Strain KO11 Research Articles

Geomicrobial Exploration of Sulfur Oxidizing Bacteria Indigenous to Kapurdi and Jalipa Lignite Mine Site, Rajasthan

Abstract The metabolic versatility and potential of microbes enables their survival and growth in harsh condition such as sites ravaged by mining activities. The Kapurdi lignite deposit of the Barmer Basin is one of the important Tertiary lignite deposits underlying the Great Indian Thar desert in the north-western part of Rajasthan. The diversity of culturable sulfur oxidizing bacteria (SOB) in Kapurdi and Jalipa lignite mine site, Barmer, Rajasthan was assessed in this study. Bacteria were isolated by plating on selective thiosulfate mineral media, and dominant isolates were identified from their physiological characteristics and 16S rRNA gene sequences. The obligate chemolithotrophic SOB were identified as Thermithiobacillus sp. strain KO11, KO12, KO13 (OP788122, OP788123, OP788124). Whereas heterotrophic SOB was identified as Bacillus sp.strain KO1 (OP788112), Bacillus sp. strain KO2 (OP788113), Bacillus sp.strain KO3 (OP788114), Enterobacter sp.strain KO4, KO5, KO6, KO9 (OP788115, OP788116, OP788117, OP788120), Leclercia sp. Strain KO7 (OP788118), Pantoea sp. strain KO8(OP788119), and Rhodococcussp.strain KO10 (OP788121). The culture condition was optimized for the two most potential SOB i.e., Thermithiobacillus sp. strain KO11 and Leclercia sp. strain KO7. Both the isolates grew well and produced maximum amount of sulfate in thiosulfate mineral media at pH 8.0 and with 40 mM of thiosulfate substrate. Thermithiobacillus sp. strain KO11 had the optimum growth at 45°C indicating its thermophillic nature whereas, Leclercia sp. strain KO7 had optimum growth at 35°C. This study significantly enhances our knowledge of native bacteria in mining impacted environments and their role in biogeochemical cycling of sulfur.

A general inhibition kinetics model for ethanol production using a novel carbon source: sodium gluconate

In this study, sodium gluconate was applied as a novel carbon source for the fuel ethanol production using an engineered Escherichia coli strain KO11 in batch fermentations. Ethanol and acetic acid were produced as two major products as well as small amount of lactic acid during the fermentation. Compared to the conventional carbon source glucose, the bioconversion of sodium gluconate possessed two distinct advantages: faster utilization rate of sodium gluconate (1.66 g/L per h) compared to glucose (0.996 g/L per h) and no requirement for pH control during fermentation. A general inhibition model including both substrate and products inhibitory effects was proposed, which adequately simulated batch fermentation kinetics at various concentrations of sodium gluconate. All of the products showed inhibitory effects on cell growth. The order of the inhibitory strength of all products and substrate was for the first time clarified in this study. Acetic acid was the most inhibitory product mitigating the cell growth, followed by ethanol and lactic acid. Sodium gluconate stimulated cell growth when its concentration was below 16 g/L, while it inhibited the cell growth when the concentration was above this concentration. It completely inhibited the cell growth when the concentration was 325 g/L. The high value of both the coefficient of determination (R (2)) and the adjusted R (2) verified the good fit of the model. This paper provides key insights into further engineering these strains to improve ethanol production.

Disruption of ptsG gene and manXYZ operon of ethanol-producing Escherichia coli KO11: Effects on glucose and xylose utilization and ethanol production

Ethanol-producing Escherichia coli strain KO11 consumed 99% of the glucose and only 13% of the xylose in a mixture of glucose (60g/L) and xylose (40g/L) during the 72-h fermentation at 30°C. The deletion mutants ΔptsG, ΔmanXYZ, and ΔptsG/manXYZ utilized 42%, 78%, and 35% of the glucose and 50%, 32%, and 32% of the xylose, respectively.

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (micro) and the cell yield (Y(X/S)). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down-regulation of genes in the ethanol-producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in-phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y(P/S)) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described.

