Optimization of rice wine fermentation process based on the simultaneous saccharification and fermentation kinetic model☆

Chinese rice wine making is a typical simultaneous saccharification and fermentation(SSF) process.During the fermentation process,temperature is one of the key parameters which decide the quality of Chinese rice wine.To optimize the SSF process for Chinese rice wine brewing,the effects of temperature on the kinetic parameters of yeast growth and ethanol production at various temperatures were determined in batch cultures using a mathematical model.The kinetic parameters as a function of temperature were evaluated using the software Origin8.0.Combing these functions with the mathematical model,an appropriate form of the model equations for the SSF considering the effects of temperature were developed.The kinetic parameters were found to fit the experimental data satisfactorily with the developed temperature-dependent model.The temperature profile for maximizing the ethanol production for rice wine fermentation was determined by genetic algorithm.The optimum temperature profile began at a low temperature of 26 °C up to 30 h.The operating temperature increased rapidly to 31.9 °C,and then decreased slowly to 18 °C at 65 h.Thereafter,the temperature was maintained at18 °C until the end of fermentation.A maximum ethanol production of 89.3 g·L~(-1)was attained.Conceivably,our model would facilitate the improvement of Chinese rice wine production at the industrial scale.     

Enhanced cold active lipase production by metagenomic library recombinant clone CALIP3 with a step-wise temperature and dissolved oxygen level control strategy

A metagenomic library recombinant clone CAPL3, an Escherichia coli strain generated by transformed with metagenomic library from deep-sea sediments, can efficiently produce cold active lipase. The effects of both temperature and dissolved oxygen(DO) on cold active lipase production by batch culture of metagenomic library recombinant clone(CAPL3) from deep-sea sediment were investigated. First, a two-stage temperature control strategy was developed, in which the temperature was kept at 34 °C for the first 15 h, and then switched to30 °C. The cold active lipase activity and productivity reached 315.2 U·ml~(-1)and 8.08 U·ml~(-1)·h~(-1), respectively,increased by both 14.5% compared to the results obtained with temperature controlled at 30°C. In addition, different DO control modes were conducted, based on the data obtained from the different DO control strategies and analysis of kinetics parameters at different DO levels. A step-wise temperature and DO control strategy were developed to improve lipase production, i.e., temperature and DO level were controlled at 34 °C, 30% during 0–15 h;30 °C, 30% during 15–18 h, and 30 °C, 20% during 18–39 h. With this strategy, the maximum lipase activity reached 354.6 U·ml~(-1)at 39 h, which was 28.8% higher than that achieved without temperature and DO control(275.3 U·ml~(-1)).     

Anaerobic hydrogen production of molasses from mixed microbial communities immobilized by activated granular carbon

Molasses wastewater was evaluated as substrate for biohydrogen production by anaerobic fermentation in a novel continuous mixed attached growth reactor ( CMAGR ) with aeration pretreated sludge attached onto granular activated carbon under continuous flow condition.It was indicated that the CMAGR system was operated at the conditions of influent COD of 2000～6000mg / L , hydraulic retention time ( HRT ) of 6hand temperature of 35 ℃ , when the pH value and oxidation-reduction potential ( ORP ) ranged from 4.16and-434 mV respectively , stable ethanol-type fermentation was formed with the sum of ethanol and acetate concentration ratio of 89.3%to the total liquid products after 40days operation.The H 2 content in biogas and chemical oxygen demand ( COD ) removal were estimated to be 46.6% and 13% , respectively.It was also investigated that the effects of organic loading rates ( OLRs ) on CMAGR hydrogen production system.It was found that hydrogen production yield increased from 3.72 mmol / hL to 12.51 mmol / hL as OLRs increased from 8 kg / m 3 d to 32 kg / m 3 d.The maximum hydrogen production rate of 12.51mmol / hL at a OLR of 32kg / m 3 d and the maximum hydrogen yield by substrate consumed was 130.57 mmol / mol happened at OLR of 16 kg / m 3 d.Greater pHs appeared to be favour to butyrate production and the maximum of 0.51mol / mol was obtained at pH of 4.14.However , ethanol / acetate ratio was greater than 1.1at pH fluctuated between 3.4 - 3.6and 4.1 - 4.4which indicated that these pHs were favour to ethanol type fermentation.Therefore , the continuous mixed attached growth reactor ( CMAGR ) could be a promising attached growth system for biohydrogen fermentation.     

