鸡尾酒δ整合策略构建表达三类纤维素酶的酿酒酵母工程菌株及初步评价

木质纤维素乙醇具有替代化石燃料的潜力,其生产过程包括生物质预处理、纤维素酶生产、水解和发酵等多个步骤。将纤维素酶生产、水解和发酵组合在一起的统合生物加工过程（consolidated bioprocessing,CBP）由于能降低水解和发酵成本而具有应用于纤维素乙醇生产的潜力,该技术的关键是构建能有效降解纤维素的工程菌株,而构建表达纤维素酶的酿酒酵母即是其中一种选择。采用鸡尾酒多拷贝δ整合的策略将7种纤维素酶基因（Trichoderma reesei cbh1、cbh2和egl2,Aspergillus aculeatus cbh1、egl1和bgl1）表达盒整合至酿酒酵母W303-1A染色体上,经4轮整合筛选得到菌株LA1、LA2、LA3和LA4。对这4个菌株进行纤维素酶活性测定,结果表明从LA1到LA3各种纤维素酶活性呈递增趋势,而LA4的酶活性与LA3的酶活水平相当。对菌株LA3进行酸碱预处理玉米芯料的发酵评价,结果表明：①在外加商品化纤维素酶的情况下,与对照菌株W303-1A和AADY相比,LA3能有效利用纤维素料发酵产醇;②与分步整合的菌株W3相比,发酵性能更优;③培养基中的营养成分影响菌株发酵性能。这些结果表明,鸡尾酒δ整合是一种有效的构建酿酒酵母CBP菌株的方法。     

Production of citric acid using its extraction wastewater treated by anaerobic digestion and ion exchange in an integrated citric acid-methane fermentation process

In order to solve the problem of extraction wastewater pollution in citric acid industry, an integrated citric acid-methane fermentation process is proposed in this study. Extraction wastewater was treated by mesophilic anaerobic digestion and then used to make mash for the next batch of citric acid fermentation. The recycling process was done for seven batches. Citric acid production (82.4 g/L on average) decreased by 34.1?% in the recycling batches (2nd–7th) compared with the first batch. And the residual reducing sugar exceeded 40 g/L on average in the recycling batches. Pigment substances, acetic acid, ammonium, and metal ions in anaerobic digestion effluent (ADE) were considered to be the inhibitors, and their effects on the fermentation were studied. Results indicated that ammonium, Na⁺ and K⁺ in the ADE significantly inhibited citric acid fermentation. Therefore, the ADE was treated by acidic cation exchange resin prior to reuse to make mash for citric acid fermentation. The recycling process was performed for ten batches, and citric acid productions in the recycling batches were 126.6 g/L on average, increasing by 1.7 % compared with the first batch. This process could eliminate extraction wastewater discharge and reduce water resource consumption.     

Dry biorefining maximizes the potentials of simultaneous saccharification and co‐fermentation for cellulosic ethanol production

Despite the well‐recognized merits of simultaneous saccharification and co‐fermentation (SSCF) on relieving sugar product inhibition on cellulase activity, a practical concomitance difficulty of xylose with inhibitors in the pretreated lignocellulose feedstock prohibits the essential application of SSCF for cellulosic ethanol fermentation. To maximize the SSCF potentials for cellulosic ethanol production, a dry biorefining approach was proposed starting from dry acid pretreatment, disk milling, and biodetoxification of lignocellulose feedstock. The successful SSCF of the inhibitor free and xylose conserved lignocellulose feedstock after dry biorefining reached a record high ethanol titer at moderate cellulase usage and minimum wastewater generation. For wheat straw, 101.4 g/L of ethanol (equivalent to 12.8% in volumetric percentage) was produced with the overall yield of 74.8% from cellulose and xylose, in which the xylose conversion was 73.9%, at the moderate cellulase usage of 15 mg protein per gram cellulose. For corn stover, 85.1 g/L of ethanol (equivalent to 10.8% in volumetric percentage) is produced with the overall conversion of 84.7% from cellulose and xylose, in which the xylose conversion was 87.7%, at the minimum cellulase usage of 10 mg protein per gram cellulose. Most significantly, the SSCF operation achieved the high conversion efficiency by generating the minimum amount of wastewater. Both the fermentation efficiency and the wastewater generation in the current dry biorefining for cellulosic ethanol production are very close to that of corn ethanol production, indicating that the technical gap between cellulosic ethanol and corn ethanol has been gradually filled by the advancing biorefining technology.     

