Microelements preparations obtained during processing of natural flint affect the physiology and biochemistry of bifidobacteria]

Microelement preparations obtained in the course of processing of flint powder stimulate the biological activity of Bifidobacterium adolescentis 94 BIM, grown on complex and synthetic nutritive media. The composition of the microelement preparations differed in the content of cations and anions. Introduction of the preparations into the cultures of physiologically active or anabiotic forms of bifidobacteria changed the parameters of exponential growth: compared to controls, the cultures were characterized by increased specific growth rate and decreased generation time. In the presence of microelements, the development of populations of bifidobacteria was associated with more pronounced accumulation of metabolic products (acetate, lactate, and ethanol). Introduction of microelement preparations increased the rate of synthesis of the extracellular proteinase (maximum content of the enzyme was observed after 3 h, whereas control cultures attained this level only after 6 h).     

Synthesis and release of proteases byBacteroides fragilis

Protease production byBacteroides fragilis ATCC 25285 was determined in batch and continuous cultures. During exponential growth in batch culture, the majority of proteolysis was cell associated. However, as the bacteria reached stationary phase, most of the intracellular proteases were released into the culture medium. Measurements of alkaline phosphatase and -galactosidase, which are respectively periplasmic and cytoplasmic marker enzymes inB. fragilis, showed that secretion of proteases in the stationary phase was a discrete event and was not associated with a general release of cytoplasmic contents. When the bacterium was grown in continuous culture, cell-associated protease activity increased concomitantly with dilution rate (D=0.03–0.23/h). The ratio of intracellular to whole cell protease activity also increased with growth rate (11 at D=0.03/h; 11.7 at D=0.23/h). Extracellular protease activity was detected only in trace amounts in continuous cultures at the lowest dilution rate. Determinations of the distribution of extracellular protease activity in batch culture after 48 h incubation showed that the majority of proteolysis (ca. 90%) was soluble. Nevertheless, a proportion was associated with particulate fractions, which had high specific activities.     

The role of energy-spilling reactions in the growth ofKlebsiella aerogenes NCTC 418 in aerobic chemostat culture

When cell-saturating amounts of glucose and phosphate were added to steady state cultures ofKlebsiella aerogenes that were, respectively, glucose-and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must posses effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved.Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and there-fore expressed a higherY _o value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of maintenance and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment.The maintenance rates of oxygen consumption ofK. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source.The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures ofK. aerogenes is discussed.     

Growth and photosynthetic response of the cyanobacterium Microcystis aeruginosa in relation to photoperiodicity and irradiance

Growth and photosynthetic characteristics, P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), of Microcystis aeruginosa were studied in continuous cultures under a range of photoperiod lengths and growth irradiances. Microcystis showed a low specific maintenance rate constant and a high growth affinity for light (typical cyanobacterial features), but required a dark period to obtain maximum growth rate. P _max and per unit dry weight increased, as did pigment content, when less light became available. By regulation in and P _max (crucial in light-limiting and high-light conditions, respectively) this buoyant species can flourish in low light, but also in high-light environments which may arise when buoyancy is lost.The two different types of light conditions affected growth, and photosynthesis, in different ways. One needs thus to discriminate between photoperiod- and irradiance-limitation, which restricts the utility of simple algal growth models. It was emphasized that photosynthetic adaptation patterns of light-limited species may resemble short-term nutrient uptake kinetics of nutrient-limited organisms.With prior knowledge of the growth limitation, we were able to assess the growth rate of a natural population of Microcystis from its photosynthetic response and from data of laboratory cultures of a known physiological state.     

The effect of glucose on 11β- and 19-hydroxylation of reichstein's substance S by Pellicularia filamentosa

Summary Experiments in batch-fermenters have demonstrated that the 11- and 19-hydroxylation of Reichstein's Substance S by Pellicularia filamentosa ceases in the absence of glucose. The effects of glucose consumption rate and growth rate on hydroxylation have been investigated using chemostat cultures. With glucose-limited cultures, increased hydroxylation rates were observed with increased glucose consumption rates. With nitrogen-limited cultures, however, some form of glucose-repression exists. The maximum rate of hydroxylation occurred at a glucose consumption rate at which the culture was just nitrogen-limited. The growth rate had no major importance.     

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.     

