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.     

Effect of physical factors on the production of bacteriocin from Pediococcus acidilactici ITV 26

The effect of pH, temperature and agitation on growth and bacteriocin production by Pediococcus acidilactici ITV 126 was investigated. Experiments were made in flasks containing MRS medium at 30 to 40°C, pH 5 to 7 and agitation 0 to 200 rpm. Factor levels were arranged in a 2³ factorial design with central and axial points. Anova and Tukey paired comparison tests showed that a temperature of 35°C favored bacteriocin production, whereas 40°C was best for cell growth. A statistical interaction of temperature and agitation was observed affecting microbial growth. pH 5 favored both cell growth and bacteriocin production. Journal of Industrial Microbiology & Biotechnology (2001) 26, 191–195. Received 30 October 1999/ Accepted in revised form 31 January 2001     

Combined effects of fermentation temperature and pH on kinetic changes of chemical constituents of durian wine fermented with Saccharomyces cerevisiae

This study investigated the effects of temperature (20 and 30 °C) and pH (pH 3.1, 3.9) on kinetic changes of chemical constituents of the durian wine fermented with Saccharomyces cerevisiae. Temperature significantly affected growth of S. cerevisiae EC-1118 regardless of pH with a higher temperature leading to a faster cell death. The pH had a more significant effect on ethanol production than temperature with higher production at 20 °C (5.95%, v/v) and 30 °C (5.56%, v/v) at pH 3.9, relative to that at pH 3.1 (5.25 and 5.01%, v/v). However, relatively higher levels of isobutyl alcohol and isoamyl alcohol up to 64.52 ± 6.39 and 56.27 ± 3.00 mg/L, respectively, were produced at pH 3.1 than at pH 3.9 regardless of temperature. In contrast, production of esters was more affected by temperature than pH, where levels of ethyl esters (ethyl esters of octanoate, nonanoate, and decanoate) and acetate esters (ethyl acetate and isoamyl acetate) were significantly higher up to 2.13 ± 0.23 and 4.61 ± 0.22 mg/L, respectively, at 20 °C than at 30 °C. On the other hand, higher temperature improved the reduction of volatile sulfur compounds. This study illustrated that temperature control would be a more effective tool than pH in modulating the resulting aroma compound profile of durian wine.

     

Phosphate solubilisation and plant growth promoting potential by stress tolerant Bacillus sp. isolated from rhizosphere of apple orchards in trans Himalayan region of Himachal Pradesh

Phosphate‐solubilising ability and co‐production of plant growth promoting traits of stress tolerant Bacillus subtilis CB₈A isolated from apple rhizosphere was tested under in vitro conditions against a wide range of temperature (30–45°C), pH (7–9) and salt (0–5%) stresses. Under the extremes of temperature (45°C), pH‐9 and salt concentration (5%), production of soluble phosphate, indole acetic acid, siderophore and antifungal activity against Dematophora necatrix were reduced by 71.09%, 75.29%, 90.3% and 88.47%, respectively. Per cent decrease in P‐solubilisation at extreme temperature (45°C) and normal pH (7) without salt concentration was 36.23%; at extreme pH (9) and normal temperature (37°C) without salt concentration was 23.45% and at extreme salt concentration (5%), optimum temperature (37°C) and pH (7) was 36.7%. P‐solubilisation by CB₈A was inversely correlated with pH (r = ?0.78) and positively correlated with siderophore production (r = 0.81), indole acetic acid (r = 0.58) and antifungal activity (r = 0.63). Gluconic acid (1.43%) and citric acid (0.67%) were detected as major organic acids. P‐solubilisation and nitrogen fixing abilities of B. subtilis CB₈A were confirmed by amplification of gdh and nifH genes. The ability of CB8A showing plant growth promoting rhizobacteria (PGPR) traits at a wide range of temperature, pH and varying salt concentration can be exploited for developing multifunctional biofertiliser in apple orchards.     

Factors Affecting the Production of Antifungal Compounds by Enterobacter aerogenes and Bacillus subtilis,Antagonists of Phytophthora cactorum1

The effects of temperature and pH on the production of antifungal compounds and growth in sterilized soil of Enterobacter aerogenes and Bacillus subtilis, antagonists of Phytophthora cactorum, the cause of crown rot of apple trees, were studied. With E. aerogenes maximum amounts of antifungal compounds were produced between 14 and 21°C and at pH levels between 3.5 and 5.0. Bacillus subtilis produced maximum amounts of antifungal compounds between 21 and 28°C andwith pH levels between 5.0 and 8.0. P. cactorum inoculum, produced in the presence of E. aerogenes or B. subtilis at optimum temperature and pH levels, was significantly less virulent compared with controls. The optimum temperature of maximum population growth in sterilized soil for E. aerogenes was 18°C and for B. subtilis 25°C. The population growth of B. subtilis was much slower than that of E. aerogenes. Fosetyl Al stimulated the multiplication of bacteria at lower temperatures while metalaxyl had the same effect at higher temperatures. These results indicate that populations of these antagonistic bacteria increase in sterile soil up to the 33rd day from inoculation and that the fungicides fosetyl, Al and metalaxyl did not limit their multiplication and production of antifungal compounds.     

