Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

Biobutanol production from sugarcane bagasse by Clostridium beijerinckii strains.

Acetone-butanol-ethanol (ABE) fermentation was performed with sugarcane bagasse (SCB) hydrolysate using Clostridium beijerinckii strains. A cost-effective SCB medium was developed with no enzymatic hydrolysis and no supplementation of extra carbon source or expensive nitrogen source. One of the C. beijerinckii strains studied was able to produce butanol with butanol productivity of 1.23 g/L/day with butanol yield of 0.18 g/g of sugars from the developed medium. High utilization rate of both glucose and xylose was observed in SCB medium during ABE fermentation. This study shows that SCB is a promising substrate for cellulosic biobutanol production.

Utilization of banana crop residue as an agricultural bioresource for the production of acetone-butanol-ethanol by Clostridium beijerinckii YVU1.

This study aimed to produce acetone-butanol-ethanol (ABE) using lignocellulosic crop residues as renewable bioresources. Butanol production from banana crop residue (BCR) was studied using a newly isolated solventogenic Clostridium beijerinckii YVU1. BCR is one of the abundant lignocellulosic substrates available in tropical countries containing 4·3 ± 3·5% cellulose, 28·5 ± 3·0% hemicellulose and 20·3 ± 2·6% lignin. The sequential dilute alkali and acid pretreatments solubilized 69% of lignin and 73% of hemicellulose. Ten percent (w/v) of pretreated substrate was subjected to enzymatic saccharification with cellulase, and it was found to release 0·481 ± 0·035 g glucose per g pretreated biomass. In the batch fermentation process, 20·5 g l-1 ABE (14·0 g l-1 of butanol, 5·4 g l-1 of acetone and 1·1 g l-1 of ethanol) was obtained. The executed fermentation process yielded 0·39 g ABE per g hydrolysate with 0·14 g l-1  h-1 of volumetric productivity. On the basis of the results, we believe that sequential alkali and acid pretreatment on the enzymatic hydrolysis for butanol production is indeed a technology with the potential to be applied and newly isolated. C. beijerinckii YVU1 is also a potential candidate organism for butanol production agricultural residues. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a banana crop residue (BCR) can be successfully utilized as an inexpensive and alternative bioresource for the production of acetone-butanol-ethanol (ABE). The sequential pretreatment of BCR with alkali and acid solubilized lignin and hemicellulose leading to high glucose release during enzymatic hydrolysis. A newly isolated Clostridium beijerinckii YVU1 was able to produce comparable amount of ABE with previous reports. Therefore, we can state that the utilization of BCR as substrate for C. beijerinckii YVU1 leads to an economical bioprocess for the microbial production of ABE.

Enhanced phenolic compounds tolerance response of Clostridium beijerinckii NCIMB 8052 by inactivation of Cbei_3304.

BACKGROUND: Phenolic compounds generated in hydrolysis of lignocellulosic materials are major limiting factors for biological production of solvents by Clostridia, but it lacks the attention on the study of adaptation or resistance mechanisms in response to phenolic compounds. RESULTS: Gene Cbei_3304, encoding a hypothetical membrane transport protein, was analyzed by bioinformatic method. After insertional inactivation of the functionally uncertain gene Cbei_3304 in Clostridium beijerinckii NCIMB 8052, resulted in enhanced phenolic compounds tolerance. Compared to the parent strain C. beijerinckii NCIMB 8052, evaluation of toxicity showed the recombination stain C. beijerinckii 3304::int had a higher level of tolerance to four model phenolic compounds of lignocellulose-derived microbial inhibitory compounds. A comparative transcriptome analysis showed that the genes were involved in membrane transport proteins (ABC and MFS family) and were up-regulated expression after disrupting gene Cbei_3304. Additionally, the adaptation of C. beijerinckii NCIMB 8052 in response to non-detoxified hemicellulosic hydrolysate was improved by disrupting gene Cbei_3304. CONCLUSION: Toxicity evaluation of lignocellulose-derived phenolic compounds shows that Cbei_3304 plays a significant role in regulating toxicities tolerance for ABE fermentation by C. beijerinckii, and the adaptation of non-detoxified hemicellulosic hydrolysate is significantly improved after inactivation of Cbei_3304 in wild-type strain C. beijerinckii NCIMB 8052. It provided a potential strategy for generating high inhibitor tolerance strains for using lignocellulosic materials to produce solvents by clostridia in this study.

