Lignocellulose-derived Sugars Research Articles

Simultaneous enhancement of lignin-derived inhibitor tolerance and lipid accumulation of oleaginous yeast Trichosporon cutaneum by adaptive evolution

Inhibitory compounds should be removed from the pretreated lignocellulose feedstock before fermentative production of microbial lipid by oleaginous yeast. Biodetoxification effectively degrades furan aldehydes and weak organic acids, but is less efficient against complex and various lignin-derived phenolic aldehydes. Residual phenolics in lignocellulose hydrolysates require a tolerant fermentation strain. This study adopted the long-term adaptive evolution combined with centrifugal enrichment in corn stover hydrolysate to simultaneously improve the phenolic inhibitors tolerance and lipid synthesis of Trichosporon cutaneum. The obtained mutant T. cutaneum MS28 showed significant improved performances of phenolic aldehydes tolerance and lipid accumulation. The final cellulosic lipid production of T. cutaneum MS28 utilizing corn stover reached 33.8 ± 0.1 g/L, approximately 6-fold higher than that of the starting strain. Transcriptional analysis suggested that T. cutaneum MS28 had more active lignocellulose-derived sugars metabolism and lipid synthesis shunts. The intracellular contents of lipid synthesis precursors including NADPH and acetyl-CoA increased 1.7–3.0 folds. This adaptive evolution in lignocellulose hydrolysate combined with centrifugal enrichment is an efficient tool to targeted enhancement of inhibitors tolerance and cellulosic lipid production of oleaginous yeast.

Modulated Arabinose Uptake and cAMP Signaling Synergistically Improve Glucose and Arabinose Consumption in Recombinant Yeast.

During the production of ethanol from lignocellulose-derived sugars, recombinant yeasts tend to utilize xylose and arabinose after glucose exhaustion. So far, many glucose-insensitive pentose transporters have been reported to counteract this phenomenon, but few studies have described intracellular factors. In this study, the combination of adaptive evolution, comparative genomics, and genetic complementation revealed that the hexokinase-deficient (Hxk0) arabinose-fermenting Saccharomyces cerevisiae requires the arabinose transporter variant Gal2-N376T and the mutations of guanine nucleotide exchange factor Cdc25 to overcome glucose restriction during arabinose assimilation. The results showed that the Hxk0 recombinant yeasts could lower the metabolic/physiological threshold of cell proliferation by downregulating the intracellular cAMP levels, resulting in smaller cells and increased arabinose assimilation under glucose restriction. In the medium containing 80 g/L glucose and 20 g/L arabinose, the evolved strain restoring the hexokinase activity completed fermentation at 22 h, compared to 24 h for the parental strain. Overall, the experimental results provide new insights into glucose repression of biorefinery yeasts.

Coproduction of single cell protein and lipid from lignocellulose derived carbohydrates and inorganic ammonia salt with soluble ammonia recycling

Co-production of single cell protein (SCP) and lipid from lignocellulose-derived carbohydrates and inorganic ammonia offers a promising alternative for poultry or aquaculture feeds. An engineered oleaginous yeast Trichosporon cutaneum MP11 showed great potential for producing SCP and lipid from wheat straw and ammonia sulfate with minimum nutrient input. Trichosporon cutaneum MP11 showed stronger SCP and lipid fermentability using dry acid pretreated and biodetoxified wheat straw than using pure sugars. The residual ammonium sulfate in fermentation broth was recycled up to five times, resulting in ∼70% of nitrogen fixation into SCP. The overall yield of SCP and lipid from lignocellulose-derived sugars was 0.15 g/g and 0.11 g/g, respectively. This translates to the production of one ton of SCP (0.56 ton) and lipid (0.44 ton) from 6.6 tons of wheat straw, or one ton of SCP and lipid containing yeast cells (dry) from 4.8 tons of wheat straw.

Development of a novel methanotrophic platform to produce ectoine from methane and lignocellulose-derived sugars

