Optimization Of Ethanol Production Research Articles

Optimization of ethanol production from microwave alkali pretreated rice straw using statistical experimental designs by Saccharomyces cerevisiae

Ethanol production from agro-waste provides an alternative energy-production system. Statistical experimental designs were used for optimization of critical nutrients and process variables for ethanol production. The critical nutrients and process variables were initially selected according to a Placket–Burman (PB) design. Selected factors (inoculum level 1–5%, pH 4.5–7, temperature 25–35°C and urea concentration 0.25–0.75g/L) were optimized by response surface methodology (RSM) based on a three-level four-factor Box–Behnken design (BBD). Under optimum conditions of inoculum level 3%, pH 5.75, temperature 30°C and urea concentration 0.50g/L maximum ethanol production obtained 13.2g/L from microwave alkali pretreated rice straw with ethanol productivity 0.33g/L/h. Under optimum conditions ethanol production studied at fermenter level and obtained ethanol concentration 19.2g/L, ethanol productivity 0.53g/L/h and ethanol yield to consumed sugar 0.50g/g. These results indicated that ethanol production can be enhanced by optimization of nutritional and process variables.

Designing optimal cell factories: integer programming couples elementary mode analysis with regulation.

BackgroundElementary mode (EM) analysis is ideally suited for metabolic engineering as it allows for an unbiased decomposition of metabolic networks in biologically meaningful pathways. Recently, constrained minimal cut sets (cMCS) have been introduced to derive optimal design strategies for strain improvement by using the full potential of EM analysis. However, this approach does not allow for the inclusion of regulatory information.ResultsHere we present an alternative, novel and simple method for the prediction of cMCS, which allows to account for boolean transcriptional regulation. We use binary linear programming and show that the design of a regulated, optimal metabolic network of minimal functionality can be formulated as a standard optimization problem, where EM and regulation show up as constraints. We validated our tool by optimizing ethanol production in E. coli. Our study showed that up to 70% of the predicted cMCS contained non-enzymatic, non-annotated reactions, which are difficult to engineer. These cMCS are automatically excluded by our approach utilizing simple weight functions. Finally, due to efficient preprocessing, the binary program remains computationally feasible.ConclusionsWe used integer programming to predict efficient deletion strategies to metabolically engineer a production organism. Our formulation utilizes the full potential of cMCS but adds additional flexibility to the design process. In particular our method allows to integrate regulatory information into the metabolic design process and explicitly favors experimentally feasible deletions. Our method remains manageable even if millions or potentially billions of EM enter the analysis. We demonstrated that our approach is able to correctly predict the most efficient designs for ethanol production in E. coli.

Optimization of ethanol production by Saccharomyces cerevisiae UFPEDA 1238 in simultaneous saccharification and fermentation of delignified sugarcane bagasse

Ethanol production by Saccharomyces cerevisiae UFPEDA1238 was performed in simultaneous saccharification and fermentation of delignified sugarcane bagasse. Temperature (32°C, 37°C), agitation (80; 100rpm), enzymatic load (20 FPU/g cellulose and 10%, v/v β-glucosidase or 10 FPU/g cellulose and 5% β-glucosidase) and composition of culture medium were evaluated. Ethanol concentration, enzymatic convertibility of cellulose and volumetric productivity were higher than 25g/L, 72% and 0.70g/Lh, respectively, after 30h, when the culture medium 1 and 20 FPU/g cellulose/10%, v/v β-glucosidase or the culture medium 2 and 10 FPU/g cellulose/5% β-glucosidase were used in SSF at 37°C and 80rpm. In the SSF with culture medium 2 (supplemented with ammonium, phosphate, potassium and magnesium), 150L ethanol/t bagasse was achieved, with minimum enzyme loading (10 FPU/g cellulose and 5%, v/v β-glucosidase) for 8%, w/v of solids, which is often an important requirement to provide cost-efficient second generation ethanol processes.

Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK17

The ethanol production capacity from sugars and lignocellulosic biomass hydrolysates (HL) by Thermoanaerobacterium strain AK(17) was studied in batch cultures. The strain converts various carbohydrates to, acetate, ethanol, hydrogen, and carbon dioxide. Ethanol yields on glucose and xylose were 1.5 and 1.1 mol/mol sugars, respectively. Increased initial glucose concentration inhibited glucose degradation and end product formation leveled off at 30 mM concentrations. Ethanol production from 5 g L(-1) of complex biomass HL (grass, hemp, wheat straw, newspaper, and cellulose) (Whatman paper) pretreated with acid (0.50% H(2) SO(4)), base (0.50% NaOH), and without acid/base (control) and the enzymes Celluclast and Novozyme 188 (0.1 mL g(-1) dw; 70 and 25 U g(-1) of Celluclast and Novozyme 188, respectively) was investigated. Highest ethanol yields (43.0 mM) were obtained on cellulose but lowest on hemp leafs (3.6 mM). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The influence of various factors (HL, enzyme, and acid/alkaline concentrations) on end-product formation from 5 g L(-1) of grass and cellulose was further studied to optimize ethanol production. Highest ethanol yields (5.5 and 8.6 mM ethanol g(-1) grass and cellulose, respectively) were obtained at very low HL concentrations (2.5 g L(-1)); with 0.25% acid/alkali (v/v) and 0.1 mL g(-1) enzyme concentrations. Inhibitory effects of furfural and hydroxymethylfurfural during glucose fermentation, revealed a total inhibition in end product formation from glucose at 4 and 6 g L(-1), respectively.

Optimization of ethanol production from thick juice: A response surface methodology approach

The course of fermentation of thick juice as intermediate product of sugar beet processing by yeast Saccharomyces cerevisiae was studied in the paper. The feasibility of ethanol production from thick juice was experimentally confirmed in batch fermentation in a bioreactor of 1.5L working volume. Important parameters for modelling of the process using response surface methodology were defined by analyzing the results of the course of fermentation. For description of the response function of the number of yeast cell number, ethanol volume fraction and total sugar mass fraction during fermentation, the effects of initial sugar content in the range 5–25% w/v and duration of fermentation in the range 0–48h, were examined. Obtained models have contributed to a better understanding of the impact of different initial sugar mass fractions, fermentation time and interactions of these factors on the selected responses. Optimization of multiple responses was also simultaneously performed i.e. maximization of both yeast cell number and ethanol volume fraction while residual total sugar mass fraction was minimized. Results of optimization suggest that the optimal conditions are fermentation time of 46h and initial sugar mass fraction of 20.67%. Further research was performed to validate the obtained results and confirm their applicability in the enlarged scale. The results obtained during the fermentation in bioreactor of 10L working volume under optimal conditions defined for fermentation in the bioreactor of 1.5L working volume were in good correlation.

Optimization of Ethanol Production from Palmyra Sap by Zymomonas mobilis using Response Surface Methodology (RSM)

Ethanol is believed to be one of the best alternatives to replace gasoline, because ethanol is a renewable energy source and environmentally friendly. The present study focuses on the optimization of palmyra sap as a source for ethanol production. Statistical experimental design using Box-Wilson central composite design (CCD) was used to optimize the quantitative effects of sugar, urea, and inoculum concentration on ethanol production. It was found that palmyra sap could be used as a substrate for ethanol production using Zymomonas mobilis (NRRL B-14234). A maximum ethanol concentration of 58.97 g L-1 was obtained after optimizing the parameters of fermentation. The optimum values of sugar, urea, and inoculums concentration were 206.01 g L-1, 3.16 g L-1, and 23.05% (v v-1), respectively, with ethanol yield of 0.3039 g g-1. A high similarity was observed between the predicted and experimental results, which reflected the accuracy and applicability of RSM to optimize the process for ethanol production.