Modulation of the Acetone/Butanol Ratio during Fermentation of Corn Stover-Derived Hydrolysate by Clostridium beijerinckii Strain NCIMB 8052.

Producing biobutanol from lignocellulosic biomass has shown promise to ultimately reduce greenhouse gases and alleviate the global energy crisis. However, because of the recalcitrance of a lignocellulosic biomass, a pretreatment of the substrate is needed which in many cases releases soluble lignin compounds (SLCs), which inhibit growth of butanol-producing clostridia. In this study, we found that SLCs changed the acetone/butanol ratio (A/B ratio) during butanol fermentation. The typical A/B molar ratio during Clostridium beijerinckii NCIMB 8052 batch fermentation with glucose as the carbon source is about 0.5. In the present study, the A/B molar ratio during batch fermentation with a lignocellulosic hydrolysate as the carbon source was 0.95 at the end of fermentation. Structural and redox potential changes of the SLCs were characterized before and after fermentation by using gas chromatography/mass spectrometry and electrochemical analyses, which indicated that some exogenous SLCs were involved in distributing electron flow to C. beijerinckii, leading to modulation of the redox balance. This was further demonstrated by the NADH/NAD+ ratio and trxB gene expression profile assays at the onset of solventogenic growth. As a result, the A/B ratio of end products changed significantly during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source compared to glucose as the carbon source. These results revealed that SLCs not only inhibited cell growth but also modulated the A/B ratio during C. beijerinckii butanol fermentation.IMPORTANCE Bioconversion of lignocellulosic feedstocks to butanol involves pretreatment, during which hundreds of soluble lignin compounds (SLCs) form. Most of these SLCs inhibit growth of solvent-producing clostridia. However, the mechanism by which these compounds modulate electron flow in clostridia remains elusive. In this study, the results revealed that SLCs changed redox balance by producing oxidative stress and modulating electron flow as electron donors. Production of H2 and acetone was stimulated, while butanol production remained unchanged, which led to a high A/B ratio during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source. These observations provide insight into utilizing C. beijerinckii to produce butanol from a lignocellulosic biomass.

Biobutanol production from apple pomace: the importance of pretreatment methods on the fermentability of lignocellulosic agro-food wastes.

Apple pomace was studied as a possible raw material for biobutanol production. Five different soft physicochemical pretreatments (autohydrolysis, acids, alkalis, organic solvents and surfactants) were compared in a high-pressure reactor, whose working parameters (temperature, time and reagent concentration) were optimised to maximise the amount of simple sugars released and to minimise inhibitor generation. The pretreated biomass was subsequently subjected to a conventional enzymatic treatment to complete the hydrolysis. A thermal analysis (DSC) of the solid biomass indicated that lignin was mainly degraded during the enzymatic treatment. The hydrolysate obtained with the surfactant polyethylene glycol 6000 (PEG 6000) (1.96% w/w) contained less inhibitors than any other pretreatment, yet providing 42 g/L sugars at relatively mild conditions (100 °C, 5 min), and was readily fermented by Clostridium beijerinckii CECT 508 in 96 h (3.55 g/L acetone, 9.11 g/L butanol, 0.26 g/L ethanol; 0.276 gB/gS yield; 91% sugar consumption). Therefore, it is possible to optimise pretreatment conditions of lignocellulosic apple pomace to reduce inhibitor concentrations in the final hydrolysate and perform successful ABE fermentations without the need of a detoxification stage.

Use of Cupriavidus basilensis-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by Clostridium beijerinckii.

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC(®)BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p-coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii: 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.

Enhanced isopropanol and n-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate.

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

Elucidating and alleviating impacts of lignocellulose-derived microbial inhibitors on Clostridium beijerinckii during fermentation of Miscanthus giganteus to butanol.

