Fermentation pattern of sucrose to ethanol conversions by Zymomonas mobilis

General patterns of sucrose fermentation by two strains of Zymomonas mobilis, designated Z7 and Z10, were established using sucrose concentrations from 50 to 200 g/liter. Strain Z7 showed a higher invertase activity than Z10. Strain Z10 showed a reduced specific growth rate at high sucrose concentration while Z7 was unaffected. High sucrose hydrolyzing activity in strain Z7 lead to glucose accumulation in the medium at high sucrose concentrations. Ethanol production and fermentation time depend on the rate of catabolism of the products of sucrose hydrolysis, glucose and fructose. The metabolic quotients for sucrose utilization, q_s, and ethanol production, q_p (g/g·hr), are unsuitable for describing sucrose utilization by Zymomonas mobilis, as the logarithmic phase of growth precedes the phase of highest substrate utilization (g/liter·hr) and ethanol production (g/liter·hr) in batch culture.     

Switching the mode of sucrose utilization by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H⁺ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.     

Substrates’ and products’ inhibition in fructanase production by a new Kluyveromyces marxianus CF15 from Agave tequilana fructan in a batch reactor

This study focuses on fructanase production in a batch reactor by a new strain isolated from agave juice (K. marxianus var. drosophilarum) employing different Agave tequilana fructan (ATF) concentrations as substrate. The experimental data suggest that the fructanase production may be inhibited or repressed by high substrate (50 g/L) and ethanol (20.7 g/L) concentrations present in culture medium. To further analyze these phenomena an unstructured kinetic mathematical model taking into account substrate and products inhibition was proposed and fitted. The mathematical model considers six reaction kinetics and the ethanol evaporation, and predicts satisfactorily the biomass, fructan, glucose, fructose, ethanol, and fructanase behavior for different raw material initial concentrations. The proposed model is the first to satisfactorily describe the production of fructanase from branched ATF with a new strain of K. marxianus.

     

Der Einfluß der Prozeßparameter auf die Ethanolausbeute in Batchprozessen bei Verwendung von Saccharomyces cerevisiae

The production costs of ethanol are dependent on the efficiency of the substrate-ethanol conversion to a high degree. The more the substrate used during the fermentation is converted into alcohol the better is the economy of the process. Therefore the ethanol yield Y _S^P is an important object of the process optimization. In batch fermentation processes the most essential influence factors are the initial biomass concentration X₀, the initial substrate concentration S₀, the temperature T, and the pH-value. A model reflecting the complex relationships between these influence factors and the ethanol yield could be obtained by regression. It allows an exact valuation of these optimum process parameters which are necessary for realizing high ethanol yields in the batch fermentation. For the strain Saccharomyces cerevisiae Sc 5 used in this research was found an ethanol yield maximum Y_S^P = 0˙5384 at the parameters X₀ = 64.61 g/l S₀ = 82.91 g/l T = 36.45°C pH = 6.54.     

Modelling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations

Aim: To study the effect of glucose concentrations on the growth by Brettanomyces bruxellensis yeast strain in batch experiments and develop a mathematical model for kinetic behaviour analysis of yeast growing in batch culture. Methods and Results: A Matlab algorithm was developed for the estimation of model parameters. Glucose fermentation by B. bruxellensis was studied by varying its concentration (5, 9·3, 13·8, 16·5, 17·6 and 21·4%). The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol and biomass production; at a substrate concentration of 50–138 g l^?1, the ethanol and biomass production were 24, 59 and 6·3, 11·4 g l^?1, respectively. However, an increase in glucose concentration to 165 g l^?1 led to a drastic decrease in product formation and substrate utilization. Conclusions: The model successfully simulated the batch kinetic observed in all cases. The confidence intervals were also estimated at each phase at a 0·95 probability level in a t‐Student distribution for f degrees of freedom. The maximum ethanol and biomass yields were obtained with an initial glucose concentration of 138 g l^?1. Significance and Impact of the Study: These experiments illustrate the importance of using a mathematical model applied to kinetic behaviour on glucose concentration by B. bruxellensis.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Ethanol fermentation by an efficient strain,NRRL B-4286, of Zymomonas mobilis

We studied the effect of the initial substrate concentration over the range of 100–250 g·l⁻¹ on the fermentation kinetics in batch cultures of Zymomonas mobilis NRRL B-4286 on glucose, fructose, and sucrose, using an adapted initial inoculum. With increasing concentrations of substrate, parameters related to growth were more rapidly and strongly affected than those related to ethanol production. This strain produced 94.0 g·l⁻¹, 76.9 g·l⁻¹, and 66.5 g·l⁻¹ of ethanol at glucose, fructose, and sucrose concentrations of 200 g·l⁻¹, respectively, more than the amount produced by the efficient strain ZM4 (NRRL B-14023).     

