Batch Culture Conditions Research Articles

Stationary phase in gram-negative bacteria

Conditions that sustain constant bacterial growth are seldom found in nature. Oligotrophic environments and competition among microorganisms force bacteria to be able to adapt quickly to rough and changing situations. A particular lifestyle composed of continuous cycles of growth and starvation is commonly referred to as feast and famine. Bacteria have developed many different mechanisms to survive in nutrient-depleted and harsh environments, varying from producing a more resistant vegetative cell to complex developmental programmes. As a consequence of prolonged starvation, certain bacterial species enter a dynamic nonproliferative state in which continuous cycles of growth and death occur until 'better times' come (restoration of favourable growth conditions). In the laboratory, microbiologists approach famine situations using batch culture conditions. The entrance to the stationary phase is a very regulated process governed by the alternative sigma factor RpoS. Induction of RpoS changes the gene expression pattern, aiming to produce a more resistant cell. The study of stationary phase revealed very interesting phenomena such as the growth advantage in stationary phase phenotype. This review focuses on some of the interesting responses of gram-negative bacteria when they enter the fascinating world of stationary phase.

Methods for imaging Shewanella oneidensis MR-1 nanofilaments

Nanofilament production by Shewanella oneidensis MR-1 was evaluated as a function of lifestyle (planktonic vs. sessile) under aerobic and anaerobic conditions using different sample preparation techniques prior to imaging with scanning electron microscopy. Nanofilaments could be imaged on MR-1 cells grown in biofilms or planktonically under both aerobic and anaerobic batch culture conditions after fixation, critical point drying and coating with a conductive metal. Critical point drying was a requirement for imaging nanofilaments attached to planktonically grown MR-1 cells, but not for cells grown in a biofilm. Techniques described in this paper cannot be used to differentiate nanowires from pili or flagella.

Production and genetic improvement of a novel antimycotic agent, Saadamycin, against Dermatophytes and other clinical fungi from Endophytic Streptomyces sp. Hedaya48

As a part of our ongoing efforts towards finding novel antimycotic agents from marine microflora of the Red Sea, vanillin, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and the new antimycotic compound saadamycin were isolated from endophytic Streptomyces sp. Hedaya48. The producing strain was isolated from the Egyptian sponge Aplysina fistularis and subjected to different UV irradiation doses. A mutant strain Ah22 with 10.5-fold (420 mg/l as compared to 40 mg/l produced by the parental strain) improved saadamycin production was isolated. Production of saadamycin from mutant Ah22 was enhanced to 2.26-fold (950 mg/l) and 2.38-fold (1000 mg/l) under optimized culture conditions in batch culture and bioreactors, respectively. Both saadamycin and 5,7-dimethoxy-4-p-methoxylphenylcoumarin exhibited significant antimycotic activity against dermatophytes and other clinical fungi.

Bioprocessing of Human Glioblastoma Brain Cancer Tissue

Solid cancer tumors are thought to arise from aberrant stem cell populations, called cancer stem cells (CSCs). Hence, the development of effective cancer therapies may rely on developing methods that specifically target these cells. However, the scarcity of CSCs in vivo represents a major impediment to such research, as there is an insufficient supply for basic biochemical and genetic analyses. It is therefore necessary to develop methods to expand reproducibly CSC tissue in vitro in a controlled environment. To date, we have developed bioreactor protocols for the suspension culture of an aggressive and deadly type of brain cancer called glioblastoma multiforme (GBM). Human GBM-derived cells achieved a maximum cell density of 2.4 x 10(6) cells/mL after 24 days under high shear conditions in batch culture conditions. In comparison, fed-batch cultures achieved 4.5 x 10(6) cells/mL after 32 days. Characterization of bioreactor-expanded cells using both flow cytometry and a differentiation assay indicated that bioreactor-generated human GBM-derived cells have similar characteristics to the initial cell population and achieve >90% CD133 expression. Additionally, genomic characterization indicated that a very small number of key genes were differentially expressed in the bioreactor-expanded GBM-derived cells, thereby conserving the basic nature of the brain cancer tissue in the cell expansion process.

