Batch Culture In Medium Research Articles

Isolation, characterization and screening of actinomycetes associated with fijian ant-plant symbioses.

In the search for novel therapeutics to combat the ongoing antimicrobial resistance crisis, scientists are turning to underexplored environments. Defensive mutualisms between hymenopteran insects and actinomycetes represent important reservoirs for bioactive compounds. In this study, we examined the association between actinomycetes and Squamellaria ant-plants spanning three different ant and plant species combinations (Squamellaria imberbis-Philidris nagasau, Squamellaria tenuiflora- Technomyrmex vitiensis, and Squamellaria tenuiflora-Tetramorium insolens). Eight Squamellaria plants were sampled including four containing T. vitiensis, three containing P. nagasau, and a single plant containing T. insolens. A total of 47 actinomycetes were obtained from the sampled material, with 5, 16, and 26 isolates originating from cuticle, tissue, and nest samples, respectively. Cross-streaking tests showed that 12 out of 47 isolates inhibited bacterial pathogens. The most frequently inhibited pathogens in the cross-streaking tests were S. aureus and E. coli while S. enterica was the least inhibited. Among the three primary screening media used, ISP2 agar was the most suitable for secondary metabolism as more isolates exhibited antibacterial activity when grown on the medium. TFS2010 and TFS2003, which matched to Streptomyces gramineus (>99% similarity), were the most bioactive isolates in cross-streaking tests. TFS2010 displayed the strong antibacterial on Nutrient agar, Mueller Hinton agar, and ISP2 agar while TFS2003 only exhibited strong antibacterial activity on Nutrient agar. Furthermore, a difference in potency of extracts based on batch culture medium was noted in TFS2010. DNA was extracted from 19 isolates and followed by 16SrRNA gene sequencing. Analysis of sequence data revealed the presence of six genera, including Amycolatopsis, Asanoa, Jiangella, Nocardia, Nocardiopsis, and Streptomyces, with the latter being the most abundant taxon. Among these, three isolates (PNS3002, PNS3005, and TFS3001) are likely to represent new species while one (TFS2015) is likely to be a member of a novel genus. Our work represents the first attempt to study actinomycetes from Squamellaria-ant mutualisms.

The effect of nitrogen limitation on carbohydrates and β-glucan accumulation in Nannochloropsis oculata

Nitrogen (N) is the second most abundant element in microalgae biomass, following carbon. Microalgae have a high carbon and nitrogen metabolism system, making nitrogen limitation (N-limitation) a crucial factor that modifies their biochemical composition. In Nannochloropsis genus cultures, N-limitation can cause lipid or carbohydrate accumulation. This research aims to evaluate the effect of N-limitation on carbohydrate and β-glucan accumulation in Nannochloropsis oculata during maximum biomass production (i.e., finished logarithmic phase and early start stationary phase in batch culture) in the two-stage process context. To achieve this goal, we tested the effect of five levels of N availability (treatments 100, 75, 50, 25, and 0 % of N, concerning F medium) in batch culture using sodium nitrate (NaNO3) as the N source. The initial availability of N in each treatment was 1.7, 1.3, 0.8, 0.4, and 0 mmol∙l−1. The results showed that the highest concentration of N. oculata biomass, carbohydrate, and β-glucans content was produced during the logarithmic phase at 0.8 and 1.32 mmol∙l−1 N initial batch culture concentration (50 and 75 % N treatments), without a significant difference (P > 0.05) from the F medium used. Total carbohydrates obtained were 15.2 %, 19.7 %, 17.2 %, 12.7 %, and 14.2 % (dry weight) for the 100 %, 75 %, 50 %, 25 %, and 0 % N treatments, respectively. Similarly, the β-glucans in microalgal biomass were 5.0 %, 7.4 %, 6.7 %, 4.4 %, and 1.7 % (dry weight) for treatments 100 %, 75 %, 50 %, 25 %, and 0 % N, respectively. The 25 % and 0 % N treatments had a negative effect (P < 0.05) on cell population growth, carbohydrates, and β-glucans accumulation. Based on these results, we recommended conducting studies on the effect of N-limitation in N. oculata culture primarily at 50 % and 75 % N treatments, during the stationary phase and/or second stage of the two-stage process context, such as a chronic or and long-term effect, along with a partial supply of N or testing other inductors might also be explored. This knowledge can help to optimize the industrial production of microalgae β-glucans.

