APPLICATION OF EXPERIMENTAL DESIGN METHOD FOR ETHANOL PRODUCTION BY FERMENTATION OF SUNFLOWER SEED HULL HYDROLYSATE USING PICHIA STIPITIS NRRL-124

The lignocellulosic hydrolysates provide a rich medium for fermentation of sugars into ethanol. The potential use of sunflower seed hull hemicellulose hydrolysate in ethanol fermentation was evaluated by using the Experimental Design method in this study. A 2² Box-Wilson experimental design was used to develop a statistical model. The effects of shaking rate (55–145 rpm) and initial pH (4.6–7.4) on the fermentation of hydrolysate with Pichia stipitis yeast were studied in shaking bath experiments at 30°C. Model equations that represent maximum ethanol concentration E_max and ethanol yield Y_P/S in terms of shaking rate and pH were developed by using the Design-Expert package program. From response surfaces, optimum variables were calculated numerically by the Design-Expert computer program. Such a procedure provided the optimum values of the parameters with respect to both Y_P/S and E_max as X₁ = 5.89 pH and X₂ = 114.6 rpm. The maximum ethanol concentration of 11.04 g/L and ethanol yield of 0.32 were obtained with these optimized parameters.     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.     

冰糖橙皮渣发酵产燃料乙醇的工艺优化

采用纤维素酶、果胶酶和β-葡萄糖苷酶对冰糖橙皮渣进行水解,所得还原糖液接种异常毕赤酵母进行发酵,考察了酵母接种量、发酵时间、pH和发酵温度等单因素对乙醇得率的影响。单因素结果表明:接种量为12%、发酵时间72 h、pH 4.5、发酵温度33℃时乙醇得率最高。在此基础上设计L9(34)正交实验。结果表明,最佳工艺条件为pH 4.5,接种量12%,发酵时间72 h,发酵温度30℃。在此条件下乙醇产率为0.2451 g/g,显著高于单因素实验(0.2263 g/g)和正交实验结果(0.2329 g/g)。     

Batch kinetics and modelling of propionic acid fermentation

Batch propionic acid fermentation kinetics was studied using five different initial concentrations of lactose (i.e., 37 g/L, 45g/L, 50g/L, 57 g/L and 73 g/L) at constant temperature (30°C) and pH (6.5) under anaerobic conditions using Propionibacterium acidipropionici (ATCC 4875). When the initial substrate concentration was 37 g/L, 45 g/L, 50 g/L, 57 g/L and 73 g/L, then, correspondingly, 16 g/L, 19 g/L, 22.25 g/L, 25.3 g/L and 26.3 g/L of propionic acid was accumulated in the fermentation broth. Increasing the supply of lactose in the fermentation medium led to the accumulation of by products succinate, acetate and pyruvate. Maximum propionate yield (0.44 g/g) and comparatively lesser impurities (byproducts) were achieved with 57 g/L initial lactose concentration. The batch growth kinetics was eventually used to develop and test a mathematical model for propionic acid fermentation by P. acidipropionici at pH 6.5 and S_o = 57 g/L. Y_X/S^max was found to be the most sensitive parameter of the model. The same model also successfully simulated the batch kinetics observed at S_o = 37 g/L. However the model failed to simulate the fermentation kinetics observed at S_o = 73 g/L. The developed model can be used for process optimization studies.     

