Xylose Utilization Rate Research Articles

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8g/L vs. 19.4g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28g/L·h vs. 0.16g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53g/L·h vs. 0.26g/L·h) and yield (0.32g/g vs. 0.28g/g). When the initial total sugar concentration was ~120g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4g/L, yield of 0.43g/g sugar consumed, productivity of 0.87g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.

Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003.

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72°C, Geobacillus LC300 has a growth rate of 2.15h−1 on glucose and 1.52h−1 on xylose (doubling time less than 30min). The corresponding specific glucose and xylose utilization rates are 5.55g/g/h and 5.24g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio׳s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.

Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate.

BackgroundComplete conversion of the major sugars of biomass including both the C5 and C6 sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to ‘hydrolysate toxicity,’ a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate.ResultsA turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several ‘sub-strains’ were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanol yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3’s improved performance.ConclusionWe have adapted/evolved Z. mobilis strain 8b for enhanced tolerance to the toxic compounds present in corn stover hydrolysates. The adapted strain SS3 has higher xylose utilization rate and produce more ethanol than the parent strain. We have identified transcriptional changes which may be responsible for these phenotypes, providing foundations for future research directions in improving Z. mobilis as biocatalysts for the production of ethanol or other fuel precursors.

Expression of a xylose-specific transporter improves ethanol production by metabolically engineered Zymomonas mobilis.

Zymomonas mobilis is a promising organism for biofuel production as it can produce ethanol from glucose at high rates. However, Z. mobilis does not natively ferment C5 sugars such as xylose. While it has been engineered to do so, the engineered strains do not metabolize these sugars at high rates. Previous research has identified some of the bottlenecks associated with xylose metabolism in Z. mobilis. In this work, we investigated transport as a possible bottleneck. In particular, we hypothesized that the slow uptake of xylose through the promiscuous Glf transporter may limit the efficiency of xylose metabolism in Z. mobilis. To test this hypothesis, we expressed XylE, the low-affinity xylose transporter from Escherichia coli, in a xylose-utilizing strain of Z. mobilis. Our results show that the expression of this pentose-specific transporter improves the rate of xylose utilization in Z. mobilis; however, this enhancement is seen only at high xylose concentrations. In addition, we also found that overexpression of the promiscuous Z. mobilis transporter Glf yielded similar results, suggesting that the transport bottleneck is not due to the specificity, but rather the capacity for sugar uptake.

Pulsed addition of HMF and furfural to batch-grown xylose-utilizing Saccharomyces cerevisiaeresults in different physiological responses in glucose and xylose consumption phase

BackgroundPretreatment of lignocellulosic biomass generates a number of undesired degradation products that can inhibit microbial metabolism. Two of these compounds, the furan aldehydes 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), have been shown to be an impediment for viable ethanol production. In the present study, HMF and furfural were pulse-added during either the glucose or the xylose consumption phase in order to dissect the effects of these inhibitors on energy state, redox metabolism, and gene expression of xylose-consuming Saccharomyces cerevisiae.ResultsPulsed addition of 3.9 g L-1 HMF and 1.2 g L-1 furfural during either the glucose or the xylose consumption phase resulted in distinct physiological responses. Addition of furan aldehydes in the glucose consumption phase was followed by a decrease in the specific growth rate and the glycerol yield, whereas the acetate yield increased 7.3-fold, suggesting that NAD(P)H for furan aldehyde conversion was generated by acetate synthesis. No change in the intracellular levels of NAD(P)H was observed 1 hour after pulsing, whereas the intracellular concentration of ATP increased by 58%. An investigation of the response at transcriptional level revealed changes known to be correlated with perturbations in the specific growth rate, such as protein and nucleotide biosynthesis. Addition of furan aldehydes during the xylose consumption phase brought about an increase in the glycerol and acetate yields, whereas the xylitol yield was severely reduced. The intracellular concentrations of NADH and NADPH decreased by 58 and 85%, respectively, hence suggesting that HMF and furfural drained the cells of reducing power. The intracellular concentration of ATP was reduced by 42% 1 hour after pulsing of inhibitors, suggesting that energy-requiring repair or maintenance processes were activated. Transcriptome profiling showed that NADPH-requiring processes such as amino acid biosynthesis and sulfate and nitrogen assimilation were induced 1 hour after pulsing.ConclusionsThe redox and energy metabolism were found to be more severely affected after pulsing of furan aldehydes during the xylose consumption phase than during glucose consumption. Conceivably, this discrepancy resulted from the low xylose utilization rate, hence suggesting that xylose metabolism is a feasible target for metabolic engineering of more robust xylose-utilizing yeast strains.

