Extracellular protease production fromXenorhabdus nematophilus, a symbiotic bacterium of entomopathogenic nematodes

Effects of nutrients and growth temperature on the production of an extracellular protease fromXenorhabdus nematophilus, a symbiotic bacterium of entomopathogenic nematodes were studied for batch culture. Tryptone and fructose were most effective nitrogen and carbon sources to produce high level of the protease within 24–28 hr of incubation. The stability of protease was poor during the incubation of the bacteria. The protease activity increased in parallel with cell growth then decreased significantly for extended culture periods of the bacteria over 48 hr at 22–30°C. Autolysis of the protease was not a major cause for the decreased protease activity since no decrease in the protease activity was observed for the whole culture broth of the bacteria which was stored up to 80 hr at 4°C.     

Temperature and incubation time effects on growth and ochratoxin A production by Aspergillus sclerotioniger and Aspergillus lacticoffeatus on culture media

Aims: As there is no knowledge of the influence of abiotic factors on the two new ochratoxin A (OTA)‐producing species Aspergillus sclerotioniger and Aspergillus lacticoffeatus, the aim of this study was to evaluate the effect of temperature and incubation time on growth and OTA production by these species on culture media. Methods and Results: The study was carried out on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) incubated at ten different temperatures from 5 to 50°C (at 5°C intervals). Growth assessment and OTA production were determined after 5, 10, 15, 20 and 30 days of incubation at each temperature. Aspergillus sclerotioniger grew from 10 to 35°C; OTA was detected from 10 to 35°C and the highest concentration was achieved at 15°C in CYA. Aspergillus lacticoffeatus grew from 10 to 45°C; OTA was detected from 15 to 45°C, and the maximum concentration was produced after 5 days at 25°C in YES. Conclusions: The studied species can produce OTA over a wide range of temperatures and significant amounts can be produced in only 5 days. Significance and Impact of the Study: This is the first report on the influence of ecophysiological factors on these two ochratoxigenic species. The pattern of effects of temperature on growth and OTA production by A. sclerotioniger and A. lacticoffeatus was similar to those reported for the closely related species Aspergillus carbonarius and Aspergillus niger, respectively. The two new OTA‐producing species have both been isolated from coffee beans, and the closely related ochratoxigenic species of section Nigri, A. carbonarius and A. niger are important sources of OTA in this substrate.     

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.     

Effect of culture conditions on the production of an extracellular proteinase byThermus sp. Rt41A

Thermus sp. Rt41A produced a single extracellular proteinase, as determined by fast protein liquid chromatography and isoelectric focusing. Proteinase activity was expressed from very early in the log phase, and halted when the growth substrate was exhausted. There was no continued proteinase production in the stationary phase. Proteinase production was not stimulated by O₂ limitation, not repressed by amino acid growth substrates, and its production could not be correlated to the type or oxidation state of the carbon and energy source or the growth rate on different carbon and energy sources. Growth on certain substrates, e.g. glutamate and glucose, resulted in production of high levels of proteinase, whereas others, such as acetate, resulted in low proteinase levels. Acetate repressed proteinase production in cultures growing on L-glutamate. In continuous culture on L-glutamate, acetate or pyruvate, proteinase production was highest at higher growth (dilution) rates. The kinetics of proteinase production in continuous culture on L-glutamate can be interpreted as evidence for the constitutive nature of proteinase expression byThermus sp. Rt41A. The data obtained show that the control of proteinase production is different to that postulated forThermus sp. Ok6.A1.     

Suitability of poor medium in counting total viable airborne bacteria

The purpose of this study was to test the effect of incubation temperature and culture medium on viable counts of airborne bacteria. The incubation temperature had different effect on indoor and outdoor air bacteria. Indoor air bacteria grew as well at 20°C as 37°C, but less at 10°C. Outdoor air bacteria grew equally well at 10°C and 20°C, but less at 37°C. Both indoor and outdoor air bacteria grew differently on poor and rich media. The counts of both indoor and outdoor air bacteria were higher on poor R2A medium (low nutrient concentration) than on rich TYG and blood media (high nutrient concentration). The results indicate that a poor medium incubated at 20°C is adequate for counting viable airborne bacteria.     

