Impact of hfq and sigE on the tolerance of Zymomonas mobilis ZM4 to furfural and acetic acid stresses.

Zymomonas mobilis, as an ethanologenic microorganism with many desirable industrial features, faces crucial obstacles in the lignocellulosic ethanol production process. A significant hindrance occurs during the pretreatment procedure that not only produces fermentable sugars but also releases severe toxic compounds. As diverse parts of regulation networks are involved in different aspects of complicated tolerance to inhibitors, we developed ZM4-hfq and ZM4-sigE strains, in which hfq and sigE genes were overexpressed, respectively. ZM4-hfq is a transcription regulator and ZM4-sigE is a transcription factor that are involved in multiple stress responses. In the present work, by overexpressing these two genes, we evaluated their impact on the Z. mobilis tolerance to furfural, acetic acid, and sugarcane bagasse hydrolysates. Both recombinant strains showed increased growth rates and ethanol production levels compared to the parental strain. Under a high concentration of furfural, the growth rate of ZM4-hfq was more inhibited compared to ZM4-sigE. More precisely, fermentation performance of ZM4-hfq revealed that the yield of ethanol production was less than that of ZM4-sigE, because more unused sugar had remained in the medium. In the case of acetic acid, ZM4-sigE was the superior strain and produced four and two-fold more ethanol compared to the parental strain and ZM4-hfq, respectively. Comparison of inhibitor tolerance between single and multiple toxic inhibitors in the fermentation of sugarcane bagasse hydrolysate by ZM4-sigE strain showed similar results. In addition, ethanol production performance was considerably higher in ZM4-sigE as well. Finally, the results of the qPCR analysis suggested that under both furfural and acetic acid treatment experiments, overproduction of both hfq and sigE improves the Z. mobilis tolerance and its ethanol production capability. Overall, our study showed the vital role of the regulatory elements to overcome the obstacles in lignocellulosic biomass-derived ethanol and provide a platform for further improvement by directed evolution or systems metabolic engineering tools.

The roles and applications of chaotropes and kosmotropes in industrial fermentation processes.

Chaotropicity has long been recognised as a property of some compounds. Chaotropes tend to disrupt non-covalent interactions in biological macromolecules (e.g. proteins and nucleic acids) and supramolecular assemblies (e.g. phospholipid membranes). This results in the destabilisation and unfolding of these macromolecules and assemblies. Unsurprisingly, these compounds are typically harmful to living cells since they act against multiple targets, comprising cellular integrity and function. Kosmotropes are the opposite of chaotropes and these compounds promote the ordering and rigidification of biological macromolecules and assemblies. Since many biological macromolecules have optimum levels of flexibility, kosmotropes can also inhibit their activity and can be harmful to cells. Some products of industrial fermentations, most notably alcohols, are chaotropic. This property can be a limiting factor on rates of production and yields. It has been hypothesised that the addition of kosmotropes may mitigate the chaotropicity of some fermentation products. Some microbes naturally adapt to chaotropic environments by producing kosmotropic compatible solutes. Exploitation of this in industrial fermentations has been hampered by scientific and economic issues. The cost of the kosmotropes and their removal during purification needs to be considered. We lack a complete understanding of the chemistry of chaotropicity and a robust, quantitative framework for estimating overall chaotropicities of mixtures. This makes it difficult to predict the amount of kosmotrope required to neutralise the chaotropicity. This review considers examples of industrial fermentations where chaotropicity is an issue and suggests possible mitigations.

Physiological effects of overexpressed sigma factors on fermentative stress response of Zymomonas mobilis.

Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z. mobilis ZM4 presents five different sigma factors identified in the genome annotation, but the absence of sigma 38 leads to the questioning of which sigma factors are responsible for its mechanism of fermentative stress response. Thus, in this study, factors sigma 32 and sigma 24, traditionally related to heat shock, were tested for their influence on the response to osmotic and ethanol stress. The overexpression of these sigma factors in Z. mobilis ZM4 strain confirmed that both are associated with heat shock response, as described in other bacteria. Moreover, sigma 32 has also a role in the adaptation to osmotic stress, increasing both growth rate and glucose influx rate. The same strain that overexpresses sigma 32 also showed a decrease in ethanol tolerance, suggesting an antagonism between these two mechanisms. It was not possible to conclude if sigma 24 really affects ethanol tolerance in Z. mobilis, but the overexpression of this sigma factor led to a decrease in ethanol productivity.

Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis.

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial-derived liposomes showed that hopanoids protect against several ethanol-driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter-lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.

Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. The software has also successfully processed data from Schizosaccharomyces pombe, Escherichia coli, and Zymomonas mobilis mutant collections. We provide general recommendations for the design of large-scale, multiplexed barcode sequencing experiments. The procedure outlined here was used to score interactions for ~4 million chemical-by-mutant combinations in our recently published chemical-genetic interaction screen of nearly 14,000 chemical compounds across seven diverse compound collections. Here we selected a representative subset of these data on which to demonstrate our analysis pipeline. BEAN-counter is open source, written in Python, and freely available for academic use. Users should be proficient at the command line; advanced users who wish to analyze larger datasets with hundreds or more conditions should also be familiar with concepts in analysis of high-throughput biological data. BEAN-counter encapsulates the knowledge we have accumulated from, and successfully applied to, our multiplexed, pooled barcode sequencing experiments. This protocol will be useful to those interested in generating their own high-dimensional, quantitative characterizations of gene or chemical function in a high-throughput manner.

Effect of Brönsted acidic ionic liquid 1-(1-propylsulfonic)-3-methylimidazolium chloride on growth and co-fermentation of glucose, xylose and arabinose by Zymomonas mobilis AX101.

The potential of large-scale lignocellulosic biomass hydrolysis to fermentable sugars using ionic liquids has increased interest in this green chemistry route to fermentation for fuel-ethanol production. The ionic liquid 1-(1-propylsulfonic)-3-methylimidazolium chloride compared to other reported ionic liquids has the advantage of hydrolysing lignocellulosic biomass to reducing sugars at catalytic concentrations (≤0·032 mol l-1 ) in a single step. However, effects of this ionic liquid on co-fermentation of glucose, xylose and arabinose to ethanol by recombinant Zymomonas mobilisAX101 has not been studied. Authentic glucose, xylose and arabinose were used to formulate fermentation media at varying catalytic 1-(1-propylsulfonic)-3-methylimidazolium chloride concentrations for batch co-fermentation of the sugars using Z. mobilisAX101. The results showed that at 0·008, 0·016 and 0·032 mol l-1 ionic liquid in the culture medium, cell growth decreased by 10, 27 and 67% respectively compared to the control. Ethanol yields were 62·6, 61·8, 50·5 and 23·1% for the control, 0·008, 0·016 and 0·032 mol l-1 ionic liquid respectively. The results indicate that lignocellulosic biomass hydrolysed using 0·008 mol l-1 of 1-(1-propylsulfonic)-3-methylimidazolium chloride would eliminate an additional separation step and provide a ready to use fermentation substrate. SIGNIFICANCE AND IMPACT OF STUDY: This is the first reported study of the effect of the Brönsted acidic ionic liquid 1-(1-propylsulfonic)-3-methylimidazolium chloride on growth and co-fermentation of glucose, xylose and arabinose by Zymomonas mobilisAX101 in batch culture. Growth on and co-fermentation of the sugars by Z. mobilisAX 101 with no significant inhibition by the ionic liquid at the same catalytic amounts of 0·008 mol l-1 used to hydrolyse lignocellulosic biomass to reducing sugars overcome two major hurdles that adversely affect the process economics of large-scale industrial cellulosic fuel ethanol production; the energy-intensive hydrolysis and ionic liquid separation steps.

Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism.

Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses.

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5-HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC25 ), 50% (IC50 ), 75% (IC75 ), and 100% (IC100 ). Z. mobilis strains ZM4 and TISTR 551 biofilm were two- to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5-HMF. The IC25 of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5-HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5-HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up-regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up-regulated.

Effect of acetic acid on ethanol production by Zymomonas mobilis mutant strains through continuous adaptation.

BACKGROUND: Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. RESULTS: In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. CONCLUSION: The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.

Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis.