Citric acid production by recycling its wastewater treated with anaerobic digestion and nanofiltration

We have developed an integrated citric acid-methane fermentation process to solve the problem of wastewater pollution in the citric acid industry. Citric acid wastewater was initially treated by anaerobic digestion. After subsequent ultrafiltration and nanofiltration, the anaerobic digestion effluent (ADE) could be recycled as process water for the next fermentation, maintaining excellent citric acid production efficiency while eliminating wastewater discharge and reducing water consumption. Untreated ADE was not suitable for direct recycling. The effects on citric acid fermentation of components in the ADE were investigated. Production was inhibited when Na⁺ and Mg²⁺ concentrations in recycled ADE were >200 mg/L and >40 mg/L, respectively. This problem was resolved by treating the ADE using ultrafiltration and nanofiltration to reduce Na⁺ and Mg²⁺ concentrations to acceptable levels. Our results prove the technical feasibility of cleaner production in the citric acid industry and provide a strategy for management of wastewater in other submerged fermentation industries.     

Statistical optimization of process conditions for cellulase production by liquid state bioconversion of domestic wastewater sludge

A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.     

Effect of low pH start-up on continuous mixed-culture lactic acid fermentation of dairy effluent

Mixed-culture fermentation that does not require an energy-intensive sterilization process is a viable approach for the economically feasible production of lactic acid (LA) due to the potential use of organic waste as feedstock. This study investigated mixed-culture LA fermentation of whey, a high-strength organic wastewater, in continuous mode. Variations in the hydraulic retention time (HRT) from 120 to 8 h under different pH regimes in two thermophilic reactors (55 °C) were compared for their fermentation performance. One reactor was maintained at a low pH (pH 3.0) during operation at HRTs of 120 to 24 h and then adjusted to pH 5.5 in the later phases of fermentation at HRTs of 24 to 8 h (R1), while the second reactor was maintained at pH 5.5 throughout the experiment (R2). Although the LA production in R1 was negligible at low pH, it increased dramatically after the pH was raised to 5.5 and exceeded that in R2 when stabilized at HRTs of 8 and 12 h. The maximum yield (0.62 g LA/g substrate fed as the chemical oxygen demand (COD) equivalent), the production rate (11.5 g/L day), and the selectivity (95 %) of LA were all determined at a 12-h HRT in R1. Additionally, molecular and statistical analyses revealed that changes in the HRT and the pH significantly affected the bacterial community structure and thus the fermentation characteristics of the experimental reactors. Bacillus coagulans was likely the predominant LA producer in both reactors. The overall results suggest that low pH start-up has a positive effect on yield and selectivity in mixed-culture LA fermentation.     

威兰胶生产废液循环利用工艺

为减少威兰胶生产过程中的废水排放量,针对威兰胶生产中废水形成的主要环节,对乙醇蒸馏母液的循环利用工艺进行初步研究。在分析乙醇蒸馏母液主要成分的基础上,对比威兰胶发酵过程的物质耗用量,以蒸馏母液作为配制培养基用水,确定威兰胶循环发酵培养基的组成,构建威兰胶生产废液循环利用的闭路工艺,达到清洁生产的目的。研究表明,与常规威兰胶发酵相比,采用闭路循环发酵工艺可节省30％KH2PO4及60％MgSO4·7H2O用量,同时产品质量浓度维持在16．8g/L左右,其黏度及流态特性指数n值分别维持在5．1 Pa·s和0．275左右。     

Effective ethanol production by reutilizing waste distillage anaerobic digestion effluent in an integrated fermentation process coupled with both ethanol and methane fermentations

An integrated ethanol–methane fermentation coupled system characterized with full wastewater reutilization was proposed. The waste distillage originated from ethanol distillation was treated with anaerobic digestion and then recycled for medium preparation in the next ethanol fermentation run. This process could enhance wastewater reutilization, save fresh water and reduce energy consumption in the cassava-based ethanol production. The results indicated that, when using anaerobic effluents from the digestion process with only one tank, an ethanol concentration of 10.5% (v/v) compatible with that of conventional one could be achieved, but ethanol fermentation was partially inhibited and operation time gradually prolonged from 48 to 105 h. Using anaerobic effluents from the digestion process with two subsequently connected tanks, ethanol fermentation performance could be largely improved, and the fermentation lag could be completely eliminated. The performance enhancement was due to the concentrations reduction in organic acids, such as acetic and propionic acids in the digestion effluents using two digestion tanks in-series.     

Effect of propionic acid on citric acid fermentation in an integrated citric acid–methane fermentation process

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.     

