Presence of low concentrations of acetic acid improves yeast tolerance to hydroxymethylfurfural (HMF) and furfural

BackgroundHydrolysates derived from lignocellulosic material contain a complex mix of inhibitory compounds dependant on the type of biomass and the pre-treatment process employed. These inhibitors prevent the subsequent fermentation of available sugars by yeast into ethanol.ResultsInhibitory compounds normally work synergistically to reduce metabolic output, rates of budding and viability; however, it was observed in this study that the presence of weak acids actually improved tolerance to hydroxymethyl furfural (HMF) and furfural in Saccharomyces cerevisiae. The protective role of weak acids in HMF or furfural stressed cells was only apparent with relatively low concentrations of acetic acid (20 mM), however, there was an improvement in glucose utilisation and ethanol production when compared with HMF or furfural stressed cells. Focusing on HMF stressed cells quantitative trait loci (QTL) analysis identified a region on chromosome VI related to the enhanced tolerance to HMF in the presence of acetic acid. Two genes FET5 and HAC1 located in this region were up-regulated under the combined stress of acetic acid with HMF stress and null mutants exhibited a return to HMF sensitivity.ConclusionsPresence of acetic acid helps yeast cells overcome HMF stress, QTL analysis identified two genes on a loci on chromosome VI, knocking out these genes returns the cell to HMF sensitivity.     

Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood

In order to evaluate the potential of an adapted inhibitor-tolerant yeast strain developed in our lab to produce ethanol from softwood, the effect of furfural and HMF presented in defined medium and pretreatment hydrolysate on cell growth was investigated. And the efficiency of ethanol production from enzymatic hydrolysate mixed with pretreatment hydrolysate of softwood by bisulfite and sulfuric acid pretreatment process was reported. The results showed that in the combined treatments of the two inhibitors, cell growth was not affected at 1 g/L each of furfural and HMF. When 3 g/L each of furfural and HMF was applied, the adapted strain responded with an extended lag phase of 24 h. Both in batch and fed-batch runs of combined hydrolysate fermentation, the final ethanol concentrations were above 20.0 g/L and the ethanol yields (Y_p/s) on the total amount of fermentable sugar presented in the pretreated materials were above 0.40 g/g. It implies the great promise of the yeast strain for improving ethanol production from softwood due to its high ability of metabolizing inhibitor compounds of furfural and HMF.     

Effects of furfural and acetic acid on growth and lipid production from glucose and xylose by Rhodotorula glutinis

Microbial conversion of lignocellulosic sugars to triacylglycerols (a biodiesel or renewable diesel feedstock) was investigated using the oleaginous yeast Rhodotorula glutinis (ATCC 15125). In the shake flask experiments, R. glutinis was first grown in a nitrogen-rich medium utilizing an artificial acid hydrolysate of lignocellulosic biomass switchgrass as the sole carbon and energy source. Once the culture had reached the stationary phase, the cells were harvested and transferred to a fresh nitrogen-free media containing artificial acid hydrolysate sugars for lipid accumulation. Analysis of the data collected showed that the yeast were able to grow in the medium containing artificial acid hydrolysate sugars as the carbon and energy source. The net specific Growth rate(s) indicated that the presence of acetic acid and furfural in the artificial acid hydrolysate inhibited the growth of R. glutinis on glucose, but not the growth on xylose. The lipid accumulated in the cells, determined by gravimetrical method, increased from initial 4.3%-39.0% of dry cell mass weight. The major fatty acids of the accumulated lipids were palmitic acid, stearic acid, oleic acid, linoleic acid and ??-linoleic acid. These results indicate that it is feasible to convert the sugars in acid hydrolysate of lignocellulosic biomass to triacylglycerols using R. glutinis.     

Effect of furans and linoleic acid on hydrogen production

The effects of furans (furfural and 5-hydroxymethylfurfural (HMF)) on hydrogen (H₂) production using mixed anaerobic cultures were evaluated by conducting batch experiments. Two mixed anaerobic cultures (culture A and B) fed furans plus glucose and treated with and without linoleic acid (LA) at pH 5.5 were maintained at 37 °C. In the LA inhibited cultures A and B fed 0.75 g L⁻¹ furfural and 0.25 g L⁻¹ HMF, the maximum H₂ yields observed were 1.89 ± 0.27 mol mol⁻¹ glucose and 1.75 ± 0.22 mol mol⁻¹ glucose, respectively. In cultures with maximum H₂ yields, Clostridium sp. and Flavobacterium sp. were dominant. Acetate, butyrate and ethanol were the major soluble metabolites detected in cultures A and B whereas propionate was also dominant in culture B. A canonical correspondence analysis based on the byproducts and the relative abundance of the terminal-restriction fragments revealed less variation between cultures treated with LA and low correlation value between the factors and the species composition.     

