Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF

Steam-exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam-explosion pretreatment process. The SE temperature and time conditions were 214 degrees C and 6 min, resulting in a log R(0) of 4.13. In enzymatic hydrolysis tests at 45 degrees C, the biomass loading in the bioreactor was 100 g(DW)/L (dry weight) and the enzyme-to-biomass ratio 0.06 g/g(DW). The enzyme activities for endo-glucanase, exo-glucanase, and beta-glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively. The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5-hydroxymethyl furfural (5-HMF), syringaldehyde, 4-hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate. Acetic acid (2 g/L), furfural, 5-HMF, syringaldehyde, 4-hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase. Synergism and cumulative concentration effects of these compounds were not detected. SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used. The presence of acetic acid, vanillin, and 5-HMF (0.5 g/L) in SSF of 100 g(DW) /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.     

The effect of sugar decomposed on the ethanol fermentation and decomposition reactions of sugars

A batch reactor was used to investigate the dilute acid hydrolysis reaction of alpha-cellulose and sugar decomposition reactions. Varying the sulfuric acid concentration from 0.07 to 5.0% for reaction temperatures between 180 and 220°C significantly affected glucose yields, which ranged from about 70% to below 10%. Increasing the reaction temperature enhanced this effect. Similar experimental results were obtained for the decomposition of xylose. For sugar decomposition reactions, less than 0.3 g/L of furfural and 5-hydroxymethylfurfural (5-HMF) were produced from glucose and xylose in the absence of sulfuric acid at 190°C and 15 min of reaction time, but adding a small amount of sulfuric acid (0.5%) dramatically increased the decomposition rate and led to the formation of four undesireable products: formic acid, 5-HMF, acetic acid, and furfural. In both hydrolysis and fermentation reactions formic acid, acetic acid, and 5-HMF severely inhibited ethanol fermentation, while furfural had less of an inhibition effect.     

Study on solvent extraction of propionic acid from simulated discharged water in vitamin B12 production by anaerobic fermentation

The potential of recovering propionic acid from discharged water in vitamin B₁₂ production by anaerobic fermentation was investigated in this paper. A primary amine, N₁₉₂₃, was used as the extractant, kerosene as diluter and n-octanol as modifier. The influences of the content of N₁₉₂₃ in the organic phase, the phase ratio and the pH of aqueous phase on the extraction yield of propionic acid were studied. The organic phase composition with the volume ratio was proposed of N₁₉₂₃:kerosene:n-octanol as 45:35:20. Under conditions of the phase ratio (o/w) as 1:4, the pH of aqueous phase of 3.0 and after 5 min extraction, the extraction yield of propionic acid can be over 97%.     

Onsite bio-detoxification of steam-exploded corn stover for cellulosic ethanol production

In the process of ethanol production from steam-exploded corn stover (SECS), a cellulose-degradation strain of Aspergillus nidulans (FLZ10) was investigated whether it could remove the inhibitors released from steam exploded pretreatment , and thereby be used for biological detoxification on Saccharomycescerevisiae. The results showed that FLZ10 removed 75.2% formic acid, 53.6% acetic acid, and 100% hydroxymethyl furfural (5-HMF) and furfural from the hydrolysate washed from SECS after 72 h cultivation. A cellulase activity of 0.49 IU/ml was simultaneously produced while the biological detoxification occurred. An ethanol yield of 0.45 g/g on glucose was obtained in the hydrolysate biodetoxified by FLZ10. The glucose consumption rate of FLZ10 was much lower than that of S. cerevisiae, thereby it had little competition with S. cerevisiae on glucose consumption. Based on SECS to ethanol mass balance analysis, with the onsite bio-detoxification, fermentation using S. cerevisiae effectively converted monomeric glucose with 94.4% ethanol yield.     

Effects of decomposed products from Japanese cedar hydrolyzates on acetic acid fermentation by Clostridium thermocellum and Moorella thermoacetica (C. thermoaceticum)

Japanese cedar (Cryptomeria japonica) was treated with hot-compressed water and as decomposed products, the following compounds were recovered: furfural, 5-hydroxymethyl furfural, levoglucosan, lactic acid, glycolic acid, coniferyl alcohol, coniferyl aldehyde and vanillin. The impacts and fermentability of these compounds were studied on acetic acid fermentation by the co-culture of Clostridium thermocellum and Moorella thermoacetica. It was found that furfural, 5-HMF and lignin-derived products strongly limited acetic acid production by free cells. Importantly, co-immobilized C. thermocellum and M. thermoacetica expressed increased tolerance towards the decomposed products and successfully provided acetic acid corresponding to 93% of the theoretical maximum from Japanese cedar hydrolyzates.     