一株分离自绿化废弃物堆肥枯草芽孢杆菌的常压室温等离子诱变及其发酵条件

【背景】为了提高堆肥降解有机废弃物的效率,高效堆肥菌剂成为了研究热点,其中以真菌应用的研究为多,但真菌也有对氧气和底物敏感等缺点,细菌对堆肥的作用开始被研究。本实验室以羧甲基纤维素钠(CMC-Na)为底物,从绿化废弃物堆肥中筛选得到枯草芽孢杆菌(Bacillussubtilis,B.subtilis) BL03,它具有较好的纤维素分解能力,能提高绿化废弃物堆肥中纤维素降解和腐殖质合成的速度。【目的】进一步提高B.subtilisBL03的纤维素酶生产能力。【方法】利用常压室温等离子(Atmospheric and room temperature plasma,ARTP)诱变BL03菌,通过CMC-刚果红固体培养基观察水解透明圈,以及液体发酵后检测酶活力的方法进行3轮筛选;通过连续多代培养观察突变株的遗传稳定性;通过梯度温度、p H培养研究突变株发酵的最适生长温度、培养基初始pH;利用正交设计方法研究适合突变株发酵培养的工业级原料配方。【结果】筛选到2株正突变株,酶活力分别提高了69%和72%;连续10代培养稳定,验证了突变株的遗传稳定性;其中酶活力最高的突变株BLA3890最适培养温度为37°C、培养基初始pH为5.0-6.5,研究得到较经济的发酵培养基配方。【结论】ARTP诱变B. subtilis BL03后得到的突变株BLA1973和BLA3890在绿化废弃物堆肥或其他纤维素降解行业具有进一步研究和应用的价值。     

Production of xylitol by Saccharomyces cerevisiae using waste xylose mother liquor and corncob residues

Exorbitant outputs of waste xylose mother liquor (WXML) and corncob residue from commercial-scale production of xylitol create environmental problems. To reduce the wastes, a Saccharomyces cerevisiae strain tolerant to WXML was conferred with abilities to express the genes of xylose reductase, a xylose-specific transporter and enzymes of the pentose phosphate pathway. This strain showed a high capacity to produce xylitol from xylose in WXML with glucose as a co-substrate. Additionally, a simultaneous saccharification and fermentation (SSF) process was designed to use corncob residues and cellulase instead of directly adding glucose as a co-substrate. Xylitol titer and the productivity were, respectively, 91.0 g l^-1 and 1.26 ± 0.01 g l^-1 h^-1 using 20% WXML, 55 g DCW l^-1 delignified corncob residues and 11.8 FPU g_cellulose^-1 cellulase at 35° during fermentation. This work demonstrates the promising strategy of SSF to exploit waste products to xylitol fermentation process.     

Comprehensive utilization of waste hemicelluloses during ethanol production to increase lactic acid yield: from pretreatment to fermentation

Background

Reducing the cost of producing cellulosic ethanol is essential for the industrialization of biorefinery. Several processes are currently under investigation, but few of these techniques are entirely satisfactory in terms of competitive cost or environmental impact. In this study, a new ethanol and lactic acid (LA) coproduction is proposed. The technique involved addition of waste alkaline peroxide pretreated hydrolysate (mainly LA and hemicelluloses) to the reaction mixture after ethanol fermentation (mainly LA and xylose) to reduce the ethanol production cost.

Results

The following processes were investigated to optimize LA production: no addition of hemicelluloses or hydrolysate, addition of recycled hemicelluloses, and addition of concentrated hydrolysate. The addition of concentrated hydrolysate at 48 hours, which resulted in a maximum LA concentration of 22.3 g/L, was the most environment-friendly and cost-effective process. After the improved fermentation, 361 mg LA and 132 mg ethanol were produced from 1 g of raw poplar wood. That is, the production of one gallon of ethanol produced $9 worth of LA.

Conclusions

The amount of LA produced from the pretreated hydrolysate and reaction mixture after ethanol fermentation cannot be underestimated. The recovery of hydrolysate rich in LA and hemicelluloses (or xylose) significantly improved LA yield and further reduced the ethanol production cost.

     

Effect of ferrous chloride on biogas production and enzymatic activities during anaerobic fermentation of cow dung and <Emphasis Type="Italic">Phragmites</Emphasis> straw

The effect of ferrous (added as FeCl₂) on the anaerobic co-digestion of Phragmites straw and cow dung was studied by investigating the biogas properties, pH values, organic matter degradation (COD) and enzyme activities (cellulase, protease and dehydrogenase) at different stages of mesophilic fermentation. The results showed that Fe²⁺ addition increased the cumulative biogas yields by 18.1 % by extending the peak period with high daily biogas yields. Meanwhile, the methane (CH₄) contents in the Fe²⁺ added groups were generally higher than the control group before the 15th day. The pH values were not significantly impacted by Fe²⁺ concentrations during the fermentation process. The COD concentrations, cellulase, protease and dehydrogenase activities varied with the added Fe²⁺ concentrations and the stages of the fermentation process. At the beginning stage of fermentation (4th day), Fe²⁺ addition increased the biogas production by improving the cellulase and dehydrogenase activities which caused a decline in COD. At the peak stage of fermentation (8th day), Fe²⁺ addition enhanced the cellulase and protease activities, and resulted in lower COD contents than the control group. When the biogas yields decreased again (13th day), the COD contents varied similar with the protease and dehydrogenase activities, whilst cellulase activities were not sensitive to Fe²⁺ concentrations. At the end of fermentation (26th day), Fe²⁺ addition decreased the cellulase activities, led to lower COD contents and finally resulted the lower biogas yields than the control group. Taking the whole fermentation process into account, the promoting effect of Fe²⁺ addition on biogas yields was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase activities.     

