Mass Production Potential of the Bacto-Helminthic Biocontrol Complex Heterorhabditis indica - Photorhabdus luminescens

Heterorhabditis indica is a potential agent for the biological control of grubs in sugarcane fields in India. The type strain LN 2 was transferred to monoxenic cultures on its symbiont Photorhabdus luminescens and successfully produced on solid media. In liquid cultures, a mean dauer juvenile yield of 457 000 was obtained with a maximum of 648 000 per ml. Comparatively high yields have not been reported before. Therefore, costs related to the liquid culture production of H. indica will be lower than for other entomopathogenic nematodes currently used in biocontrol. Different bacterial clones had no significant influence on the dauer juvenile yields in liquid media. The exit from the dauer juvenile stage (recovery) after inoculation and the number of hermaphrodites significantly decreased when culture temperature was increased from 25-30 ° C; the dauer juvenile yields were not affected. The cell density of P. luminescens in batch cultures was higher at 25 and 30 ° C than at growth temperatures of 35 and 37 ° C. In continuous culture, the bacterial growth was inhibited when the growth temperature reached 38 ° C. After approximately 60 h, the bacteria adapted to higher temperature and the growth rate increased again. When the temperature was further increased to 40 ° C, the bacterial growth was inhibited.     

The effect of growth temperature on the thermotropic behavior of the membranes of a thermophilic Bacillus. Composition-structure-function relationships

The following study was carried out with the aim of widening our understanding of the thermoadaptive mechanisms of the membrane of thermophiles, using Bacillus stearothermophilus var. nondiastaticus as test-organism. The phospholipids and their acyl chain composition of this Bacillus studied in relation to the physical properties of its membrane from bacteria grown at various temperatures. Phospholipids account for 68-75 weight% of the total lipid in cells grown at 45, 55 or 65 degrees C. Phosphatidylglycerol and diphosphatidylglycerol constitute up to 90% of the total phospholipids; no amino phospholipids were found. Increasing the growth temperatures from 45 degrees to 65 degrees C caused an approximately 4-fold decrease in the proportion of the branched-chain fatty acids and a 2-fold increase in the amount of the saturated acyl chains. The reduced proportion of the branched fatty acids was mainly due to a decrease in their anteiso forms. Unsaturated fatty acids were not produced by cells grown at 65 degrees C. In accordance with the fatty acid composition, the molecular packing of phospholipids in monolayers was more expanded with phospholipids from 45 degrees C grown cells as compared with cultures grown at 55 degrees C. The thermotropic gel to liquid-crystalline phase transition of the membrane lipids was monitored by differential scanning calorimetry and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. With increase of the growth temperature the phase transition was progressively shifted to higher but narrower range of temperatures. Completion of the lipid melting occurred always at temperatures below those employed for growth. A constructed phase diagram enabled to relate the growth temperature, the fatty acid composition and the lipid apparent microviscosity at temperatures not used in the present study for growth of the thermophile. The minimum temperature for growth and the upper boundary temperature of the least saturated lipid crystallization were extrapolated in this manner; they correspond to the experimentally determined minimal growth temperature. The apparent microviscosity, a measure of membrane order, decreased gradually and conspicuously as the growth temperature was elevated. The delimiting apparent microviscosity values, at the maximal (65 degrees C) and minimal (41 degrees C) growth temperatures were 0.8 and 1.8 poise, respectively. This lack of rigorous homeostatic control of the bulk lipid viscosity prompted reevaluation of the physiological significance of 'homeoviscous adaptation' in Bacillus stearothermophilus.     

