Lignocellulose-derived Inhibitors Research Articles

Adaptive evolution of Paecilomyces variotii enhanced the biodetoxification of high-titer inhibitors in pretreated lignocellulosic feedstock

High inhibitor concentrations in lignocellulose feedstock negatively affect the degradation rate of biodetoxification strains. This study designed two adaptive laboratory evolutions in solid substrate and liquid medium to boost the biodetoxification capacity of P. variotii to high titers of lignocellulose-derived inhibitors, resulting in two evolved strains AC70 and ZW70. The results showed that the evolutionary adaptation in liquid medium could better boost the acetic acid assimilation compared to that on solid substrate. Transcriptional analysis revealed that the evolved strains exhibited a significant upregulation of adh, acs, ach1, and ackA directly related to the initial steps of acetate and furan aldehydes metabolisms. ZW70 strain can effectively remove the high concentration inhibitors cocktail from the hydrolysates derived from pretreated wheat straw and furfural residues. The biodetoxified hydrolysates by ZW70 were successfully used for cellulose chiral L-lactic acid production with the titers of ∼110 g/L, which were over 20 % higher than that detoxified by parental strain.

Application of fed-batch strategy to fully eliminate the negative effect of lignocellulose-derived inhibitors in ABE fermentation

BackgroundInhibitors that are released from lignocellulose biomass during its treatment represent one of the major bottlenecks hindering its massive utilization in the biotechnological production of chemicals. This study demonstrates that negative effect of inhibitors can be mitigated by proper feeding strategy. Both, crude undetoxified lignocellulose hydrolysate and complex medium supplemented with corresponding inhibitors were tested in acetone–butanol–ethanol (ABE) fermentation using Clostridium beijerinckii NRRL B-598 as the producer strain.ResultsFirst, it was found that the sensitivity of C. beijerinckii to inhibitors varied with different growth stages, being the most significant during the early acidogenic phase and less pronounced during late acidogenesis and early solventogenesis. Thus, a fed-batch regime with three feeding schemes was tested for toxic hydrolysate (no growth in batch mode was observed). The best results were obtained when the feeding of an otherwise toxic hydrolysate was initiated close to the metabolic switch, resulting in stable and high ABE production. Complete utilization of glucose, and up to 88% of xylose, were obtained. The most abundant inhibitors present in the alkaline wheat straw hydrolysate were ferulic and coumaric acids; both phenolic acids were efficiently detoxified by the intrinsic metabolic activity of clostridia during the early stages of cultivation as well as during the feeding period, thus preventing their accumulation. Finally, the best feeding strategy was verified using a TYA culture medium supplemented with both inhibitors, resulting in 500% increase in butanol titer over control batch cultivation in which inhibitors were added prior to inoculation.ConclusionProperly timed sequential feeding effectively prevented acid-crash and enabled utilization of otherwise toxic substrate. This study unequivocally demonstrates that an appropriate biotechnological process control strategy can fully eliminate the negative effects of lignocellulose-derived inhibitors.Graphical

Mining novel gene targets for improving tolerance to furfural and acetic acid in Yarrowia lipolytica using whole-genome CRISPRi library

Abundant renewable resource lignocellulosic biomass possesses tremendous potential for green biomanufacturing, while its efficient utilization by Yarrowia lipolytica, an attractive biochemical production host, is restricted since the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate. Given deficient understanding of inherent interactions between inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using clustered regularly interspaced short palindromic repeats interference library in Y. lipolytica, achieving tolerance to 0.35 % (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15 % (v/v) acetic acid. The tolerance mechanism might involve improvement of cell division and decrease of reactive oxygen species level. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for improving microbial tolerance to lignocellulose-derived inhibitors and revealing underlying mechanism.

