the parental microorganism can acquire new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular metabolite. The genetic material introduced into the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of n-butanol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences. An engineered or modified microorganism can also include in the alternative or in addition to the introduction of a genetic material into a host or parental microorganism, the disruption, deletion, or knocking out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the reduction, disruption or knocking out of a gene or polynucleotide the microorganism acquires new or improved properties (e.g., the ability to produce a new or greater quantities of an intracellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of undesirable by-products). For example, the microorganism may be modified to express one or more exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. A microorganism Attorney Dkt.10620-156WO1 may also be modified to modulate the expression level of an endogenous gene. In particular, a genetic modification of a microorganism may be carried out by using techniques known in the art, such as CRISPR-Cas systems described in U.S. Patent No. 11,142,751, which is incorporated by reference herein. As used herein, the term “conditions effective” as it relates to the inoculation of a fermentation mixture refers to a set of adjustable process parameters, such as pH, temperature, metabolite environment, and time, which can produce a desired product. For example, the incubation of a fermentation mixture under conditions effective can convert a compound or compounds, such as single carbon compounds, to a target product, such as acetate, at an efficiency. While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only, and one of ordinary skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. The present invention may be understood more readily by reference to the following detailed description of various aspects of the invention and the examples included therein and to the Figures and their previous and following description. Engineered Bacteria Disclosed herein are engineered bacteria useful for the conversion of a biomass to a carbonaceous product. In various aspects, described herein is a genetically modified extremely thermophilic bacterium, wherein the genetically modified extremely thermophilic bacterium comprises a heterologous gene encoding a bifunctional alcohol dehydrogenase. As used herein, the term “bifunctional” is intended to include enzymes that catalyze more than one biochemical reaction step. For example, the bifunctional enzyme used herein is an enzyme (adhE) that catalyzes both the alcohol dehydrogenase and acetaldehyde dehydrogenase reactions. Attorney Dkt.10620-156WO1 The genetically modified bacterium described herein may be extremely thermophilic. The term “extremely thermophilic,” which is synonymous with the term “hyperthermophilic,” refers to an organism which grows optimally at temperatures at or above 70° C. In some aspects, the engineered bacterium is a bacterium from the order Caldicellulosiruptorales. In some aspects, the bacterium comprises Anaerocellum (e.g., Anaerocellum bescii), Acetivibrio (e.g., Acetivibrio thermocellus Caldicellulosiruptor, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium. In some aspects, the bacterium is a genetically modified strain. In some aspects, the genetically modified bacterium comprises A. bescii. In some aspects, the heterologous gene comprises an AdhE gene from a moderately thermophilic bacterium. As used herein, the term “moderately thermophiles,” also called “facultative thermophiles,” refers to bacterial strains, which are capable of growing at temperatures between 30-65° C, typically having an optimum between 40-60° C. In some aspects, the heterologous gene comprises an AdhE gene from Thermoclostridium stercorarium, e.g., SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 80% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 85% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 90% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 95% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 97% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 99% or more sequence identity to SEQ ID NO:1. In some aspects, the AdhE gene from Thermoclostridium stercorarium comprises an Asp492Gly mutation. In some aspects, the bifunctional alcohol dehydrogenase has 80% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 85% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 90% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 95% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 97% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 99% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase comprises SEQ ID NO:2. In some aspects, a gene encoding lactate dehydrogenase expression has been inactivated. In some aspects, a gene encoding lactate dehydrogenase has been inactivated by Attorney Dkt.10620-156WO1 homologous recombination. In some aspects, the heterologous gene bifunctional AdhE is expressed from a plasmid. In some aspects, the heterologous gene encoding the bifunctional alcohol dehydrogenase is inserted into a genomic locus of the bacterium. In some aspects, the engineered bacterium further includes a promoter operably linked to the heterologous gene encoding the bifunctional alcohol dehydrogenase, wherein the promoter is configured to function at elevated temperatures. In some aspects, the heterologous bifunctional alcohol dehydrogenase gene is inserted downstream of Athe_0949 in the A. bescii genome. In some aspects, described herein are genetically modified A. bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C, e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C. In some aspects, described herein is a method for enhancing ethanol production. In some aspects, the method includes: obtaining a biomass sample; contacting the plant biomass sample with a genetically modified bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase under conditions effective to convert the plant biomass to ethanol. In some aspects, the conditions effective include one or more of: (i) maintaining pH between 5.8 and 7.0, and/or (ii) maintaining an operational temperature of 55-75°C. In some aspects, the genetically modified bacterium comprising A. bescii that has been genetically modified with the insertion of alcohol dehydrogenase gene. In some aspects, described herein are genetically modified A. bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C, e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C. In some aspects, also described herein are genetically modified A. bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C, e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C. Methods and Systems Also described herein is a bioreactor system for enhancing ethanol production and selectivity relative to other fermentation byproducts from plant biomass, comprising: a temperature control system configured to maintain a fermentation temperature (e.g., from Attorney Dkt.10620-156WO1 55°C to 75°C) for a genetically modified A. bescii strain, the genetically modified A. bescii bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase; a pH control system configured to maintain a pH between 5.8-7.0 (e.g., using sodium bicarbonate and sodium hydroxide); a pressurization system configured to control carbon dioxide head pressure; and an agitation system configured to mitigate cell aggregation. In some aspects, the fermentation byproducts comprise acetate, lactate, hydrogen, carbon dioxide, acetoin, 2,3-butanediol, pyruvate, butanol, isopropanol, and/or higher chain alcohols. As described above, bioreactor systems may typically include one or more vessels, towers, or piping arrangements, such as a continuous stirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bed reactor (TBR), bubble column, gas lift fermenter, static mixer, or other vessel or other device suitable for gas-liquid contact. In some aspects, one or more of the bioreactors can include a growth reactor which can be used to seed a fermentation reactor. Bioreactors can range in size from a few liters to several cubic meters (i.e. several 1000 liters) or larger and can be formed using a number of different materials, e.g. stainless steel or glass. Based on the mode of operation, a bioreactor may be classified as batch, fed-batch or continuous. The bioreactor is typically equipped with one or more inlets for supplying culture medium to the cells, and with one or more outlets for harvesting product or emptying the bioreactor. Additionally, the bioreactor may be equipped with at least one outlet constructed in such a way that a separation device can be attached to the bioreactor. Typically, the bioreactor's environmental conditions such as gas (i.e., air, oxygen, nitrogen, carbon dioxide) flow rates, temperature, pH and dissolved oxygen levels, and agitation speed/circulation rate can be closely monitored and controlled. In various aspects, the fermentation mixture(s) are maintained in an aqueous culture medium including nutrients, vitamins, and/or minerals sufficient to permit growth of the microorganism(s). Suitable media are well known in the art as instructed by this disclosure. In some aspects, described is a method of producing ethanol from a plant biomass sample. The method can include: obtaining a plant biomass sample; contacting the plant biomass sample with any of the genetically modified thermophilic bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample; and fermenting the fermentable carbohydrate to produce ethanol. In some aspects, the plant biomass sample comprises a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. Attorney Dkt.10620-156WO1 In some aspects, the poplar tree is a genetically modified poplar tree. In some aspects, the plant biomass comprises a plant biomass that has been genetically modified to have a lower methoxy content compared to a wild-type plant biomass. In some aspects, the mixing of the plant biomass sample with the genetically modified bacterium occurs at about pH 5.8-7.0. In some aspects, the pH is maintained using NaOH or NaHCO₃. In some aspects, contacting the plant biomass sample with the genetically modified bacterium occurs at about 55-75˚C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample. In some aspects, described herein is a method of degrading lignin in a plant biomass sample, comprising: obtaining a plant biomass sample; mixing the plant biomass sample with the genetically modified bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample. In some aspects, the method further includes: determining a methoxy content number in the plant biomass sample; wherein the methoxy content number is determined using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample. In some aspects, a value of less than or equal to 0.2 is indicative that the plant biomass can be solubilized without chemical pretreatment. In some aspects, the plant biomass sample is from a poplar tree. In some aspects, the poplar tree is a genetically modified poplar tree. In some aspects, mixing the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about pH 5.8-7.0. In some aspects, the pH is maintained using sodium hydroxide. In some aspects, the mixing of the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about 55-75˚C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample. In some aspects, the fermentable carbohydrate is converted to ethanol, e.g., at a high titer. As used herein, the term “titer” refers to the quantity of targeted product (e.g., ethanol) produced per unit volume of host cell culture. In some aspects, a titer of the ethanol is 25 mM or more, such as 30 mM or more, 35 mM or more, 40 mM or more, 45 mM or more, 50 mM or more, 55 mM or more, 60 mM or more, 65 mM or more, 70 mM or more, 75 mM or more, Attorney Dkt.10620-156WO1 80 mM or more, 85 mM or more, 90 mM or more, 95 mM or more, 100 mM or more, 105 mM or more, 110 mM or more, 115 mM or more, 120 mM or more, 125 mM or more, 130 mM or more, 135 mM or more, 140 mM or more, 145 mM or more, or 150 mM or more. In some aspects, a titer of ethanol is produced is from 25 mM to 150 mM, such as from 35 mM to 150 mM, from 50 mM to 150 mM, from 65 mM to 150 mM, from 75 mM to 150 mM, from 95 mM to 150 mM, from 25 mM to 135 mM, from 45 mM to 135 mM, from 65 mM to 135 mM, from 75 mM to 135 mM, from 85 mM to 135 mM, from 95 mM to 135 mM, from 100 mM to 135 mM, or about 135 mM. In various aspects, described herein is a method of degrading lignocellulose in a plant biomass sample, comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; based on the methoxy content number being below a threshold value, contacting the plant biomass sample with a bacterium, wherein the bacterium degrades the lignocellulose; and forming a fermentable carbohydrate from the plant biomass sample. In some aspects, the threshold value is 0.17 or less. In some aspects, the bacterium is an extremely thermophilic bacterium. In some aspects, the bacterium is Anaerocellum (e.g., A. bescii), Acetivibrio e.g., Acetivibrio thermocellus Caldicellulosiruptor, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium. In some aspects, the bacterium is a genetically modified strain. In various examples, the biomass includes a lignocellulose hydrolysate. As used herein, the term “lignocellulose hydrolysate” refers to hydrolysis products of lignocellulose or lignocellulosic material comprising cellulose and/or hemicellulose, oligosaccharides, mono- and/or disaccharides, acetic acid, formic acid, other organic acids, furfural, hydroxymethyl furfural, levulinic acid, phenolic compounds, other hydrolysis and/or degradation products formed from lignin, cellulose, hemicellulose and/or other components of lignocellulose, nitrogen compounds originating from proteins, metals and/or non- hydrolyzed or partly hydrolyzed fragments of lignocellulose. In some examples, lignocellulose hydrolysates are obtained from a lignocellulosic biomass such as