ADAPTIVE PHOTOSYNTHESIS RESPONSES TO TEMPERATURE EXTREMES BY THE THERMOPHILIC CYANOPHYTE SYNECHOCOCCUS LIVIDUS1

The physiological and biochemical changes associated with and resulting in adaptation to both sub- and supra-optimal temperatures are presented for the thermophilic cyanophyte Synechococcus lividus Copeland. The optimum temperature for growth was 45 C. An increase in the optimum temperature of photosynthesis from 50 to 55 C was shown for cells grown at the supra-optimum temperature of 57 C; whereas, cells grown at the sub-optimal temperature of 35 C exhibited a decrease in the optimal temperature from 50 to 45 C for ¹⁴CO₂ uptake. These changes in optimal temperatures are interpreted as adaptive. Associated with the 5 C increase in optimal temperature for photosynthesis was an increase in chlorophyll a, plastoquinone A, and activity of ribulose-1,5-diphosphate carboxylase (RuDP carboxylase). However, the increase in the temperature optimum for 57 C grown cells was associated with a reduced O₂ yield correlated with a reduced ferricyanide photoreduction capacity. RuDP carboxylase activity decreased rapidly above 55 C. Therefore reduced rates above 55 C resulted from damage to ferricyanide reducing systems and reduced RuDP-carboxylase activity, whereas low photosynthesis rates at sub-optimal temperatures were probably due to rate limiting effect of low temperatures on RuDP carboxylase activity with no evidence of damage to ferricyanide photoreducing systems.     

Influence of white light on production of aflatoxins and anthraquinones in Aspergillus parasiticus.

The effect of continuous light and continuous darkness on the growth of Aspergillus parasiticus and on the production of aflatoxin, averufin, versicolorin A, and versicolorin C by Aspergillus parasiticus were determined at six different temperatures with six replicates for each experiment. No growth was observed at 15 degrees C in the light, although slight growth was observed at this temperature in the dark. No aflatoxins or anthraquinones were produced in the light or dark at 35 and 40 degrees C, although growth was good at these temperatures. Differences in aflatoxins and anthraquinones for cultures grown in light and in dark were consistent at each temperature. Higher mean quantities of these secondary metabolites were produced in the light at 20 and 25 degrees C; lower mean quantities were produced in the light at 30 degrees C. The ranges of values overlapped considerably, but in all cases the differences between temperatures were significant.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. Effect of culture temperature

A three year old, alkaloid producing cell line of Catharanthus roseus, maintained at 25°C, was grown on 2% sucrose at various temperatures from 10° to 45°C. Growth rates were maximal at 35°C but declined rapidly above 35°C and below 25°C. Maximum serpentine yields reached a peak at between 20°C and 25°C and fell sharply above and below these temperatures, while ajmalicine showed a sharp peak of accumulation at 20°C. The variable serpentine/ajmalicine ratio at different growth temperatures suggests that lower temperatures may favour ajmalicine accumulation. Both the growth rate and the rate of alkaloid accumulation at 25°C were therefore sensitive to small changes in average culture temperature.     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

The effects of digestion temperature and temperature shock on the biogas yields from the mesophilic anaerobic digestion of swine manure

In order to obtain basic design criteria for anaerobic digesters of swine manure, the effects of different digesting temperatures, temperature shocks and feed loads, on the biogas yields and methane content were evaluated. The digester temperatures were set at 25, 30 and 35 degrees C, with four feed loads of 5%, 10%, 20% and 40% (feed volume/digester volume). At a temperature of 30 degrees C, the methane yield was reduced by only 3% compared to 35 degrees C, while a 17.4% reduction was observed when the digestion was performed at 25 degrees C. Ultimate methane yields of 327, 389 and 403 mL CH(4)/g VS(added) were obtained at 25, 30 and 35 degrees C, respectively; with moderate feed loads from 5% to 20% (V/V). From the elemental analysis of swine manure, the theoretical biogas and methane yields at standard temperature and pressure were 1.12L biogas/g VS(destroyed) and 0.724 L CH(4)/g VS(destroyed), respectively. Also, the methane content increased with increasing digestion temperatures, but only to a small degree. Temperature shocks from 35 to 30 degrees C and again from 30 to 32 degrees C led to a decrease in the biogas production rate, but it rapidly resumed the value of the control reactor. In addition, no lasting damage was observed for the digestion performance, once it had recovered.     