Fuel alcohol production by Zymomanas anaerobia: Kinetics

Process kinetics of a strain of Zymomonas anaerobia,which recently was shown to posses a high potential for ethanol production,are described applying the formal macroapproach The effect of ethanol inhibition on microbial growth,substrate utilization and alcohol production was investigated in detail. Growth rate and yield coefficients were affected by the ethanol concentration Several mathematical model functions from literature were tested for the quantification of experimental batch data. As none of these models was able to describe the entire growth phase a modified model using formal kinetics was developed for the rates of growth,substrate utilization and product formation (see Eqs. 10, 14, 18).     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G

A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying α-amylase and glucoamylase activities. Cell growth is modulated mainly by intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular substrate and biomass concentrations. Extracellular α-amylase and glucoamylase activities are taken to depend on biomass concentration. The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, α-amylase and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement. Journal of Industrial Microbiology & Biotechnology (2002) 29, 111–116 doi:10.1038/sj.jim.7000281 Received 07 January 2002/ Accepted in revised form 22 May 2002     

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (µ) and the cell yield (Y_X/S). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down‐regulation of genes in the ethanol‐producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in‐phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y_P/S) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described. Biotechnol. Bioeng. 2010;106: 721–730. © 2010 Wiley Periodicals, Inc.     

Dynamic flux balance modeling of S. cerevisiae and E. coli co-cultures for efficient consumption of glucose/xylose mixtures

Current researches into the production of biochemicals from lignocellulosic feedstocks are focused on the identification and engineering of individual microbes that utilize complex sugar mixtures. Microbial consortia represent an alternative approach that has the potential to better exploit individual species capabilities for substrate uptake and biochemical production. In this work, we construct and experimentally validate a dynamic flux balance model of a Saccharomyces cerevisiae and Escherichia coli co-culture designed for efficient aerobic consumption of glucose/xylose mixtures. Each microbe is a substrate specialist, with wild-type S. cerevisiae consuming only glucose and engineered E. coli strain ZSC113 consuming only xylose, to avoid diauxic growth commonly observed in individual microbes. Following experimental identification of a common pH and temperature for optimal co-culture batch growth, we demonstrate that pure culture models developed for optimal growth conditions can be adapted to the suboptimal, common growth condition by adjustment of the non-growth associated ATP maintenance of each microbe. By comparing pure culture model predictions to co-culture experimental data, the inhibitory effect of ethanol produced by S. cerevisiae on E. coli growth was found to be the only interaction necessary to include in the co-culture model to generate accurate batch profile predictions. Co-culture model utility was demonstrated by predicting initial cell concentrations that yield simultaneous glucose and xylose exhaustion for different sugar mixtures. Successful experimental validation of the model predictions demonstrated that steady-state metabolic reconstructions developed for individual microbes can be adapted to develop dynamic flux balance models of microbial consortia for the production of renewable chemicals.     

Fermentation of molasses using a thermotolerant yeast,Kluyveromyces marxianus IMB3: simplex optimisation of media supplements

 The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45°C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308–313, 1965) simplex optimisation method. The optimum concentrations of the supplements were 0.576 g l^-1 magnesium sulphate, 0.288 g l^-1 potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate. Received: 29 January 1996/Received revision: 23 April 1996/Accepted: 29 April 1996     

Production of ethanol from sugar cane molasses by Zymomonas mobilis

Summary Zymomonas mobilis strains were compared with each other and with a Saacharomyces cerevisiae strain for the production of ethanol from sugar cane molasses in batch fermentations. The effect of pH and temperature on ethanol production by Zymomonas was studied. The ability of Z. mobilis to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae. However, at higher sugar concentrations the yeast produced considerably more ethanol than Zymomonas.     

Rapid ethanol production from sucrose without by-product formation

Summary Non-sorbitol-producing Zymomonas mobilis ACM 3963 was developed from Z. mobilis UQM 2716. This strain was co-immobilised with invertase in alginate and incubated on sucrose-based media. This combination allowed theoretical yields of ethanol to be produced from 100 and 150 g/l sucrose, using both semi-defined media and sugar-cane syrup. No sorbitol or fructo-oligosaccharides were formed in either fermentation. Increased biomass concentrations immobilised in alginate reduced the batch fermentation times of 100 and 150 g/l sucrose by 50–70%, to 3 and 5 hours respectively. This strain also improved the efficiency of the fed-batch fermentation of sucrose.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

Simultaneous production of sugars and ethanol from inulin rich-extracts in a chemostat

Summary Incomplete fermentation of inulin-containing extracts by Saccharomyces diastaticus allows the simultaneous production of ethanol and syrups with increased fructose content. The yeast strain used ferments sucrose and inulin small polymers but does not easily ferment inulin large polymers. After batch fermentation a production of 62.5 g/L ethanol and 75 g/L of sugars containing up to 94 % fructose can be obtained. A continuous fermentation was performed in a chemostat permitting the adjustment of both productions according to the dilution rate with a maximal ethanol productivity of 3.9 g/L.h.     

The kinetics of ethanol production by Zymomonas mobilis on fructose and sucrose media

Summary Z.mobilis is strain ZM4 was grown on 250 g/l fructose and sucrose media in batch culture and on 100 and 150 g/l sucrose media in continuous culture. With fructose, a significant reduction in the growth rate and the cell yield was apparent although the other kinetic parameters were similar to those previously reported for fermentation of glucose. With sucrose the major differences were a reduction in ethanol yield, (due to levan formation) and a lower final ethanol concentration. Ethanol inhibition of sucrose metabolism occurred at relatively low ethanol concentrations compared to those inhibiting glucose metabolism.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.