paper, paper Attorney Dkt.10620-156WO1 products, wood, wood-related materials, particle board, grasses, rice hulls, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, coconut hair, algae, seaweed, altered celluloses, e.g., cellulose acetate, regenerated cellulose, and the like, or combinations thereof. In some aspects, the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. In some aspects, the poplar tree comprises a genetically modified poplar tree. In some aspects, contacting the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about pH 5.0-7.2. In some aspects, the pH is maintained with NaOH. In some aspects, the contacting of the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about 55˚C-85˚C, e.g., from 55°C to 75°C, from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample. In some aspects, the fermentable carbohydrate is converted to ethanol. In some aspects, described herein is a method of determining an amount of a chemical pretreatment of a plant biomass sample, the method comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; and determining, based on the methoxy content number, an amount of a chemical pretreatment needed for the plant biomass sample, wherein a higher methoxy content number indicates more chemical pretreatment and a lower methoxy content number indicates a less or no chemical pretreatment. In some aspects, the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. In some aspects, the poplar tree comprises a genetically modified poplar tree. Attorney Dkt.10620-156WO1 EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by volume, the temperature is in degrees C or is at ambient temperature, and pressure is at or near atmospheric. Example 1: Identifying the primary barrier to plant biomass conversion by fermentative bacteria Introduction. Renewable alternatives for non-electrifiable fossil-derived chemicals are needed and plants, the most abundant biomass on earth, provide an ideal feedstock. However, the heterogeneous polymeric composition of lignocellulose makes conversion difficult. Lignin presents a formidable barrier to plant biomass conversion. Extensive chemical and enzymatic treatments can liberate fermentable carbohydrates from plant biomass for subsequent processing, but microbial routes offer many advantages, including concomitant conversion to industrial chemicals. Here, fermentation of plant biomass, with varying lignin content and composition, revealed that the primary microbial degradation barrier relates to methoxy substitutions on phenolic rings of lignols. This contrasts with optimal lignin composition for chemical pretreatment that favors high S/G ratio and low H lignin. Genetically modified poplar trees with diverse lignin compositions confirm these findings. Additionally, poplar trees with low methoxy content achieve industrially relevant levels of microbial solubilization without any pretreatments and with no impact on tree fitness in greenhouse. The environmental impact of climate change motivates reduced dependence on non- renewable fossil feedstocks for producing fuels and chemicals. Plant biomass, as the most abundant renewable material on Earth, is the primary candidate for replacing fossil feedstocks, but this requires efficient deconstruction and subsequent conversion of the polymers contained within lignocellulose to be economically viable. Lignocellulose is composed of polysaccharides cross-linked with the phenolic polymer lignin such that processes utilizing lignocellulose need to liberate carbohydrates from lignin. Mechanical, Attorney Dkt.10620-156WO1 chemical, and enzymatic pretreatments, alone or in combination, have been used to achieve this. However, microbial routes, which can solubilize the carbohydrate content of plant biomass and concomitantly produce fuels and chemicals (i.e., consolidated bioprocessing), are particularly attractive. Metabolic engineering has been used to create microbial strains that convert simple fermentable sugars to industrial products, but there are significant advantages if the microbes can also catabolize carbohydrate polymers from plant biomass, especially microcrystalline cellulose. Certain thermophilic bacteria (i.e. genera Caldicellulosiruptor, Anaerocellum, Acetivibrio) natively solubilize and utilize a wide range of plant polysaccharides employing large sets of extracellular enzymes and have been metabolically engineered to make fuels and chemicals. Among these, Anaerocellum (f. Caldicellulosiruptor) bescii, belonging to the extremely thermophilic Caldicellulosiruptorales (T_opt > 70°C), has demonstrated the capacity to degrade a wide range of plant biomasses, resist contamination, and to produce industrially relevant products. However, it is clear that lignin remains a barrier to highly efficient biomass degradation by A. bescii, which is reflected in the disparate levels of carbohydrate solubilization of low and high lignin plant biomasses (such as soybean hulls as compared to poplar wood). The lignin barrier extends to other microbes, such as Acetivibrio thermocellus as well. The key to expanding the use of plant feedstocks is to make them less recalcitrant and easier to degrade without compromising fitness. An ideal plant biomass feedstock should be genetically tractable, fast growing, require minimal use of pesticides, and grow on marginal lands in order to not compete with food crops; Populus trichocarpa (black cottonwood) fits these requirements. A. bescii fermentation of genetically modified P. trichocarpa lines has been used to screen trees for improved feedstock qualities (i.e., more efficient deconstruction); A. bescii reached nearly 90% carbohydrate solubilization of certain low-lignin poplar lines. Unfortunately, poplar lines that were the most amenable to degradation had significant fitness issues. But, as recently reported, multiplexed CRISPR edited poplar trees can have superior wood properties for fiber pulping, producing trees with lower lignin, higher carbohydrate to lignin ratio, and increased S/G ratio all while preserving the overall fitness of the trees. The issue considered here is whether plant biomasses, including poplar, best for fiber pulping are also amenable to microbial conversion and what features are most significant for solubilization to fermentable sugars. Attorney Dkt.10620-156WO1 Impact of lignin composition on plant biomass fermentation by Anaerocellum bescii. Wild-type A. bescii fermentations of various plant biomasses were evaluated against lignin properties. This includes previously published data on Frasier fir, corn stover, sugarcane bagasse, wheat straw, Cave-in-Rock switchgrass, spent coffee beans, and crystalline cellulose (Avicel), as well as not previously published hemp fiber (Table 1). Lignin compositional data (monolignols and hydroxycinnamic acids) were taken from literature sources, as indicated in Table 1. As might be expected, the ability of A. bescii to solubilize and convert carbohydrates in these plant biomasses has an inverse correlation with total lignin content; this is seen for total fermentation products (acetate and lactate) (Figure 1, panel A) and total mass fraction of the plant matter that is solubilized (Figure 1, panel B). However, linear regression analysis of these data suggests that these relationships are not highly significant (R² = 0.59, 0.46). Some biomasses with similar total lignin content have very different microbial carbohydrate accessibility. Comparing switchgrass, sugarcane bagasse, wild-type poplar, and corn stover, which are all 20-23% total lignin, A. bescii mass solubilization ranged from 26 – 41%. This raises the question of what differentiates these lignocellulosic substrates. Table 1. Composition and fermentation data for selected plant biomasses Attorney Dkt.10620-156WO1 PB: p-hydroxybenzoic acid; H: H-subunits; G: G-subunits; S: S-subunits; Other Lignin Acids; %: percentage volume of total lignin.*Other reports may suggest up to 100% mass solubilization and >30 mM products, these numbers are used for culture condition consistency Lignin is a highly complex polymer, containing a wide variety of subunits and chemical linkages, making direct quantification of their structures difficult. However, the majority of the subunits are derived from monolignol precursors: p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S). These monolignols differ only by the number of methoxy (-OCH3) substitutions on their phenol rings. Where H-units have no methoxy substitutions, G-units have one methoxy substitution at phenol carbon 3, and S-units have two methoxy substitutions at phenol carbons 3 and 5 (Figure 2). Weighting the total lignin content by the number of methoxy substitutions greatly improves the correlation with fermentation products and microbial mass solubilization (Figure 1, panels C, D); methoxy content is estimated by the following dimensionless equation: ^_{^^^^^^^^^^^ℎ^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ = (^^^^ + 2^^^^)(^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^) (Eq. 1)} Methoxy substitutions of H, G, and S lignin units were represented as 0, 1, and 2 respectively. Equation 1 weights monolignols (as percent of monolignols, H + G + S from 2D-NMR data) by their number of methoxy substitutions, then multiplies this by total lignin content (mass fraction of total plant biomass). Linear regression of A. bescii fermentation products and mass solubilization resulted in R² values of 0.66 and 0.91, respectively (Figure 1, panels C, D). Here, mass solubilization is the primary measurement of microbial ability to solubilize polysaccharides contained in the lignocellulose, and fermentation products measure both solubilization and subsequent conversion. While mass solubilization and fermentation products generally follow similar trends, more variation is seen in fermentation products due to the variety of possible metabolic outcomes (some of which are not quantified, such as cell mass and amino acids). The variation in product distribution is affected by divergence in culture conditions (for example: high carbohydrate solubilization and consumption will lead to a larger pH drop as organic acids are formed). As such, the Attorney Dkt.10620-156WO1 large improvement in the mass solubilization linear regressions from Figure 1, panel B to panel D suggests methoxy content is the primary driver of plant biomass recalcitrance to microbial solubilization by A. bescii. Screening gene-edited poplar lines for fermentation products and mass solubilization by the extremely thermophilic bacterium A. bescii. Due to the small number of data points (ten) in Figure 1 and the limited availability of lignin composition for plant biomasses, further validation of the methoxy content correlation was addressed with A. bescii fermentation of genetically modified poplar lines with highly variable lignin content and composition. Two strains of A. bescii (wild-type strain DSM6725 and recombinant strain MACB1058) were used to screen wild-type poplar and 133 genetically modified poplar lines (including 18 lines from previous studies ) for conversion of plant carbohydrates into primary fermentation products (acetate, ethanol, lactate) as a function of tree fitness (Figure 3, panel A). MACB1058 is an A. bescii strain engineered to produce ethanol by insertion of the gene encoding Alcohol Dehydrogenase E (adhE) from Acetivibrio thermocellus. Previous work showed that the carbohydrate content of Lines 54 and 80 of RNAi-modified lines could be extensively fermented by MACB1058, although these poplar lines had significant growth defects. The carbohydrate content of Line 54 was fermented to a greater extent than Line 80, up to 30 mM and 27 mM, respectively. The carbohydrate content of RNAi-modified Line i20-3-1 could also be fermented to a high degree (21 mM), albeit below the levels observed for Lines 54 and 80, but with no significant impact on tree fitness; Line i20-3-1 actually grew better than wild-type. CRISPR-edited Lines H-4-1 and E-3-1 had similar lignin levels (~ 15% compared to ~ 22% for WT), but H-4-1 generated 22.1 mM fermentation products compared to 13.0 mM for E- 3-1. H-4-1 had no significant fitness issues, in contrast to E-3-1 which did (Figure 3, panel A). Note that fermentation of wild-type poplar lines tested (WT) generated less fermentation products (7-11 mM) than most of the genetically modified lines. Total fermentation products tracked with biomass solubilization. Relative tree growth (% stem volume as compared to wild-type poplar) multiplied by the total mass solubilization was used to represent the total fermentable mass of a 6-month poplar tree, relative to a wild-type tree (Figure 3, panel B x-axis). Total fermentation products are plotted as the y-axis (Figure 3, panel B). Line H-4-1, at 6-months, had fermentable mass equivalent to 67% of a wild-type tree, producing 22.1 mM products from 5 g/L mass loading. Line 80 had fermentable mass equivalent to 20.5% of a wild-type tree, even though at 5 g/L mass Attorney Dkt.10620-156WO1 loading more fermentation products were generated (i.e., lower microbial recalcitrance, but poor growth). The most improved lines for microbial conversion (H-4-1, i20-3-1) are represented in the upper right of Figure 3, panel B. While total fermentation products are inversely proportional to poplar total lignin content, these data had a linear correlation coefficient (R²) of 0.5 (Figure 3, panel C), in line with data shown in Figure 1, panel A for non-genetically edited plant biomasses. High S/G ratio and low H is desirable for pulp and paper feedstocks. Here, however, for microbial conversion, the relationship between total fermentation products and S/G ratio of lignin in the poplar samples showed no statistically significant relationship (Figure 3, panel D), as the S/G ratio is calculated independently of total lignin content. Comparisons of selected poplar line fermentations by wild-type and engineered strains of A. bescii are summarized in Figure 4, panel A and panel B. To assess possible difference between A. bescii strains, wild-type and Line 54 poplar