Phytophthora cryptogea root rot of tomato in rockwool nutrient culture:

The effect of root temperature on growth and yield of rockwool-grown tomato plants infected with Phytophthora cryptogea was investigated. Measurements of shoot and root growth were taken at high (25^oC) and low (15^oC) root temperatures during the generative phase of growth. The growth of roots of healthy and P. cryptogea-infected tomato plants in rockwool blocks was higher in plants grown with roots at 25^oC than at 15^oC after 60 days and a similar effect was found in slabs after 98 days. Under sub-optimal conditions for growth the disease became severe when root temperatures were low. Growth of roots was greatest when roots were maintained at a high temperature in combination with an ambient air temperature of c. 15^oC and the response was greater in cv. Counter than cvs Calypso and Marathon. Water-soluble carbohydrates of roots were higher in those produced in blocks than slabs and were reduced by infection compared to healthy plants with roots at 15^oC and 25^oC. Reduced transpiration rates were found 17 days after inoculation in symptomless plants grown at a root temperature of 25^oC. Infection, regardless of the temperature of the roots or cultivar, led to reduced stem growth. The plants grown at 25^oC were taller than those with a root temperature of 15^oC. After 9 wk of harvest, the cumulative fruit yields in infected cvs Counter and Calypso grown at 25^oC were comparable to that in healthy plants grown at either temperature and cumulative fruit numbers followed a similar pattern. High root temperatures led to delayed fruit ripening between weeks 3–10 and a larger number of unripe fruit. The weight of unripe fruit from infected plants grown at 25^oC at the terminal harvest was higher than from healthy plants with roots maintained at 15^oC.     

Acclimation to the growth temperature and thermosensitivity of photosystem II in a mesophilic cyanobacterium, Synechocystis sp. PCC6803

Differences in the temperature dependence and thermosensitivities of PSII activities in Synechocystis sp. PCC6803 grown at 25 and 35 degrees C were studied. Hill reactions in cells, thylakoid membranes and purified PSII core complexes were measured at high temperatures or at their growth temperatures after high-temperature treatments. In the presence of 2,5-dichloro-p-benzoquinone as an electron acceptor, which can accept electrons directly from Q(A), the temperature dependence of the oxygen-evolving activity was almost the same in thylakoid membranes and in the purified PSII complexes from cells grown at 25 or 35 degrees C. When duroquinone, which accepts electrons only through Q(B) plastoquinone, was used as an electron acceptor, the temperature dependence was the same for purified PSII core complexes but was different between thylakoids isolated from the cells grown at 25 and 35 degrees C. No remarkable difference was observed in protein compositions between thylakoids and between purified PSII complexes from cells grown at 25 or 35 degrees C. However, the fluidity of thylakoids, measured by electron flow to P700, was affected by the growth temperature. These results suggest that one of the major factors which cause the changes in the thermosensitivity of PSII is the change in the fluidity of thylakoid membranes. As for the acclimation of PSII in thylakoids to high temperatures, one of the main causes is the decrease in the high-temperature-induced formation of non-Q(B) PSII due to the decreased fluidity in the cells grown at 35 degrees C.     

Comparative studies on the role of nitrogenous compounds in the growth and perithecial development ofChaetomium aureum

Sodium nitrate is a suitable nitrogen source supporting growth and perithecial formation in Chaetomium aureum. 31 inorganic and organic nitrogen sources at a concentration of 0.033% were used at different incubating temperatures (25, 30 and 35 degrees C). The cultures were grown in Petri dishes, culture tubes and Erlenmeyer flasks. An incubating temperature of 30 degrees C was found most suitable for perithecial formation. In general, a nitrogen source in a solid medium could induce better perithecial production than in liquid media.     