Kinetic study on ethanol production using Saccharomyces cerevisiae ITV‐01 yeast isolated from sugar cane molasses

BACKGROUND: Bio‐ethanol production from renewable sources, such as sugar cane, makes it a biofuel that is both renewable and environmentally friendly. One of the strategies to reduce production costs and to make ethanol fuel economically competitive with fossil fuels could be the use of wild yeast with osmotolerance, ethanol resistance and low nutritional requirements. The aim of this work was to investigate the kinetics of ethanol fermentation using Saccharomyces cerevisiae ITV‐01 yeast strain in a batch system at different glucose and ethanol concentrations, pH values and temperature in order to determine the optimum fermentation conditions. RESULTS: This strain showed osmotolerance (its specific growth rate (µ_max) remained unchanged at glucose concentrations between 100 and 200 g L^?1) as well as ethanol resistance (it was able to grow at 10% v/v ethanol). Activation energy (Ea) and Q₁₀ values calculated at temperatures between 27 and 39 °C, pH 3.5, was 15.6 kcal mol^?1 (with a pre‐exponential factor of 3.8 × 10¹² h^?1 (R² = 0.94)) and 3.93 respectively, indicating that this system is biologically limited. CONCLUSIONS: The optimal conditions for ethanol production were pH 3.5, 30 °C and initial glucose concentration 150 g L^?1. In this case, a maximum ethanol concentration of 58.4 g L^?1, ethanol productivity of 1.8 g L^?1 h^?1 and ethanol yield of 0.41 g g^?1 were obtained. Copyright © 2010 Society of Chemical Industry     

蒸汽爆破麦草同步糖化发酵转化乙醇的研究

近年来对木质生物资源同步糖化发酵转化乙醇的研究较多,但是,麦草同步糖化发酵转化乙醇的最佳工艺条件还未确定。文中采用正交试验设计的方法,对在混合酶(纤维素酶Celluclast 1.5 1,β-葡萄糖苷酶Novozym 188)与酿酒酵母菌作用下,稀硫酸催化的蒸汽爆破麦草原料同步糖化发酵转化乙醇的工艺条件进行研究,详细讨论了反应温度、底物质量浓度、发酵液pH值、纤维素酶浓度对乙醇质量浓度和得率的影响。结果表明,工艺条件对乙醇质量浓度和得率的影响程度由高到低依次为:底物质量浓度、纤维素酶浓度、发酵液pH值、反应温度。最佳工艺条件为反应温度35℃,底物质量浓度100 g/L,发酵液pH值5.0,纤维素酶浓度30 FPU/g。在此条件下,随着反应时间的延长,乙醇质量浓度持续上升。反应72 h后,乙醇质量浓度和得率分别达到22.7 g/L和65.8%。     

Evaluation of process conditions in the performance of yeast on alcoholic fermentation

This work studied the resistance of Saccharomyces cerevisiae Y904 to ethanol on an alcoholic fermentation process operated in fed-batch. The effect of temperature, inoculum size and substrate concentration on fermentation yield, productivity and residual sugars concentration was studied by a central composite design (CCD). Based on the CCD study, it was determined the optimum values of 240, 35?g/L, and 26°C for total reducing sugars, inoculum concentration and temperature, respectively. This set of conditions experimentally enabled a productivity of 6.0?g/L?h, a yield of 93% and an alcohol content of 113.6?g/L, after 10?h of fermentation. When yeast cells were adapted at 4°C, the inoculum pH adjusted to 2.5 and sugarcane broth used as substrate, a 94% yield and a 10.1?g/L.?h productivity were achieved.     

玉米芯HNO_3/HCl预处理及同步糖化发酵制乙醇

采用正交实验对玉米芯在2%HNO3/HCl中的水解条件进行优化,得出最适宜的预处理条件为：反应温度120℃,反应时间30 min,固含量15%。将经过预处理的玉米芯作为同步糖化发酵的底物,采用单因素实验考查影响发酵的因素,结果表明：在底物浓度为150 g/L、37℃、pH值为5.0、纤维素酶用量为30 FPU/g底物、酵母接种量10%、发酵周期72 h时,乙醇的产率可达到76.8%,此时乙醇溶液的浓度为41.4 g/L。     

