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.     

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars

Currently, microbial conversion of lignocellulose‐derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome‐scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole‐genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations’ involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next‐generation biofuels.     

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.     

Succinate production from xylose‐glucose mixtures using a consortium of engineered Escherichia coli

The conversion of variable sugar mixtures into biochemicals poses a challenge for a single microorganism. For example, succinate has not been effectively generated from mixtures of glucose and xylose. In this work, a consortium of two Escherichia coli strains converted xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, the optimal pathway from xylose or glucose to succinate was determined by expressing either heterologous pyruvate carboxylase or heterologous adenosine triphosphate‐forming phosphoenol pyruvate (PEP) carboxykinase. Expression of PEP carboxykinase (pck) resulted in higher yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase (pyc) resulted in higher productivity (76 mg/gh) for glucose conversion. Then, processes using consortia of the two optimal xylose‐selective and glucose‐selective strains were designed for two different feed ratios of glucose/xylose. In each case the consortia generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of microbial consortia for the conversion of sugar mixtures: each sugar‐to‐product pathway can be optimized independently, and the volumetric consumption rate for each sugar can be controlled independently, for example, by altering the biomass concentration of each consortium member strain.     

Microbial removal of acetate selectively from sugar mixtures

Acetic acid is an unavoidable constituent of the biomass hydrolysates generated from acetylated hemicellulose and lignin, and acetate affects the performance of microbes used to convert these hydrolysates into biofuels or other biochemicals. In this study, acetate was selectively removed from synthetic mixtures of glucose and xylose using metabolically engineered Escherichia coli strains having mutations in the glucose phosphotransferase system (PTS) genes (ptsG, manZ, crr), glucokinase (glk), and xylose (xylA). In batch culture, ALS1060 (ptsG manZ glk xylA) consumed exclusively acetate to depletion, and then consumed the two sugars only at a very slow rate (a growth rate of about 0.01 h⁻¹). We also examined the effects of an additional knockout of either malX, fruA, fruB, bglF, or crr, genes that are involved in other PTSs, and a batch process using KD840 (ptsG manZ glk crr xylA) demonstrated a further reduction in glucose or xylose consumption by E. coli. These results demonstrate the feasibility of using a substrate-selective approach for the pre-treatment of biomass hydrolysate for microbial processes.     

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.     

Fermentation performance and intracellular metabolite patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae

Heterologous genes for xylose utilization were introduced into an industrial Saccharomyces cerevisiae, strain A, with the aim of producing fuel ethanol from lignocellulosic feedstocks. Two transformants, A4 and A6, were evaluated by comparing the performance in 4-l anaerobic batch cultivations to both the parent strain and a laboratory xylose-utilizing strain: S. cerevisiae TMB 3001. During growth in a minimal medium containing a mixture of glucose and xylose (50 g/l each), glucose was preferentially consumed. During the first growth phase on glucose, the specific growth rates were 0.26, 0.32, 0.27 and 0.30 h^–1 for strains TMB 3001, A (parental strain), A4, and A6, respectively. The specific ethanol productivities were 0.04, 0.13, 0.04 and 0.03 g/g^.per hour, for TMB 3001, A, A4 and A6, respectively. The specific xylose consumption rates were 0.06, 0.21 and 0.14 g/g^.per hour, respectively for strains TMB 3001, A4 and A6. Xylose consumption resulted mainly in the formation of xylitol, with biomass and ethanol being minor products. The metabolite profile of intermediates in the pentose phosphate pathway and key glycolytic intermediates were determined during growth on glucose and xylose, respectively. The metabolite pattern differed depending on whether glucose or xylose was utilized. The levels of intracellular metabolites were higher in the industrial strains than in the laboratory strain during growth on xylose. Electronic Publication     

Modeling alcoholic fermentation of glucose/xylose mixtures by ethanologenic Escherichia coli as a function of pH

In order to understand the effect of pH on growth and ethanol production in ethanologenic Escherichia coli, we investigated the kinetic behavior of ethanologenic E. coli during alcoholic fermentation of glucose or xylose in a controlled pH environment and the fermentation of glucose, xylose, or their mixtures without pH control. Based on the Monod equation, an unstructured and unsegregated kinetic model was proposed as a function of the pH of the fermentation medium. The pH effects on cell growth, sugar consumption, and ethanol production were taken into account in the proposed model. Both cell growth and ethanol production were found to be significantly influenced by the pH of the fermentation medium. The optimal pH range for ethanol production by ethanologenic E. coli on either glucose or xylose was 6.0–6.5. The highest value of the maximum specific growth rate (μ _m) was obtained at pH 7.0. In the kinetic model of the fermentations of the sugar mixture, two inhibition terms related to glucose concentrations were included in both the cell growth and ethanol production equations because of the strong inhibitions of glucose and glucose metabolites on xylose metabolism. A good fit was found between model predictions and experimental data for both single-sugar and mixed-sugar fermentations without pH control within the experimental domain.     

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.     

