Direct bioethanol production from wheat straw using xylose/glucose co-fermentation by co-culture of two recombinant yeasts

Journal of Industrial Microbiology & Biotechnology - To achieve a cost-effective bioconversion of lignocellulosic materials, a novel xylose/glucose co-fermentation process by co-culture of...     

In silico analysis of bioethanol production from glucose/xylose mixtures during fed-batch fermentation of co-culture and mono-culture systems

This study presents a detailed in silico analysis of bioethanol production from glucose/xylose mixtures of various compositions by fed-batch co-culture and mono-culture fermentation of specialized microbes. The mono-culture consists of recombinant Saccharomyces cerevisise that can metabolize both hexose and pentose sugars while the co-culture system consists of substrate-selective microbes. Dynamic flux balance models based on available genome-scale reconstructions of the microorganisms have been used to analyze bioethanol production in fed-batch culture with constant feed rates and the maximization of ethanol productivity is addressed by computing optimal aerobic-anaerobic switching times. The simulation results clearly point to the superior performance of fed-batch fermentation of microbial co-culture against fed-batch fermentation of mono-culture for bioethanol production from glucose/xylose mixtures. A set of potential genetic engineering strategies for enhancement of S. cerevisiae and Escherichia coli strains performance have been identified. Such in silico predictions using genome-scale models provide valuable guidance for conducting in vivo metabolic engineering experiments.     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Bench-scale bioethanol production from eucalyptus by high solid saccharification and glucose/xylose fermentation method

In the bioethanol production process, high solid saccharification and glucose/xylose co-fermentation are important technologies for obtaining increased ethanol concentrations; however, bench-scale studies using combinations of these methods are limited. In this study, we hydrolyzed high solid concentration of milled eucalyptus using commercial enzymes and obtained 138.4 g/L total monomeric sugar concentration. These sugars were fermented to 53.5 g/L of ethanol by a xylose-utilizing recombinant Saccharomyces cerevisiae strain, MA-R4. These experiments were performed in bench scale (using 50 L scale solid mixer and 70 L scale fermenter). The results obtained in this study were comparable to our previous results in laboratory scale, indicating that we successfully achieved an efficient high solid saccharification and glucose/xylose co-fermentation system in bench scale.     

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49–0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

木糖发酵生产乙醇的研究

选育出一株优良的木糖发酵菌株树干毕赤酵母菌7124,并利用纯木糖优化了木糖发酵条件,利用海藻酸钠固定化树干毕赤酵母菌增殖细胞,不仅能较好满足限氧发酵条件,而且能耐较高糖浓度,使乙醇发酵浓度提高到20g/L。利用半纤维素水解液进行了乙醇发酵的初步研究,基本达到了纯木糖发酵的效果。     

Ethanol production from xylose by a recombinant Candida utilis strain expressing protein-engineered xylose reductase and xylitol dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD(+)-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD(+)-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.     

Ethanol production from xylose by Fusarium oxysporum and the optimization of culture conditions

Bioethanol is the most commonly used renewable biofuel as an alternative to fossil fuels. Many microbial strains can convert lignocellulosics into bioethanol. However, very few natural strains with a high capability of fermenting pentose sugars and simultaneously utilizing various sugars have been reported. In this study, fermentation of sugar by Fusarium oxysporum G was performed for the production of ethanol to improve the performance of the fermentation process. The influences of pH, substrate concentration, temperature, and rotation speed on ethanol fermentation are investigated. The three significant factors (pH, substrate concentration, and temperature) are further optimized by quadratic orthogonal rotation regression combination design and response surface methodology (RSM). The optimum conditions are pH 4, 40?g/L of xylose, 32?°C, and 110?rpm obtained through single factor experiment design. Finally, it is found that the maximum ethanol production (10.0?g/L) can be achieved after 7 d of fermentation under conditions of pH 3.87, 45.2?g/L of xylose, and 30.4?°C. Glucose is utilized preferentially for the glucose–xylose mixture during the initial fermentation stage, but glucose and xylose are synchronously consumed without preference in the second period. These findings are significant for the potential industrial application of this strain for bioethanol production.     

Electricity production from xylose using a mediator-less microbial fuel cell

Electricity generation integrated with xylose degradation was investigated in a two-chamber mediator-less microbial fuel cell (MFC). Voltage output followed saturation kinetics as a function of xylose concentration for concentration below 9.7 mM, with a predicted maximum of 86 mV (6.3 mW m(-2) or 116 mW m(-3)) and half-saturation constant (K(s)) of 0.29 mM. Xylose concentrations from 0.5 mM to 1.5 mM resulted in coulombic efficiencies and maximum voltage ranging from 41+/-1.6% to 36+/-1.2% and 55+/-2.0 mV to 70+/-3.0 mV respectively. Xylose degradation rate increased with increasing xylose concentration up to 9.7 mM and the predicted maximum degradation rate was 0.13 mM h(-1) and K(s) of 3.0 mM. Stirring by nitrogen in the anode chamber led to 99+/-2.3 mV maximum voltage (8.4+/-0.4 mW m(-2) or 153+/-7.1 mW m(-3)) and 5.9+/-0.3% coulombic efficiency at MFC running time 180 h, which were respectively 17+/-1.2% and 37+/-1.8%, higher than those without stirring. The COD removal under stirring was 22.1+/-0.3%, which was slightly lower than that of 23.7+/-0.4% under no stirring. However, stirring resulted in 59% lower xylose degradation rate. This work demonstrates that xylose can be used in the MFC for electricity production. Comparatively higher electricity generation and coulombic efficiency can be obtained by adjusting initial xylose concentration and applying stirring in the anode chamber.     

High cell density co-culture for production of recombinant hydrolases

Sugarcane bagasse is a residue with great potential as a feedstock for second-generation ethanol production. One of the approaches studied for making use of this material is the utilization of enzymes to hydrolyze the cell wall carbohydrates and generate fermentable sugars. These enzymes can be produced by cultivation of filamentous fungi or bacteria; however, the high production cost still represents a bottleneck to second-generation ethanol production. Expression of recombinant hydrolases through a co-culture strategy could be an interesting alternative for producing a recombinant cocktail at high levels of productivity that is tailor-made for each material to be hydrolyzed. In this study we evaluate the production of hydrolases by co-culturing two recombinant Escherichia coli, each expressing a specific hydrolase, β-1,3-1,4-glucanase or β-1,4-xylanase, both isolated from Bacillus subtilis. The cultures were conducted in bioreactors in batch and fed-batch mode in order to reach high cell densities. Co-culture in batch cultivation reached a dry cell weight of 10.4 g/L and volumetric activities of 31.96 U/mL and 11.89 U/mL for xylanase and endoglucanase, respectively. Fed-batch cultivation reached a dry cell weight of 60 g/L and the volumetric activities of xylanase and endoglucanase were respectively up to 5 and 1.3 times higher than those in batch mode. A competition assay indicates that no clone predominates over the other during cultivation. These results suggest that co-culture is a potential technique for producing recombinant hydrolase cocktails at lower cost than those associated with the production of a single culture.     

Ethanol production by In situ xylose isomerization using recombinant Escherichia coli and fermentation using conventional Saccharomyces cerevisiae

Xylose isomerase from Geobacillus kaustophilus HTA426 was functionally expressed in Escherichia coli BL21 (DE3) and the recombinant E. coli cells were used together with conventional Saccharomyces cerevisiae to produce ethanol from xylose by simultaneous xylose isomerisation and fermentation. When recombinant E. coli cells were used as the source of xylose isomerase, a significant amount of ethanol was produced from xylose, whereas the control without recombinant E. coli cells did not produce any detectable amount of ethanol from xylose. Ethanol production was increased by 38% by feeding more recombinant E. coli at 48 h compared to adding recombinant E. coli only in the beginning, resulting in more ethanol production than P. stipitis CBS6054 under the same conditions. The xylitol accumulation by the in situ process was only 57% of that produced by the P. stipitis CBS6054.     