Phenotypic and comparative transcriptomics analysis of RDS1 overexpression reveal tolerance of Saccharomyces cerevisiae to furfural

The yeast Saccharomyces cerevisiae able to tolerate lignocellulose-derived inhibitors like furfural. Yeast strain performance tolerance has been measured by the length of the lag phase for cell growth in response to the furfural inhibitor challenge. The aims of this work were to obtain RDS1 yeast tolerant strain against furfural through overexpression using a method of invivo homologous recombination. Here, we report that the overexpressing RDS1 recovered more rapidly and displayed a lag phase at about 12h than its parental strain. Overexpressing RDS1 strain encodes a novel aldehyde reductase with catalytic function for reduction of furfural with NAD(P)H as the co-factor. It displayed the highest specific activity (24.8 U/mg) for furfural reduction using NADH as a cofactor. Fluorescence microscopy revealed improved accumulation of reactive oxygen species resistance to the damaging effects of inhibitor in contrast to the parental. Comparative transcriptomics revealed key genes potentially associated with stress responses to the furfural inhibitor, including specific and multiple functions involving defensive reduction-oxidation reaction process and cell wall response. A significant change in expression level of log2 (fold change >1) was displayed for RDS1 gene in the recombinant strain, which demonstrated that the introduction of RDS1 overexpression promoted the expression level. Such signature expressions differentiated tolerance phenotypes of RDS1 from the innate stress response of its parental strain. Overexpression of the RDS1 gene involving diversified functional categories is accountable for stress tolerance in yeast S.cerevisiae to survive and adapt the furfural during the lag phase.

Enhancing tolerance of Kluyveromyces marxianus to lignocellulose-derived inhibitors and its ethanol production from corn cob via overexpression of a nitroreductase gene

Despite its potential as a sustainable feedstock for producing renewable fuels and chemicals, lignocellulosic biomass (LCB) remains largely untapped due to problems associated with its deconstruction and the toxicity of its sugar-rich hydrolysates in microbial conversion processes. Using genetic modification to facilitate inhibitor tolerance, the present study aimed to construct a robust yeast cell factory for cellulosic ethanol bioproduction from non-detoxified LCB. To this effect, two putative nitroreductase genes (KmHBN1 and KmFRM2) hypothesized to be possible mediators of oxidative stress response were separately overexpressed and disrupted in Kluyveromyces marxianus. In contrast to KmFRM2 modified strains (disrupted or overexpressed) with no significant change, the inhibitor tolerance to phenols, furfural, and acetic acid cocktail was improved in YKmHBN1-URA3 strain overexpressing KmHBN1. Although the recombinant KmHBN1 and KmFRM2 had nitroreductase activity, they did not degrade or convert inhibitors. The change of intracellular reactive oxygen species level (ROS) of KmHBN1 disrupted or overexpressed strains indicated that KmHBN1 affected intracellular ROS elimination. Cells with disrupted KmHBN1 failed to respond to ROS created during inhibitor metabolism, and this disruption also led to reduced expression of respiratory chain genes which translates to a decreased inhibitor tolerance. The overexpression of KmHBN1 not only enhanced the ethanol production from glucose in the presence of inhibitors but also improved ethanol productivity (18.75%) from simultaneously-saccharified and co-fermented inhibitor-ladened corn cob. These findings unlock a new pathway for the creation of robust yeast strains to aid in the biorefinery conversion of lignocellulosic feedstock.

Contribution of YPRO15C Overexpression to the Resistance of Saccharomyces cerevisiae BY4742 Strain to Furfural Inhibitor.

Lignocellulosic biomass is still considered a feasible source of bioethanol production. Saccharomyces cerevisiae can adapt to detoxify lignocellulose-derived inhibitors, including furfural. Tolerance of strain performance has been measured by the extent of the lag phase for cell proliferation following the furfural inhibitor challenge. The purpose of this work was to obtain a tolerant yeast strain against furfural through overexpression of YPR015C using the in vivo homologous recombination method. The physiological observation of the overexpressing yeast strain showed that it was more resistant to furfural than its parental strain. Fluorescence microscopy revealed improved enzyme reductase activity and accumulation of oxygen reactive species due to the harmful effects of furfural inhibitor in contrast to its parental strain. Comparative transcriptomic analysis revealed 79 genes potentially involved in amino acid biosynthesis, oxidative stress, cell wall response, heat shock protein, and mitochondrial-associated protein for the YPR015C overexpressing strain associated with stress responses to furfural at the late stage of lag phase growth. Both up- and down-regulated genes involved in diversified functional categories were accountable for tolerance in yeast to survive and adapt to the furfural stress in a time course study during the lag phase growth. This study enlarges our perceptions comprehensively about the physiological and molecular mechanisms implicated in the YPR015C overexpressing strain's tolerance under furfural stress. Construction illustration of the recombinant plasmid. a) pUG6-TEF1p-YPR015C, b) integration diagram of the recombinant plasmid pUG6-TEF1p-YPR into the chromosomal DNA of Saccharomyces cerevisiae.

