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Batch Culture (batch + culture)
Selected AbstractsCARBON SOURCES AND THEIR EFFECT ON GROWTH, ACETIC ACID AND ETHANOL PRODUCTION BY BRETTANOMYCES BRUXELLENSIS IN BATCH CULTUREJOURNAL OF FOOD PROCESS ENGINEERING, Issue 1 2007M.G. AGUILAR USCANGA ABSTRACT The influence of available low-cost carbohydrates as carbon sources on Brettanomyces bruxellensis growth, acetic acid and ethanol production was studied in order to ascertain the viability of this yeast to eventually become an industrial acetic acid producer. Six different raw materials were included as carbon sources (glucose, sugarcane molasses, refined cane sugar, pineapple, sugarcane and beet juices). B. bruxellensis develops in a complex culture medium like plant juices and sugarcane molasses better than in a medium with a simple carbohydrate such as glucose. The maximum acid acetic yield (0.24 g/g) and productivity (0.14 g/L/h) were attained in tests carried out with sugarcane molasses containing 60 g/L sucrose. The strain produced low levels of ethanol in a refined sugarcane medium, but was able to produce a substantial quantity of acetic acid (13 g/L). [source] The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009A.A.T. De Carvalho Abstract Aims:, To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5. Methods and Results:, Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l,1 of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l,1, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation. Conclusions:,Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem. Significance and Impact of the Study:, The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria. [source] A novel finding that Streptomyces clavuligerus can produce the antibiotic clavulanic acid using olive oil as a sole carbon sourceJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2008G. Efthimiou Abstract Aims:, This study aims to establish whether commercially available food oils can be used by Streptomyces clavuligerus as sole carbon sources for growth and clavulanic acid production. Methods and results:, Batch cultures in bioreactors showed that Strep. clavuligerus growth and clavulanic acid yields in a P-limited medium containing 0.6% (v/v) olive oil were respectively 2.5- and 2.6-fold higher than in a glycerol-containing medium used as control. Glycerol- and olive oil-grown cells present different macromolecular composition, particularly lipid and protein content. Conclusions:,Streptomyces clavuligerus uses olive oil as the sole carbon and energy source for growth and clavulanic acid production. Yields and production rates in olive oil are comparable to those reported for oil-containing complex media. Differences in yields and in the macromolecular composition indicate that different metabolic pathways convert substrate into product. Significance and impact of the study:, This is the first report of oils being used as the sole carbon source by Strep. clavuligerus. Apart from economic benefits, interesting questions are raised about Strep. clavuligerus physiology. Defined culture media allow physiological studies to be performed in the absence of interference by other compounds. Understanding how Strep. clavuligerus catabolises oils may have an economic impact in clavulanic acid production. [source] Cyst Formation in a Freshwater Strain of the Choanoflagellate Desmarella moniliformis KentTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2000BARRY S. C. LEADBEATER ABSTRACT. Cyst formation in a freshwater strain of the colonial freshwater Choanoflagellate Desmarella moniliformis Kent (Protozoa; Choanoflagellida) has been studied with light and electron microscopy for the first time. Batch cultures inoculated with motile vegetative cells start to produce cysts within 3 days during the exponential phase of growth. Cyst production proceeds until in late stationary phase there is a preponderance of cysts. Transfer of cysts to fresh medium results in limited excystment. Encystment involves the production of electron-dense fibrillar wall material, firstly around the neck of the cell and then around the posterior end. As the wall material is deposited the neck of the cell elongates and the dictyosome rotates from the horizontal to vertical plane. The number of mitochondrial profiles seen in individual sections of cells increases. Finally the neck of the cell is retracted, the flagellum and collar tentacles are withdrawn, and the bottom of the neck of the cyst wall is sealed with a diaphragm of wall material. Excystment, which has not been observed directly, appears to involve the disruption of the wall at the base of the neck, the remainder of the cyst wall remains intact. Comparisons are made between encystment in Desmarella and cyst development in other protists. [source] Effects of Cloned Gene Dosage on the Response of Recombinant CHO Cells to Hyperosmotic Pressure in Regard to Cell Growth and Antibody ProductionBIOTECHNOLOGY PROGRESS, Issue 6 2001Joon Soo Ryu