Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.     

Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase

A derivative of Klebsiella oxytoca M5A1 containing chromosomally integrated genes for ethanol production from Zymomonas mobilis (pdc, adhB) and endoglucanase genes from Erwinia chrysanthemi (celY, celZ) produced over 20 000 U endoglucanase l^–1 activity during fermentation. In combination with the native ability to metabolize cellobiose and cellotriose, this strain was able to ferment amorphous cellulose to ethanol (58–76% of theoretical yield) without the addition of cellulase enzymes from other organisms.     

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.Nomenclature S glucose concentration (g/L) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - Q_p volumetric ethanol productivity (g/L.h) - Q_X volumetric biomass productivity (g/L.h) - Y_X/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

A kinetic model and simulation of starch saccharification and simultaneous ethanol fermentation by amyloglucosidase and Zymomonas mobilis

A mathematical model is described for the simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and Zymomonas mobilis. By introducing the degree of polymerization (DP) of oligosaccharides produced from sago starch treated with -amylase, a series of Michaelis-Menten equations were obtained. After determining kinetic parameters from the results of simple experiments carried out at various substrate and enzyme concentrations and from the subsite mapping theory, this model was adapted to simulate the SSF process. The results of simulation for SSF are in good agreement with experimental results.List of Symbols g/g rate coefficient of production - _max 1/h maximum specific growth rate - E %, v/w AMG concentration - G ₁ mmol/l glucose concentration - G _c mmol/l glucose concentration consumed - G _f mmol/l glucose concentration formed - G _n mmol/l n-mer maltooligosaccharide concentration - K _i g/l ethanol inhibition constant for ethanol production - K _g mmol/l glucose inhibition constant for glucose production - K _p mmol/l glucose limitation constant for ethanol production - K _x mmol/l glucose limitation constant for cell growth - K _m,n mmol/l Michaelis-Menten constant for n-mer oligosaccharide - k _e %, v/w enzyme limitation constant - k _es proportional constant - k _{max, n} 1/s maximal velocity for n-mer digestion - k _s g/l substrate limitation constant - m _s g/g maintenance energy - MW _n g/mol molecular weight of n-mer oligosaccharide - P g/l ethanol concentration - P ₀ g/l initial ethanol concentration - P _m g/l maximal ethanol concentration - Q _pm g/(g · h) maximum specific ethanol production rate - S _n mmol/h branched n-mer oligosaccharide concentration - S ₀ g/l initial starch concentration - S _sta g/l starch concentration - S _tot g/l total sugar concentration - V _{max, n} 1/h maximum digestion rate of n-mer oligosaccharide - V ₀ g/(l · h) initial glucose formation rate - X g/l cell mass - X ₀ g/l initial cell mass - Y _p/s g/g ethanol yield - Y _x/s g/g cell mass yield     

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.     

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.     

Continuous ethanol production by a flocculent strain of Zymomonas mobilis

Summary Cell retention and ethanol production using the flocculent bacterium Zymomonas mobilis NRRL B-12526 were studied in three bioreactor configurations. The flocculent growth characteristic of this strain and a special reactor design were combined to achieve relatively high cell concentrations in a continuous bioreactor for the conversion of glucose to ethanol.Research sponsored by the Office of Energy Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.     

Modelling ethanol production from cellulose: separate hydrolysis and fermentation versus simultaneous saccharification and fermentation

In ethanol production from cellulose, enzymatic hydrolysis, and fermentative conversion may be performed sequentially (separate hydrolysis and fermentation, SHF) or in a single reaction vessel (simultaneous saccharification and fermentation, SSF). Opting for either is essentially a trade-off between optimal temperatures and inhibitory glucose concentrations on the one hand (SHF) vs. sub-optimal temperatures and ethanol-inhibited cellulolysis on the other (SSF). Although the impact of ethanol on cellobiose hydrolysis was found to be negligible, formation of glucose and cellobiose from cellulose were found to be significantly inhibited by ethanol. A previous model for the kinetics of enzymatic cellulose hydrolysis was, therefore, extended with enzyme inhibition by ethanol, thus allowing a rational evaluation of SSF and SHF. The model predicted SSF processing to be superior. The superiority of SSF over SHF (separate hydrolysis and fermentation) was confirmed experimentally, both with respect to ethanol yield on glucose (0.41 g g^?1 for SSF vs. 0.35 g g^?1 for SHF) and ethanol production rate, being 30% higher for an SSF type process. High conversion rates were found to be difficult to achieve since at a conversion rate of 52% in a SSF process the reaction rate dropped to 5% of its initial value. The model, extended with the impact of ethanol on the cellulase complex proved to predict reaction progress accurately.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