were assessed with both A. bescii strains (DSM6725, MACB1058), with similar results. It is clear that wild-type poplar (WT) was more recalcitrant to fermentation than the genetically modified lines that had lower lignin and significant fitness issues (Lines 54, 80 and E-3-1). The other reduced lignin lines (i20-3-1 and H-4-1) were solubilized and fermented to a greater extent than wild-type poplar, but with no fitness issues. Lines i20-3-1 and H-4-1 had 2.7 and 3 times, respectively, more fermentable mass in a 6-month-old tree than wild-type poplar (Figure 4, panel B, relative growth multiplied by mass solubilization), and the carbohydrate content was 2-2.5 times more accessible (Figure 4, panel B, total fermentation products). Experimental scale, degree of mixing, and pH control were not significant factors. For example, Line H-4-1 was compared at 50 mL serum bottle scale with no pH control to 1L bioreactor scale with pH control; no substantial differences in solubilized carbohydrate content was noted (Figure 4, panel C, panel D). Wild-type A. bescii solubilized carbohydrates relatively uniformly from Line H-4-1, with slightly more glucose and mannose solubilized than xylose and galactose (Figure 4, panel C); overall ~80% of total carbohydrate and 11% of the total lignin were solubilized (Figure 4, panel D). How lignin was solubilized is unclear, but lignin fragments or oligomers could be released due to high carbohydrate solubilization levels. Poplar lignin content and composition impacts microbial solubilization and conversion. Even though CRISPR-edited Lines E-3-1 and H-4-1 had similar reduced lignin content (15% and 15.5%, respectively), the microbial total mass solubilization (35.3% vs. Attorney Dkt.10620-156WO1 63.4%, respectively) and fermentation products (13.0 mM vs.22.1 mM, respectively) differed significantly (two-way t-test, ≥ 99.5% confidence). This result parallels the data seen in Figure 1, panels A, B for non-genetically modified plants with lignin ~20-22%. This difference was examined in greater detail. E-3-1 genetic edits resulted in loss of function for gene targets PtrPAL2 (81.8%), PtrPAL4 (55.3%), and PtrPAL5 (55.3%), while in H-4-1 loss of function was for PtrC3H3 (98.2%), PtrAldOMT2 (40.3%) and PtrCAD1 (12.9%). H-4-1 had better growth characteristics than E-3-1 as indicated by stem volume as percent of wild-type (106% vs.42%, respectively). Although total lignin content was comparable for H-4-1 and E-3-1, lignin composition differed significantly between the two lines (Table 2). Clearly, the S/G ratio is the most striking difference between H-4-1 and E-3-1 (0.21 vs.2.19, respectively), with E- 3-1 being comparable to wild-type poplar (S/G of 2.1-2.6) (Figure 3, panel D). Figure 5 shows SEM, widefield and confocal images of wild-type, E-3-1 and H-4-1. It is clear from these images that A. bescii reduced the carbohydrate content of the poplar lines through fermentation. This is seen in the thinning of cell walls (SEM) and reduction of cellulose content (blue in fluorescence widefield and confocal). Lignin (red autofluorescence in widefield and confocal) is relatively unchanged before and after fermentation. It was also clear that polysaccharide solubilization occurred in a uniform way, suggesting microbial and extracellular enzymatic access to carbohydrate was not localized. In other words, physical restrictions did not prevent microbes from solubilizing the residual carbohydrate, at least at this particle size. Nonetheless, the carbohydrate content of Line H-4-1 was significantly reduced as is evident from imaging results. Table 2. Characteristics of selected CRISPR-edited poplar lines E-3-1 and H-4-1. , Attorney Dkt.10620-156WO1 *Expressed as percent of (H+G+S) Methoxy content correlates with fermentation products and mass solubilization for all plant biomasses. As shown in Figure 1, panels A,B and Figure 3, panel C, total lignin content does not fully explain the differences among all plant biomasses tested (poplar and otherwise), as this relates to recalcitrance (i.e., E-3-1 vs. H-4-1 or wild-type poplar vs. switchgrass). S/G ratio also had no significant correlation to microbial recalcitrance, as seen in Figure 3, panel D. The S/G ratio for H-4-1 was very small compared to a value of ~10 for the highly solubilized Line 54; note that E-3-1 had an S/G ratio close to wild-type poplar. Several poplar lines with improved solubilization were relatively high in H-lignin (i.e., Line 54) or G-lignin (i.e., H-4-1). Lignin compositions were compiled for 32 poplar lines (including data from this work and previously published work, and used to solve for their methoxy contents (Equation 1). This yielded methoxy content coefficients as 0.357 for wild-type, 0.248 for E-3-1, but only 0.170 for H-4-1 (Figures 6, panels A and B). Total fermentation products and mass solubilization of all plant biomasses tested (from Table 1 and the 32 poplar lines) linearly correlate with methoxy content (R² = 0.66 and 0.76, respectively). Validation of the methoxy content coefficient weighting factors, for G and S-units, in Equation 1 (i.e.1 for G and 2 for S) was done using Excel Solver to optimize these coefficients for the data in Figure 6, panels A and B. This resulted in (G, S) coefficients of (1.05, 1.89) for Figure 6, panel A and (0.99, 2.03) for Figure 6, panel B. Both very close to the true methoxy coefficients. As noted in the results from Figure 1, panels C, D, mass solubilization correlates better than fermentation products (Figure 6, panels A, B). While wild-type, E-3-1, H-4-1, and Line 54 poplar all fall well within this correlation, Line 80 does not. Exclusion of Line 80 from the data improves the mass solubilization linear regression fit from an R² of 0.76 to 0.82. Clearly, an unaccounted for factor is improving microbial solubilization of Line 80. Line 80 is 30% lignin aldehydes Attorney Dkt.10620-156WO1 (G and S unit monolignol precursors). Weighting the methoxy content (Equation 1) by the alcohol content of lignin (accounting for acids and aldehydes) improves the fit for Line 80 and grasses containing ferulic and coumaric acid (sugarcane, wheat straw), but worsens the fits for other high aldehyde lines (E and F CRISPR-edit poplar lines targeting PtrPAL genes). Thus, no modifications are made to Equation 1, as the secondary effects of specific lignin aldehydes and acids remains unclear. Further evaluation beyond A. bescii of the non-genetically engineered plant biomasses (including wildtype poplar and crystalline cellulose) with the moderately thermophilic Acetivibrio thermocellus supported the methoxy content correlations (using the side-by-side fermentation data, previously published (Figure 6, panels C,D). Linear correlation between total mass solubilization and methoxy content is again more robust (R² = 0.69), despite the limited quantity of data for Ac. thermocellus (Figure 6, panel D). While total fermentation products trend well for the extreme thermophile A. bescii (Figure 1, panel C), they are less consistent for the moderate thermophile Ac. thermocellus (Figure 6, panel C). This is due to contamination from indigenous microbial life that grows only under 75°C, as seen for the autoclaved versus not autoclaved sugarcane bagasse and Avicel (reported to have no contaminants). As Ac. thermocellus is unable to consume pentoses (even though it solubilizes them from hemicellulose), and does not grow above the thermophilic threshold to resist contamination, fermentation products produced from non- pretreated plant biomass are inconsistent; this emphasizes the need to use sterilizing pretreatments with moderately thermophilic Ac. thermocellus. However, as Ac. thermocellus is still the primary lignocellulose degrader, the mass solubilization (Figure 6, panel D) has a better linear fit with methoxy content. Relationship between lignin content and composition. The results here provide insights into the relationship between lignin content and composition as this relates to recalcitrance to A. bescii and Ac. thermocellus carbohydrate solubilization and conversion, for not just poplar but also for a wide variety of plant biomasses. The extension of these findings to other (hemi)cellulolytic, thermophilic bacteria (and less thermophilic microorganisms) needs to be determined but likely follows the findings here, based on other studies on biomass solubilization and conversion. These results likely only apply to microbes using carbohydrate active enzymes, and not to lignin degrading aerobic microorganisms. The challenge of developing modified biomasses with good growth and Attorney Dkt.10620-156WO1 fitness characteristics and low recalcitrance has been met for genetically modified poplar lines, but targeting low methoxy content provides a clear-cut objective. Correlation between methoxy content and microbial access to carbohydrates explains the variability reported previously for lignin impact on solubilization; data either poorly correlate with total lignin (as seen in Figure 1, panels A, B; Figure 3, panel C) or are inconsistent on the impact of S/G ratio. Here, incorporating the large number of poplar lines with significantly different lignin contents and compositions, the unifying variable, methoxy content, was validated as the primary barrier for microbial plant biomass solubilization. For lignocellulose with the same lignin compositions, total lignin and methoxy content correlate linearly. However, this correlation breaks down when lignin compositions differ. S/G ratio does not correlate with microbial solubilization and conversion because the ratio fails to account for the absolute quantities of S or G lignin in relation to total plant biomass. For microbial solubilization, monolignol preference appears to be H > G > S. This allows lines like H-4-1 (high G) and Line 54 (high H) to have low methoxy content at higher total lignin levels than if lignin composition was identical to wild-type poplar. In other words, for poplar with wild-type lignin composition to have the same methoxy content as H-4-1 or Line 54, ~10.1% or ~6.2% total lignin would be required, respectively (undoubtedly impacting tree fitness). This observation also extends to other biomasses. Grasses (such as wheat straw, corn stover, and switchgrass) are higher in H lignin and G lignin compared to hardwoods like wild-type poplar. These lignin compositions contribute less methoxy content per total lignin (Tables 1, 2) when compared to the S-unit rich wild-type poplar. Softwoods, like Fraser fir examined here, are difficult for anaerobic microbes to access carbohydrates; despite being high in G lignin and low in S lignin, their methoxy content remains high due to high total lignin (37.8% for Fraser Fir). Conversely, spent coffee beans, which are high in H lignin (60%) and total lignin (32.8%), can still have low methoxy content, enabling efficient microbial access to carbohydrates. H- 4-1 poplar would be economically and technologically relevant in an industrial biorefinery setting (≥ 65% substrate utilization ). However, opportunities remain for further optimization. As H-4-1 is predominantly G lignin, a shift to higher H and even lower S lignin could generate poplar with even lower methoxy content. The relationship between low lignin and methoxy content relates to plant biomass recalcitrance is not completely clear. Bonds associated with methoxy groups may result in a higher degree of carbohydrate-lignin cross-links that are less enzymatically available. Attorney Dkt.10620-156WO1 Higher S/G ratio has been associated with less condensed lignin polymer harboring lower levels of carbon-carbon linkages between subunits of lignin, which may reduce interference during chemical or enzymatic deconstruction. It is clear from Figure 5 that the residual cellulose (blue) is not localized, suggesting a chemical rather than physical reason from the recalcitrant residual carbohydrate; this is consistent with the carbohydrate-lignin crosslinking hypothesis. Interestingly, high S lignin is favorable for alkaline and chemical treatments (i.e., pretreatments or pulp and paper), in contrast to conditions that favor microbial solubilization and conversion. This explains why alkaline pre-treatments work well with A. bescii, as it likely breaks these recalcitrant lignin-carbohydrate linkages. Furthermore, methoxy content could be used to guide the intensity of chemical pretreatments required. Where low methoxy biomasses (≤ ~0.17) do not require pretreatment with A. bescii, higher methoxy substrates may benefit from pretreatments. The exact methoxy content cut-off is likely dependent on processes and microbes selected. This also suggests post-fermentation chemical treatments may liberate the residual, recalcitrant carbohydrates. Without wishing to be bound by theory, other factors beyond methoxy content likely influence microbial solubilization of lignocellulose. For example, when A. bescii was compared with Ac. thermocellus for solubilization of certain agricultural wastes (corn fiber, soybean hulls), significant differences were noted. The carbohydrate composition of these biomasses is significantly different than the cellulose- and xylan-rich lignocellulose examined here. Most likely, A. bescii and A. thermocellus lack carbohydrate active enzyme(s) needed to deconstruct specific carbohydrates, within corn fiber and soybean hulls, respectively. Additionally, deviation of Line 80 poplar from the fit in Figure 6, panels A, B is likely due to the incorporation of specific monolignol intermediates (most likely aldehydes) into the final lignin polymers. How hydroxycinnamaldehyde units in lignin contribute to the overall methoxy content correlation remains unclear from this data. Further evaluation of these aldehyde units in lignin may be needed. Despite this, most lignocellulosic substrates are primarily cellulose, xylan, and lignols. For these substrates, the methoxy content correlation is highly predictive. As methoxy content represents the primary barrier to microbial solubilization and conversion of cellulose- and xylan-rich lignocellulose, the