Production of naringenin from D‐xylose with co‐culture of E. coli and S. cerevisiae

Heterologous production of naringenin, a valuable flavonoid with various biotechnological applications, was well studied in the model organisms such as Escherichia coli or Saccharomyces cerevisiae. In this study, a synergistic co‐culture system was developed for the production of naringenin from xylose by engineering microorganism. A long metabolic pathway was reconstructed in the co‐culture system by metabolic engineering. In addition, the critical gene of 4‐coumaroyl‐CoA ligase (4CL) was simultaneously integrated into the yeast genome as well as a multi‐copy free plasmid for increasing enzyme activity. On this basis, some factors related with fermentation process were considered in this study, including fermented medium, inoculation size and the inoculation ratio of two microbes. A yield of 21.16 ± 0.41 mg/L naringenin was produced in this optimized co‐culture system, which was nearly eight fold to that of the mono‐culture of yeast. This is the first time for the biosynthesis of naringenin in the co‐culture system of S. cerevisiae and E. coli from xylose, which lays a foundation for future study on production of flavonoid.     

Engineering of carbon catabolite repression in recombinant xylose fermenting<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (mig1, XYL1, XYL2, XKS1) and CPB.MBH2 (mig1, mig2, XYL1, XYL2, XKS1) were analysed for changes in xylose consumption rate and ethanol production rate during anaerobic batch and chemostat cultivations on a mixture of 20 g l^–1 glucose and 50 g l^–1 xylose, and their characteristics were compared to the parental strain S. cerevisiae TMB3001 (XYL1, XYL2, XKS1). Improvement of xylose utilisation was limited during batch cultivations for the constructed strains compared to the parental strain. However, a 25% and 12% increased xylose consumption rate during chemostat cultivation was achieved for CPB.CR2 and CPB.MBH2, respectively. Furthermore, during chemostat cultivations of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting that xylose is a repressive sugar for S. cerevisiae.     

Dynamic flux balance modeling of microbial co-cultures for efficient batch fermentation of glucose and xylose mixtures

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture.     

Fermentation of xylose into ethanol by a new fungus strain <Emphasis Type="Italic">Pestalotiopsis</Emphasis> sp. XE-1

A new fungus, Pestalotiopsis sp. XE-1, which produced ethanol from xylose with yield of 0.47 g ethanol/g of consumed xylose was isolated. It also produced ethanol from arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, and sucrose with yields of 0.38, 0.47, 0.45, 0.46, 0.31, 0.25, 0.31, and 0.34 g ethanol/g of sugar consumed, respectively. It produced maximum ethanol from xylose at pH 6.5, 30°C under a semi-aerobic condition. Acetic acid produced in xylose fermenting process inhibited ethanol production of XE-1. The ethanol yield in the pH-uncontrolled batch fermentation was about 27% lower than that in the pH-controlled one. The ethanol tolerance of XE-1 was higher than most xylose-fermenting, ethanol-producing microbes, but lower than Saccharomyces cerevisiae and Hansenula polymorpha. XE-1 showed tolerance to high concentration of xylose, and was able to grow and produce ethanol even when it was cultivated in 97.71 g/l xylose.     

Microbial consortium-mediated reprogramming of defence network in pea to enhance tolerance against Sclerotinia sclerotiorum

Aims: To evaluate the potentiality of three rhizosphere microorganisms in suppression of Sclerotinia rot in pea in consortia mode and their impact on host defence responses. Methods and Results: Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 from rhizospheric soils were selected based on compatibility, antagonistic and plant growth promotion activities. The microbes were used as consortia to assess their ability to trigger the phenylpropanoid and antioxidant activities and accumulation of proline, total phenol and pathogenesis‐related (PR) proteins in pea under the challenge of the soft‐rot pathogen Sclerotinia sclerotiorum. The triple‐microbe consortium and single‐microbe treatments showed 1·4–2·3 and 1·1–1·7‐fold increment in defence parameters, respectively, when compared to untreated challenged control. Activation of the phenylpropanoid pathway and accumulation of total phenolics were highest at 48 h, whereas accumulation of proline and PR proteins along with activities of the antioxidant enzymes was highest at 72 h. Conclusions: The compatible microbial consortia triggered defence responses in an enhanced level in pea than the microbes alone and provided better protection against Sclerotinia rot. Significance and Impact of the Study: Rhizosphere microbes in consortium can enhance protection in pea against the soft‐rot pathogen through augmented elicitation of host defence responses.     

Conversion of glucose‐xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose‐xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed‐batch process, both sugars in a glucose‐xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.     

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.     

A co-fermentation strategy to consume sugar mixtures effectively

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates.     

Kinetic models for growth and product formation on multiple substrates

Hydrolyzates from lignocellulosic biomass contain a mixture of simple sugars; the predominant ones being glucose, cellobiose and xylose. The fermentation of such mixtures to ethanol or other chemicals requires an understanding of how each of these substrates is utilized.Candida lusitaniae can efficiently produce ethanol from both glucose and cellobiose and is an attractive organism for ethanol production. Experiments were performed to obtain kinetic data for ethanol production from glucose, cellobiose and xylose. Various combinations were tested in order to determine kinetic behavior with multiple carbon sources. Glucose was shown to repress the utilization of cellobiose and xylose. However, cellobiose and xylose were simultaneously utilized after glucose depletion. Maximum volumetric ethanol production rates were 0.56, 0.33, and 0.003 g/L-h from glucose, cellobiose and xylose, respectively. A kinetic model based on cAMP mediated catabolite repression was developed. This model adequately described the growth and ethanol production from a mixture of sugars in a batch culture.