一株葡萄糖和木糖共发酵高效产乙醇重组运动发酵单胞菌的性能评价

木糖是木质纤维素原料水解液中的第二大组分,木糖和葡萄糖的充分利用是有经济性地生产纤维素乙醇的关键。通过基因克隆手段构建了一株可以高效利用木糖产乙醇的重组运动发酵单胞菌Zymomonas mobilis TSH01,并进行了利用单糖溶液、混合糖溶液及玉米秸秆水解液发酵产乙醇效率的研究。结果表明,利用单一葡萄糖或单一木糖溶液发酵时,当糖浓度为8%、发酵72 h后,糖利用率分别为100%和98.9%,乙醇代谢收率分别为87.8%和78.3%;利用8%葡萄糖和8%木糖的混合溶液发酵时,72 h后,葡萄糖和木糖的利用率分别为98.5%和97.4%,乙醇代谢收率为94.9%。利用含3.2%葡萄糖和3.5%木糖的玉米秸秆水解液发酵72 h后,葡萄糖和木糖的利用率分别为100%和92.3%,乙醇代谢收率为91.5%。此外,磷酸二氢钾对发酵过程中木糖利用率以及乙醇收率的提高有明显促进作用。     

High productivity continuous ethanol fermentation with a flocculating mutant strain of Zymomonas mobilis

High fermenter (volumetric) ethanol productivities (80 g/lh^–1) were attained in a simple single-stage continuous-stirred-tank-reactor (CSTR) employing a flocculent mutant of Zymomonas mobilis with a feed containing 100g/l glucose. Under these conditions a final ethanol concentration of 47.6 g/l was obtained, representing a maximum conversion efficiency of 97% of theoretical.Nomenclature SR = Medium glucose concentration (g/l)X Biomass concentration (g/l) - P Ethanol concentration (g/l) - VP Volumetric productivity (g ethanol/l/h) - Yp/s Product yield coefficient (g ethanol/g glucose consumed) - Qp Specific rate of ethanol formation (g ethanol/g cells/h) - D Dilution rate (h^–1) - Dmax Maximum dilution rate: ie., highest dilution rate at which the effluent glucose concentration 4g/l (h^–1)     

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.     

Saccharification and fermentation of Sugar Cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway

Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter papaer units (FPU) g(-1) acid-treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid-treated bagasse L(-1)), a combination of 20 FPU cellulase g(-1) bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L(-1). Alternatively, almost 40 g ethanol L(-1) was produced with 10 FPU cellulase g(-1) bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU(-1) of commercial cellulase. (c) 1994 John Wiley & Sons, Inc.     

Studies on the use of a thermotolerant strain of Kluyveromyces marxianus in simultaneous saccharification and ethanol formation from cellulose

 The thermotolerant yeast strain, Kluyveromyces marxianus IMB3, was found to be capable of ethanol production during growth at 45°C on media containing milled paper and exogenously added commercial cellulase. At maximum achievable cellulose concentrations in shake-flask cultures, ethanol production increased to 6.6 g/l at 45°C, representing an overall level of conversion of 21% of the maximum theoretical yield. Subsequent studies involving variations in added cellulase concentrations to the batch systems demonstrated that ethanol yields could be increased to 10 g/l at 45°C, which represented 39% of the maximum theoretical yield. As a result of ethanol production at 45°C in the systems examined, we suggest that the thermotolerant ethanol-producing yeast strain K. marxianus represents a novel candidate for use in simultaneous saccharification and conversion of the resulting substrates to ethanol. Received: 9 June 1994/Received revision: 8 August 1994/Accepted: 12 August 1994     

Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis

A model has been developed for the fermentation of mixtures of glucose and xylose by recombinant Zymomonas mobilis strain ZM4(pZB5), containing additional genes for xylose assimilation and metabolism. A two-substrate model based on substrate limitation, substrate inhibition, and product (ethanol) inhibition was evaluated, and experimental data was compared with model simulations using a Microsoft EXCEL based program and methods of statistical analysis for error minimization. From the results it was established that the model provides good predictions of experimental batch culture data for 25/25, 50/50, and 65/65 g l^–1 glucose/xylose media.     

Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.     

Development of new unstructured model for simultaneous saccharification and fermentation of starch to ethanol by recombinant strain

The development of simultaneous saccharification and fermentation of starch to ethanol (SSFSE) by genetically modified microbial strains has been studied intensively [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445; G. Birol, Z.Ï. Önsan, B. Kirdar, S.G. Oliver, Ethanol production and fermentation characteristics of recombinant Saccharomyces cerevisiae strains grown on starch, Enzyme Microb. Technol. 22 (1998) 672–677; F. Kobayashi, Y. Nakamura, Effect of repressor gene on stability of bioprocess with continuous conversion of starch into ethanol using recombinant yeast, Biochem. Eng. J. 18 (2004) 133–141; F. Kobayashi, Y. Nakamura, Mathematical model of direct ethanol production from starch in immobilized recombinant yeast culture, Biochem. Eng. J. 21 (2004) 93–101; M.M. Altintas, K.Ö. Ülgen, B. Kirdar, Z.Ï. Önsan, S.G. Oliver, Improvement of ethanol production from starch by recombinant yeast through manipulation of environmental factors, Enzyme Microb. Technol. 31 (2002) 640–647; K.Ö. Ülgen, B. Saygili, Z.Ï. Önsan, B. Kirdar, Bioconversion of starch into ethanol by a recombinant Saccharomyces cerevisiae strain YPG-AB, Process Biochem. 37 (2002) 1157–1168]. Saccharomyces cerevisiae YPB-G strain secretes a bifunctional fusion protein containing enzymatic activity of the B. subtilis alpha-amylase and of the Aspergillus awamori glucoamylase [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445], and therefore is distinguished in relation to SSFSE step. In this work we have used the experimental data, presented in the paper [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445] to develop two-hierarchic-level unstructured mathematical model describing kinetics of direct bioconversion of starch to ethanol. The first level has modeled enzymatic hydrolysis of starch to glucose by bifunctional protein and the second level includes utilization and bioconversion of glucose to ethanol by yeasts. The second level has unified the enzymatic degradation of starch, and glucose metabolization to ethanol by microorganisms. The response surface analysis was used to develop the rates models. A hybrid genetic algorithm and a decomposition approach were used in the nonlinear parameters identification procedure. The proposed model demonstrated excellent flexibility for different operational conditions of SSFSE process, and can be used successfully to describe microbial physiology of genetically modified strains.     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

Ethanol production in simultaneous saccharification and fermentation of cellulose with temperature profiling

The effects of temperature on enzymatic saccharification of cellulose and simulataneous saccharification and fermentation (SSF) were investigated with 100 g·l⁻¹ Solka Floc, 5g·l⁻¹Trichoderma reesei cellulase, and Zymomonas mobilis ATCC 29191. The following results were obtained: 1) Ethanol fermentation under glucose dificient conditions can proceed for more than 100 h at 30°C but gradually ceases after 50 h of operation at 40°C. 2) Equivalent glucose yield based on cellulose for SSF operated at its optimum temperature (37°C) is higher than that for enzymatic saccharification of cellulose at the same temperature by 32%. However, the same equivalent glucose yields were obtained for both processes if they were operated at their respective optimum temperature. 3) SSF with temperature cycling increased the ethanol productivity but gave similar ethanol yield to SSF at 37°C. 4) SSF with temperature profiling gave an ethanol yield of 0.32 g·g⁻¹ and cellulose use of 0.86 g·g⁻¹ which were increased by 39% and 34% over SSF with temperature cycling and at 37°C.