recalcitrance of plant biomasses can now be predicted. This establishes specific lignin compositional goals for feedstock engineering for microbial biorefineries. While low methoxy content substrates do not Attorney Dkt.10620-156WO1 require chemical pre- or post-treatments, opportunity exists to reduce recalcitrance in higher methoxy substrates through chemical means that leverage the divergent lignin composition preferences for chemical and microbial solubilization. Materials and Methods Bacterial strains and culture conditions. Anaerocellum (f. Caldicellulosiruptor) bescii wild-type strain (DSM6725) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and maintained, as previously described. A. bescii strain MACB1058 was developed as reported previously. Fermentations with wild-type A. bescii were conducted at 75°C, while MACB1058 was fermented at 65°C to allow ethanol production. All fermentations were conducted at 5 g/L substrate loading in modified D671 medium (without MOPS) at 50 mL culture volume in 125 mL sealed serum bottles in New Brunswick Innova 42 incubator shakers at 150 rpm as previously described. Poplar Source. The details on most of the CRISPR-edited poplar lines were previously described. Additional lines used here are described in Bing, Ryan G., et al., “Beyond low lignin: Identifying the primary barrier to plant biomass conversion by fermentative bacteria.” Science Advances 10.42 (2024): eadq4941, which is herein expressly incorporated by reference in its entirety.This includes additional CRISPR-edited and non-CRISPR edited lines (RNAi) not previously published. RNAi lines were generated, as previously described. Preparation of plant biomass for cultures. Poplar biomass was processed, as previously described. Briefly, poplar biomass from 6-month-old trees were prepared with a Wiley mill and sieved to collect #40/#80 fraction. Poplar biomass was washed with 65°C water (30 g/L) 3 times, then dried at the same temperature in an oven. Hemp fiber was processed in a similar manner as previously described. Bacterial solubilization of plant biomass. A. bescii strains were adapted to plant biomass (poplar lines or hemp fiber), as previously described, with 5 g/L mass loading of substrate. Briefly, A. bescii freezer stocks were revived on medium containing 1 g/L cellobiose, 2 g/L Avicel (PH-101), and 2 g/L beechwood xylan and allowed to grow for 48 h. Cultures were passaged to medium containing 5 g/L milled and washed substrate, 0.1 g/L cellobiose, 0.2 g/L Avicel, and 0.2 g/L beechwood xylan, and allowed to grow for 48 h. Cultures were then passaged to medium containing 5 g/L substrate at and allowed to grow for ~3 days or until cell density reached ≥ 5x10⁸ cells/mL. Biological replicate cultures were Attorney Dkt.10620-156WO1 started with 5 g/L substrate (exact amount recorded) at 1x10⁷ cells/mL and allowed to ferment for 7 d. Cell densities were determined by epifluorescence microscopy, as previously described. Most experiments were conducted in triplicate, however some were conducted in duplicate due to limited poplar biomass availability and to enable more rapid screening of the large number of poplar lines. For mass solubilization calculations, residual plant biomass particles were collected, washed, dried, and their mass recorded to calculate total mass solubilized by fermentation, as previously described. Quantification of fermentation products. Water soluble fermentation products in post-fermentation culture broth were quantified by High Performance Liquid Chromatography (HPLC) with a Phenomenex Rezex-ROA Column (300mm x 7.8mm) with a Waters 2414 refractive index detector, as previously described. Bioreactor culture conditions. Line H-4-1 poplar was fermented at 5 g/L loading at 1 L culture volume in 3 L glass bioreactors (Chem Glass CLS-1380-01) at 75°C. A double impeller structure was used with an uplift marine style impeller (Chem Glass CLS-1380-07) at the bottom of the shaft and a Rushton style impeller (Chem Glass CLS-1380-08) located 2 cm below the liquid surface. Reactors were agitated at 150 rpm, sparged at 25 SCCM with N₂/CO₂ (80/20 v/v) mix, and heat supplied via an electrical heating mantel. pH 6.5 was maintained with 1 M sodium hydroxide. An Applikon Bio Console was used to control the bioreactors. Inocula for the bioreactors were prepared in the same manner, as described above, except that the final culture passage was inoculated into a single bioreactor. Bioreactor experiments were conducted in duplicate, each with separately adapted cultures. Fermentations were allowed to progress for 7 d, and processed and analyzed identical to the 50 ml solubilization cultures. Quantitative saccharification. Plant biomass compositions were determined using the Klason lignin method with sugar HPLC analysis conducted with a Shodex SP0810 column, as previously described. Microscopy of poplar particles. For scanning electron microscopy (SEM), dry poplar particles before and after fermentation were sputter-coated with Au/Pd for 60 seconds. A HITACHI SU3900 SEM instrument was used to obtain SEM images. To visualize the distribution of lignin and cellulose in samples, several cross-sections of the poplar particles were prepared. Sample preparation and embedding was carried out per LR White manufacturer recommendations (Electron Microscopy Sciences, USA). Poplar particles were chemically fixed using a solution of 4% paraformaldehyde and 0.05% Attorney Dkt.10620-156WO1 glutaraldehyde. Dehydration and embedding were carried out sequentially as follows: 50% and 70% ethanol in water for 15 min, 80% ethanol in water for 10 min, 2:1 LR white resin to 70% ethanol, and 100% LR white resin for 1 h, 100% LR white resin overnight, and 100% LR white resin for 30 min. The North Carolina State University Analytical Instrumentation Facility (NCSU AIF) polymerized the resin, cut ~250 µm thick cross sections with an ultramicrotome, and placed the cross sections on microscope slides. Immediately prior to imaging, cross sections on slides were washed with distilled water once (~ 1 mL), incubated with 0.02% Calcofluor White for 5 min (~1 mL), a few drops of 1M NaOH were added (~100 µL), and then cross sections were washed twice with distilled water (~2 mL). Excess liquid was decanted from slides. Samples were briefly air-dried before imaging. Widefield and confocal microscopy was conducted on a Leica Mica microscope. Widefield images used the CS220x objective (no cover slip). For widefield, Calcofluor White was excited with 365 nm LED (0.3 %) to visualize cellulose; images were collected using the DAPI filter with 100 ms exposure, gain setting of 4.4 / 5.0 / 5.0 / 5.0. Lignin autofluorescence was excited with 555 nm LED (100.0%); images were collected using the Texas Red filter with 500 ms exposure and gain setting of 5.0. Image histograms were set for blue (0 to 750) and red (150 to 500). Figure 5 images are the composite images of blue and red channels. Confocal images used the CS263X water immersion objective with a glass coverslip. For confocal, Calcofluor White was excited with 405 nm laser and blue channel images captured with the DAPI filter. Lignin autofluorescence was excited with 561 nm laser and red channel images captured with the Texas Red filter. Confocal settings used 600 Hz bidirectional scan speed and line averaging with 2 repetitions. Image histograms were set for blue (0 to 170) and red (0 to 30). Figure 5 shows composite images of blue and red channels. All image processing used Leica LAS X Office (1.4.4.26810) software. 2D-NMR. Poplar lignin composition was determined with 2-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), as previously described. Extractive-free wood samples were ground to 40-60 mesh as woodmeal using Willey mill and dried over P₂O₅. The woodmeal (~2 g) was further milled at 600 rpm using 17 ZrO₂ balls for a duration of 6 h using a Pulverisette 7 Planetary ball mill. Each milling cycle comprised a 15-minute milling period followed by a 30-minute pause. Subsequent to the ball milling process, the samples were stored under vacuum conditions with P₂O₅ prior to their utilization. Attorney Dkt.10620-156WO1 For 2D-NMR ¹³C-¹H (heteronuclear single quantum coherence; HSQC), ~ 40 mg of each ball milled sample was directly introduced into a 5 mm NMR tube with addition of 500 µL of premixed DMSO-d6/Pyriding-d5 (4:1). The NMR sample was sonicated for a duration ranging from 1 to 6 h in an ultrasonic bath until achieving a uniform gel consistency. The 2D HSQC spectra was recorded in a 700 MHz Bruker Avance NEO magnet equipped with a 5 mm TCI helium-cooled probe. The pulse program hsqcetgpsisp.2 was used to acquire the spectra with 2,048 points in F2 and 512 points in F1 for acquisition times of 125 ms and 6.6 ms, D1 delay of 1 s and 32 scans. The spectra were processed using Topspin 4.1.1. The S/G/H units were quantified by integration of contours of S2/6, G2 and H2/6, and expressed on an S + G + H = 100% basis. The relative abundances of interunit linkages were measured by integrating the Cα/Hα contours and expressed on the basis of the sum of (β-O-4’) + (β-5’) + (β-β’) + (β-1’) levels 2D-NMR lignin. Additional experimental results are illustrated in Bing, Ryan G., et al., “Beyond low lignin: Identifying the primary barrier to plant biomass conversion by fermentative bacteria.” Science Advances 10.42 (2024): eadq4941, which is herein expressly incorporated by reference in its entirety. Example 2: Engineering ethanologenicity into the extremely thermophilic bacterium Anaerocellum The anaerobic bacterium Anaerocellum (f. Caldicellulosiruptor) bescii natively ferments the carbohydrate content of plant biomass (including microcrystalline cellulose) into predominantly acetate, H₂, and CO₂, and smaller amounts of lactate, alanine and valine. While this extreme thermophile (growth Topt 78°C) is not natively ethanologenic, it has been previously metabolically engineered with this property, albeit initially yielding low solvent titers (~ 15 mM). The current application shows significant progress on improving ethanologenicity in A. bescii, such that titers above 130 mM have now been achieved, while concomitantly improving selectivity by minimizing acetate formation. Metabolic engineering progress has benefited from improved molecular genetic tools and better understanding of A. bescii growth physiology. Heterologous expression of a mutated thermophilic alcohol dehydrogenase (AdhE) modified for co-factor requirement, coupled with bioreactor operation strategies related to pH control, have been key to enhanced ethanol generation and fermentation product specificity. Insights gained from metabolic Attorney Dkt.10620-156WO1 modeling of A. bescii set the stage for its further improvement as a metabolic engineering platform. Over the past two decades, there have been significant efforts aimed at converting renewable lignocellulosic feedstocks into fuels and chemicals by microbial metabolic engineering. Several strategies have been pursued. Microorganisms that natively produce ethanol, such as Saccharomyces cerevisiae, have been engineered to enhance ethanol titers, yields and productivities. However, yeast cannot natively convert the entire carbohydrate content of lignocellulose into fermentable sugars, such that chemical and enzymatic biomass pretreatments are required. Given the challenges in engineering cellulose and hemicellulose hydrolysis capability into non-native (hemi)cellulose degraders, microorganisms that do this natively are particularly attractive. The moderately thermophilic bacterium Acetivibrio thermocellus (f. Clostridum thermocellum) (T_opt 60°C) is an option here with the additional advantage that it also natively produces ethanol; in fact, strains of A. thermocellus have been developed that can generate high ethanol titers. A concern for metabolically engineered moderate thermophiles, however, is that native biomasses contain indigenous moderate thermophiles that could interfere with engineered microorganisms by concomitantly converting plant biomass carbohydrates into undesirable fermentation byproducts; industrially, this forces the use of sterilizing pretreatments to avoid out-competition of indigenous microbes. In fact, a thermophilic threshold was determined such that temperatures approach 75°C this issue can be mitigated by precluding growth and metabolism of less thermophilic fermentative microorganisms. In view of this, extremely thermophilic bacteria belonging to the order Caldicellulosiruptorales, which grow optimally at or above 75°C, have been considered. These bacteria were initially of interest for their capacity to produce molecular hydrogen from carbohydrates at yields that approach the Thauer limit. Caldicellulosiruptorales strains and enzymes have been engineered to produce industrial chemicals, such as lactate, ethanol, acetone, 2,3-butanediol, and acetoin, albeit at low titers. A. bescii is the most studied of the Caldicellulosiruptorales, capable of fermenting microcrystalline cellulose and other biomass polysaccharides, primarily generating hydrogen, acetate, and CO₂, with smaller amounts of lactate and amino acids. Importantly, A. bescii is capable of solubilizing native and transgenic biomasses, allowing them to remain compatible with metabolic engineering efforts to improve biomass feedstock species. However, A. bescii natively produces neither ethanol nor any other carbon-based industrially relevant chemical in significant amounts. However, meaningful Attorney Dkt.10620-156WO1 progress has been made towards establishing A. bescii as a metabolic engineering platform (Figure 15). Technical economic analysis of A. bescii strains engineered to produce acetone and molecular hydrogen from plant biomass showed economic viability if certain biomass solubilization and metabolic engineering targets could be met. This motivates further improvements in titer, selectivity, and productivity of industrial chemicals to fully leverage A. bescii’s capabilities for lignocellulose solubilization and conversion. The A. bescii genetic toolkit relies on uracil