The effect of temperature and pH on the growth of aerobic alkalithermophilic bacteria from hot springs in Buryatia

Growth parameters (temperature and pH) were determined for collection cultures of aerobic heterotrophic bacteria. Analysis of the experimental data with the use of the Rosso model made it possible to calculate the extreme values of temperature and pH permissive for culture growth. The examined cultures were subdivided into three groups with respect to their growth temperature and pH. The first group is represented by the cultures with minimum, maximum, and optimal growth temperatures of < 20, 60-64, and 38-40 degrees C, respectively, and with the optimal growth pH 8.0-8.5. Bacteria of the second group are true alkalithermophilic organisms with a temperature optimum of 45-50 degrees C and pH optimum of 8.5-9.0. The third group includes a culture of a thermophilic alkalitolerant bacterium.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

Studies on the alkalophilicStreptomyces with extracellular xylanolytic activity

Summary An alkalophilicStreptomyces which produced xylanase, isolated from soil, grew in a temperature range of 15–37°C. The pH optimum for growth was 10 and no growth occurred at pH 7. On a simple wheat bran medium the microorganism exhibited maximum enzyme secretion of 12 U/ml at pH 10. The enzyme had a broad pH optimum of 4.8–10 and the optimum temperature of 50°C. It was completely inactivated at 60°C in 2 h. The enzyme hydrolyzed xylan to a mixture of oligomeric products indicating that the main activity was of the endoxylanase type. The culture filtrate had no cellulase activity.     

A new control strategy for yeast production based on the L/A* approach

Summary We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h. Offprint requests to: J. Chaloupka     

A novel alkaline, thermostable, protease-free lipase from Pseudomonas sp.

An extracellular, alkali-tolerant, thermostable lipase was from a Pseudomonas sp. It had optimal activity at 65 °C and retained 75% of its activity at 65 °C for 90 min. The pH optimum was 9.6 and it retained more than 70% activity between pH 5 and 9 for 2 h. The culture broth was free of protease and, at 30 °C, the culture filtrate retained all the activity for at least 7 days, without any stabilizer. In shake flask culture, addition of groundnut oil (3 g l^–1) towards the end of growth phase increased the activity from 4 U ml^–1 to 8 ml^–1.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers

Optimization of culture conditions such as the dissolved O₂ (DO) concentration, temperature and pH was attempted regarding both cell growth and the production of tissue plasminogen activator (TPA) in a microcarrier cell culture of human embryo lung cells. The growth rate was suppressed at a DO concentration below 30% saturation. From the pH range 7.2–7.6, both the specific growth rate and maximal cell concentration decreased. At a lower temperature than 37°C, although both the specific growth rate and the maximal cell concentration decreased, the cell concentration was maintained for a longer time during the production period, high TPA productivity being maintained. As the optimal conditions for culture growth, a DO concentration of 30% saturation or over, temperature of 37°C and pH of 7.4 are recommended. However, for TPA production after cell culture growth, the DO concentration should be in the range 20–30% O₂ saturation, and the temperature and pH should be lowered to 33°C and 6.8, respectively.     

Isolation of thermophilic iron-oxidising bacteria from sulfidic waste rock

Summary A thermophilic, rod-shaped, iron-oxidising bacterium was isolated by enrichment culture of rock samples from an overburden dump at the Rum Jungle mine site in Australia's Northern Territory. Oxidation of ferrous iron and sulfur occurred at 50–55°C, with a temperature maximum of 60°C. The isolate required yeast extract for growth. The pH optimum for iron oxidation at 50°C was 1.4. Rapid iron-oxidation occurred at a pH as low as 0.35, but little or no oxidation occurred at or above pH 2.2.     

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

With a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Y _glucose ^max and values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h^–1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K⁺ or Mg²⁺)-limited cultures expressed the highest metabolic rates and with the K⁺ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K⁺-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K⁺ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.     

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.     

A cellulase-free xylanase from alkali-tolerantAspergillus fischeri Fxn1

Summary An alkali-tolerant fungusAsperqillus fischeri Fxn1 isolated from xylan enrichment grew in the pH range 5–10 and secreted an extracellular cellulase-free xylanase. Arabinose, lactose, maltose, cellobiose and glucose induced low levels of xylanase (1.8–9.0 IU/ml), whereas xylose, xylan and wheat bran induced higher level (34–45 IU/ml).CMcellulose and FPcellulose did not support growth. The optimum pH of xylanase was 6.0–6.5 and it was stable in a wide range of pH 5–9.5. The optimum temperature was 60°C and it was stable upto 55°C. The half-lives at 50 and 55 °C were 240 and 40 min. respectively. This enzyme released reducing sugars from pulp at pH 9.0 and 40°C.     

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.     

