Detoxification of a lignocellulosic biomass slurry by soluble polyelectrolyte adsorption for improved fermentation efficiency

This study investigated the detoxification of a dilute acid pretreated Ponderosa pine slurry using the polyelectrolyte polyethyleneimine (PEI). The addition of polyelectrolyte to remove enzymatic and/or fermentation inhibitory compounds, that is, acetic acid, furfural, and 5-hydroxymethylfurfural (HMF), was performed either before or after enzymatic hydrolysis to determine the optimal process sequence. Negligible acetic acid, glucose, and xylose were removed regardless of where in the process the polymer addition was made. Maximum furfural and HMF separation was achieved with the addition of PEI to a clarified pre-enzymatic hydrolysis liquor, which showed that 88.3% of furfural and 66.4% of HMF could be removed. On the other hand, only 23.1% and 13.4% of furfural and HMF, respectively, were removed from a post-enzymatic hydrolysis sample; thus, the effects of enzymes, glucose, and wood solids on inhibitor removal were also investigated. The presence of solid particles >0.2 μm and unknown soluble components <10 kDa reduced inhibitory compound removal, but the presence of elevated glucose levels and enzymes (cellulases) did not affect the separation. The fermentability of detoxified versus undetoxified hydrolysate was also investigated. An ethanol yield of 92.6% of theoretical was achieved with Saccharomyces cerevisiae fermenting the detoxified hydrolyzate, while no significant ethanol was produced in the undetoxified hydrolyzate. These results indicate that PEI may provide a practical alternative for furan removal and detoxification of lignocellolosic hydrolysates, and that application before enzymatic hydrolysis minimizes separation interferences.     

Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation

During pretreatment and hydrolysis of fiber-rich agricultural biomass, compounds such as salts, furfural, hydroxymethyl furfural (HMF), acetic, ferulic, glucuronic, rho-coumaric acids, and phenolic compounds are produced. Clostridium beijerinckii BA101 can utilize the individual sugars present in lignocellulosic [e.g., corn fiber, distillers dry grain solubles (DDGS), etc] hydrolysates such as cellobiose, glucose, mannose, arabinose, and xylose. In these studies we investigated the effect of some of the lignocellulosic hydrolysate inhibitors associated with C. beijerinckii BA101 growth and acetone-butanol-ethanol (ABE) production. When 0.3 g/L rho-coumaric and ferulic acids were introduced into the fermentation medium, growth and ABE production by C. beijerinckii BA101 decreased significantly. Furfural and HMF are not inhibitory to C. beijerinckii BA101; rather they have stimulatory effect on the growth of the microorganism and ABE production.     

Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.     

Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors

One major barrier to the economic conversion of biomass to ethanol is inhibitory compounds generated during biomass pretreatment using dilute acid hydrolysis. Major inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) inhibit yeast growth and subsequent fermentation. The ethanologenic yeast Saccharomyces cerevisiae demonstrated a dose-dependant inhibition by the inhibitors and has the potential to transform furfural and HMF into less toxic compounds of furfuryl alcohol and 2,5-bis-hydroxymethylfuran (also termed as furan-2,5-dimethanol (FDM)), respectively. For a sustainable and cost-competitive biomass-to-ethanol industry, it is important to develop more tolerant yeast strains that can, in situ, detoxify the inhibitors and produce ethanol. This study summarizes current knowledge and our understanding of the inhibitors furfural and HMF and discusses metabolic conversion pathways of the inhibitors and the yeast genomic expression response to inhibitor stress. Unlike laboratory strains, gene expression response of the ethanologenic yeast to furfural and HMF was not transient, but a continued dynamic process involving multiple genes at the genome level. This suggests that during the lag phase, ethanologenic yeasts undergo a genomic adaptation process in response to the inhibitors. The findings to date provide a strong foundation for future studies on genomic adaptation and manipulation of yeast to aid more robust strain design and development.The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Removal of enzymatic and fermentation inhibitory compounds from biomass slurries for enhanced biorefinery process efficiencies