Factors affecting intra- and extracellular phospholipase A1 production bySalmonella newport

The effect of various physico-chemical factors on production of intra- and extracellular phospholipase A₁ bySalmonella newport was investigated. Maximum intracellular enzyme levels were observed when cells were grown in brain heart infusion broth, after 12 h of incubation at 37°C. Highest level of extracellular phospholipase A₁, however, was seen in synthetic medium (pH 7.0) after 24 h of incubation at 37°C. Agitation during incubation had no effect on the intracellular enzyme synthesis but enhanced extracellular enzyme levels. Addition of surfactants to the growth media significantly decreased both intra- and extracellular phospholipase A₁ production.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus

Summary Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c₁. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32°, 34° or 37° C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32° and 34° C than at 37° C. Decreased levels of RDDP were attributed to enzyme thermolability at 37° C incubation. Research sponsored by the National Cancer Institute under Contract No. N01-CO-25423 with Litton Bionetics, Inc., and Contract No. N01-CP-33253 with the University of California.     

The Influence of pH and Temperature on Cellulolytic and Pectolytic Activity of Cylindrocarpon destructans (Zins.) Scholt.

In studies on the effect of pH and temperature on cellulolytic and pectolytic activity of C. destructans, it was found that the isolates used produced only endoglucanases. The temperature and pH affected the synthesis of these enzymes. Fungi cultured at 26°C produced more of these enzymes than those grown at the two other temperatures. At 10°C, only one isolate produced minute amounts of endoglucanases. None of fungi studied exhibited cellulolytic activity in cultures grown at 20°C. Cellulolytic activity was found only in acidic media (pH 5.0). The fungi studied exhibited higher pectolytic than cellulolytic activity. In the post culture liquids of these organisms, both types of pectolytic enzymes (exo- and endo-PMG) were detected. Different temperature and pH values affected the production of these enzymes differently in various isolates.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

A unique thermostable and organic solvent tolerant lipase from newly isolated <Emphasis Type="Italic">Aneurinibacillus thermoaerophilus</Emphasis> strain HZ: physical factor studies

A newly isolated thermophilic bacterium, Aneurinibacillus thermoaerophilus strain HZ, from a hot spring recreational area (Sungai Kelah, Malaysia), showed an extracellular lipase activity. It was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its homology to Aneurinibacillus thermoaerophilus. The strain produced a lipase that was stable in various organic solvents such as dimethyl sulfoxide, toluene, p-xylene, and hexane. In order to increase lipase production, optimization of physical factors which affected the growth and lipase production was studied. The optimal growth was obtained at 50°C and pH 8.0; while the maximal lipase production was achieved in the logarithmic decline phase at 60°C and pH 7.5 with 7% starting inoculum and 150 rev/min shaking rate for 48 h incubation.     

Sporulation and synthesis of extracellular proteinases inBacillus subtilis are more temperature-sensitive than growth

Bacillus subtilis 115 grew in a medium with amino acids and glucose with the maximum specific growth rates μ of 1.20-1.10/h in the temperature range of 45–48°C. Activity of the extracellular neutral proteinase excreted by 1.3 mg/mL dry mass during 8 h of the postexponential and stationary growth phases decreased from its maximum value of 0.23 TU/mL at 40°C to 0.13 and 0.06 TU/mL at 45 and 48°C, respectively. Formation of the extracellular serine proteinase decreased even more—from 0.18 TU/mL at 40°C to 0.06 and 0.03 TU/mL at 45 at 48°C, respectively. Sporulation, expressed as the portion of sporangia rith refractile spores at the 6th h of the stationary phase decreased from 46% at 40°C to 17 and 3% at 45 and 48°C, respectively.     

Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Summary An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.     

Effect of culture conditions on growth and esterase production by the moderate thermophile Bacillus circulans MAS2

Growth and esterase production (activity on p-nitrophenyl caprylate) by the newly isolated Bacillus circulans MAS2 bacterial strain were studied. The growth rate at 50°C was high (0.9 h^-1) on LB medium with glucose added. Esterase production followed growth with the majority of activity being intracellular during exponential growth phase. During stationary phase, the esterase activity was released in the culture medium. The strain was able to grow at 35– 55°C with maximum growth rate at 50°C, showing a pattern typical of a moderate thermophile. Growth occurred at pH 6–9 with a maximum at 8, with a similar pattern for the esterase production. Addition of glucose, fructose, sucrose or sodium acetate greatly promoted both growth and esterase production while starch, inulin, tributyrin or glycerol showed no effect. Complex nitrogen sources such as tryptone or yeast extract increased growth and esterase production while mineral sources (ammonium chloride or sulfate), glycine or glutamate showed no effect. An increase of tryptone plus yeast extract and glucose concentrations stimulated growth and esterase production which reached 160 U L⁻¹. Received 17 March 1999/ Accepted in revised form 25 June 1999     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Effect of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates of Phoma bakeriana