Adaptation of Leishmania to In Vitro Cultivation at 37 C*

SYNOPSIS Promastigotes of Leishmania donovani 3S grown in Tobie's modified medium (Tm) at 25 C multiplied at 37 C after short periods of growth at 32 and 35 C. Only inocula from logarithmic phase cultures grew when placed at 32 C. Amastigotes placed at 32, 35, or 37 C became promastigotes but did not multiply upon transfer. The shortest period required for adaptation of 3S promastigotes to 37 C was 44 days (∽66 generations requiring 18 serial transfers). Addition of chick embryo extract to the medium was unnecessary for growth at elevated temperatures. Promastigotes of the Khartoum strain could not be acclimated to temperatures above 35 C. The long-lasting nature of the adaptation was indicated by (a) ability of 37 C-acclimated promastigotes to grow at this temperature after 14 serial transfers (∽100 generations) at 25 C, and (b) immediate growth of promastigotes at 37 C in cultures inoculated with homogenized hamster spleen previously infected with 37 C acclimated cells. The ability of the temperature-adapted promastigotes to grow at 37 C was lost after 30 serial transfers (224 generations) at 25 C. Since sexual reproduction has not been demonstrated in Leishmania, it was impossible to ascertain whether acclimation was a consequence of mutation or represented a dauermodification.     

Penicillic acid production in submerged culture.

Twenty known penicillic acid (PA)-producing Aspergillus and Penicillium cultures were grown under various conditions in shaken flasks to determine the highest yielding strains and their requirements for maximum toxin production. Abilities of the cultures to utilize eight different carbon sources in Raulin-Thom medium for mycotoxin synthesis were determined at four different incubation temperatures: 15, 20, 25, and 28 degrees C. Of the 20 cultures, P. cyclopium NRRL 1888 was superior, yielding up to 4 mg of PG per ml, with mannitol as the carbon source and 25 degrees C as the incubation temperature. Fifteen of the cultures elaborated lesser amounts of PA, whereas four strains yielded none under the test conditions. Whey from the manufacture of cottage cheese by the cultured process was also a satisfactory medium for PA production. In whey medium, yields up to 3 mg/ml were obtained with P. cyclopium NRRL 1888.     

Sister-chromatid exchange formation at supra-optimal growth temperatures

The present research was mainly focused on characterizing the formation of sister-chromatid exchanges at both optimal and supra-optimal growth temperatures. Under these conditions (25, 30 and 35 degrees C) meristem cells of Allium cepa L. exhibited a roughly constant cell-cycle time, and modifications in other cell-cycle parameters were negligible. Second-division chromosomes of cells incubated at 30 and 35 degrees C showed increased SCE yields as compared with those detected in cells maintained at the optimal temperature (25 degrees C). When cells with unifilarly BrdUrd-substituted DNA was damaged by irradiation with visible light, we obtained almost the same SCE yields at the various temperatures tested. We suggest that this production of SCEs could be the result of a reduced number of lesions produced by light (perhaps as a consequence of reduced intracellular free oxygen at high temperature) and/or of an increased efficiency in the repair capacity of the cells at these temperatures. The analysis of SCE formation in undamaged cells incubated at different temperatures during BrdUrd treatment has shown that the optimal growth temperature appears to be the experimental condition in which the cells are able to exhibit the lowest frequency of SCE.     

Effect of Decreasing Growth Temperature on Cell Yield of Escherichia coli

Studies of the relationship between yield coefficient and growth rate, as affected by temperature of growth, in Escherichia coli have shown that, over a wide range of temperature, yield is relatively constant until the specific growth rate falls below about 0.2 hr(-1), at which point the yield begins to fall off precipitously. No intermediates of glucose metabolism in a form utilizable at higher temperatures could be found in the medium, and no toxic product was produced which limited growth. At 10 C, 37% of the carbon from glucose-UL-(14)C was assimilated into cellular material, whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbohydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells grown at 10 C was approximately three times higher than that of cells grown at 37 C. Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar. Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at 35 C growth was most rapid on glucose and slowest on succinate. The data suggest that the decrease in yield with decrease in temperature is a result of uncoupling of energy production from energy utilization.     

Penicillic acid production in submerged culture.