Within the biorefinery paradigm, many non-monomeric sugar compounds have been shown to be inhibitory to enzymes and microbial organisms that are used for hydrolysis and fermentation. Here, two novel separation technologies, polyelectrolyte polymer adsorption and resin-wafer electrodeionization (RW-EDI), have been evaluated to detoxify a dilute acid pretreated biomass slurry. Results showed that detoxification of a dilute acid pretreated ponderosa pine slurry by sequential polyelectrolyte and RW-EDI treatments was very promising, with significant removal of acetic acid, 5-hydroxymethyl furfural, and furfural (up to 77%, 60%, and 74% removed, respectively) along with >97% removal of sulfuric acid. Removal of these compounds increased the cellulose conversion to 94% and elevated the hydrolysis rate to 0.69 g glucose/L/h. When using Saccharomyces cerevisiae D₅A for fermentation of detoxified slurry, the process achieved 99% of the maximum theoretical ethanol yield and an ethanol production rate nearly five-times faster than untreated slurry.     

Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol.     

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose

The rates and extents of enzymatic cellulose hydrolysis of dilute acid pretreated corn stover (PCS) decline with increasing slurry concentration. However, mass transfer limitations are not apparent until insoluble solids concentrations approach 20% w/w, indicating that inhibition of enzyme hydrolysis at lower solids concentrations is primarily due to soluble components. Consequently, the inhibitory effects of pH-adjusted pretreatment liquor on the enzymatic hydrolysis of PCS were investigated. A response surface methodology (RSM) was applied to empirically model how hydrolysis performance varied as a function of enzyme loading (12-40mg protein/g cellulose) and insoluble solids concentration (5-13%) in full-slurry hydrolyzates. Factorial design and analysis of variance (ANOVA) were also used to assess the contribution of the major classes of soluble components (acetic acid, phenolics, furans, sugars) to total inhibition. High sugar concentrations (130g/L total initial background sugars) were shown to be the primary cause of performance inhibition, with acetic acid (15g/L) only slightly inhibiting enzymatic hydrolysis and phenolic compounds (9g/L total including vanillin, syringaldehyde, and 4-hydroxycinnamic acid) and furans (8g/L total of furfural and hydroxymethylfurfural, HMF) with only a minor effect on reaction kinetics. It was also demonstrated that this enzyme inhibition in high-solids PCS slurries can be approximated using a synthetic hydrolyzate composed of pure sugars supplemented with a mixture of acetic acid, furans, and phenolic compounds, which indicates that generally all of the reaction rate-determining soluble compounds for this system can be approximated synthetically.     

Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds

Synthetic mixtures of predominant lignocellulosic hexose sugars were supplemented with separate aliquots of three inhibitory compounds (furfural, hydroxymethylfurfural (HMF), and acetic acid) in a series of concentrations and fermented by the spent sulfite liquor (SSL)-adapted yeast strain Tembec T1 and the natural isolate Saccharomyces cerevisiae (S. cerevisiae) Y-1528 to compare tolerance and assess fermentative efficacy. The performance of Y-1528 exceeded that of Tembec T1 by a significant margin, with faster hexose sugar consumption, higher ethanol productivity, and in the case of furfural and HMF, faster inhibitor consumption. Nevertheless, furfural had a dose-proportionate effect on sugar consumption rate and ethanol productivity in both strains, but did not substantially affect ethanol yield. HMF had a similar effect on sugar consumption rate and ethanol productivity, and also lowered ethanol yield. Surprisingly, acetic acid had the least impact on sugar consumption rate and ethanol productivity, and stimulated ethanol yield at moderate concentrations. Sequential iterations of softwood (SW) and hardwood (HW) SSL were subsequently inoculated with the two yeast strains in order to compare adaptation to, and performance in lignocellulosic substrates in a cell recycle batch fermentation (CRBF) regime. Both strains were severely affected by the HW SSL, which was attributed to specific syringyl lignin-derived degradation products and synergistic interactions between inhibitors. Though ethanologenic capacity was preserved, a net loss of performance was evident from both strains, indicating the absence of adaptation to the substrates, regardless of the sequence in which the SSL types were employed.     