重组尿苷磷酸化酶的诱导及其在嘧啶核苷生物合成中的应用

Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase(UPase),was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth.The lactose-induced UPase was stable at 50°C.Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L 1uridine and 5-fluorouracil in phosphate buffer(pH 7.0)catalyzed at 50°C for 1.5 h and the yield of 5-fluorouridine was higher than 68%.Under the optimum reaction conditions for production of 5-fluorouridine,5-methyluridine and azauridine were synthesized from uridine by pUDP,the yield was 61.7%and 47.2%respectively.Deoxynucleosides were also synthesized by pUDP,but the yield was only about 20%.     

枯草芽孢杆菌SL-13的生防性能及其抗菌几丁质酶特性研究

The seed germination and tomato seedling tests showed that Bacillus subtilis SL-13 could promote the sprouting and seedling growth of tomato.The fresh and dry weight of tomato seedlings increased 42.86%and 18.75%,respectively.The control efficacies of the SL-13 to tomato Rhizoctonia rot were 20.65%and 35.23%in the greenhouse and field,respectively.The growth of the plant-pathogenic fungus Rhizoctonia solani was considerably inhibited in the presence of the strain SL-13 culture supernatant.The main antifungal protein was detected to be chitinase through vitro assay.The chitinase was purified with DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration for further characterization.The optimal pH and temperature for the chitinase activity were 7.0 and 50°C,respectively.It was demonstrated that the enzyme was stable at pH 5-9 and 40-60°C.70%of the enzyme activity was retained when incubated at 121°C and 0.11 MPa for 20 min,and the enzyme was not sensitive to protease K and ultraviolet radiation.Thus it is suitable for effective biological control in relatively unstable environment.     

The influence mechanism of solvent on the hydrogenation of dimethyl oxalate

The hydrogenation of dimethyl oxalate(DMO) for the producing of C2-C4 alcohols with methanol as solvent was researched at the temperature of 270 °C to 310 °C. Ethylene glycol(EG) was the main product at low temperature and the selectivity of which was 61.9% at 230 °C. However, EG selectivity decreased sharply with the increase of temperature while ethanol became the main liquid products with the selectivity of 43.5% at 270 °C. It can be ascribed to a thorough hydrogenation of DMO at a high temperature. In addition, the promotion of Guerbet reaction led to the production of propanol and butanol. Simultaneously, the amount of gas products including CO, CO_2 and dimethyl ether(DME) also increased, which became a competition factor in the conversion of DMO to liquid products including C2-C4 alcohols. The blank test was carried out with pure methanol as feedstock with and without Cu/SiO_2 catalyst, which revealed that methanol was involved in the formation of gas products and higher alcohols on Cu-based catalyst, and the main gas product was CO.     

葡萄糖及磷酸盐对刺糖多孢菌多杀菌素发酵的影响

The effects of glucose and inorganic phosphate on mycelium growth and spinosad production with Saccharopolyspora spinosa were studied. The results showed that the increase of glucose concentration from 18.6g·L-1 to 58.8g·L-1 could promote the mycelium growth and spinosad production. And when the glucose concentration increased from 58.8g·L-1 to 79.6g·L-1, no obvious change was detected but a slight drop in spinosad production was observed, whereas, when the glucose concentration increased from 79.6g·L-1 to l15.3g·L-1, substantial decrease in both mycelium growth and spinosad production occurred. The increase of phosphate conccntration from 3.68mmol· L-1 to 29.41mmol·L-1 rendered corresponding increase in mycelium growth and spinosad production. When phosphate concentration increased from 29.41mmol· L-1 to 44.12mmol·L-1, mycelium growth slightly increased and spinosad production dropped, while when phosphate concentration increased from44.12mmol·L-1 to 57.62mmol·L-1, both mycelium growth and spinosad production decreased sharply. Conclusively,the optimal initial concentration of glucose and phosphate is 58.8g·L-1 and 29.41mmol·L-1, respectively. The spinosad fermentation in the production medium containing 58.8g·L-1 glucose and 29.41mmol·L-1 phosphate was scaled up in 5-L fermentation and the spinosad production reached 507mg·L-1, which was 28% higher than that in the flask fermentation.     