The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (,) and increased specific antibody productivity (qAb) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in qAb and less reduction in , at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and qAb, indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G1 -phase arrest, suggesting that the increase of cell population in G1 -phase may contribute in part to enhanced qAb at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in qAb at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage. [source] Dynamics of Benzene and Toluene Degradation in Pseudomonas putida F1 in the Presence of the Alternative Substrate SuccinateENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007I. Rüegg Abstract In batch and continuous culture, the regulation of benzene and toluene degradation by Pseudomonas putida,F1 was investigated in the presence of the alternative carbon and energy source succinate. In batch culture, benzene and toluene were used simultaneously, whereas succinate suppressed benzene consumption under carbon excess conditions resulting in diauxic growth. In carbon-limited continuous culture mixed substrate growth was observed. Since in nature low substrate concentrations and ever changing conditions prevail, this paper focuses on the dynamics of benzene/toluene degradation, biomass synthesis, and the regulation of benzene/toluene-degrading enzymes in cultures growing continuously at a dilution rate of 0.1,h,1, when shifting the supply of the carbon and energy source from succinate to various mixtures of succinate and benzene/toluene, or to benzene only. When the succinate concentration was kept constant (1.25,mM) and the medium was supplemented with benzene (2,mM), growth with benzene began already two hours after the shift. In contrast, replacing succinate with benzene only led to a wash out of biomass for more then ten hours, before biomass production from benzene started. A striking and reproducible transition pattern was observed for all shifts where the succinate concentration was reduced or succinate was omitted. After an initial period of biomass production from benzene, the culture collapsed and a wash-out of biomass was observed. However, this wash-out was not accompanied by an increase in benzene in the cultivation liquid, indicating a benzene uptake without conversion into biomass. Another possibility is that in phases of low biomass concentrations, cells were only able to use the low amounts of benzene/toluene dissolved in the cultivation liquid yielding low biomass concentrations whereas in phases of high biomass concentrations, they were able to rapidly utilize the aromatic solvents so that additional benzene from the gas phase diffused into the cultivation liquid resulting in more biomass production. In most cases, growth resumed again after 10 to 80,h. Currently, the reasons for the decrease in biomass after the first rise are unknown. However, several indications rule out intoxication of the cells by either the solvents benzene or toluene themselves, or by toxic degradation intermediates, or by-products. [source] Toxicity of arsenic species to Lemna gibba L. and the influence of phosphate on arsenic bioavailabilityENVIRONMENTAL TOXICOLOGY, Issue 1 2004Martin Mkandawire Abstract The toxicity of arsenic (As) species to Lemna gibba L. and the influence of PO on As bioavailability and uptake were tested in batch culture. L. gibba were exposed to six test concentrations of NaHAsO4 · 7H2O and NaAsO3, with 0, 0.0136, 13.6, and 40 mg L,1 KH2PO4. In batch culture As toxicity to L. gibba did not relate linearly to As concentration. The growth rate, related to frond number as recommended by OECD and ISO/DIN, was significantly inhibited in fronds exposed to 20,50 ,g L,1 As(III) compared with fronds exposed to As(V). The growth rate was stimulated when plants were exposed to 50,250 ,g L,1 of both As(III) and As(V). After exposure to 300,800 ,g L,1 growth inhibition was significantly higher for As(III) than for As(V), whereas above 800 ,g L,1 As(V) was inhibited the most. The bioaccumulation of As(III) and As(V) was significantly higher for P-deficient cultures (0.98 ± 0.08 and 1.02 ± 0.19 g kg,1, respectively for 0.0136 mg L,1 PO) than for P-sufficient cultures (243 and 343 mg kg,1 for 40 mg L,1, respectively). Plants exposed to As(V) had uptake and accumulation values slightly higher than did plants exposed to As(III). No significant differences in bioaccumulation were found between plants exposed to a concentration of As(III) >1 mg L,1 and those exposed to As(V) at the same concentration. This indicates a direct relationship to P content in the culture. Toxicity may result from the uptake of As(V) instead of PO as a result of ion competition during uptake because of close thermodynamic properties, which may change the interaction among components in the media. The toxicity pattern is interpreted as a manifestation of changing speciation in the batch culture and of the oxidation of As(III) to As(V) in an oxygen-rich environment. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 26,34, 2004. [source] Phenotypical variation in a toxic strain of the phytoplankter, Cylindrospermopsis raciborskii (nostocales, cyanophyceae) during batch cultureENVIRONMENTAL TOXICOLOGY, Issue 6 2001Peter R. Hawkins Abstract A nonaxenic strain of Cylindrospermopsis raciborskii Woloszynska (AWT 205) was grown in batch culture, with and without nitrate as the primary N source. Rapid log-phase growth with nitrate was 1.0 doubling/day versus 0.3 doubling/day without nitrate. Cylindrospermopsin (CYN) production was measured by HPLC. The rate of intracellular CYN production matched cell division rate for both the diazotrophies at cell densities less than 107 cell/ml. At cell density >107 cell/ml, additional resource limitation in batch culture slowed log-phase growth to 0.04 division/day and cell division and CYN production decoupled. Intracellular CYN concentration increased at a rate of 0.08 doubling/day, twice the cell division rate. Extracellular CYN as a proportion of the total CYN increased from 20% during the rapid growth phase, to 50% during the slow growth phase. The total CYN yield from cultures grown out to stationary phase (55 days) exceeded 2 mg CYN/l. C. raciborskii cells in log-phase growth, exposed to 1 ppm copper (as copper sulphate), lysed within 24 hours. After copper treatment, all CYN was in the filterable fraction. These findings imply that in naturally occurring blooms of C. raciborskii, the movement of intracellular CYN into solution will be the greatest during stationary phase, when intracellular concentrations are highest and cell lysis is more frequent. The application of algicides that promote cell lysis will exacerbate this effect. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 460,467, 2001 [source] Aminopeptidase and phosphatase activities in basins of Lake Hiidenvesi dominated by cyanobacteria and in laboratory grown AnabaenaFRESHWATER BIOLOGY, Issue 9 2002JAANA VAITOMAA 1.,Extracellular enzyme activities were examined in freshwater basins representing a transition from hypertrophy to mesotrophy and in axenic cyanobacterial cultures to evaluate the ecological role of extracellular enzyme activities of cyanobacteria. 2.,Aminopeptidase activity was related to the trophic status of the lake basins. The activity was highest in the most eutrophic basin and decreased in the less nutrient-rich basins. Cyanobacteria were the most important autotrophic organisms and aminopeptidase activity was positively associated with cyanobacterial biomass. 3.,In an axenic Anabaena batch culture, nitrogenase activity was several orders of magnitude higher than leucine aminopeptidase activity. Nitrate did not have an effect on aminopeptidase activity or growth, but significantly reduced the rate of nitrogen fixation. A high phosphorus concentration at the beginning of the Anabaena batch-culture experiment resulted in reduced phosphatase activity. 4.,In Lake Hiidenvesi, aminopeptidase activity probably originated mostly from attached bacteria and less so from cyanobacteria. [source] Sequential secretion of collagenolytic, elastolytic, and keratinolytic proteases in peptide-limited cultures of two Bacillus cereus strains isolated from woolJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009A.C. Ad, güzel Abstract Aims:, To characterize the secretion of proteolytic activities against keratin, collagen and elastin in liquid cultures of Bacillus cereus IZ-06b and IZ-06r isolated from wool. Methods and Results:, Growth of B. cereus IZ-06b and IZ-06r were characterized in batch culture. Both strains needed an organic nitrogen source, were able to grow on wool or peptone as sole carbon and nitrogen sources, and metabolized glucose, maltose and other simple sugars. Proteolytic activities were investigated in batch cultures grown in peptide-restricted, carbon-sufficient medium. Secretion of proteases was induced by peptide limitation while different proteolytic activities appeared sequentially in the growth medium. When the most available components of the peptone were depleted, collagenolytic and elastolytic proteases were produced. These were later replaced by the production of keratinolytic protease. Conclusions:,B. cereus can adjust its proteolytic affinity profile in response to the supply of organic nitrogen and sequentially secrete proteases with activities targeted against increasingly inaccessible proteinous substrates as the nutritional availability in the environment deteriorates. Significance and Impact of the Study:, Peptide-limited, carbon-sufficient growth media containing no proteinous substrates are well suited for protease production in B. cereus while growth conditions can be adjusted to optimize the proteolytic affinity profiles. [source] Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistanceJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000J.P. Grill Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N,-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (Z,pH) was