Adaptive laboratory evolution boosts Yarrowia lipolytica tolerance to vanillic acid

Microbial tolerance to lignocellulose-derived inhibitors, such as aromatic acids, is critical for the economical production of biofuels and biochemicals. Here, adaptive laboratory evolution was applied to improve the tolerance of Yarrowia lipolytica to a representative aromatic acid inhibitor vanillic acid. The transcriptome profiling of evolved strain suggested that the tolerance could be related to the up-regulation of RNA processing and multidrug transporting pathways. Further analysis by reverse engineering confirmed that the amplification of YALI0_F13475g coding for transcriptional coactivator and YALI0_E25201g coding for multidrug transporter conferred tolerance not only to vanillic acid but also towards ferulic acid, p-coumaric acid, p-hydroxybenzoic acid and syringic acid. These findings suggested that regulation of RNA processing and multidrug transporting pathways may be important for enhanced aromatic acid tolerance in Y. lipolytica. This study provides valuable genetic information for robust strain construction for lignocellulosic biorefinery.

Utilization of lignocellulosic biofuel conversion residue by diverse microorganisms

BackgroundLignocellulosic conversion residue (LCR) is the material remaining after deconstructed lignocellulosic biomass is subjected to microbial fermentation and treated to remove the biofuel. Technoeconomic analyses of biofuel refineries have shown that further microbial processing of this LCR into other bioproducts may help offset the costs of biofuel generation. Identifying organisms able to metabolize LCR is an important first step for harnessing the full chemical and economic potential of this material. In this study, we investigated the aerobic LCR utilization capabilities of 71 Streptomyces and 163 yeast species that could be engineered to produce valuable bioproducts. The LCR utilization by these individual microbes was compared to that of an aerobic mixed microbial consortium derived from a wastewater treatment plant as representative of a consortium with the highest potential for degrading the LCR components and a source of genetic material for future engineering efforts.ResultsWe analyzed several batches of a model LCR by chemical oxygen demand (COD) and chromatography-based assays and determined that the major components of LCR were oligomeric and monomeric sugars and other organic compounds. Many of the Streptomyces and yeast species tested were able to grow in LCR, with some individual microbes capable of utilizing over 40% of the soluble COD. For comparison, the maximum total soluble COD utilized by the mixed microbial consortium was about 70%. This represents an upper limit on how much of the LCR could be valorized by engineered Streptomyces or yeasts into bioproducts. To investigate the utilization of specific components in LCR and have a defined media for future experiments, we developed a synthetic conversion residue (SynCR) to mimic our model LCR and used it to show lignocellulose-derived inhibitors (LDIs) had little effect on the ability of the Streptomyces species to metabolize SynCR.ConclusionsWe found that LCR is rich in carbon sources for microbial utilization and has vitamins, minerals, amino acids and other trace metabolites necessary to support growth. Testing diverse collections of Streptomyces and yeast species confirmed that these microorganisms were capable of growth on LCR and revealed a phylogenetic correlation between those able to best utilize LCR. Identification and quantification of the components of LCR enabled us to develop a synthetic LCR (SynCR) that will be a useful tool for examining how individual components of LCR contribute to microbial growth and as a substrate for future engineering efforts to use these microorganisms to generate valuable bioproducts.

Formate Dehydrogenase Improves the Resistance to Formic Acid and Acetic Acid Simultaneously in Saccharomyces cerevisiae.

Bioethanol from lignocellulosic biomass is a promising and sustainable strategy to meet the energy demand and to be carbon neutral. Nevertheless, the damage of lignocellulose-derived inhibitors to microorganisms is still the main bottleneck. Developing robust strains is critical for lignocellulosic ethanol production. An evolved strain with a stronger tolerance to formate and acetate was obtained after adaptive laboratory evolution (ALE) in the formate. Transcriptional analysis was conducted to reveal the possible resistance mechanisms to weak acids, and fdh coding for formate dehydrogenase was selected as the target to verify whether it was related to resistance enhancement in Saccharomyces cerevisiae F3. Engineered S. cerevisiae FA with fdh overexpression exhibited boosted tolerance to both formate and acetate, but the resistance mechanism to formate and acetate was different. When formate exists, it breaks down by formate dehydrogenase into carbon dioxide (CO2) to relieve its inhibition. When there was acetate without formate, FDH1 converted CO2 from glucose fermentation to formate and ATP and enhanced cell viability. Together, fdh overexpression alone can improve the tolerance to both formate and acetate with a higher cell viability and ATP, which provides a novel strategy for robustness strain construction to produce lignocellulosic ethanol.