Evaluation of mycophenolic acid production by Penicillium brevicompactum MUCL 19011 in batch and continuous submerged cultures

Mycophenolic acid (MPA) is a secondary metabolite produced by Penicillium brevicompactum, which has antibiotic and immunosuppressive properties. In this study, production of MPA by P. brevicompactum MUCL 19011 in shake flask and a 2 L bench-scale stirred tank bioreactor under batch and continuous culture conditions was evaluated. The maximum concentration of MPA, its yield and rate of production in continuous culture were 1.46 g L −1, 29 mg g −1 glucose and 6.8 mg L −1 h −1, respectively. Growth kinetics in batch culture was investigated with five models of Monod, Moser, Verhulst, Contois and Exponential. The compatibility of the experimental data fitted well with the Contois model and the regression value was 0.98. The maximum specific growth rate and saturation constant were 0.07 h −1 and 0.61 g L −1, respectively. Glucose, biomass and MPA concentration profiles were evaluated during the course of fermentation. Under steady-state conditions, glucose consumption, biomass formation and MPA production fluxes were 9.2, 22.35 and 0.194 mmol L −1 h −1, respectively. In batch culture, enzymatically hydrolyzed casein and glycine increased MPA production by 165 and 112%, respectively.

The effect of temperature on the growth of Hantzchia amphioxys (Ehrenberg) grunow isolated from the Homa Lagoon, Izmir

The aim of this study was to determine the parameters of growth kinetics (μmax Ks), carrying capacity (Pchla μmaxi, Ksi) and temperature of the benthic diatom Hantzschia amphioxys (Ehrenberg) Grunow. In this research, the impacts of light, temperature and nutrients on the growth of H. amphioxys were investigated under continuous-light period and batch culture conditions. As a results, the optimum light 52 μmol/m2 s (saturasyon light),intensities were found as 18°C.

In vitro colonization of an experimental silicone by Candida albicans

Denture soft-lining materials are exposed to the oral cavity for long periods and are in continuous contact with saliva, subject to inhibition of liquid molecules, and susceptible to colonization by microorganisms. The opportunist yeast Candida albicans is of particular concern in this context, being associated with denture plaque and denture-related stomatitis. In this study, penetration of C. albicans into an experimental silicone elastomer soft-lining material was investigated under batch and continuous culture conditions. A model "denture plaque" microcosm was also used. Increasing the filler concentration within the elastomer decreased penetration under both batch and continuous culture conditions. In continuous culture, C. albicans penetration was significantly greater in pure culture than in the presence of bacteria after 72-h incubation (p < 0.05).

Growth and toxin production in the ciguatera-causing dinoflagellate Gambierdiscus polynesiensis (Dinophyceae) in culture

The growth and toxin production in a clonal strain of Gambierdiscus polynesiensis, TB-92, was examined in batch culture conditions. The mean growth rate at exponential phase was (0.13 ± 0.03) division day −1. Regardless of the age of cultures, all mice injected with dichloromethanolic and methanolic extracts showed symptoms specific to ciguatoxin (CTX) and maitotoxin (MTX) bioactivity, respectively. The highest total toxicity assessed in TB-92 cultures was 10.4 × 10 −4 mouse unit cell −1. The toxin production pattern reveals an enhanced cellular toxin content with the age of the culture. CTX- and MTX-like compounds each accounted for approx. 50% of the total toxicity of TB-92 cultures, except in aged cells where CTXs were dominant. The high ciguatoxic activity of TB-92 was further confirmed in dichloromethanolic extracts by means of the receptor-binding assay. The highest CTX level monitored at late stationary phase was (11.9 ± 0.4) pg P-CTX-3C equiv cell −1. Further HPLC and LC-MS analysis revealed the presence of five CTXs congeners in lipid-soluble extracts, i.e. CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C, and of new CTX congeners. Toxin composition comparison between two G. polynesiensis strains suggests that the toxin profile is a stable characteristic in this species. G. polynesiensis clones also proved inherently more toxic than other Gambierdiscus species isolated from other geographical areas.