involved in Lactobacillus bile salt resistance. [source] Effect of loading rate on TOC consumption efficiency in a sulfate reducing process: sulfide effect in batch cultureJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 12 2008Citlali García-Saucedo Abstract BACKGROUND: The sulfate reducing process (SRP) was analyzed in order to identify factors that diminish the effectiveness of the SRP during wastewater treatment. The effect of different sulfate loading rates (SLR, 290 to 981 mg SO4 -S L,1d,1) and lactate at a stoichiometric C/S ratio of 0.75 on SRP was studied in an upflow anaerobic sludge blanket (UASB) reactor. The effect of sulfide concentration (0 to 200 mg sulfide-S L,1) on SRP in batch culture was evaluated. RESULTS: When the SLR was increased, the total organic carbon (TOC) and sulfate consumption efficiencies decreased from 93% ± 3 to 66% ± 2 and 60% ± 5 to 45% ± 4, respectively. Acetate and propionate were accumulated. Microbial analysis showed the presence of microorganisms related with the SRP, fermentation and methanogenesis. In batch culture, when lactate and sulfate were present, SRP and fermentation were observed. When sulfide was added only SRP was observed. At concentrations higher than 150 mg sulfide-S L,1 the efficiencies, yields and specific consumption rates (q) decreased. CONCLUSION: Based on the sulfide-S/volatile suspended solid ratio, it was found that the decrease in efficiency and accumulation of acetate and propionate in the UASB reactor was not related to sulfide inhibition but to the q of acetate and propionate, which were up to 11 times lower than lactate. Copyright © 2008 Society of Chemical Industry [source] Fermentative production of L(+)-lactic acid from starch hydrolyzate and corn steep liquor as inexpensive nutrients by batch culture of Enterococcus faecalis RKY1JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2008Young-Jung Wee Abstract BACKGROUND: Attempts were made to determine the lactic acid production efficiency of novel isolate, Enterococcus faecalis RKY1 using four different starches (corn, tapioca, potato, and wheat starch) with different concentrations (50, 75, 100, and 125 g L,1) and corn steep liquor as an inexpensive nitrogen source. RESULTS: The yield of lactic acid from each starch was higher than 95% based on initial starch concentrations. High lactic acid concentration (129.9 g L,1) and yield (1.04 g-lactic acid g,1 -starch) were achieved faster (84 h) from 125 g L,1 of corn starch. Among the starches used, tapioca starch fermentation usually completed in a shorter incubation period. The final dry cell weight was highest (7.0 g L,1) for the medium containing 75 g L,1 of corn starch, which resulted in maximum volumetric productivity of lactic acid (3.6 g L,1 h,1). The addition of 30 g L,1 corn steep liquor supplemented with a minimal amount of yeast extract supported both cell growth and lactic acid fermentation. CONCLUSION:Enterococcus faecalis RKY1 was found to be capable of growing well on inexpensive nutrients and producing maximum lactic acid from starches and corn steep liquor as lower-cost raw materials than conventionally-used refined sugars such as glucose, and yeast extract as an organic nitrogen source in laboratory-scale studies. These fermentation characteristics are prerequisites for the industrial scale production of lactic acid. Copyright © 2008 Society of Chemical Industry [source] Experimentation of a new mode of batch culture for lactic acid bacteria: cell reuse with an initial period of cell reactivation at acidic pHJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2001Abdeltif Amrane Abstract Cell reuse was compared with conventional batch culture for lactic acid fermentation, the objective was to simplify the batch process and to alleviate the need for added nitrogen. At high levels of nitrogen supplementation to the culture medium (20,g,dm,3 yeast extract and 5,g,dm,3 each of tryptic and pancreatic casein peptones), similar mean production rates were obtained with partial cell reuse and the conventional batch process, without any additional gain when cells were initially reactivated at acidic pH. On the other hand, cell reuse with an initial period without pH control appeared particularly effective for low levels of nitrogen supplementation (5,g,dm,3 yeast extract): a 57% increase in the mean production rate with regard to the conventional batch process was obtained. © 2001 Society of Chemical Industry [source] FLUORESCENCE-BASED MAXIMAL QUANTUM YIELD FOR PSII AS A DIAGNOSTIC OF NUTRIENT STRESSJOURNAL OF PHYCOLOGY, Issue 4 2001Jean-Paul Parkhill In biological oceanography, it has been widely accepted that the maximum quantum yield of photosynthesis is influenced by nutrient stress. A closely related parameter, the maximum quantum yield for stable charge separation of PSII, (,PSII)m, can be estimated by measuring the increase in fluorescence yield from dark-adapted minimal fluorescence (Fo) to maximal fluorescence (Fm) associated with the closing