Highly efficient production of an influenza H9N2 vaccine using MDCK suspension cells

The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Statistical optimization of lipid production by the diatom Gyrosigma sp. grown in industrial wastewater

The marine diatom Gyrosigma sp. was cultured in a medium comprised of inorganic nutrients dissolved in palm oil mill effluent (POME) wastewater. The production of lipids in the biomass was optimized using a statistical design of experiments in combination with the response surface method. The experimental factors were incident light level and initial concentrations of nitrate, phosphate, and silicate in the medium in batch culture. At 25 ± 2 °C, the maximum lipid content in the biomass harvested at the end of a 12-day batch culture was 70.7 ± 6.0% by dry weight for the following values of the experimental factors: an incident light level of 131 μmol photons m−2 s−1, a nitrate concentration of 1.8 mg L−1 (29.0 μM), a phosphate concentration of 6.8 mg L−1 (71.6 μM), and a silicate concentration of 10.1 mg L−1 (132.7 μM). Under the optimized conditions, the maximum dry mass concentration of the diatom was 560 mg L−1 on day 8 of a batch culture, declining to ~409 mg L−1 on day 12. For the 12-day batch operation, the final average productivities of the biomass and the lipids were 34.1 ± 5.5 mg L−1 day−1 and 24.1 ± 0.2 mg L−1 day−1, respectively. The fatty acids in the algal lipids were found to be as follows (%, w/w of total lipids): palmitic acid (48.6%), eicosapentaenoic acid (10.6%), myristic acid (8.1%), stearic acid (8.0%), linoleic acid (7.5%), oleic acid (6.4%), and linolenic acid (5.8%). The response surface model predicted the lipid content in the biomass with a high degree of confidence.

Physical interactions between marine phytoplankton and PET plastics in seawater

Plastics are the most abundant marine debris globally dispersed in the oceans and its production is rising with documented negative impacts in marine ecosystems. However, the chemical-physical and biological interactions occurring between plastic and planktonic communities of different types of microorganisms are poorly understood. In these respects, it is of paramount importance to understand, on a molecular level on the surface, what happens to plastic fragments when dispersed in the ocean and directly interacting with phytoplankton assemblages. This study presents a computer-aided analysis of electron paramagnetic resonance (EPR) spectra of selected spin probes able to enter the phyoplanktonic cell interface and interact with the plastic surface. Two different marine phytoplankton species were analyzed, such as the diatom Skeletonema marinoi and dinoflagellate Lingulodinium polyedrum, in absence and presence of polyethylene terephthalate (PET) fragments in synthetic seawater (ASPM), in order to in-situ characterize the interactions occurring between the microalgal cells and plastic surfaces. The analysis was performed at increasing incubation times. The cellular growth and adhesion rates of microalgae in batch culture medium and on the plastic fragments were also evaluated. The data agreed with the EPR results, which showed a significant difference in terms of surface properties between the diatom and dinoflagellate species. Low-polar interactions of lipid aggregates with the plastic surface sites were mainly responsible for the cell-plastic adhesion by S. marinoi, which is exponentially growing on the plastic surface over the incubation time.

The Potential of Algae Spyrogyra peipingensis to Produce Nanogold in Batch Culture Medium

The biosynthesis of gold nanoparticles by using algae Spirogyra peipingensis was conducted. This research aimed to determine biosynthesis of gold nanoparticles by using algae Spirogyra peipingensis with different concentration and incubation time. The synthesis of gold nanoparticles using HauCl4 solution with variations of concentration 5 ppm, 10 ppm, 15 ppm and 20 ppm respectively in 1 liter of aquabidest. Algae Spirogyra peipingensis was grown in HauCl4 medium with the addition of 0.2 gr Sulfahri-01 nutrient. Then each 5 gram Spirogyra algae incubated in HAuCl4 medium with the addition of Sulfahri-01 0.2 gram nutrients. 4 hours long incubation under exposure to sunlight. Nanoparticle size determination is done by looking at the color that appears in the solution. From the results of the research, it is known that Spirogyra peipingensis algae is able to synthesize gold nanoparticles characterized by the color change in algae biomass from green to purple color after being treated and forming gold nanoparticles with size 40-60 NM. The best-used HAuCl4 consent is 5 ppm with the smallest particle size. KeywordIntracelullar, Spirogyra peipingensis,HAuCl4,Biosynthesis. Article HistoryReceived 8 May 2017 Accepted 3 July 2017

Symbiodinium-Induced Formation of Microbialites: Mechanistic Insights From in Vitro Experiments and the Prospect of Its Occurrence in Nature.

