Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at −1°C and on PDA containing potassium chloride (KCl) (water potential, −4.27 to −0.85 MPa) or d(−) sorbitol (−3.48 to −0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.     

The interactive effects of temperature and osmotic potential on the growth of marine isolates of <Emphasis Type="Italic">Fusarium solani</Emphasis>

The mycelial growth of 18 Fusarium solani strains isolated from sea beds of the south-eastern coast of Spain was tested on potato-dextrose-agar adjusted to different osmotic potentials with either KCl or NaCl (−1.50 to −144.54 bars) in 10 °C intervals ranging from 15 to 35 °C. Fungal growth was determined by measuring colony diameter after 4 days of incubation. Mycelial growth was maximal at 25 °C. The quantity and frequency pattern of mycelial growth of F. solani differ significantly at 15 and 25 °C, with maximal growth occurring at the highest water potential tested (−1.50 bars); and at 35 °C, with a maximal mycelial growth at −13.79 bars. The effect of water potential was independent of salt composition. The general growth pattern of F. solani showed declining growth at potentials below −41.79 bars. Fungal growth at 35 °C was always higher than that grow at 15 °C, of all the water potentials tested. Significant differences observed in the response of mycelia to water potential and temperature as main and interactive effects. The viability of cultures was increasingly inhibited as the water potential dropped, but some growth was still observed at −99.56 bars. These findings could indicate that marine strains of F. solani have a physiological mechanism that permits survival in environments with low water potential. The observed differences in viability and the magnitude of growth could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Influence of nutritional conditions on exopolysaccharide production by submerged cultivation of the medicinal fungus <Emphasis Type="Italic">Shiraia bambusicola</Emphasis>

Maltose and yeast extract were the most favourable carbon and nitrogen sources for exopolysaccharide production by submerged culture of Shiraia bambusicola WZ-003, and initial maltose and yeast extract concentrations were at 30 and 3 g l⁻¹, respectively. Plant oils could increase the mycelial growth and exopolysaccharide production in tested concentration. K⁺ and Mg²⁺ could enhance the mycelial growth and exopolysaccharide biosynthesis. The optimal cultivation temperature and initial pH were found to be 26°C and 6.0, respectively. Exopolysaccharide concentration reached 0.53 g l⁻¹ in 15-l fermenter under optimal nutritional conditions.     

<Emphasis Type="Italic">Neochloris oleabundans</Emphasis> UTEX #1185: a suitable renewable lipid source for biofuel production

Energy crises, global warming, and climatic changes call for technological and commercial advances in manufacturing high-quality transportation fuels from unconventional feedstocks. Microalgae is one of the most promising sources of biofuels due to the high yields attained per unit area and because it does not displace food crops. Neochloris oleabundans (Neo) microalga is an important promising microbial source of single-cell oil (SCO). Different experimental growth and lipid production conditions were evaluated and compared by using optical density (540 nm), dry-weight determination, and flow cytometry (FC). Best Neo average biomass productivity was obtained at 30°C under conditions of nitrogen-sufficiency and CO₂ supplementation (N+/30°C/CO₂), with an average doubling time of 1.4 days. The second and third highest productivities occurred with N-sufficient cultures without CO₂ supplementation at 26°C (N+/26°C) and at 30°C (N+/30°C), with doubling times of 1.7 and 2.2 days, respectively. Microbial lipid production was monitored by flow cytometry using Nile red (NR), a lipophilic fluorochrome that possesses several advantageous characteristics for in situ screening near real time (at line). Results showed maximum lipid content (56%) after 6 days of nitrogen depletion under nitrogen starvation without CO₂ supplementation (N−/30°C), followed by N−/30°C/CO₂ and N−/26°C conditions with 52% lipid content, after 5 and 6 days of N starvation, respectively. The adequate fatty acid profile and iodine value of Neo lipids reinforced this microalga as a good source of SCO, in particular for use as biodiesel.     

Characterization of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from a sub-alpine soil in the North Western Indian Himalayas

Pantoea dispersa strain 1A is a Gram-negative rod-shaped, yellow-pigmented bacterium isolated on nutrient agar plates incubated at 4°C. The identity of the bacterium was confirmed by sequencing of the 16 S rRNA gene. It was capable of growing at temperatures ranging from 4 to 42°C, but maximum growth was observed at 30°C. It is endowed with multiple plant growth promotion attributes such as phosphate solubilization, IAA production, siderophore production and HCN production, which are expressed differentially at sub-optimal temperatures (15 and 4°C). It was able to solubilize phosphate (17.6 μg of P₂O₅ ml⁻¹ day⁻¹), and produce IAA (3.7 μg ml⁻¹ day⁻¹), at 15°C. Qualitative detection of siderophore production and HCN were also observed at 15°C. At 4°C it was found to express all the plant growth promotion attributes. This bacterial isolate was able to positively influence and promote the growth and nutrient uptake parameters of wheat (cv. VL.802) under glasshouse conditions. Hence in the context, of cold wheat-growing environments, it is proposed that Pantoea dispersa 1A (MTCC 8706), could be deployed as an inoculant to attain the desired results of bacterization.     

