Effects of inoculum size on ethanol inhibition modeling and other fermentation parameters

The effects of inoculum size on the kinetics of ethanol fermentation are not well defined in the literature. The purpose of this article is to examine the influence of the initial cell concentration on the modeling of ethanol inhibition. Experimental results show that increasing the inoculum level decreases the severity of ethanol inhibition. The effect of cell concentration can be related to the different inhibitory effects of autogeneously produced versus extracellularly added ethanol. On this basis, it is concluded that the extracellular ethanol concentration in the fermentation media is not the only variable to account for product inhibition modeling. Other fermentation parameters, such as yields and maintenance coefficients, are presented at different levels of initial cell concentration.     

Production of high concentrations of ethanol from inulin by simultaneous saccharification and fermentation using Aspergillus niger and Saccharomyces cerevisiae.

Pure nonhydrolyzed inulin was directly converted to ethanol in a simultaneous saccharification and fermentation process. An inulinase-hyperproducing mutant, Aspergillus niger 817, was grown in a submerged culture at 30 degrees C for 5 days. The inulin-digestive liquid culture (150 ml) was supplemented with 45 g of inulin, 0.45 g of (NH4)2SO4, and 0.15 g of KH2PO4. The medium (pH 5.0) was inoculated with an ethanol-tolerant strain, Saccharomyces cerevisiae 1200, and fermentation was conducted at 30 degrees C. An additional 20 g of inulin was added to the culture after 15 h of fermentation. S. cerevisiae 1200 utilized 99% of the 65 g of inulin during the fermentation, and produced 20.4 and 21.0% (vol/vol) ethanol from chicory and dahlia inulins, respectively, within 3 days of fermentation. The maximum volumetric productivities of ethanol were 6.2 and 6.0 g/liter/h for chicory and dahlia inulins, respectively. The conversion efficiency of inulin to ethanol was 83 to 84% of the theoretical ethanol yield.     

Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10.

The metabolism of strain H10, a cellulolytic mesophilic Clostridium sp., was studied on glucose and cellobiose as energy and carbon sources. The main products of fermentation of both sugars were acetate, lactate, and ethanol. At low sugar levels, molar growth yields were better for cellobiose than for glucose. In both cases, an inhibition of growth was observed between 1 and 2 g/liter and a total inhibition after the latter concentration. Inhibition was not the result of low pH due to acid formation; growth under static pH conditions was limited in the same way. On the other hand, acetate and lactate had no inhibitory effect when added at concentrations equal to the final titers. Concomitant with the inhibition of growth was a change in metabolic pathways for sugar concentrations between 1 and 2 g/liter, i.e., the production of lactate was higher. After complete inhibition of growth, an accumulation of carbohydrates which were neither glucose nor cellobiose was observed.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Elucidation of Growth Inhibition and Acetic Acid Production by Clostridium thermoaceticum

The production of acetic acid by Clostridium thermoaceticum was studied by using batch fermentations. In a pH-controlled fermentation with sodium hydroxide (pH 6.9), this organism was able to produce 56 g of acetic acid per liter. On the other hand, when the pH was not controlled and was decreased during fermentation to 5.4, the maximum attainable acetic acid concentration was only 15.3 g/liter. To obtain a better understanding of the end product inhibition, various salts were tested to determine their effect on the growth rate of C. thermoaceticum. An inverse linear relationship between the growth rate and the final cell concentration to the sodium acetate concentration was found. By using different concentrations of externally added sodium salts, the relative growth inhibition caused by the anion was found to be in the order of acetate > chloride > sulfate. Various externally added cations of acetate were also examined with respect to their inhibitory effects on growth. The relative magnitude of inhibition on the growth rate was found to be ammonium > potassium > sodium. The combined results have shown that the undissociated acetic acid was much more inhibitory than the ionized acetate ion. Complete growth inhibition resulted when the undissociated acetic acid concentration was between 0.04 and 0.05 M and when the ionized acetate concentration was 0.8 M. Therefore, at low pH (below 6.0), undissociated acetic acid is responsible for growth inhibition, and at high pH (above 6.0), ionized acetate ion is responsible for growth inhibition.     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose.

The efficient diversion of pyruvate from normal fermentative pathways to ethanol production in Klebsiella oxytoca M5A1 requires the expression of Zymomonas mobilis genes encoding both pyruvate decarboxylase and alcohol dehydrogenase. Final ethanol concentrations obtained with the best recombinant, strain M5A1 (pLOI555), were in excess of 40 g/liter with an efficiency of 0.48 g of ethanol (xylose) and 0.50 g of ethanol (glucose) per g of sugar, as compared with a theoretical maximum of 0.51 g of ethanol per g of sugar. The maximal volumetric productivity per hour for both sugars was 2.0 g/liter. This volumetric productivity with xylose is almost twice that previously obtained with ethanologenic Escherichia coli. Succinate was also produced as a minor product during fermentation.     

Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose.

The efficient diversion of pyruvate from normal fermentative pathways to ethanol production in Klebsiella oxytoca M5A1 requires the expression of Zymomonas mobilis genes encoding both pyruvate decarboxylase and alcohol dehydrogenase. Final ethanol concentrations obtained with the best recombinant, strain M5A1 (pLOI555), were in excess of 40 g/liter with an efficiency of 0.48 g of ethanol (xylose) and 0.50 g of ethanol (glucose) per g of sugar, as compared with a theoretical maximum of 0.51 g of ethanol per g of sugar. The maximal volumetric productivity per hour for both sugars was 2.0 g/liter. This volumetric productivity with xylose is almost twice that previously obtained with ethanologenic Escherichia coli. Succinate was also produced as a minor product during fermentation.     

Effect of temperature on ethanol tolerance of thermotolerant Isshatchenkia orientalis IPE100

Tolerance to high temperature and ethanol is a major factor in high‐temperature bio‐ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L^?1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (P_m) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L^?1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high‐temperature bio‐ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature‐dependent inhibitory effects of ethanol on yeast growth.     

Acetic Acid Production by an Electrodialysis Fermentation Method with a Computerized Control System

In acetic acid fermentation by Acetobacter aceti, the acetic acid produced inhibits the production of acetic acid by this microorganism. To alleviate this inhibitory effect, we developed an electrodialysis fermentation method such that acetic acid is continuously removed from the broth. The fermentation unit has a computerized system for the control of the pH and the concentration of ethanol in the fermentation broth. The electrodialysis fermentation system resulted in improved cell growth and higher productivity over an extended period; the productivity exceeded that from non-pH-controlled fermentation. During electrodialysis fermentation in our system, 97.6 g of acetic acid was produced from 86.0 g of ethanol; the amount of acetic acid was about 2.4 times greater than that produced by non-pH-controlled fermentation (40.1 g of acetic acid produced from 33.8 g of ethanol). Maximum productivity of electrodialysis fermentation in our system was 2.13 g/h, a rate which was 1.35 times higher than that of non-pH-controlled fermentation (1.58 g/h).     

Effects of alcohols on the respiration and fermentation of aerated suspensions of baker's yeast.

The immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker's yeast were studied by stopped-flow membrane inlet mass spectrometry. Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0-20%, v/v). The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects. For five different alkanols (methanol, ethanol, 1-propanol, 2-propanol and 1-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu. This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane. Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.     

A general inhibition kinetics model for ethanol production using a novel carbon source: sodium gluconate

In this study, sodium gluconate was applied as a novel carbon source for the fuel ethanol production using an engineered Escherichia coli strain KO11 in batch fermentations. Ethanol and acetic acid were produced as two major products as well as small amount of lactic acid during the fermentation. Compared to the conventional carbon source glucose, the bioconversion of sodium gluconate possessed two distinct advantages: faster utilization rate of sodium gluconate (1.66 g/L per h) compared to glucose (0.996 g/L per h) and no requirement for pH control during fermentation. A general inhibition model including both substrate and products inhibitory effects was proposed, which adequately simulated batch fermentation kinetics at various concentrations of sodium gluconate. All of the products showed inhibitory effects on cell growth. The order of the inhibitory strength of all products and substrate was for the first time clarified in this study. Acetic acid was the most inhibitory product mitigating the cell growth, followed by ethanol and lactic acid. Sodium gluconate stimulated cell growth when its concentration was below 16 g/L, while it inhibited the cell growth when the concentration was above this concentration. It completely inhibited the cell growth when the concentration was 325 g/L. The high value of both the coefficient of determination (R ²) and the adjusted R ² verified the good fit of the model. This paper provides key insights into further engineering these strains to improve ethanol production.     

The ethanol tolerance of Pichia stipitis Y 7124 grown on a d-xylose,d-glucose and l-arabinose mixture

The tolerance of Pichia stipitis Y 7124 to initial added ethanol was evaluated in anaerobic and microaerobic conditions, during the fermentation of a sugar mixture (d-glucose 20%, d-xylose 75%, l-arabinose 5%). The ethanol tolerance depends on the presence of oxygen. In microaerobiosis, the fermentative capacity of P. stipitis is not inhibited when the initial ethanol concentration does not exceed 20 g/l; in this added ethanol range, the strain produced ethanol with a yield up to 0.40 g/g and a specific rate of 0.1 g/g·h. An increase of the initial ethanol level decreases the rate of ethanol production but the ethanol yield appears to be less sensitive to ethanol inhibition. In anaerobiosis, maximum fermentative performances are obtained in the zero initial ethanol culture. When initial ethanol increases, growth and ethanol production decline gradually. But P. stipitis produces ethanol at an initial ethanol level of 50 g/l, even though this totally inhibits the strain activity in microaerobiosis.     