Production of L-aminoacylase by fermentation of Pseudomonas sp. BA2

The growth and enzymatic production of Pseudomonas sp. BA2 a new L -aminoacylase-producing microorganism, were studied in a bench-top fermenter. Multiple fermentations were carried out in order to determine the optical pH and temperature values. The influence of the substrate concentration on both growth and L aminoacylase activity was also investigated. The maximum growth rate and the greatest yield of enzyme were obtained when the fermentation was carried out at pH 7·5, 25°C and DOT ≥ 50%. N-Acetyl-DL -alanine, at a concentration 20 g dm^?3, was used as the sole carbon and nitrogen source. The fermentation process provided a maximum biomass concentration of 3·36 g dry weight dm^?3. The highest L -aminoacylase production (11429 U g^?1 dry weight) was obtained after 39 h of cultivation. The results were a significant improvement over those previously reported.     

橡子粉同步液化糖化产燃料乙醇的发酵条件优化

以除去单宁的橡子粉为原料,应用活性干酵母同步液化糖化发酵(SLSF)制备燃料乙醇,并通过单因素试验和正交试验优化发酵条件。结果表明,同步液化糖化发酵技术适用于橡子粉发酵制备燃料乙醇;发酵的最佳条件为:除去单宁的橡子粉20 g,料液比为1:3(g:mL),淀粉酶100 U/g,糖化酶3 750 U/g,活性干酵母1.50%;在30 ℃静止发酵120 h,发酵液中的乙醇质量浓度达到106.5 g/L,橡子淀粉的乙醇转化率达到89.36 %。采用橡子粉发酵法制备燃料乙醇与以玉米等粮食作物为原料制备的燃料乙醇质量浓度相当,可以替代粮食作物生产燃料乙醇。     

Improvement of fermentative production of exopolysaccharides from Aureobasidium pullulans under various conditions

The optimization of exopolysaccharide (EPS) production was investigated in the polymorphic fungal strain of Aureobasidium pullulans (KCTC 6081) with varying pH, nutrients concentration, and mixing parameters in batch fermentation condition. The maximum production of EPS (～7.5 g/L) was observed at pH 4, while optimum nutrient concentration of carbon (sucrose), nitrogen (NaNO₃), phosphorous (K₂HPO₄), and ascorbic acid was 50 g/L, 5 g/L, 1 g/L and 2 g/L, respectively. Interestingly, EPS productivity under non pH controlled fermentation conditions was 0.12 g/L/h with 400 rpm mixing, while under a controlled pH of 4, the EPS productivity was 0.21 g/L/h with 600 rpm, respectively. The fed-batch fermentation increased the EPS productivity up to 0.345 g/L/h with changing mixing conditions from 200 to 600 rpm and reached 47 g/L with 88% pullulan. Thus, pH and mixing were the key parameters for enhancing EPS production from A. pullulans. It is expected that these optimized parameters can be well used for enhanced industrial production of pullulan.     

Continuous ethanol production by the synchronous saccharification and fermentation using food wastes

The synchronous saccharification and fermentation (SSF) by continuous fill and draw method was investigated in order to develop a continuous ethanol fermentation process using the food wastes (FW) available among Korea’s organic wastes. The activity of the hydrolytic enzymes was maintained constantly in the continuous culture by their intermittent addition together with medium exchange. The concentrations of reducing sugar in the culture were maintained at a steady state by regulating supplemented enzyme concentration and exchange rate of medium, reflecting on the consumption rate of reducing sugar caused by the fermentation. When the temperature of the SSF was maintained at the fixation of 35 °C, which enabled us to perform both enzymatic hydrolysis and enzyme fermentation simultaneously, the rate of reducing sugar consumption was 3.61 g/L-hr. For the enzymatic saccharification of FW, when 0.01 BGU as Viscozyme/g-FW and 0.05 AGU as Spirizyme Plus/g-FW were used, the production rate of reducing sugar was 3.93 g/L-hr, indicating a little higher rate of production than that of consumption. A decompression device with ethanol condensing ability was used to continuously pull out ethanol from the culture broth at −600 mmHg, where the ethanol evaporation ability would be maximized and the water evaporation minimized during the process. As a result of the continuous SSF performance, the reducing sugar concentration was maintained at around 30 g/L. The amylase activity was maintained at 8.93±2.17 U/mL. During a 352 hour culture, the whole ethanol productivity was 2.24 g/L-hr, indicating a considerable productivity compared with the other result reported in the continuous SSF.     