Production and characterization of a collagenolytic serine proteinase by <Emphasis Type="Italic">Penicillium aurantiogriseum</Emphasis> URM 4622: A factorial study

A 2⁴ full factorial design was used to identify the main effects and interactions of the initial medium pH, soybean flour concentration, temperature and orbital agitation speed on extracellular collagenase production by Penicillium aurantiogriseum URM4622. The most significant variables for collagenase production were soybean flour concentration and initial medium pH that had positive main effects, and temperature that had a negative one. Protein concentration in soybean flour revealed to be a significant factor for the production of a collagenase serine proteinase. The most favorable production conditions were found to be 0.75% soybean flour, pH 8.0, 200 rpm, and 28°C, which led to a collagenase activity of 164 U. The enzyme showed an optimum activity at 37°C and pH 9.0, was stable over wide ranges of pH and temperature (6.0 ∼ 10.0 and 25 ∼ 45°C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonylfluoride. The firstorder rate constants for collagenase inactivation in the crude extract, calculated from semi-log plots of the residual activity versus time, were used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* _d = 107.4 kJ/mol and ΔH* _d = 104.7 kJ/mol). The enzyme is probably an extracellular neutral serine collagenase effective on azocoll, gelatin and collagen decomposition.     

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion‐weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high‐temperature exotherm (?3°C to ?5°C) corresponded with ice formation and high ice‐nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (?6°C and ?8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low‐temperature exotherm corresponded with SAM injury and the killing temperature in wheat (?21°C) and rye (?27°C). The SAM had lower ice‐nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.     

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.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

Isolation and characterization of hydroxylamine-induced mutations in the Erwinia herbicola ice nucleation gene that selectively reduce warm temperature ice nucleation activity

Cells of ice nucleation active bacterial species catalyse ice formation over the temperature range of -2 to -12°C. Current models of ice nucleus structure associate the size of ice nucleation protein aggregates with the temperature at which they catalyse ice formation. To better define the structural features of ice nucleation proteins responsible for the functional heterogeneity of ice nuclei within a genetically homogeneous collection of cells we used in vitro chemical mutagenesis to isolate mutants with reduced ability to nucleate ice at warm assay temperatures but which retain normal or near normal nucleation activity at cold temperatures (WIND, i.e. w arm i ce n ucleus-d eficient mutants). Nearly half of the mutants obtained after hydroxylamine mutagenesis of the iceE gene from Erwinia herbicola had this phenotype. The phenotypes and location of lesions on the genetic map of iceE were determined for a number of mutants. All WIND mutations were restricted to the portion of iceE encoding the repetitive region of the poty peptide. DNA sequencing of two WIND mutants revealed single nucleotide substitutions changing a conserved serine or glycine residue to phenylalanine and serine, respectively. The implications of these findings in structure/function models for the ice nucleation protein are discussed.     

Application of a mathematical model and Differential Evolution algorithm approach to optimization of bacteriocin production by Lactococcus lactis C7

The effect of pH and temperature on cell growth and bacteriocin production in Lactococcus lactis C7 was investigated in order to optimize the production of bacteriocin. The study showed that the bacteriocin production was growth-associated, but declined after reaching the maximum titer. The decrease of bacteriocin was caused by a cell-bound protease. Maximum bacteriocin titer was obtained at pH 5.5 and at 22°C. In order to obtain a global optimized solution for production of bacteriocin, the optimal temperature for bacteriocin production was further studied. Mathematical models were developed for cell growth, substrate consumption, lactic acid production and bacteriocin production. A Differential Evolution algorithm was used both to estimate the model parameters from the experimental data and to compute a temperature profile for maximizing the final bacteriocin titer and bacteriocin productivity. This simulation showed that maximum bacteriocin production was obtained at the optimal temperature profile, starting at 30°C and terminating at 22°C, which was validated by experiment. This temperature profile yielded 20% higher maximum bacteriocin productivity than that obtained at a constant temperature of 22°C, although the total amount of bacteriocin obtained was slightly decreased.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Effects of process errors on the production of ethanol by Escherichia coli KO11

