玉米秸秆酸解副产物对重组酿酒酵母6508-127发酵的影响

将木质纤维素类生物质如玉米秸秆等用稀酸水解预处理,在半纤维素水解为单糖的同时,水解液中还会产生一些可能对后续发酵有影响的副产物。本实验分别考查了在玉米秸秆稀酸水解液中检测出的乙酸、甲酸、香草醛、糠醛和羟甲基糠醛对重组木糖发酵菌株S. cerevisiae 6508-127生长和发酵的影响。结果表明,甲酸和乙酸对菌体生长的抑制强于乙醇生成,且甲酸的抑制程度远大于乙酸;2g/L香草醛可使菌体生长延滞期明显延长,而在较低浓度（≤1.2g/L）此现象不明显。糠醛在0.5-1.5g/L范围内对菌体生长有抑制作用,但使乙醇得率提高;羟甲基糠醛在0.2g/L浓度存在就使乙醇得率有明显降低,但使生物量得率提高;研究中还发现,糠醛、羟甲基糠醛和香草醛可被S. cerevisiae 6508-127代谢。     

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.     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。     

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.     

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.     

Effect of binary combinations of selected toxic compounds on growth and fermentation of Kluyveromyces marxianus

The inhibitory effects of various lignocellulose degradation products on glucose fermentation by the thermotolerant yeast Kluyveromyces marxianus were studied in batch cultures. The toxicity of the aromatic alcohol catechol and two aromatic aldehydes (4-hydroxybenzaldehyde and vanillin) was investigated in binary combinations. The aldehyde furfural that usually is present in relatively high concentration in hydrolyzates from pentose degradation was also tested. Experiments were conducted by combining agents at concentrations that individually caused 25% inhibition of growth. Compared to the relative toxicity of the individual compounds, combinations of furfural with catechol and 4-hydroxybenzaldehyde were additive (50% inhibition of growth). The other binary combinations assayed (catechol with 4-hydroxybenzaldehyde, and vanillin with catechol, furfural, or 4-hydroxybenzaldehyde) showed synergistic effect on toxicity and caused a 60-90% decrease in cell mass production. The presence of aldehydes in the fermentation medium strongly inhibited cell growth and ethanol production. Kluyveromyces marxianus reduces aldehydes to their corresponding alcohols to mitigate the toxicity of these compounds. The total reduction of aldehydes was needed to start ethanol production. Vanillin, in binary combination, was dramatically toxic and was the only compound for which inhibition could not be overcome by yeast strain assimilation, causing a 90% reduction in both cell growth and fermentation.     

EXTRACTIVE FERMENTATION OF GIBBERELLIC ACID WITH FREE AND IMMOBILIZED Gibberella fujikuroi

Gibberelic acid fermentation using extractive methods was carried out in the presence of corn oil and Alamine 336. Gibberella fujikuroi fungus (NRRL 2278) was used to produce gibberellic acid. Oleyl alcohol was a diluting agent for Alamine 336. The effects of oleyl alcohol (100%, v/v), corn oil (5–25%, v/v), the concentration of Alamine 336 in oleyl alcohol, and feeding air were examined in this study. According to the results, oleyl alcohol was not effective on the production. On the other hand, oleyl alcohol solutions containing 15–30% (v/v) Alamine 336 showed effects as a toxic substance. In order to reduce solvent toxicity, corn oil was used. Addition of corn oil increased the concentration of gibberellic acid 1.3-fold compared to the control. Then the effects of immobilization and co-immobilization on extractive gibberelic acid fermentation were investigated. The highest total gibberellic acid concentration of 158.9 mg/L was produced with immobilized cells and feeding air by using extractive fermentation. The yield of gibberellic acid increased about 2.6-fold compared with the shake-flask fermentation (60.5 mg/L) without organic solutions.     

Selection of organic solvents for in situ extraction of fermentation products fromClostridium thermohydrosulfuricum cultures

Summary Fifteen organic solvents were examined to determine their biocompatibility for in situ extraction of fermentation products from cultures of the thermophilic anaerobeClostridium thermohydrosul furicum. Five solvents (hexadecane, isooctane, kerosene, oleyl alcohol, Shellsol TD) were found to be non-toxic toClostridium thermohydrosul furicum. Interfacial tensions, phase separation and partition coefficients for ethanol of the biocompatible solvents were compared. With the exception of kerosene, these solvents showed good separation from the aqueous phase. Oleyl alcohol had the highest partition coefficient for ethanol (K_D=0.34 at 65°C) and appears to be suitable for extractive ethanol fermentation.     