Establishment of Kluyveromyces marxianus as a Microbial Cell Factory for Lignocellulosic Processes: Production of High Value Furan Derivatives.

The establishment of lignocellulosic biorefineries is dependent on microorganisms being able to cope with the stressful conditions resulting from the release of inhibitory compounds during biomass processing. The yeast Kluyveromyces marxianus has been explored as an alternative microbial factory due to its thermotolerance and ability to natively metabolize xylose. The lignocellulose-derived inhibitors furfural and 5-hydroxymethylfurfural (HMF) are considered promising building-block platforms that can be converted into a wide variety of high-value derivatives. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. The potential of this yeast to reduce furfural and HMF at high inhibitory loads was disclosed and characterized. Our results associated HMF reduction with NADPH while furfural-reducing activity was higher with NADH. In addition, furans’ inhibitory effect was higher when combined with xylose consumption. The furan derivatives produced by K. marxianus in different conditions were identified. Furthermore, one selected isolate was efficiently used as a whole-cell biocatalyst to convert furfural and HMF into their derivatives, furfuryl alcohol and 2,5-bis(hydroxymethyl)furan (BHMF), with high yields and productivities. These results validate K. marxianus as a promising microbial platform in lignocellulosic biorefineries.

Lipid production by Mortierella isabellina from pretreated corn cobs and effect of lignocellulose derived inhibitors on growth and lipid synthesis

High production costs of microbial lipids can be reduced by using available and cheap lignocellulosic biomass. The present study optimizes the cultivation conditions of oleaginous fungus Mortierella isabellina and lipid production in corn cobs hydrolysate regarding initial carbon to nitrogen ratio (C:N) in the growth medium, a pretreatment method and, inoculum type. Additionally, we studied the effect of the most abundant lignocellulose-derived inhibitors on lipid synthesis and fungus growth. Low C:N ratios from 5 to 25 mol mol−1 stimulated the fungus growth, while C:N ratios above 50 mol mol−1 gave rise to lipid accumulation. Corn cobs were subjected to dilute acid and alkali pretreatment, using 0.369 g of sulphuric acid and 0.16 g of sodium hydroxide per gram of lignocellulosic biomass, respectively. Growth media based on hydrolysate of alkaline pretreated corn cobs has proved suitable for growth and lipid production. Lipid yield and lipid concentration was 0.13 g g−1 and 8.40 g L−1, respectively. The most toxic inhibitors generated during pretreatment were phenolics, followed by furan aldehydes and aliphatic acids. Using vegetative mycelium instead of spores as inoculum helped cells to overcome the inhibition effect by 5-hydroxymethylfurfural and furan.

Enhanced ethanol production from lignocellulosic hydrolysates by Thermoanaerobacterium aotearoense SCUT27/ΔargR1864 with improved lignocellulose-derived inhibitors tolerance

The transcriptome data of Thermoanaerobacterium aotearoense SCUT27 (SCUT27) and Thermoanaerobacterium aotearoense SCUT27/ΔargR1864 (SCUT27/ΔargR1864) under xylose showed that ArgR1864 was the negative regulator for chaperonin synthesis. With argR1864 knockout, the mutant showed up-regulated expression of DnaK-DnaJ-GrpE system and GroEL-GroES chaperonin, which not only endowed the strain with the ability to scavenge the reactive oxygen species (ROS), but also gave the mutant the capability to maintain better cell growth, xylose consumption, acetic acid and ethanol production under the stress of various lignocellulose-derived inhibitors. Diluted acid pretreated soybean straw, sorghum stalk and wheat straw hydrolysates were used to evaluate the ethanol production of SCUT27/ΔargR1864 and results showed that the mutant definitely had advantages over SCUT27 for ethanol production under lignocellulosic hydrolysates, with the xylose consumption rate increased by 25.00%–44.83%, the ethanol production improved by 113.86%–366.36% and ethanol yield enhanced by 88.89%–208.33%.