of photosynthetic reaction centers with saturating light or with a photosynthetic inhibitor such as 3,-(3,4-dichlorophenyl)-1,,1,-dimethyl urea (DCMU). The ratio Fv/Fm (= (Fm, Fo)/Fm) is thus used as a diagnostic of nutrient stress. Published results indicate that Fv/Fm is depressed for nutrient-stressed phytoplankton, both during nutrient starvation (unbalanced growth) and acclimated nutrient limitation (steady-state or balanced growth). In contrast to published results, fluorescence measurements from our laboratory indicate that Fv/Fm is high and insensitive to nutrient limitation for cultures in steady state under a wide range of relative growth rates and irradiance levels. This discrepancy between results could be attributed to differences in measurement systems or to differences in growth conditions. To resolve the uncertainty about Fv/Fm as a diagnostic of nutrient stress, we grew the neritic diatom Thalassiosira pseudonana (Hustedt) Hasle et Heimdal under nutrient-replete and nutrient-stressed conditions, using replicate semicontinuous, batch, and continuous cultures. Fv/Fm was determined using a conventional fluorometer and DCMU and with a pulse amplitude modulated (PAM) fluorometer. Reduction of excitation irradiance in the conventional fluorometer eliminated overestimation of Fo in the DCMU methodology for cultures grown at lower light levels, and for a large range of growth conditions there was a strong correlation between the measurements of Fv/Fm with DCMU and PAM (r2 = 0.77, n = 460). Consistent with the literature, nutrient-replete cultures showed consistently high Fv/Fm (,0.65), independent of growth irradiance. Under nutrient-starved (batch culture and perturbed steady state) conditions, Fv/Fm was significantly correlated to time without the limiting nutrient and to nutrient-limited growth rate before starvation. In contrast to published results, our continuous culture experiments showed that Fv/Fm was not a good measure of nutrient limitation under balanced growth conditions and remained constant (,0.65) and independent of nutrient-limited growth rate under different irradiance levels. Because variable fluorescence can only be used as a diagnostic for nutrient-starved unbalanced growth conditions, a robust measure of nutrient stressed oceanic waters is still required. [source] FATTY ACIDS IN PHOTOTROPHIC AND MIXOTROPHIC GYRODINIUM GALATHE-ANUM (DINOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 2000J.E. Adolf Fatty acids were measured in G. galatheanum grown either phototrophically, or mixotrophically with Storeatula major (Cryptophyceae) as prey. G. galatheanum, like many photosynthetic dinoflagellates, contains high amounts of n-3 long-chain-polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA, 22:6n-3) and the hemolytic toxic fatty acid 18:5n-3. We hypothesize that a benefit of phagotrophy in G. galatheanum is the acquisition of precursor linolenic acid (18:3n-3) that fuels LC-PUFA synthesis. Phototrophs grew at 0.37 d,1, while mixotrophs grew at 0.40 d,1 with a feeding rate of 0.62 d,1. Photosynthesis was lower in mixotrophs (3.7 pg C cell,1 h,1) than phototrophs (4.9 pg C cell,1 h,1). DHA levels were higher in mixotrophs [3.7 (+/, 0.11) pg cell,1] than phototrophs [3.0 (+/, 0.16) pg cell,1] and prey [0.4 (+/, 0.01) pg cell,1]. 18:5n,3 levels [1.7 (+/, 0.03) pg cell,1] were similar in phototrophs and mixotrophs. An intermediate in n-3 LC-PUFA synthesis, 20:4n-3, accumulated in mixotrophs [0.6 (+/, 0.27) pg cell,1] relative to phototrophs (not detected) and prey [0.03 (+/, 0.002) pg cell,1]. Low ratios of linolenic acid to DHA in phototrophic G. galatheanum (0.14) relative to mixotrophic G. galatheanum (0.29) and prey (2.14) are consistent with substrate limitation of LC-PUFA synthesis in phototrophs. Accumulation of 20:4n-3 suggests incomplete conversion of linolenic acid to DHA, possibly due to conditions in batch culture. We conclude that precursors for n-3 LC-PUFA biosynthesis in G. galatheanum may be acquired through ingestion of S. major, and may partially control feeding/photosynthesis in mixotrophic populations. [source] GENETIC AND PHYSIOLOGICAL VARIATION IN PIGMENT COMPOSITION OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AND THE POTENTIAL USE OF ITS PIGMENT RATIOS AS A QUANTITATIVE PHYSIOLOGICAL MARKERJOURNAL OF PHYCOLOGY, Issue 3 2000Willem Stolte Genetic variation of pigment composition was studied in 16 different strains of Emiliania huxleyi (Lohm.) Hay et Mohler in batch culture. Distinct strain-dependent differences were found in the ratios of fucoxanthin, 19,-hexanoyloxyfucoxanthin, and 19,-butanoyloxyfucoxanthin, hampering the use of these individual pigments as a taxonomic marker at the species level. The molar ratio of total carotenoids to chl a, however, was constant for all strains tested. In addition, the pigment composition of one axenic strain (L) of E. huxleyi at different growth rates in light-, nitrate-, and phosphate-limited continuous cultures was analyzed quantitatively. The pigments