Improvement of Ethanol Production Using Saccharomyces cerevisiae by Enhancement of Biomass and Nutrient Supplementation

Optimization of ethanol production through addition of substratum and protein-lipid additives was studied. Oilseed meal extract was used as protein lipid supplement, while rice husk was used as substratum. The effect of oil seed meal extract and rice husk was observed at varying concentration of medium sugar from 8% to 20%. Of the three oil seed meal extracts used, viz. groundnut, safflower, and sunflower, safflower was found to be most efficient. The use of oilseed meal extract at 4% was found to enhance ethanol production by almost 50% and enhanced sugar tolerance from 8% to 16%. A further increase of almost 48% ethanol was observed on addition of 2 g of rice husk per 100 ml of medium. An increase in cell mass with better sugar attenuation was observed. Further optimization was sought through use of sugarcane juice as the sugar source. While 8.9% ethanol yield with 75% sugar attenuation was observed at 20% sucrose concentration, it was found to increase to 12% (v/v) with almost complete utilization of medium sugar when sugarcane juice was used. Cell weight was also observed to increase by 26%.

Optimization of ethanol production from mango pulp using yeast strains isolated from taberna: A Mexican fermented beverage

Undamaged, uninfected and ripe mango fruits were collected from mango trees grown in Tuxtla Gutierrez, Chiapas, Mexico. In a first experiment, a Plackett-Burman statistical experimental design was used to study the effect of KH2PO4 (0.5 to 6.0 g/ll); (NH4)2SO4(0.5 to 4.0 g/l); ZnSO4 (0.05 to 1.0 g/l); MgSO4 (0.4 to 2 g/l); CaCl2 (0.1 to 1.0 g/l); CoSO4(0.1 to 1.0 g/l) and the total sugar content of mango pulp (Mangifera indica var. criollo) (80 to 120 g/l) on ethanol production using two yeasts strains (denominated TL-ITTG-01 and TL-ITTG-06) isolated from a Mexican fermented native beverage. Saccharomyces cerevisiae NRRL-Y-2034 was used as reference strain. The variables studied had no significant effect on ethanol production by the yeast strain TL-ITTG-01 and S. cerevisiaeNRRL-Y-2034. Highest ethanol productions were 24.89±0.17 for TL-ITTG-01 and 51.24±3.40 g/l for S. cerevisiae NRRL-Y-2034. Substrate concentration, that is, sugars in the mango pulp, and CoSO4, however, had a significant effect on the ethanol production by strain TL-ITTG-06 and the highest ethanol production for this strain was 47.92±0.82 g/l. Ethanol production by the strain TL-ITTG-06 was further optimized using a factorial design 22. The maximum ethanol concentration predicted by the model was of 48.56 g/l with 210.0 g/l sugar and 0.1 g/l of CoSO4, but a fermentation with the aforementioned concentrations resulted in an ethanol production of 52.60±0.77 g/l. Key words: Ethanol, Plackett-Burman design, mango pulp.

Evaluation and optimization of ethanol production from carob pod extract by Zymomonas mobilis using response surface methodology

In this research, ethanol production from carob pod extract (extract) using Zymomonas mobilis with medium optimized by Plackett-Burman (P-B) and response surface methodologies (RSM) was studied. Z. mobilis was recognized as useful for ethanol production from carob pod extract. The effects of initial concentrations of sugar, peptone, and yeast extract as well as agitation rate (rpm), pH, and culture time in nonhydrolyzed carob pod extract were investigated. Significantly affecting variables (P = 0.05) in the model obtained from RSM studies were: weights of bacterial inoculum, initial sugar, peptone, and yeast extract. Acid hydrolysis was useful to complete conversion of sugars to glucose and fructose. Nonhydrolyzed extract showed higher ethanol yield and residual sugar compared with hydrolyzed extract. Ethanol produced (g g(-1) initial sugar, as the response) was not significantly different (P = 0.05) when Z. mobilis performance was compared in hydrolyzed and nonhydrolyzed extract. The maximum ethanol of 0.34 ± 0.02 g g(-1) initial sugar was obtained at 30°C, initial pH 5.2, and 80 rpm, using concentrations (g per 50 mL culture media) of: inoculum bacterial dry weight, 0.017; initial sugar, 5.78; peptone, 0.43; yeast extract, 0.43; and culture time of 36 h.

Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol

Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.