Increased phenotypic stability and ethanol tolerance of recombinant Escherichia coli KO11 when immobilized in continuous fluidized bed culture

The recombinant Escherichia coli B strain KO11, containing chromosomally-integrated genes for ethanol production, was developed for use in lignocellulose-to-ethanol bioconversion processes but suffers from instability in continuous culture and a low ethanol tolerance compared to yeast. Here we report the ability cell immobilization to improve its phenotypic stability and ethanol tolerance during continuous culture on a 50 g/L xylose feed. Experiments conducted in a vertical tubular fermentor operated as a liquid-fluidized bed with the cells immobilized on porous glass microspheres were compared to control experiments in the same reactor operated as a chemostat without the support particles. Without cell immobilization the ethanol yield fell sharply following start-up, declining to 60% of theoretical after only 8-9 days of continuous fermentation. While immobilizing the cells did not prevent this decline, it delayed its onset and slowed its rate. With immobilization, a stable high ethanol yield (>85%) was maintained for at least 10 days, thereafter declining slowly, but remaining above 70% even after up to 40 days of fermentation. The ethanol tolerance of E. coli KO11 cells was substantially increased by immobilization on the glass microspheres. In ethanol tolerance tests, immobilized cells released from the microspheres had survival rates 2.3- to 15-fold higher than those of free cells isolated from the same broth. Immobilization is concluded to be an effective means of increasing ethanol tolerance in E. coli KO11. While immobilization was only partially effective in combating its phenotypic instability, further improvements can be expected following optimization of the immobilization conditions.

Specific Ethanol Production Rate in Ethanologenic Escherichia coli Strain KO11 Is Limited by Pyruvate Decarboxylase

Modification of ethanol productivity and yield, using mineral medium supplemented with glucose or xylose as carbon sources, was studied in ethanologenic Escherichia coli KO11 by increasing the activity of five key carbon metabolism enzymes. KO11 efficiently converted glucose or xylose to ethanol with a yield close to 100% of the theoretical maximum when growing in rich medium. However, when KO11 ferments glucose or xylose in mineral medium, the ethanol yields decreased to only 70 and 60%, respectively. An increase in GALP_Ec (permease of galactose-glucose-xylose) or PGK_Ec (phosphoglycerate kinase) activities did not change xylose or glucose and ethanol flux. However, when PDC_Zm (pyruvate decarboxylase from Zymomonas mobilis) activity was increased 7-fold, the yields of ethanol from glucose or xylose were increased to 85 and 75%, respectively, and organic acid formation rates were reduced. Furthermore, as a response to a reduction in acetate and ATP yield, and a limited PDC_Zm activity, an increase in PFK_Ec (phosphofructokinase) or PYK_Bs (pyruvate kinase from Bacillus stearothermophilus) activity drastically reduced glucose or xylose consumption and ethanol formation flux. This experimental metabolic control analysis showed that ethanol flux in KO11 is negatively controlled by phosphofructokinase and pyruvate kinase, and positively influenced by the PDC_Zm activity level.

Ethanolic fermentation of sucrose, sugarcane juice and molasses by Escherichia coli strain ko11 and Klebsiella oxytoca strain P2