培养基优化以促进超高浓度发酵生产乙醇(英文)

An optimal medium (300 g·L-1 initial glucose) comprising 6.3 mmol·L-1 Mg2+, 5.0 mmol·L-1 Ca2+, 15.0 g·L-1 peptone and 21.5 g·L-1 yeast extract was determined by uniform design to improve very high gravity (VHG) ethanol fermentation, showing over 30% increase in final ethanol (from 13.1% to 17.1%, by volume), 29% decrease in fermentation time (from 84 to 60 h), 80% increase in biomass formation and 26% increase in glucose utilization. Experiments also revealed physiological aspects linked to the fermentation enhancements. Compared to the control, trehalose in the cells grown in optimal fermentation medium increased 17.9-, 2.8-, 1.9-, 1.8- and 1.9-fold at the fermentation time of 12, 24, 36, 48 and 60 h, respectively. Its sharp rise at the early stage of fermentation when there was a considerable osmotic stress suggested that trehalose played an important role in promoting fermentation. Meanwhile, at the identical five fermentation time, the plasma membrane ATPase activity of the cells grown in optimal medium was 2.3, 1.8, 1.6, 1.5 and 1.3 times that of the control, respectively. Their disparities in enzymatic activity became wider when the glucose levels were dramatically changed for ethanol production, suggesting this enzyme also contributed to the fermentation improvements. Thus, medium optimization for VHG ethanol fermentation was found to trigger the increased yeast trehalose accumulation and plasma membrane ATPase activity.     

牛奶中分离的乳酸菌CGMCC1306中谷氨酸脱羧酶的分离纯化及酶学性质研究

A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase （GAD） activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps： 30%-90% saturation （NH4）2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50rain at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22.5＇-pyridoxal phosphate （PLP） had little effect on the regulation of its activity.     

重组大肠杆菌高密度发酵生产类人胶原蛋白的过程控制研究

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)     

pH值控制下γ-氨基丁酸分批发酵动力学模型

在3.7 L发酵罐中研究短乳杆菌CGMCC NO.1306控制pH=5.0发酵生产γ-氨基丁酸(GABA)的过程。结果表明,γ-氨基丁酸的发酵过程大致分为菌体生长和产物合成2个阶段。基于Logistic方程和改进的Luedek ing-P iret方程,分2个阶段建立了控制pH=5.0的细胞生长、底物消耗以及GABA合成动力学模型,运用MATLAB 6.0软件处理实验数据和模型,拟合出描述发酵过程的模型参数。经验证,模型的拟合结果和实验值吻合较好,表明此动力学模型对指导GABA的发酵生产具有实际意义。     

零价铝在碱性条件下还原吡虫啉

采用零价铝（ZVAl）在碱性条件下直接还原吡虫啉,考察了NaOH浓度、反应时间、温度对吡虫啉降解的影响。使用高效液相色谱仪（HPLC）检测。加入过量ZVAl和NaOH的水溶液中,吡虫啉的降解率随NaOH浓度的升高而增大,随反应时间的增长和温度的升高,吡虫啉的降解率增大。采用高效液相色谱-质谱联用仪（HPLC-MS）检测,结果表明吡虫啉的可能降解产物为1-（6-氯吡啶-3-甲基（-2-亚硝基亚氨基吡咯烷、1-（6-氯-3-吡啶甲基（-2-咪唑、2-氯-5-乙基吡啶和2-氯-5-甲基吡啶,并对产物的降解途径进行了分析。     

生物法拆分外消旋甲霜灵制备R-甲霜灵

从土壤和污泥样品中分离得到一株能不对称水解甲霜灵的革兰阴性菌。通过分子生物学鉴定,命名该菌株为Albibacters sp.zjut528。当底物浓度为50 g·L^-1,湿菌体（含水量81%）：底物=0.3：1（质量比）,反应28 h,产物得率为47.1%,主产物为R-甲霜灵酸,对映体过量值ee_p > 99.9%。将菌株细胞破碎得到的粗酶液分离纯化得到一个SDS-PAGE电泳纯的酯酶,分子量约为40×10³,酶的N端10个氨基酸序列为NH₂-Ala-Ala-Lys-Ala-Pro-Leu-Arg-Leu-Lys-Glu。该酶最适温度为40℃,20~40℃稳定性较好。最适pH为9.0,在pH 5.0~10.0范围内稳定。Fe²⁺、Mn²⁺、Zn²⁺对酶活有促进作用,Fe³⁺对酶活有一定的抑制作用。在含有20%的乙腈、异丙醇、丙酮、二异丙醚、环己烷、正己烷的水溶液中,酶活均比在纯水相中有明显提高。该酶反应动力学符合米氏方程,V_m为0.18 mmol·L^-1·min^-1,K_m为2.29 mmol·L^-1,K_cat为0.85 min^-1。建立了一条利用Albibacters sp.zjut528细胞作催化剂拆分甲霜灵制备R-甲霜灵的工艺路线,终产品R-甲霜灵的纯度是96.2%,ee值为99.3%。     