Fermentation of liquid hot water (LHW) pretreated Miscanthus giganteus (MG) by Clostridium beijerinckii NCIMB 8052 was investigated towards understanding the toxicity of lignocellulose-derived inhibitors to solventogenic Clostridium species vis-à-vis butanol production. While C. beijerinckii NCIMB 8052 did not grow in undiluted MG hydrolysate-based fermentation medium, supplementation of this medium with Calcium carbonate enabled the growth of C. beijerinckii NCIMB 8052 and production of butanol. Using high-performance liquid chromatography (HPLC) and spectrophotometric assays, LHW-pretreated MG was found to contain lignocellulose-derived microbial inhibitory compounds; some of which were transformed by exponentially growing C. beijerinckii to less inhibitory compounds during fermentation. Contrary to all expectations, the reduction product of furfural, furfuryl alcohol, inhibited butanol production by C. beijerinckii by more than 16 %. Collectively, these results provide new insights into why lignocellulosic biomass hydrolysates are recalcitrant to fermentation to biofuels and chemicals.

Butanol production from corncob residue using Clostridium beijerinckii NCIMB 8052.

AIMS: To determine whether corncob residue (CCR) could be a good substrate for butanol production. METHODS AND RESULTS: In this study, Ca(OH)2 detoxification technique was used to remove inhibitors of lignocellulose enzymatic hydrolysis. During fermentation of untreated corncob residue hydrolysate (CCRH) by Clostridium beijerinckii NCIMB 8052, cell growth was inhibited and only 3·8 g l⁻¹ acetone, butanol and ethanol (ABE) was produced. After pretreatment with Ca(OH)2, enzymatic hydrolysis of CCR resulted in 49·3 g l⁻¹ total sugars, about twofold of that of untreated one. In the fermentation of the Ca(OH)2-detoxified CCRH, sugar utilization ratio was increased by 27·3%. When using the Ca(OH)2-detoxified CCRH supplemented with 10 g l⁻¹ glucose, 16·0 g l⁻¹ ABE was produced, resulting in an ABE yield of 0·32 and a productivity of 0·33 g l⁻¹ h⁻¹. CONCLUSION: The results in this study suggest that CCR was a good carbon source for ABE fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first time to use CCR as substrate for butanol production. Ca(OH)2 detoxification pretreatment was proved to be an effective method to improve enzymatic digestibility of lignocellulose.

Sugarcane bagasse hydrolysates as feedstock to produce the isopropanol-butanol-ethanol fuel mixture: Effect of lactic acid derived from microbial contamination on Clostridium beijerinckii DSM 6423.

Enzymatic hydrolysis of lignocellulose under industrial conditions is prone to contamination by lactic acid bacteria, and in this study, a cellulose hydrolysate produced from dilute-acid pretreatedsugarcane bagasse contained 13 g/L lactic acid and was used for IBE production by Clostridium beijerinckii DSM 6423. In fermentation of the cellulose hydrolysate supplemented with sugarcane molasses for nutrients and buffering of the medium (40 g/L total sugar), 92% of the lactic acid was consumed, and the butanol yield was as high as 0.28 (7.9 g/L butanol), suggesting that lactic acid was preferentially metabolized to butanol. When the hydrolysate was mixed with a detoxified bagasse hemicellulose hydrolysate and supplemented with molasses (35 g/L total sugar), the culture was able to exhaust glucose and utilized sucrose (by 38%), xylose (31%), and lactic acid (70%). Overall, this study shows that C. beijerinckii DSM 6423 can co-ferment first- and second-generation sugars while consuming lactic acid.

Metabolic Engineering and Adaptive Evolution of Clostridium beijerinckii To Increase Solvent Production from Corn Stover Hydrolysate.

The production of acetone-butanol-ethanol by solventogenic Clostridium using lignocellulosic biomass can be a potential alternative to petroleum-based butanol. However, previous studies on nondetoxified lignocellulose hydrolysate could not provide better results when compared to those in synthetic medium. In this study, we engineered the pentose pathway of Clostridium beijerinckii NCIMB 8052, which was then subjected to adaptive laboratory evolution in the gradient mixture of synthetic medium and pretreated corn stover enzymatic hydrolysate (CSH) prepared according to the National Renewable Energy Laboratory (NREL) standard. The final resultant strain CIBTS1274A produced 20.7 g/L of total solvents in NREL CSH diluted to 6% initial total sugars, supplemented with ammonium acetate. This performance was comparable with that of corn-based butanol. In addition, this strain was successfully used in the scale-up operation using nondetoxified corn stover and corncob hydrolysate at Lignicell Refining Biotechnologies Ltd., which once was the only commercial biobutanol industry in the world.