auxotrophy, initially generated as a random deletion in the pyrimidine biosynthesis locus (strain JWCB005, ΔpyrFA). Later introduction of a thermostable kanamycin resistance gene allowed for targeted knock out of the pyrE gene (strain MACB1018). MACB1018 and its derived strains have improved genomic stability over strains belonging to the JWCB005 lineage. Both lineages contain strains with disruption or deletion of the gene encoding lactate dehydrogenase (ldh) that diverts carbon flux towards acetate and electron flux towards H₂. Subsequent genomic insertion of the gene encoding a bifunctional acetaldehyde-CoA /alcohol dehydrogenase (AdhE) from the moderate thermophile A. thermocellus in ldh disrupted strains, enabled and enhanced ethanol production. Figure 15 summarizes reported fermentation performance of ethanol producing strains. Figure 7 displays the central metabolism for A. bescii and highlights the main genetic modifications mentioned (ethanol producing AdhE and the deletion of the ldh). A. bescii strain JWCB032 (derivative of JWCB005) produced ethanol from simple sugars and plant biomass at measurable but still low titers (15 mM) at 65°C. Swapping out the AdhE from A. thermocellus for the AdhE or AdhB from Thermoanaerobacter pseudoethanolicus 39E allowed ethanol production at temperatures closer to the thermophilic contamination threshold of 75°C, but resulted in lower titers (~ 2 mM) and lower ethanol selectivity (higher amounts of acetate) (JWCB049, JWCB054). Increased titers were achieved in bioreactors through use of the more genetically stable parent strain MACB1034 (ΔpyrE, Δldh, derivative of MACB1018) expressing the A. thermocellus AdhE from a different locus (MACB1058) to produced up to 61 mM ethanol and 45 mM acetate at 60°C. Further insertion of genes encoding the Rnf complex from Thermoanaerobacter sp. X14 to improve redox balancing (MACB1062) resulted in up to 76 mM ethanol and 52 mM acetate at 60°C. Focus then turned to selectivity and investigating strategies to redirect carbon flux going to acetate, the primary ATP-generating bioenergetic pathway in A. bescii. While MACB ethanologenic strains demonstrated major improvement in ethanol titers Attorney Dkt.10620-156WO1 compared to JWCB ethanologenic strains, ethanol to acetate selectivity was 1.4-1.5 compared to 2.8 in JWCB032. Significant amounts of pyruvate were also reported in the MACB strains, resulting in 50-60% water-soluble fermentation products as ethanol (molar basis). The strain RKCB87, engineered for acetone production, was the first to demonstrate redirection of metabolism away from acetate. This engineered strain produced acetone (9 mM) and ethanol (3 mM) while consuming acetate from exogenous sources. Improved ethanologenicity in A. bescii, in terms of titer, productivity, and selectivity is demonstrated in the current application. This is achieved by redirecting carbon away from acetate through modulation of growth rate via optimized bioreactor operation strategies and strains engineered for improved redox control and thermophilicity. Disruption of acetate formation in A. bescii improves ethanol: acetate selectivity. Initial efforts to improve ethanol selectivity focused on knocking out genes in the acetate formation pathway (phosphate acetyltransferase (pta) and acetate kinase (ack), Figure 7) in A. bescii. Several non-replicating vectors were designed to knock out one, both, or partial versions of pta and ack. Over 1,000 colonies were screened for an acetate knockout, across MACB1018, MACB1058 and RKCB87; first crossover vector integration in the 3’ (downstream of ack) orientation disrupted acetate production and was obtained in MACB1058 and RKCB87. However, only 5’ (upstream) crossovers were obtained in MACB1018 with no acetate disruption, indicating homolactate fermentation may not be possible in A. bescii. Across all colonies, a clean second crossover was never found. However, co-selection with chloroacetate and 5-fluoroorotic acid (5-FOA) led to an acetate knockdown strain, RKCB89 from MACB1058, that produced up to 71 mM ethanol from cellobiose at 60°C and pH 6.3 (ethanol:acetate = 3.4:1) (Figure 16). Bottle cultures of RKCB89 demonstrated up to >90:1 ethanol:acetate selectivity at 24 h (Figure 16). RKCB89 was determined to be a permanent 3’ (downstream of ack) first crossover. This strain had major genomic mutations, formed aggregates, and grew poorly above 70°C, likely related to the thermostability of the inserted AdhE from A. thermocellus and the reduced ability to produce acetate for bionenergetic benefit. Further analysis of RKCB89 was done to explore the impact of media composition (e.g., varying amounts of uracil, NH4, cellobiose, trace elements, vitamins, phosphate, cysteine), degree of mixing, pH, and temperature (Figure 16). At the 50 mL scale in closed bottles containing 5 psig N₂/CO₂ (80/20) head space and buffered medium, ethanol production and selectivity at 60°C was best when pHinitial was 6.4, producing 24 mM ethanol Attorney Dkt.10620-156WO1 at ethanol:acetate of 93:1. Later evaluation of pHinitial 5.2 – 6.0 cultures showed up to 25 mM acetoin accumulation. Acetoin production was minimal (< 3 mM above pH 6.0). At bioreactor scale (1 L), the best results obtained were at pH 6.3 (controlled by NaOH addition) and 60°C, producing 71 mM ethanol (ethanol:acetate = 3.4) from cellobiose. Note, by comparison, MACB1058 at 65°C and pH 6.2 produced 20.2 mM ethanol (ethanol:acetate = 1.0) from cellobiose (Figure 16). The best previously reported results for MACB1058 were from Avicel (crystalline cellulose) at 60°C and pH 7.0, producing 61 mM ethanol (ethanol:acetate = 1.4) (Figure 15). In all RKCB89 bioreactor runs, the highest ethanol selectivity is seen in very late log phase, up to 8.7:1 ethanol:acetate; this is still significantly lower than the 93:1 peak selectivity observed in bottle cultures. Clearly, pH (measured at room temperature) between 6.2-6.4 improved ethanol selectivity. At bioreactor scale, nutrient limitations were identified and overcome by supplementing uracil (up to 180 µM consumed), NH₄ (additions above 48 mM yielded no improvement), and sugar (up to 17.8 g/L cellobiose consumed). Addition of extra vitamins, phosphate, cysteine, and trace minerals did not further improve ethanol production or increase cell densities. Improving A. bescii ethanol production using a more thermostable AdhE. Although RKCB89 significantly improves ethanol:acetate selectivity, it is limited to temperatures below the optimum for A. bescii and the thermophilic contamination threshold. Additionally, as RKCB89 is a permanent first crossover with genomic mutations, further genetic edits are not possible (i.e., it is kanamycin and 5-FOA resistant). Here, the aim was for improved thermophilicity and better ethanol:acetate selectivity along with high ethanol titers. Thermophilic Thermoclostridium stercorarium subsp. strain RKWS1 (Tster) (Topt 68°C) that was isolated from wheat straw provided a path to improved thermophilicity. Since this bacterium grows optimally at about 10°C above A. thermocellus, the AdhE encoded in its genome was likely more thermostable than A. thermocellus AdhE. Amino acid alignment of the two enzymes (Figure 8) showed they are 80% identical and 90% similar for the aligned region. RKWS1 AdhE contains 36 extra residues on the C-terminus compared to A. thermocellus AdhE. Reports on thermophilic AdhE’s revealed differences in cofactor requirements (NADH/NAPDH); Asp to Gly mutations in A. thermocellus and Thermoanaerobacterium saccharolyticum allowed both cofactor formats (NADH and NAPDH) to be used by the enzyme for the alcohol dehydrogenase step. Recombinant versions of the more thermoactive RKWS1 AdhE, with and without the Asp492Gly mutation, were generated and alcohol dehydrogenase activities (65°C) of purified proteins Attorney Dkt.10620-156WO1 were examined. Wildtype RKWS1 AdhE had no detectable activity (< 0.1 U/mg) with NADPH, but the Asp492Gly mutant activity with NADPH was confirmed (15.8 U/mg). Specific activities with NADH were similar for both the wildtype and mutant enzymes (8.9 and 8.7 U/mg, respectively). RKWS1 AdhE Asp492Gly differs from A. thermocellus and T. saccharolyticum AdhEs where NADPH-linked activities were substantially higher than NADH-linked activities in Gly-containing mutant AdhEs (by 1-2 orders of magnitude). Based on this, the Tster-AdhE-Asp492Gly strain (RKCB92) was created with the mutated Tster adhE gene inserted downstream of Athe_0949 locus in the A. bescii genome. RKCB92 was able to produce significant amounts of ethanol up to 75°C, but growth and fermentation products at 50 ml scale in sealed culture bottles varied greatly across different temperatures (Figure 9). The final OD680 was similar across all conditions despite differing growth rates at 65°C (t_d ~2.7 h), 70°C (t_d ~ 2.1 h) and 75°C (t_d ~ 1.9 h), which reflects the optimum temperature of A. Additionally, fermentation product distributions were significantly affected, with ethanol titer and selectivity inversely correlating with temperature (ethanol:acetate (mM:mM) 65°C - 54:17, 70°C - 24:26, 75°C - 22:26). The optimal temperature of Tster AdhE (Asp492Gly) conversion of acetaldehyde to ethanol was determined to be between 70-75°C, with slight differences for NADH and NADPH usage (Figure 18). This contrasts with in vivo conditions for optimal ethanol selectivity, this may be due to lower AdhE thermostability near its maximum activity temperature, or an increase in activity of native enzymes that divert metabolic flux away from ethanol.. Interestingly, RKCB92 (as well as RKCB89 and MACB1058) cultures were observed to flocculate significantly (Figure 10); this behavior was not seen in the parent strain (MACB1034). The material associated with flocs was determined to be mostly nucleic acids by DNAse I digest. This phenotype manifested most strongly in RKCB89, which grew nearly as flocs in the absence of agitation. The floccing phenotype of both RKCB89 and RKCB92 was dependent on temperature and correlated with ethanol selectivity: for RKCB92, no flocking at ≥ 78°C (no ethanol produced) and peak floccing at 65-68°C (highest ethanol selectivity). With sufficient agitation, this phenotype manifests as formation of 1-3 large white clumps amongst otherwise planktonic cells. Flocculation has been observed as a response to external stressors in Anaerocellum and Caldicellulosiruptor species. The potential of ethanol damage was examined by inducing this phenotype in the parent strain (MACB1034), but exogenous ethanol failed to induce floccing. Interestingly, floccing is observed to correlate with ethanol production in mesophilic organisms such as Attorney Dkt.10620-156WO1 Zymomonas sp., though the mechanism is not known. This phenotype presented in A. bescii may be due to a similar fermentation-related process, though other unexplored stressors cannot be ruled out. In context of industrial ethanol fermentation, floccing can be advantageous, as it helps reduce down-stream process intensity required to separate cells from fermentation broth. The impact of fermentation conditions on ethanol titer and selectivity was investigated with the RKCB92 strain (Figure 17). Main products formed were ethanol and acetate, with small amounts of amino acids (valine, alanine), pyruvate, and acetoin also detected. Significant differences were seen between bottle scale, 1.5 L bioreactor scale, and 17 L bioreactor scale. While buffering and pH control (with NaOH) consistently led to higher ethanol titers, selectivity was variable across scales. Sealed bottles (pressurized up to > 40 psig by fermentation product gases H₂, CO₂) had the highest selectivity, followed by the 17 L (~5 psig) and vented bottles (~1 psig), and lastly sparged 1.5 L bioreactors (~1 psig). This suggested that dissolved H2 and/or CO2 due to head-pressure was driving ethanol selectivity. Addition of H₂ (20-100%) into bottle headspace resulted in significant lag (24- 72 h) and low (< 1) ethanol:acetate selectivity. As such, increased solubility of CO₂ under higher head pressure was attributed to the observed selectivity differences. Carbonic acid may induce cellular responses affecting redox balancing and cell growth that could lead to improved ethanol selectivity. Impact of pH control on ethanologenicity and selectivity: NaOH vs. NaHCO_3. The observed influence of dissolved CO2 (and by proxy carbonic acid) led to experiments at bioreactor scale (~1 psig, sparged with N₂/CO₂ mix (80/20 v/v)) in which pH control was affected by either NaOH or NaHCO3 addition; sodium bicarbonate was employed to increase carbonic acid concentrations without high head pressures. As shown in Figure 11, significant differences in growth physiology, ethanol production, and ethanol:acetate selectivity were observed. At pH 6.0 and 65°C with 50 g/L Avicel (microcrystalline cellulose), both cultures formed aggregates and produced similar amounts of acetoin (9-12 mM), pyruvate (3 mM), alanine (8 mM) and valine (2-3 mM). More importantly though, the NaHCO₃ case produced 132 mM ethanol:48 mM acetate compared to 21 mM ethanol:55 mM acetate with NaOH. The stark contrast between NaOH- and NaHCO3-controlled bioreactor cultures of A. bescii RKCB92 further supports the carbonic acid hypothesis. It is known that bicarbonate impacts growth of mesophilic bacteria and is believed to be the result of an increased driving force for bicarbonate uptake. Bicarbonate Attorney Dkt.10620-156WO1 deprotonation in the cytoplasm is mitigated through reversal of ATP synthase exporting the excess protons, and the resulting ATP consumption slows growth. Similarly, though both conditions had cultures grow to comparable maximum cell densities (~4E9 cell/mL, planktonic), the doubling times were 1.4 h^-1 with NaOH, but much slower at 6.2 h^-1 with NaHCO₃. In both cases, ethanol selectivity peaked at the end of exponential phase (ethanol mole % of water-soluble products at 27 h NaOH was 54% and at 72.5 h NaHCO₃ was 75%), although ethanol was still produced in stationary phase albeit with diminishing selectivity. Additionally, there