Inhibitors In Lignocellulosic Hydrolysates Research Articles

Dark Fermentation of Arundo donax: Characterization of the Anaerobic Microbial Consortium

The dark fermentation of lignocellulose hydrolysates is a promising process for the production of hydrogen from renewable sources. Nevertheless, hydrogen yields are often lower than those obtained from other carbohydrate sources due to the presence of microbial growth inhibitors in lignocellulose hydrolysates. In this study, a microbial consortium for the production of hydrogen by dark fermentation has been obtained from a wild methanogenic sludge by means of thermal treatments. The consortium has been initially acclimated to a glucose-based medium and then used as inoculum for the fermentation of Arundo donax hydrolysates. Hydrogen yields obtained from fermentation of A. donax hydrolysates were lower than those obtained from glucose fermentation using the same inoculum (0.30 ± 0.05 versus 1.11 ± 0.06 mol of H2 per mol of glucose equivalents). The hydrogen-producing bacteria belonged mainly to the Enterobacteriaceae family in cultures growing on glucose and to Clostridium in those growing on A. donax hydrolysate. In the latter cultures, Lactobacillus outcompeted Enterobacteriaceae, although Clostridium also increased. Lactobacillus outgrowth could account for the lower yields observed in cultures growing on A. donax hydrolysate.

CRISPRi screen highlights chromatin regulation to be involved in formic acid tolerance in Saccharomyces cerevisiae

Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in Saccharomyces cerevisiae. To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Cysteine supplementation enhanced inhibitor tolerance of Zymomonas mobilis for economic lignocellulosic bioethanol production

Inhibitors in lignocellulosic hydrolysates are toxic to Zymomonas mobilis and reduce its bioethanol production. This study revealed cysteine supplementation enhanced furfural tolerance in Z. mobilis with a 2-fold biomass increase. Transcriptomic study illustrated that cysteine biosynthesis pathway was down-regulated while cysteine catabolism was up-regulated with cysteine supplementation. Mutants for genes involved in cysteine metabolism were constructed, and metabolites in cysteine metabolic pathway including methionine, glutathione, NaHS, glutamate, and pyruvate were supplemented into media. Cysteine supplementation boosted glutathione synthesis or H2S release effectively in Z. mobilis leading to the reduced accumulation of reactive oxygen species (ROS) induced by furfural, while pyruvate and glutamate produced in the H2S generation pathway promoted cell growth by serving as the carbon or nitrogen source. Finally, cysteine supplementation was confirmed to enhance Z. mobilis tolerance against ethanol, acetate, and corncob hydrolysate with an enhanced ethanol productivity from 0.38 to 0.55 g−1∙L−1∙h−1.

Biotransformation of 5-hydroxymethylfurfural and furfural with bacteria of bacillus genus

Furfural and 5-hydroxymethylfurfural (5-HMF) are considered to be the most toxic compounds among fermentation inhibitors in lignocellulosic hydrolysates. These compounds raise severe problems for the bioproduction of fuels and chemicals. In this study, we describe the detoxification of furfural and 5-HMF using bacterial isolates from residues of sugarcane bagasse, identified as Bacillus pasteuri, Bacillus toyonensis, Bacillus megaterium, Bacillus cereus, Bacillus subtilis, and Bacillus spp. by 16S rDNA sequencing. Under aerobic culture conditions, 2-furoic acid and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) were produced as the main degradation product in the detoxification of furfural and 5-HMF, respectively. In both cases these acids were produced in greater concentrations when bacteria were incubated in nutrient broth. B. toyonensis in nutrient broth with 20 mM of furfural after 120 h produced the highest amount of 2-furoic acid. A concentration of 15.2 mM was achieved using B. toyonensis followed by B. cereus with a concentration of 13.1 mM 2-Furoic acid production for other strains did not exceed 6.8 mM. B. toyonensis was the strain with highest degradation capacity of 5-HMF, following the oxidation pathway to obtain HMFCA and 2,5-furan dicarboxylic acid (FDCA) which rose to a final concentration of 21.3 mM and 1.5 mM, respectively, in a nutrient broth with 25 mM of 5-HMF at 120 h of fermentation. B. toyonensis is a useful strain to detoxify lignocellulosic residues and to produce furans with a high added value. • First comparative study of strains belonging to genus Bacillus in the biotransformation of furans. • First study on B. pasteuri and B. toyonensis used in the biodegradation of furfural and 5-HMF. • The novel B. toyonensis is a native strain that is highly tolerant to 5-HMF/furfural (>60 mM) with the ability to produce 2-furoic acid and HMFCA in nutrient broths with yields greater than 76 and 85%, respectively.