The effects of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates (PB1, PB2, PB3) of Phoma bakeriana were investigated in vitro. There was no isolate effect on growth and pycnidium production but some evidence of a medium effect. Exceptionally high growth rate on two media corresponded with high pycnidium production; while low growth rates on other media were generally associated with high sporulation. There was a large temperature effect and an isolate effect on growth with a significant temperature by isolate interaction. Temperatures of 5°C, 10°C and 35°C inhibited pycnidium formation completely while the range 15–30°C had no effect on sporulation. Maximum growth and pycnidium production occurred at 25°C. Near-ultraviolet radiation seemed to have caused higher pycnidium counts than the other light sources.     

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.     

Production of ethanol by the thermotolerant yeastKluyveromyces marxianus IMB3 during growth on lactose-containing media

Summary The thermotolerant yeast strain,Kluyveromyces marxianus IMB3 was shown to be capable of growth and ethanol production on lactose containing media at 45°C. On media containing 4% (w/v) lactose, ethanol production increased to 6.0g/l within 50h and this represented 29% of theoretical yield. During growth on lactose containing media the organism was shown to produce a cell-associated β-galactosidase and no significant enzyme could be detected in the extracellular culture filtrate. Addition of β-galactosidase, released fromKluyveromyces marxianus IMB3 cells, to active fermentations, resulted in increasing ethanol production to 53% of theoretical yield at 45°C.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.     

Optimization of Culture Conditions for the Production of Extracellular Agarases from Newly Isolated Pseudomonas Aeruginosa AG LSL-11

Summary An agar-degrading bacterium capable of utilizing agar as sole source of carbon and energy was isolated from sea water by enrichment culture technique. The bacterium was identified as Pseudomonas aeruginosa and the culture conditions were standardized for the maximal production of extracellular agarases. The bacterium grew in the pH range 5.0–11.0, optimal between pH 7.0 and 8.0; temperature between 25 °C and 37 °C, optimal at 30 °C and sodium chloride concentration 0–8% and optimal at 2% respectively. The agarases secreted by Pseudomonas aeruginosa AG LSL-11 were inducible by agar and not by any other simple sugars tested. Maximal agarase production was observed at pH 8.0, and temperature 30 °C. The bacterium had no requirement for NaCl for both growth and production of agarases. The bacterium did not utilize other polysaccharides like ĸ-carrageenan, alginate, cellulose and CMC. The activity staining of partially purified agarase preparation after native-PAGE revealed the presence of three different agarases, agarase LSL-11a, LSL-11b and LSL-11c, whose molecular weights were estimated to be 76, 64 and 46 kDa respectively.     

Fungi of the Windmill Islands, continental Antarctica. Effect of temperature, pH and culture media on the growth of selected microfungi

Studies were conducted on microfungi isolated from soils in the Windmill Islands, continental Antarctica. Growth responses of Alternaria alternata, Chrysosporium pannorum, Nectria peziza, Thelebolus microsporus, Mycelia sterile and Phoma cf. herbarum to temperature, pH and culture media were investigated. Maximum growth occurred after 16 days, except in Nectria peziza and Thelebolus microsporus, where maximum growth occurred 12 days after inoculation. All isolates showed poor growth at 0°C. Maximum growth was obtained with temperatures ranging from 15 to 25°C. The optimum temperature for all fungi was 20°C. An acid medium favoured growth. Chrysosporium pannorum, Phoma cf. herbarum and Nectria peziza grew best at pH 3–4, whereas Mycelia sterile, Alternaria alternata and Thelebolus microsporus grew best at pH 5–6. The culture medium had little effect on growth, except for nutrient agar, which showed poor growth against all isolates with the exception of Thelebolus microsporus. Received: 11 September 1996 / Accepted: 13 January 1997