Effect of dilution rate on eicosapentaenoic acid productivity ofPhaeodactylum tricornutum utex 640 in outdoor chemostat culture

Summary Eicosapentaenoic acid (EPA) volumetric productivity from an outdoor chemostat culture ofPhaeodactylum tricornutum UTEX 640 in a 50-l tubular photobioreactor varies with dilution rate, reaching a maximum of 47.8 mg l^–1 d^–1 at D=0.36 d^–1. Continuous culture at high dilution rates' is proposed as the most adequate operating mode to maximize polyunsaturated fatty acid production.     

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1). However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.     

Complete genome sequence of Methanosaeta concilii, a specialist in aceticlastic methanogenesis

The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined and exhibits considerable differences in gene content from that of Methanosaeta thermophila.     

Effect of temperature,light intensity and dilution rate on the cellular composition of red algaPorphyridium cruentum in light-limited chemostat cultures

Summary The fatty acid, polysaccharide and pigment contents ofPorphyridium cruentum at different growth temperatures, light intensities and dilution rates were studied in light-limited chemostat. The highest biomass yield was obtained at 25°C over the range of dilution rate studied (0.079 to 0.30 day^–1). The production of fatty acids increased with increasing dilution rates. At low dilution rates, the contents of fatty acids at 20°C, 25°C and 30°C were comparable. Temperature did not affect the content of linolenic acid (182) though fatty acids with less than two double bonds predominated at higher temperatures, those with more than two double bonds accumulated at lower temperatures. The yields of arachidonic acid (204) increased under light of higher intensity. The maximum production of extracellular and cellular polysaccharides occured at low dilution rates under high light at 25°C. The amounts of chlorophylla, carotenoids and phycoerythrin all increased in the same proportions with dilution rate, and were highest at 25°C.

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Resumen Se estudió el contenido en polisaccaridos, pigmentos y ácidos grasos dePorphyridium cruentum a distintas temperaturas, intensidades lumínicas y porcentajes de dilución en un quimiostato con luz limitada. La mayor cantidad de biomasa para todas las diluciones estudiadas (0.079 a 0.30 día^–1) se obtuvo a 25°C. A bajas diluciones los contenidos en ácidos grasos a 20°C, 25°C y 30°C eran comparables. La temperatura no afectó al contenido en ácido linolénico (182), sin embargo los ácidos grasos con menos de dos dobles enlaces se acumularon a temperaturas más bajas. La producción de ácido arachidónico (204) se incrementó bajo la mayor intensidad lumínica ensayada. La producción máxima de polisaccaridos celulares y extracelulares se dió a bajas diluciones en condiciones de alta iluminación a 25°C. Las cantidades de clorofila a, carotenoides y ficoeritrina se incrementaron paralelamente al porcentaje de dilución siendo más elevadas a 25°C.

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Résumé On a étudié le contenu dePorphyridium cruentum en acides gras, en polysaccharides et en pigments, en fonction de la température de croissance, de l'intensité lumineuse et du taux de dilution dans un chémostat limité par la lumière. Le rendement le plus élevé en biomasse a été obtenu à 25°C quel que soit le taux de dilution (compris entre 0.079 et 0.30 jours^–1). La production d'acides gras augmente avec l'augmentation du taux de dilution. A des taux de dilution faibles, le contenu en acides gras est indépendant de la température (comprise entre 20 et 30°C). La température n'influence pas le contenu en acide linolénique (182) bien que les acides gras à deux ou plusieurs doubles liaisons s'accumulent à température plus basse. Les rendements en acide arachidonique (204) augmentent sous une lumière de plus forte intensité. La production maximum de polysaccharides tant extracellulaires que cellulaires a lieu à un taux faible de dilution sous une lumière intense à 25°C. Les teneurs en chlorophylle a, caroténoïdes et en phycoerythrine augmentent toutes les trois dans les mêmes proportions avec le taux de dilution et sont les plus élevées à 25°C.