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products

Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 10¹ CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (10¹ and 10⁶ CFU/ml) after storage at 4° C and −30° C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at −30° C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (10⁶ CFU/ml) at 4° C. The titers of viable organisms were diminished over 8 wk at 4° C under aerobic and anaerobic conditions.     

Maintenance ofRhodotorula rubra isolated from liquid samples of gold mine effluents

TheRhodotorula rubra strain isolated from waste waters of a gold mining plant has demonstrated the ability to grow in the presence of cyanide. The maintenance of this strain in complex organic media leads to a loss of this ability. To preserve the cyanide resistance ofR. rubra we tested the following maintenance methods: subculturing in sterile distilled water, freezing at −20, −40, −70°C, liquid nitrogen freezing and the paper replica method. The ability to grow in the presence of cyanide was preserved and a higher viability level was observed for cells maintained frozen at −70°C, in liquid nitrogen and by the paper replica method. Preservation in distilled water resulted in the lowest viability after twelve months of storage.     

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

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

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

Growth requirements for production of stable cells of the bioherbicidal bacterium Xanthomonas campestris

Xanthomonas campestris MB245, a specific pathogen of the weedy grass Poa annua (annual bluegrass), is being developed as a bioherbicide to control this pest in turf. Nutritional and environmental factors were evaluated based on their ability to support rapid submerged culture growth and high cell yield. Temperature optima for the growth of X. campestris cells in submerged culture were between 27 and 30°C. At 30°C, optimal nutritional conditions for X. campestris growth supported generation times of 150–175 min and cell yields after 24 h growth of 1–2 × 10¹⁰ cells ml⁻¹. Media containing sucrose or glucose as the carbon source and various organic nitrogen sources supported optimal X. campestris growth and cell yield. The addition of vitamin mixtures to complex and defined media had no significant effect on growth or cell yield. The age of X. campestris cultures had a significant impact on cell survival after freeze drying. Following freeze drying, log phase cell survival (44%) was significantly lower than early and late stationary phase cell survival, 62% and 68%, respectively. Cells harvested in stationary phase, freeze dried and stored under vacuum at 4°C, showed no significant loss in viability after 6 months. Thus, high cell concentrations of the bioherbicide X. campestris can be rapidly produced in submerged culture and stabilized as freeze-dried preparations. Received 14 August 1998/ Accepted in revised form 8 October 1998     

A new method for the preservation of fungus stock cultures by deep-freezing

Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at −85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures. Received: December 7, 2000 / Accepted: December 19, 2001     

Growth promotion of wheat seedlings by <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 μg of P ml⁻¹ day⁻¹), and produced IAA (14.9 μg ml⁻¹ day⁻¹) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Temperature profiles of cellular growth and exopolysaccharide synthesis by Botryococus braunii Kütz. UC 58

Temperature profiles (range 20–33 °C) were obtained for growth and exopolysaccharide (EPS) biosynthesis of the microalga Botryococcus braunii strain UC 58 under photoautotrophic conditions. The maximum temperature for growth was 32 °C and the temperature dependence of the specific growth rate was described by the Hinshelwood equation based on the Arrhenius relationship. The optimal range of temperatures for growth and extracellular EPS synthesis (25–30 °C) concurred and production of 4.5–5 g l⁻¹ of EPS was obtained routinely, leading to high broth viscosities. Below 23 °C EPS biosynthesis was negligible, although the specific growth rate maintained high values. At supraoptimal temperatures EPS biosynthesis decreased, accompanying the increase in doubling time. The polymers formed at temperatures within the optimal range for production, when dissolved in water, produced solutions (2 gl⁻¹) with the highest viscosity, suggesting that their molecular weight showed the highest values. The degree of polymerization of the EPS synthesized at suboptimal and supraoptimal temperatures was significantly below the values within the optimal range.     