Fine-tuning of xylose metabolism in genetically engineered Saccharomyces cerevisiae by scattered integration of xylose assimilation genes

Manipulation of multiple genes is a common experience in metabolic engineering and synthetic biology studies. Chromosome integration of multiple genes in one single position is always performed, however, there is so far no study on the integration of multiple genes separately in various positions (here in after referred to as “scattered integration”) and its effect on fine-tuning of cellular metabolism. In this study, scattered integration of the xylose assimilation genes PsXR, PsXDH and ScXK was investigated in Saccharomyces cerevisiae, and transcription analysis of these genes as well as their enzyme activities were compared with those observed when the genes were integrated into one single site (defined as “tandem integration” here). Not only notable differences in transcription levels and enzyme activities were observed when the genes were integrated by the two strategies, but also change of the cofactor preference of PsXR gene was validated. Xylose fermentation was further studied with the strains developed with these strategies, and elevated xylose utilization rate was obtained in the scattered integration strain. These results proved that by positioning multiple genes on different chromosomes, fine-tuning of cellular metabolism could be achieved in recombinant S. cerevisiae.

Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source.

Overcoming inhibitors in a hemicellulosic hydrolysate: improving fermentability by feedstock detoxification and adaptation of Pichia stipitis

In order to improve the fermentative efficiency of sugar maple hemicellulosic hydrolysates for fuel ethanol production, various methods to mitigate the effects of inhibitory compounds were employed. These methods included detoxification treatments utilizing activated charcoal, anion exchange resin, overliming, and ethyl acetate extraction. Results demonstrated the greatest fermentative improvement of 50% wood hydrolysate (v/v) by Pichia stipitis with activated charcoal treatment. Another method employed to reduce inhibition was an adaptation procedure to produce P. stipitis stains more tolerant of inhibitory compounds. This adaptation resulted in yeast variants capable of improved fermentation of 75% untreated wood hydrolysate (v/v), one of which produced 9.8g/l±0.6 ethanol, whereas the parent strain produced 0.0g/l±0.0 within the first 24h. Adapted strains RS01, RS02, and RS03 were analyzed for glucose and xylose utilization and results demonstrated increased glucose and decreased xylose utilization rates in comparison to the wild type. These changes in carbohydrate utilization may be indicative of detoxification or tolerance activities related to proteins involved in glucose and xylose metabolism.

Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V(max) of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] × h versus 0.007 g/g [DW] × h), and the anaerobic growth rate was increased from ∼0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H 2 production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H 2 yield and specific H 2 production rates with xylose and glucose are 1.63 mol-H 2/mol xylose and 11.14-H 2 mmol/h g-cdw, and 2.02 mol-H 2/mol-glucose and 9.37 mmol-H 2/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H 2 yield and specific H 2 production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H 2/mol-mixed sugar and 12.56 mmol-H 2/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.

Multi-stage continuous culture fermentation of glucose–xylose mixtures to fuel ethanol using genetically engineered Saccharomyces cerevisiae 424A

Multi-stage continuous (chemostat) culture fermentation (MCCF) with variable fermentor volumes was carried out to study the utilization of glucose and xylose for ethanol production via mixed sugar fermentation (MSF). Variable fermentor volumes were used to enable enhanced sugar utilization, accounting for differences in glucose and xylose utilization rates. Saccharomyces cerevisiae 424A-LNH-ST was used for fermentation of glucose–xylose mixtures. The dilution rates employed for continuous fermentation were based on earlier batch kinetic studies of ethanol production and sugar utilization. With a feed containing approximately 30 g L −1 glucose and 15 g L −1 xylose, cell washout was observed at a dilution rate of 0.8 h −1. At dilution rates below 0.5 h −1, complete glucose utilization was observed. Xylose consumption in the first-stage 1 L reactor was only 37% at the lowest dilution rate studied, 0.05 h −1. At this same flow rate, xylose consumption rose to 69% after subsequently passing through 3 and 1 L reactors in series, primarily due to the longer residence time in the 3 L reactor (0.0167 h −1 dilution rate).

The Ethanol Tolerance of Pachysolen tannophilus in Fermentation on Xylose

The influence of ethanol on fermentation by Pachysolen tannophilus was studied. When xylose utilization rate was 80%, ethanol concentration began to decline. Fermentation of P. tannophilus was affected by ethanol addition in the beginning of fermentation; average xylose consumption rate was 0.065 g.l(-1).h(-1), and maximum specific growth rate was 0.07 h(-1) at 28 g.l(-1) ethanol, comparing with the average xylose consumption rate of 0.38 g.l(-1).h(-1) and maximum specific growth rate of 0.14 h(-1) in fermentation with no ethanol addition; P. tannophilus stopped growth at 40 g.l(-1) ethanol. When the initial ethanol concentration was 30 g.l(-1), the addition of glucose in xylose media made the growth of P. tannophilus better, and the most favorable glucose concentration was 15 g.l(-1) with the highest biomass of 1.51 g.l(-1) as compared with that of 0.95 g.l(-1) in pure xylose media.