Escherichia coli KO11 was previously constructed for the production of ethanol from both hexose and pentose sugars in hemicellulose hydrolysates by inserting the Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). This biocatalyst appears relatively resistant to potential process errors during fermentation. Antibiotics were not required to maintain the maximum catabolic activity of KO11 even after deliberate contamination with up to 10% soil. Fermentations exposed to extremes of temperature (2 h at 5°C or 50°C) or pH (2 h at pH 3 or pH 10) recovered after re-adjustment to optimal fermentation conditions (35°C, pH6) although longer times were required for completion in most cases. Ethanol yields were not altered by exposure to extremes in temperature but were reduced by exposure to extremes in pH. Re-inoculation with 5% (by volume) from control fermentors reduced this delay after exposure to pH extremes. Received 24 July 1997/ Accepted in revised form 16 April 1998     

Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047

An endosymbiont Halobacterium salinarum MMD047, which could produce high yields of amylase, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% sucrose. The enzyme was found to be produced constitutively even in the absence of starch. The optimum temperature and pH for the enzyme production was 40°C and 8.0, respectively. The enzyme exhibited maximum activity in pH range of 6∼10 with an optimum pH of 9.0. The enzyme was stable at 40°C and the enzyme activity decreased dramatically above 50°C. Based on the present findings, the enzyme was characterized as relatively heat sensitive and alkalophilic amylase which can be developed for extensive industrial applications.     

A novel low-temperature-active β-glucosidase from symbiotic <Emphasis Type="Italic">Serratia</Emphasis> sp. TN49 reveals four essential positions for substrate accommodation

To maximize the production of flag-tagged cartilage oligomeric matrix protein angiopoietin-1 (FCA1) from Chinese hamster ovary (CHO) cells, the effects of culture pH and temperature on cell growth and FCA1 production were investigated. Cells were cultivated in a bioreactor at different culture pH (6.7, 6.9, 7.2, and 7.5) and temperatures (33 and 37 °C). Lowering the culture temperature suppressed cell growth while allowing maintenance of high cell viability for a longer culture period. The specific FCA1 productivity (q _FCA1) was increased at low culture temperature. Accordingly, the highest FCA1 concentration was obtained at pH 7.2 and 33 °C, and was approximately 4.0-fold higher than that at pH 7.2 and 37 °C. However, aggregates and a monomeric form of FCA1, which are undesirable due to reduced biological activity or immunogenicity, were significant at pH 7.2 and 33 °C. It was also found that the expression pattern of FCA1 was affected more significantly by culture pH than by the culture temperature. FCA1 aggregation dramatically decreased at culture pH 7.5 regardless of the culture temperature. Furthermore, the monomeric form of FCA1 was not observed. Taken together, optimization of culture temperature and culture pH (33 °C and pH 7.5) significantly improves the production of biologically active FCA1 with tetrameric or pentameric forms from CHO cells.     

Measurement of cooling and warming rates in vitrification‐based plant cryopreservation protocols

Cryopreservation protocols include the use of additives and pretreatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro‐viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (T_f) and the glass transition (T_G) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice‐derived damage. Vitrification and droplet‐vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between ?20°C and ?120°C, ranged from ca. 5°C s^?1 to 400°C s^?1, while warming rates spanned from ca. 2°C s^?1 to 280°C s^?1, for the different protocols and conditions studied. A wider measuring window (0°C to ?150°C) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1177–1184, 2014     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Freezing tolerance and avoidance in high-elevation Hawaiian plants

Freezing resistance mechanisms were studied in five endemic Hawaiian species growing at high elevations on Haleakala volcano, Hawaii, where nocturnal subzero (°C) air temperatures frequently occur. Extracellular freezing occurred at around -5°C in leaves of Argyroxiphium sandwicense and Sophora chrysophylla, but these leaves can tolerate extracellular ice accumulation to -15°C and -12°C, respectively. Mucilage, which apparently acted as an ice nucleator, comprised 9 to 11% of the dry weight of leaf tissue in these two species. Leaves of Vaccinium reticulatum and Styphelia tameiameiae were also found to tolerate substantial extracellular freezing. Dubautia menziesii, on the other hand, exhibited the characteristics of permanent supercooling; a very rapid decline in liquid water content associated with simultaneous intracellular and extracellular freezing. However, in those species that tolerate extracellular freezing, the decline in liquid water content during freezing is relatively slow. Osmotic potential was lower at pre-dawn than at midday in four of the species studied. Nocturnal production of osmotically active solutes may have helped to prevent intracellular freeze dehydration as well as to provide non-colligative protection of cell membranes. Styphelia tameiameiae supercooled to -9·3°C and tolerated tissue freezing to below -15°C, a unique combination of physiological characteristics related to freezing. Tolerance of extracellular ice formation after considerable supercooling may have resulted from low tissue water content and a high degree of intracellular water binding in this species, as determined by nuclear magnetic resonance studies. The climate at high elevations in Hawaii is relatively unpredictable in terms of the duration of subzero temperatures and the lowest subzero temperature reached during the night. It appears that plants growing in this tropical alpine habitat have been under selective pressures for the evolution of freezing tolerance mechanisms.