Simultaneous effect of divalent cation in hydrolyzed cassava starch medium used by immobilized yeast for ethanol production

Response surface methodology was adopted in a central composite design to optimize ethanol production from cassava starch hydrolysate medium. Starch hydrolyzate was prepared from TMS 98/0581, a genetically developed cassava mosaic disease-resistant variety. The yeast whole cell, Saccharomyces pastorianus, a lager brewing strain (726 x 106 cells/ml, 98.78% viability) and fungamyl and termamyl (a-amylase enzymes), used for the 120 h fermentation, were immobilized by entrapment in calcium alginate gel. Effects of three divalent cationic concentrations Mg2+, Zn2+ and Ca2+ on ethanol yield were investigated at five variable combinations in 20 experimental runs in accordance with the experimental design. Maximum ethanol concentration of 12.53 %v/v was produced in the 96 h of fermentation when the divalent cationic combination was 64, 0.48 and 30 mg/l (Mg2+, Zn2+ and Ca2+), respectively. The study showed that effect of Zn2+ on ethanol yield was significantly (P≤0.05) quadratic. Keywords: Ethanol, immobilization, Saccharomyces pastorianus, divalent cations, optimization, response surface methodology, cassava mosaic disease.

Optimization of ethanol production from hot-water extracts of sugar maple chips

Hot-water extracts from sugar maple chips prior to papermaking was employed in this study to produce ethanol by Pichia stipitis 58784. The effects of several factors, seed culture age, fermentation time, inoculum quantity, agitation rate, percent extract, concentration of inorganic nitrogen source (NH 4) 2SO 4 and pH value, on ethanol production were investigated by orthogonal experiments. Orthogonal analysis shows that the optimal fermentation was obtained in the condition of 48-h seed culture, 120-h fermentation, 16% inoculum, 180 rpm, containing 30% extracts, 8% ammonium sulphate supplement and pH 5. This optimal condition was verified at 800-mL level in a 1.3 L fermentor. The ethanol yield reached 82.27% of the theoretical (20.57 g/L) after 120 h.

Optimization of Fermentation Conditions for the Production of Ethanol from Stalk Juice of Sweet Sorghum by Immobilized Yeast Using Response Surface Methodology

Optimization of three parameters, including initial total sugar concentration, supplement rate of (NH4)2SO4, and particles stuffing rate, was attempted using response surface methodology based on a Box−Behnken design for the optimal production of ethanol by immobilized yeast fermentation of stalk juice of Liaotian number 1 sweet sorghum cultivar in shaking flasks. The correlation analysis of the mathematical regression model indicated that the quadratic polynomial model could be employed to optimize ethanol production. The optimum conditions were found to be an initial total sugar concentration of 22.88%, supplement rate of (NH4)2SO4 of 0.244%, and particles stuffing rate of 25.15%. At the optimum conditions, the maximum predicted ethanol yield of 93.83% was obtained. The ethanol yield and fermentation time of verification experiments in the shaking flask were 92.37% and 14 h at the corresponding parameters, respectively, while they were 93.23% and 13 h, respectively, in a 5 L bioreactor, in which the pre...

English

  Optimization options for production of ethanol from bitter kola (Garcinia kola) pulp wastes were investigated. The methods are degumming, saccharification, acid hydrolysis, alkaline hydrolysis. Degumming was effectively achieved at 96 h of saccaharification. The concentration of reducing sugar for the treated sample (acid hydrolysed and saccharification) and control sample (saccharification) was maximum at 144 h (86.2 g/100g) and 96 h (31.5 g/100 g), respectively. Ethanol yield from treated sample and control sample using baker’s yeast was maximum at 120 h (70.7 g/L) and 192 h (29.3 g/L), respectively. Alkaline hydrolysis with 0.25 M sodium hydroxide has no significant effect on concentration of reducing sugar and ethanol yield. Acid hydrolysis with 2.5 M sulphuric acid and saccharification using Aspergillus niger are better methods for optimizing ethanol production from bitter kola pulp waste. Solar drying of the bitter kola pulp waste significantly enhanced ethanol production.   Key words: Bitter kola pulp wastes, degumming, saccharification, acid hydrolysis, alkaline hydrolysis, fermentation.