Dinoflagellates in the genus Symbiodinium exhibit a variety of life styles, ranging from mutualistic endosymbioses with animal and protist hosts to free-living life styles. In culture, Symbiodinium spp. and naturally associated bacteria are known to form calcifying biofilms that produce so-called symbiolites, i.e., aragonitic microbialites that incorporate Symbiodinium as endolithic cells. In this study, we investigated (i) how algal growth and the combined physiological activity of these bacterial-algal associations affect the physicochemical macroenvironment in culture and the microenvironment within bacterial-algal biofilms, and (ii) how these interactions induce the formation of symbiolites. In batch culture, calcification typically commenced when Symbiodinium spp. growth approached stationary phase and when photosynthetic activity and its influence on pH and the carbonate system of the culture medium had already subsided, indicating that symbiolite formation is not simply a function of photosynthetic activity in the bulk medium. Physical disturbance of bacteria-algal biofilms, via repeated detaching and dispersing of the developing biofilm, generally impeded symbiolite formation, suggesting that the structural integrity of biofilms plays an important role in generating conditions conducive to calcification. Microsensor measurements of pH and O2 revealed a biofilm microenvironment characterized by high photosynthetic rates and by dynamic changes in photosynthesis and respiration with light intensity and culture age. Ca2+ microsensor measurements confirmed the significance of the biofilm microenvironment in inducing calcification, as photosynthesis within the biofilm induced calcification without the influence of batch culture medium and under environmentally relevant flow conditions. Furthermore, first quantitative data on calcification from 26 calcifying cultures enabled a first broad comparison of Symbiodinium-induced bacterial-algal calcification with other calcification processes. Our findings support the idea that symbiolite formation is a typical, photosynthesis-induced, bacterial-algal calcification process that is likely to occur under natural conditions.

Effective role of medium supplementation in microalgal lipid accumulation.

The present study investigated the interaction between starch and lipid accumulation in a green microalgae enrichment culture. The objective was to optimize the lipid content by manipulation of the medium in regular batch culture. Two medium designs were evaluated: First a high ortho-P concentration with vitamin supplementary (Pi-vitamins supplemented medium), second normal growth medium (control). Both media contained a low amount of nitrogen which was consumed during batch growth in three days. The batch experiments continued for another 4 days with the absence of soluble nitrogen in the medium. When the mixed microalgal culture was incubated in the Pi-vitamin supplemented medium, the lipid, and starch content of the culture increased within the first 3 days to 102.0 ± 5.2 mg/L (12.7 ± 0.6% of DW) and 31.7 ± 1.6 mg/L (4.0 ± 0.2% of DW), respectively. On the last day of the experiment, the lipid, and starch content in Pi-vitamin medium increased to 663.1 ± 32.5 mg/L (33.4 ± 1.6% of DW) and 127.5 ± 5.2 mg/L (6.4 ± 0.3% of DW). However, the lipid and starch content in the control process, reached to 334.7 ± 16.4 mg/L (20.1 ± 1.0% of DW) and 94.3 ± 4.6 mg/L (5.7 ± 0.3% of DW), respectively. The high Pi-vitamin medium induced storing lipid formation clearly while the starch formation was not affected. The lipid contents reported here are among the high reported in the literature, note that already under full growth conditions significant lipid levels occurred in the algal enrichment culture. The high lipid productivity of the reported mixed microalgae culture provides an efficient route for efficient algal biodiesel production.