The Structure and Biogeochemical Activity of the Phototrophic Communities from the Bol'sherechenskii Alkaline Hot Spring

Microbial communities growing in the bed of the alkaline, sulfide hot spring Bol'sherechenskii (the Baikal rift area) were studied over many years (1986–2001). The effluent water temperature ranged from 72 to 74°C, pH was from 9.25 to 9.8, and sulfide content was from 12 to 13.4 mg/ml. Simultaneous effects of several extreme factors restrict the spread of phototrophic microorganisms. Visible microbial mat appears with a decrease in the temperature to 62°C and in sulfide content to 5.9 mg/l. Cyanobacteria predominated in all biological zones of the microbial mat. The filamentous cyanobacteria of the genus Phormidium are the major mat-forming organisms, whereas unicellular cyanobacteria and the filamentous green bacterium Chloroflexus aurantiacus are minor components of the phototrophic communities. No cyanobacteria of the species Mastigocladus laminosus, typical of neutral and subacid springs, were identified. Seventeen species of both anoxygenic phototrophic bacteria and cyanobacteria were isolated from the microbial mats, most of which exhibited optimum growth at 20 to 45°C. The anoxygenic phototrophs were neutrophiles with pH optimum at about 7. The cyanobacteria were the most adapted to the alkaline conditions in the spring. Their optimum growth was observed at pH 8.5–9.0. As determined by the in situ radioisotope method, the optimal growth and decomposition rates were observed at 40–32°C, which is 10–15°C lower than the same parameter in the sulfide-deficient Octopus Spring (Yellowstone, United States). The maximum chlorophyll a concentration was 555 mg/m² at 40°C. The total rate of photosynthesis in the mats reached 1.3 g C/m² per day. The maximum rate of dark fixation of carbon dioxide in the microbial mats was 0.806 g C/m² per day. The maximum rate of sulfate reduction comprised 0.367 g S/m² per day at 40°C. The rate of methanogenesis did not exceed 1.188 g C/m² per day. The role of methanogenesis in the terminal decomposition of the organic matter was insignificant. Methane formation consumed 100 times less organic matter than sulfate reduction.     

Characterization of polysaccharidase activity optima in the anaerobic digestion of municipal solid waste

Summary Cellulolytic enzymes from a laboratory anaerobic digester fed municipal solid waste were examined with respect to pH and temperature. The pH optimum was pH 6.6, considerably lower than the pH range in which digesters are normally operated (pH 7.2–7.6). The optimum temperature was between 50 and 60°C, rather than the 35–37°C range in which most digesters are controlled.     

Optimization of fermentation conditions for thermostable cellulase production byThielavia terrestris

Summary Of the eighteen different carbon sources, solka floc was optimal for the induction of cellulases by the thermophilic fungusThielavia terrestris. The temperature optimum for growth was between 44–52°C. The effect of initial and controlled pH on fungal growth and cellulase production was investigated and the results obtained showed that the maximum volumetric productivity (6.07 I.U./1 per h) of filter paper activity was achieved when the pH was controlled at 4.5–5.0.     

Production of laccase byBotrytis cinerea and fermentation studies with strain F226

After induction, seven strains ofBotrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L^–1 of glucose and 2 g L^–1 of yeast extract at pH 3.5, 2 g L^–1 gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24°C. By optimizing the culture conditions, the enzyme activity reached 28 U ml^–1 in 5 days with a specific activity of 560 U mg^–1 protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40°C, respectively, with syringaldazine as the substrate.     

Decolorization of azo dyes by Rhodobacter sphaeroides

Rhodobacter sphaeroides AS1.1737 decolorized more than 90% of several azo dyes (200 mg dyes l^–1) in 24 h. The optimal culture conditions were: anaerobic illumination (1990 lx), peptone as carbon source, temperature 35–40 °C and pH 7–8. Intracellular crude enzyme from this strain had azoreductase activity, optimized temperature as 45–50 °C, and decolorization kinetics which were consistent with a ping-pong mechanism.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Production and characterization of extracellular lipase from Calvatia gigantea

Summary A number of factors affecting production of extracellular lipase by the edible fungus Calvatia gigantea were investigated. Consecutive optimization of carbon and nitrogen sources, initial pH of culture medium and growth temperature resulted in an increase in lipase activity of 87%. Under optimum conditions, activities as high as 22.4 units ml^–1 of culture medium were obtained, competing favourably with most activities reported for other lipase hyperproducing microorganisms. The enzyme was optimally active at pH 7.0 and 30°C and had, at optimum pH, half-lives of 75.7 and 22.9 min at 45 and 55°C. Both high activity and kinetic characteristics of the enzyme make this process worthy of further investigation.Correspondence to: B. J. Macris     

Study of physicochemical factors limiting the growth ofKluyveromyces marxianus

Summary The effects of various physicochemical parameters on the growth of twoKluyveromyces marxianus strains were investigated, including: pH values, sodium chloride, water activity in the medium and temperature. Both yeast strains were unaffected by pH changes. Optimal pH for growth was found to be 4 with both strains, but they were able to develop within the pH 3–8 range. Suitable growth was obtained at temperatures of 4–44°C and the optimal temperature for growth was 36°C for both strains. Modelling of this latter parameter is described. Growth of both microorganisms was considerably modified by increased NaCl or decreased water activity in the medium.