High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Isolation and characterization of Deinococcus geothermalis T27, a slightly thermophilic and organic solvent-tolerant bacterium able to survive in the presence of high concentrations of ethyl acetate

A solvent-tolerant, slightly thermophilic bacterium was isolated at 45 degrees C in the presence of toluene vapor provided as the sole carbon source. Strain T27 was identified as Deinococcus geothermalis T27. It could tolerate high concentrations of solvent provided as a nonaqueous layer (5% and 20%, v/v) to a cell suspension and had a remarkable ability to tolerate a broad range of solvents having log P(ow) values ranging from 5.6 of n-decane to as low as 0.7 of ethyl acetate. It was also able to utilize some of the solvents tested as a growth substrate at 45 degrees C. The addition of Ca(2+) ion, glucose and fructose partially promoted solvent tolerance. Cells exposed to ethyl acetate appeared to have a smaller size; however, the cell structure was not altered and was apparently well defined even after solvent shock. The tolerance of D. geothermalis T27 in the presence of high levels of toxic solvent stress at a comparatively high temperature indicated its potential use in biotechnological applications as well as bioremediation of xenobiotics.     

Chronic functional ethanol tolerance in mice influenced by body temperature during acquisition

Previous studies have found that body temperature during intoxication influences brain sensitivity to ethanol with the sensitivity being less at cool than at warm body temperatures. If this effect of temperature reflects alterations in the acute membrane perturbing action of ethanol, as suggested by in vitro studies, then body temperature reduction (hypothermia) during tolerance acquisition should reduce the effectiveness of a given ethanol concentration and, in turn, should reduce the development of chronic functional ethanol tolerance. To test this hypothesis, adult drug-naive C57BL/6J mice were injected i.p. once daily for five days with 3.6 g/kg ethanol (20% w/v) and were exposed to 34 degrees C or 25 degrees C for five hours following injection. On day 6, both ethanol acquisition groups and naive mice were injected i.p. with 4.0 g/kg ethanol and exposed to 25 degrees C. During acquisition, the group exposed to 34 degrees C had significantly higher body temperatures than the mice exposed to 25 degrees C, and there were no statistically significant differences in blood ethanol concentrations between treatment conditions. The extent of tolerance on day 6, measured by sleep-times and wake-up blood and brain ethanol concentrations versus naive mice, was significantly greater in the 34 degrees C acquisition group than in the 25 degrees C acquisition group. The results demonstrate that body temperature influences tolerance development in the manner predicted by membrane perturbation theories of anesthesia and adaptation based tolerance theories.     

Inactivation of animal viruses during sewage sludge treatment

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Inactivation of animal viruses during sewage sludge treatment.

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Genome shuffling to improve thermotolerance,ethanol tolerance and ethanol productivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we improved the thermotolerance and ethanol tolerance of an industrial yeast strain SM-3 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by protoplast ultraviolet irradiation and then subjected for the recursive protoplast fusion. The positive colonies from the library, created by fusing the inactivated protoplasts were screened for growth at 35, 40, 45, 50 and 55°C on YPD-agar plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of three phenotypes of thermotolerance, ethanol tolerance and ethanol yields enhancement. After three rounds of genome shuffling, the best performing strain, F34, which could grow on plate cultures up to 55°C, was obtained. It was found capable of completely utilizing 20% (w/v) glucose at 45–48°C, producing 9.95% (w/v) ethanol, and tolerating 25% (v/v) ethanol stress.     

Effect of temperature on ethanol tolerance of thermotolerant Isshatchenkia orientalis IPE100

Tolerance to high temperature and ethanol is a major factor in high‐temperature bio‐ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L^?1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (P_m) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L^?1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high‐temperature bio‐ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature‐dependent inhibitory effects of ethanol on yeast growth.     

High ethanol tolerance of new isolates of Clostridium thermocellum strains SS21 and SS22

Clostridium thermocellum strains SS21 and SS22, producing high yields of ethanol, were tolerant to 4.0 and 5.0% (v/v) ethanol, respectively. This is the highest ethanol tolerance so far reported by wild type strains of C. thermocellum. In the presence of added ethanol, both the strains had extended period of growth arrest. On addition of ethanol at different culture ages increase in ethanol tolerance upto 7.0 and 8.0% (v/v) by strains SS21 and SS22, respectively was observed. The optimum growth temperature for strain SS21 decreased as the concentration of ethanol in the medium increased and remained constant for strain SS22. Both the strains were tolerant to various solvents and acetic acid indicating that high ethanol tolerance of the strains is due to the general solvent tolerance of the organisms.     

Methanol utilization by a novel thermophilic homoacetogenic bacterium,Moorella mulderi sp. nov., isolated from a bioreactor

A thermophilic, anaerobic, spore-forming bacterium (strain TMS) was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as the energy source. Cells were gram-positive straight rods, 0.4-0.6 microm x 2-8 microm, growing as single cells or in pairs. The temperature range for growth was 40-70 degrees C with an optimum at 65 degrees C. Growth was observed from pH 5.5 to 8.5, and the optimum pH was around 7. The salinity range for growth was 0-45 g NaCl l(-1 )with an optimum at 10 g l(-1). The isolate was able to grow on methanol, H(2)-CO(2 )(80/20%, v/v), formate, lactate, pyruvate, glucose, fructose, cellobiose and pectin. The bacterium reduced thiosulfate to sulfide. The G+C content of the DNA was 53 mol%. Comparison of 16S rRNA genes revealed that strain TMS is related to Moorella glycerini (96%, sequence similarity), Moorella thermoacetica (92%) and Moorella thermoautotrophica (92%). On the basis of physiological and phylogenetic differences, strain TMS is proposed as a new species within the genus Moorella, Moorella mulderi sp. nov. (=DSM 14980, =ATCC BAA-608).     

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

Aims: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. Methods and Results: A solvent‐tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram‐positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p‐xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p‐xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron‐transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. Conclusions: The thermophilic, Gram‐positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. Significance and Impact of the Study: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.     

Conditions for forming ethanol during bioconversion of cellulose-containing raw material

Several natural associations composed by thermophilic anaerobic bacteria capable of utilizing various cellulose materials at 60 +/- 2 degrees C and pH 6.0-7.0 were isolated from the sludge of Kamchatka geothermal springs. The rate of ethanol production (up to 1.7 g/l per day) and the concentration of ethanol in the medium (up to 1.2%), as well as the fermentation period (10-15 days) were determined under anaerobic conditions in the presence of cellulose, coniferous sawdust, newsprint, or paper pulp as a carbon source. Microorganisms were found that inhibited the production of ethanol. The initial pH value was found to influence both the ethanol production rate and ethanol/acetate ratio. A pH decrease from 7.0 to 5.0 led to 6.7-fold increased the ethanol production and caused a 23.8-fold increase in the ethanol/acetate ratio.     

Effects of temperature and medium composition on the ethanol tolerance ofBacillus stearothermophilus LLD-15

The effects of temperature (60°–70°C) and medium composition (complex and defined) on ethanol tolerance ofBacillus stearothermophillus LLD-15, an L-lactate dehydrogenase mutant have been determined in shake flasks under aerobic conditions. In all cases, there was complete inhibition of growth in the presence of 6%v/v ethanol.B. stearothermophillus LLD-15 was found to be less tolerant to ethanol at 70°C than at 60°C and also less tolerant to ethanol in a defined medium than in a complex medium.     

A study of producing ethanol from cellulose using Clostridium thermocellum

The feasibility of producing ethanol in a continuous system from cellulose using Clostridirrrn thermocellum was investigated. This anaerobic and therniophilic bacterium was able to degrade cellulose directly into ethanol with acetic acid, hydrogen. and carbon dioxide as by-products of this fermentation. The fermentation was first carried out in a batch mode to study the effects of buffers, temperature, and agitation on microbial growth and ethanol production. From the compounds used to control pH. sodium bicarbonate had the most preferred effects on generation time and ethanol production. As expected, there was a positive relationship between temperature and growth rate. On the other hand, agitation did not benefit from ethanol production or microbial growth. The possibility of noncompetitive inhibition within such a system was deduced from the calculation of the kinetic constants K(m) and V(max). Continuous fermentations were carried out at 60 degrees C and pH 7.0 using 1.5 and 3% pure cellulose as a limiting substrate. The maximum ethanol concentration reached during the 1.5% cellulose fermentation was 0.3%. and 0.9% during the 3% cellulose fermentation. The yield of ethanol was about 0.3 grams per gram of consumed cellulose. The overall yield in both schemes was around 0.45 and 0.75 grams per gram of cellulose degraded. It was concluded that cellulose could be degraded continuously in a system with C. thermocellum for production of ethanol. While the continuous system like the batch method is feasible, it may not be promising as yet because of the slow generation time of this microorganism.     

Enhancement of apparent resistance to ethanol in Lactobacillus hilgardii

The survival of Lactobacillus hilgardii, a highly ethanol-tolerant organism, after an ethanol challenge at 25% (v/v) for 10 min, increased by several log cycles when cells, grown in the absence of ethanol, were pre-treated with 10% (v/v) ethanol, 15% (v/v) methanol or 2% (v/v) butanol for 4 h. A temperature upshift (25 to 40°C) before ethanol challenge demonstrated a similar enhancement of apparent resistance to ethanol. Ethanol shock enhanced apparent resistance to methanol, butanol and heat challenges. The addition of chloramphenicol to cells prior to any pre-treatment did not significantly diminish the increase in ethanol tolerance, suggesting that de novo protein synthesis is not required for induced tolerance in this organism. This revised version was published online in November 2006 with corrections to the Cover Date.     

Effects of acetic acid on the temperature profile of ethanol tolerance inSaccharomyces cerevisiae

Summary In a strain ofSaccharomyces cerevisiae, acetic acid at concentrations up to 1% (v/v) depressed the tolerance to added ethanol, from 11% (v/v) down to zero, and simultaneously narrowed the temperature range of growth from 3–42°C to 19–26°C. In addition, acetic acid shifted the associative temperature profile of growthand death to lower temperatures, and depressed the growth yield on glucose.     

Improvements in ethanol tolerance of Kluyveromyces fragilis in jerusalem artichoke juice

Alcoholic fermentation of Jerusalem artichoke juice, a natural complex medium, allowed the production of 13% (v/v) ethanol utilizing an inulin-fermenting strain of Kluyveromyces fragilis, strongly sensitive to ethanol. However, the fermentation of a simple medium with a similar concentration of fermentable sugars (235 g/L) as saccharose stopped prematurely when only 7% (v/v) ethanol had been produced. Differences in the two fermentation profiles were attributed to the significantly lower ethanol tolerance of K. fragilis IGC 2671 in the simple medium with 2% saccharose as compared with diluted J.a. juice with a similar sugar concentration, in fact, (1) in diluted J. a. juice, growth was possible up to 8% (v/v) added ethanol compared with 6% (v/v) in simple medium and (2) ethanol-induced inhibition of the specific growth and fermentation rate as well as ethanol-induced stimulation of the specific death rate were much more drastic in simple medium. Present results show that (1) the complex composition of the medium used for alcoholic fermentation plays a marked role in the ability of the yeast to tolerate and produce ethanol; (2) J. a. juice proved a very appropriate medium for a productive alcoholic fermentation, namely, in processes based on strains with a low ethanol resistance; and (3) to characterize and compare the ethanol tolerance of fermenting yeasts, the standardization of the medium composition must be taken in consideration.     

Effect of process temperature on bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste

The bacterial and archaeal community structure was examined in two methanogenic anaerobic digestion processes degrading organic household waste at mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Analysis of bacterial clone libraries revealed a predominance of Bacteroidetes (34% of total clones) and Chloroflexi (27%) at the mesophilic temperature. In contrast, in the thermophilic clone library, the major group of clones were affiliated with Thermotogae (61%). Within the domain Archaea, the phyla Euryarchaeota and Crenarchaeota were both represented, the latter only at the mesophilic temperature. The dominating archaeons grouped with Methanospirillum and Methanosarcina species at the mesophilic and thermophilic temperature, respectively. Generally, there was a higher frequency of different sequences at the lower temperature, suggesting a higher diversity compared to the community present at the thermophilic temperature. Furthermore, it was not only the species richness that was affected by temperature, but also the phylogenetic distribution of the microbial populations.     

Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, central Mongolia

A Gram reaction positive, spore-forming, facultative anaerobic bacterium belonging to the Phylum Firmicutes, was isolated from alkaline hot (80 degrees C, pH 9.8 spring Tsenher, central Mongolia. The cells were rod shaped, feebly motile, peritrichously flagellated. Strain T4 was moderately thermophilic with optimum growth at 60 degrees C. Maximum temperature for growth was between 70 and 75 degrees C; minimum temperature for growth was between 35 and 30 degrees C. Alkalitolerant, optimum pH for growth was 8.0; minimum pH for growth was between 5.0 and 5.5 and maximum was between 10.5 and 10.8. The growth was observed at NaCl concentrations of 0-5% (w/v) with the optimum at 0.2-0.5%. No growth was observed at 6% NaCl (w/v). Aerobically, the strain utilized proteinaceous substrates, organic acids and a range of carbohydrates including glucose, ribose, sucrose and xylose as well. Anaerobically, only glucose and sucrose were utilized. Strain T4T produced thermostable alkaline subtilisin-like serine proteinase. The G + C content was 44.2 mol. % (td). On the basis of 16S rRNA gene sequence similarity strain T4(T) was shown to be closely related to the members of the genus Anoxybacillus (family Bacillaceae, class "Bacilli"). DNA-DNA hybridization data revealed that strain T4T had only 38% relatedness to A. flavithermus and 28% relatedness to A. pushchinoensis. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA relatedness values with validly published species of Anoxybacillus, it is proposed that strain T4T represents a novel species Anoxybacillus mongoliensis sp. nov., with the type strain T4(T) (=DSM 19169 = VKM 2407).     

Bactericidal activity of ethanol against glucose nonfermentative Gram-negative bacilli

The bactericidal effect of ethanol on glucose nonfermentative Gram-negative bacilli (nonfermentative bacilli) and other species of micro-organisms was studied with emphasis on the former. At 20 degrees C, 10 to 20% v/v ethanol took 1 h or more to kill thirteen strains of nonfermentative bacilli while 40 to 99.5% concentrations produced a bactericidal effect within 1 min of exposure. Eleven strains of glucose fermentative organisms showed a similar tendency to that noted with nonfermentative bacilli, except that S. aureus was a little resistant to 99.5% ethanol. Using several strains of nonfermentative bacilli, the effects of temperature and equine serum on the bactericidal action of ethanol were determined. The bactericidal action of ethanol increased with rising of temperature (10-30 degrees C), and it was sufficient even at 10 degrees C provided that the concentration of ethanol was over 50%. The equine serum produced little effect on the bactericidal action of ethanol.     

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.