湿式过氧化氢催化氧化降解喹啉及其机理

采用浸渍法制备了负载型铜铁氧化物催化剂,并以喹啉为目标污染物,建立了湿式过氧化氢催化氧化(CWPO)体系.研究了反应温度、初始pH、H₂O₂和催化剂投加量对喹啉去除效果的影响,并分析了CWPO体系中 的作用及喹啉的降解路径.结果表明,CWPO对喹啉具有很好的去除效果,反应30 min喹啉的去除率可达到100%,反应60 min矿化率可达到88.34%.确定了最佳反应温度为80℃,初始pH为7,H₂O₂和催化剂投加量分别为29.15 mmol·L^-1和4 g·L^-1. 氧化在CWPO降解喹啉体系中起主导作用,其平均产生速率为1.69×10^-6 mmol·L^-1·min^-1.推测了CWPO降解喹啉的4种可能路径,在中性和酸性条件下,分别生成以吡啶环或苯环为主的中间产物.     

“活性污泥-生物膜”杂合ABR制氢系统的启动与运行

对ABR系统进行改良,建立新型的“活性污泥-生物膜”杂合厌氧折流板生物制氢反应器（SMHABR）,研究其乙醇型发酵的形成及其产氢及COD处理能力。反应器分为5个格室,有效容积43.2 L,实验共进行180 d。系统以红糖废水为原料,在HRT为12 h,温度为（35±1）℃,通过分阶段提高进水COD的方式,可使ABR系统在35 d内培育驯化形成乙醇型发酵菌群体系。进水COD在约3500 mg·L^-1时产氢量最大,总产氢量可达到44.75 L·d^-1。进水COD浓度达到约7100 mg·L^-1时COD去除率最大,平均总去除率可达到49.33%。COD去除率最大值并未与产氢量最大值同时出现,说明产氢最适进水浓度与COD去除最适进水浓度并非相同。     

Enhanced production of glycyrrhetic acid 3-O-mono-β-D-glucuronide by fed-batch fermentation using p H and dissolved oxygen as feedback parameters

Glycyrrhetic acid 3-O-mono-β-D-glucuronide(GAMG), the major functional ingredient in licorice, has widespread applications in food, pharmacy and cosmetics industry. The production of GAMG through Penicillium purpurogenum Li-3 cultivation was for the first time performed through both batch and fed-batch processes in bioreactors. In batch process, under optimal conditions(p H 5.0, temperature 32 °C, agitation speed 100 r·min-1), 3.55 g·L-1GAMG was obtained in a 2.5 L fermentor. To further enhance GAMG production, a fine fed-batch process was developed by using p H and DO as feedback parameters. Starting from 48 h, 100 ml 90 g·L-1substrate Glycyrrhizin(GL) was fed each time when p H increased to above 5.0 and DO was increased to above 80%. This strategy can significantly enhance GAMG production: the achieved GL conversion was 95.34% with GAMG yield of 95.15%, and GAMG concentration was 16.62 g·L-1which was 5 times higher than that of batch. Then, a two-step separation strategy was established to separate GAMG from fermentation broth by crude extraction of 15 ml column packed with D101 resin followed by fine purification with preparative C18 chromatography. The obtained GAMG purity was 95.79%. This study provides a new insight into the industrial bioprocess of high-level GAMG production.     

对比分析进水基质浓度对乙醇型和丁酸型发酵制氢系统的影响

以糖蜜废水为基质,将两套厌氧接触式发酵制氢反应器(ACR)出水pH分别控制在4.5～5.0、5.5～6.0的水平,通过逐级提升进水COD浓度方式,系统对比分析基质浓度对乙醇型和丁酸型发酵制氢系统的影响。结果显示,对于乙醇型发酵制氢系统而言,当HRT 6 h,进水COD从5000逐步提升至12000 mg·L^-1时,反应器的产氢效能逐步得到增强,但当COD进一步提升至15000 mg·L^-1时,底物反馈抑制作用开始显现,因而在进水COD为12000 mg·L^-1时,ACR产氢性能最佳,系统的产氢速率、污泥比产氢速率和单位基质氢气转化率分别为68.8 L·d^-1、744.5 ml H₂·(g VSS·d)^-1、2.3 mol H₂·(mol葡萄糖)^-1。对于丁酸型发酵制氢系统而言,当HRT 8 h,在进水COD从5000提升至20000 mg·L^-1过程中,ACR产氢效能总体呈下降趋势,在进水COD为5000 mg·L^-1时,系统的污泥比产氢速率和单位基质氢气转化率最大,分别为159.6 ml H₂·(g VSS·d)^-1、1.0 mol H₂·(mol葡萄糖)^-1。研究结果表明,在进水COD为500～20000 mg·L^-1的运行中,ACR乙醇型发酵系统的产氢效能优于丁酸型发酵制氢系统。     

