Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1–2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

The effect of ethanol on the phase behavior of membrane lipids extracted from Clostridium thermocellum strains

The phase behavior of aqueous dispersions of extracted lipids from Clostridium thermocellum wild-type and ethanol-tolerant C919 cells has been examined by DSC. The optimum growth temperature of this anaerobe is 60°C. The wild-type lipids exhibit a broad phase transition centered at 30°C; the C919 mutant lipids show a 10°C lower T_m. The direct addition of growth inhibiting concentrations of ethanol has no significant effect on T_m or headgroup mobility (monitored by ²H-NMR) of either set of lipids. In contrast, wild-type cells adapted to growth in ethanol exhibit a broadened and lower T_m (15–25°C plateau); C919 membrane lipids do not exhibit significantly altered phase behavior when adapted to growth in ethanol. Both wild-type and mutant membranes have fatty acid composition changes upon growth in ethanol, which increases lower-melting components. It is concluded that fatty acid changes which occur upon adaptation of the organism to growth in ethanol are secondary responses and not necessarily direct responses to alter membrane fluidity.     

Physiology,Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.     

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Objective

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential.

Results

Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain.

Conclusion

A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

Background

An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.

Methodology/Principal Findings

In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v).

Conclusion/Significance

The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.     

Adaptation of Yeast Cell Membranes to Ethanol

A highly ethanol-tolerant Saccharomyces wine strain is able, after growth in the presence of ethanol, to efficiently improve the ethanol tolerance of its membrane. A less-tolerant Saccharomyces laboratory strain, however, is unable to adapt its membrane to ethanol. Furthermore, after growth in the presence of ethanol, the membrane of the latter strain becomes increasingly sensitive, although this is a reversible process. Reversion to a higher tolerance occurs only after the addition of an energy source and does not take place in the presence of cycloheximide.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via ¹³C-metabolic flux analysis (¹³C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated ¹³C labeling data were used with ¹³C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.     

Screening and Mutation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> UV-20 with a High Yield of Second Generation Bioethanol and High Tolerance of Temperature,Glucose and Ethanol

A wild-type strain was isolated from slightly rotted pears after three rounds of enrichment culture, identified as Saccharomyces cerevisiae 3308, and evaluated for its fermentation capability of second generation bioethanol and tolerance of temperature, glucose and ethanol. S. cerevisiae 3308 was mutated by using the physical and chemical mutagenesis methods, ultraviolet (UV) and diethyl sulfate (DES), respectively. Positive mutated strains were mainly generated by the treatment of UV, but numerous negative mutations emerged under the treatment of DES. A positive mutated strain, UV-20, produced ethanol from 62.33?±?1.34 to 122.22?±?2.80 g/L at 30–45 °C, and had a maximum yield of ethanol at 37 °C. Furthermore, UV-20 produced 121.18?±?2.51 g/L of second generation bioethanol at 37 °C. Simultaneously, UV-20 exhibited superior tolerance to 50% of glucose and 21% of ethanol. In a conclusion, all of these results indicated that UV-20 has a potential industrial application value.     

Physiological Function of Alcohol Dehydrogenases and Long-Chain (C30) Fatty Acids in Alcohol Tolerance of Thermoanaerobacter ethanolicus

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C₃₀ fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).     

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol), whereas when ethanol concentration was changed from 0.1% to 1.6% (wt/vol), the ethanol/glucose ratio for the parent strain changed from 1.6 to 0.6. These data indicate that C. thermohydrosulfuricum strains are tolerant of solvents and that low ethanol tolerance is not a result of disruption of membrane fluidity or glycolytic enzyme activity.     

Continuous Cellulosic Bioethanol Fermentation by Cyclic Fed-Batch Cocultivation

Cocultivation of cellulolytic and saccharolytic microbial populations is a promising strategy to improve bioethanol production from the fermentation of recalcitrant cellulosic materials. Earlier studies have demonstrated the effectiveness of cocultivation in enhancing ethanolic fermentation of cellulose in batch fermentation. To further enhance process efficiency, a semicontinuous cyclic fed-batch fermentor configuration was evaluated for its potential in enhancing the efficiency of cellulose fermentation using cocultivation. Cocultures of cellulolytic Clostridium thermocellum LQRI and saccharolytic Thermoanaerobacter pseudethanolicus strain X514 were tested in the semicontinuous fermentor as a model system. Initial cellulose concentration and pH were identified as the key process parameters controlling cellulose fermentation performance in the fixed-volume cyclic fed-batch coculture system. At an initial cellulose concentration of 40 g liter⁻¹, the concentration of ethanol produced with pH control was 4.5-fold higher than that without pH control. It was also found that efficient cellulosic bioethanol production by cocultivation was sustained in the semicontinuous configuration, with bioethanol production reaching 474 mM in 96 h with an initial cellulose concentration of 80 g liter⁻¹ and pH controlled at 6.5 to 6.8. These results suggested the advantages of the cyclic fed-batch process for cellulosic bioethanol fermentation by the cocultures.     

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H₂), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.