Effects of light intensity and light quality on growth and circadian rhythm of saxitoxins production in Cylindrospermopsis raciborskii (Cyanobacteria)

Effects of light intensity and light quality on saxitoxin and neosaxitoxin production by Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju (strain T3) were investigated in batch culture conditions. Three light intensities (50, 100 and 150 μmol photons m−2 s−1) and three light qualities (white and blue 470 nm and red 630 nm) were used. Growth rates were higher under 100 and 150 when compared to 50 μmol photons m−2 s−1 (control condition). Saxitoxin (STX) and neosaxitoxin (NSTX) production showed an increase during the early stationary phase under 50 μmol photons m−2 s−1 (12 days). Higher concentrations of STX and NSTX were observed in cells under 100 μmol photons m−2 s−1. Production of these toxins showed a circadian rhythm synchronized with light:dark (L:D) cycle and also with a period of 24 h under the three light intensities tested. The circadian rhythm of STX and NSTX production under white light conditions was confirmed. Red light induced the loss of circadian rhythm in NSTX production. These data demonstrate a circadian rhythm in saxitoxins production modulated by light.

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species ( Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation; cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms caused by two species in the Yangtze River Estuary.

In vitroeffects of phosphatidylcholine andtransgalactooligosaccharides on the production of 1,2-sn-diacylglycerol byBifidobacterium longumbiovarinfantis

To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.

NMR bioreactor development for live in-situ microbial functional analysis

A live, in-situ metabolomics capability was developed for prokaryotic cultures under controlled growth conditions. Toward this goal, a radiofrequency-transparent bioreactor was developed and integrated with a commercial wide-bore nuclear magnetic resonance (NMR) imaging spectrometer and a commercial bioreactor controller. Water suppressed 1H NMR spectroscopy was used to monitor glucose and fructose utilization and byproduct excretion by Eubacterium aggregans (an anaerobic bacterial species relevant for biofuel production) under controlled batch and continuous culture conditions. The resulting metabolite profiles (short chain organic acids and ethanol) and trends are consistent with existing knowledge of its metabolism. However, our study also showed that E. aggregans produces lactate end product in significant concentrations—a result not previously reported. The advantages of live in-situ microbial metabolomics analysis and its complementariness with functional genomics/systems biology methods are discussed.

石炭灰造粒物を用いた沿岸底質環境改善材開発のための基礎的研究〜栄養塩溶出試験およびＳｋｅｌｅｔｏｎｅｍａ ｃｏｓｔａｔｕｍの増殖試験〜

The Characterization of cement-granulated coal ash was carried out for the purpose of remediation of organically rich sediment by controlling the nutrient balance of ecosystems. The granulated coal ash used in this study is composed of SiO2, CO3, Al2O3, CaO with quartz and aluminum silicate as the main phases. Moderate dissolutions of phosphate, silicate and calcium were observed. This may be useful for the neutralization of the acidified sediment and effective for controlling the balance of nutrients in coastal seas. Although the growth of the diatom S. costatum was inhibited under the batch culture condition owing to pH increase, such a pH increase would not occur under natural conditions because of magnesium precipitates and carbonate equilibrium.

Acid–base properties of cyanobacterial surfaces I: Influences of growth phase and nitrogen metabolism on cell surface reactivity