Engineering prokaryotic regulator IrrE to enhance stress tolerance in budding yeast

BackgroundStress tolerance is one of the important desired microbial traits for industrial bioprocesses, and global regulatory protein engineering is an efficient approach to improve strain tolerance. In our study, IrrE, a global regulatory protein from the prokaryotic organism Deinococcus radiodurans, was engineered to confer yeast improved tolerance to the inhibitors in lignocellulose hydrolysates or high temperatures.ResultsThree IrrE mutations were developed through directed evolution, and the expression of these mutants could improve the yeast fermentation rate by threefold or more in the presence of multiple inhibitors. Subsequently, the tolerance to multiple inhibitors of single-site mutants based on the mutations from the variants were then evaluated, and 11 mutants, including L65P, I103T, E119V, L160F, P162S, M169V, V204A, R244G, Base 824 Deletion, V299A, and A300V were identified to be critical for the improved representative inhibitors, i.e., furfural, acetic acid and phenol (FAP) tolerance. Further studies indicated that IrrE caused genome-wide transcriptional perturbation in yeast, and the mutant I24 led to the rapid growth of Saccharomyces cerevisiae by primarily regulating the transcription level of transcription activators/factors, protecting the intracellular environment and enhancing the antioxidant capacity under inhibitor environments, which reflected IrrE plasticity. Meanwhile, we observed that the expression of the wild-type or mutant IrrE could also protect Saccharomyces cerevisiae from the damage caused by thermal stress. The recombinant yeast strains were able to grow with glucose at 42 ℃.ConclusionsIrrE from Deinococcus radiodurans can be engineered as a tolerance-enhancer for Saccharomyces cerevisiae. Systematic research on the regulatory model and mechanism of a prokaryotic global regulatory factor IrrE to increase yeast tolerance provided valuable insights for the improvements in microbial tolerance to complex industrial stress conditions.

A C6/C5 co‐fermentingSaccharomyces cerevisiaestrain with the alleviation of antagonism between xylose utilization and robustness

AbstractDuring second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such asSaccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermentingS. cerevisiaestrain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g−1 h−1) in high‐level mixed sugars (80 g L−1glucose and 40 g L−1xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g−1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.

Furfural and 5-(Hydroxymethyl) furfural Tolerance Candida strains in Bioethanol Fermentation

The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl)furfural (HMF) and furfural are specifically well known. This study investigated the furfural and 5-HMF tolerance in Candida strains, which could be used for the development of advanced generation bioethanol processes. The 10 isolates of Candida were selected based on the cell growth and bioethanol production on YPD medium containing several concentrations of furfural and 5-HMF by using spectrophotometer and HPLC. Candida parapsilosis Y80 could grow and produce bioethanol in the medium that contains furfural and 5-HMF with a concentration of 60 mM and 40 mM, respectively. Based on the results, C. parapsilosis Y80 has potential activity in the development of bioethanol fermentation.

Overexpression of delta-12 desaturase in the yeast Schwanniomyces occidentalis enhances the production of linoleic acid

The oleaginous yeast Schwanniomyces occidentalis was previously isolated because of its excellent suitability to convert lignocellulosic hydrolysates into triacyl glycerides: it is able to use a broad range of sugars and is able to tolerate high concentrations of lignocellulosic hydrolysate inhibitors. Compared to other oleaginous yeasts S. occidentalis however produces a low content of unsaturated fatty acids. We show here that the linoleic acid content can be significantly improved by (over)expression Δ12-desaturases derived from S. occidentalis and Fusarium moniliforme. Expression was stable for the homologous expression but decreased during heterologous expression. Both homologous and heterologous expression of mCherry-Δ12-desaturase led to a 4-fold increase in linoleic acid from 0.02 g/g biomass to 0.08 g/g biomass resulting in the production of 2.23 g/L and 2.05 g/L of linoleic acid.