Fermentable sugars recovery from grape stalks for bioethanol production

Three different processes were investigated for the recovery of fermentable sugars from grape stalks: autohydrolysis at 121 °C before and after a pre-washing step and acid hydrolysis (2% H₂SO₄ w/w) after a pre-washing step. Moreover, optimal conditions of a charcoal-based purification process were determined by experimental design. All hydrolysates, with their corresponding synthetic liquors were used as fermentation substrates for the production of metabolites by the yeast: Debaryomyces nepalensis NCYC 1026. The main fermentation product was ethanol, where a maximum production of 20.84 g/l, a conversion yield of 0.35 g ethanol/g monomeric sugars and a productivity of 0.453 g/l h were obtained from non-purified autohydrolysate liquor. In all cases, ethanol production and cell growth were better in non-purified liquors than in synthetic liquors. These results could be influenced by the presence of other sugars in the hydrolysates, with higher concentration in non-purified ones.     

Distinct effects of furfural,hydroxymethylfurfural and its mixtures on dark fermentation hydrogen production and microbial structure of a mixed culture

Past research has suggested that furfural and hydroxymethylfurfural (HMF) present in lignocellulosic hydrolyzates exert a synergistic effect on dark fermentative H₂ production, which has not adequately proven in microbial consortia. It is possible that some members of a consortium experience less inhibition than others, helping to the entire consortium to overcome the inhibition. To elucidate the type of inhibitory effect that these agents exert, the objective of this study was to contrast the individual impacts of furfural and HMF with the corresponding mixtures at the same concentration threshold on the hydrogen production and the relative abundance of different members in the consortium. Heat-treated anaerobic granules served as inoculum to ferment xylose for hydrogen production in presence of furfural (0.10, 0.50, and 1.00 g/L), HMF (0.02, 0.09, 0.19 g/L) and the corresponding mixtures in comparison with a control in absence of these agents. Furfural alone did not inhibit the hydrogen production; indeed, the inoculum completely degraded furfural at all its concentrations with the presence of furoic acid.HFM was partially degraded in the treatments with the lowest/middle concentrations, resulting in higher hydrogen production than the control. In contrast, at the highest HMF concentration, the inoculum was unable to remove it resulting in the strongest inhibition of hydrogen production. All the F/HMF mixtures had an inhibitory effect on the hydrogen production. A log2 fold change analysis of pyrosequencing reads evidenced that these agents promoted the growth of Lactobacillus, and clostridia species such as Clostridium butyricum, Clostridium bifermentans, and Clostridium sartagoforme, suggesting their active participation in the detoxification process.     

Detection of furfural and 5-hydroxymethylfurfural with a yhcN::luxCDABE bioreporter strain

RT-qPCR data demonstrated that the yhcN gene was highly expressed when Escherichia coli was exposed to 500 ppm of ferulic acid (9.8-fold), vanillin (7.3-fold) or furfural (2.2-fold). Consequently, an E. coli yhcN::lux bioreporter strain (DMY1) was constructed. This strain is very responsive to furfural and 5-hydroxymethylfurfural, showing a maximum induced bioluminescence of 29.4- and 17.8-fold, respectively. The responses to different phenolics were also significant, with relative responses of between 4- and 16-fold. In tests with rice straw hydrolysate samples, a dual induction of DMY1 was observed and it was demonstrated that this corresponded to the activities of the phenolics and HMF within the sample. The maximum response seen with the hydrolysate sample was 3.5 and the minimum phenolic concentration detected was 39 mg/L. These results illustrate that this strain can be used to monitor for the presence of furans and phenolics commonly found within plant hydrolysate samples.     

