Effects of growth conditions on production of methyl selenides in cultures of Rhodobacter sphaeroides

Rhodobacter sphaeroides 2.4.1 exposed to selenate or selenite produced volatile selenium compounds. Total amounts of dimethyl selenide, dimethyl diselenide, dimethyl sulfide and dimethyl disulfide in culture medium and headspace were determined. The highest selenate volatilization occurred in the late stationary phase of growth. However, cultures deprived of light in the stationary phase of growth produced much less of the volatile organo-selenium compounds. Lower culture pHs increased the rate of selenium volatilization. Low sulfate concentration limited biomass production and selenium volatilization; high sulfate concentrations had an enhancing effect on the release of organo-selenium compounds. Cultures of R. sphaeroides reacted very differently to amendments with increasing amounts of selenate and selenite. Only small amounts of selenite were volatilized; meanwhile high amounts of methylated selenides were found in selenate-poisoned cultures. Received 03 February 1997/ Accepted in revised form 16 May 1997     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.     

GROWTH AND DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA SERIATA (BACILLARIOPHYCEAE) UNDER PHOSPHATE AND SILICATE LIMITATION1

Pseudo‐nitzschia seriata (Cleve) H. Peragallo isolated from Scottish west coast waters was studied in batch culture with phosphate (P) or silicate (Si) as the yield‐limiting nutrient at 15°C. This species produced the neurotoxin domoic acid (DA) when either nutrient was limiting but produced more when stressed by Si limitation during the stationary phase. Under P‐limiting conditions, exponential growth stopped after P was reduced to a low threshold concentration. Under Si‐limiting conditions, fast exponential growth was followed by a period of slower exponential growth, until Si became exhausted. A stationary phase was observed in the P‐limited but not the Si‐limited cultures, the latter showing a rapid decrease in cell density after the second exponential growth phase. Si‐limited cultures exhibited a further period of active metabolism (as indicated by increases in chl and carbon per cell) late in the experiment, presumably fueled by regenerated Si. DA production was low in exponential phase under both conditions. In P‐limited cultures, most DA was produced during the immediate postexponential phase, with little or no new DA produced during later cell senescence. In contrast, although a substantial amount of DA was produced during the slower exponential phase of the Si‐limited cultures, DA production was even greater near the end of the experiment, coincident with the period of chl synthesis and increase in carbon biomass. Comparison of the magnitude of toxin production in the two nutrient regimes indicated a greater threat of P. seriata‐generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.     

Effects of oxygen and nitrate on growth of Escherichia coli and Pseudomonas aeruginosa in the presence of organic solvents

Escherichia coli and Pseudomonas aeruginosa grown in the presence of certain harmful organic solvents become susceptible to these solvents during the cultivation. This susceptibility is conspicuous in the stationary phase of growth. The organic solvent tolerance levels of these microorganisms were maintained when the oxygen concentration was kept high. The tolerance levels were maintained also when these organisms were grown with nitrate present under anaerobic respiratory conditions. Received: 21 March, 1997 / Accepted: July 20, 1997     

On-line monitoring of important organoleptic methyl-branched aldehydes during batch fermentation of starter culture Staphylococcus xylosus reveal new insight into their production in a model fermentation

A small fermentor (55 mL) was directly interfaced to a membrane inlet mass spectrometer for continuous on-line monitoring of oxygen and volatile metabolites during batch fermentations of the starter culture Staphylococcus xylosus. Using this technique, we were able to correlate production of the very important flavor compounds 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal with various growth conditions. We found that the aldehydes were present in the culture broth only as transient metabolites. They were produced in the exponential growth phase, reached a maximum concentration when the culture became anaerobic, and then they rapidly disappeared from the culture medium. This general pattern was observed for three different strains of S. xylosus and S. carnosus. Small amounts of inoculum or increased exposure to oxygen were found to favor production of the aldehydes as a result of a longer aerobic growth period. Growing S. xylosus under conditions resembling those in a fermented sausage revealed that NaCl (5%) increased aldehyde production considerably, whereas KNO(3) (0.03%) or NaNO(2) (0.03%) had little effect. A lowering of pH from 7.2 to 6.0 reduced cell density, but had a minor affect on aldehyde production.     

