Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

An artificial neural network for biomass estimation from automatic pH control signal

This study developed an artificial neural network (ANN) to estimate the growth of microorganisms during a fermentation process. The ANN relies solely on the cumulative consumption of alkali and the buffer capacity, which were measured on-line from the on/off control signal and pH values through automatic pH control. The two input variables were monitored on-line from a series of different batch cultivations and used to train the ANN to estimate biomass. The ANN was refined by optimizing the network structure and by adopting various algorithms for its training. The software estimator successfully generated growth profiles that showed good agreement with the measured biomass of separate batch cultures carried out between at 25 and 35_C.     

微生物木糖发酵产乙醇的代谢工程

利用木质纤维素发酵生产乙醇具有广泛的应用前景。而自然界中缺少有效转化木糖为乙醇的微生物是充分利用纤维素水解产物、提高乙醇产率、降低生产成本的关键因素。多年来研究者利用分子生物学技术对微生物菌株进行了代谢工程改造,使其能更有效地利用木糖生产乙醇。以下主要对运动发酵单胞菌、大肠杆菌和酵母等候选产乙醇微生物的木糖代谢工程研究进展进行了概述。     

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.     

High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast?

The process of ethanol fermentation has a long history in the production of alcoholic drinks, but much larger scale production of ethanol is now required to enable its use as a substituent of gasoline fuels at 3%, 10%, or 85% (referred to as E3, E10, and E85, respectively). Compared with fossil fuels, the production costs are a major issue for the production of fuel ethanol. There are a number of possible approaches to delivering cost-effective fuel ethanol production from different biomass sources, but we focus in our current report on high-temperature fermentation using a newly isolated thermotolerant strain of the yeast Kluyveromyces marxianus. We demonstrate that a 5°C increase only in the fermentation temperature can greatly affect the fuel ethanol production costs. We contend that this approach may also be applicable to the other microbial fermentations systems and propose that thermotolerant mesophilic microorganisms have considerable potential for the development of future fermentation technologies.     

Method of culturing microorganisms at constant concentrations of the nutrient components

A method for batch cultivation of microorganisms in a flow medium is described, characterized by slight changes in concentrations of medium components in time and by the absence of products of vital activity of microorganisms in the fermentation medium. The conditions are achieved due to application of a fermentation installation with a microfiltrative membrane that separates the cells of cultivated microorganisms from the culture fluid and due to increasing the flow rate to a value at which the inlet and outlet concentrations of the medium components are almost equal. The cells of cultivated microorganisms under such conditions remain in the fermentation medium volume. The system was called "Ekostat". If the process is performed in "Ekostat" system, a positive deviation from the logarithmic law is observed for the growth rate of the yeast Candida utilis VSB-651 on ethanol cultivation.     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I – Yeasts in general

There are a number of process advantages which could be exploited through the use of thermophilic microorganisms for ethanol production. Energy savings through reduced cooling costs, higher saccharification and fermentation rates, continuous ethanol removal and reduced contamination have stimulated a search for routes to thermophilic or thermotolerant yeasts. These routes have included screening existing culture collections, temperature adaptation, mutagenesis and molecular techniques and finally isolating new strains. Varying success has been achieved, however, the most thermotolerant yeasts have come from fresh isolations from environments which experience high temperatures. Thermotolerant yeasts have been investigated for the following potential applications: simultaneous saccharification and fermentation of cellulose, where the high fermentation temperature allows more rapid and efficient enzymatic cellulose hydrolysis; whey fermentation, where high salt and low fermentable substrate concentrations make conditions difficult; and fermentation of D-xylose and cellobiose, which is essential for efficient conversion of woody biomass to ethanol. Ethanol and temperature tolerance are important characteristics for commercial yeast strains. Both characteristics are interactive and generally decrease with increasing temperature and ethanol concentration. Considerable research has been directed towards investigation of fatty acid composition changes in response to these stresses and the role of heat shock proteins in tolerance mechanisms. If thermotolerant yeasts are to be used in commercial processes, bioreactor configuration will play an important part in the design of production processes. Batch and fed-batch systems have been shown to be useful in some circumstances as have continuous flow systems, however, some of the newly isolated thermotolerant yeasts such as Kluyveromyces marxianus do not show the high growth rate under anaerobic conditions that is characteristic of Saccharomyces cerevisiae. Various immobilization techniques appear to offer a means of presenting and maintaining high biomass in anaerobic continuous flow reactors.     