Escherichia coli KO11 and Klebsiella oxytoca P2 recombinants fermented sucrose to ethanol. In minimal medium with 2% or 12% added sucrose KO11 produced 75% and 41%, respectively, of the maximum theoretical yield (0.54g ethanol/g sucrose). In Luria-Bertani (LB) broth with up to 8% sucrose, KO11 presented a 94-96% yield and with 12% sucrose, KO11 presented about 69% yield (44.5g ethanol/L). P2 presented 55% of the theoretical maximum yield in minimal medium supplemented with 2% sucrose and 47% of the maximum in 12% sucrose. In LB broth with 2 or 4% sucrose, P2 presented 94-95% of the theoretical maximum yield, which fell to 73% with 8% added sucrose (31.4g ethanol/L) and 58% with 12% sucrose (37.5 g/L). Volumetric productivity in LB broth containing 2% sucrose was 0.41 g/L/h for KO11 and 1.1 g/L/h for P2, while in LB broth with 12% added sucrose, productivity was 0.96 g/L/h (KO11) and 1.4 g/L/h (P2). During fermentation of sugar cane juice by E. coli KO11 and K. oxytoca P2 produced 39.4 g/L and 42.1 g/L ethanol when supplemented with 0.5% yeast extract, micronutrients and thiamine. In sugar cane juice supplemented with LB broth ingredients, KO11 ethanol fermentation was inhibited with production of only 23.0 g ethanol/L, while P2 produced 44.2 g/L. Ethanol production by KO11 and P2, respectively, in sugarcane juice was a) 25.3 and 30.2 g/L with 0.2% ammonium sulfate, b) 24.9 and 31.6 g/L with ammonium sulfate and micronutrients, c) 25.6 and 37.5 g/L with ammonium sulfate, micronutrients and thiamine. During molasses fermentation E. coli KO11 presented low ethanol production and high lactic acid production. K. oxytoca P2 produced 25 g ethanol/L in molasses diluted 10-fold in water, with or without addition of 0.5% yeast extract, and 27.8 g/L with addition of LB broth ingredients after 96h. P2 produced 24.5, 26.9, and 28.0 g ethanol/L in molasses diluted 10-fold in vinasse, vinasse with 0.5% added yeast extract and with LB broth ingredients, respectively.

Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol.

Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion to ethanol using recombinant bacteria as biocatalysts. Hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not fermentable to ethanol by these organisms. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. E. coli KO11 is able to convert pure galacturonic acid to ethanol with minimal acetate production. Using an enzyme loading of 10.5 filter paper units of cellulase, 120.4 polygalacturonase units of pectinase, and 6.4 g of cellobiase (per gram of dry wt sugar beet pulp), with substrate addition after 24 h of fermentation, 40 g of ethanol/L was produced. Other recombinants exhibited lower ethanol yields with increases in acetate and succinate production.

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.

Ethanol production from AFEX pretreated corn fiber by recombinant bacteria

Fermentation of an enzymatic hydrolyzate of ammonia fiber explosion (AFEX) pretreated corn fiber (containing a mixture of different sugars including glucose, xylose, arabinose, and galactose) by genetically-engineered Escherichia coli strain SL40 and KO11 and Klebsiella oxytoca strain P2 was investigated under pH-controlled conditions. Both E. coli strains (SL40 and KO11) efficiently utilized most of the sugars contained in the hydrolyzate and produced a maximum of 26.6 and 27.1 g/l ethanol, respectively, equivalent to 90 and 92% of the theoretical yield. Very little difference was observed in cell growth and ethanol production between fermentations of the enzymatic hydrolyzate and mixtures of pure sugars, simulating the hydrolyzate. These results confirm the fermentability of the AFEX-treated corn fiber hydrolyzate by ethanologenic E. coli. K.oxytoca strain P2, on the other hand, showed comparatively poor growth and ethanol production (maximum 20 g/l) from both enzymatic hydrolyzate and simulated sugar mixtures under the same fermentation conditions.

Factors contributing to the loss of ethanologenicity of Escherichia coli B recombinants pL0I297 and KO11.