碱性条件促进纺织印染污泥厌氧发酵产挥发性脂肪酸

系统研究了酸性（pH值5、pH值6）、中性（pH值7）和碱性（pH值8～10）条件下,纺织印染污泥厌氧发酵产挥发性脂肪酸（VFA）的发酵类型,比较了总酸及主要酸的最高发酵浓度及分布、总酸及乙酸的产率和生产速率,并揭示了碱性条件有利于有机酸发酵的机制。研究结果表明,不同pH值条件下,乙酸型均为主要发酵类型,但较低的pH值利于丁酸累积。总酸及乙酸的浓度随着pH值的升高而增加,且碱性条件能够促进纺织印染污泥发酵产酸,原因为高pH值能促进有机物的降解并提高转化效率,同时较高的pH值抑制了有机酸的降解。pH值为10是厌氧发酵产酸并累积乙酸的最佳pH值。该条件下,乙酸和总酸的质量浓度达最高,为8.21 g/L和14.08 g/L;乙酸和总酸产率也达最高,为34.97%和75.72%;同时乙酸和总酸的生产速率达最高值,为2.41 g/(L·d)和3.48 g/(L·d)。pH值为6比较适合丁酸发酵,丁酸的最高浓度为1.89 g/L。     

An integrated process for continuous cellulosic bioethanol production

A high-efficiency, integrated bioethanol production process was developed in this study, using Miscanthus as lignocellulosic biomass. The continuous process involved a twin-screw extruder, a pretreated biomass washing/dewatering process, and a saccharification/fermentation process. In addition, the integration process was designed for the reuse of pretreatment solution and the production of highly concentrated bioethanol. Pretreatment was performed with 0.72 M NaOH solution at 95 °C using an 80 rpm twin-screw speed and a flow rate of 90mL/min (18 g/min of raw biomass feeding). Following washing and dewatering steps, the pretreated biomass was subjected to simultaneous saccharification and bioethanol fermentation processes. The maximum ethanol concentration, yield from biomass, and total volume obtained were 59.3 g/L, 89.9%, and 60 L, respectively, using a pretreated biomass loading of 23.1% (w/v) and an enzyme dosage of 30 FPU/g cellulose. The results presented here constitute an important contribution toward the production of bioethanol from Miscanthus.     

A novel aeration strategy in repeated-batch fermentation for efficient ethanol production from sweet sorghum juice

To improve the efficiency of ethanol production in a batch fermentation from sweet sorghum juice under a very high gravity(VHG) condition(～ 290 g/L of total sugar) by Saccharomyces cerevisiae NP01, repeatedbatch fermentation under an aerated condition(2.5 vvm for the first 4 h during every cycle) was done in a5-L fermenter. The average ethanol concentration(P), productivity(Qp) and yield(Yp/s) for five successive cycles were 112.31 g/L, 1.55 g/L·h~(-1) and 0.44, respectively with 80.97% sugar consumption. To complete sugar consumption, the total sugar of the juice was reduced to a high gravity(HG) level(～240 g/L). The results showed that yeast extract was not necessary for ethanol production, and aeration during every other cycle i.e., alternating cycles, was sufficient to promote both yeast growth and ethanol production.The average P, Qpand Yp/svalues for eight successive cycles with aeration during alternating cycles were97.58 g/L, 1.98 g/Láh and 0.41, respectively with 91.21% sugar consumption. The total fatty acids in the yeast cells under the aerated condition were ～ 50% higher than without aeration, irrespective the initial sugar concentration, whereas the ergosterol contents under aeration condition were ～ 29% to 49% higher than those without aeration.     

重组人uPA_(17)-KPI大规模发酵及纯化工艺的建立

目的建立重组人uPA17-KPI(rhuPA17-KPI)毕赤酵母工程菌在80L发酵罐中的大规模发酵及纯化工艺。方法对工程菌表达rhuPA17-KPI的pH值进行优化。在摇瓶中制备rhuPA17-KPI工程菌种子2L,接种至80L发酵罐中,采用优化的pH值,补料分批培养方式对工程菌进行高密度发酵,控制和优化各种发酵条件,经甲醇诱导48h后结束发酵。将发酵液离心,经SP Sepharose XL阳离子交换层析和SourceTM 30 RPC反相疏水柱层析纯化。结果建立的发酵工艺为:控制发酵温度为28℃,pH值为5.5,溶氧值为25%～30%之间,在酵母细胞湿重达到190g/L时开始甲醇诱导表达,诱导30h,rhuPA17-KPI的分泌达高峰,为300mg/L发酵液。发酵上清经阳离子交换层析和反相疏水层析纯化后,获得纯度为95%以上的rhuPA17-KPI,产量为180mg/L,回收率为60%。结论已建立了rhuPA17-KPI毕赤酵母工程菌在80L发酵罐中的大规模发酵及纯化工艺,为其产业化及临床应用奠定了基础。     

“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。     

Ethanol production by co-fermentation of hexose and pentose from food wastes using Saccharomyces coreanus and Pichia stipitis

To improve the conversion rate of a saccharification liquid from food wastes containing pentoses and hexoses into bioethanol, after selecting Saccharomyces coreanus and Pichia stipitis, the respective fermentation and co-fermentation properties were investigated. In the fermentation using S. coreanus, the result under anaerobic condition was better than under aerobic conditions. In the anaerobic fermentation, the concentration of the reducing sugar and glucose remaining after 24 hrs was 9.09 and 1.88 g/L, respectively, with 40.59 g/L of ethanol produced; the ethanol productivity was 1.69 g/L-h. Also, even with the fermentation using P. stipitis, the reducing sugars and glucose were rapidly reduced, with a marked production of ethanol, but the ethanol produced was lower than those under anaerobic and aerobic conditions with the use of S. coreanus. Therefore, for the production of a high concentration of bioethanol from food wastes, ethanol fermentation was induced using S. coreanus until the middle of the fermentation, with P. stipitis used during the latter stage of the fermentation, where the circumstance favored its use, and thus, the carbon source not converted by S. coreanus was later converted to ethanol. As a result, both ethanol production of 48.63 g/L and productivity of 2.03 g/L-h increased over those of the anaerobic fermentation using S. coreanus.     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.     

Improvement of ethanol fermentation from lignocellulosic hydrolysates by the removal of inhibitors

In this study, the removal efficiency of fermentation inhibitors in a lignocellulosic hydrolysate by electrodialysis (ED) and the ethanol performance of ED-treated hydrolysate were investigated. The fermentable sugars and inhibitors concentrations in the hydrolysate differed significantly depending on the kind of biomass and acid catalysts. In the mixed hardwood, acetic acid and furfural in the hydrolysate were high as 8.41–8.57 g/L and 2.68–4.23 g/L, respectively, but 5-hydroxymethylfurfural (HMF) concentration was relatively low compared with that of mixed softwood. The ED process showed the high effectiveness for removing acetic acid and total phenolic compounds in the hydrolysate without loss of fermentable sugars. However, most of the HMF and furfural remained in the hydrolysate after ED. Ethanol fermentation was not completed in untreated and mixed hardwood ED-treated hydrolysates due to the high concentration of furfural. Meanwhile, ethanol fermentation was successfully performed in a mixed softwood ED-treated hydrolysate pretreated with dicarboxylic acid. The maximum ethanol concentration attained after fermentation with an initial fermentable sugar level of 27.78 g/L was 10.12 g/L after 48 h.