Fungal metabolism of fermentation inhibitors present in corn stover dilute acid hydrolysate

Use of agricultural residues for ethanol production requires pretreatment of the material to facilitate release of sugars. Physical–chemical pretreatment of lignocellulosic biomass can, however, give rise to side-products that may be toxic to fermenting microorganisms and hinder utilization of sugars obtained from biomass. Potentially problematic compounds include furan aldehydes formed by degradation of sugars, organic acids released from hemicellulose side-groups, and aldehydes and phenolics released from lignin. A fungal isolate, Coniochaeta ligniaria NRRL30616, metabolizes furfural and 5-hydroxymethylfurfural (HMF) as well as aromatic and aliphatic acids and aldehydes. NRRL30616 grew in corn stover dilute-acid hydrolysate, and converted furfural to both furfuryl alcohol and furoic acid. Hydrolysate was inoculated with NRRL30616, and the fate of pretreatment side-products was followed in a time-course study. A number of aromatic and aliphatic acids, aldehydes, and phenolic compounds were quantitated by analytical extraction of corn stover hydrolysate, followed by HPLC–UV–MS/MS analysis. Compounds representing all of the classes of inhibitory side-products were removed during the course of fungal growth. Biological abatement of hydrolysates using C. ligniaria improved xylose utilization in subsequent ethanol fermentations.     

Isolation and characterization of Cupriavidus basilensis HMF14 for biological removal of inhibitors from lignocellulosic hydrolysate

The formation of toxic fermentation inhibitors such as furfural and 5-hydroxy-2-methylfurfural (HMF) during acid (pre-)treatment of lignocellulose, calls for the efficient removal of these compounds. Lignocellulosic hydrolysates can be efficiently detoxified biologically with microorganisms that specifically metabolize the fermentation inhibitors while preserving the sugars for subsequent use by the fermentation host. The bacterium Cupriavidus basilensis HMF14 was isolated from enrichment cultures with HMF as the sole carbon source and was found to metabolize many of the toxic constituents of lignocellulosic hydrolysate including furfural, HMF, acetate, formate and a host of aromatic compounds. Remarkably, this microorganism does not grow on the most abundant sugars in lignocellulosic hydrolysates: glucose, xylose and arabinose. In addition, C. basilensis HMF14 can produce polyhydroxyalkanoates. Cultivation of C. basilensis HMF14 on wheat straw hydrolysate resulted in the complete removal of furfural, HMF, acetate and formate, leaving the sugar fraction intact. This unique substrate profile makes C. basilensis HMF14 extremely well suited for biological removal of inhibitors from lignocellulosic hydrolysates prior to their use as fermentation feedstock.     

Microbial degradation of furanic compounds: biochemistry,genetics, and impact

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid.     

选育耐受复合抑制剂酿酒酵母提高乙醇产量

能够耐受纤维素预处理中抑制剂的酿酒酵母对高效、经济生产纤维素乙醇至关重要。利用诱变结合驯化工程选育了一株可耐受复合抑制剂(1.3g/L糠醛、5.3g/L乙酸及1.0g/L苯酚)的工业酿酒酵母YYJ003。在pH 4.0的含有抑制剂的培养基中,耐受菌株乙醇产率是原始菌株的7.8倍,糠醛转化速率提高了5倍。在pH 5.5的复合抑制剂条件下,YYJ003发酵时间(16h)比野生菌株发酵时间(22h)缩短6h。在pH 4.0的未脱毒的玉米秸秆水热法预处理水解液中YYJ003的乙醇产率达到0.50g/g(乙醇/葡萄糖),乙醇产速达到4.16g/(L·h),而对照菌株无乙醇产出。     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2^N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1^D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2^N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.     