Study on bioremediation of Lead by exopolysaccharide producing metallophilic bacterium isolated from extreme habitat

Lead released from manufacturing factories, recycling plants, automobile company and landfill leachate is abundantly found in wastewater. An efficient bioremediating agent for lead removal from wastewater is expected to ease the ever increasing problem. The present study reports Pseudomonas sp. W6 isolated from extreme habitat of hot water spring of North–East India evaluated for its Lead biosorption property. The bacterium showed capacity to resist 1.0mM lead in both solid and liquid minimal media. Epifluorescence microscopy reveal the viability of bacterial cells under metal stress condition. ICP-MS analysis revealed 65% and 61.2% removal of lead from the Synthetic Bangladesh Ground Water medium in batch culture and column study respectively which was higher when compared to biosorption capacity of P. aeruginosa MTCC2474, P. alcaligenes MJ7 from forest soil and P. ficuserectae PKRS11 from uranium rich soil. Exopolysaccharide released by the isolate which influenced biosorption revealed the presence of ligands assayed using microbial hydrophobicity and FTIR. The extremophilic isolate is proposed as a choice for efficient bioremediation of lead contaminated wastewater.

Growth, nutrient uptake and chemical composition of Chlorella sp. and Nannochloropsis oculata under nitrogen starvation

The production of microalgae biomass shows wide valuable uses, in the aquaculture, biotechnology, and food science, among others. However, microalgae show fluctuations in their chemical profile generated mainly by the culture conditions. This study was designated to assess the effects of nitrogen starvation on growth, nutrient uptake, and gross chemical composition of Chlorella sp. and Nannochloropsis oculata. The control experiments were performed with Conway culture medium in 13-day batch cultures, 12 h photoperiod, and aeration. A second experimental condition was the addition of the nutrients except nitrogen, one week after the start of growth (experiments designed as N-). Cell yield were similar in the control and in the N- experiments for both species. Cell biovolumes did not vary over growth in the control, but both microalgae exhibited larger cell biovolumes in N- experiments, probably as a consequence of the higher accumulation of storage substances. Dissolved nitrogen was exhausted before the end of the experiments, but phosphorus was not totally consumed. Protein and total carotenoid did not vary from the exponential to the stationary growth phase of the control in both species. For Chlorella sp., concentrations of lipid did not vary in the control either, but there was a significant increase of carbohydrate over growth. In the N- experiment, concentrations of all substances varied throughout growth of Chlorella sp., except lipid. For N. oculata , all substances exhibited significant variations over growth, except protein and total carotenoid in the control. Protein and chlorophyll-a concentrations decreased over growth in N- experiments for both species. In contrast, concentrations of carbohydrate increased throughout growth in N- experiments, especially in Chlorella sp. Nitrogen starvation caused accumulation of carbohydrate, but increments of lipid were restricted to N. oculata . Both species showed a fast growth, but the small content of lipid in Chlorella sp. is unfavorable for its use as a food-species in a monospecific diet in mariculture, and as a feedstock for biodiesel production. N. oculata is a lipid-rich species, and its lipid content can be successfully incremented through nitrogen starvation. This species is promising in uses that demand high concentrations of lipid, such as the production of biodiesel.

Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions

Due to their remarkable resistance to extreme conditions, Deinococcaceae strains are of great interest to biotechnological prospects. However, the physiology of the extremophile strain Deinococcus geothermalis has scarcely been studied and is not well understood. The physiological behaviour was then studied in well-controlled conditions in flask and bioreactor cultures. The growth of D. geothermalis type strains was compared. Among the strains tested, the strain from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen DSM) DSM-11302 was found to give the highest biomass concentration and growth rate: in a complex medium with glucose, the growth rate reached 0.75 h−1 at 45 °C. Yeast extract concentration in the medium had significant constitutive and catalytic effects. Furthermore, the results showed that the physiological descriptors were not affected by the inoculum preparation steps. A batch culture of D. geothermalis DSM-11302 on defined medium was carried out: cells grew exponentially with a maximal growth rate of 0.28 h−1 and D. geothermalis DSM-11302 biomass reached 1.4 g·L−1 in 20 h. Then, 1.4 gDryCellWeight of biomass (X) was obtained from 5.6 g glucose (Glc) consumed as carbon source, corresponding to a yield of 0.3 CmolX·CmolGlc−1; cell specific oxygen uptake and carbon dioxide production rates reached 216 and 226 mmol.CmolX−1·h−1, respectively, and the respiratory quotient (QR) value varied from 1.1 to 1.7. This is the first time that kinetic parameters and yields are reported for D. geothermalis DSM-11302 grown on a mineral medium in well-controlled batch culture.