Twenty known penicillic acid (PA)-producing Aspergillus and Penicillium cultures were grown under various conditions in shaken flasks to determine the highest yielding strains and their requirements for maximum toxin production. Abilities of the cultures to utilize eight different carbon sources in Raulin-Thom medium for mycotoxin synthesis were determined at four different incubation temperatures: 15, 20, 25, and 28 degrees C. Of the 20 cultures, P. cyclopium NRRL 1888 was superior, yielding up to 4 mg of PG per ml, with mannitol as the carbon source and 25 degrees C as the incubation temperature. Fifteen of the cultures elaborated lesser amounts of PA, whereas four strains yielded none under the test conditions. Whey from the manufacture of cottage cheese by the cultured process was also a satisfactory medium for PA production. In whey medium, yields up to 3 mg/ml were obtained with P. cyclopium NRRL 1888.     

Immunochemistry of Yersinia enterocolitica O3 grown at different temperatures.

Comparative agglutinations of homogeneous stable suspensions prepared with Yersinia enterocolitica growth at 37 degrees C and at 25 degrees C were performed with anti-sera prepared in rabbits with the bacteria grown at both these temperatures. Sera prepared with live Y. enterocolitica grown at 37 degrees C agglutinated both suspensions at a much lower titre than the sera prepared with formaldehyde-treated bacteria is grown at 25 degrees C. All the sera in which strongly precipitating antibodies were induced reacted, in agar-gel, against native and heated proteins. The small amounts of antipolysaccharides induced in all the sera reacted only in the ring test against the bacterial polysaccharides. The absorption of the sera prepared with live Y. enterocolitica grown at 37 degrees C, with antigens synthesized at 25 degrees C did not remove all the homologous antibodies; apparently, some determinants are specific for the bacteria grown at 37 degrees C. Morphological changes of the small rods to elongated bacilli and filamentous forms were observed in most cultures of the Y. enterocolitica grown at 37 degrees C; these changes coincided with a low yield of proteins and point to an inhibitory effect of the 37 degrees C temperature.     

Effects of elevated temperature on bacterial community structure and function in bioreactors treating a synthetic wastewater

The impact of elevated temperature on bacterial community structure and function during aerobic biological wastewater treatment was investigated. Continuous cultures, fed a complex growth medium containing gelatin and α-lactose as the principal carbon and energy sources, supported mixed bacterial consortia at temperatures ranging from 25–65°C. These temperature- and substrate-acclimated organisms were then used as inocula for batch growth experiments in which the kinetics of microbial growth and substrate utilization, efficiency of substrate removal, and mechanism of substrate removal were compared as functions of temperature. Bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) revealed that distinct bacterial consortia were supported at each temperature. The efficiency of substrate removal declined at elevated temperatures. Maximum specific growth rates and the growth yield increased with temperature from 25–45°C, but then decreased with further elevations in temperature. Thus, maximum specific substrate utilization rates did not vary significantly over the 40°C temperature range (0.64 ± 0.04 mg COD mg⁻¹ dry cell mass h⁻¹). A comparison of the degradation of the protein and carbohydrate portions of the feed medium revealed a lag in α-lactose uptake at 55°C, whereas both components were utilized simultaneously at 25°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 140–145. Received 09 August 1999/ Accepted in revised form 12 November 1999     

Growth and photosynthesis in seedlings of Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeophyta) cultured at two different temperatures

This study examined temperature acclimation, growth, and photosynthetic characteristics of the zygote-derived seedlings of Hizikia fusiformis (Harvey) Okamura (Sargassaceae). The seedlings were cultured at 15°C or 25°C for 4 weeks. The average relative growth rate was significantly higher in seedlings acclimated at 25°C. The photosynthetic rate measured at 15°C was much higher in seedlings grown at 15°C than those grown at 25°C, indicating photosynthetic acclimation to a lower temperature. At 35°C, the photosynthetic rate of 15°C-grown seedlings was drastically decreased, whereas that of 25°C-grown seedlings was significantly increased. The maximum relative electron transport rate (rETR_max) measured at the respective growth temperature was significantly higher in seedlings grown at 25°C than at 15°C. At a measuring temperature of 35°C, the rETR_max in both 15°C- and 25°C-grown seedlings were considerably reduced with regard to those measured at 15°C or 25°C. Our results suggested that, compared with the seedlings grown at 25°C, those acclimated at a lower temperature could be disadvantaged under adverse conditions such as increased temperatures.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.