Significant efforts have been made to elucidate the chemical properties of bacterial surfaces for the purposes of refining surface complexation models that can account for their metal sorptive behavior under diverse conditions. However, the influence of culturing conditions on surface chemical parameters that are modeled from the potentiometric titration of bacterial surfaces has received little regard. While culture age and metabolic pathway have been considered as factors potentially influencing cell surface reactivity, statistical treatments have been incomplete and variability has remained unconfirmed. In this study, we employ potentiometric titrations to evaluate variations in bacterial surface ligand distributions using live cells of the sheathless cyanobacterium Anabaena sp. strain PCC 7120, grown under a variety of batch culture conditions. We evaluate the ability for a single set of modeled parameters, describing acid–base surface properties averaged over all culture conditions tested, to accurately account for the ligand distributions modeled for each individual culture condition. In addition to considering growth phase, we assess the role of the various assimilatory nitrogen metabolisms available to this organism as potential determinants of surface reactivity. We observe statistically significant variability in site distribution between the majority of conditions assessed. By employing post hoc Tukey–Kramer analysis for all possible pair-wise condition comparisons, we conclude that the average parameters are inadequate for the accurate chemical description of this cyanobacterial surface. It was determined that for this Gram-negative bacterium in batch culture, ligand distributions were influenced to a greater extent by nitrogen assimilation pathway than by growth phase.

In vitro study on gas generation and prebiotic effects of some carbohydrates and their mixtures

This study was carried out to examine the effect of inulin (IN), fructooligosaccharide (FOS), polydextrose (POL) and isomaltooligosaccharides (ISO), alone and in combination, on gas production, gas composition and prebiotic effects. Static batch culture fermentation was performed with faecal samples from three healthy volunteers to study the volume and composition of gas generated and changes in bacterial populations. Four carbohydrates alone or mixed with one another (50:50) were examined. Prebiotic index (PI) was calculated and used to compare the prebiotic effect. The high amount of gas produced by IN was reduced by mixing it with FOS. No reduction in gas generation was observed when POL and ISO mixed with other substrates. It was found that the mixture of IN and FOS was effective in reducing the amount of gas produced while augmenting or maintaining their potential to support the growth of bifidobacteria in faecal batch culture as the highest PI was achieved with FOS alone and a mixture of FOS and IN. It was also found that high volume of gas was generated in presence of POL and ISO and they had lower prebiotic effect. The results of this study imply that a mixture of prebiotics could prove effective in reducing the amount of gas generated by the gut microflora.

Frequency-dependent advantages of plasmid carriage by Pseudomonas in homogeneous and spatially structured environments

The conditions promoting the persistence of a plasmid carrying a trait that may be mutually beneficial to other cells in its vicinity were studied in structured and unstructured environments. A large plasmid encoding mercury resistance in Pseudomonas fluorescens was used, and the mercury concentration allowing invasion from rare for both plasmid-bearing and plasmid-free cells was determined for different initial inoculum densities in batch-culture structured (filter surface) and unstructured (mixed broth) environments. A range of mercury concentrations were found where both cell types could coexist, the regions being relatively similar in the two types of environment although density-dependent in the unstructured environment. The coexistence is explained in terms of frequency-dependent selection of the mutually beneficial mercury resistance trait, and the dynamics of bacterial growth under batch culture conditions. However, the region of coexistence was complicated by conjugation which increased plasmid spread in the mixed broth culture but not the structured environment.

Metabolism and biochemical pathway of n-butyl benzyl phthalate by Pseudomonas fluorescens B-1 isolated from a mangrove sediment

n-Butyl benzyl phthalate (BBP) is an endocrine-disrupting chemical. Biodegradation of BBP was investigated using the bacterium Pseudomonas fluorescens B-1 isolated from mangrove sediment of Mai Po Nature Reserve of Hong Kong. The microorganism was capable of utilizing BBP as the sole source of carbon and energy while BBP was degraded in 6 days under aerobic batch culture conditions. The optimum pH, temperature, and salinity for BBP degradation by P. fluorescens B-1 was found to be 7.0, 37 °C, and 15‰, respectively. Biodegradation of BBP was fitted to the first-order kinetics model. The process of BBP biodegradation was monitored by reversed-phase high-performance liquid chromatography with ultra-violet detection after solid-phase extraction. The major metabolites of BBP degradation were identified as mono-butyl phthalate, mono-benzyl phthalate, phthalic acid, and benzoic acid by gas chromatography-mass spectrometry. BBP-degrading activity of P. fluorescens B-1 was found mostly in the soluble fraction associated with the smaller fragments of cellular membranes. Results suggest that mineralization of BBP can be achieved by microorganism of the mangrove environment.