were significant differences in cell morphologies during bioreactor growth (Figure 12). With NaOH, cell morphology was equivalent to that observed in wildtype cultures, but very different with NaHCO3. At high ethanol titers in late stationary phase with NaHCO3, cells were elongated and formed spore-like structures; no fully-formed endospores were observed and addition of exogenous ethanol (up to 400 mM) to wildtype cultures had no effect on cell morphology. Elongated cells were also observed in the 17 L bioreactor (~5 psig) at ethanol titers above 90 mM (Figure 17). Cell elongation phenotype can be indicative of redox stress, as the cells increase their surface area:volume to regulate membrane potential. This makes sense for the elongated cells in RKCB92, as this phenotype only manifests in stationary phase, after high ethanol selectivity during exponential phase. Ethanol selectivity drops during stationary phase, which likely reflects a greater redox imbalance in stationary phase. These observations combined with the improved titers and selectivity with increased head-pressure and the optimal pH (~6.0 at culture temperature, pH 6.3-6.4 room temperature) supports carbonic acid being the causative agent. The pK_a of carbonic acid with bicarbonate at 65°C is approximately 6.0. In sealed bottles, as fermentation progresses, CO2 is forced into solution, increasing carbonic acid concentrations, but also acidifying the culture, thereby limiting ethanol titer without pH control. In sparged bioreactors, an open system at lower pressures, CO₂ does not accumulate and concentrations in the liquid are substantially lower compared to sealed bottles. However, addition of bicarbonate simultaneously maintains pH and replenishes carbonic acid and CO₂ as they leave the system. This maintains a higher concentration of carbonic acid in solution and keeps the pH near the bicarbonate carbonic acid pK. The use of bicarbonate avoids the need to pressure the bioreactor to achieve this and enhances ethanol production. Carbon consumption based on the amount of Avicel utilized and fermentation products detected when input into the metabolic model for A. bescii showed 72% and 74% Attorney Dkt.10620-156WO1 carbon closures, for the NaHCO3 and NaOH cases, respectively. Note that significant soluble cellooligosaccharides from Avicel hydrolysis were present but not quantified in the terminal cultures. While the carbon balances account for residual cellobiose, glucose, and planktonic cells, any cells remaining attached to Avicel were not quantified. Also of note, the amount of acetate made per cell was similar in both cases during exponential growth (3- 4x10^-9 mM acetate / cell); the NaHCO₃ condition just took longer to grow and accumulate the acetate. Total fermentation product formation per cell was higher and more uniform during exponential growth in the NaHCO3 case (1-5x10^-8 mM / cell) which is reflected in more Avicel being consumed during fermentation compared to the NaOH case (1.2x10^-9 – 1x10^-8 mM / cell). Cell growth was likely inhibited due to loss of ATP from increased intracellular pH, resulting from increased carbonic acid. However, carbon flux did not decrease with cell growth; instead, it was higher in the NaHCO₃ case. In the NaHCO₃- controlled reactors, excess NADPH that would normally go to biomass formation is instead utilized to produce ethanol, as RKBC92 expresses the Asp492Gly-AdhE, allowing utilization of NADPH. NaHCO₃ pH controlled Avicel reactors with RKCB92 at higher temperatures (70°C – 78°C) also had reduced growth rates compared to NaOH (T_d,min 4-5 h), confirming the effect of NaHCO3 on cellular growth rate. However, ethanol selectivity reflected what was seen in bottles (Figure 9), suggesting that the thermostability of the AdhE still plays a role in maximizing ethanol selectivity. The carbonic acid effect was further examined by comparing genomic and transcriptomic differences across the engineered A. bescii strains. Genomes of ethanol-producing A. bescii strains. As the genetic heritage of engineered A. bescii has previously had severe impact on bioproduction (i.e., JWCB005 vs MACB1018 lineages), the genomes of ethanologenic A. bescii strains of the MACB1018 lineage were reexamined, in the context of their parent strains, to determine whether any unexpected modifications occurred. The genomes of wildtype (DSM6725), MACB1018, MACB1034, MACB1058, MACB1062, RKCB89 and RKCB92 were generated via de novo assembly of Nanopore long read sequence data. While for the most part the genomes examined contained the intended deletions and insertions (Figure 13), RKCB89 had other significant mutations. The Athe_0949 locus (Regions 6,7 in Figure 13) confirmed the intended insertion of genes encoding AdhEs in MACB1058, MACB1062, RKCB89, and RKCB92, as well as the additional operon encoding an Rnf in MACB1062 (Region 8). Region 12 (pyrimidine biosynthesis locus) shows the intended pyrE deletion in Attorney Dkt.10620-156WO1 MACB1018, MACB1058, and RKCB92. However, RKCB89 regained pyrE in the acetate locus (Athe_1493-1494) via integration of a non-replicating vector (permeant first crossover, Region 14) and deleted a large region of the pyrimidine biosynthesis locus (Region 11). This apparently allowed the ack knockdown strain RKCB89 to tolerate 5-FOA while retaining pyrE and resist chloroacetate from the 3’ vector integration in the acetate locus. In Region 15, lactate dehydrogenase (ldh) (Athe_1918) was deleted, as expected in MACB1034, MACB1058, MACB1062, RKCB89, RKCB92. A. bescii natively harbors two plasmids (pAthe01 and pAthe02). Here, the use of Nanopore long reads provided new insight into their structures, where both plasmids, especially the larger pAthe01, appear to exist in a multimeric state where long reads filtered to a 20kB cutoff were still able to produce circular contigs representing ≥ 4-mers of pAthe01. Monomers and dimer contigs for pAthe02 were generated in some assemblies. The final assemblies provided here list the plasmids as monomers, but further investigation is needed to determine which multimers are the most dominant in A. bescii. Additionally, in RKCB92, pAthe01 was lost, while retaining pAthe02. Note that the A. bescii genome encodes genes related to spore formation (including spore coat, Stage II, III, IV, V proteins, as well as spore associated transcriptional regulators and sigma factors). A previous report gave A. bescii an 85% likelihood of being capable of spore formation, suggesting the right conditions to induce endospore formation had just not yet been found. These genes could contribute to the spore-like formations observed during bioreactor experiments with NaHCO3 (Figure 12). Comparative transcriptomics of A. bescii strains. Figure 14 summarize transcriptional data comparing late exponential phase (corresponding to the highest ethanol selectivity) of RKCB92 for the NaOH and NaHCO3 Avicel bioreactor cases (both at 1E9 cells/mL). A total of 455 of 2,587 coding sequences in the genome were significantly differentially transcribed. The bulk of down-regulated genes in the NaHCO₃ case are growth-associated, including the entire Glucan Degradation Locus (GDL) (Figure 14 - Region J), by 5- to 13-fold. The NaHCO₃ case has a large number of upregulated iron, sulfur, and molybdopterin associated genes (Regions B, D, F, H, M, N, and Q). Region D contains 21 up-regulated genes, up to 212-fold (sulfurtransferase), related to sulfur assimilation. These include a [4Fe-4S]-containing ferredoxin-like protein and sulfonate, molybdopterin, sulfite, uroporphyrinogen, and porphobilinogen related proteins. Additionally, the cysteine synthase, up-regulated 14-fold, was the 8^th most highly expressed Attorney Dkt.10620-156WO1 gene (based on transcript counts) in the NaHCO3 case (224^th highest in NaOH). Several stress response and sporulation related genes are differential expressed and were among the highest expressed genes in NaHCO₃, including rubrerythrin (+6.3-fold, 9^th highest in NaHCO3, 98^th in NaOH), peroxiredoxin (4^th highest in NaHCO3), chaperonin GroEL (6^th highest in NaHCO₃), heat shock response proteins (Regions E, F), septation regulator SpoVG (-10 fold, Region O), and sporulation protein A (+30 fold, Region Q). The cell morphology of RKCB92 in the NaHCO3 case (Figure 12) may be related to the sporulation related proteins and the elongation factor G (-412 fold, Region I). Flagella and chemotaxis related proteins were downregulated in the NaHCO₃ case (-2.3 to -4 fold, Region L), which may reflect the higher degree of aggregation seen in the NaOH case. Notably, the acetate formation genes (pta, ack) were not responsive. The Surface Layer Protein (Slp) (highest transcript in both cases) was down-regulated slightly (1.4-fold) in the NaHCO₃ case, while the AdhE was slightly up-regulated 1.7-fold; these were the 3^rd and 7^th highest transcript in NaHCO3 and NaOH, respectively. The operon terminating with Athe_0949, immediately upstream of the adhE, was up regulated (5-30 fold in NaHCO₃ case, Region C). Carry-over transcription from Athe_0949 may explain the increased adhE transcript in the NaHCO₃ case compared to the NaOH. Additionally, the second most down-regulated gene in the NaHCO3 condition compared to NaOH encodes a predicted carbonic anhydrase. These enzymes bidirectionally convert CO₂ to carbonic acid and have been known to have regulatory roles. This difference in expression demonstrates the severe impact of NaHCO3 on metabolic regulation within the cell and the inferred associated increased carbonic acid level. Cells need less carbonic anhydrase because there is already abundant carbonic acid intracellularly. This further supports the hypothesis that increased carbonic acid is the driver for the observed changes. Clearly, this causes a cellular stress response, and the cells do not need the carbonic anhydrase when carbonic acid concentrations are already high. Several sulfur metabolism related genes (and cysteine synthase) were highly regulated. Cysteine (6.3 mM) is the sulfur source in the medium; the metabolic model for A. bescii suggests only ~1/10^th of this is required. As such, sulfur limitation is unlikely, but should be tested in the future. To produce ethanol with AdhE (NADH and NADPH consuming), reduced ferredoxin needs to be converted into NAD(P)H or disposed of in another manner. This results in redox stress to the cell. The differential regulation surrounding [4Fe-4S]-containing enzymes and molybdopterin-associated proteins point to ferredoxin electron balancing. For example, the glycolytic enzyme glyceraldehyde-3- Attorney Dkt.10620-156WO1 phosphate ferredoxin oxidoreductase contains [4Fe-4S] clusters as well as molybdopterin that binds a tungsten atom. Taken together, the stress response genes and ferredoxin-related genes are indicative of redox stress. The reoccurring aggregation phenotype seen in ethanol strains is most evident when ethanol selectivity is highest. This phenotype appears to be linked to the induced redox stress. A second transcriptome from stationary phase (~4E9 cell/ml) largely aligned with results from the exponential phase, with the exception that the sulfur assimilation genes (Region D) were not differentially regulated. In addition, the acetate formation gene, pta, was slightly up-regulated in the NaHCO₃ case (1.9-fold), while the ack gene remained unaffected. Again, the AdhE was up-regulated in the NaHCO3 case (1.8-fold, 3^rd and 5^th highest transcript in NaHCO3 and NaOH, respectively). Similarly, the surface layer protein was again down-regulated in the NaHCO₃ case (-1.8-fold, 1^st and 2^nd 5^th highest transcript in NaHCO₃ and NaOH, respectively). The differential transcriptional response between NaHCO3 and NaOH bioreactors could be directly related to ethanol production as there is slight up-regulation of AdhE (in both growth phases). The up-regulation of the pta gene in NaHCO₃ stationary phase may explain the reduced ethanol selectivity in mid- to late-stationary (but still better than the NaOH case). While these transcriptional fold-changes are relatively low, the AdhE is consistently among the highest transcripts in the genome. As such, a small increase in transcription could nonetheless generate substantial amounts of protein, thereby outcompeting the acetate formation genes. Conversely, there is consistently 10-20 times more adhE transcript than pta and ack in all cases. As such, it is unlikely that the Pta and Ack are out competing the AdhE. Rather, as the AdhE needs NAD(P)H to form ethanol, redox availability is the most likely explanation for the ethanol selectivity differences. Metabolic engineering of A. bescii as a platform microorganism. Here, the production of ~6 g/L (~130 mM) ethanol is a significant step towards industrial relevance of A. bescii as a metabolic engineering platform, especially given the gains in selectivity and productivity (up to 3.8 and 1.8 mM/h ethanol, from cellobiose and Avicel, respectively). The floccing phenotype could be useful in industrial settings, where cell aggregation and settling reduces process costs associated with centrifugation and filtration downstream of fermentation. RKCB92 under high NaHCO3 conditions is a candidate for laboratory evolution to increase cell growth and extend metabolic activity. What drives A. bescii into stationary phase, eventually ceasing ethanol generation, is not clear. While phosphate, Attorney Dkt.10620-156WO1 uracil, ammonium, carbohydrate, iron, sulfur (presumably), and vitamins were ruled out as limiting nutrients, insufficient supplies of other nutrients cannot be excluded. Beyond the ethanologenicity of A. bescii, this work informs future engineering efforts to generate other non-native products. Improved culture conditions have increased cell densities from ~5E8 to 8E9 cells/ml. Higher cell mass in combination with the bicarbonate effect to suppress native acetate formation could be leveraged to improve titers, productivities, and selectivity of other products, such as acetone. Furthermore, this work showed that A. bescii produces acetoin at pH