Primary production in ten reservoirs in southern Brazil

The growth and phosphatase activity during phosphorus starvation of cultures of Nodularia spumigera Mertens were examined. Stationary phase was reached much sooner in phosphorus-deficient cultures than in phosphorus-sufficient cultures; the growth rate did not change. Phosphatase activities were greatly increased in stationary phase. Diurnal patterns were established for phosphorus-sufficient cultures, but they were not light related. In phosphorus-deficient cultures, an increase in phosphatase activities over a 24 h period was superimposed on a diurnal pattern. Both phosphorus and nitrogen additions lowered the relative phosphatase activities in long term studies, but the effect of phosphorus was much more pronounced. In short term studies, phosphorus appeared to cause an immediate decrease in phosphatase activity, but did not affect phosphatase activity after that for up to 24 h. Nitrogen did not have any short term effect on phosphatase. Phosphatase activity was correlated with changes in the proportion of TCA-insoluble phosphorus (polyphosphates).     

Effect of the Nutritional Status of Semi-continuous Microalgal Cultures on the Productivity and Biochemical Composition of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

The rotifer Brachionus plicatilis was cultured using the microalga Isochrysis aff. galbana clone T-ISO as feed. T-ISO was cultured semi-continuously with daily renewal rates of 10%, 20%, 30%, 40%, and 50% of the volume of cultures. The increase of renewal rate led to increasing nutrient and light availability in microalgal cultures, which caused differences in the biochemical composition of microalgal biomass. Growth rate, individual dry weight, organic content, and biomass productivity of rotifer cultures increased in response to higher growth rate in T-ISO cultures. Rotifer growth rate showed a strong negative correlation (R ² = 0.90) with the C/N ratio of microalgal biomass. Rotifer dry weight was also affected by nutrient availability of T-ISO cultures, increasing up to 50% from nutrient-limited to nutrient-sufficient conditions. Consequently, biomass productivity of rotifer cultures increased more than twofold with the increase of renewal rate of T-ISO cultures. Rotifer organic content underwent the same trend of total dry weight. Maximum content of polyunsaturated fatty acids was reached in rotifers fed T-ISO from the renewal rate of 40%, with percentages of docosahexaenoic acid (22:6ω-3, DHA) and eicosapentaenoic acid (20:5ω-3, EPA) of 11% and 5% of total fatty acids, respectively. Selecting the most appropriate conditions for microalgal culture can therefore enhance the nutritive quality of microalgal biomass, resulting in a better performance of filter feeders and their nutrient content, and may constitute a useful tool to improve the rearing of fish larvae and other aquaculture organisms that require live feed in some or all the stages of their life cycle.     

REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT- OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady-state light or phosphate-limited cultures of Aphanizomenon flos-aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy-limited growth rate increased hut was the same at several phosphate-limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate-limited cultures, hut the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy-limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4-fold. This variation appears to be due to differences in gas vesicle content among individual filaments.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

A re-assessment of bacterial growth efficiency: the heat production and membrane potential of Streptococcus bovis in batch and continuous culture

Glucose-limited, continuous cultures (dilution rate 0.1 h^-1) of Streptococcus bovis JB1 fermented glucose at a rate of 3.9 mol mg protein^-1 h^-1 and produced acctate, formate and ethanol. Based on a maximum ATP yield of 32 cells/mol ATP (Stouthamer 1973) and 3 ATP/glucose, the theoretical glucose consumption for growth would have been 2.1 mol mg protein^-1 h^-1. Because the maintenance energy requirement was 1.7 mol/mg protein/h (Russell and Baldwin 1979), virtually all of the glucose consumption could be explained by growth and maintenance and the Y_ATP was 30. Glucose-limited, continuous cultures produced heat at a rate of 0.29 mW/mg protein, and this value was similar to the enthalpy change of the fermentation (0.32 mW/mg protein). Batch cultures (specific growth rate 2.0 h^-1) fermented glucose at a rate of 81 mol mg protein^-1 h^-1, and produced only lactate. The heat production was in close agreement with the theoretical enthalpy change (1.72 versus 1.70 mW/mg protein), but only 80% of the glucose consumption could be accounted by growth and maintenance. The Y_ATP of the batch cultures was 25. Nitrogen-limited, glucose-excess, non-growing cultures fermented glucose at a rate of 6.9 mol mg protein^-1 h^-1, and virtually all of the enthalpy for this homolactic fermentation could be accounted as heat (0.17 mW/mg protein). The nitrogenlimited cultures had a membrane potential of 150 mV, and nearly all of the heat production could be explained by a futile cycle of protons through the cell membrane (watts = amperes x voltage where H⁺/ATP was 3). The membrane voltage of the nitrogen-limited cells was higher than the glucose-limited continuous cultures (150 versus 80 mV), and this difference in voltage explained why nitrogen-limited cultures consumed glucose faster than the maintenance rate. Batch cultures had a membrane potential of 100 mV, and this voltage could not account for increased glucose consumption (more than growth plus maintenance). It appears that another mechanism causes the increased heat production and lower growth efficiency of batch cultures.     