Effect of fermentation inhibitors in the presence and absence of activated charcoal on the growth of Saccharomyces cerevisiae

The acidic hydrolysis of biomass generates numerous inhibitors of fermentation, which adversely affect cell growth and metabolism. The goal of the present study was to determine the effects of fermentation inhibitors on growth and glucose consumption by Saccharomyces cerevisiae. We also conducted in situ adsorption during cell cultivation in synthetic broth containing fermentation inhibitors. In order to evaluate the effect of in situ adsorption on cell growth, five inhibitors, namely 5-hydroxymethylfurfural, levulinic acid, furfural, formic acid, and acetic acid, were introduced into synthetic broth. The existence of fermentation inhibitors during cell culture adversely affects cell growth and sugar consumption. Furfural, formic acid, and acetic acid were the most potent inhibitors in our culture system. The in situ adsorption of inhibitors by the addition of activated charcoal to the synthetic broth increased cell growth and sugar consumption. Our results indicate that detoxification of fermentation media by in situ adsorption may be useful for enhancing biofuel production.     

Phosphorous pentoxide mediated synthesis of 5-HMF in ionic liquid at low temperature

A convenient, mild and environment-friendly dehydration reaction of fructose in ionic liquid using phosphorous pentoxide (P₂O₅) has been investigated. The acidic nature of P₂O₅ along with its hygroscopic properties has been successfully utilized to afford 81.2% yield of 5-hydroxymethylfurfural (5-HMF) at 50 °C in 60 mins. Phosphoric acid yielded remarkably less 5-HMF even at higher temperature and longer reaction times. The reaction was optimized by varying different parameters and the results indicated that no rehydration products, such as levulinic acid or formic acid, were formed.     

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 II: production of xylitol and ethanol in the presence of inhibitors

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30?g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5?g/l of furfural stimulated production of xylitol to 77?% of theoretical yield (10?% higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3?g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99?% of theoretical yield (15?% higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5?g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.     

Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production

Microbial lipid production using lignocellulosic biomass is considered an alternative for biodiesel production. In this study, 418 yeast strains were screened to find efficient oleaginous yeasts which accumulated large quantities of lipid when cultivated in lignocellulosic sugars. Preliminary screening by Nile red staining revealed that 142 strains contained many or large lipid bodies. These strains were selected for quantitative analysis of lipid accumulation by shaking flask cultivation in nitrogen-limited medium II containing 70 g/L glucose or xylose or mixture of glucose and xylose in a ratio of 2:1. Rhodosporidium fluviale DMKU-SP314 produced the highest lipid concentration of 7.9 g/L when cultivated in the mixture of glucose and xylose after 9 days of cultivation, which was 55.0% of dry biomass (14.3 g/L). The main composition of fatty acids were oleic acid (40.2%), palmitic acid (25.2%), linoleic acid (17.9%) and stearic acid (11.1%). Moreover, the strain DMKU-SP314 could grow and produce lipid in a medium containing predominantly lignocellulose degradation products, namely, acetic acid, formic acid, furfural, 5-hydroxymethylfurfural (5-HMF) and vanillin, with however, some inhibitory effects. This strain showed high tolerance to acetic acid, 5-HMF and vanillin. Therefore, R. fluviale DMKU-SP314 is a promising strain for lipid production from lignocellulosic hydrolysate.     

Detoxification of corn stover prehydrolyzate by trialkylamine extraction to improve the ethanol production with Pichia stipitis CBS 5776

In order to realize the separated ethanol fermentation of glucose and xylose, prehydrolysis of corn stover with sulfuric acid at moderate temperature was applied, while inhibitors were produced inevitably. A complex extraction was adopted to detoxify the prehydrolyzate before fermentation to ethanol with Pichia stipitis CBS 5776. The best proportion of mixed extractant was 30% trialkylamine-50% n-octanol -20% kerosene. Detoxification results indicated that 73.3% of acetic acid, 45.7% of 5-hydroxymethylfurfural and 100% of furfural could be removed. Compared with the undetoxified prehydrolyzate, the fermentability of the detoxified prehydrolyzate was significantly improved. After 48 h fermentation of the detoxified prehydrolyzate containing 7.80 g/l of glucose and 52.8 g/l of xylose, the sugar utilization ratio was 93.2%; the ethanol concentration reached its peak value of 21.8 g/l, which was corresponding to 82.3% of the theoretical value.     

Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniciproducens

Acetic acid is by-product from fermentation processes for producing succinic acid using Mannheimia succiniciproducens . To obtain pure succinic acid from the final fermentation broth, acetic acid was selectively removed based on the different extractability of succinic acid and acetic acid with pH using tri-n-octylamine (TOA) as extractant. When successive batch extractions were performed using 0.25 mol TOA kg(-1) dissolved in 1-octanol at pH 5, the mol ratio of succinic acid to acetic acid before extraction was 4.9 and the final ratio after the fourth batch was 9.4.     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

Enhancing Corynebacterium glutamicum robustness by over-expressing a gene,mshA, for mycothiol glycosyltransferase

Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H₂O₂ (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml^?1), streptomycin (0.005 mg ml^?1), Cd²⁺ (0.01 mM), Mn²⁺ (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals.     