below 6.0 (measured at room temperature). This can be advantageous for some strains (i.e., 2,3-butanediol producers), or unwanted (and avoidable with pH control) for other products. Additionally, accumulation of amino acids (particularly alanine and valine) occurs mostly in stationary when excess ammonium (> 10 mM) is present. A fed-batch strategy for ammonium addition mitigated amino acid production and reduced osmolarity. A fed-batch system for growth on soluble sugars (i.e., mono- and disaccharides) avoids possible osmotic stress. Improved strains and fermentation conditions established here form the basis for further development of A. bescii as a platform for industrial chemical production from lignocellulosic plant biomass, thereby exploiting thermophilicity as a bioprocessing advantage. Although the focus here was on converting plant biomass to ethanol and minimizing acetate production, future metabolic engineering efforts with A. bescii should also consider maximizing acetate production and minimizing CO2 formation as another route to valorizing thermophilic fermentation physiology. Materials and Methods Bacterial strains and culture conditions. Anaerocellum (f. Caldicellulosiruptor) bescii wild-type strain (DSM6725) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and maintained as previously described. A. bescii strains MACB1018, MACB1034, and MACB1058 were developed, as reported previously. Modified defined DSMZ 516 medium was used for all cultures, as previously described. Several additional modifications were made as indicated. These include addition of buffering agents, MOPS (3-(N-morpholino)propanesulfonic acid) and/or MES (2-(N- morpholino)ethanesulfonic acid), at combined concentrations from 20 to 60 mM. Increased ammonium chloride was routinely used at 36 mM, occasionally 48 mM, or by fed batch by addition of 3.6 M ammonium chloride in 12 mM amounts as needed in bioreactors. Vitamins and phosphate loadings were routinely doubled. Uracil was added up to 1 mM, but routinely at 0.3 mM. Uracil was prepared as a 0.25 M stock with enough sodium hydroxide Attorney Dkt.10620-156WO1 to maintain pH between 10-10.5. Carbohydrate sources were varied. Cellobiose was routinely used and sterilized by filtration of final medium. Insoluble substrates, Avicel (microcrystalline cellulose) and beechwood xylan, were sterilized by a 60 min 121°C autoclave cycle, then directly added to sterile serum bottles or bioreactors. Initial pH was 7.2 at room temperature, unless indicated otherwise. N₂/CO₂ (80/20 v/v) gas mix was used to degas or sparge cultures. All bottle cultures were conducted as 50 ml cultures in 125 ml sealed serum bottles (unless noted otherwise) with 5 psig initial head pressure. Head pressure was intermittently monitored at temperature for active cultures with a pressure gauge fitted to a syringe needle. Vented bottles, 1 L and 1.5 L bioreactors used luer-lok check valves on gas outlets (Masterflex 30505-92) resulting in ~1 psig head pressure. Culture inocula were passaged twice from revived freezer stocks on medium identical to the experimental case in serum bottles. Inocula (3%) were routinely used from cultures in late exponential phase (OD₆₈₀0.4-0.6 or 5E8 - 1E9 cells/ml). Experimental conditions were all started at ~5E6 cells/ml. For bioreactors, culture transfers were appropriately scaled to obtain enough volume for inoculation. Sealed 1 L (300 mL culture) and 2 L (600 mL culture) screw-top Erlenmeyer flasks were used for larger cultures. Chloroacetate toxicity. A. bescii chloroacetate toxicity was determined for use as a selective agent. Wildtype A. bescii was grown for 45 h in serum bottles at 75°C, with variable concentration of sodium chloroacetate: 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mM. Minimal toxicity was observed up to 1.6 mM, 3.2 mM had ~60% growth compared to 0 mM, and no growth was observed at either 6.4 or 12.8 mM sodium chloroacetate. As such, 6.4 mM was determined to be above wildtype tolerance to chloroacetate. Strain construction and bacterial genetics. Five plasmids were constructed for this work in NEB 5α E. coli (Figure 18). Primers used to construct these plasmids are reported in Table 3. All vectors were constructed via Gibson assembly of DNA fragments, except where indicated. Two E. coli pSC101 origin A. bescii targeting non-replicating vectors were created to knock out A. bescii acetate formation genes (pRGB008 and pRGB011). pRGB008 used the same flanking regions previously used to create RKCB87, but without the acetone formation genes. pRGB0085’ flanking region contains a partial pta (Athe_1494). pRGB0115’ flanking region contains the entire pta without promotor (starts at the start codon). Two E. coli expression vectors (pRGB025 and pRGB026) were constructed using the pRSF Attorney Dkt.10620-156WO1 backbone; sequence confirmed plasmid was also transformed into Rosetta (DE3) pLysS E. coli, as previously described. The sequence encoding the bifunctional alcohol dehydrogenase from Thermoanaerobacter stercorarium RKWS1 was PCR amplified from genomic DNA. This was assembled into the pRSF backbone to create pRGB025, containing a 6x histidine N-terminal tag. A KLD reaction (kinase, ligase, DPN1) was used to alter 2 base pairs to induce the Asp492Gly mutation (GAC to GGT) to create pRGB026. The mutated gene was PCR amplified and then inserted into the E. coli pSC101 origin A. bescii targeting non-replicating vector with flanking regions for the Athe_0949 locus, to generate pRGB032. A synthetic DNA fragment including the 307 bp of the AdhE and the Calkro_0402 terminator was used to aid in Gibson assembly of pRGB032. A. bescii genetics were carried out, as previously described, with a few modifications. Briefly, A. bescii cells were grown to an OD₆₈₀ of 0.06-0.07, pelleted and washed with sucrose, then electroporated at 1.8 kV, 400 Ω, 25 µF. Selection was carried out with 50 µg/mL kanamycin. Cultures were plate purified 6 times on solid kanamycin containing medium to isolate pure 1^st crossover cultures. Freshly grown 1^st crossover cultures were then passaged (3% inoculum) to non-selective medium with 80 µM uracil for 4 h, then plated on solid medium with 8 mM 5-fluoroorotic acid (5-FOA) and 80 µM uracil. Colonies were screened by PCR for 2^nd crossovers. Positive colonies were plate purified 6 times on non-selective medium with 80 µM uracil. Plasmid pRGB032 generated A. bescii strain RKCB92. For acetate knockout plasmids (pRGB008 and pRGB011), 6.4 mM chloroacetate was also added to liquid and solid 5-FOA medium. Over 1,000 colonies were screened both by colony PCR, as well as HPLC analysis, to detect acetate formation. A. bescii strain RKCB89 resulted from pRGB0113’ crossover insertion into MACB1058. RKCB89 was the end strain after passage on liquid medium containing 6.4 mM chloroacetate, 8 mM 5-FOA, followed by plating on solid medium of the same composition. Bioreactor conditions. All ethanol producing bioreactors were allowed to progress until ethanol titers peaked. All bioreactors used a 4°C condenser on gas outlets. All other ports were sealed and pressure leak tested. Bioreactor cultures (1 L and 1.5 L working volumes) were conducted in 3 L glass vessels with stainless steel head plates (Chem Glass CLS-1380-01) and double Rushton impellers (Chem Glass CLS-1380-08) located at the bottom of the shaft - 2 cm below liquid surface. Reactors were controlled with an Applikon Bio Console. Control of pH was effected by addition of 2M NaOH or ~9% saturated (at 22°C) NaHCO3. Hamilton Attorney Dkt.10620-156WO1 EasyFerm PHI K8 sensors (ChemGlass CLS-1435-P70), stable to 140°C, were used and no stability issues were noted. Bioreactor samples were routinely re-checked with a standalone pH probe to verify accuracy. Gas sparge rate of 25 SCCM of N₂/CO₂ (80/20 v/v) was used. Bioreactors were sterilized by autoclaving for 60 min at 121°C with the pH probe installed. Filter sterilized and degassed medium was added to reactors after autoclaving, sealed, heated to fermentation temperature, and sparged until anaerobic (minimum 3 h, as indicated by resazurin color). Bioreactors (17 L working volume) were conducted in a Sartorius Biostat C⁺ 20-2 bioreactor. Total vessel volume is 30 L with 2:1 height to diameter ratio. Gas sparge rate of 1 or 0.1 ± 0.1LPM of N2/CO2 (80/20 v/v) was used. Dual Rushton impellers at 100 rpm agitated the culture. Addition of 2M NaOH controlled pH. Base medium (salts, trace metals, phosphates) were sterilized in place at 121°C for 60 min. Filter sterilized solutions containing cellobiose, vitamins, resazurin, cysteine, uracil, and sodium bicarbonate were added after heat sterilization. Medium was sparged at 1 LPM until anaerobic as indicated by resazurin, for a minimum of 3 h. Cell counting and imaging. Acridine orange-stained cells were imaged on Nikon Eclipse 50i microscope to determine cell densities and micrographs were captured with an AccuScope Exelis HD camera, as previously reported. Quantification of cell mass and Avicel . Determination of dry cell weight and residual insoluble Avicel from 1.5 L bioreactor cultures was determined to allow calculation of carbon balances. Terminal bioreactor culture volumes were measured first. Avicel was then separated from cell mass via centrifugation at 200 x g for 2 min, followed by decanting of planktonic cells. Three rounds of 200 x g spins were done to collect Avicel from cells. Subsequently, cell mass was pelleted at 6,000 x g for 15 min. Avicel was washed 2x with water, vortexed to resuspend, and pelleted at 200 x g. Water supernatant was then pelleted at 6,000 x g to collect any residual cell mass that was recovered from Avicel. Avicel and cell pellets were then dried at 110°C for 24 h to determine dry mass. Recombinant expression of Thermoclostridium stercorarium RKWS1 AdhEs. E. coli Rosetta (DE3)pLysS strains harboring pRGB025 and pRGB026 were grown in ZYM- 5052 auto-induction medium, as previously described. Cells were pelleted, resuspended in IMAC buffer A (500 mM NaCl; 20 mM sodium phosphate; 20 mM imidazole, pH 8.0) at 4 mL/g-cell-wet-weight with 0.001 g lysozyme/mL. Solutions were lysed using a French Pressure Vessel, heat treated at 55°C for 30 min, then centrifuged and filtered to clarify. Attorney Dkt.10620-156WO1 Lysate was applied to a Cytiva HisTrap HP 5mL column using a Bio-Rad NGC Quest 10 Chromatography System. IMAC Buffer B (500 mM NaCl; 20 mM sodium phosphate; 500 mM imidazole, pH 8.0) was used to elute the protein. Fractions were screened by SDS- PAGE, then desired fractions pooled, concentrated, and protein concentration was determined by Qubit Total Protein Assay. Samples were then brought to 50% glycerol by addition of 100% glycerol and stored at -20°C. Enzyme assays. Alcohol dehydrogenase specific activity of Thermoclostridium stercorarium RKWS1 AdhE enzymes, with and without the Asp492Gly mutation, were determined. Purified recombinantly expressed AdhE’s were assayed under the following conditions in a Coy Anaerobic Chamber (95% N2, 5% H2 gas phase): final 200 µL reactions contained 100 mM Tris-HCl (pH 7.5), 5 µM FeSO4, 1 mM dithiothreitol (DTT), 0.25 mM NADH or NADPH, 20 mM acetaldehyde, and 10 µg/ml purified AdhE. All components except enzyme were added, heated to 65°C (unless otherwise specified) for 1 min, then enzyme was added to initiate the reaction. Absorbance (340 nm) was measured every 30 s for 3 min. NAD(P)H extinction coefficient of 6.22 mM^-1cm^-1 was used. Quantification of fermentation products and nutrients. Cellobiose, glucose, acetate, ethanol, acetoin, pyruvate, and uracil were quantified on a Waters Arc High- Performance Liquid Chromatograph (HPLC) with a Phenomenex Rezex-ROA column (300 mm x 7.8 mm) with 5 mM sulfuric acid mobile phase at 60°C. A Waters 2414 refractive index detector and Waters 2998 photodiode array detector were used to quantify products, as previously described. Ammonium was measured using reagents from API Freshwater/Saltwater Ammonia Test Kit (salicylate-based ammonia test). “Bottle 1” solution (11 µL) was added to 120 µL diluted sample or standard followed by 11 µL of “Bottle 2” solution. Samples were vortexed for 10 s, then allowed to incubate at room temperature for 10 min. Absorbance was read at 690 nm. Water was used as a blank. Culture samples were diluted 100-fold. Detection was reliable from sample concentrations of 0.5 to 24 mM NH4. Quantification above 24 mM used 300x dilutions. Quantification of amino acids was performed using a Waters AccQ Tag Amino Acids C18 Column (3.9 mm x 150 mM, WAT052885) on the Arc HPLC with a Waters 2475 Fluorescence Detector, per manufacture instructions. Derivatization of samples via the Waters AccQ-Fluor Reagent Kit (WAT052880) was done per manufacture instructions. A. bescii culture samples were diluted 1:100 in water and 10 µL input as the sample to the kit. Attorney Dkt.10620-156WO1 Serially diluted Waters Amino Acid Standard H (WAT088122) was used to quantify alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. Genomic DNA extraction, sequencing, assembly, and annotation. Genomic DNA was extracted, processed and sequenced, as previously described. Briefly, NEB Monarch Genomic DNA Purification Kit (New England Biolabs, Inc.) was used to extract gDNA. Oxford Nanopore Native Barcoding Kit 24 V14 and 10.4.1 flow cells or ONT Ligation Sequencing Kit with Native Barcoding Expansion Kit 1-12 and R9.4.1 flow cells on a MinION Mk1B device. Assembly of RKCB92 reads was done as previously described. For Wildtype A. bescii, MACB1018, MACB1058, and RKCB89, a TryCycler pipeline was employed to generate consensus genomes using 15 sub-assemblies, 5 each from Flye v2.9.1, Canu v2.2, and NECAT v0.0.1, as previously described. RKCB92 was annotated using a local instance of NCBI Prok aryotic Genome Annotation Pipeline (PGAP). Identification of structural variations >50 bp between the engineered strains and wildtype A. bescii was performed with SVIM-ASM v1.0.3 and visualized using Circos. RNA extraction. Samples from triplicate 1.5 L bioreactors on 50 g/L Avicel with RKCB92 were taken at ~1E9 