Growth In Batch Culture Research Articles

Effect of growth hormone and γ-aminobutyric acid on Brachionus plicatilis (Rotifera) reproduction at low food or high ammonia levels

Abstract Growth hormone (GH, 0.0025 and 0.025 I.U. ml −1 ) and γ-aminobutyric acid (GABA, 50 μg ml −1 ) enhance rotifer population growth in batch cultures. In order to further understand the mechanism of their actions, we conducted experiments culturing isolated females at low food and high free ammonia levels. At an optimum food level of 7×10 6 Nannochloropsis oculata cells ml −1 or at low free ammonia level of 2.4 μg ml −1 , the F 1 offspring of rotifers treated with GH at 0.0025 I.U. ml −1 had significantly higher population growth rate ( r ) and net reproduction rate (Ro), and shorter generation time than untreated rotifers. At a lower food level of 7×10 5 cells ml −1 or at high free ammonia level of 3.1 μg ml −1 , rotifers treated with GABA at 50 μg ml −1 had significantly higher r and Ro, and shorter generation time. These results indicate that GABA is effective in enhancing rotifer reproduction when rotifers are cultured under stress whereas GH enhances rotifer reproduction when culture conditions are optimal. Significant effects were also observed in F 1 and F 2 generations which were not treated with hormones. These data may be useful for treating rotifer mass cultures to mitigate the effects of stress caused by high population densities.

An investigation of non-steady-state algal growth. II. Mathematical modelling of co-nutrient-limited algal growth

The ability of mathematical models to simulate competition for nutrients between three algal species, the diatom <it>Thalassiosira pseudonana</it>, a marine raphidophyte <it>Heterosigma carterae</it> and the dinoflagellate <it>Alexandrium minutum</it>, was investigated. Transient growth models were parameterized and tested using a number of closely controlled laboratory data sets including batch monocultures, batch competition experiments and semi-continuous culture competition experiments. The cell quota model of algal growth was found to be adequate to simulate growth of both the raphidophyte and the dinoflagellate. Batch monoculture data for diatom growth obtained under either nitrogen (N) or silicon (Si) limitation could also be simulated with a quota-style model, which in this case included feedback inhibition of nutrient uptake. However, to simulate both batch and semi-continuous culture experiments (and competition between the species), it was necessary to consider diatom Si-N metabolism. A model was derived which contains a representation of both intracellular N ad Si, and of the interaction of these nutrients within the cell. The model used a co-nutrient limitation based on the perceived functional and structural role of N and Si, respectively, within the cell. Simulations indicated that models capable of adequately representing monoculture growth in batch culture may produce erroneous results when incorporated into models of competition. The co-nutrient model is a first step to producing tractable algal growth models which will represent multiple nutrient stress in transient growth conditions.

Sugar Uptake and Storage by Sugarcane Suspension Cells at Different Temperatures and High Sugar Concentrations

Experiments with sugarcane cell suspensions were performed for two purposes: (a) To reveal the temperature dependence of sugar transport systems for interpretation of sugarcane growth at suboptimal temperatures; and (b) to test whether different temperatures or high sugar concentrations elevate the intracellular sucrose level by shifting the sucrose cycle of synthesis and degradation. It was found that the hexose uptake systems (for glucose and fructose) of suspension cells were strongly inhibited at low temperature (e.g. 15°), whereas high temperatures were much less harmful. The optimum temperature was 45 °. Hexose uptake was modulated by temperature and by development during the batch culture growth period. Neither the presence of high concentrations of hexoses, sucrose or osmoticum (mannitol) nor the growth at low or high temperature did increase the intracellular sucrose concentration. The best effect was obtained at high sucrose concentration in the medium, when the cells were grown in chemostat culture, but still no sucrose accumulation relative to the concentration in the medium occurred. Analysis of enzymes involved in sucrose metabolism showed that the correlation berween internal sucrose and sucrose phosphate synthase minus acid invertase, a correlation that had been observed in sugarcane plants, did not exist in sugarcane suspension cells. Also, neutral invertase activity did not give any correlation to sucrose. It is speculated that the low sucrose level in suspension cells and the absence of correlation to acid invertase is basically due to the low degree of vacuolization and the substantially faster sucrose transport at the tonoplast of suspension cells compared with internodal storage parenchyma cells.