BACKGROUND: With the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. One of the major challenges faced during the process of biofuel production is the low tolerance of the microbial host towards increasing biofuel concentrations. RESULTS: Here, we demonstrate that the ethanol tolerance of Zymomonas mobilis can be greatly enhanced through the random mutagenesis of global transcription factor RpoD protein, (σ(70)). Using an enrichment screening, four mutants with elevated ethanol tolerance were isolated from error-prone PCR libraries. All mutants showed significant growth improvement in the presence of ethanol stress when compared to the control strain. After an ethanol (9 %) stress exposure lasting 22 h, the rate of glucose consumption was approximately 1.77, 1.78 and 1.39 g L(-1) h(-1) in the best ethanol-tolerant strain ZM4-mrpoD4, its rebuilt mutant strain ZM4-imrpoD and the control strain, respectively. Our results indicated that both ZM4-mrpoD4 and ZM4-imrpoD consumed glucose at a faster rate after the initial 9 % (v/v) ethanol stress, as nearly 0.64 % of the initial glucose remained after 54 h incubation versus approximately 5.43 % for the control strain. At 9 % ethanol stress, the net ethanol productions by ZM4-mrpoD4 and ZM4-imrpoD during the 30-54 h were 13.0-14.1 g/l versus only 6.6-7.7 g/l for the control strain. The pyruvate decarboxylase activity of ZM4-mrpoD4 was 62.23 and 68.42 U/g at 24 and 48 h, respectively, which were 2.6 and 1.6 times higher than the control strain. After 24 and 48 h of 9 % ethanol stress, the alcohol dehydrogenase activities of ZM4-mrpoD4 were also augmented, showing an approximate 1.4 and 1.3 times increase, respectively, when compared to the control strain. Subsequent quantitative real-time PCR analysis under these stress conditions revealed that the relative expression of pdc in cultured (6 and 24 h) ZM4-mrpoD4 increased by 9.0- and 12.7-fold when compared to control strain. CONCLUSIONS: Collectively, these results demonstrate that the RpoD mutation can enhance ethanol tolerance in Z. mobilis. Our results also suggested that RpoD may play an important role in resisting high ethanol concentration in Z. mobilis and manipulating RpoD via global transcription machinery engineering (gTME) can provide an alternative and useful approach for strain improvement for complex phenotypes.

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

High tolerance and physiological mechanism of Zymomonas mobilis to phenolic inhibitors in ethanol fermentation of corncob residue.

Corncob residue as the lignocellulosic biomass accumulated phenolic compounds generated from xylitol production industry. For utilization of this biomass, Zymomonas mobilis ZM4 was tested as the ethanol fermenting strain and presented a better performance of cell growth (2.8 × 10(8) CFU/mL) and ethanol fermentability (54.42 g/L) in the simultaneous saccharification and fermentation (SSF) than the typical robust strain Saccharomyces cerevisiae DQ1 (cell growth of 2.9 × 10(7) CFU/mL, ethanol titer of 48.6 g/L). The physiological response of Z. mobilis ZM4 to the twelve typical phenolic compounds derived from lignocellulose was assayed and compared with that of S. cerevisiae DQ1. Z. mobilis ZM4 showed nearly the same tolerance to the phenolic aldehydes with S. cerevisiae DQ1, but the stronger tolerance to the phenolic acids existing in corncob residue (2-furoic acid, p-hydroxybenzoic acid, p-coumaric acid, vanillic acid, ferulic acid, and syringic acid). The tolerance mechanism of Z. mobilis was investigated in terms of inhibitor degradation, cell morphology and membrane permeability under the stress of phenolics using GC-MS, scanning and transmission electron microscopies (SEM and TEM), as well as fluorescent probes. The results reveal that Z. mobilis ZM4 has the capability for in situ detoxification of phenolic aldehydes, and the lipopolysaccharide aggregation on the cell outer membrane of Z. mobilis ZM4 provided the permeable barrier to the attack of phenolic acids.

Pre-treatment step with Leuconostoc mesenteroides or L. pseudomesenteroides strains removes furfural from Zymomonas mobilis ethanolic fermentation broth.

Furfural is an inhibitor of growth and ethanol production by Zymomonas mobilis. This study used a naturally occurring (not GMO) biological pre-treatment to reduce that amount of furfural in a model fermentation broth. Pre-treatment involved inoculating and incubating the fermentation broth with strains of Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides. The Leuconostoc strains converted furfural to furfuryl alcohol without consuming large amounts of dextrose in the process. Coupling this pre-treatment to ethanolic fermentation reduced furfural in the broth and improved growth, dextrose uptake and ethanol formation. Pre-treatment permitted ethanol formation in the presence of 5.2 g L(-1) furfural, which was otherwise inhibitive. The pre-treatment and presence of the Leuconostoc strains in the fermentation broth did not interfere with Z. mobilis ethanolic fermentation or the amounts of ethanol produced. The method suggests a possible technique for reducing the effect that furfural has on the production of ethanol for use as a biofuel.