Concentration-driven reverse membrane bioreactor for the fermentation of highly inhibitory lignocellulosic hydrolysate

Optimal production of lignocellulosic bioethanol is hindered due to commonly faced issues with the presence of inhibitory compounds and sequentially consumed sugars in the lignocellulosic hydrolysate. Therefore, in order to find a robust fermentation approach, this study aimed at enhancing simultaneous co-assimilation of sugars, and inhibitor tolerance and detoxification. Therefore, fermentation of toxic wheat straw hydrolysate containing up to 20 g/l furfural, using the concentration-driven diffusion-based technique of reverse membrane bioreactor (rMBR) was studied. The rMBR fermentation of the hydrolysate led to complete furfural detoxification and the conversion of 87 % of sugars into ethanol at a yield of 0.48 g/g. Moreover, when the toxicity level of the hydrolysate was increased to 9 g/l of initial furfural, the system responded exceptionally by reducing 89 % of the inhibitor while only experiencing about 25 % drop in the ethanol yield. In addition, using this diffusion-based set-up in extremely inhibitory conditions (16 g/l furfural), cells could detoxify 40 % of the furfural at a high initial furfural to cell ratio of 9.5:1. The rMBR set-up applied proved that by properly synchronizing the medium condition, membrane area, and inhibitor to cell ratio, some of the shortcomings with conventional lignocellulosic fermentation can be tackled, guaranteeing a robust fermentation.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric 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.     

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

Stillage backset and its impact on ethanol fermentation by the flocculating yeast

The second largest cost in fuel ethanol production is from energy consumption with ethanol distillation and stillage treatment, particularly when stillage is treated by the multi-evaporation process. Therefore, stillage backset is the most economically competitive strategy for reducing discharge and saving energy consumption. In this article, continuous ethanol fermentation was performed by the flocculating yeast under stillage backset conditions. Compared to regular yeast, immobilized yeast within the fermentor through flocculation reduced byproducts formation in the stillage, since heat lysis of yeast during ethanol distillation was prevented, and many side reactions were thus eliminated, making more stillage backset within the fermentation system possible. Although pyruvic acid, succinic acid, citric acid, α-ketoglutaric acid, fumaric acid and glycerol from yeast metabolism, furfural and 5-hydroxymethyl furfural from process operations, and acetic acid and lactic acid from slight contamination were accumulated with the stillage backset, they had no significant impact on yeast growth and ethanol fermentation due to low concentrations accumulated within the fermentation system. However, propionic acid that was generated mainly during hydrolysate sterilization and distillation of the fermentation broth was detected as the major inhibitor, but this byproduct would be significantly reduced under industrial conditions without hydrolysate sterilization, making the stillage backset more reliable for industrial application.     

Ethanol recovery from corn fiber hydrolysate fermentations by pervaporation

Corn fiber, a byproduct of corn wet milling, is an attractive feedstock for biomass ethanol production. Corn fiber was hydrolyzed by dilute sulfuric acid and neutralized by one of two methods: conventional lime treatment or neutralization by strongly basic anion exchange. The anion exchange neutralized (AEN) hydrolysate contained substantially lower levels of the inhibiting compounds furfural, 5-hydroxymethylfurfural, and acetic acid compared to the lime neutralized hydrolysate. In batch fermentations the ethanol yields and final ethanol concentration of the two hydrolysates were similar at 0.32-0.43 g/g and 29-44 g/l, respectively. Sugar consumption in the AEN fermentations was superior. Coupling of a membrane pervaporation unit to a fed-batch fermentation of AEN hydrolysate maintained the ethanol concentration below 25 g/l with complete sugar utilization for approximately 5 days. A concentrated ethanol stream of 17 wt.% ethanol was produced by the pervaporation unit.     

Extractive fermentation for lactic acid production

Lactic acid extractive fermentation was demonstrated using Alamine 336 in oleyl alcohol at acidic pH. The use of an efficient extraction system was possible through employment of the cell immobilization procedure. Process modeling was performed to relate the various process parameters such as flow rate, concentration, and pH. In experiments with 15% Alamine 336/oleyl alcohol, the bioreactor operation resulted in a higher productivity (12 g/L gel h) compared to that of a control fermentation (7 g/L gel h). Strategies for optimizing the extractive fermentation process were proposed considering both productivity and product recovery.     