Optimization of ethanol production from starch by an amylolytic nuclear petite Saccharomyces cerevisiae strain

Ethanol fermentation characteristics of the 100% respiratory-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was investigated in both shake-flask and controlled bioreactor cultivation conditions, and comparison with the earlier reported results revealed 54.6% increase in ethanol yield. Efforts to improve the starch utilization rate by increasing the selection pressure or supplying the fermentation medium with glucose did not prevent the observed decrease in time-dependent amylolytic activity. Response surface methodology (RSM) was then used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both the respiratory-deficient NPB-G and the parental respiratory-sufficient WTPB-G strains, with concomitant improvement in their amylolytic potentials. High ethanol yields on substrate values of the bioreactor cultures, which were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient NPB-G strain was effective in the direct fermentation of starch into ethanol.

Nitrogen source and mineral optimization enhance d-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124

Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61+/-9 g/l) by providing C:N in the vicinity of approximately 57-126:1 and optimizing the combination of urea and amino acids to supply 40-80 % nitrogen from urea and 60-20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others.

Optimization of Ethanol Production in Saccharomyces cerevisiae by Metabolic Engineering of the Ammonium Assimilation

Ethanol is still one of the most important products originating from the biotechnological industry with respect to both value and amount. In addition to ethanol, a number of byproducts are formed during an anaerobic fermentation of Saccharomyces cerevisiae. One of the most important of these compounds, glycerol, is produced by yeast to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD+. The purpose of this study was to evaluate whether a reduced formation of surplus NADH and an increased consumption of ATP in biosynthesis would result in a decreased glycerol yield and an increased ethanol yield in anaerobic cultivations of S. cerevisiae. A yeast strain was constructed in which GLN1, encoding glutamine synthetase, and GLT1, encoding glutamate synthase, were overexpressed, and GDH1, encoding the NADPH-dependent glutamate dehydrogenase, was deleted. Hereby the normal NADPH-consuming synthesis of glutamate from ammonium and 2-oxoglutarate was substituted by a new pathway in which ATP and NADH were consumed. The resulting strain TN19 (gdh1-Δ1PGK1p-GLT1PGK1p-GLN1) had a 10% higher ethanol yield and a 38% lower glycerol yield compared to the wild type in anaerobic batch fermentations. The maximum specific growth rate of strain TN19 was slightly lower than the wild-type value, but earlier results suggest that this can be circumvented by increasing the specific activities of Gln1p and Glt1p even more. Thus, the results verify the proposed concept of increasing the ethanol yield in S. cerevisiae by metabolic engineering of pathways involved in biomass synthesis.

Experimental optimization of a real time fed-batch fermentation process using Markov decision process

This article describes a methodology that implements a Markov decision process (MDP) optimization technique in a real time fed-batch experiment. Biological systems can be better modeled under the stochastic framework and MDP is shown to be a suitable technique for their optimization. A nonlinear input/output model is used to calculate the probability transitions. All elements of the MDP are identified according to physical parameters. Finally, this study compares the results obtained when optimizing ethanol production using the infinite horizon problem, with total expected discount policy, to previous experimental results aimed at optimizing ethanol production using a recombinant Escherichia coli fed-batch cultivation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 317-327, 1997.

Experimental optimization of a real time fed‐batch fermentation process using Markov decision process

This article describes a methodology that implements a Markov decision process (MDP) optimization technique in a real time fed-batch experiment. Biological systems can be better modeled under the stochastic framework and MDP is shown to be a suitable technique for their optimization. A nonlinear input/output model is used to calculate the probability transitions. All elements of the MDP are identified according to physical parameters. Finally, this study compares the results obtained when optimizing ethanol production using the infinite horizon problem, with total expected discount policy, to previous experimental results aimed at optimizing ethanol production using a recombinant Escherichia coli fed-batch cultivation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 317–327, 1997.

A decreasing feeding profile for the optimization of ethanol production in a recombinant Escherichia coli fed-batch fermentation

A decreasing feed flowrate was used to optimize the ethanol volumetric productivity and was found to be optimal and equal to 1.8 g ethanol/h·L with a recombinant Escherichia coli with plasmid pLOI297. This productivity is more than double in previous reports. High substrate concentrations favor ethanol production when the microorganisms are in the exponential phase and when they are not inhibited.