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL⁻¹ reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL⁻¹, 0.426 gg ⁻¹ and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.     

Starch processing wastewater as a new medium for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL⁻¹ and higher entomotoxicity of 1,050 IU μL⁻¹ were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.     

The investigation of effect of organic carbon sources addition in anaerobic–aerobic (low dissolved oxygen) sequencing batch reactor for nutrients removal from wastewaters

The effect of addition of organic carbon sources (acetic acid and waste activated sludge alkaline fermentation liquid) on anaerobic–aerobic (low dissolved oxygen, 0.15–0.45 mg/L) biological municipal wastewater treatment was investigated. The results showed that carbon source addition affected not only the transformations of polyhydroxyalkanoates (PHA), glycogen, nitrogen and phosphorus, but the net removal of nitrogen and phosphorus. The removal efficiencies of TN and TP were, respectively, 61% and 61% without organic carbon source addition, 81% and 95% with acetic acid addition, and 83% and 97% with waste activated sludge alkaline fermentation liquid addition. It seems that the alkaline fermentation liquid of waste biosolids generated in biological wastewater treatment plant can be used to replace acetic acid as an additional carbon source to improve the anaerobic–aerobic (low dissolved oxygen) municipal wastewater nutrients removal although its use was observed to cause a slight increase of effluent BOD and COD concentrations.     

Cellulase production by solid state fermentation on lignocellulosic waste from the xylose industry

Cellulase production was carried out by solid state fermentation using corncob residue, a lignocellulosic waste from the xylose industry, as the substrate of Trichoderma reesei ZU-02. The effects of water content, dosage of wheat bran and initial pH value in solid substrate on cellulase synthesis were studied in shallow tray fermentors. The solid substrate could be reused in at least three batches and the highest cellulase activity (158 IFPU/g koji) was obtained in the second fermentation batch. To produce cellulase on a larger scale, a deep trough fermentor with forced aeration was used and 128 IFPU/g koji (305 IFPU/g cellulose) was reached after 5 days solid state fermentation. The enzyme koji produced in the present process can be used directly to hydrolyze corncob residue effectively, when the cellulase dosage was above 20 IFPU/g substrate, the saccharification yield could be over 84%.     

Novel inducers derived from starch for cellulase production by Trichoderma reesei

Soluble inducers derived from starch were investigated for cellulase production by T. reesei. Various methods of starch treatment were compared. Acid-hydrolysed starch was found to be most effective. When 1·0 g starch was employed for cellulase production, the cellulase so produced after 6 days of fermentation, with supplementation of 1% (0·01 ml/g paper) β-glucosidase, saccharified more than 15 g shredded waste office paper in 9·34% suspension, resulting in 10 g fermentable sugars, 72% of which was glucose. The effectiveness of the starch-derived inducers was compared with that of lactose, pure cellulose, CMC, xylan and prehydrolysate of pine wood. The effects of calcium chloride and sorbose addition on cellulase production, and the kinetics of cellulase production by the starch-derived inducers are presented.     

Microflora from leaf debris is suitable for treatment of starch industry wastewater

Biological treatment of industrial waste is a widely practiced technique that generates comparatively less environmentally hazardous waste than other chemical treatment processes. Wet milling of maize generates huge amount of wastewater (5 m³/ton) of low pH with organic matter and nutrients. Anaerobic methanogenic and aerobic bacteria are mostly highly sensitive to low pH. The treatment of wastewater causes huge cost of chemical neutralization or hydraulic recirculation for maintaining neutral pH. In the present study, different microbial consortia isolated from cow dung, active sludge from an anaerobic reactor for treatment of industrial wastewater, and leaf debris from benthic soil were screened for tolerance against low pH and for potential of chemical oxygen demand (COD) removal in order to find out an alternative microbial population for industrial water treatment at low pH. The most effective consortia found from leaf debris were further investigated for optimal operation. The microscopic analysis of leaf debris sludge showed abundance of Gram‐negative methanococci, which was found tolerant to low pH in plate culture method. On further investigation for COD removal from starch industry effluent, they were found to be most effective at pH 5 with highest COD removal rate of 70% and lowest biomass generation of 81%. Hence, it was concluded that the low pH‐tolerant methanogen bacteria, enriched from leaf debris sludge, is highly beneficial for anaerobic treatment of wastewater from several industries including corn starch industry by reducing cost of operation for neutralization to neutral pH and through reducing excess waste sludge production by the treatment system.     

Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 10⁶ spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.