Influence of glucose and oxygen on the production of ethyl acetate and isoamyl acetate by a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain during alcoholic fermentation

The effect of glucose and dissolved oxygen in a synthetic medium simulating the standard composition of grape juice on the production of ethyl acetate and isoamyl acetate by a Saccharomyces cerevisiae strain during alcoholic fermentation was studied. The specific in vitro activity of alcohol acetyltransferase (AATase, EC 2.3.1.84) and esterases (ESase, EC 3.1.1.1; hydrolysis and synthesis of esters) in cell-free extracts was also examined. The specific activity of AATase for ethyl acetate was found to peak at the beginning of the exponential growth phase and that for isoamyl acetate at its end. While the glucose concentration only affected the maximum specific activity of AATase, and only slightly, oxygen inhibited such activity, to a greater extent for isoamyl acetate than for ethyl acetate. On the other hand, esterases were found to catalyse the synthesis of ethyl acetate only at a low or medium glucose concentration (50 or 100 g l^-1, respectively), and to reach their maximum hydrolytic activity on isoamyl acetate during the stationary growth phase. The highest ethyl acetate and isoamyl acetate concentrations in the medium were obtained with a glucose concentration of 250 g l^-1 and semianaerobic conditions.     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Production of volatile organic compounds by the yeast fungus Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically in a synthetic nutrient solution and the volatile compounds produced were concentrated. Identification of the volatiles was performed by combined gas chromatographymass spectrometry or by one of these methods. The compounds identified were 11 esters, 9 alcohols, 5 acids and 3 carbonyls.The time course production of volatile neutral compounds was followed. During the phase of no apparent growth only a few substances were formed (mostly alcohols). The rapid phase of growth was characterized by an intense synthesis of many compounds in relatively high concentrations and later a sudden decrease in the number and amounts of substances. A slow successive, decline in the number and amounts of volatile components was observed during the phase of no net growth.The volatiles emitted by the fungus were concentrated, when most of the compounds were most abundant and the relative amounts of the major volatile neutral compounds were determined. The main components were ethyl acetate, ethyl propionate and ethanol.     

Degradation of Benzene,Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene

This study concerns the potential use of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene for the degradation of important harmful aromatic compounds such as benzene, toluene, and xylene (BTX). The use of these compounds by both strains was determined as the production of cell mass (viable cell number) in a minimal medium containing any one of the BTX compounds as the sole carbon and energy source. Furthermore, the BTX degradation capability of both strains was monitored by measuring the production of 3‐methylcatechol, a common intermediate. For the cells of the logarithmic phase, which were grown at high aeration/high agitation or low aeration/low agitation, the engineered strain showed a better growth rate than the host strain. With the benzene in the medium, the recombinant strain exhibited a higher (up to 4‐fold) cell density than the parental wild‐type strain at this phase. In contrast, regarding the cells of the late stationary phase under high aeration/high agitation conditions, the host strain had generally higher viable cell numbers than the recombinant strain. At this phase this difference was, however, less significant under the conditions of low aeration/low agitation. Similarly, in toluene containing medium (at high aeration/high agitation) the recombinant strain showed a higher cell density which was from a 15‐fold to almost one order of magnitude greater than its parental strain during the logarithmic phase where the cell density of P. aeruginosa remained nearly constant. Contrary to the results with benzene and toluene, both strains exhibited similar growth characteristics when they were grown in the presence of xylene. The positive effect of the oxygen uptake by the recombinant system on the BTX metabolizing activity was also apparent in a high accumulation of 3‐methylcatechol in the cultures of the recombinant strain. At certain points of incubation, the hemoglobin expressing strain showed a significantly (p < 0.05) higher 3‐methylcatechol accumulation than the host strain. These results demonstrated the possible potential of the Vitreoscilla hemoglobin as an efficient oxygen uptake system for the bioremediation of some compounds of environmental concern.     

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.     