An assessment of the usefulness of the cyanobacterium Synechococcus subsalsus as a source of biomass for biofuel production

Nowadays algal biofuels are considered one of the most promising solutions of global energy crisis and climate change for the years to come. By manipulation of the culture conditions, many algal species can be induced to accumulate high concentrations of particular biomolecules and can be directed to the desired output for each fuel. In this context, the present study involved the assessment of the effects of CO 2 availability and nitrogen starvation on growth and chemical composition of the cyanobacterium Synechococcus subsalsus , testing a fast-growing native strain. The control experiments were performed with Conway culture medium in 12-day batch cultures, in 6-liter flasks and 12 h photoperiod, with addition of 2 L min -1 filtered air to each flask. Other two experimental conditions were also tested: (i) the placement into the cultures of additional dissolved nutrients except nitrogen, one week after the start of growth (N-), and (ii) the input of pure CO 2 into the flasks from the 5th day of growth (C+). In all cultures, daily cell counts were done throughout the cultivation, as well as measurements of pH and cell biovolumes. Maximum cell yield were found in N-experiments, while cell yields of C+ and control were similar. Dissolved nitrogen was exhausted before the end of the experiments, but dissolved phosphorus was not totally consumed. Protein and chlorophyll-a concentrations decreased from the exponential to the stationary growth phase of all experiments, except for protein in the control. In all experiments, carbohydrate, lipid and total carotenoid increased from the exponential to the stationary growth phase, as an effect of nitrogen limitation. Increments in carbohydrate concentrations were remarkable, achieving more than 42% of the dry weight (dw), but concentrations of lipid were always lower than 13% dw. The addition of pure CO 2 did not cause a significant increase in biomass of S. subsalsus nor generated more lipid and carbohydrate than the other treatments. Nitrogen starvation caused an intense accumulation of carbohydrate, but the increments of lipid were small. Despite the fast growth, the cyanobacterium S. subsalsus has a virtually null potential for biodiesel production, given its low lipid concentrations. The high concentrations of carbohydrate combined with fast growth point to the potential use of this species as raw material for other possible biotechnological processes, after a demonstration of technical and economic viability.

Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley β-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3-O-β-cellotriosyl-d-glucose [LDP4] or 4-O-β-laminaribiosyl-d-cellobiose [CDP4]) present in β-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis, whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3-O-β-cellotriosyl-d-glucose released by β-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis.

The yield of experimental yeast populations declines during selection

The trade-off between growth rate and yield can limit population productivity. Here we tested for this life-history trade-off in replicate haploid and diploid populations of Saccharomyces cerevisiae propagated in glucose-limited medium in batch cultures for 5000 generations. The yield of single clones isolated from the haploid lineages, measured as both optical and population density at the end of a growth cycle, declined during selection and was negatively correlated with growth rate. Initially, diploid populations did not pay this cost of adaptation but haploidized after about 1000-3000 generations of selection, and this ploidy transition was associated with a decline in yield caused by reduced cell size. These results demonstrate the experimental evolution of a trade-off between growth rate and yield, caused by antagonistic pleiotropy, during adaptation in haploids and after an adaptive transition from diploidy to haploidy.

In glucose-limited continuous culture the minimum substrate concentration for growth, smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii, an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ(max) and K(s) were determined for growth with glucose. For both strains, μ(max) was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ(max) was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h(-1) C. heintzii grew immediately with a μ(max) of 0.17 ± 0.03 h(-1). After five transfers in batch culture, μ(max) had increased only slightly to 0.18 ± 0.03 h(-1). A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D = 0.075 h(-1) into glucose-excess batch medium E. coli grew only after an acceleration phase of ~3.5 h with a μ(max) of 0.52 h(-1). After 120 generations and several transfers into fresh medium, μ(max) had increased to 0.80 ± 0.03 h(-1). For long-term adapted chemostat-cultivated cells, a K(s) for glucose of 15 μg l(-1) for C. heintzii, and of 35 μg l(-1) for E. coli, respectively, was determined in (14)C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h(-1). Using the determined pure culture parameter values for K(s) and μ(max), it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s(min)). The values estimated for s(min) were dependent on growth rate; at D = 0.05 h(-1), it was 12.6 and 0 μg l(-1) for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h(-1), s(min) for E. coli had to be raised to 34.9 μg l(-1) whereas s(min) for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K(s) value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s(min).