Omics-based analyses revealed metabolic responses of Clostridium acetobutylicum to lignocellulose-derived inhibitors furfural, formic acid and phenol stress for butanol fermentation

BackgroundClostridium acetobutylicum is a model fermentative anaerobe for consolidated bioprocessing of lignocellulose hydrolysates into acetone–butanol–ethanol (ABE). However, the main inhibitors (acids, furans and phenols) ubiquitous in lignocellulose hydrolysates strictly limit the conversion efficiency. Thus, it is essential to understand the underlying mechanisms of lignocellulose hydrolysate inhibitors to identify key industrial bottlenecks that undermine efficient biofuel production. The recently developed omics strategy for intracellular metabolites and protein quantification now allow for the in-depth mapping of strain metabolism and thereby enable the resolution of the underlying mechanisms.ResultsThe toxicity of the main inhibitors in lignocellulose hydrolysates against C. acetobutylicum and ABE production was systematically investigated, and the changes in intracellular metabolism were analyzed by metabolomics and proteomics. The toxicity of the main lignocellulose hydrolysate inhibitors at the same dose was ranked as follows: formic acid > phenol > furfural. Metabolomic analysis based on weighted gene coexpression network analysis (WGCNA) revealed that the three inhibitors triggered the stringent response of C. acetobutylicum. Proteomic analysis based on peptide mass spectrometry (MS) supported the above results and provided more comprehensive conclusions. Under the stress of three inhibitors, the metabolites and key enzymes/proteins involved in glycolysis, reductive tricarboxylic acid (TCA) cycle, acetone–butanol synthesis and redox metabolism were lower than those in the control group. Moreover, proteins involved in gluconeogenesis, the oxidative TCA cycle, thiol peroxidase (TPX) for oxidative stress were significantly upregulated, indicating that inhibitor stress induced the stress response and metabolic regulation. In addition, the three inhibitors also showed stress specificity related to fatty acid synthesis, ATP synthesis, nucleic acid metabolism, nicotinic acid metabolism, cell wall synthesis, spore synthesis and flagellum synthesis and so on.ConclusionsIntegrated omics platforms provide insight into the cellular responses of C. acetobutylicum to cytotoxic inhibitors released during the deconstruction of lignocellulose. This insight allows us to fully improve the strain to adapt to a challenging culture environment, which will prove critical to the industrial efficacy of C. acetobutylicum.

Genomic and transcriptomic analysis of a coniferyl aldehyde-resistant Saccharomyces cerevisiae strain obtained by evolutionary engineering.

Phenolic inhibitors in lignocellulosic hydrolysates interfere with the performance of fermenting microorganisms. Among these, coniferyl aldehyde is one of the most toxic inhibitors. In this study, genetically stable Saccharomyces cerevisiae mutants with high coniferyl aldehyde resistance were successfully obtained for the first time by using an evolutionary engineering strategy, based on the systematic application of increasing coniferyl aldehyde stress in batch cultures. Among the selected coniferyl aldehyde-resistant mutants, the highly resistant strain called BH13 was also cross-resistant to other phenolic inhibitors, vanillin, ferulic acid and 4-hydroxybenzaldehyde. In the presence of 1.2 mM coniferyl aldehyde stress, BH13 had a significantly reduced lag phase, which was less than 3 h and only about 25% of that of the reference strain and converted coniferyl aldehyde faster. Additionally, there was no reduction in its growth rate, either. Comparative transcriptomic analysis of a highly coniferyl aldehyde-resistant mutant revealed upregulation of the genes involved in energy pathways, response to oxidative stress and oxidoreductase activity in the mutant strain BH13, already under non-stress conditions. Transcripts associated with pleiotropic drug resistance were also identified as upregulated. Genome re-sequencing data generally supported transcriptomic results and identified gene targets that may have a potential role in coniferyl aldehyde resistance.

New technologies provide more metabolic engineering strategies for bioethanol production in Zymomonas mobilis.