fucoxanthin and 19,-hexanoyloxyfucoxanthin correlated closely under all conditions. From steady-state rate calculations, it is hypothesized that 19,-hexanoyloxyfucoxanthin is synthesized from fucoxanthin, with light as a modulating factor. The net rate of synthesis of diatoxanthin depended both on the concentration of diadinoxanthin (its partner in the xanthophyll cycle) and on light, illustrating its photoprotective function in the xanthophyll cycle. In axenic strain L, the ratio of total fucoxanthins to chl a correlated strongly with photon flux density and can potentially be used to assess the physiological status with respect to irradiance in field populations. In multispecific bloom situations, the ratio of diadinoxanthin plus diatoxanthin to total fucoxanthins could be used as an alternative indicator for the light-dependent physiological state of E. huxleyi, provided that no other chromophytes are present. Application of these correlations to mesocosm data from the literature has so far provided no evidence that E. huxleyi blooms form only at inhibiting light levels, as previously suggested. [source] Effect of medium molecular weight xanthan gum in rheology and stability of oil-in-water emulsion stabilized with legume proteinsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2005Evdoxia M Papalamprou Abstract Xanthan gum is a water-soluble extracellular polysaccharide that has gained widespread commercial use because of its strong pseudoplasticity and tolerance to high ionic strength, which bring unique rheological properties to solutions. This study compares and evaluates the emulsifying properties of oil-in-water (30:70 v/v) emulsions stabilized with lupin and soya protein isolates and medium molecular weight xanthan gum. The protein was obtained by an isoelectric precipitation method and the polysaccharide was produced by Xanthomonas campestris ATCC 1395 in batch culture in a laboratory fermenter (LBG medium) without pH control. The addition of xanthan gum in the emulsion formulation enhances emulsion stability through the phenomenon of thermodynamic incompatibility with the legume protein, resulting in an increase of the adsorbed protein at the interface. The emulsion stability is also enhanced by a network structure built by the polysaccharide in the bulk phase. Copyright © 2005 Society of Chemical Industry [source] Effect of temperature and soluble reactive phosphorus on abundance of Aphanizomenon flos-aquae (Cyanophyceae)PHYCOLOGICAL RESEARCH, Issue 1 2000Keishi Takano SUMMARY Filament density of Aphanizomenon flos-aquae (Lemmerm.) Ralfs, water temperature and soluble reactive phosphorus (SRP) were measured from April to August in 1993,1996 in Lake Barato, Hokkaido, Japan. In addition, growth characteristics and internal phosphorus (P) utilization of Aph. flos-aquae were evaluated under P limitation at three temperatures (15, 20 and 25,C) to clarify the role of internal accumulated P for its growth in the incubation experiment. The filament density was highest in early July 1994, when SRP concentration had not yet decreased and the water temperature was high. These are important factors favoring an increase in abundance of this species in L. Barato. During batch culture, the time course of the stationary phase was shortest at 25,C and longest at 15,C; the cellular C:P molar ratio was 111 under P sufficiency and increased eight- to 12-fold under P limitation. As the C:P ratio was significantly higher in the decreasing phase at 15,C, Aph. flos-aquae may be more adaptable to Plimitation at 15,C than at 20,C and 25,C. However, the low temperatures did not favor the abundance of Aph. flos-aquae in 1996. This indicates that the filament density of Aph. flos-aquae decreases before it reaches the maximum value for some reason under P limitation in L. Barato. [source] Profiling of N -glycosylation gene expression in CHO cell fed-batch culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Danny Chee Furng Wong Abstract One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N -glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N -glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed-batch culture of a CHO cell line producing recombinant human interferon-, (IFN-,). Of the 24 N -glycosylation genes examined, 21 showed significant up- or down-regulation of gene expression as the fed-batch culture progressed from exponential, stationary and death phase. As the fed-batch culture progressed, there was also an increase in less sialylated IFN-, glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-,. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed-batch strategy appears to need a 0.5,mM glutamine threshold to maintain similar N -glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible "bottlenecks" or "compromised" pathways in N -glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516,528. © 2010 Wiley Periodicals, Inc. [source] Metabolism of PER.C6TM cells cultivated under fed-batch conditions at low glucose and glutamine levelsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Luis Maranga Abstract This is the first study to examine PER.C6TM cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6TM cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced qGln and q by approximately 30%, qAla 85%, and qNEAA 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced qGlc about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y of approximately 6 mol/mol. However, despite no increase in qGln, an increased ammonium ion accumulation and Y were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6TM cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The qGln value was again reduced and, despite an increased q compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6TM cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6TM cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism. © 2006 Wiley Periodicals, Inc. [source] Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Zhi-Gang Qian Abstract A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 ,M DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L,1 sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 ,M DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 ± 29 mg L,1, which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 ± 41 mg L,1 and 33.2 ± 1.9 mg L,1 d,1, respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites. © 2005 Wiley Periodicals, Inc. [source] Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2002Idsada Lengwehasatit Abstract Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells. This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis. During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells. This event was paralleled by a simultaneous increase in GADD153 expression. The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented. In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress. However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment. Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death. These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 719,730, 2002. [source] Development of a large-scale biocalorimeter to monitor and control bioprocessesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2002D. Voisard Abstract Calorimetry has shown real potential at bench-scale for chemical and biochemical processes. The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor. To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation implemented to enable on-line monitoring and dynamic heat balance estimation. Providing that the signals are sufficiently precise, such a heat balance would enable calculation of the heat released or taken up during an operational (bio)process. Two electrical Wattmeters were developed, the first for determination of the power consumption by the stirrer motor and the second for determination of the power released by an internal calibration heater. Experiments were designed to optimize the temperature controller of the bioreactor such that it was sufficiently rapid so as to enable the heat accumulation terms to be neglected. Further calibration experiments were designed to correlate the measured stirring power to frictional heat losses of the stirrer into the reaction mass. This allows the quantitative measurement of all background heat flows and the on-line quantitative calculation of the (bio)process power. Three test fermentations were then performed with B. sphaericus 1593M, a spore-forming bacterium pathogenic to mosquitoes. A first batch culture was performed on a complex medium, to enable optimization of the calorimeter system. A second batch culture, on defined medium containing three carbon sources, was used to show the fast, accurate response of the heat signal and the ability to perfectly monitor the different growth phases associated with growth on mixed substrates, in particular when carbon sources became depleted. A maximum heat output of 1100 W was measured at the end of the log-phase. A fed-batch culture on the same defined medium was then carried out with the feed rate controlled as a function of the calorimeter signal. A maximum heat output of 2250 W was measured at the end of the first log-phase. This work demonstrates that real-time quantitative calorimetry is not only possible at pilot-scale, but could be readily applied at even larger scales. The technique requires simple, readily available devices for determination of the few necessary heat flows, making it a robust, cost-effective technique for process development and routine monitoring and control of production processes. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 125,138, 2002 [source] Expression of the transcription factor GADD153 is an indicator of apoptosis for recombinant chinese hamster ovary (CHO) cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2001Tracy C. Murphy Abstract Loss of cell viability, through engagement of apoptotic cell death, represents a limitation to maintenance of high levels of productivity of recombinant animal cells in culture. The ability to monitor the status of recombinant cells, and to define indicators of their "well-being," would present a valuable approach to permit a rational intervention at appropriate times during culture. Growth arrest and DNA damage gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors and has been associated with apoptosis. We have examined the expression of GADD153 in conditions associated with apoptosis of recombinant CHO cells in batch culture. GADD153 expression is very low in CHO cells growing in the exponential phase of batch culture but is activated as cells enter the decline phase. Depletion of nutrients (glucose or glutamine) causes activation of GADD153 expression as does the imposition of endoplasmic reticulum stress. In all cases, there is a good relationship between the extent of apoptosis that occurs in response to each stress and the degree of GADD153 expression. In addition, nutrient refeeding or reversal of stress produces a concomitant decrease in expression of GADD153 and the susceptibility to apoptosis. Thus, GADD153 appears to offer a valid indicator of apoptosis and illustrates the potential for definition of monitors of cellular status related to the likelihood of apoptosis of cell populations. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 621,629, 2001. [source] Enhanced interferon-, production by CHO cells through elevated osmolality and reduced culture temperatureBIOTECHNOLOGY PROGRESS, Issue 5 2009Young Kue Han Abstract For efficient production of native interferon-, (IFN-,) in recombinant CHO cell culture, the IFN-, molecular aggregation that occurs during culture needs to be minimized. To do so, we investigated the effect of hyperosmolality and hypothermia on IFN-, production and molecular aggregation in rCHO cell culture. Both hyperosmolality (470 mOsm/kg) and hypothermia (32°C) increased specific native INF-, productivity qIFN-,. Furthermore, they decreased the IFN-, molecular aggregation, although severe IFN-, molecular aggregation could not be avoided in the later phase of culture. To overcome growth suppression at hyperosmolality and hypothermia, cells were cultivated in a biphasic mode. Cells were first cultivated at 310 mOsm/kg and 37°C for 2 days to rapidly obtain a reasonably high cell concentration. The temperature and osmolality were then shifted to 32°C and 470 mOsm/kg, respectively, to achieve high qIFN-, and reduced IFN-, molecular aggregation. Due to the enhanced qIFN-, and delayed molecular aggregation, the highest native IFN-, concentration achieved on day 6 was 18.03 ± 0.61 mg/L, which was 5.30,fold higher than that in a control batch culture (310 mOsm/kg and 37°C). Taken together, a combination of hyperosmolality and hypothermia in a biphasic culture is a useful strategy for improved native IFN-, production from rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor-Perfusion Culture SystemBIOTECHNOLOGY PROGRESS, Issue 1 2007Ya-Jie Tang A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 ,m was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 × 105 and 200.5 × 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 × 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L·day) in perfusion culture were much higher than those (i.e., 22.3 × 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L·day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale. [source] Optimization of the Human Adenosine A2a Receptor Yields in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 5 2006Alison Wedekind G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A2a receptor (A2aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A2aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD600) and per cell A2aR expression levels. For these studies, the A2aR expression levels were determined using a previously described A2aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times (,60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A2aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points (,60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization. [source] Enhancement of Monascus Pigment Production by the Culture of Monascus sp.BIOTECHNOLOGY PROGRESS, Issue 1 2006J101 at Low Temperature In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 °C. In a batch culture at 30 °C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 °C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 °C was 10 times greater than at 30 °C. [source] Defined Protein-Free NS0 Myeloma Cell Cultures: Stimulation of Proliferation by Conditioned Medium FactorsBIOTECHNOLOGY PROGRESS, Issue 1 2005Erika Spens A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, ,-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 × 106 cells mL,1 in this medium. The antibody yield was 195 mg L,1 in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 × 106 cells mL,1, had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20,25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes. [source] | |||||||||||||||||||||||||||||||