Susceptibility of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> strains different in oxalate production to infection by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

Three strains of Sclerotinia sclerotiorum, namely Ep-1PB (PB), Ep-1PK (PK) and Ep-1PNA5 (A5), were compared for the production of oxalic acid (OA) on potato dextrose agar (PDA) and Maxwell agar medium (MAM) and for mycelial susceptibility to infection by the mycoparasite Coniothyrium minitans on PDA. Results showed that strain PB produced negligible oxalate, whereas strain PK was detected to produce oxalate, but much less than that produced by strain A5. The three investigated strains differed slightly in mycelial growth rates and mycelial biomass on PDA. However, colonies of strains PB and PK formed on PDA were more susceptible to invasion by C. minitans than colonies of strain A5. Meanwhile, amendment of synthetic oxalate in PDA at 0.25–2.00 mg g⁻¹ medium suppressed aggressiveness of C. minitans in invasion of colonies of S. sclerotiorum strain PB developed on this medium. These results suggest that infection of hyphae of S. sclerotiorum is negatively affected by the presence of oxalate. The importance of oxalate degradation by C. minitans in its mycoparasitism on hyphae of S. sclerotiorum provides a clue for improvement of the biocontrol efficacy of C. minitans in the future.     

The influence of temperature and nitrogen source on growth and nitrogen uptake of two polar-desert species,Saxifraga caespitosa and Cerastium alpinum

Polar-desert plants experience low average air temperatures during their short growing season (4–8 °C mean July temperature). In addition, low availability of inorganic nitrogen in the soil may also limit plant growth. Our goals were to elucidate which N sources can be acquired by polar-desert plants, and how growth and N-uptake are affected by low growth temperatures. We compared rates of N-uptake and increases in mass and leaf area of two polar-desert species (Cerastium alpinum L. and Saxifraga caespitosa L.) over a period of 3 weeks when grown at two temperatures (6 °C vs. 15 °C) and supplied with either glycine, NH₄ ⁺ or NO₃ ⁻. At 15 °C, plants at least doubled their leaf area, whereas there was no change in leaf area at 6 °C. Measured mean N-uptake rates varied between 0.5 nmol g⁻¹ root DM s⁻¹ on glycine at 15 °C and 7.5 nmol g⁻¹ root DM s⁻¹ on NH₄ ⁺ at 15 °C. Uptake rates based upon increases in mass and tissue N concentrations showed that plants had a lower N-uptake rate at 6 °C, regardless of N source or species. We conclude that these polar-desert plants can use all three N sources to increase their leaf area and support flowering when grown at 15 °C. Based upon short-term (8 h) uptake experiments, we also conclude that the short-term capacity to take up inorganic or organic N is not reduced by low temperature (6 °C). However, net N-uptake integrated over a three-week period is severely reduced at 6 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of Water Potential on Growth, Enzyme Secretion and In Vitro Enzyme Activities of Trichoderma harzianum at Different Temperatures

The influence of water potential on linear mycelial growth, secretion, and the in vitro activities of enzymes β-glucosidase, cellobiohydrolase, β-xylosidase, exochitinase, and chymotrypsin of Trichoderma harzianum strain T66 was studied at different temperatures. Nearly linear correlation was found between water potential and colony growth rate at both 25°C and 10°C, with higher growth rates at the higher temperature and higher water potentials. The amounts of enzyme secretion depended on the water potential and not on the quality of salt (NaCl or KCl) used as osmoticum. Enzyme activities were significantly affected by water potential. Significant enzyme activities were measured for most of the enzymes even at −14.800 megapascal (MPa), which is below the water potential where mycelial growth ceased. These results suggest the possibility of using mutants with improved xerotolerance for biocontrol purposes in soils with lower water potentials. Received: 5 October 1999 / Accepted: 22 November 1999     

Genetic and physiological alterations occurring in a yeast population continuously propagated at increasing temperatures with cell recycling

This work investigated the effects of increasing temperature from 30°C to 47°C on the physiological and genetic characteristics of Saccharomyces cerevisiae strain 63M after continuous fermentation with cell recycling in a system of five reactors in series. Steady state was attained at 30°C, and then the temperature of the system was raised so it ranged from 35°C in the last reactor to 43°C in the first reactor or feeding reactor with a 2°C difference between reactors. After 15 days at steady state, the temperature was raised from 37°C to 45°C for 25 days at steady state, then from 39°C to 47°C for 20 days at steady state. Starter strain 63M was a hybrid strain constructed to have a MAT a/α, LYS/lys, URA/ura genotype. This hybrid yeast showed vigorous growth on plates at 40°C, weak growth at 41°C, positive assimilation of melibiose, positive fermentation of galactose, raffinose and sucrose. Of 156 isolates obtained from this system at the end of the fermentation process, only 17.3% showed the same characteristics as starter strain 63M. Alterations in mating type reaction and in utilization of raffinose, melibiose, and sucrose were identified. Only 1.9% of the isolates lost the ability to grow at 40°C. Isolates showing requirements for lysine and uracil were also obtained. In addition, cell survival was observed at 39–47°C, but no isolates showing growth above 41°C were obtained.     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.