Bioethanol has been considered as a potentially renewable energy source, and metabolic engineering plays an important role in the production of biofuels. As an efficient ethanol-producing bacterium, Zymomonas mobilis has garnered special attention due to its high sugar uptake, ethanol yield, and tolerance. Different metabolic engineering strategies have been used to establish new metabolic pathways for Z. mobilis to broaden its substrate range, remove competing pathways, and enhance its tolerance to ethanol and lignocellulosic hydrolysate inhibitors. Recent advances in omics technology, computational modeling and simulation, system biology, and synthetic biology contribute to the efficient re-design and manipulation of microbes via metabolic engineering at the whole-cell level. In this review, we summarize the progress of some new technologies used for metabolic engineering to improve bioethanol production and tolerance in Z. mobilis. Some successful examples of metabolic engineering used to develop strains for ethanol production are described in detail. Lastly, some important strategies for future metabolic engineering efforts are also highlighted.

Polygenic analysis and targeted improvement of the complex trait of high acetic acid tolerance in the yeast Saccharomyces cerevisiae.

BackgroundAcetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of second-generation bioethanol. Although several genes have been identified in laboratory yeast strains that are required for tolerance to acetic acid, the genetic basis of the high acetic acid tolerance naturally present in some Saccharomyces cerevisiae strains is unknown. Identification of its polygenic basis may allow improvement of acetic acid tolerance in yeast strains used for second-generation bioethanol production by precise genome editing, minimizing the risk of negatively affecting other industrially important properties of the yeast.ResultsHaploid segregants of a strain with unusually high acetic acid tolerance and a reference industrial strain were used as superior and inferior parent strain, respectively. After crossing of the parent strains, QTL mapping using the SNP variant frequency determined by pooled-segregant whole-genome sequence analysis revealed two major QTLs. All F1 segregants were then submitted to multiple rounds of random inbreeding and the superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance in the QTL mapping with the F7 segregants of a major F1 QTL, in which we identified HAA1, a known regulator of high acetic acid tolerance, as a true causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, and a previously identified component, VMA7, were identified as causative alleles in the second major F1 QTL and in three newly appearing F7 QTLs, respectively. The superior HAA1 allele contained a unique single point mutation that significantly improved acetic acid tolerance under industrially relevant conditions when inserted into an industrial yeast strain for second-generation bioethanol production.ConclusionsThis work reveals the polygenic basis of high acetic acid tolerance in S. cerevisiae in unprecedented detail. It also shows for the first time that a single strain can harbor different sets of causative genes able to establish the same polygenic trait. The superior alleles identified can be used successfully for improvement of acetic acid tolerance in industrial yeast strains.

Short-term adaptation during propagation improves the performance of xylose-fermenting Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation.

BackgroundInhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation.ResultsThe presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield.ConclusionsShort-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.

Identification of benzoquinones in pretreated lignocellulosic feedstocks and inhibitory effects on yeast.

Pretreatment of lignocellulosic biomass under acidic conditions gives rise to by-products that inhibit fermenting microorganisms. An analytical procedure for identification of p-benzoquinone (BQ) and 2,6-dimethoxybenzoquinone (DMBQ) in pretreated biomass was developed, and the inhibitory effects of BQ and DMBQ on the yeast Saccharomyces cerevisiae were assessed. The benzoquinones were analyzed using ultra-high performance liquid chromatography-electrospray ionization-triple quadrupole-mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Pretreatment liquids examined with regard to the presence of BQ and DMBQ originated from six different lignocellulosic feedstocks covering agricultural residues, hardwood, and softwood, and were produced through impregnation with sulfuric acid or sulfur dioxide at varying pretreatment temperature (165–204 °C) and residence time (6–20 min). BQ was detected in all six pretreatment liquids in concentrations ranging up to 6 mg/l, while DMBQ was detected in four pretreatment liquids in concentrations ranging up to 0.5 mg/l. The result indicates that benzoquinones are ubiquitous as by-products of acid pretreatment of lignocellulose, regardless of feedstock and pretreatment conditions. Fermentation experiments with BQ and DMBQ covered the concentration ranges 2 mg/l to 1 g/l and 20 mg/l to 1 g/l, respectively. Even the lowest BQ concentration tested (2 mg/l) was strongly inhibitory to yeast, while 20 mg/l DMBQ gave a slight negative effect on ethanol formation. This work shows that benzoquinones should be regarded as potent and widespread inhibitors in lignocellulosic hydrolysates, and that they warrant attention besides more well-studied inhibitory substances, such as aliphatic carboxylic acids, phenols, and furan aldehydes.