Removal of furfural and HMF from monosaccharides by nanofiltration and reverse osmosis membranes

Ethanol can be produced from lignocellulosic biomass through fermentation. However, some inhibitory compounds are also generated, and can substantially reduce the efficiency of ethanol production. In the present work, two commercial nanofiltration (NF) membranes and two commercial reverse osmosis (RO) membranes were utilized to remove the model fermentation inhibitors, furfural and 5-hydroxymethyl-2-furaldehyde (HMF), from the synthetic xylose-glucose-furfural-HMF model solution. The influences of operating parameters such as pH, pressure, temperature and solute concentration in feed on the separation performance of the tested membranes were investigated. The selectivities of furfural and HMF from monosaccharides by NF/RO membranes were compared to evaluate the feasibility of simultaneous inhibitor removal and sugar concentration. Results showed that RO membranes are more efficient than NF membranes for retaining monosaccharides under all tested conditions. RO membranes are more suitable than NF membranes for sugar concentration and removal of some inhibitors from lignocellulose hydrolyzate simultaneously.     

Biohydrogen production from pretreated corn cobs

In this study, the co-fermentability of four different pretreated corn cob streams at different mixing ratios was assessed. The four streams, denoted DP, DS, HP, and HS, were: two dilute acid pretreatment comprising one purge and one squeeze and two high pressure autohydrolysis comprising one purge and one squeeze. The “Purge” stream was taken from the steam percolation reactor during cooling and the “Squeeze” stream was recovered from the cooked biomass with a pressing step. In addition, the impact of furfural and 5-hydroxymethylfurfural (HMF) on biohydrogen production potential was evaluated. The DP:DS mix at 50:50 by volume achieved the maximum H₂ yield of 265 (mL/gCOD sugars consumed). Furfural at concentrations of 0.21–1.09 g/L had no impact on H₂ production rates and yields and HMF was below the inhibitory threshold of 0.14 g/L. A positive correlation was observed between the monomeric-to-polymeric sugars ratio and H₂ production rates and yields.     

Investigating how biomass hydrolysis derivatives inhibit H2 production by an isolated Clostridium beijerinckii

This study evaluates how fermentation inhibitors derived from biomass, namely 5-hydroxymethylfurfural (HMF), levulinic acid (LA), and formic acid (FA), affect H2 production by a Clostridium beijerinckii strain. The specific fermentative H2 production rate (μH2), bacterial cell growth rate (μ), and substrate (glucose) consumption rate (μS) during fermentation helped to estimate which HMF, LA, and FA concentrations inhibited 50% of the rates (IC50). IC50 for μ was 2.4, 2.7, and 1.4 g/L for HMF, LA, and FA, respectively. HMF inhibited H2 production the most potently and favored the lactate and ethanol pathways. Butyric acid was the only metabolite to be detected in the presence of LA or FA, which attested that these inhibitors completely inhibited the acetate pathway. The glucose consumption rate was the least affected by the inhibitors, and FA was more potent than HMF and LA. This information should be useful for more appropriate biomass feedstock application in fermentative H2 production.     

Empty fruit bunches from oil palm as a potential raw material for fuel ethanol production

In this paper the fuel ethanol production from empty fruit bunches was experimentally evaluated using alkaline pretreatment and enzymatic hydrolysis for sugars release. Fermentation was accomplished using a native Saccharomyces cerevisiae strain. Ethanol concentration was carried on using a glass bench-scale distillation column. Experimental results were used for planning and designing the process scheme using Aspen Plus. Process simulation allowed calculating the mass and energy balances. It was found that coupling alkaline pretreatment with a later autoclaving improved the sugars yield in enzymatic hydrolysis. However, the use of the remaining soaking solution from pretreatment as hydrolysis medium had negative effects on sugars yield suggesting that there exist inhibit substance for the enzyme. Better results for enzymatic hydrolysis were obtained when sodium acetate buffer was used. Ethanol yield obtained from both experiments and simulation were very similar (66.50 and 65.84 dm³ of ethanol per each t of empty fruit bunches, respectively). These low ethanol yields were obtained because the native S. cerevisiae does not assimilate all reducing sugars, suggesting that those sugars were pentoses. Simulated alkaline and autoclaving pretreatment contributed only with 2% of the total energy consumption (198.4 GJ m⁻³ ethanol) while product recovery represented 57% of the total energy.     