Influence of growth rate on the accumulation of ergosterol in yeast-cells

Summary The influence of growth rate on the accumulation of ergosterol inSaccharomyces cerevisiae was studied with glucose, maltose, ethanol and acetic acid as substrates under C- and N-limitations in chemostat experiments. In carbon limited cultures an decrease in ergosterol content with rising dilution rate was observed, whereas in nitrogen limited cells an quite opposite behaviour was attained. A maximum specific rate of ergosterol synthesis of about 2 mg per h per g dry cell mass was calculated for nitrogen limited cultures.     

Investigation of the Physiological and Biochemical Characteristics of Bifidobacteria at the Late Stages of Their Development

An investigation of the physiological and biochemical characteristics of the Bifidobacterium bifidumno. 1, B. adolescentisMC-42, and B. adolescentis94-BIM strains showed that bifidobacteria with a higher growth rate produced greater amounts of the end fermentation products, acetate and lactate. The growth of the strains in batch cultures was found to be inhibited by acidic fermentation products. The growth of B. bifidumno. 1 in a batch mode lasted 100 h at a population density of 10⁶CFU/ml and the growth of B. adolescentisMC-42 and 94-BIM lasted 96–120 h at population densities from 10⁴to 10⁷CFU/ml. Analysis of the bifidobacterial populations by light and electron microscopy showed that they represent conglomerates of cells with a lysed cytoplasm in the cell center and an intact cytoplasm in the apical parts of the cells. The maximum production of extracellular and cell-bound proteinases was observed in the logarithmic growth phase. By the 120th h of cultivation, the metabolic activity of cells, the production of proteinases, and the protein content of bifidobacterial cultures considerably decreased. In the first, second, and third subcultures of 96-h-old bifidobacterial cells on fresh nutrient media, the population density of bifidobacteria and their normal physiological and biochemical characteristics were restored after 48 to 72 h of cultivation.     

The effect of peptone on the growth of heterotrophic cultures of Chlorella vulgaris (Emerson strain)

Summary Peptone is more effective than nitrate as a nitrogen source for heterotrophic cultures of Chlorella vulgaris (Emerson strain). It allows the production of an increased amount of algal material and supports an enhanced rate of cell division. Peptone-supplied heterotrophic cultures have a significantly higher content of protein and of soluble nitrogenous substances. The relation between these observations and the growth behaviour of this strain is referred to.The experimental work described in this communication was carried out whilst the author was on the staff of the Botany Department, University College of North Wales, Bangor.     

Effects of temperature,photoperiod and culture vessel size on adventitious shoot production of in vitro propagated <Emphasis Type="Italic">Huernia hystrix</Emphasis>

High production costs due to low growth rate in vitro and high labour costs are among factors limiting commercial application of micropropagation techniques. The low growth rate could be due to unfavourable or sub-optimal environmental and chemical conditions of the cultures. The effects of temperature, photoperiod and culture vessel size were investigated on adventitious shoot production of Huernia hystrix. There were significant increases in shoot proliferation with increased temperature in cultures maintained under a 16 h photoperiod. Slow growth observed at low temperatures (15 and 20°C) offers a potential strategy for cost-effective in vitro storage of H. hystrix germplasm. The maximum adventitious shoots produced per explant and percentage of explants producing shoots (4.2 ± 0.74 and 94% respectively) were observed in cultures maintained at 35°C, the optimum temperature for photosynthesis in plants possessing crassulacean acid metabolism (CAM). The nocturnal accumulation of organic acids in cultures incubated under a 16 h photoperiod further suggest the presence of CAM in this species. On the other hand, cultures kept under continuous light appear to shift to a C-3 photosynthetic pathway. There was a significant decrease in fresh weight of adventitious shoots regenerated per explant as temperature increased. The use of larger culture vessels further increased the shoot proliferation to 5.6 shoots per explant with a potential production of 3,429 shoots per m² in the growth room compared to 2,750 shoots per m² using culture tubes.     