     

Evidence for aceticlastic methanogenesis in the presence of sulfate in a gas condensate-contaminated aquifer

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-(14)C]acetate or [U-(14)C]acetate produced higher quantities of CH(4) compared to CO(2) in the presence or absence of sulfate.(14)CH(4) accounted for 70 to 100% of the total labeled gas in the [(14)C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.     

The effect of the dilution rate on CHO cell physiology and recombinant interferon-gamma production in glucose-limited chemostat culture

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (mu) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (mu(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 muM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma. (c) 1993 John Wiley & Sons, Inc.     

Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day⁻¹. Butyrate, fed into the chemostat, was almost completely mineralized to CH₄ and CO₂ at dilution rates below 0.5 day⁻¹. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day⁻¹ were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day⁻¹, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.     

Simultaneous utilization of methanol-glucose mixtures by Hansenula polymorpha in chemostat: Influence of dilution rate and mixture composition on utilization pattern

The utilization of mixtures of methanol (C(1)) and glucose (C(6)) of different composition by the methylotrophic yeast Hansenula polymorpha was studied in carbon-limited chemostat culture. For all mixtures tested a similar utilization pattern was observed: At low dilution rates both carbon sources were utilized simultaneously, but at high dilution rates the cells used glucose only and the unutilized methanol accumulated in the culture medium. When grown with C(1) only, the cells exhibited a critical dilution rate D(c)(C(1)) of 0.19 h(-1), but when C(1)-C(6) mixtures were used as the carbon and energy substrate, the yeast was able to completely utilize C(1) at dilution rates considerably higher than D(c)(C(1)). The dilution rate at which the transition from C(1)-C(6) growth to C(6) growth occurred (D(t)) was strictly dependent on the composition of the C(1)-C(6) mixture in the feed, and D(t) increased with decreasing proportions of C(1) in the mixture. During mixed substrate growth the formation of biomass from the two substrates was additive. The results reported indicate that the utilization of C(1)-C(6) mixtures and hence D(t) in H. polymorpha are subject to two different regulatory regimes. When the cells were growing with C(1)-C(6) mixtures containing more than 60% C(1), the transition form C(1)-C(6) to C(6) growth was most probably influenced by the maximum C(1) oxidizing capacity of the cells, whereas for growth with mixtures containing less than 40% C(1), a growth rate of 0.28-0.30 h(-1) seemed to be the limiting barrier for the simultaneous utilization of the components of the binary carbon and energy substrate mixture.     

Effect of dilution rate and carbon availability on Bifidobacterium breve fermentation

Bifidobacterium breve NCFB 2257 was grown in glucose-limited and nitrogen (N)-limited chemostats at dilution rates (D) from 0.04 to 0.60 h^–1, to study the effect of nutrient availability on carbohydrate metabolism. The results showed that D had little effect on fermentation product formation, irrespective of the form of nutrient limitation. However, marked differeces were observed in the distribution of fermentation products, that were attributable to glucose availability. In glucose-limited cultures, formate and acetate were the principal end-products of metabolism. Lactate was never detected under these growth conditions. In contrast, lactate and acetate were mainly formed when glucose was in excess, and formate was not produced. These results are explained by the metabolic fate of pyruvate, which can be dissimilated by either phosphoroclastic cleavage to acetyl phosphate and formate, or alternatively, it may be reduced to lactate. Enzymic studies were made to establish the mechanisms that regulated pyruvate metabolism. The data demonstrated that control was not exercised through regulation of the synthesis and activity of lactate dehydrogenase (LDH), phosphofructokinase or alcohol dehydrogenase. It is possible however, that there was competition for pyruvate by LDH and the phosphoroclastic enzyme, which would determine the levels of lactate and formate produced respectively. These results demonstrate the metabolic flexibility of B. breve, which preferentially uses lactate as an electron sink during N-limited growth, whereas under energy-limitation, carbon flow is directed towards acetyl phosphate to maximise ATP synthesis. Correspondence to: B. A. Degnan     

Effect of prolonged cultivation in a chemostat under nitrogen limitation on the morphology and ultrastructure ofBacillus subtilis

Cells ofBacillus subtilis A32 grown in a chemostat under nitrogen limitation display morphological changes after long-term cultivation. Bacillary rods are transformed into irregular spherical forms. A general chemical analysis of the normal and of the altered cells was carried out. The spherical cells were studied by electron microscopy in ultrathin sections. Differences in the arrangement of the superficial layers and a damaged mechanism of regular division were observed. The spherical forms revert to rods after removing the limitation.     