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from Clostridium beijerinckii.

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H(2)O(2) to each compound up to 0.5-1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 microM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64-74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.

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.

Fermentative butanol production by Clostridia.

Butanol is an aliphatic saturated alcohol having the molecular formula of C(4)H(9)OH. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. Moreover, butanol has been considered as a potential fuel or fuel additive. Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century. However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes. Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production. This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes. The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed.

Efficient butanol recovery from acetone-butanol-ethanol fermentation cultures grown on sweet sorghum juice by pervaporation using silicalite-1 membrane.

We investigated butanol recovery by pervaporation separation, using a silicalite-1 membrane, from batch cultures of butanol-producing Clostridium beijerinckii SBP2 grown on sweet sorghum juice as a fermentation medium. The pervaporation system yielded 73% (w/v) butanol from intact feed cultures containing 1% (w/v) butanol, and had a butanol permeation flux of 11 g m(-2) h(-1). Upon neutralization and activated charcoal treatment of the feed cultures, butanol yield and total flux increased to 82% (w/v) and 40 g m(-2) h(-1), respectively. This system is applicable to refining processes for practical biobutanol production from a promising energy crop, sweet sorghum.

A biorefining process: Sequential, combinational lignocellulose pretreatment procedure for improving biobutanol production from sugarcane bagasse.

Here, for the first time, we designed a sequential, combinatorial lignocellulose pretreatment procedure (SCLPP) for microbial biofuel fermentation to reduce generation of microbial growth inhibitors and furthermore increase sugar yields. We tested this pretreatment process using sugarcane bagasse as substrate and assessed the effectiveness by analysis of biobutanol production through microbial clostridium beijerinckii NCIMB 8052 conversion. Our results showed that there were no inhibitory effects when using the hydrolysates as fermentation substrate. Under the SSF scheme, we observed the highest concentrations of butanol (6.4g/L) and total ABE (11.9g/L), resulting in a higher ABE productivity, compared with the SHF method. These findings suggest that the SCLPP is a feasible method for improving ABE production, lowering microbial inhibitor generation, and ensuring success in the subsequent fermentation process. Therefore, our work demonstrated developing a tractable integrated process that facilitates to increase biofuel production from agricultural residues rich in lignocellulose is feasible.

Pretreatment of lignocellulosic biomass using Fenton chemistry.

In an attempt to mimic white-rot fungi lignin degradation via in vivo Fenton chemistry, solution phase Fenton chemistry (10 g biomass, 176 mmol hydrogen peroxide and 1.25 mmol Fe(2+) in 200 mL of water) was applied to four different biomass feedstocks. An enzymatic saccharification of Fenton pretreated biomass showed an average 212% increase relative to untreated control across all four feedstocks (P<0.05, statistically significant). A microbial fermentation of the same Fenton pretreated biomass showed a threefold increase in gas production upon a sequential co-culture with Clostridium thermocellum and Clostridium beijerinckii. These results demonstrate the use of solution phase Fenton chemistry as a viable pretreatment method to make cellulose more bioavailable for microbial biofuel conversion.

Lignocellulosic hydrolysates and extracellular electron shuttles for H2 production using co-culture fermentation with Clostridium beijerinckii and Geobacter metallireducens.

A co-culture of Clostridium beijerinckii and Geobacter metallireducens with AH2QDS produced hydrogen from lignocellulosic hydrolysates (biomass of Miscanthus prepared by hydrothermal treatment with dilute acids). This co-culture system enhanced hydrogen production from lignocellulosic hydrolysates by improving substrate utilization and diminishing acetate accumulation, despite the presence of fermentation inhibitors in the hydrolysates. The improvements were greater for xylose-rich hydrolysates. The increase in maximum cumulative hydrogen production for hydrolysates with glucose:xylose mass ratios of 1:0.2, 1:1 and 1:10 g/g was 0%, 22% and 11%, respectively. Alternative extracellular electron shuttles (EES), including indigo dye, juglone, lawsone, fulvic acids and humic acids, were able to substitute for AH2QDS, improving hydrogen production in the co-culture system using xylose as model substrate. Increased utilization of xylose-rich hydrolysates and substitution of alternative EES make the co-culture with EES system a more attractive strategy for industrial biohydrogen production.