Interpreting the role of phosphorus and growth rate in enhanced fungal induction of sesquiterpenes from Hyoscyamus muticus root cultures

The effect of thiamine limitation in combination with fungal elicitation on sesquiterpene (solavetivone) production was studied in Agrobacterium-transformed hairy-root cultures of Hyoscyamus muticus as a potential means of manipulating the growth rate independent of phosphorus availability. Limiting the initial supply of thiamine did not affect the growth of these cultures compared to growth at the control level of thiamine (0.01 g/l). There was also no enhancement in sesquiterpene production when thiamine supply was limited. Serial culturing in thiamine-free media suggests that these root cultures are not strictly auxotrophic for thiamine, in contrast to previously published results for untransformed root culture. The effect of phosphate limitation combined with elicitation on the production of solavetivone was examined at constant media volume to provide a constant elicitor concentration and to eliminate feedback-inhibition effects. Limiting the initial supply of phosphate to elicited cultures resulted in a twofold increase in solavetivone production as compared to the elicitation at control media phosphate levels (1.1mm). Because growth was attenuated, production per unit cell mass increased 11-fold compared to the control. The effect of phosphate limitation on solavetivone production at constant cell mass and elicitor per root mass was studied. Limiting the initial supply of phosphate to elicited cultures under these conditions did not result in enhanced production of solavetivone. The initially observed enhanced production of solavetivone at limiting initial phosphate concentrations is therefore due to factors other than the growth rate or phosphate involvement in secondary metabolism. Correspondence to: W. R. Curtis     

Growth and aflatoxin production by Aspergillus parasiticus when in the presence of Streptococcus lactis

Streptococcus lactis was grown with Aspergillus parasiticus in modified APT broth. Three inoculation procedures were used: (a) S. lactis was grown 3 days, then conidia of A. parasiticus were added (SLAP), (b) both organisms were added simultaneously (ST) and (c) A. parasiticus was grown 3 days, then S. lactis was added (APSL). At 3, 6 and 10 days of incubation, contents of flasks were analyzed for growth of each organism, pH of broth and aflatoxin content. S. lactis did not survive past 3 days when grown alone. In ST cultures, S. lactis grew to the same extent as in the control at 3 days; it remained viable at a low level through 10 days. In APSL cultures, S. lactis growth was inhibited at 3 days but the bacterium survived through 7 days (10 days of mold growth) at reduced numbers. At 3 days there were no appreciable differences in growth of A. parasiticus. At 6 days, in ST and SLAP cultures, growth of the mold was inhibited, while in the APSL culture growth increased over that in the control. At 10 days, growth of mold was somewhat increased over the control in all test conditions. The pH of broth in the A. parasiticus control and APSL culture was 6 at 3 days, dropped to 4.5–4.6 at 6 days and rose to 7 by 10 days. In ST and SLAP cultures, the pH was at 4.1 at 3 days and rose to pH 7 by 10 days. Aflatoxin (B₁ plus G₁) content was lowest at 3 days and increased at 6 days. Between 6 and 10 days two patterns were observed. In APSL and SLAP cultures, aflatoxin content decreased, while it increased in the ST culture. These patterns occurred when aflatoxin content was expressed on a total or per gram of dried mycelium basis. At 3 days the amounts of aflatoxin B₁ and G₁ were approximately equal. Between 3–6 days the amount of G₁ increased more rapidly than that of B₁. Between 6 and 10 days in the ST culture, the amount of G₁ increased at a slower rate than that of B₁ while in SLAP and APSL cultures, the amount of G₁ decreased more rapidly than that of B₁. When a different lot of the same medium was used, aflatoxin production was greatly reduced. The pH of broth at all test conditions rose through the incubation period.     

Effects of silicate resupply to silicate‐deprived Thalassiosira weissflogii (Bacillariophyceae) in stationary or senescent phase: short‐term patterns of growth and cell death

The ability of nutrient‐deprived phytoplankton to recover in the short term when nutrients are resupplied has been studied for nitrogen and phosphorus, but the case for silicate (Si) is poorly understood. Si‐limited Thalassiosira weissflogii (Grunow) Fryxell et Hasle (grown in batch culture) was harvested in stationary phase (when cell numbers stopped increasing ~2 d after Si depletion) and senescence (when cell numbers declined ~4 d after Si depletion) and Si was resupplied at different concentrations (from 0 to 100 μM). Cell numbers, proportion of dead cells, variable fluorescence emissions (F_v/F_m), and activities of proteases were measured during Si depletion and for 24 h after Si resupply. As Si was depleted, the specific growth rate declined, dead cells increased from ~2% in log phase, to ~25% in stationary phase to over 35% in senescence, and activities of proteases associated with cell death increased several‐fold. Concentration‐dependent recovery of growth rate was seen after 24 h for cultures resupplied with Si in stationary phase but not in senescence. However, resupply of Si at 100 μM to stationary phase cultures alone increased protease activity to nearly the levels seen in senescence. Differences in the responses to Si resupply suggest that the ability and time to recover from Si depletion depend not only on the growth phase but also on the concentration resupplied.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate