Development and optimization of a defined medium for aerobic growth ofBacillus stearothermophilus LLD-15

Methionine, biotin, nicotinic acid and thiamine were found to be absolute requirements for growth ofBacillus stearothermophilus strain LLD-15, an L-lactate dehydrogenase mutant, at 70°C. A defined medium for aerobic growth did not support anaerobic growth. Physiological and subsequent taxonomic studies showed that strain LLD-15 is not a derivative ofB. stearothermophilus NCA 1503 as originally thought, but bears some similarity toB. caldotenax.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Ethanol production from glucose byClostridium thermosaccharolyticum strains: Effect of pH and temperature

Summary The fermentation of glucose byClostridium thermosaccharolyticum strains IMG 2811^T, 6544 and 6564 was studied in batch culture in a complex medium at different temperatures in defined and free-floating pH conditions. All the strains ferment 5 g glucose.l^–1 completely. The yield of the fermentation products turned out to be independent of the incubation temperature for strain IMG 2811^T. Strain IMG 6544 produced at 60°C significantly more ethanol and less acetic acid, butyric acid, hydrogen gas and biomass than at lower temperatures. With strain IMG 6564, the opposite effect occurred: ethanol appeared to be the main fermentation product at 45°C; at 60°C less ethanol and more acetic acid, butyric acid and hydrogen gas was formed.Experiments, carried out with strain IMG 6564, at defined pH conditions (between 5.5 and 7) and different temperatures (45, 55 and 60°C) revealed no effect of the incubation temperature, but an important effect of the pH on the product formation. At pH 7, ethanol was the main fermentation product while minor amounts of hydrogen gas, acetic and butyric acid were produced. Lowering the pH gradually to 5.5 resulted in a decrease of ethanol and an increase of biomass, hydrogen gas, acetic, butyric and lactic acids. At pH higher than 7 no growth occurred. Similar conclusions could be drawn for strains IMG 2811^T and 6544.     

Development of a synthetic medium for continuous anaerobic growth and ethanol production with a lactate dehydrogenase mutant of Bacillus stearothermophilus.

A synthetic medium was developed by the pulse and medium-shift technique for the continuous cultivation of Bacillus stearothermophilus strain LLD-15 (NCIMB 12428) under anaerobic conditions. This mutant strain lacks L-lactate dehydrogenase activity, and is a promising candidate for the production of ethanol from pentoses and hexoses, using a high-temperature two-stage process. The final medium contained four amino acids and five vitamins, and growth characteristics in this medium compared well with those in complex medium containing yeast extract and tryptone. At 70 degrees C, the medium was capable of supporting good anaerobic and aerobic growth at 10 g input sucrose l-1. High ethanol production indicated that pyruvate metabolism probably occurred via the combined activity of the pyruvate-formate-lyase pathway and pyruvate dehydrogenase.     

Capsaicin hydrolysis by Candida antarctica lipase

Capsaicin was hydrolysed by lipase B from Candida antarctica into vanillylamine and 8-methyl-6-trans-nonenoic acid. Conversions of 70% were obtained after 72 h at 70 °C in water but decreased to only 15% when capsaicin was solubilized in 15% (v/v) ethanol/water after 72 h at 45 °C. No activity occurred in chloroform/water mixtures. According to our knowledge, this is the first report concerning amide hydrolysis by a lipase.     

Extreme temperatures and thermal tolerances for seedlings of desert succulents

Summary Extreme temperatures near the soil surface, which can reach 70°C at the main study site in the northwestern Sonoran Desert, markedly affect seedling survival. Computer simulations indicated that for the rather spherical barrel cactus Ferocactus acanthodes (Lem.) Britt. & Rose the maximum surface temperature decreased 8°C and the minimum temperature increased 3°C as the seedling height was increased from 1 mm up to 50 mm. Simulated changes in shortwave and longwave irradiation alone showed that shading could decrease the maximum temperature by about 5°C for the common desert agave, Agave deserti Engelm., and raise the minimum 1°C. Actual field measurements on seedlings of both species, where shading would affect local air temperatures and wind speeds in addition to irradiation, indicated that shading decreased the average maximum surface temperature by 11°C in the summer and raised the minimum temperature by 3°C in winter.Seedlings grown at day/iight air temperatures of 30°C/20°C tolerated low temperatures of about -7°C and high temperatures of about 56°C, as measured by the temperature where stain uptake by chlorenchyma cells was reduced 50%. Seedling tolerance to high temperatures increased slightly with age, and F. acanthodes was more tolerant than A. deserti. Even taking the acclimation of high temperature tolerance into account (2.7°C increase per 10°C increase in temperature), seedlings of A. deserti would not be expected to withstand the high temperatures at exposed sites, consistent with previous observations that these seedlings occur only in protected microhabitats. Based primarily on greater high temperature acclimation (4.3°C per 10°C), seedlings of F. acanthodes have a greater high temperature tolerance and can just barely survive in exposed sites. Wide ranges in photoperiod had little effect on the thermal sensitivities of either species. When drought increased the chlorenchyma osmotic pressure from about 0.5 MPa to 1.3 MPa, seedlings of both species became about 2°C less tolerant of high temperatures, which would be nonadaptive in a desert environment, and 2°C more tolerant of low temperatures, which also occurs for other species.In conclusion, seedlings of A. deserti and F. acanthodes could tolerate tissue temperatures over 60°C when acclimated to high temperatures and below -8°C when acclimated to low temperatures. However, the extreme environment adjacent to desert soil requires sheltered microhabitats to protect the plants from high temperature damage and also to protect them from low temperature damage at their upper elevational limits.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Thermostability of l-phenylalanine aminotransferase from thermophilic bacteria

Summary Various mesophilic and thermophilic bacteria were screened for the presence of thermostable l-phenylalanine aminotransferases. With organisms from culture collections best results were obtained with Thermus aquaticus and Bacillus caldolyticus. Cell-free extracts of these bacteria contained enzymes which did not lose activity by heat treatment at 60°C for 25 min, although they became rapidly inactivated during incubation at 70°C. Bacillus species able to grow at 70–75°C in mineral medium with phenylalanine as the sole carbon- and energy source were subsequently isolated in pure culture. At 70°C Bacillus strain IS1 grew on phenylalanine with a doubling time of 35 min and synthesized a phenylalanine aminotransferase which only slowly lost activity when incubated at 70°C and was stable at 60°C for at least 7 h.During the purification of the phenylalanine aminotransferase from Bacillus IS1 only a single peak of activity was observed consistently. This enzyme showed activity with phenylalanine and tyrosine but not with aspartate. The apparent K _m values for phenylalanine and tyrosine were 0.95 and 0.77 mM, respectively. The enzyme had an optimum pH of 6.4 and a temperature optimum of 71.5°C for the deamination of phenylalanine. Similar levels of the enzyme were synthesized during growth of Bacillus IS1 on a variety of substrates, suggesting that it functions in phenylalanine (and tyrosine) biosynthesis rather than in phenylalanine catabolism.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32°C; several isolates grew at 8 and 37 °C. None of the isolates grew at 40 °C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 °C with several isolates exhibiting optimal growth at temperatures as high as 32 °C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 °C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 °C. However, the two most cold tolerant isolates at 8 °C were M. flavoviride and the three most heat tolerant at 35 °C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid     

Oviposition-site preference and ADH activity in Drosophila melanogaster

Four D. melanogaster strains characterized by different alcohol dehydrogenase (ADH) activities were compared for their oviposition site preferences. The comparison was made between different temperatures (15 °C, 20 °C, 25 °C, 30 °C), day and night, replicates, and different concentrations in ethanol (00%, 5%, 10%, 15%) in the medium. All these factors influence behaviour. ADH activities seem to affect the oviposition site preferences as they directly affect ethanol tolerance and the ability to use alcohol as a source of metabolic energy.     

Biosynthesis of extracellular low-molecular-mass papain and trypsin inhibitors by Streptomyces sp. 22: effect of cultural conditions

The production of extracellular inhibitors of papain and trypsin by Streptomyces sp. 22 was studied under different cultural conditions including complex and defined media, temperatures ranging from 18 °C to 37 °C and a variety of sole carbon and nitrogen sources. In complex nutritionally rich medium, maximal specific growth rates were obtained at 37 °C, whereas the highest specific production rates for both papain and trypsin inhibitors were registered at 18 °C. Studies on the effect of different carbon and nitrogen sources in defined media underline the importance of the nitrogen source as a strong regulator of the biosynthesis of both inhibitors. Enhanced formation of the inhibitory compounds occurred in the presence of casein. The dynamics of the formation of both inhibitors in defined media showed close association with growth. However, a partial separation of production phases for papain and trypsin inhibitors was observed in complex medium. The results imply differences in the regulation of biosynthesis of the two inhibitors.     

Production of ethanol at high temperatures in the fermentation of Jerusalem artichoke juice and a simple medium byKluyveromyces marxianus

Summary Temperatures as high as 36°C and 40°C did not negatively affect the ethanol productivity of Jerusalem artichoke (J.a.) juice batch fermentation and the final concentrations of ethanol were close to those produced at lower temperatures. At higher process temperatures (36–40°C), ethanol toxicity inKluyveromyces marxianus was less important during the fermentation of J.a. juice as compared with a simple medium. In simple medium, the heat-sticking of fermentation was observed and the percentage of unfermented sugars steeply increased from 28°C up to 40°C.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Ethanol production from starch by recombinantEscherichia coli containing integrated genes for ethanol production and plasmid genes for saccharification

Ethanologenic strains ofEscherichia coli have been developed which can express thermostable enzymes for starch saccharification as intracellular products. These enzymes can be harvested within cells at the end of fermentation and liberated by heating to the temperature at which they exhibit maximal activity (60°C to 70°C). Organisms such as these could be used to supply enzymes for yeast-based fermentations while producing ethanol as a co-product.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.