Phosphoric acid activation of recalcitrant biomass originated in ethanol production from banana plants

Biomass samples have been studied as activated carbon precursors. Two, denoted as recalcitrant biomass, are ethanol production wastes, one from acid hydrolysis of starch-rich banana (Musa acuminate) fruit and one from enzymatic hydrolysis of banana pseudostem (lignocellulosic material) and a third one is raw banana tree pseudostem. The samples were characterized by proximate, ultimate, forage, morphological and structural analyses. They all have volatile matter contents above 50% but present some remarkable differences. The sample coming from the acid hydrolysis has too high ash content (mostly calcium sulfate) and low cellulose and lignin contents that prevents its use as an activated carbon precursor. Contrarily, the other two samples have lower ash contents, higher carbon percentages and higher cellulose and lignin contents. The activation with phosphoric acid at 450 °C has been investigated. The results are quite reproducible, showing good porosity developments and a strong dependence on the precursor, the H₃PO₄ concentration and the H₃PO₄/sample weight ratio used. The activated carbons have quite high apparent surface area, porosity and yield. Interestingly, the phosphoric acid activated carbons prepared in this work have quite well developed mesoporosity which is especially useful for some applications (e.g. for gasoline evaporative control). Our preliminary comparative study, carried out with a well known commercial activated carbon used to control automobile hydrocarbons emissions, has shown that these activated carbons perform very well, being their performance a function of their mesoporosity development. Their oxygenated surface complexes (assessed from TPD experiments) are considerably high.     

Preparation of activated carbon with highly developed mesoporous structure from Camellia oleifera shell through water vapor gasification and phosphoric acid modification

Using Camellia oleifera shell as starting material, production of activated carbon rich in mesoporous structure has been investigated with water vapor gasification followed by phosphoric acid modification. This method is found to be very effective in increasing the mesoporous ratio of the activated carbon. The micropores of the activated carbon gasified by water vapor (raw activated carbon) serve as channels for phosphoric acid impregnation to enlarge the pore size. Results of the activated carbon modified by phosphoric acid (final activated carbon) show a high mesoporous ratio of 61%, which is twice as high as that of raw activated carbon. Such modification also increases the BET surface area and total pore volume to 1608 m²/g and 1.17 cm³/g, respectively. The final activated carbon exhibits high adsorption capacity for methylene blue and iodine, with the adsorption values of methylene blue and iodine increasing from 180 to 1012 mg/g to 330 and 1326 mg/g, respectively. The present preparation is a convenient yet promising method to combine gasification and modification to obtain activated carbon with highly developed mesoporous structures.     

Valorization of Leucaena leucocephala for energy and chemicals from autohydrolysis

In this work, Leucaena leucocephala K366 was characterized chemical and energy terms, and assessed its potential as a lignocellulosic raw material and energetic and industrial crop specie, and its integral fractionation by autohydrolysis by evaluating its calorific value, holocellulose, glucan, xylan, arabinan, lignin and oligomers and monomers contents in autohydrolysis liquor and solid phase. Also, this paper will consider the influence of the temperature and time of autohydrolysis process from L. leucocephala K366 to obtain valuable liquor and a suitable solid phase to produce energy by combustion.Valuable liquor was obtained from the autohydrolysis of L. leucocephala by simultaneously using operating temperatures and times in the medium-high ranges studied, namely: 172-184 °C and 15-30 min. The optimum processing conditions provided an acceptable yield (16-26%), and high xylose and xylo-oligomer contents in the liquor (10.0 and 58.6%, respectively, of the amounts present in the starting raw material when operating at 184 °C for 30 min) in comparison with other raw materials. The arabinan fraction was extracted virtually completely —only 8.3% remained in the solid fraction—, and the acetyl group fraction was recovered in full. In addition, these conditions reduced the glucose content of the liquor to 2.9% of the amount present in the raw material while largely preserving the integrity of cellulose fibers.Klason lignin was scarcely dissolved under the operating conditions of the autohydrolysis process. This increased the calorific value of the solid phase by 9% (under the most drastic operating conditions) with respect to the starting raw material.     

Influence of activated carbon porous texture on catalyst activity for ethanol electro-oxidation

Direct ethanol fuel cells are based in ethanol electro-oxidation at low temperature and they constitute an alternative energy conversion system. However, they need catalysts in order to improve their efficiency, given that ethanol electro-oxidation is a slow-kinetic reaction. Among those catalysts platinum and platinum alloys play an important role in the increase of the ethanol cleavage kinetics for fuel cell application. However, to maximize the catalyst performance, support materials are needed in order to reduce the catalyst load considering its high cost. One of the more versatile catalyst supports is activated carbon. Recently, attention has focused on wood as carbon material precursor, because of its sustainability and also because the obtained carbons have excellent final properties. In the present work, activated carbon obtained by physical activation of Eucalyptus grandis wood (biocarbon) was tested as Pt and PtSn catalyst support, for ethanol electro-oxidation reaction. For comparison purposes, commercial activated carbon Vulcan XC72 was also tested. The catalyst supports were characterized by textural analysis, elemental analysis and infrared spectroscopy. The obtained catalysts were characterized regarding structure by XRD and their electrochemical behavior was evaluated by cyclic voltammetry. Biocarbon-supported PtSn electrocatalysts showed better electrochemical performance related to the commercial activated carbon (Vulcan XC72)-supported ones, since its developed current density and potential were the highest and its onset potential was the lowest. However, pure platinum showed better values for current density, potential and onset potential in Vulcan XC72-based activated carbons, being the untreated one the best support in this case.     

Cross-polymerisation between the model furans and carbohydrates in bio-oil with acid or alkaline catalysts

Understanding the potential cross-polymerisation between the main components in bio-oil is essential to develop the method to minimize the coking in hydrotreatment of bio-oil or to maximize the production of carbon material from bio-oil. In this study, the potential cross-polymerisation between furfural and the sugars (xylose and glucose) and the impacts of acid/base catalysts on the polymerisation reactions were investigated. The results indicated that the cross-polymerisation of furfural/xylose and furfural/glucose existed in absence of catalyst, and the extent was more significant for that between furfural and glucose. The strong acid promoted the cross-polymerisation while the weak organic acid like acetic acid and the strong base like NaOH did not. The cross-polymerisation of furfural/sugars produced the more hydrophilic carbonaceous spheres that tended to merge to form bigger particles. The polymerisation of furfural involved opening of the furan ring via hydrolysis, forming oxygen-containing intermediates, while glucose/xylose were more reactive towards polymerisation reaction via dehydration route. The polymers from the sugars were more aliphatic than that from furfural, especially for that from glucose, leading to their lower thermal stabilities. The cross-polymerisation also affected the size/abundance of the π-conjugated structures in the soluble polymers and the relative ratio of the CO/CC in the resulting insoluble polymer.     

Development of an integrated pretreatment fractionation process for fermentable sugars and lignin: Application to almond (Prunus dulcis) shell

An environmentally friendly pretreatment process was developed to fractionate cellulose, hemicellulose and lignin from almond (Prunus dulcis) shells, consisting of hot water pretreatment (HWP) coupled with organic solvent (organosolv) pretreatment of water/ethanol (OWEP). This integrated pretreatment process proved more effective on the basis of yield of fermentable sugar and lignin separation compared with HWP alone, dilute acid pretreatment (DAP), ammonia pretreatment (AP), lime pretreatment LP, organosolv water/ethanol pretreatment (OWEP), and organosolv water/acetone pretreatment (OWAP). In the coupled hot water-organosolv process, hemicellulose sugars were recovered in the first residual liquid while varying amounts of cellulose was retained in the residual solid. The lignin fraction was obtained by simply adjusting the pH from the second liquid. The optimal two-stage process consisted of first HWP stage at 195 °C for 30 min, resulting in w_glucose = 95.4% glucose recovery yield and w_xylose = 92.2% xylose removal. The second organosolv OWEP stage was operated at 195 °C for 20 min, in ethanol in water mixtures of <phi>_ethanol = 50% and resulted in nearly w_glucose = 100% glucose recovery yield, w_xylose = 90% xylose and w_lignin = 61% lignin removal. After enzymatic hydrolysis, glucose yield was up to w_glucose = 95%, compared to 61% yield from untreated almond. Images obtained via scanning electron microscopy (SEM) highlighted the differences in almond structure from the varying pretreatment methods during biomass fractionation.     