Potential inhibitors from wet oxidation of wheat straw and their effect on ethanol production of Saccharomyces cerevisiae: wet oxidation and fermentation by yeast

Alkaline wet oxidation (WO) (using water, 6.5 g/L sodium carbonate and 12 bar oxygen at 195 degrees C) was used as pretreatment method for wheat straw (60 g/L), resulting in a hydrolysate and a cellulosic solid fraction. The hydrolysate consisted of soluble hemicellulose (8 g/L), low-molecular-weight carboxylic acids (3.9 g/L), phenols (0.27 g/L = 1.7 mM) and 2-furoic acid (0.007 g/L). The wet oxidized wheat straw hydrolysate caused no inhibition of ethanol production by Saccharomyces cerevisiae ATCC 96581. Nine phenols and 2-furoic acid, identified to be present in the hydrolysate, were each tested in concentrations of 50-100 times the concentration found in the hydrolysate for their effect on fermentation by yeast. At these high concentrations (10 mM), 4-hydroxybenzaldehyde, vanillin, 4-hydroxyacetophenone and acetovanillone caused a 53-67% decrease in the volumetric ethanol productivity in S. cerevisiae compared to controls with an ethanol productivity of 3.8 g/L. The phenol acids (4-hydroxy, vanillic and syringic acid), 2-furoic acid, syringaldehyde and acetosyringone were less inhibitory, causing a 5-16% decrease in ethanol productivity. By adding the same aromatic compounds to hydrolysate (10 mM), it was shown that syringaldehyde and acetovanillone interacted negatively with hydrolysate components on the ethanol productivity. Fermentation in WO hydrolysate, that had been concentrated 6 times by freeze-drying, lasted 4 hours longer than in regular hydrolysate; however, the ethanol yield was the same. The longer fermentation time could not be explained by an inhibitory action of phenols alone, but was more likely caused by inhibitory interactions of phenols with carboxylic acids, such as acetic and formic acid.     

Effect of selected aldehydes found in the corncob hemicellulose hydrolysate on the growth and xylitol fermentation of Candida tropicalis

The effects of four aldehydes (furfural, 5‐hydroxymethylfurfural, vanillin and syringaldehyde), which were found in the corncob hemicellulose hydrolysate, on the growth and xylitol fermentation of Candida tropicalis were investigated. The results showed that vanillin was the most toxic aldehyde for the xylitol fermentation, followed by syringaldehyde, furfural and 5‐hydroxymethylfurfural. Moreover, the binary combination tests revealed that furfural amplified the toxicity of other aldehydes and the toxicities of other binary combinations without furfural were simply additive. Based on the fermentation experiments, it was demonstrated that the inhibition of aldehydes could be alleviated by prolonging the fermentation incubation, increasing the initial cell concentration, enhancing the initial pH value and minimizing the furfural levels in the hydrolysate evaporation process. The strategies that we proposed to suppress the inhibitory effects of the aldehydes successfully avoided the complicated and costly detoxifications. Our findings could be potentially adopted for the industrial xylitol fermentation from hydrolysates. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1181–1189, 2013     

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。     

用非常规培养方法提高乳酸菌产酸率的研究

以德氏乳酸杆菌为研究对象,考察了八种有机溶剂分别加入培养基对细菌生长及产酸的影响。结果表明,采用油醇和三辛胺混合溶剂时,既能降低对细胞生长的毒性,又保持了较强的萃取能力。对悬浮细胞发酵和萃取整合的方法和固定化细胞发酵和萃取整合的方法进行了比较,表明这两种方法均较常规培养方法提高乳酸产率60％以上。     

Effect of furfural addition on validamycin-A production in fermentation of Streptomyces hygroscopicus 5008

Furfural is one of main inhibitors in hemicellulose hydrolysates such as xylose mother liquor, but its positive effect on the production of validamycin-A (VAL-A), a widely used agricultural antibiotic, was interestingly found in fermentation of Streptomyces hygroscopicus 5008. The furfural level in medium up to 1 g/L was effectively converted to furfuryl alcohol and furoic acid by the microorganism. Both intracellular H₂O₂ level and ValG enzyme activity of the cells were enhanced by furfural addition. Xylose mother liquor medium with supplementation of about 1 g/L furfural could enhance the VAL-A titer by 39 %. This work is helpful to VAL-A fermentation using the hemicellulose hydrolysate.