To be economic and to be compatible with modern continuous bioconversion systems, it is imperative that the process organism exhibits an extremely high degree of stability. In the case of ethanol production from lignocellulosic biomass, functional stability of the potential process biocatalyst can be assessed in terms of the capacity to sustain high-performance fermentation during the continuous fermentation of biomass-derived sugars. This investigation employed glucose- or xylose-limited chemostat culture to examine the functional stability of two patented, genetically engineered E. coli-namely E. coli B (ATCC 11303) carrying the Zymomonas genes for pyruvate decarboxylase and alcohol dehydrogenase II on a multicopy plasmid pLOI297 and a chromosomal pet integrant of strain 11303, designated as strain KO11. Both recombinants carry markers for antibiotic resistance and have been reported to exhibit genetic stability in the absence of antibiotic selection. Chemostats were fed with Luria broth (LB) (with 25 g/L sugar) at a dilution rate of 0.14 and 0.07/h when the feed medium was glucose-LB and xylose-LB, respectively. They pH was controlled at 6.3. With glucose, both recombinants exhibited a rapid loss of ethanologenicity even when selection pressure was imposed by the inclusion of antibiotics in the feed medium. With strain KO11, increasing the concentration of chloramphenicol from 40 to 300 mg/L, resulted in a retardation in the rate of loss of ethanologenicity, but it did not prevent it. Under xylose limitation, the plasmid-bearing recombinant appeared to be stabilized by antibiotics, but this did not reflect genetic stability, since the slower-growing revertant was washed out at a dilution rate of 0.07/h. With both recombinants, interpretation of functional stability with xylose was complicated by the inherent ethanologenicity associated with the host culture. Based on an average cost for large bulk quantities of antibiotics at $55/kg and an amendment level of 40 g/m3, the estimated economic impact regarding the potential requirement for operational stabilization by antibiotics in a plant operating in batch mode varied from a maximum of 29 cents/gal of E95 ethanol for antibiotic amendment of all fermentation media to a minimum of 0.45 cents/gal where antibiotics were used exclusively for the preparation of the inocula for every fourth batch fermentation cycle. The high degree of instability observed in these continuous fermentations does not auger well for the proposed potential industrial utility of these patented, genetically engineered constructs for the production of fuel ethanol from biomass and wastes.

Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineeredEscherichia coli strain KO11

Hemicellulose hydrolysates of the agricultural residues bagasse, corn stover, and corn hulls plus fibers were readily fermented to ethanol by recombinantEscherichia coli strain KO11. Corn steep liquor and crude yeast autolysate served as excellent nutrients. Fermentations were substantially complete within 48 h, often achieving over 40 g ethanol L−1. Ethanol yields ranged from 86% to over 100% of the maximum theoretical yield (0.51 g ethanol g sugar−1.

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl-1 ethanol from 120 gl-1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.

Cost analysis of ethanol production from willow using recombinant Escherichia coli.

This study comprises a technical and economic analysis of the production of fuel ethanol by fermentation of a pentose-rich hydrolysate with recombinant Escherichia coli, strain KO11. Hydrolysate from steam-pretreated willow was used as raw material in calculations regarding the fermentation. The calculations were based on a feed capacity of 10 metric tons of dry willow per hour to the pretreatment stage, providing 35 metric tons of hydrolysate per hour, consisting of 45 g of sugars/L, to the pentose fermentation plant. A detoxification step was included, since the hydrolysate has been shown to have an inhibitory effect on the E. coli KO11. The technical data used in the calculations were based on a kinetic fermentation model, which was developed from laboratory-scale experiments in a previous study. The economic analysis predicted an ethanol production cost of 48/L in the pentose fermentation plant, indicating potentially good economy. The detoxification cost constitutes 22% of this cost. Sensitivity analyses revealed that if the concentration of sugars in the feed to the fermentation was decreased by 40% to 27 g/L, the ethanol production cost was increased to 54/L. The production cost was increased to 50/L ethanol if the cell mass was recirculated to the fermentation stage 5 times instead of 20.

Conversion of hydrolysates of corn cobs and hulls into ethanol by recombinantEscherichia coli B containing integrated genes for ethanol production

Hemicellulose and residual starch in corn hulls from wet milling and hemicellulose in corn cobs were hydrolyzed by incubation in dilute sulfuric acid at 140°C to 160°C. These hydrolysates were efficiently fermented to ethanol by a genetically engineered derivative ofE. coli B, strain KO11. Fermentation of com hull hydrolysate was complete after 48 h with a final ethanol concentration of 38 grams per liter. Fermentation of corn cob hydrolysate was essentially complete after 24 h due to a lower concentration of sugars and higher levels of inocula. In both cases, ethanol produced was equivalent to 100% of the maximum theoretical yield (0.51 grams ethanol/gram sugar) based on momoner sugar content. ThusE. coli B strain KO11 appears to be an excellent candidate for the efficient production of ethanol from hydrolysates of corn residues.

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.