Cultivation of the hyperthermophilic archaeonSulfolobus solfataricus in low-salt media

Two low-salt complex media, bactopeptone and desalted yeast extract, were used for high density cultivation of the hyperthermophilic archaeonSulfolobus solfataricus (DSM 1617). Bactopeptone, which has low mineral ion content among various complex media, was good for cell growth in batch cultures; the maximal cell density in bactopeptone was comparable to that in yeast extract. Howver, cell growth was rather poor when bactopeptone was added by the fed-batch procedure. Since several vitamins are deficient in bactopeptone, the effect of vitamins on cell growth was examined. Among the vitamins tested, pyridoxine was found to improve the growth rate ofS. solfataricus. To reduce the growth inhibition caused by mineral ions, yeast extract was dialyzed against distilled water and then fed-batch cultures were carried out using a feed medium containing desalted yeast extract. Although the concentrations of mineral ions in yeast extract were significantly lowered by the dialysis procedure, fed-batch cultivation with desalted yeast extract was unsatisfactory. To examine whether low molecular weight solutes in yeast extract are crucial for cell growth, we investigated the effect of trehalose, a most abundant compatible solute in yeast extract, on the growth pattern. Cell densities were increased and the length of the lag phase was markedly shortened by the presence of trehalose, indicating that trehalose plays an important role in the growth ofS. solfataricus.

Interactions between nitrate and ammonium in Emiliania huxleyi

The coccolithophorid Emiliania huxleyi was grown with ammonium and nitrate in “stretched-batch” culture. This provided a continuous input of fresh media at a dilution rate of 0.1 d −1 and maintained the derepression of N-assimilatory systems during increasing N-stress, while exhibiting batch culture growth dynamics. Estimates of Kt (half-saturation constant for transport) for both N nutrients were less than 0.4 μM, with a median of 0.2 μM. During the development of N-stress, the maximum rate of N-specific transport ( Vt max) for ammonium increased while that for nitrate more closely matched the concurrent rate of N-specific growth as N-stress developed. When both N nutrients were added together, respective Kt values showed no significant change, suggesting the operation of discrete nitrate and ammonium porters. In contrast, there were changes in Vt max when both nutrients were assimilated concurrently. However, higher concentrations of nitrate (4 μM) were required to achieve a 50% depression of ammonium Vt max compared to the concentration of ammonium (0.5 μM) required to bring about a similar depression of nitrate Vt max. This is consistent with the regulation of transport of both nutrients by an organic product of N-assimilation, which accumulates more rapidly following ammonium addition to N-stressed cells. Ammonium-grown cells contained higher concentrations of internal free amino acids, such as glutamine, than nitrate-grown cells. Interactions between ammonium and nitrate transport and assimilations could be simulated using the Ammonium–Nitrate Interaction Model, which contains constants for Kt and transports regulated by the level of the early product of N-assimilation, glutamine. There were no indications of a significant loss of N from the [dissolved inorganic N plus particulate organic N] fraction for exponentially growing Emiliania huxleyi in experiments, and there was no need to include such a process in the model for it to simulate the experiments.

Relationship between glycosyl hydrolase inventory and growth physiology of the hyperthermophile Pyrococcus furiosus on carbohydrate-based media.

Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several alpha-linked carbohydrates and cellobiose but not on glucose or other beta-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98 degrees C occurred on beta-1,3- and beta-1,3-beta-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of beta-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98 degrees C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism.

Hybridoma growth and productivity: effects of conditioned medium and of inoculum size.