Effect of some antineoplastics on metabolic heme pathway

1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.     

Regulatory flexibility of methylotrophic yeasts in chemostat cultures: Simultaneous assimilation of glucose and methanol at a fixed dilution rate

The influence of the composition of methanol/glucose-mixtures as only sources of carbon and energy on growth and regulation of the synthesis of enzymes involved in methanol-dissimilation was studied under chemostat conditions at a fixed dilution rate with the methylotrophic yeasts Hansenula polymorpha and Kloeckera sp. 2201. Both carbon sources were found to be utilized completely independently of the composition of the C₁/C₆ mixture. Using mixtures of ¹⁴C-labelled methanol and glucose the growth yield for glucose was found to be constant for all C₁/C₆-mixtures tested and both yeasts. The growth yield for methanol, however, was reduced by up to 25% when the proportion of methanol in the inflowing medium was lower than 20% (w/w with respect to glucose) for H. polymorpha and 50% (w/w with respect to glucose) for Kloeckera sp. 2201 respectively. During growth with C₁/C₆-mixtures containing higher C₁-proportions of methanol regular growth yields for methanol were recorded which corresponded to the growth yields found with methanol as the only carbon source.The regulation of the synthesis of the enzymes of the dissimilatory pathway for methanol was found to be under multiple control. Although glucose was present in the medium methanol had a positive effect on the synthesis of these enzymes. Thus, in addition to derepression induction by methanol was also observed. This inductive effect was found to increase with increasing proportions of methanol in the mixture. Depending on the enzyme, 10–40% methanol in the mixture resulted in a maximal induction with enzyme specific activities equal to those found in cells grown with methanol as the only carbon source. No further enhancements in enzyme specific activities were observed during growth on mixtures containing more than 40% methanol.Abbreviations and terms C₁ Methanol - C₆ glucose - C₁/C₆ mixture compositions are given in % (w/w) - C₀ concentration of ¹⁴C in the inflowing medium (DPM ml^-1) - C(t) concentration of ¹⁴C incorporated in cells as a function of time t (DPM ml^-1) - d dilution rate (h^-1) - DPM disintegrations per minute - q _s q _C1 and q _C6 are specific rates of consumption of substrate, methanol and glucose respectively [g (g cell dry weight)^-1 h^-1] - q _O2 and q _CO2 are the specific rates of oxygen consumption and carbon dioxide release [mmol (g cell dry weight)^-1 h^-1] - RQ respiration quotient (q _CO2 q _O2 ^-1) - s _C1 and s _C6 are the residual concentrations of methanol and glucose in the culture liquid (g l^-1) - s _O/C1 and s _O/C6 are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - Sp.A. enzyme specific activity - x cell dry weight concentration (g l^-1) - Y _X/C1 and Y _X/C6 are growth yields on methanol and glucose respectively (g cell dry weight (g substrate)^-1 - Y _C/C1 growth yield with methanol with respect to carbon (g carbon assimilated (g carbon supplied)^-1 - _m maximum specific growth rate (h^-1)     

Hybridoma antibody content and production rate in continuous culture: Effect of dilution rate

Summary Hybridoma IND1 viability was 95% for dilution rates (D) ranging from 45 to 100% of _max (0.037 hr^–1). Over this range, the cell concentration and total protein content increased with D. Washout occurred at D=0.041 hr^–1, but the intracellular protein content continued to increase. The high- and low-content modes of the intracellular antibody distribution did not vary with D. The fraction of cells with high antibody content decreased with time, except for an increase at D=0.041 hr^–1. This decrease did not affect the specific antibody production rate, which, like the high- and low-content modes, was independent of D.     

The response of a nitrogen-limited chemostat culture of Escherichia coli B/r to pH and dilution rate steps

The effects of four pH steps and one dilution rate step are described for an ammonia-nitrogen-limited continuous culture of Escherichia coli B/r. Two of the pH steps, 6.06-5.49 and 5.96-5.60, led to prolonged transients in both cell density and rate-limiting nutrient concentration. The other two pH steps, 6.20-5.96 and 5.60-6.20, had almost no effect on the culture. The dilution rate step led to a sharp transition in the steady-state external pyruvate concentration. Monitoring of the external pyruvate concentration for the pH 5.96-5.60 step revealed that the transient phase continued long after the cell density and rate-limiting nutrient concentration returned to steady-state values. The implications for industrial and laboratory fermentations are discussed.     

Effect of glucose analog supplementation on metabolic flux distribution in anaerobic chemostat cultures of Escherichia coli

Previous work in our laboratories investigated the use of methyl alpha-glucoside (alpha-MG), a glucose analog that shares a phosphotransferase system with glucose, to modulate glucose uptake and therefore reduce acetate accumulation. The results of that study showed a significant improvement in batch culture performance and a reduction in acetate excretion without any significant effect on the growth rate in complex medium. The current study investigates the effect of supplementing the culture medium with the glucose analog alpha-MG on the metabolic fluxes of Escherichia coli under anaerobic chemostat conditions at two different dilution rates. Anaerobic chemostat studies utilizing complex media supplemented with glucose or glucose and alpha-MG at dilution rates of 0.1 and 0.4 h(-1), were performed, and the metabolic fluxes were analyzed. It was found that the addition of the glucose analog alpha-MG has an effect on the specific production rate of various extracellular metabolites. This effect is slightly greater at the higher dilution rate of 0.4 h(-1). However, the glucose analog does not cause any major shift in the central metabolic patterns. It was further observed that alpha-MG supplementation does not result in the reduction in specific acetate synthesis rate in anaerobic chemostat cultures. These results emphasize the importance of testing different strategies for metabolic manipulation under the actual operating conditions.     

Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture

The optimum dilution rate for 2, 3-butanediol (BDL) production by Paenibacillus polymyxa was 0.2 h1 and the optical purity of BDL remained above 98 % at all dilution rates. With decreasing culture pH, ethanol and BDL production increased, whereas the optical purity of BDL decreased to 94 % at pH 5.7. In the chemostat culture at pH 6.3 and a 0.1 h1 dilution rate, the optimum air supply for BDL production was 200 ml min–1 in which the O2 uptake rate was 6.7 mmol l–1 h–1. Under this condition, the optical purity of BDL decreased to 93 %. © Rapid Science Ltd. 1998     

Energetic and rate effects on methanogenesis of ethanol and propionate in perturbed CSTRs

Energetic and reaction-rate interactions between hydrogenic (hydrogen-producing) and hydrogenotrophic (hydrogen-consuming) bacteria were investigated in five perturbation experiments performed on steady-state, mixed-culture methanogenic CSTRs receiving ethanol, propionate, or both hydrogenic substrates. When a large quantity of propionate was suddenly added to a propionatefed CSTR, P(H(2) ) increased to 10(-4) atm and propionate oxidation remained energetically favorable. When ethanol was added to a CSTR receiving ethanol, P(H(2) ) rose to 6.3 x 10(-3) atm within 5 h. In both perturbations, P(H(2) ) remained at levels such that oxidation of the hydrogenic substrate remained energetically favorable throughout the transient. Sudden increase in ethanol concentration in the ethanol- and propionate-fed CSTR resulted in an increase in P(H(2) ) such that propionate oxidation became energetically unfavorable and was blocked. Propionate utilization resumed when the added ethanol was depleted and P(H(2) ) returned to its previous steady-state levels. Ethanol perturbation of ethanol- and propionate-fed CSTRs led to the formation of reduced products, including n-propanol and four-through seven-carbon n-carboxylic acids, when P(H(2) ) was elevated; these products disappeared after P(H(2) ) returned to previous, steady-state levels. The transformations were consistent with reaction energetics. Reduced product formation may have been a sink for reducing equivalents, as an alternative to oxidation for propionate utilization, as indicated by an electron equivalents balance over the time course of experiments.