Adaptive laboratory evolution of Yarrowia lipolytica improves ferulic acid tolerance

Yarrowia lipolytica strain is a promising cell factory for the conversion of lignocellulose to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to lignocellulose-derived inhibitors toxicity tolerance of Y. lipolytica are also required to achieve industrial application. Here, adaptive laboratory evolution was employed with increasing concentrations of ferulic acid. The adaptive laboratory evolution experiments led to evolve Y. lipolytica strain yl-XYL + *FA*4 with increased tolerance to ferulic acid as compared to the parental strain. Specifically, the evolved strain could tolerate 1.5 g/L ferulic acid, whereas 0.5 g/L ferulic acid could cause about 90% lethality of the parental strain. Transcriptome analysis of the evolved strain revealed several targets underlying toxicity tolerance enhancements. YALI0_E25201g, YALI0_F05984g, YALI0_B18854g, and YALI0_F16731g were among the highest upregulated genes, and the beneficial contributions of these genes were verified via reverse engineering. Recombinant strains with overexpressing each of these four genes obtained enhanced tolerance to ferulic acid as compared to the control strain. Fortunately, recombinant strains with overexpression of YALI0_E25201g, YALI0_B18854g, and YALI0_F16731g individually also obtained enhanced tolerance to vanillic acid. Overall, this work demonstrated a whole strain improvement cycle by "non-rational" metabolic engineering and presented new targets to modify Y. lipolytica for microbial lignocellulose valorization. KEY POINTS: • Adaptive evolution improved the ferulic acid tolerance of Yarrowia lipolytica • Transcriptome sequence was applied to analyze the ferulic acid tolerate strain • Three genes were demonstrated for both ferulic acid and vanillic acid tolerance.

Evaluating the Effect of Lignocellulose-Derived Microbial Inhibitors on the Growth and Lactic Acid Production by Bacillus coagulans Azu-10

Effective lactic acid (LA) production from lignocellulosic biomass materials is challenged by several limitations related to pentose sugar utilization, inhibitory compounds, and/or fermentation conditions. In this study, a newly isolated Bacillus coagulans strain Azu-10 was obtained and showed homofermentative LA production from xylose with optimal fermentation conditions at 50 °C and pH 7.0. Growth of strain Azu-10 and LA-fermentation efficiency were evaluated in the presence of various lignocellulose-derived inhibitors (furans, carboxylic acids, and phenols) at different concentrations. Furanic lignocellulosic-derived inhibitors were completely detoxified. The strain has exhibited high biomass, complete xylose consumption, and high LA production in the presence of 1.0–4.0 g/L furfural and 1.0–5.0 g/L of hydroxymethyl furfural, separately. Moreover, strain Azu-10 exhibited high LA production in the presence of 5.0–15.0 g/L acetic acid, 5.0 g/L of formic acid, and up to 7.0 g/L of levulinic acid, separately. Besides, for phenolic compounds, p-coumaric acid was most toxic at 1.0 g/L, while syringaldehyde or p-hydroxybenzaldehyde, and vanillin at 1.0 g/L did not inhibit LA fermentation. The present study provides an interesting potential candidate for the thermophilic LA fermentation from lignocellulose-derived substrates at the industrial biorefinery level.

Physiological characterization and transcriptome analysis of Pichia pastoris reveals its response to lignocellulose-derived inhibitors

The negative effects of lignocellulose-derived inhibitors such as acetic acid and furaldehydes on microbial metabolism constitute a significant drawback to the usage of biomass feedstocks for the production of fuels and chemicals. The yeast Pichia pastoris has shown a great biotechnological potential for producing heterologous proteins and renewable chemicals. Despite its relevance, the performance of P. pastoris in presence of lignocellulose-derived inhibitors remains unclear. In this work, our results show for the first time the dose-dependent response of P. pastoris to acetic acid, furaldehydes (HMF and furfural), and sugarcane biomass hydrolysate, both at physiological and transcriptional levels. The yeast was able to grow in synthetic media with up to 6 g.L−1 acetic acid, 1.75 g.L−1 furaldehydes or hydrolysate diluted to 10% (v/v). However, its metabolism was completely hindered in presence of hydrolysate diluted to 30% (v/v). Additionally, the yeast was capable to co-consume acetic acid and glucose. At the transcriptional level, P. pastoris response to lignocellulose-derived inhibitors relays on the up-regulation of genes related to transmembrane transport, oxidoreductase activities, RNA processing, and the repression of pathways related to biosynthetic processes and central carbon metabolism. These results demonstrate a polygenetic response that involves detoxification activities, and maintenance of energy and cellular homeostasis. In this context, ALD4, OYE3, QOR2, NTL100, YCT1, and PPR1 were identified as target genes to improve P. pastoris tolerance. Altogether, this work provides valuable insights into the P. pastoris stress tolerance, which can be useful to expand its use in different bioprocesses.