Ethanol production from sugarcane bagasse holocellulose. Coupled systems for saccharification,xylose isomerization and yeast fermentation

Summary Ethanol production from sugarcane bagasse holocellulose. Coupled systems for saccharification, xylose isomerization and yeast fermentation.An isomerization step was added to the coupled system for ethanol production from sugar cane bagasse in order to allow the alcoholic fermentation of xylose as xylulose bySaccharomyces cerevisiae. The yield (g ethanol/g pretreated bagasse) was improved according to the method of pretreatment: 0.185 to 0.24 with NaOH pretreatment, 0.11 to 0.18 for Ca(OH)₂ and 0.26 to 0.29 for a combined Ca(OH)₂/NaCl method.

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Resumen Se estudio la inculsión de una etapa de isomerización en el sistema de columnas acopladas, de producción de etanol a partir del bagazo de caña de azúcar, con el fin de permitir asi la fermentación alcohólica de la xilosa. El rendimiento expresado como g etanol/g bagazo pretratado, aumentó dependiendo del tipo de pretratamiento: 0.185 a 0.24 para el pretratamiento con NaOH; 0.11 a 0.18 para el correspondiente a Ca(OH)₂ y 0.26 a 0.29 para un tratamiento combinado de Ca(OH)₂-NaCl.

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Résumé L'ajout d'une étape d'isomérisation à un système couplé pour la production d'éthanol à partir de bagasse de cane à sucre a permis la fermentation éthanolique tant du xylose que du xylulose parSaccharomyces cerevisiae. Le rendement en g d'éthanol par g de bagasse prétraitée a crû selon la méthode de prétraitement: de 0.185 à 0.24 en cas de prétraitement à la soude caustique, de 0.11 à 0.18 dans le cas de Ca(OH)₂ et de 0.26 à 0.29 dans le cas de la méthode combinée Ca(OH)₂-NaCl.

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Based on a paper presented at the First Latin American Congress on Biotechnology, Tucumán, Argentina, October 4–8, 1987     

Modeling and estimation of production rate for the production phase of non-growth-associated high cell density processes

Bioprocess and Biosystems Engineering - This paper addresses the estimation of the specific production rate of intracellular products and the modeling of the bioreactor volume dynamics in high cell...     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Alcohol production from glucose and xylose usingEscherichia coli containingZymomonas mobilis genes

Summary AnEscherichia coli strain containing a recombinant plasmid encoding the pyruvate decarboxylase and alcohol dehydrogenase genes fromZymomonas mobilis metabolized glucose and xylose to near theoretical yields of ethanol. Enzyme activity measurements indicate high expression levels of both plasmid-encodedZymomonas proteins in the recombinantE. coli. The expression inE. coli is under the control of a promoter in theZymomonas sequence upstream of the pyruvate decarboxylase gene. The maximum ethanol level, using 4% glucose as substrate, was 1.8% (w/v) in anaerobic conditions. In aerobic conditions the natural repression ofE. coli alcohol dehydrogenase results in less ethanol production from clones expressing onlyZymomonas pyruvate decarboxylase.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae

The performance of encapsulated Saccharomyces cerevisiae CBS 8066 in anaerobic cultivation of glucose, in the presence and absence of furfural as well as in dilute-acid hydrolyzates, was investigated. The cultivation of encapsulated cells in 10 sequential batches in synthetic media resulted in linear increase of biomass up to 106 g/L of capsule volume, while the ethanol productivity remained constant at 5.15 (+/-0.17) g/L x h (for batches 6-10). The cells had average ethanol and glycerol yields of 0.464 and 0.056 g/g in these 10 batches. Addition of 5 g/L furfural decreased the ethanol productivity to a value of 1.31 (+/-0.10) g/L x h with the encapsulated cells, but it was stable in this range for five consecutive batches. On the other hand, the furfural decreased the ethanol yield to 0.41-0.42 g/g and increased the yield of acetic acid drastically up to 0.068 g/g. No significant lag phase was observed in any of these experiments. The encapsulated cells were also used to cultivate two different types of dilute-acid hydrolyzates. While the free cells were not able to ferment the hydrolyzates within at least 24 h, the encapsulated yeast successfully converted glucose and mannose in both of the hydrolyzates in less than 10 h with no significant lag phase. However, since the hydrolyzates were too toxic, the encapsulated cells lost their activity gradually in sequential batches.