Apart from gas concentrations, temperature, and pH, generally only the initial conditions can be manipulated in batch culture. Inoculum size and initial conditioned medium concentration represent two important considerations for optimal batch production. Two hybridoma cell lines were used to assess the impact of these initial conditions on population growth and monoclonal antibody productivity in suspension batch culture. Varying initial cell concentration over the range of 1.0 x 105 cells mL-1 to 3.0 x 105 cells mL-1 did not affect maximum product titre or maximum volumetric cell-hours attained. Initial percent of conditioned medium up to 40 percent strongly impacted on population growth and productivity, with initial levels of 30 to 40% conditioned medium reducing or eliminating lag phase and increasing average viable cell density. However, specific productivity and product titre declined with increasing initial percent conditioned medium, even on a per volume of fresh medium basis. Glutamine and glucose depletion or ammonia toxicity could cause depressed product titres when conditioned medium is used. Glutamine and glucose levels can easily be replenished in conditioned medium at minimal cost, and ammonia can be removed. Specific productivity was higher during cyclic batch operating mode than during batch operating mode. This may be because cyclic batch operating mode results in an incidental volume of conditioned medium at the beginning of each cycle. A two stage, cyclic-batch/batch operating mode can be employed to fully utilize medium and maximize product titre.

Growth and lactic acid production in batch culture of Lactobacillus rhamnosus in a defined medium

To facilitate metabolic analysis, batch fermentations of Lactobacillus rhamnosus were carried out in a new defined medium. Biomass at 10.5 g/l and lactic acid at 67 g/l with a Y-P/S of 0.84 were achieved. The maximum specific growth rate and the average productivity were 0.49/h and 2.48 g/l.h, respectively. These are comparable to those of this organism and related organisms in complex media. Preliminary amino acid studies were also conducted, highlighting the importance of serine, asparagine, glutamine and cysteine. Kinetic analysis revealed that lactic acid production was predominantly growth-associated with growth associated and non-growth associated lactic acid constants of 0.389 mol/g-cell and 0.0025 mol/g-cell.h, respectively. Finally a kinetic model has been included to describe the fermentation of L. rhamnosus.

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.

Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose.

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate-decarboxylase-negative (Pdc-) mutant lacking all three PDC genes exhibited a three-fold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc- strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc- and wild-type S. cerevisiae were identical. However, Pdc- S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0 center dot 10 h-1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.

A bacteriocin-mediated antagonism by Enterococcus faecium BC25 against ruminal Streptococcus bovis

A bacteriocin-like activity produced by Enterococcus faecium BC25, isolated from the rumen of a cow, was partially purified and characterized. The active substance was prepared by ammonium sulfate and chloroform/methanol precipitation of culture supernatant. The bacteriocin was a protein of molecular mass 17.5 kDa. Activity was inactivated by trypsin and proteinase K. The bacteriocin BC25 inhibited growth of amylolytic ruminal strains of Streptococcus bovis, including S. bovis AO 24185. Results showed that bacteriocine substance BC25 has a bacteriostatic effect when present in concentrations exceeding 125 AU/ml. Agar overlays and batch culture growth experiments proved that E. faecium BC25 was producing bacteriocin that inhibited the growth of S. bovis.

Dielectric spectroscopy in the cultivation of suspended and immobilized hybridoma cells

Dielectric spectroscopy, also referred to as capacitance measurement, was evaluated as a tool for on-line and real-time monitoring of hybridoma cell growth in suspension batch culture and in immobilized cell culture in porous glass carriers. The capacitance signal proved to be a lumped parameter influenced by cell concentration, cell size and culture conditions. During a batch culture the cell specific capacitance signal changes by about 45% having a maximum value at the maximum growth rate. An excellent correlation between the specific capacitance and the specific amount of nucleotidetriphosphates in the cells could be shown. Dielectric spectroscopy proved to be a useful tool for on-line monitoring of cell attachment and growth in open porous microcarriers in fluidized bed fermenters. Also, in this system only an approximate correlation with viable cell concentration appeared, whereas an exact correlation with the glutamine consumption rate, a measure of the metabolic activity of the cells, could be shown. This allowed a closed loop control of the medium feed rate, which was directly linked to the capacitance signal during the entire course of a continuous fermentation.

Investigation of three wastewaters entering Izmit Bay (Turkey) by means of batch and chemostat culture algal bioassays

The bay of Ízmit is the focus of much of Turkey's industrial development and three of the most important of the wastes discharged into its surface waters have been studied as a necessary step in minimising their environmental impact. Gas chromatography/mass spectrometry of the extracted wastes revealed the presence of organic xenobiotics listed as priority pollutants. Nevertheless, measurements of cell populations, chlorophyll fluorescence and the consumption of nutrients demonstrated that most of the wastes stimulated the growth of both batch and chemostat cultures of Phaeodactylum tricornutum (Bohlin). Experiments showed the growth of the cultures to be phosphate limited and it appears that the major effect of the wastes was due to their excessive loading of phosphate.