Variations in exopolysaccharide production by Rhizobium tropici

Rhizobium tropici, a legume-symbiont soil bacterium, is known for its copious production of exopolysaccharide (EPS). Many aspects of this organism's growth and EPS production, however, remain uncharacterized, including the influence of environment and culturing conditions upon EPS. Here, we demonstrate that R. tropici EPS chemical composition and yield differ when grown with different substrates in a defined minimal medium in batch culture. Exopolysaccharide was quantified from R. tropici grown using arabinose, glucose, sucrose, mannitol, fructose, or glutamate as a sole carbon source. All tested substrates produced plenteous amounts of exopolysaccharide material. Variations in pH and carbon-to-nitrogen (C/N) ratio also resulted in assorted cell growth and exopolysaccharide production differences. We found that optimizing the C/N ratio has a greater impact upon R. tropici EPS production than upon R. tropici growth. A maximum EPS yield of 4.08g/L was realized under optimized conditions, which is large even in comparison with other known rhizobia. We provide evidence that the chemical composition of R. tropici EPS can vary with changes to the growth environment. The composition of glucose-grown EPS contained rhamnose-linked residues that were not present in arabinose-grown EPS.

Study of the growth of Escherichia coli on mixed substrates using dynamic flux balance analysis

Escherichia coli produces acetate as the major by-product when grown in a batch culture medium of glucose under aerobic conditions. The secreted acetate can be used as an additional substrate for cell growth leading to a mixed-substrate growth pattern of diauxic growth. However the acetate formed can limit growth of the organism if found in high amounts in the culture medium and such a scenario necessitates the study of the growth of microorganisms on mixed substrates. Dynamic flux balance analysis has turned out to be a promising tool in capturing such scenarios of growth on mixed substrates. In this study we carry out a dynamic flux balance analysis study of the growth of Escherichia coli in a batch culture medium of glucose and acetate where the organism encounters the presence of mixed substrates. Additionally we have used a detailed metabolic network to describe the process of metabolism in the organism Escherichia coli. DFBA combined with such detailed metabolic networks can open up possibilities of preventing such harmful growth scenarios for biotechnologically useful micro-organisms.

Monitoring of sorbitol in Pichia pastoris cultivation applying sequential injection analysis

A simple and robust analyser system based on sequential injection analysis for the determination of sorbitol in Pichia pastoris Mut S phenotype batch fermentation is described. Applicability for the bioprocess monitoring of sorbitol in batch culture medium in a concentration range of 8–1.5 g l −1 with satisfying sorbitol recovery was demonstrated during 60 h. Optimization of physical and chemical parameters is further described. The method proved to be reproducible (R.S.D. < 3%), robust, and fast (17 injections per hour). The SIA sorbitol analyzer showed to be applicable for the monitoring of sorbitol concentration in a mixed substrate P. pastoris fermentation strategy. No interference of methanol was observed. The method is suited for the implementation of sorbitol concentration closed-loop control strategies and for the study of the effect of sorbitol concentration in heterologous protein productivity.

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.

Application of sodium propionate to the suspension culture of Chinese hamster ovary cells for enhanced production of follicle-stimulating hormone

In this study, we found that adding sodium propionate to batch culture medium resulted in cell growth suppression in a dosedependent manner, while it enhanced follicle-stimulating hormone (FSH) production in Chinese hamster ovary (CHO) cells. In a pseudo-perfusion culture, with the exchange of fresh medium containing 2.5 mM sodium propionate, a final FSH of 532 μg was obtained, which was approximately 1.5-fold higher than the control. Overall, the results demonstrate that the application of sodium proplonate for the commercial production of recombinant proteins in rCHO cells is feasible.