Forced modulation of biological processes: A review

Cell culture or biochemical processes utilizing free or immobilized cells or enzymes are candidates for periodic operation. Even though these processes are isothermal, inhibition frequently arises giving their kinetics a push–pull character of the Turing type so that excited states or multiplicity are observed. Modulation of an input such as a substrate or a nutrient concentration or the cell environment such as the pH can enhance the rates of biochemical reactions that are occurring. This possibility was realized by explorers of reactor periodic operation in the 1980s. However, the use of rotating disc biological filters for aerobic waste treatment precedes these activities by at least a decade. The biologically active slime on the disc surface is exposed alternately to air and to an aqueous media containing dissolved and/or suspended waste matter. Indeed, periodic variation of reactor volume in a CSTR, which is equivalent to periodic variation of space velocity, was explored in the early 1970s. The application of this was to activated sludge waste treatment. Periodic operation has been used to address the problem of plasmid loss in continuous fermenters to produce genetically altered microorganisms. A twofold increase in the concentration of plasmid bearing cells using square wave modulation of the dilution rate has been found in a simulation using a model validated by experimental data. These results are in agreement with other studies attempting to control species populations in chemostats. Improvement in the rate of fermentation of a substrate to form a metabolite has been addressed by modulation. It has been shown that the rate of xylose utilization by a yeast is affected by a periodic variation of pH. These were lowered, but the metabolite yield, ethanol, remained the same. With secondary metabolites associated with the non-growth period in fermentation, modulation of the growth-limiting substrate concentration fed to immobilized live cells resulted in significantly higher yields. Differences in the effect of cycling on primary and secondary metabolites has been observed in several biochemical systems, such as those used for penicillin and monoclonal antibody production or in citric acid fermentation.

The effect of initial cell concentration on xylose fermentation by Pichia stipitis

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R(2) of 0.99.

Conversion of Xylose to Ethanol by Recombinant Saccharomyces cerevisiae: Importance of Xylulokinase (XKS1) and Oxygen Availability

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4gL−1 ethanol was produced from 47gL−1 xylose during 100h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP:ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).

Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4(pZB5).

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in kappa-carrageenan beads having a diameter of 1.5-2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2-4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5-15.3 g/L.h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars.

Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugar D-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design.Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinant Z. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a "benchmark" yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5-2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.

Effect of inoculum level of xylitol production from rice straw hemicellulose hydrolysate by Candida guilliermondii.

The effect of inoculum level on xylitol production by Candida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L-1 to 2.41 g L-1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g-1) and volumetric productivity (0.56 g L-1 h-1) were reached with an inoculum level of 0.9 g L-1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeast C. guilliermondii with efficiency values as high as 77% of the theoretical maximum.

Relative rates of sugar utilization by an ethanologenic recombinant Escherichia coli using mixtures of glucose, mannose, and xylose.

The volumetric rates of glucose (G), mannose (M), and xylose (X) utilization by recombinant Escherichia coli B (pLO1297) were compared in pH-stat batch fermentations with Luria broth containing various combinations of two of these sugars at differing mass ratios. Using single substrate media, the rates of glucose, mannose, and xylose utilization were 3.0, 0.8, and 1.5 g/L/h, respectively. With all two substrate media, hexose and pentose sugars were consumed simultaneously. At a mass ratio of 2:1 (M or X:G), the rate of glucose utilization was reduced to 1.7 and 1.2 g/L/h by mannose and xylose, respectively. In media containing glucose and xylose, the rate of xylose utilization was inhibited when the glucose component exceeded about 40% of the total sugar mass in the medium. At a mass ratio of 2M:1X, mannose did not inhibit the rate of xylose utilization. At a mass ratio of 1:2 (G or X:M), the rate of mannose utilization was unaffected by either glucose or xylose. Synthetic media containing a mixture of hexose and pentose sugars were formulated to mimic different biomass hemicellulose hydrolysates. Relative to the rate in a single substrate medium, the respective rates of glucose and xylose utilization were 70% (2.1 g/L/h) and 40% (0.6 g/L/h) in a synthetic softwood prehydrolysate (SW) medium with a total reducing sugar (TRS) content of 45.7 g/L (20 wt% glucose, 30% xylose, and 50% mannose). However, the rate of mannose utilization in the SW medium was not inhibited. The respective rates of glucose and xylose utilization were 30% (0.9 g/L/h) and > 90% (1.4 g/L/h) in a synthetic crop residue prehydrolysate (CR) medium with a TRS content of 46.9 g/L (10 wt% glucose, 73% xylose, and 17% arabinose). Based on the results of this study, we suggest that the apparent "preference" for fermentation of hexose sugars by recombinant E. coli may be owing to the decreased rate of xylose transport caused by hexose sugars. Glucose is a more potent modulator of xylose utilization than mannose, but since xylose affects the rate of glucose utilization, this study also points to the importance of the concentration of the different sugars in terms of the relative rates of utilization by recombinant E. coli.