High‐solids biphasic CO2–H2O pretreatment of lignocellulosic biomass

A high pressure (200 bar) CO₂–H₂O process was developed for pretreating lignocellulosic biomass at high‐solid contents, while minimizing chemical inputs. Hardwood was pretreated at 20 and 40 (wt.%) solids. Switchgrass, corn stover, big bluestem, and mixed perennial grasses (a co‐culture of big bluestem and switchgrass) were pretreated at 40 (wt.%) solids. Operating temperatures ranged from 150 to 250°C, and residence times from 20 s to 60 min. At these conditions a biphasic mixture of an H₂O‐rich liquid (hydrothermal) phase and a CO₂‐rich supercritical phase coexist. Following pretreatment, samples were then enzymatically hydrolyzed. Total yields, defined as the fraction of the theoretical maximum, were determined for glucose, hemicellulose sugars, and two degradation products: furfural and 5‐hydroxymethylfurfural. Response surfaces of yield as a function of temperature and residence time were compared for different moisture contents and biomass species. Pretreatment at 170°C for 60 min gave glucose yields of 77%, 73%, and 68% for 20 and 40 (wt.%) solids mixed hardwood and mixed perennial grasses, respectively. Pretreatment at 160°C for 60 min gave glucan to glucose yields of 81% for switchgrass and 85% for corn stover. Biotechnol. Bioeng. 2010;107: 451–460. © 2010 Wiley Periodicals, Inc.     

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.     

Comparative evaluations of cellulosic raw materials for second generation bioethanol production

Aims: To evaluate sugar recoveries and fermentabilities of eight lignocellulosic raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis. Methods and Results: Dilute acid pretreatment (2% H₂SO₄) with 10% (w/v) substrate loading was performed at 134°C for 60 min followed by enzyme hydrolysis at 60°C. The results demonstrated that hydrolysis of herbaceous raw materials resulted in higher sugar recoveries (up to 60–75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 (pZB5) demonstrated that final ethanol concentrations and yields were also higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50°C compared to 60°C following acid pretreatment. Conclusions: Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates. Significance and Impact of the Study: The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural/forestry biomass rather than food‐based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.     

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.     

Pseudo reaction kinetics of organic degradation products in dilute-acid-catalyzed corn stover pretreatment hydrolysates

A variety of degradation products are produced upon pretreatment of lignocellulosic biomass with dilute acid. To date, the complexity of these samples has significantly limited the scope of efforts to perform summative analyses of degradation products. Qualitative and quantitative interrogation of hydrolysates is also paramount to identifying potential correlations between pretreatment chemistry and microbial inhibition in downstream bioconversion processes. A recently developed reversed-phase high performance liquid chromatography technique with UV detection has been applied to perform quantitative assessments of a variety of hydrolysate components as a function of pretreatment time and temperature. Correlations of product concentrations to the pretreatment severity function indicate differing responses of various compounds to the kinetic influences of temperature and reaction time. Of the compounds measured, four demonstrated initial accumulation rates were sufficiently linear over the time period tested to enable determination of activation energy E(a). All four compounds appear to demonstrate higher E(a) than that assumed in the commonly applied severity function. Overall accumulation trends for most compounds indicated similar under-estimation of apparent activation energy by the severity function. Biotechnol. Bioeng. 2007;98: 1135-1145. (c) 2007 Wiley Periodicals, Inc.     

Butanol production from sweet sorghum bagasse with high solids content: Part I—comparison of liquid hot water pretreatment with dilute sulfuric acid

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H₂SO₄ (2 g L^?1) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250–300 g L^?1. This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L^?1 sugars, 7.60 g L^?1 acetic acid, 0.33 g L^?1 furfural, and 0.07 g L^?1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L^?1 additional sugars, 2.40 g L^?1 acetic acid, zero g L^?1 furfural, and zero g L^?1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone‐butanol‐ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L^?1 ABE of which butanol was the main product. Use of 2 g L^?1 H₂SO₄ as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6–93.8% (w/w) sugars from 250 to 300 g L^?1 SSB, respectively. LHW or dilute H₂SO₄ were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960–966, 2018     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

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     

Cofactor Dependence in Furan Reduction by Saccharomyces cerevisiae in Fermentation of Acid-Hydrolyzed Lignocellulose

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.     

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.     

Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain

The production of fuel ethanol from low‐cost lignocellulosic biomass currently suffers from several limitations. One of them is the presence of inhibitors in lignocellulosic hydrolysates that are released during pre‐treatment. These compounds inhibit growth and hamper the production of ethanol, thereby affecting process economics. To delineate the effects of such complex mixtures, we conducted a chemical analysis of four different real‐world lignocellulosic hydrolysates and determined their toxicological effect on yeast. By correlating the potential inhibitor abundance to the growth‐inhibiting properties of the corresponding hydrolysates, we identified furfural as an important contributor to hydrolysate toxicity for yeast. Subsequently, we conducted a targeted evolution experiment to improve growth behaviour of the half industrial Saccharomyces cerevisiae strain TMB3400 in the hydrolysates. After about 300 generations, representative clones from these evolved populations exhibited significantly reduced lag phases in medium containing the single inhibitor furfural, but also in hydrolysate‐supplemented medium. Furthermore, these strains were able to grow at concentrations of hydrolysates that effectively killed the parental strain and exhibited significantly improved bioconversion characteristics under industrially relevant conditions. The improved resistance of our evolved strains was based on their capacity to remain viable in a toxic environment during the prolonged, furfural induced lag phase.     

Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.     

Differential effects of mineral and organic acids on the kinetics of arabinose degradation under lignocellulose pretreatment conditions

Sugar degradation occurs during acid-catalyzed pretreatment of lignocellulosic biomass at elevated temperatures, resulting in degradation products that inhibit microbial fermentation in the ethanol production process. Arabinose, the second most abundant pentose in grasses like corn stover and wheat straw, degrades into furfural. This paper focuses on the first-order rate constants of arabinose (5 g/L) degradation to furfural at 150 and 170 °C in the presence of sulfuric, fumaric, and maleic acid and water alone. The calculated degradation rate constants (k_d) showed a correlation with the acid dissociation constant (pK_a), meaning that the stronger the acid, the higher the arabinose degradation rate. However, de-ionized water alone showed a catalytic power exceeding that of 50 mM fumaric acid and equaling that of 50 mM maleic acid. This cannot be explained by specific acid catalysis and the shift in pK_w of water at elevated temperatures. These results suggest application of maleic and fumaric acid in the pretreatment of lignocellulosic plant biomass may be preferred over sulfuric acid. Lastly, the degradation rate constants found in this study suggest that arabinose is somewhat more stable than its stereoisomer xylose under the tested conditions.     

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.     

Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid

This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.     

Hydrolysates of lignocellulosic materials for biohydrogen production

Lignocellulosic materials are commonly used in bio-H₂ production for the sustainable energy resource development as they are abundant, cheap, renewable and highly biodegradable. In the process of the bio-H₂ production, the pretreated lignocellulosic materials are firstly converted to monosaccharides by enzymolysis and then to H₂ by fermentation. Since the structures of lignocellulosic materials are rather complex, the hydrolysates vary with the used materials. Even using the same lignocellulosic materials, the hydrolysates also change with different pretreatment methods. It has been shown that the appropriate hydrolysate compositions can dramatically improve the biological activities and bio-H₂ production performances. Over the past decades, hydrolysis with respect to different lignocellulosic materials and pretreatments has been widely investigated. Besides, effects of the hydrolysates on the biohydrogen yields have also been examined. In this review, recent studies on hydrolysis as well as their effects on the biohydrogen production performance are summarized. [BMB Reports 2013; 46(5): 244-251]     

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.     

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions

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

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.     

Temporal quantitative proteomics of Saccharomyces cerevisiae in response to a nonlethal concentration of furfural

Furfural, one of the main inhibitory compounds in lignocellulosic hydrolytes, inhibits the growth and ethanol production rate of yeast. To get a global view of the dynamic expression pattern of proteins in Saccharomyces cerevisiae during the fermentation with the introduction of 8 g/L furfural, the protein samples were taken before the addition of furfural, during the initial phase of furfural conversion and immediately after the conversion of furfural for comparative proteomic analysis with iTRAQ on a LC‐ESI‐MS/MS instrument. A comparison of the temporal expression pattern of 107 proteins related to protein synthesis between the reference cultures and the furfural‐treated cultures showed that a temporal downregulation of these proteins was retarded after the addition of furfural. The expression levels of 20 enzymes in glucose fermentation and 5 enzymes in the tricarboxylic acid cycle were reduced by furfural, with notably delayed temporal downregulations of Glk1p, Tdh1p, Eno1p and Aco1p, which is correlated to the reduced ethanol formation rate and glucose consumption rate by 66.7 and 60.4%, respectively. In the presence of furfural, proteins catalyzing the upper part of the super pathway of sulfur amino acid biosynthesis were repressed at all time points, which is related to the inhibited growth of furfural‐treated yeast. The expressions of 18 proteins related to stress response showed increased trends, including several highly induced heat shock proteins and proteins related to cellular signaling pathways.