控制发酵过程氧化还原电位优化酿酒酵母乙醇生产

利用氧化还原电极,研究了在厌氧条件下将氧化还原电位值(ORP)控制在不同水平(-50mV、-100mV、-150mV、-230mV)对乙醇发酵过程的影响。试验结果表明,不同的ORP值水平对乙醇得率,甘油形成、有机酸分泌、生物量和菌体死亡率的影响有明显的差异。当ORP为-50mV时的生物量是ORP为-100mV时的1.26倍、ORP为-150mV时的1.86倍、ORP为-230mV时的2.59倍,甘油浓度分别是后三者的1.2倍、1.1倍、1.7倍,而乙醇浓度却分别只有后三者的0.87倍、0.49倍、0.51倍。综合考虑生物量、乙醇浓度、甘油产量、残糖的测定结果,表明将ORP控制在-150mV时对乙醇发酵极为有利。说明可以用ORP电极来精确控制厌氧发酵条件,从而为酵母细胞合理分配代谢流以实现乙醇生产最优化的宏观控制提供了一种有效的手段。     

Repeated use of yeast biomass in ethanol fermentation of juniper berry sugars

Summary The object of this study was to establish the possibility of using the yeast biomass separated from the fermentation broth at the end of ethanol fermentation of juniper berry sugars as an inoculum in successive batch fermentation processes. A part of the fermentation broth (10% v/v) and a suspension of yeast biomass (separated from the same broth) into the water extract of juniper berries (2 g of wet yeast biomass per liter of water extract) were used as inocula. It was shown that the suspension of yeast biomass could be used as inoculum in successive batch processes without negative effects on the kinetics and ethanol yield, but with positive effects on the capacity and economy of the bioprocess. The addition of ammonium salts at optimum levels did not affect the final ethanol concentrations (4.3–4.4% v/v), but enhanced the specific rate of ethanol production, thus reducing the process duration by about five times.     

基于人工神经网络-遗传算法的樟芝发酵培养基优化

采用优化模型对药用丝状真菌樟芝的复杂发酵过程进行建模,并获得最优发酵培养基组成.对樟芝发酵过程中的形态变化过程进行了观察,并分别采用人工神经网络(ANN)和响应面法(RSM)对樟芝发酵过程进行建模,同时采用遗传算法(GA)优化了发酵培养基组成.结果表明,ANN模型比RSM模型具有更好的实验数据拟合能力和预测能力,GA计算得到樟芝生物量理论最大值为6.2 g/L,并获得发酵最佳接种量及培养基组成:孢子浓度1.76× 105个/mL,葡萄糖29.1 g/L,蛋白胨9.4 g/L,黄豆粉2.8 g/L.在最佳培养条件下,樟芝生物量为(6.1±0.2)g/L.基于ANN-GA的优化方法可用于优化其他丝状真菌的复杂发酵过程,从而获得生物量或活性代谢产物.     

An economic comparison of different fermentation configurations to convert corn stover to ethanol using Z. mobilis and Saccharomyces

Numerous routes are being explored to lower the cost of cellulosic ethanol production and enable large‐scale production. One critical area is the development of robust cofermentative organisms to convert the multiple, mixed sugars found in biomass feedstocks to ethanol at high yields and titers without the need for processing to remove inhibitors. Until such microorganisms are commercialized, the challenge is to design processes that exploit the current microorganisms' strengths. This study explored various process configurations tailored to take advantage of the specific capabilities of three microorganisms, Z. mobilis 8b, S. cerevisiae, and S. pastorianus. A technoeconomic study, based on bench‐scale experimental data generated by integrated process testing, was completed to understand the resulting costs of the different process configurations. The configurations included whole slurry fermentation with a coculture, and separate cellulose simultaneous saccharification and fermentation (SSF) and xylose fermentations with none, some or all of the water to the SSF replaced with the fermented liquor from the xylose fermentation. The difference between the highest and lowest ethanol cost for the different experimental process configurations studied was $0.27 per gallon ethanol. Separate fermentation of solid and liquor streams with recycle of fermented liquor to dilute the solids gave the lowest ethanol cost, primarily because this option achieved the highest concentrations of ethanol after fermentation. Further studies, using methods similar to ones employed here, can help understand and improve the performance and hence the economics of integrated processes involving enzymes and fermentative microorganisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Comparison of SHF and SSF processes from steam-exploded wheat straw for ethanol production by xylose-fermenting and robust glucose-fermenting Saccharomyces cerevisiae strains

In this study, bioethanol production from steam-exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentoses. Red Star is a robust hexose-fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. The highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) in an SSF process, it showed an ethanol yield on consumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/L h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furthermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced.     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

Bacterial fermentation of cellulose: Effect of physical and chemical parameters

Acetivibrio celluloyticus converts cellulose directly to ethanol, acetate, H(2), and CO(2). The effects of various physical and chemical parameters, and their interdependence, including pH, temperature, redox, and ethanol toxicity on this fermentation, were studied. Controlling pH at 6.8 favored a predominance of ethanol over acetate. Supplementation of the medium with additional reductant, concomitant with a lower redox potential, incresed ethanol formation. Results from ethanol-challenged cultures indicated that cell lysis occurs with growing but not with nongrowing cells. A stable strain was adapted for growth in ethanol concentrations almost sevenfold greater than the parent organism.     

Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h⁻¹. The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-¹³C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-¹³C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (~95%) and redox (~96%) balances. On the other hand, cultivation conditions that prevent H₂ accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H₂ is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.     

Application of Artificial Neural Network for predicting biomass growth during domestic wastewater treatment through a biological process

The biological treatment process is responsible for removing organic and inorganic matter in wastewater. This process relies heavily on microorganisms to successfully remove organic and inorganic matter. The aim of the study was to model biomass growth in the biological treatment process. Multilayer perceptron (MLP) Artificial Neural Network (ANN) algorithm was used to model biomass growth. Three metrics: coefficient of determination (R²), root mean squared error (RMSE), and mean squared error (MSE) were used to evaluate the performance of the model. Sensitivity analysis was applied to confirm variables that have a strong influence on biomass growth. The results of the study showed that MLP ANN algorithm was able to model biomass growth successfully. R² values were 0.844, 0.853, and 0.823 during training, validation, and testing phases, respectively. RMSE values were 0.7476, 1.1641, and 0.7798 during training, validation, and testing phases respectively. MSE values were 0.5589, 1.3551, and 0.6081 during training, validation, and testing phases, respectively. Sensitivity analysis results showed that temperature (47.2%) and dissolved oxygen (DO) concentration (40.2%) were the biggest drivers of biomass growth. Aeration period (4.3%), chemical oxygen demand (COD) concentration (3.2%), and oxygen uptake rate (OUR) (5.1%) contributed minimally. The biomass growth model can be applied at different wastewater treatment plants by different plant managers/operators in order to achieve optimum biomass growth. The optimum biomass growth will improve the removal of organic and inorganic matter in the biological treatment process.     

Modeling and static optimization of the ethanol production in a cascade reactor. I. Modeling

Let us consider the modeling of a cascade reactor for the production of ethanol. The rates of reaction in alcoholic fermentation are modeled so that it can assume both ethanol and substrate inhibition, in relation to the observed results.A nonstructured model, based on biomass, substrate, and ethanol concentrations, is developed and identified. It is a complex model, this being due to the nonlinearity between the specific rate of ethanol production and the growth rate and, on the other hand, the study of the static optimization of ethanol fermentation is performed.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.