Study of simultaneous saccharification and fermentation for steam exploded wheat straw to ethanol

Although simultaneous saccharification and fermentation (SSF) has been investigated extensively, the optimum condition for SSF of wheat straw has not yet been determined. Dilute sulfuric acid impregnated and steam explosion pretreated wheat straw was used as a substrate for the production of ethanol by SSF through orthogonal experiment design in this study. Cellulase mixture (Celluclast 1.5 l and ?-glucosidase Novozym 188) were adopted in combination with the yeast Saccharomyces cerevisiae AS2.1. The effects of reaction temperature, substrate concentration, initial fermentation liquid pH value and enzyme loading were evaluated and the SSF conditions were optimized. The ranking, from high to low, of influential extent of the SSF affecting factors to ethanol concentration and yield was substrate concentration, enzyme loading, initial fermentation liquid pH value and reaction temperature, respectively. The optimal SSF conditions were: reaction temperature, 35°C; substrate concentration, 100 g·L^-1; initial fermentation liquid pH, 5.0; enzyme loading, 30 FPU·g^-1. Under these conditions, the ethanol concentration increased with reaction time, and after 72 h, ethanol was obtained in 65.8% yield with a concentration of 22.7 g·L^-1.     

Fe(Ⅱ)活化过硫酸盐氧化破解剩余污泥

采用 Fe(Ⅱ)活化过硫酸盐,产生强氧化性硫酸根自由基(SO₄^-·),以污泥释放的溶解性COD(SCOD)、相对疏水性(RH)和污泥比阻(SRF)为表征,考察了SO₄^-·氧化破解剩余污泥强化脱水的影响因素,并解析了机理。结果表明:当初始pH为4.5,n(S₂O₈^2-)=2.2 mmol·(g VSS)^-1,n(Fe²⁺)=1.32 mmol·(g VSS)^-1,常温下反应3 h后,SCOD由66.5 mg·L^-1增加到472.3 mg·L^-1,RH由26.9%升高到41.1%,SRF由24.9×10⁸ S²·g^-1降低至4.5×10⁸ S²·g^-1;在此基础上利用响应面法,根据Box-Benhnken中心组合试验设计,以SRF为响应指标,优化条件为pH 4.27,n(S₂O₈^2-)=2.6 mmol·(g VSS)^-1,n(Fe²⁺)=1.59 mmol·(g VSS)^-1时,SRF为3.8×10⁸ S²·g^-1,泥饼含水率为72.7%。镜检发现,破解后污泥变为颗粒碎片状;傅里叶红外光谱显示污泥表面官能团对应的特征吸收峰强度有一定程度的减弱;热重分析表明无明显物理吸附水失重区。证实了在SO₄^-·作用下,污泥菌胶团结构破坏,溶胞释放了有机物,使表观疏水性更强,与水结合力明显减弱,脱水性得到了较大提高,有利于污泥减量化应用。     

自产气气提与纤维床反应器耦合发酵生产丁醇

采用发酵产物中的二氧化碳（CO₂）和氢气（H₂）作为循环气提气源,对丙酮丁醇梭菌（Clostridium acetobutylicum CGMCC 5234）发酵产物进行原位气提,实现丙酮、丁醇和乙醇混合物（ABE）的连续纤维床固定化发酵生产。连续发酵实验进行了12批次共309 h,总溶剂ABE当量浓度为133.3 g·L^-1（其中丁醇 83.5 g·L^-1,丙酮38.4 g·L^-1,乙醇11.4 g·L^-1）,葡萄糖消耗率为1.29 g·（L·h） ^-1,总溶剂ABE产率为0.431 g·（L·h） ^-1,转化率为0.333 g·g^-1,其中丁醇产率为0.270 g·（L·h） ^-1,转化率为 0.209 g·g^-1,发酵液中丁醇浓度控制在8～12 g·L^-1,显著优于游离发酵的结果。气提提取之后冷凝的ABE溶液出现分层现象,其中丁醇相丁醇浓度高达603.7 g·L^-1,极大地减缓后续分离提纯的负担。结果表明,自产气循环气提与纤维床固定化耦合连续发酵生产ABE（特别是丁醇）的工艺具有可行性和竞争力。