Tolerance of S. cerevisiae and Z. mobilis to inhibitors produced during dilute acid hydrolysis of soybean meal.

The objective of this research was to determine the minimum inhibitory concentration of 5-hydroxymethyl furfural, furfural, and acetic acid on Saccharomyces cerevisiae (NRRL Y-2233) and Zymomonas mobilis subspecies mobilis (NRRL B-4286) in both detoxified hydrolyzed soybean meal and synthetic YM broth spiked with the three compounds. Soybean meal was hydrolyzed with dilute sulfuric acid (0.0, 0.5, 1.25, and 2.0% wt v(-1)) at three temperatures (105, 120, and 135°C) and three durations (15, 30, and 45 min) followed by detoxification with activated carbon. Of all the combinations, only the treatments obtained at 135°C, 2.0% H2SO4, and 45 min and the one at 135°C, 1.25% H2SO4, and 45 min showed inhibition in the growth of the tested microorganisms. Spiked YM broths showed inhibition for the highest levels of inhibitors, either applied individually or in combination.

Systems biology analysis of Zymomonas mobilis ZM4 ethanol stress responses.

BACKGROUND: Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. METHODOLOGY/PRINCIPAL FINDINGS: In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. CONCLUSIONS: Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated "omics" approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress.

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.

Bioethanol production using carbohydrate-rich microalgae biomass as feedstock.

This study aimed to evaluate the potential of using a carbohydrate-rich microalga Chlorella vulgaris FSP-E as feedstock for bioethanol production via various hydrolysis strategies and fermentation processes. Enzymatic hydrolysis of C. vulgaris FSP-E biomass (containing 51% carbohydrate per dry weight) gave a glucose yield of 90.4% (or 0.461 g (g biomass)(-1)). The SHF and SSF processes converted the enzymatic microalgae hydrolysate into ethanol with a 79.9% and 92.3% theoretical yield, respectively. Dilute acidic hydrolysis with 1% sulfuric acid was also very effective in saccharifying C. vulgaris FSP-E biomass, achieving a glucose yield of nearly 93.6% from the microalgal carbohydrates at a starting biomass concentration of 50 g L(-1). Using the acidic hydrolysate of C. vulgaris FSP-E biomass as feedstock, the SHF process produced ethanol at a concentration of 11.7 g L(-1) and an 87.6% theoretical yield. These findings indicate the feasibility of using carbohydrate-producing microalgae as feedstock for fermentative bioethanol production.

Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors.

BACKGROUND: Zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions. RESULTS: We generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions. CONCLUSIONS: We have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

Ethanol production from paper sludge by simultaneous saccharification and co-fermentation using recombinant xylose-fermenting microorganisms.

Simultaneous saccharification and co-fermentation (SSCF) of waste paper sludge to ethanol was investigated using two recombinant xylose-fermenting microbes: Zymomonas mobilis 8b and Saccharomyces cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/L ethanol with a yield of 0.39 g ethanol/g carbohydrate on paper sludge at 37 degrees C, while similar titers and yields were achieved by Z. mobilis 8b at 30 degrees C. Both S. cerevisiae RWB222 and Z. mobilis 8b exhibited decreasing cell viability at 37 degrees C when producing over 40 g/L ethanol. A high ethanol concentration can account for S. cerevisiae RWB222 viability loss, but ethanol concentration was not the only factor influencing Z. mobilis 8b viability loss at 37 degrees C. Over 3 g/L residual glucose was observed at the end of paper sludge SSCF by Z. mobilis 8b, and a statistical analysis revealed that a high calcium concentration originating from paper sludge, a high ethanol concentration, and a high temperature were the key interactive factors resulting in glucose accumulation. The highest ethanol yields were achieved by SSCF of paper sludge with S. cerevisiae RWB222 at 37 degrees C and Z. mobilis 8b at 30 degrees C. With good sugar consumption at 37 degrees C, S. cerevisiae RWB222 was able to gain an improvement in the polysaccharide to sugar yield compared to that at 30 degrees C, whereas Z. mobilis 8b at 30 degrees C had a lower polysaccharide to sugar yield, but a higher sugar to ethanol yield than S. cerevisiae. Both organisms under optimal conditions achieved a 19% higher overall conversion of paper sludge to ethanol than the non-xylose utilizing S. cerevisiae D5A at its optimal process temperature of 37 degrees C.