Effect of nutrient availability on the C, N, and P elemental ratios in the cyanobacterium Microcystis aeruginosa

To clarify whether nutrients limit the growth of Microcystis aeruginosa (Kutz.) Kutz during the growing season in Lake Yogo, we examined the cellular ratios of carbon (C), nitrogen (N), and phosphorus (P) in the populations of M. aeruginosa from August to December 2001. We also measured cellular C, N, and P ratios of M. aeruginosa under batch culture conditions. The cellular levels of N and P of M. aeruginosa in natural population changed more than twofold. The atomic N: C ratio of natural populations of Microcystis fluctuated from 0.11 to 0.26. The atomic P: C ratio fluctuated from 0.0080 to 0.024. The N: C, P: C, and N: P ratios of exponentially growing M. aeruginosa in N-and P-rich medium were 0.19, 0.013, and 15 on average. The growth of M. aeruginosa was suppressed below the N: C ratio of 0.13 under the N-free condition and below the P: C ratio of 0.0026 in the P-free condition. In the natural population, the N: C ratio was low on August 1-2 (0.11) and the P: C ratio was low (less than 0.011) until September. The Microcystis population on August 1-2 was N limited, judging from the results of the culture experiment. In other periods, the population seemed to be supplied with a sufficient amount of N. Although the P: C ratio was low (approximately 0.01) during August and September, it was several times larger than the value of the reduction of growth rate that occurred in culture. P limitation did not occur during the study period. N became more of a limiting factor than P for the formation of blooms of Microcystis. No blooms were observed in August and September, in spite of the increase of cellular levels of N. The formation of Microcystis blooms in Lake Yogo seems to be affected by artificial manipulations such as pumping from Lake Biwa and outflow.

A comparison of direct infusion MS and GC‐MS for metabolic footprinting of yeast mutants

Recent technical advances in mass spectrometry (MS) have propelled this technology to the forefront of methods employed in metabolome analysis. Here, we compare two distinct analytical approaches based on MS for their potential in revealing specific metabolic footprints of yeast single-deletion mutants. Filtered fermentation broth samples were analyzed by GC-MS and direct infusion ESI-MS. The potential of both methods in producing specific and, therefore, discriminant metabolite profiles was evaluated using samples from several yeast deletion mutants grown in batch-culture conditions with glucose as the carbon source. The mutants evaluated were cat8, gln3, ino2, opi1, and nil1, all with deletion of genes involved in nutrient sensing and regulation. From the analysis, we found that both methods can be used to classify mutants, but the classification depends on which metabolites are measured. Thus, the GC-MS method is good for classification of mutants with altered nitrogen regulation as it primarily measures amino acids, whereas this method cannot classify mutants involved in regulation of phospholipids metabolism as well as the direct infusion MS (DI-MS) method. From the analysis, we find that it is possible to discriminate the mutants in both the exponential and stationary growth phase, but the data from the exponential growth phase provide more physiological relevant information. Based on the data, we identified metabolites that are primarily involved in discrimination of the different mutants, and hereby providing a link between high-throughput metabolome analysis, strain classification, and physiology.

Screening of static culture and comparison of batch and continuous culture for the textile dye biological decolorization by Phanerochaete chrysosporium

The production of manganese dependent peroxidase (MnP) by Phanerochaete chrysosporium and the level of decolorization of 13 dyes were evaluated using static and agitated batch cultures and continuous cultures. A screening carried out under static conditions showed that the oxidative system has a certain affinity for azoic structures. For concentrations of 100 mg l-1 of Acid Black 1, Reactive Black 5, Reactive Orange 16 and Acid Red 27, decolorization percentages higher than 90% were obtained. In batch cultures with Acid Black 1 and Reactive Black 5 a significant increment in primary post-metabolism biomass was observed. For these last two dyes, it was possible to explore the response of the continuous system during 32 to 47 days, with concentrations between 25 to 400 mg l-1, obtaining decolorization percentages greater than 70% for 400 mg l-1.