Evidence for the importance of catechol-type siderophores in the iron-limited growth of a cyanobacterium

To compensate for low levels of available iron, cyanobacteria may produce siderophores to assist in the scavenging of iron from the environment. In this paper we examine the role of catechol -type siderophores produced by the halotolerant cyanobacterium Synechococcus sp. PCC 7002 in the acquisition of iron from a chelated source. To inhibit catecholtype siderophore mediated iron transport, bovine serum albumin (BSA) was added to iron -deficient and replete cultures. Batch culture growth rates and cellular photosynthetic pigments decreased markedly in iron -limited populations in the presence of BSA, with no apparent decreases in growth rate in the iron-replete cultures. These results are supported by experiments with continuous culture chemostats where the addition of BSA to steady -state cultures leads to the washout of cells from low -iron chemostats, indicating that the cellular growth rate was reduced. The addition of BSA to short-term iron assimilation experiments further demonstrates that the presence of BSA can induce uptake kinetics consistent with the activity of an “iron -shuttle,” while BSA itself has no affinity for iron. These results demonstrate that catechol -type siderophores associated with the surface of the cell play an important role as “iron custodians.” While the presence of these catechols introduces complexity in the iron -transport mechanism and decreases the maximum velocity of iron uptake during episodic pulses of iron, the presence of the catechol associated with the cell surface functions to increase the overall cellular affinity for iron in low-iron environments.

Estimating growth kinetics of Penicillium chrysogenum by nonlinear regression

A simple unstructured microbial growth model was used to describe the dynamics of fungal growth in batch cultures. Biokinetic parameters were evaluated using the integral form of the Contois equation by nonlinear regression. Monte Carlo simulations and sensitivity analyses were performed to evaluate the effectiveness of the nonlinear parameter estimation technique for obtaining unique and accurate estimates of the Contois equation parameters. This technique was subsequently used to estimate growth kinetic parameters of Penicillium chrysogenum grown on glucose. Microbial growth and substrate depletion were accurately described by the Contois equation and the kinetic parameters were estimated as μ max=0.103 h −1, K c=0.145 and Y= 0.445. The applicability of the Contois equation to describe both pellet and filamentous fungal growth was verified using previously published data. The use of the integrated Contois equation is advantageous as biokinetic parameters can be estimated from a single substrate depletion curve.

Reduction of soluble iron and reductive dissolution of ferric iron-containing minerals by moderately thermophilic iron-oxidizing bacteria

Five moderately thermophilic iron-oxidizing bacteria, including representative strains of the three classified species (Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, and Acidimicrobium ferrooxidans), were shown to be capable of reducing ferric iron to ferrous iron when they were grown under oxygen limitation conditions. Iron reduction was most readily observed when the isolates were grown as mixotrophs or heterotrophs with glycerol as an electron donor; in addition, some strains were able to couple the oxidation of tetrathionate to the reduction of ferric iron. Cycling of iron between the ferrous and ferric states was observed during batch culture growth in unshaken flasks incubated under aerobic conditions, although the patterns of oxidoreduction of iron varied in different species of iron-oxidizing moderate thermophiles and in strains of a single species (S. acidophilus). All three bacterial species were able to grow anaerobically with ferric iron as a sole electron acceptor; the growth yields correlated with the amount of ferric iron reduced when the isolates were grown in the absence of oxygen. One of the moderate thermophiles (identified as a strain of S. acidophilus) was able to bring about the reductive dissolution of three ferric iron-containing minerals (ferric hydroxide, jarosite, and goethite) when it was grown under restricted aeration conditions with glycerol as a carbon and energy source. The significance of iron reduction by moderately thermophilic iron oxidizers in both environmental and applied contexts is discussed.