Determinants of tolerance to inhibitors in hardwood spent sulfite liquor in genome shuffled Pachysolen tannophilus strains.

Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to the HW SSL UV mutant (70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70-0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to the acetic acid UV mutant, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp-1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates.

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme–inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors.

BackgroundLignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels. A significant technical challenge to using lignocellulose is the presence of microbial growth inhibitors generated during pretreatment processes. Triacylglycerols (TAGs) are potential precursors for lipid-based biofuel production. Rhodococcus opacus MITXM-61 is an oleaginous bacterium capable of producing large amounts of TAGs on high concentrations of glucose and xylose present in lignocellulosic hydrolysates. However, this strain is sensitive to ligonocellulose-derived inhibitors. To understand the toxic effects of the inhibitors in lignocellulosic hydrolysates, strain MITXM-61 was examined for tolerance toward the potential inhibitors and was subjected to adaptive evolution for the resistance to the inhibitors.ResultsWe investigated growth-inhibitory effects by potential lignocellulose-derived inhibitors of phenols (lignin, vanillin, 4-hydroxybenzaldehyde (4-HB), syringaldehyde), furans (furfural and 5-hydroxymethyl-2-furaldehyde), and organic acids (levulinic acid, formic acid, and acetic acid) on the growth and TAG production of strain MITXM-61. Phenols and furans exhibited potent inhibitory effects at a concentration of 1 g L−1, while organic acids had insignificant impacts at concentrations of up to 2 g L−1. In an attempt to improve the inhibitor tolerance of strain MITXM-61, we evaluated the adaptation of this strain to the potential inhibitors. Adapted mutants were generated on defined agar media containing lignin, 4-HB, and syringaldehyde. Strain MITXM-61SHL33 with improved multiple resistance of lignin, 4-HB, and syringaldehyde was constructed through adaptive evolution-based strategies. The evolved strain exhibited a two- to threefold increase in resistance to lignin, 4-HB, and syringaldehyde at 50% growth-inhibitory concentrations, compared to the parental strain. When grown in genuine lignocellulosic hydrolysates of corn stover, wheat straw, and hardwood containing growth inhibitors, strain MITXM-61SHL33 exhibited a markedly shortened lag phase in comparison with that of strain MITXM-61.ConclusionThis study provides important clues to overcome the negative effects of inhibitors in lignocellulosic hydrolysates on TAG production of R. opacus cells. The findings can contribute to significant progress in detoxified pretreatment of hydrolysates and development of more efficient strains for industrial TAG fermentations of R. opacus using lignocellulosic biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0258-3) contains supplementary material, which is available to authorized users.

Current Trends in Bioethanol Production by Saccharomyces cerevisiae: Substrate, Inhibitor Reduction, Growth Variables, Coculture, and Immobilization.

Bioethanol is one of the most commonly used biofuels in transportation sector to reduce greenhouse gases. S. cerevisiae is the most employed yeast for ethanol production at industrial level though ethanol is produced by an array of other yeasts, bacteria, and fungi. This paper reviews the current and nonmolecular trends in ethanol production using S. cerevisiae. Ethanol has been produced from wide range of substrates such as molasses, starch based substrate, sweet sorghum cane extract, lignocellulose, and other wastes. The inhibitors in lignocellulosic hydrolysates can be reduced by repeated sequential fermentation, treatment with reducing agents and activated charcoal, overliming, anion exchanger, evaporation, enzymatic treatment with peroxidase and laccase, in situ detoxification by fermenting microbes, and different extraction methods. Coculturing S. cerevisiae with other yeasts or microbes is targeted to optimize ethanol production, shorten fermentation time, and reduce process cost. Immobilization of yeast cells has been considered as potential alternative for enhancing ethanol productivity, because immobilizing yeasts reduce risk of contamination, make the separation of cell mass from the bulk liquid easy, retain stability of cell activities, minimize production costs, enable biocatalyst recycling, reduce fermentation time, and protect the cells from inhibitors. The effects of growth variables of the yeast and supplementation of external nitrogen sources on ethanol optimization are also reviewed.

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions.

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.