Comparison of glucose/xylose cofermentation of poplar hydrolysates processed by different pretreatment technologies

The inhibitory effects of furfural and acetic acid on the fermentation of xylose and glucose to ethanol in YEPDX medium by a recombinant Saccharomyces cerevisiae strain (LNH‐ST 424A) were investigated. Initial furfural concentrations below 5 g/L caused negligible inhibition to glucose and xylose consumption rates in batch fermentations with high inoculum (4.5–6.0 g/L). At higher initial furfural concentrations (10–15 g/L) the inhibition became significant with xylose consumption rates especially affected. Interactive inhibition between acetic acid and pH were observed and quantified, and the results suggested the importance of conditioning the pH of hydrolysates for optimal fermentation performance. Poplar biomass pretreated by various CAFI processes (dilute acid, AFEX, ARP, SO₂‐catalyzed steam explosion, and controlled‐pH) under respective optimal conditions was enzymatically hydrolyzed, and the mixed sugar streams in the hydrolysates were fermented. The 5‐hydroxymethyl furfural (HMF) and furfural concentrations were low in all hydrolysates and did not pose negative effects on fermentation. Maximum ethanol productivity showed that 0–6.2 g/L initial acetic acid does not substantially affect the ethanol fermentation with proper pH adjustment, confirming the results from rich media fermentations with reagent grade sugars. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis

The inhibitory effect of the main inhibitors (acetic acid, furfural and 5-hydroxymethylfurfural) formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis. Experimental results showed that an increase in acetic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF together with solid fraction. When using only the solid fraction from steam explosion, hydrolysates presented 0.5 g/L of acetic acid, whose fermentations have submitted promising results, providing an ethanol yield of 0.45 g ethanol/g sugars and the final ethanol concentration reached was 12.2 g/L (10.9 g ethanol/100 g DM).     

High temperature dilute acid pretreatment of coastal Bermuda grass for enzymatic hydrolysis

Dilute sulfuric acid was used to pretreat coastal Bermuda grass at high temperature prior to enzymatic hydrolysis. After both pretreatment and enzymatic hydrolysis processes, the highest yield of total sugars (combined xylose and glucose) was 97% of the theoretical value. The prehydrolyzate liquor was analyzed for inhibitory compounds (furfural, hydroxymethylfurfural (HMF)) in order to assess potential risk for inhibition during the following fermentation. Accounting for the formation of the inhibitory compounds, a pretreatment with 1.2% acid at 140 °C for 30 min with a total sugar yield of 94% of the theoretical value may be more favorable for fermentation. From this study, it can be concluded that dilute sulfuric acid pretreatment can be successfully applied to coastal Bermuda grass to achieve high yields of monomeric glucose and xylose with acceptable levels of inhibitory compound formation.     

Effect of pretreatment severity on the conversion of barley straw to fermentable substrates and the release of inhibitory compounds

The production of fermentable substrates from barley straw under various process conditions was studied. Pretreatment included chemical pretreatment with dilute-acid followed by enzymatic hydrolysis; the pretreatment conditions were expressed in a combined severity factor, CS, which ranged in the present study from −1.6 to 1.1. Considering the production of fermentable sugars and the release of inhibitory compounds, the optimal pretreatment conditions were 170 °C, 0% sulfuric acid and 60 min, corresponding to CS −0.4. Under these conditions, 21.4 g glucose/L, 8.5 g xylose/L, and 0.5 g arabinose/L were produced, while 0.1 g HMF/L, 0.4 g furfural/L, 0.0 g levulinic acid/L, 0.0 g formic acid/L, and 2.1 g acetic acid/L were released. The ratio of Σsugars/Σinhibitors proved to be a good tool for evaluating the suitability of a hydrolysate for fermentation purposes.