Effect of a Novel Alpha/Beta Hydrolase Domain Protein on Tolerance of K. marxianus to Lignocellulosic Biomass Derived Inhibitors.

The multiple inhibitors tolerance of microorganism is important in bioconversion of lignocellulosic biomass which is a promising renewable and sustainable source for biofuels and other chemicals. The disruption of an unknown α/β hydrolase, which was termed KmYME and located in mitochondria in this study, resulted in the yeast more susceptible to lignocellulose-derived inhibitors, particularly to acetic acid, furfural and 5-HMF. The KmYME disrupted strain lost more mitochondrial membrane potential, showed increased plasma membrane permeability, severer redox ratio imbalance, and increased ROS accumulation, compared with those of the non-disrupted strain in the presence of the same inhibitors. The intracellular concentration of ATP, NAD and NADP in the KmYME disrupted strain was decreased. However, disruption of KmYME did not result in a significant change of gene expression at the transcriptional level. The KmYME possessed esterase/thioesterase activity which was necessary for the resistance to inhibitors. In addition, KmYME was also required for the resistance to other stresses including ethanol, temperature, and osmotic pressure. Disruption of two possible homologous genes in S. cerevisiae also reduced its tolerance to inhibitors.

Exploring the Lignin Catabolism Potential of Soil-Derived Lignocellulolytic Microbial Consortia by a Gene-Centric Metagenomic Approach.

An exploration of the ligninolytic potential of lignocellulolytic microbial consortia can improve our understanding of the eco-enzymology of lignin conversion in nature. In this study, we aimed to detect enriched lignin-transforming enzymes on metagenomes from three soil-derived microbial consortia that were cultivated on "pre-digested" plant biomass (wheat straw, WS1-M; switchgrass, SG-M; and corn stover, CS-M). Of 60 selected enzyme-encoding genes putatively involved in lignin catabolism, 20 genes were significantly abundant in WS1-M, CS-M, and/or SG-M consortia compared with the initial forest soil inoculum metagenome (FS1). These genes could be involved in lignin oxidation (e.g., superoxide dismutases), oxidative stress responses (e.g., catalase/peroxidases), generation of protocatechuate (e.g., vanAB genes), catabolism of gentisate, catechol and 3-phenylpropionic acid (e.g., gentisate 1,2-dioxygenases, muconate cycloisomerases, and hcaAB genes), the beta-ketoadipate pathway (e.g., pcaIJ genes), and tolerance to lignocellulose-derived inhibitors (e.g., thymidylate synthases). The taxonomic affiliation of 22 selected lignin-transforming enzymes from WS1-M and CS-M consortia metagenomes revealed that Pseudomonadaceae, Alcaligenaceae, Sphingomonadaceae, Caulobacteraceae, Comamonadaceae, and Xanthomonadaceae are the key bacterial families in the catabolism of lignin. A predictive "model" was sketched out, where each microbial population has the potential to metabolize an array of aromatic compounds through different pathways, suggesting that lignin catabolism can follow a "task division" strategy. Here, we have established an association between functions and taxonomy, allowing a better understanding of lignin transformations in soil-derived lignocellulolytic microbial consortia, and pinpointing some bacterial taxa and catabolic genes as ligninolytic trait-markers.

Multiomic Fermentation Using Chemically Defined Synthetic Hydrolyzates Revealed Multiple Effects of Lignocellulose-Derived Inhibitors on Cell Physiology and Xylose Utilization in Zymomonas mobilis.

Utilization of both C5 and C6 sugars to produce biofuels and bioproducts is a key goal for the development of integrated lignocellulosic biorefineries. Previously we found that although engineered Zymomonas mobilis 2032 was able to ferment glucose to ethanol when fermenting highly concentrated hydrolyzates such as 9% glucan-loading AFEX-pretreated corn stover hydrolyzate (9% ACSH), xylose conversion after glucose depletion was greatly impaired. We hypothesized that impaired xylose conversion was caused by lignocellulose-derived inhibitors (LDIs) in hydrolyzates. To investigate the effects of LDIs on the cellular physiology of Z. mobilis during fermentation of hydrolyzates, including impacts on xylose utilization, we generated synthetic hydrolyzates (SynHs) that contained nutrients and LDIs at concentrations found in 9% ACSH. Comparative fermentations of Z. mobilis 2032 using SynH with or without LDIs were performed, and samples were collected for end product, transcriptomic, metabolomic, and proteomic analyses. Several LDI-specific effects were observed at various timepoints during fermentation including upregulation of sulfur assimilation and cysteine biosynthesis, upregulation of RND family efflux pump systems (ZMO0282-0285) and ZMO1429-1432, downregulation of a Type I secretion system (ZMO0252-0255), depletion of reduced glutathione, and intracellular accumulation of mannose-1P and mannose-6P. Furthermore, when grown in SynH containing LDIs, Z. mobilis 2032 only metabolized ∼50% of xylose, compared to ∼80% in SynH without LDIs, recapitulating the poor xylose utilization observed in 9% ACSH. Our metabolomic data suggest that the overall flux of xylose metabolism is reduced in the presence of LDIs. However, the expression of most genes involved in glucose and xylose assimilation was not affected by LDIs, nor did we observe blocks in glucose and xylose metabolic pathways. Accumulations of intracellular xylitol and xylonic acid was observed in both SynH with and without LDIs, which decreased overall xylose-to-ethanol conversion efficiency. Our results suggest that xylose metabolism in Z. mobilis 2032 may not be able to support the cellular demands of LDI mitigation and detoxification during fermentation of highly concentrated lignocellulosic hydrolyzates with elevated levels of LDIs. Together, our findings identify several cellular responses to LDIs and possible causes of impaired xylose conversion that will enable future strain engineering of Z. mobilis.

Insoluble solids at high concentrations repress yeast\u2019s response against stress and increase intracellular ROS levels

Lignocellulosic ethanol production requires high substrate concentrations for its cost-competitiveness. This implies the presence of high concentrations of insoluble solids (IS) at the initial stages of the process, which may limit the fermentation performance of the corresponding microorganism. The presence of 40–60% IS (w/w) resulted in lower glucose consumption rates and reduced ethanol volumetric productivities of Saccharomyces cerevisiae F12. Yeast cells exposed to IS exhibited a wrinkled cell surface and a reduced mean cell size due to cavity formation. In addition, the intracellular levels of reactive oxygen species (ROS) increased up to 40%. These ROS levels increased up to 70% when both lignocellulose-derived inhibitors and IS were simultaneously present. The general stress response mechanisms (e.g. DDR2, TPS1 or ZWF1 genes, trehalose and glycogen biosynthesis, and DNA repair mechanisms) were found repressed, and ROS formation could not be counteracted by the induction of the genes involved in repairing the oxidative damage such as glutathione, thioredoxin and methionine scavenging systems (e.g. CTA1, GRX4, MXR1, and TSA1; and the repression of cell cycle progression, CLN3). Overall, these results clearly show the role of IS as an important microbial stress factor that affect yeast cells at physical, physiological, and molecular levels.

Chicken feather and wheat straw hydrolysate for direct utilization in biobutanol production

Lignocellulosic material possesses a source of fermentable saccharides for biofuel production, however it must be supplemented with other nutrients for microbial growth. We have investigated the use of chicken feather as a source of amino acids and peptides. Alkaline hydrolysis of feather under mild conditions (80 °C, 0.6% NaOH) provided a highly solubilized preparation of assimilable nitrogen. Similar conditions were applicable for pre-treatment of wheat straw. The concentration of lignocellulose-derived inhibitors was low so washing or detoxification steps were unnecessary. Therefore, hydrolysis of both materials was carried out simultaneously, resulting in a cultivation medium that was suitable for direct use in biobutanol production. Thirteen strains of solventogenic clostridia were tested for their ability to produce butanol on this mixed hydrolysate; strains of Clostridium beijerinckii and Clostridium saccharoperbutylacetonicum produced the highest butanol concentrations. The proposed process of simultaneous hydrolysis followed by direct inoculation was then demonstrated by experimental production of butanol in a laboratory bioreactor, using C. beijerinckii strain NCIMB 8052 and producing 4.6 g/L of butanol. Although process optimization is still required, we have shown that feather and wheat straw could be co-processed, forming a convenient mixed substrate for biobutanol production.