Methane is considered a promising carbon source in the field of biotechnology. Using methane as the carbon source for producing chemicals reduces manufacturing costs and tackles the issue of global warming. Like methane, lignocellulose biomass is also an inexpensive and ample carbon source, which could be used for industrial biomanufacturing. In this study, Methylotuvimicrobium alcaliphilum 20Z was genetically modified to simultaneously consume methane and lignocellulose-derived sugars (glucose and xylose) to produce ectoine, a native osmoprotectant. The xylose utilization pathway, comprising xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli, was first integrated into the chromosome of M. alcaliphilum 20Z. Further expression of the glucose utilization, consisting of glf (glucose facilitator) from Zymmonas mobilis, glk (endogenous glucokinase), and pgi (glucose 6-phosphate isomerase) from E. coli, made the engineered strain 20ZXG consume methane, xylose, and glucose concurrently. Cultivation on three substrates upregulated the Embden–Meyerhof–Parnas and ectoine biosynthesis pathways of 20Z. As a result, the ectoine content produced by 20ZXG grown on the three substrates was 1.7-fold enhanced compared with that obtained on methane solely. Furthermore, the knock-out of ectoine biosynthesis repressor (ectR1) led to an output of 37.93 ± 3.27 mg/g-DCW, which was the highest quantity of ectoine produced after 48 h of cultivation in the presence of three substrates and was 2.3-fold higher than the initial output.

Increased Butyrate Production in Clostridium saccharoperbutylacetonicum from Lignocellulose-Derived Sugars.

Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase (pta) and butyraldehyde dehydrogenase (bld) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis (bcs) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and β, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_PbgaL]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L-1 and 14.3 g·L-1, respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).

Co-utilization of multiple lignocellulose-derived sugars and inhibitors by the robust mutant Kluyveromyces marxianus through adaptive laboratory evolution

Economical bioethanol production using lignocellulosic biomass requires robust microorganisms with strong inhibitor resistance, thermotolerance and multi-carbons uptake. To obtain a desirable microbial platform, Kluyveromyces marxianus 1727 was employed to conduct adaptive laboratory evolution (ALE) in mixed inhibitors of increasing concentrations about 90 days, a robust mutant K. marxianus 1727-5 was gained with enhanced inhibitors tolerance and improved utilization of the inhibitors. The uptake rate of high concentration acetate, formate, furfural, vanillin increased by 25%, 32%, 65% and 48%, respectively. Besides, K. marxianus 1727-5 had great competence in utilizing glucose, xylose, arabinose, acetate, formate, furfural, vanillin in non-detoxified hydrolysates simultaneously at 40 °C with 80% sugars used and produced 14.2 g/L ethanol with the yield 0.46 g/g. It’s a breakthrough in lignocellulosic biomass bioconversion which multiple carbon sources were used with high product yield. To find the relevant mechanism, genome resequencing was carried and then SNPs correlate to the phenotypes were revealed with potential targets for gene modification in K. marxianus. This study provides a new choice for the construction of robust strains to facilitate utilization of lignocellulosic biomass.

Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition

Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l–1 glucose–xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.

A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose.

SummaryHigh‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50.

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr-1 . The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr-1 , strain QU 50 produced 42.6 g L-1 of l-LA with a yield of 0.912 g g-1 -consumed sugars, LA yield of 0.655 g g-1 based on mixed sugar-loaded, and a productivity of 4.31 g L-1 hr-1 from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L-1 with LA yield of 0.944 g g-1 -consumed sugar and LA productivity of 6.49 ± 0.357 g L-1 hr-1 . Besides this, a dramatic increase in LA yield of 0.927 g g-1 based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.

Co-fermentation of a sugar mixture for microbial lipid production in a two-stage fermentation mode under non-sterile conditions

The economical production of biodiesel from lignocellulose requires an effective co-fermentation of lignocellulose-derived sugars, such as glucose and xylose.

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose-Derived Sugars.

Currently, microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome-scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole-genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations' involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next-generation biofuels.

Adaptive evolution of Gluconobacter oxydans accelerates the conversion rate of non-glucose sugars derived from lignocellulose biomass

Gluconobacter oxydans is capable of oxidizing various lignocellulose derived sugars into the corresponding sugar acids including glucose, xylose, arabinose, galactose and mannose, but simultaneous utilization of these sugars is difficult. This study attempted an adaptive evolution of G. oxydans by alternate transfer in inhibitors containing hydrolysate and inhibitors free hydrolysate for intensifying sugars simultaneous utilization. After 420 days’ continuous culture, the conversion rate of all non-glucose sugars significantly improved by several folds and achieved complete conversion of lignocellulose-derived sugars to the corresponding sugar acids. The significant up-regulation of mGDH gene in the adapted G. oxydans strain (more than 40-fold greater than the parental) was considered as the decisive factor for the improvement of strain performance. This evolution adaptation strategy also could be used to accelerate robust sugars utilization for other fermented strains in lignocellulose biorefinery.

Chromosomal integration of aldo-keto-reductase and short-chain dehydrogenase/reductase genes in Clostridium beijerinckii NCIMB 8052 enhanced tolerance to lignocellulose-derived microbial inhibitory compounds

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.