Regulation of formation of proteases inBacillus megaterium

Protease was formed only at the end of the growth phase and its synthesis continued in the stationary phase during the growth of the sporulatingBacillus megaterium KM Sp⁺ in complex media with amino acids or peptone and glucose. The enzyme was also not formed during the growth phase in the glucose containing mineral medium and was detected only later during the stationary phase, smaller quantities being observed than those formed in the complex medium. The addition of glucose at the beginning of the synthesis of protease inhibited the production of the enzyme. On the other hand, the addition of the mixture of amino acids under the same conditions stimulated its formation several fold. Cysteine blocked the synthesis of the enzyme unlike other amino acids. Within a certain range the stimulatory effect of amino acids was related to their concentration, being manifested only after a lag period of several hours. The ability to form protease dissappeared after the formation of refractile spores in sporangial cells. Preliminary communication published in Biochem. biophys. Res. Commun. 37: 233, 1969.     

Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression

In the present study, a food-borne pathogen strain of Bacillus cereus (F4430/73) was anaerobically grown in controlled-batch conditions under low initial oxidoreduction potential (ORP=–148 mV) using hydrogen gas as reducing agent. Its physiological characteristics, including growth, glucose fermentation capacity and enterotoxin production, were compared with anaerobic conditions generated by nitrogen gas (ORP=+ 45 mV). The results showed that low ORP affected growth mainly during the early stages. Maximal specific rates of growth and glucose consumption were reduced, and drastic changes in time profiles of fermentation product concentration were observed. Production of lactate was promoted at the expense of acetate. Nevertheless, low ORP did not affect final biomass yield. Under both ORP conditions, Non-haemolytic enterotoxin (Nhe) was produced early during the exponential growth phase as a first enterotoxin and Haemolysin BL (Hbl) later during the early stationary growth phase as a second enterotoxin. The major effect of low ORP was the strong stimulation of Hbl production and, to a lesser extent, Nhe production. This control was complex, involving different levels of regulation. We discussed the regulation of enterotoxin expression and the involvement of the pleiotropic regulator PlcR.     

Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria.

The stringent halobacterial strain Haloferax volcanii was subjected to a set of physiological conditions different from amino acid starvation that are known to cause production of guanosine polyphosphates [(p)pp Gpp] in eubacteria via the relA-independent (spoT) pathway. The conditions used were temperature upshift, treatment with cyanide, and total starvation. Under none of these conditions were detectable levels of (p)ppGpp observed. This result, in conjunction with our previous finding that (p)ppGpp synthesis does not occur under amino acid starvation, leads to the conclusion that in halobacteria both growth rate control and stringency are probably governed by mechanisms that operate in the absence of ppGpp. During exponential growth, a low level of phosphorylated compounds with electrophoretic mobilities similar, but not identical, to that of (p)ppGpp were observed. The intracellular concentration of these compounds increased considerably during the stationary phase of growth and with all of the treatments used. The compounds were identified as short-chain polyphosphates identical to those found under similar conditions in Saccharomyces cerevisiae.     

Growth kinetics under adenine-limiting conditions of Bacillus subtilis KYA 741, an adenine-requiring strain

The growth kinetics of Bacillus subtilis KYA 741, an adenine-requiring strain, was investigated under adenine-limiting conditions. The concentration of adenine (the limiting substrate for cell growth) in the culture filtrate remained constant during the stationary phase. In this phase, DNA turnover was active and the DNA content per cell was constant throughout the cultivation period. When cells were transferred to medium without adenine, the cell concentration began to decrease immediately and then reached a constant level due to the supply of adenine from lysing to growing cells. The rates of degradation of cells and DNA were both found to be 0.2 hr^?1. An equation for cell growth in this pseudostationary phase was obtained by combining Contois' equation, in which the apparent saturation constant was a function of the cell concentration, with a term for cell degradation. This equation satisfactorily expressed the feature of cell growth and adenine consumption by B. subtilis KYA 741 under adenine-limiting conditions.     

Induction of Hsp104 synthesis in Saccharomyces cerevisiae in the stationary growth phase is inhibited by the petite mutation

The elevation of Hsp104 (heat shock protein) content under heat stress plays a key role in the development of thermotolerance in yeast (Saccharomyces cerevisiae) cells. Hsp104 synthesis is increased under heat stress and in the stationary growth phase. The loss of mitochondrial DNA (petite mutation) was shown to inhibit the induction of Hsp104 synthesis under heat stress (39°C) and during the transition to the stationary growth phase. Also, the petite mutation suppressed the increase in activity of antioxidant enzymes in the stationary phase, which accompanied by decrease in thermotolerance. At the same time, mutation inhibited production of reactive oxygen species and prevented cell death under heat shock in the logarithmic growth phase. The results of this study suggest that disruption of the mitochondrial functional state suppresses the expression of yeast nuclear genes upon upon entry into the stationary growth phase.     

Volatile Ketone Formation in Bacteria: Release of 3-Oxopentanoate by Soil Pseudomonads During Growth on Heptanoate

During enrichments to search for soil bacteria that form the volatile ketones, acetone and butanone, we isolated pure cultures of Pseudomonas aureofaciens, P. fluorescens, and P. putida that excrete the β-keto-acid, 3-oxopentanoate (3-OPA), when grown on heptanoic acid as carbon source. Analysis of 3-OPA used enzymatic decarboxylation by acetoacetate decarboxylase to yield butanone, which was detected by headspace gas chromatography. The formation of 3-OPA was strongly dependent on heptanoic acid concentration, the level of oxygen, and the state of growth, and was not seen with even-chain or other odd-chain fatty acids. Uptake of 3-OPA during stationary phase of growth is probably related to polyhydroxyalkanoate (PHA) formation in these isolates. A model for formation and release of 3-OPA is proposed. Received: 28 August 2000 / Accepted: 2 October 2000     

ORGANIC EXCRETION BY DUNALIELLA TERTIOLECTA1

Excretion of C¹⁴-labeled organic material by Dunaliella tertiolecta was observed under a variety of conditions. In healthy cultures, percent excretion tints related to cell density bill independent of light and preconditioning of the medium. Exaction was lower in nutrient-limited culture than in an enriched medium, and in both cases was minimal during logarithmic growth. Increases in dissolved carbohydrate during stationary phase represented an accumulation of high molecular weight compounds. The general composition of labeled excretory products was similar in both actively growing and stationary cultures. The data suggest that excretion may involve different processes at high and low cell densities.     

Studies of fungal growth and intermediary carbon metabolism under steady and non-steady state conditions

The physiology of Aspergillus nidulans strain 224 has been studied under conditions of batch- and glucose-limited chemostat-culture and the effect of different steady state growth rates and dissolved oxygen tensions (DOT) examined. Measurements of the specific activities of selected glucose enzymes, the extent of oxygen uptake inhibition by glycolytic inhibitors, and radiorespirometric analyses were made in order to follow the variations in glucose catabolism, which occurred under these conditions. Greatly increased activity of the hexosemonophosphate (HMP) pathway was found during: (i) exponential growth of batch cultures; (ii) at near maximum specific growth rates (μ = 0.072 hr^?1) (DOT = 156 mm Hg); and (iii) at low DOT levels (<30 mm Hg) (μ = 0.050 hr^?1) in chemostat cultures. These changes in glucose eatabolism have been discussed in terms of the biosynthetic demands of the fungus under the influence of changing growth pressures. Preliminary studies also have been made of transition state behavior following stepwise alteration of the DOT. A new steady state was established after 4–5 culture doublings during which period an “overshoot” in HMP pathway activity occurred; these kinetics are indicative of a derepression of certain glucose enzymes. Low molecular weight phenols are synthesized during the exponential phase in batch cultures and these are further metabliized to a major secondary metabolite, melanin, at the onset of stationary phase conditions. The kinetics of tyrosinase production in steady state chemostats differs from those that might be predicted for an enzyme associated solely with secondary metabolism. A primary physiological role for this oxidase in Aspergillus nidulans has been postulated.