cells/ml (10 mL, late log) and ~4E9 cells/ml (5 mL, stationary). Samples were immediately quenched in a dry ice ethanol bath, spun at 200 x g for 1 min to settle Avicel. Avicel was washed with 1 mL sterile phosphate buffered saline (PBS), vortexed, and pelleted again at 200 x g, 1 min. Pooled supernatants were pelleted at 12,500 x g for 10 min. Stationary phase cells were stored in 800 µL RNA Protection Reagent (NEB Monarch Total RNA Miniprep Kit, New England Biolabs, Inc.) and stored at -80°C. Frozen cells were stored until processed (2 weeks); cells were pelleted from protection reagent, and then processed the same as log phase cells as follows. Cells (immediately for log phase cells), were resuspended in 240 µL PBS, 60 µL of 25 mg/mL lysozyme was added, vortexed briefly, then 300 µL of Tissue Lysis Buffer (from NEB Monarch Genomic DNA Purification Kit) and vortexed. Solution was incubated at 37°C for 25 min. Two volumes of RNA Lysis Buffer were added, then proceeded as per manufacture instruction for Monarch Total RNA Miniprep Kit, including the on-column DNAse I digest. Quibit Broad Range RNA Kit was used to quantify RNA. Ribosomal RNA depletion, dscDNA synthesis, and sequencing. Total RNA samples were depleted of ribosomal RNA by RNAse H digest. A mixture of ssDNA probes targeting A. bescii ribosomal RNA (120 probes at 2 µM, Table 3) each, were hybridized Attorney Dkt.10620-156WO1 with 10 µg of total RNA at 95°C for 5min with 40U of murine RNAse Inhibitor (NEB); 10 mM tris-HCl pH 8.0, 100 mM NaCl, and 1 mM EDTA. Thermostable RNAse H and buffer were added (31.25U, NEB) then incubated at 50°C for 30 min. TurboDNAse and buffer were added (7.36U, Invitrogen) then incubated at 37°C for 30 min. A 1.8x RNAClean XP Bead cleanup was used to purify ribodepleted RNA per manufacture instructions with 16 µL elution. Modified protocols from Oxford Nanopore Technologies (ONT) were used in the following steps. RNAClean XP and AMPure XP beads were used per manufacture and ONT instructions where required. Ribodepleted RNA was polyadenylated with E. coli Poly(A) Polymerase (NEB). Reverse Transcription and second strand synthesis were carried out per ONT instructions except custom primers were used: 2 µM Oligo(dt)VN primer, 10 µM Template Switching Oligo (TSO-1), and 10 µM second strand synthesis primer (S3P) (Table 3), resulting in double stranded cDNA (dscDNA). ONT Native Barcoding Kit 24 V14 and 10.4.1 flowcells on a MinION Mk1B with high-accuracy model, 400 bps base- calling (MinKNOW v23.11.5) were used per manufacture instructions to sequence dscDNA starting at the End-prep step, multiplexing 6 samples at a time. Transcriptomics analysis. MinKNOW trimmed reads were filtered to Q9 and 200bp with Nanofilt v2.8.0. Reads were aligned to RKCB92 genome with BowTie2 local alignment and then counted with HTSeq. Differential expression levels were generated from read counts using a generalized linear model in EdgeR with RStudio. Metabolic modeling. Metabolic modeling was performed using the A. bescii genome scale metabolic model. Model simulations were performed with the PSAMM software v1.2.1, using IBM ILOG CPLEX Optimizer version 22.1.0 and Python version 3.9.15. The model was modified to enable the accumulation of amino acids (i.e by adding sink reactions for Gly, Glu, Ile, Leu, Pro, Met) following experimental observations. Flux constraints were calculated for cellobiose consumption and for amino acid, product, and protein yields by taking the difference between the highest and lowest measured concentrations during RKCB92 bioreactor experiments (Table 4). The modeled maximum Avicel consumption was calculated using fba while fixing the constraints of production and protein yields. Carbon closure was calculated by dividing the maximum Avicel consumption in the model by the experimentally measured Avicel consumption. The minimum required Cysteine consumption was calculated using fva while fixing the constraints of production and protein yields. Table 3. Primers and Synthetic Gene Sequences Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1

Attorney Dkt.10620-156WO1 Table 4. Modeling Inputs and Outputs Additional experimental results are illustrated in Bing, Ryan G., et al. "Engineering ethanologenicity into the extremely thermophilic bacterium Anaerocellum (f. Caldicellulosiriuptor) bescii." Metabolic Engineering 86 (2024): 99-114, which is herein expressly incorporated by reference in its entirety. Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub- combinations are of utility and may be employed without reference to other features and sub- combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope Attorney Dkt.10620-156WO1 thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein. 1. J. Nielsen, C. Larsson, A. van Maris, J. Pronk, Metabolic engineering of yeast for production of fuels and chemicals. Curr Opin Biotechnol 24, 398-404 (2013). 2. B. R. Papanek B, Rydzak, Guss AM, Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum. Metabolic Eng 32, 49-54 (2015). 3. R. G. Bing, D. J. Willard, J. R. Crosby, M. W. W. Adams, R. M. Kelly, Whither the genus Caldicellulosiruptor and the order Thermoanaerobacterales: phylogeny, taxonomy, ecology, and phenotype. Front Microbiol 14, 1212538 (2023). 4. E. Byrne et al., Characterization and adaptation of Caldicellulosiruptor strains to higher sugar concentrations, targeting enhanced hydrogen production from lignocellulosic hydrolysates. Biotechnol Biofuels 14, 210 (2021). 5. M. 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Claims

Attorney Dkt.10620-156WO1 WHAT IS CLAIMED IS: 1. A genetically modified extremely thermophilic bacterium, wherein the genetically modified thermophilic bacterium comprises a heterologous gene encoding a bifunctional alcohol dehydrogenase. 2. The genetically modified bacterium of claim 1, comprising a bacterium from the order Caldicellulosiruptorales. 3. The genetically modified bacterium of claim 1, wherein the genetically modified bacterium comprises Anaerocellum (f. Caldicellulosiruptor) bescii. 4. The genetically modified bacterium of any one of claims 1-3, wherein the heterologous gene comprises an AdhE gene from Thermoclostridium stercorarium. 5. The genetically modified bacterium of claim 4, wherein the AdhE gene from Thermoclostridium stercorarium comprises an Asp492Gly mutation. 6. The genetically modified bacterium of any one of claims 1-5, wherein a gene encoding lactate dehydrogenase expression has been inactivated. 7. The genetically modified bacterium of any one of claims 1-6, wherein a gene encoding lactate dehydrogenase has been inactivated by homologous recombination. 8. The genetically modified bacterium of any one of claims 1-7, wherein the heterologous gene bifunctional AdhE is expressed from a plasmid. 9. The genetically modified bacterium of any one of claims 1-8, wherein the heterologous gene encoding the bifunctional alcohol dehydrogenase is inserted into a genomic locus of the bacterium. 10. The genetically modified bacterium of any one of claims 1-9, further comprising a promoter operably linked to the heterologous gene encoding the bifunctional alcohol Attorney Dkt.10620-156WO1 dehydrogenase, wherein the promoter is configured to function at elevated temperatures. 11. The genetically modified bacterium of claims 4-10, wherein the heterologous bifunctional alcohol dehydrogenase gene is inserted downstream of Athe_0949 in the Anaerocellum bescii genome. 12. A method for enhancing ethanol production, comprising: obtaining a biomass sample; contacting the plant biomass sample with a genetically modified bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase under conditions effective to convert the plant biomass to ethanol. 13. The method of claim 12, wherein the conditions effective comprise one or more of: (i) maintaining pH between 5.8 and 7, and/or (ii) maintaining an operational temperature of 55-75°C. 14. The method of any one of claims 12-13, wherein the genetically modified bacterium comprising A. bescii that has been genetically modified with the insertion of alcohol dehydrogenase gene. 15. A genetically modified Anaerocellum (f. Caldicellulosiruptor) bescii strain configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C. 16. A bioreactor system for enhancing ethanol production and selectivity relative to other fermentation byproducts from plant biomass, comprising: a temperature control system configured to maintain a fermentation temperature (e.g., from 55°C to 75°C) for a genetically modified Anaerocellum (f. Caldicellulosiruptor) bescii strain, the genetically modified A. bescii bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase; a pH control system configured to maintain a pH between 5.8-7.0 (e.g., using sodium bicarbonate and sodium hydroxide); a pressurization system configured to control carbon dioxide head pressure; and Attorney Dkt.10620-156WO1 an agitation system configured to mitigate cell aggregation. 17. The bioreactor system of claim 16, wherein the fermentation byproducts comprise acetate, lactate, hydrogen, carbon dioxide, acetoin, 2,3-butanediol, pyruvate, butanol, isopropanol, and/or higher chain alcohols. 18. A method of producing ethanol from a plant biomass sample, comprising: obtaining a plant biomass sample; contacting the plant biomass sample with the genetically modified extremely thermophilic bacterium of any one of claims 1-11; and releasing a fermentable carbohydrate from the plant biomass sample; and fermenting the fermentable carbohydrate to produce ethanol. 19. The method of claim 18, wherein the plant biomass sample comprises a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. 20. The method of claim 19, wherein the poplar tree is a genetically modified poplar tree. 21. The method of any one of claims 18-20, wherein the mixing of the plant biomass sample with the genetically modified bacterium occurs at about pH 5.8-7.0. 22. The method of claim 21, wherein the pH is maintained using NaOH or NaHCO3. 23. The method of any one of claims 18-22, wherein contacting the plant biomass sample with the genetically modified bacterium occurs at about 55-75˚C. 24. The method of any one of claims 18-23, wherein the method does not comprise a chemical pretreatment of the plant biomass sample. 25. A method of degrading lignin in a plant biomass sample, comprising: obtaining a plant biomass sample; Attorney Dkt.10620-156WO1 mixing the plant biomass sample with the genetically modified bacterium of any one of claims 1-11; and releasing a fermentable carbohydrate from the plant biomass sample. 26. The method of claim 25, further comprising the step: determining a methoxy content number in the plant biomass sample; wherein the methoxy content number is determined using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample. 27. The method of claim 26, wherein a value of less than or equal to 0.2 is indicative that the plant biomass can be solubilized without chemical pretreatment. 28. The method of any one of claims 25-27, wherein the plant biomass sample is from a poplar tree. 29. The method of claim 28, wherein the poplar tree is a genetically modified poplar tree. 30. The method of any one of claims 25-29, wherein mixing the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about pH 5.8-7.0. 31. The method of claim 30, wherein the pH is maintained using sodium hydroxide. 32. The method of any one of claims 25-31, wherein the mixing of the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about 55- 75˚C. Attorney Dkt.10620-156WO1 33. The method of any one of claims 25-32, wherein the method does not comprise a chemical pretreatment of the plant biomass sample. 34. The method of any one of claims 25-33, wherein the fermentable carbohydrate is converted to ethanol. 35. A method of degrading lignocellulose in a plant biomass sample, comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; based on the methoxy content number being below a threshold value, contacting the plant biomass sample with a bacterium, wherein the bacterium degrades the lignocellulose; and forming a fermentable carbohydrate from the plant biomass sample. 36. The method of claim 35, wherein the threshold value is 0.17 or less. 37. The method of any one of claims 35-36, wherein the bacterium is a thermophilic bacterium. 38. The method of any one of claims 35-37, wherein the bacterium is Anaerocellum (e.g., A. bescii), Acetivibrio (e.g., Acetivibrio thermocellus Caldicellulosiruptor, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium. 39. The method of any one of claims 35-38, wherein the bacterium is a genetically modified strain. Attorney Dkt.10620-156WO1 40. The method of any one of claims 35-39, wherein the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. 41. The method of claim 40, wherein the poplar tree comprises a genetically modified poplar tree. 42. The method of any one of claims 35-41, wherein contacting the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about pH 5.0- 7.2. 43. The method of claim 42, wherein the pH is maintained with NaOH. 44. The method of any one of claims 35-43, wherein the contacting of the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about 55˚C- 85˚C. 45. The method of any one of claims 35-44, wherein the method does not comprise a chemical pretreatment of the plant biomass sample. 46. The method of any one of claims 35-45, wherein the fermentable carbohydrate is converted to ethanol. 47. A method of determining an amount of a chemical pretreatment of a plant biomass sample, the method comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; and Attorney Dkt.10620-156WO1 determining, based on the methoxy content number, an amount of a chemical pretreatment needed for the plant biomass sample, wherein a higher methoxy content number indicates more chemical pretreatment and a lower methoxy content number indicates a less or no chemical pretreatment. 48. The method of claim 47, wherein the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose. 49. The method of claim 48, wherein the poplar tree comprises a genetically modified poplar tree.