Engineering a Synthetic, Catabolically Orthogonal Coculture System for Enhanced Conversion of Lignocellulose-Derived Sugars to Ethanol.

Fermentation of lignocellulosic sugar mixtures is often suboptimal due to inefficient xylose catabolism and sequential sugar utilization caused by carbon catabolite repression. Unlike in conventional applications employing a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting the unique community features, including modularity, division of labor, and facile tunability. A series of synthetic, catabolically orthogonal coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W or ethanologenic LY180. Net catabolic activities were effectively balanced by simple tuning of the inoculum ratio between specialist strains, which enabled coutilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) for both culture systems in simple batch fermentations. The engineered ethanologenic cocultures achieved ethanol titer (46 g L-1), productivity (488 mg L-1 h-1), and yield (∼90% of theoretical maximum), which were all significantly increased compared to LY180 monocultures.

Sustainable production of glutathione from lignocellulose-derived sugars using engineered Saccharomyces cerevisiae.

Glutathione has diverse physiological functions, and therefore, the demand for it has increased recently. Currently, industrial mass production of glutathione is performed from D-glucose via fermentation by the budding yeast Saccharomyces cerevisiae. However, use of D-glucose often competes with demands for various other industries, leading to high production costs. To affordably produce glutathione, we aimed to produce high amounts of glutathione from D-glucose and D-xylose, which are the main constituents of lignocellulosic biomass pre-treated with acids. Genetically engineered S. cerevisiae strains that can produce high amounts of glutathione and assimilate D-xylose were constructed and cultured in media containing D-xylose. Among these recombinant strains, a S. cerevisiae GCI (XR/XDH/XK) strain over-expressing γ-glutamylcysteine synthetase, glutathione synthetase, D-xylose reductase, xylitol dehydrogenase, and xylulokinase genes successfully consumed D-xylose in the medium and produced the highest amount of glutathione. When strains were grown in media containing D-glucose and D-xylose, the GCI (XR/XDH/XK) strain showed 4.6-fold higher volumetric glutathione production (mg/L-broth), 2.2-fold higher glutathione content (%), and 2.1-fold higher cell growth (g-cell/L-broth) than the vector control strain of YPH499 (Vector). Furthermore, when recombinant S. cerevisiae strains were grown in medium containing fermentation inhibitory materials, the GCI (XR/XDH/XK) strain produced 5.8- and higher volumetric glutathione, 2.6-fold higher intracellular glutathione, and 2.9-fold higher cell growth than the vector control YPH499 (Vector) strain. The gradual sugar consumption by recombinant S. cerevisiae strains in medium containing D-glucose and D-xylose leads to high yields of glutathione. These results indicate the potential for glutathione production from lignocellulosic materials.

Proteomic and metabolomic analysis of the cellular biomarkers related to inhibitors tolerance in Zymomonas mobilis ZM4

BackgroundToxic compounds present in both the hydrolysate and pyrolysate of lignocellulosic biomass severely hinder the further conversion of lignocellulose-derived fermentable sugars into useful chemicals by common biocatalysts like Zymomonas mobilis, which has remarkable advantages over yeast. Although the extra detoxification treatment prior to fermentation process can help biocatalysts to eliminate the inhibitory environment, it is not environment friendly and cost effective for industrial application. As also reported by previous studies, an ideal and holistic approach to solve this issue is to develop microbial strains with inhibitor tolerance. However, previously engineered strains had the limitation that they could not cope well with the synergistic effect of multiple inhibitors as they are resistant only to a single inhibitor. Hence, understanding the universal cellular responses of Z. mobilis to various inhibitors may guide the designing of rational strategies to obtain more robust engineered strains for biofuel production from lignocellulosic biomass.ResultsQuantitative proteomics and metabolomics approaches were used to determine the cellular responses of Z. mobilis ZM4 to representative biomass-derived inhibitors like formic acid, acetic acid, furfural, 5-hydroxymethylfurfural, and phenol. The differentially expressed proteins identified under the challenge of single and combined inhibitors were involved in cell wall/membrane biogenesis, energy production, DNA replication, DNA recombination, DNA repair, DNA transcription, RNA translation, posttranslational modification, biosynthesis of amino acids, central carbon metabolism, etc. Metabolomics analysis showed that the up- or down-regulation pattern of metabolites was changed consistently with that of relevant proteins.ConclusionFifteen up-regulated proteins (e.g., Isopropylmalate isomerase LeuC, transcription-repair-coupling factor Mfd, and phosphoglucose isomerase PGI) and thirteen down-regulated proteins (e.g., TonB-dependent transporter ZMO1522, transcription termination factor Rho, and S1/P1 nuclease ZMO0127) were identified as candidate proteins related to all the stress conditions, implying that these proteins are potential biomarkers for the improvement of Z. mobilis ZM4 to resist complex biomass-derived inhibitors. These data can be used to generate a database of inhibitor-tolerance biomarkers, which could provide a basis for engineering Z. mobilis that would be able to grow in the presence of multiple inhibitors and directly ferment the biomass-derived sugars into biofuels.

