Lonnie O. Ingram

Effects of Alcohols on Micro-Organisms

Publisher Summary This chapter reviews the effects of alcohols on microorganisms. Alcohols are ubiquitous small molecules, which are produced both chemically and as products of microbial fermentation. Accumulation of alcohols in the microbial environment represents a form of environmental stress, analogous to extremes in pH value and temperature. The chapter discusses the action of ethanol and other alcohols on microorganisms, explains several important mechanisms of action. Alcohols have been employed for many years both as a disinfectant and as a preservative. Concentrations of ethanol above 15% result in immediate inactivation of most vegetative organisms, with spores being considerably more resistant. Low concentrations of ethanol also render bacteria more sensitive to inactivation by ionizing radiation and by lipophilic acids. The chapter concludes that the basic actions of alcohols on both eukaryotic and prokaryotic organisms share the same general principles. These effects appear to be dominated by the physicochemical properties of alcohols rather than involving specific receptors. All hydrophobic and electrostatic interactions in the cytosolic and envelope components of cells can potentially be affected. These include membranes, conformations of enzymes and macromolecules, activity coefficients of metabolites, permitivity, ionization potentials, pK values of functional groups, and pH value.

Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli.

Bioethanol production from lignocellulosic raw-materials requires the hydrolysis of carbohydrate polymers into a fermentable syrup. During the hydrolysis of hemicellulose with dilute acid, a variety of toxic compounds are produced such as soluble aromatic aldehydes from lignin and furfural from pentose destruction. In this study, we have investigated the toxicity of representative aldehydes (furfural, 5-hydroxymethlyfurfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) as inhibitors of growth and ethanol production by ethanologenic derivatives of Escherichia coli B (strains KO11 and LY01). Aromatic aldehydes were at least twice as toxic as furfural or 5-hydroxymethylfurfural on a weight basis. The toxicities of all aldehydes (and ethanol) except furfural were additive when tested in binary combinations. In all cases, combinations with furfural were unexpectedly toxic. Although the potency of these aldehydes was directly related to hydrophobicity indicating a hydrophobic site of action, none caused sufficient membrane damage to allow the leakage of intracellular magnesium even when present at sixfold the concentrations required for growth inhibition. Of the aldehydes tested, only furfural strongly inhibited ethanol production in vitro. A comparison with published results for other microorganisms indicates that LY01 is equivalent or more resistant than other biocatalysts to the aldehydes examined in this study.

Metabolic engineering of bacteria for ethanol production

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.

Detoxification of dilute acid hydrolysates of lignocellulose with lime.

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis is accompanied by the production of inhibitors that retard microbial fermentation. Treatment of hot hydrolysate with Ca(OH)2 (overliming) is an effective method for detoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our results indicate that the optimal lime addition for detoxification varies and depends on the concentration of mineral acids and organic acids in each hydrolysate. This optimum was shown to be readily predicted on the basis of the titration of hydrolysate with 2 N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average composition of 15 hydrolysates prior to treatment was as follows (per L): 95.24 ± 7.29 g sugar, 5.3 ± 2.99 g acetic acid, 1.305 ± 0.288 g total furans (furfural and hydroxymethylfurfural), and 2.86 ± 0.34 g phenolic compounds. Optimal overliming resulted in a 51 ± 9% reduction of total furans, a 41 ± 6% reduction in phenolic compounds, and a 8.7 ± 4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarity of microorganisms, it is possible that the titration method described here may also prove useful for detoxification and fermentation processes using other microbial biocatalysts.

Enteric bacterial catalysts for fuel ethanol production

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose‐derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.

Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production.

Genetically engineered Escherichia coli KO11 is capable of efficiently producing ethanol from all sugar constituents of lignocellulose but lacks the high ethanol tolerance of yeasts currently used for commercial starch-based ethanol processes. Using an enrichment method which selects alternatively for ethanol tolerance during growth in broth and for ethanol production on solid medium, mutants of KO11 with increased ethanol tolerance were isolated which can produce more than 60 g ethanol L−1 from xylose in 72 h. Ethanol concentrations and yields achieved by the LY01 mutant with xylose exceed those reported for recombinant strains of Saccharomyces and Zymomonas mobilis, both of which have a high native ethanol tolerance.

Eliminating Side Products and Increasing Succinate Yields in Engineered Strains of Escherichia coli C

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA‐pflB::FRT, mgsA, poxB) with growth‐based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co‐products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(ΔldhA ΔadhE ΔackA ΔfocA‐pflB ΔmgsA ΔpoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2‐ketobutyrate formate‐lyase (tdcE; pyruvate formate‐lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol−1 glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD+‐linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol−1 glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L−1 h−1). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (ΔldhA ΔadhE ΔfocA‐pflB ΔmgsA ΔpoxB ΔtdcDE ΔcitF ΔaspC ΔsfcA Δpta‐ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate. Biotechnol. Bioeng.

Advances in ethanol production

Barriers to the commercialization of lignocellulosic ethanol include the development of more robust biocatalysts, reduction of cellulase costs, and high capital cost associated with a complex process. Improvements have been made in all areas during the past two years. Oxidoreductases, transporters, and regulators have been identified that can increase the tolerance of biocatalysts to inhibitors formed during pretreatment. Biocatalysts are being developed that grow under conditions that are optimal for cellulase activity and others have been engineered to produce glycoside hydrolases. Ethanol yields resulting from most current process configurations are similar, approximately 0.21 g ethanol/g dry cellulosic feedstock. Potentially, this can be increased to at least 0.27 g ethanol/g biomass (83 gal/ton) using simpler processes.

Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli

During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzyme-dominated CO2 fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.

Ethanol Tolerance in Bacteria

The adverse effects of ethanol on bacterial growth, viability, and metabolism are caused primarily by ethanol-induced leakage of the plasma membrane. This increase in membrane leakage is consistent with known biophysical properties of membranes and ethanolic solutions. The primary actions of ethanol result from colligative effects of the high molar concentrations rather than from specific interactions with receptors. The ethanol tolerance of growth in different microorganisms appears to result in large part from adaptive and evolutionary changes in cell membrane composition. Different cellular activities vary in their tolerance to ethanol. Therefore, it is essential that the aspect of cellular function under study be specifically defined and that comparisons of ethanol tolerance among systems share this common definition. Growth is typically one of the most sensitive cellular activities to inhibition by ethanol, followed by survival, or loss of reproductive ability. Glycolysis is the most resistant of these three activities. Since glycolysis is an exergonic process, a cell need not be able to grow or remain viable for glycolysis to occur.