Inactivation of the transcription factor <Emphasis Type="Italic">mig1</Emphasis> (<Emphasis Type="Italic">YGL035C</Emphasis>) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves tolerance towards monocarboxylic weak acids: acetic,formic and levulinic acid

Toxic concentrations of monocarboxylic weak acids present in lignocellulosic hydrolyzates affect cell integrity and fermentative performance of Saccharomyces cerevisiae. In this work, we report the deletion of the general catabolite repressor Mig1p as a strategy to improve the tolerance of S. cerevisiae towards inhibitory concentrations of acetic, formic or levulinic acid. In contrast with the wt yeast, where the growth and ethanol production were ceased in presence of acetic acid 5 g/L or formic acid 1.75 g/L (initial pH not adjusted), the m9 strain (Δmig1::kan) produced 4.06?±?0.14 and 3.87?±?0.06 g/L of ethanol, respectively. Also, m9 strain tolerated a higher concentration of 12.5 g/L acetic acid (initial pH adjusted to 4.5) without affecting its fermentative performance. Moreover, m9 strain produced 33% less acetic acid and 50–70% less glycerol in presence of weak acids, and consumed acetate and formate as carbon sources under aerobic conditions. Our results show that the deletion of Mig1p provides a single gene deletion target for improving the acid tolerance of yeast strains significantly.     

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.     

发酵抑制物对絮凝酵母戊糖发酵的影响

将絮凝剂加入酵母溶液中,使酵母絮凝成颗粒以此作为固定化酵母进行戊糖发酵。研究了常见发酵抑制物(甲酸、乙酸、糠醛和乳酸等)对絮凝酵母发酵木糖的影响。结果表明:在60.0g/L木糖发酵液中,经过24h发酵,木糖利用率达94.6%,当分别添加抑制物甲酸、乙酸、糠醛、乙醇和乳酸时,聚氧乙烯絮凝酵母分别对其的耐受浓度为0.5、0.5、1.0、30.0和8.0g/L。当抑制物添加量超过各自的耐受浓度后,对絮凝酵母发酵会产生明显的抑制作用。     

Effects of Ca(OH)(2) treatments ("overliming") on the composition and toxicity of bagasse hemicellulose hydrolysates

Hemicellulose syrups from dilute sulfuric acid hydrolysates of hemicellulose contain inhibitors that prevent efficient fermentation by yeast or bacteria. It is well known that the toxicity of these hydrolysate syrups can be ameliorated by optimized "overliming" with Ca(OH)(2). We have investigated the optimization of overliming treatments for sugar cane bagasse hydrolysates (primarily pentose sugars) using recombinant Escherichia coli LY01 as the biocatalyst. A comparison of composition before and after optimal overliming revealed a substantial reduction in furfural, hydroxymethylfurfural, and three unidentified high-performance liquid chromatography (HPLC) peaks. Organic acids (acetic, formic, levulinic) were not affected. Similar changes have been reported after overliming of spruce hemicellulose hydrolysates (Larsson et al., 1999). Our studies further demonstrated that the extent of furan reduction correlated with increasing fermentability. However, furan reduction was not the sole cause for reduced toxicity. After optimal overliming, bagasse hydrolysate was rapidly and efficiently fermented (>90% yield) by LY01. During these studies, titration, and conductivity were found to be in excellent agreement as methods to estimate sulfuric acid content. Titration was also found to provide an estimate of total organic acids in hydrolysate, which agreed well with the sum of acetic, levulinic, and formic acids obtained by HPLC. Titration of acids, measurement of pH before and after treatment, and furan analyses are proposed as relatively simple methods to monitor the reproducibility of hydrolysate preparations and the effectiveness of overliming treatments.     

Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria

Summary A survey was conducted with seventeen enteric bacterial strains (including the generaKlebsiella, Enterobacter, Escherichia, Citrobacter, Edwardsiella andProteus) to examine their ability to transform furfural and 5-hydroxymethyl furfural (5-MHF). The enteric bacteria were able to convert furfural to furfuryl alcohol under both aerobic and anaerobic conditions in a relatively short incubation time of 8 h. 5-HMF was transformed by all the enteric bacteria studied to an unidentified compound postulated to be 5-hydroxymethyl furfuryl alcohol, which had an absorbance maximum of 222 nm. These bacteria did not transform furfuryl alcohol or 2-furoic acid. The enteric bacteria did not use furfural, 5-HMF, furfuryl alcohol or 2-furoic acid as sole source of carbon and energy. Biotransformation of furfural and 5-HMF was accomplished by co-metabolism in the presence of glucose and peptone as main substrates. The rate of transformation was similar under both aerobic and anaerobic conditions. These transformations are likely to be of value in the detoxification of furfurals, and in their ultimate conversion to methane and CO₂ by anaerobic digestion.     

A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in <Emphasis Type="Italic">Thermoanaerobacter pseudethanolicus</Emphasis> 39E

Background

Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.

Results

Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

Conclusions

T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.