Evaluation of dilute acid and alkaline pretreatments,enzymatic hydrolysis and fermentation of napiergrass for fuel ethanol production

Napiergrass (Pennisetum purpureum Schum.) is a promising low cost raw material which does not compete with food prices, has attractive yields and an environmentally friendly farming. Dilute sulfuric acid pretreatment of napiergrass was effective to obtain high yields of sugars and low level of degradation by-products from hemicellulose. Detoxification with Ca(OH)₂ removed inhibitors but showed sugars loss. An ethanol concentration of 21 g/L after 176 h was found from the hydrolyzate using Pichia stipitis NBRC 10063 (fermentation efficiency 66%). An additional alkaline pretreatment applied to the solid fraction remaining from the diluted acid pretreatment improved the lignin removal. The highest cellulose hydrolysis values were found with the addition of β-glucosidase and PEG 6000. The simultaneous hydrolysis and fermentation of the cellulosic fraction with Saccharomyces cerevisiae, 10% (w/v) solid concentration, β-glucosidase and PEG 6000, showed the highest ethanol concentration (24 g/L), and cellulose hydrolysis values (81%). 162 L ethanol/t of dry napiergrass were produced (overall efficiency of 52%): 128 L/t from the cellulosic fraction and 34 L/t from the hemicellulosic fraction.     

Bio-hydrogen production from tequila vinasses: Effect of detoxification with activated charcoal on dark fermentation performance

Hydrogen production from dark fermentation is a potential source of sustainable fuel when it is generated from waste. This study compared hydrogen production resulting from fermentation using raw and detoxified tequila vinasse. Vinasse was detoxified with granular activated charcoal, which was used to adsorb compounds that could inhibit the production of hydrogen by dark fermentation. In batch cultures detoxification of vinasse led to up to 20% higher maximum velocities of hydrogen production, a 5.4 h reduction in the lag phase and an 11% higher molar yield, compared to results obtained with raw vinasse. Losses of sugars after detoxification provoked that the specific hydrogen volumetric yields obtained with detoxified vinasse were 30–40% lower with 5 g COD/L and 15 g COD/L initial concentrations, compared to the ones obtained with raw vinasse. For an initial 30 g COD/L no differences in specific hydrogen yields were observed between raw or detoxified vinasse in batch fermentation. Continuous culture fermentation of vinasse showed hydrogen production rates between 1.32 ± 0.07 to 1.39 ± 0.14 NL H₂/L-d when extra nutrients were added, while a stable production of hydrogen through fermentation of detoxified vinasse could not be maintained despite nutrient addition. Production of hydrogen from vinasse diluted with water with no additional nutrients was assessed and rates close to 0.42 ± 0.02 NL H₂/L-d and hydrogen content close to 37% were obtained. Accumulation of lactic acid and a predominant production of butyric acid over acetic acid suggested that the fermentation dynamics of vinasse with no supplementary nutrients were especially susceptible to high substrate loading rates and prolonged hydraulic retention times.     

Mixtures of 5-hydroxymethylfurfural,levulinic acid,and formic acid have different impact on H2-producing Clostridium strains

Fermentative hydrogen production allows the use of renewable biomasses as feedstocks. However, biomass saccharification results not only in carbohydrates, but also in products that can inhibit fermentation. Although biomass hydrolysates contain mixtures of inhibitors, most studies are performed with a single inhibitor. This study evaluates how 5-hydroxymethylfurfural (HMF, 0.60 g/L), levulinic acid (LA, 2.10 g/L), and/or formic acid (FA, 0.80 g/L) mixtures affect two H₂-producing clostridia, Clostridium beijerinckii Br21 and Clostridium acetobutylicum ATCC 824. Fermentation assays with and without (control) the inhibitors helped to calculate the specific H₂ production, substrate consumption, and bacterial cell growth rates for Clostridium beijerinckii Br21 or Clostridium acetobutylicum ATCC 824. HMF + AL, HMF + AF, AL + AF, and HMF + AL + AF mixtures inhibited H₂ production by C. beijerinckii Br21 by 58.7, 60.0, 46.9, and 83.0%, respectively, and by C. acetobutylicum ATCC 824 by 68.1, 71.4, 58.2, and 89.0%, respectively. Clostridium acetobutylicum ATCC 824 metabolized HMF more efficiently. However, organic acids and their combination with HMF inhibited H₂ production by C. beijerinckii Br21 to a lesser extent, which highlighted that this microorganism is robust for H₂ production from biomass hydrolysates.