Retraction Note to: Engineering Bacillus licheniformis as a thermophilic platform for the production of l-lactic acid from lignocellulose-derived sugars

The authors are retracting this article because the biological materials were used and the experiments were conducted without proper authorization from the laboratory where these data were obtained [1].

RETRACTED ARTICLE: Engineering Bacillus licheniformis as a thermophilic platform for the production of l-lactic acid from lignocellulose-derived sugars

BackgroundBacillus licheniformis MW3 as a GRAS and thermophilic strain is a promising microorganism for chemical and biofuel production. However, its capacity to co-utilize glucose and xylose, the major sugars found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, a “dual-channel” process was implemented to engineer strain MW3 for simultaneous utilization of glucose and xylose, using l-lactic acid as a target product.ResultsA non-phosphotransferase system (PTS) glucose uptake route was activated via deletion of the glucose transporter gene ptsG and introduction of the galactose permease gene galP. After replacing the promoter of glucokinase gene glck with the strong promoter Pals, the engineered strain recovered glucose consumption and utilized glucose and xylose simultaneously. Meanwhile, to improve the consumption rate of xylose in this strain, several measures were undertaken, such as relieving the regulation of the xylose repressor XylR, reducing the catabolite-responsive element, and optimizing the rate-limiting step. Knockout of ethanol and acetic acid pathway genes further increased lactic acid yield by 6.2%. The resultant strain, RH15, was capable of producing 121.9 g/L l-lactic acid at high yield (95.3%) after 40 h of fermentation from a mixture of glucose and xylose. When a lignocellulosic hydrolysate was used as the substrate, 99.3 g/L l-lactic acid was produced within 40 h, with a specific productivity of 2.48 g/[L h] and a yield of 94.6%.ConclusionsOur engineered strain B. licheniformis RH15 could thermophilically produced l-lactic acid from lignocellulosic hydrolysate with relatively high concentration and productivity at levels that were competitive with most reported cases of l-lactic acid-producers. Thus, the engineered strain might be used as a platform for the production of other chemicals. In addition to engineering the B. licheniformis strain, the “dual-channel” process might serve as an alternative method for engineering a variety of other strains.

Isolation of lignocellulose-derived sugars, co-fermentation of lactic acid bacteria strains, and evaluation of L-lactic acid productivity

High-productivity lactic acid bacteria (LAB) strains were screened and their capability to ferment lignocellulose-derived sugars into lactic acid were evaluated. Fifteen LAB strains were successfully isolated from cow dung, haystack, and sheep manure, respectively. Four relatively good strains were selected based on Gram stain, colony morphology, and catalase activity tests. The four strains and commercial inoculants (Lactobacillus pentosus and Enterococcus faecalis) were used to ferment cellobiose/ glucose/xylose to produce high-purity L-lactic acid. One of the strains (N4) presented the highest production of L-lactic acid after fermentation for 12 h and showed a L-lactic acid production of 15.1 g/L, 18.5 g/L, and 2.8 g/L and a productivity of 1.01 g∙L−1∙h−1, 3.68 g∙L−1∙h−1, and 0.47 g∙L−1∙h−1 by metabolizing cellobiose, glucose, and xylose, respectively. Through a phylogenetic tree analysis, strain N4 was identified as Enterococcus faecium and named Enterococcus faecium N4. Enterococcus faecium N4 has a great potential to ferment lignocellulose-derived sugars into L-lactic acid.

Bacillus coagulans MA-13: a promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate

BackgroundThe transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed.ResultsA thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans, named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 °C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-)hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 °C), still retaining an optimal operational activity at 50 °C.ConclusionsThe above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto employed for the depolymerisation of lignocellulosic biomasses to fermentable sugars. Moreover, the robustness of B. coagulans MA-13 is a desirable trait, given the presence of microbial growth inhibitors in the pre-treated biomass hydrolysate.