Bacterial-fungal biofilms in flowing water photo-processing tanks

A complex seven species model community, including bacteria and fungi, was selected from organisms isolated from the walls of an industrial flowing water system. Growth rates of the species were determined in single and mixed batch culture growth. The rates were found to be significantly higher in mixed culture for Pseudomonas alcaligenes and Flavobacterium indologenes and higher in single culture for Xanthomonas maltophilia, Rhodotorula glutinis and Fusarium solani, whereas no significant difference was recorded for Alcaligenes denitrificans and Fusarium oxysporum. All species attached to PVC in single and mixed culture to form biofilms. Xanthomonas maltophilia, Alc. denitrificans, Ps. alcaligenes and F. solani biofilm cell densities cm-2 were significantly higher than attachment of the component species in mixed culture. Statistical analyses showed a significant difference in rate of colonization between single and mixed cultures for some species. No significant difference was noted between mixed culture cell densities cm-2 at laminar flows of Reynolds number 2.7 and 5.4.

Fractionation of sulfur isotopes during thiosulfate reduction by desulfovibrio desulfuricans

Sulfur isotope fractionation during reduction of thiosulfate was investigated with growing batch cultures of Desulfovibrio desulfuricans CSN (DSM 9104) at 30 degreesC. The sulfide produced was depleted in 34S by 10 per thousand as compared to total thiosulfate sulfur. The depletion was equal to that during sulfate reduction under similar conditions. The two sulfur atoms of the thiosulfate molecule were affected differently by fractionation. Sulfide produced from sulfonate sulfur was depleted by 15.4 per thousand, sulfide produced from sulfane sulfur by 5.0 per thousand.

The Steady States of Microbial Growth on Mixtures of Substitutable Substrates in a Chemostat

Microbes growing on mixtures of substrates in a chemostat exhibit different substrate utilization patterns, depending on the dilution rate and feed concentrations. For instance, when supplied with high feed concentrations of a binary mixture, both substrates are consumed at low dilution rates, but only one of the substrates is consumed at high dilution rates. The goal of this work is to explain the onset of such transitions, which play a very significant role in ecology and bioengineering. In previous work, we formulated a mathematical model of mixed-substrate growth in batch cultures. We use the extension of this model to continuous cultures as the framework for understanding substrate utilization patterns in continuous cultures. Our explanation rests upon the existence of two special types of dilution rates predicted by the model. The first is the so-calledcriticaldilution rate at which the growth rate becomes zero, leading to cell washout. The existence of the critical dilution rate obtains from the simplest models of microbial growth, and is rooted in the fact that growth is inherently autocatalytic. The second type of special dilution rate, a unique feature of our model, stems from the recognition that synthesis of the enzymes catalysing the uptake of substrates is also autocatalytic. Hence, associated with each substrate is atransitiondilution rate at which the synthesis rate of the transport enzyme becomes zero. We show that: (1) the substrate utilization patterns in continuous cultures are completely determined by the relative magnitudes of the critical and transition dilution rates; and (2) the critical and transition dilution rates are in turn determined by the feed concentrations. This allows us to construct anoperating diagram, which yields the substrate utilization pattern for any given dilution rate and feed concentrations. The theory explains most of the mixed-substrate phenomena summarized in a recent review article by Egli (1995,Adv. Microbiol. Ecol.14,305–386).

Altered adherence properties of a Streptococcus gordonii hppA (oligopeptide permease) mutant result from transcriptional effects on cshA adhesin gene expression.

Cell-surface polypeptide CshA (259 kDa) mediates multiple adherence interactions of Streptococcus gordonii. By generating a chromosomal cshA promoter (p-cshA)-cat gene fusion and measuring both CAT enzyme activity and cat mRNA levels, it was shown that cshA is expressed maximally in cells in the late exponential phase of growth in batch culture. The expression of CAT enzyme activity from the p-cshA-cat promoter fusion was 28% decreased in early stationary phase cell extracts of mutant strain OB528 in which the hppA (oligopeptide-binding lipoprotein) gene was insertionally inactivated. This effect was correlated with proportionally reduced cell-surface expression of CshA protein and with impaired adherence of hppA mutant cells to cells of an oral Actinomyces naeslundii strain. cshA promoter activity was enhanced in streptococcal cells that were incubated in conditioned culture medium as opposed to fresh medium, but this did not occur in an hppA genetic background. It is suggested that HppA is necessary for the response of cells to an extracellular factor that modulates cshA transcription, and hence affects cell-surface CshA expression and streptococcal cell adherence properties.