Modeling and advanced control of recombinant Zymomonas mobilis fed-batch fermentation

This work presents the development of an unstructured kinetic model incorporating the differing degrees of product, substrate, and pH inhibition on the kinetic rates of ethanol fermentation by recombinant Zymomonas mobilis CP4:pZB5 for growth on two substrates. Product inhibition was observed to start affecting the specific growth rate at an ethanol concentration of 20 g/L and the specific productivity at about 35-40 g/L. Specific growth rate was also shown to be more sensitive to inhibition by lowered pH as well. A model for the inhibition of two competing substrates' cellular uptake via membrane transport is proposed. Inhibition functions and model parameters were determined by fitting experimental data to the model. The model was utilized in a nonlinear model predictive control (NMPC) algorithm to control the product concentration during fed-batch fermentation to offset the inhibitory effects of product inhibition. Using the optimal feeding policy determined online, the volumetric productivity of ethanol was improved 16.6% relative to the equivalent batch operation when the final ethanol concentration was reached.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Bio-ethanol Production from Green Onion by Yeast in Repeated Batch

Considered to be the cleanest liquid fuel, bio-ethanol can be a reliable alternative to fossil fuels. It is produced by fermentation of sugar components of plant materials. The common onions are considered to be a favorable source of fermentation products as they have high sugar contents as well as contain various nutrients. This study focused on the effective production of ethanol from Green onion (Allium fistulosum L.) by the yeast “Saccharomyces cerevisiae” in repeated batch. The results showed that the total sugar concentration of onion juice was 68.4 g/l. The maximum rate of productivity, ethanol yield and final bio-ethanol percentage was 7 g/l/h (g ethanol per liter of onion juice per hour), 35 g/l (g ethanol per liter of onion juice) and 90 %, respectively.     

Cofermentation of sweet sorghum juice and grain for production of fuel ethanol and distillers' wet grain

In an attempt to reduce the costs associated with fuel ethanol production from grain, the authors used sweet sorghum juice as a partial or complete replacement for tap-water in mash preparation and fermentation. This juice, which was an unutilized by-product of sweet sorghum silage preservation by the Ag-Bag method, contained 6·5–7·6% (wt/wt) reducing sugar and produced up to 3·51% (v/v) ethanol beers after fermentation. Varying amounts of this juice were mixed with water and corn or wheat, either before or after liquefaction (front-end or back-end loading, respectively). When over 60% juice replacement was used in front-end loading trials, salt buildup, due to required pH adjustments during cooking, inhibited yeast metabolism and thereby reduced yields. This inhibition was not observed during back-end loading trials since acid and base usage during cooking were reduced. However, in all trials we noted yeast inhibition by some factor(s) present in juice from sweet sorghum variety NK 8368. This inhibition was not observed with variety NK 405. If sweet sorghum juice is used to replace 40% of the water and either 12·5% of the corn or 12% of the wheat in mash preparation, production costs can be reduced by $0.032/liter ($0.12/US gallon) for corn and $0.040/liter ($0.15/US gallon) for wheat.     

Rapid ethanol fermentations using vacuum and cell recycle

Cell recycle and vacuum fermentation systems were developed for continuous ethanol production. Cell recycle was employed in both atmospheric pressure and vacuum fermentations to achieve high cell densities and rapid ethanol fermentation rates. Studies were conducted with Saccharomyces cerevisiae (ATCC No. 4126) at a fermentation temperature of 35°C. Employing a 10% glucose feed, a cell density of 50 g dry wt/liter was obtained in atmospheric-cell recycle fermentations which produced a fermentor ethanol productivity of 29.0 g/liter-hr. The vacuum fermentor eliminated ethanol inhibition by boiling away ethanol from the fermenting beer as it was formed. This permitted the rapid and complete fermentation of concentrated sugar solutions. At a total pressure of 50 mmHg and using a 33.4% glucose feed, ethanol productivities of 82 and 40 g/liter-hr were achieved with the vacuum system with and without cell recycle, respectively. Fermentor ethanol productivities were thus increased as much as twelvefold over conventional continuous fermentations. In order to maintain a viable yeast culture in the vacuum fermentor, a bleed of fermented broth had to be continuously withdrawn to remove nonvolatile compounds. It was also necessary to sparge the vacuum fermentor with pure oxygen to satisfy the trace oxygen requirement of the fermenting yeast.     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.