Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

Amino acid consumption by Chloroflexus aurantiacus in batch and continuous cultures

Amino acid consumption was studied with batch and continuous chemostat cultures of Chloroflexus aurantiacus grown phototrophically in complex medium with casamino acids (Pierson and Castenholz 1974). Amino acids like Arg, Asx, Thr, Ala, Tyr, which were utilized during the early exponential phase by cells grown in batch cultures were consumed in chemostat cultures essentially at any of the dilution rates employed (0.018–0.104 h^-1). Those amino acids which were taken up during subsequent phases of growth were consumed in chemostat cultures preferentially at low dilution rates. For example, the consumption of Glx was enhanced during the late exponential phase and at low dilution rates. At high dilution rates Glx was not consumed at all. Since Glx utilization largely paralleled bacteriochlorophyll formation, it is discussed that formation of the photopigment depends on the intracellular availability of Glu as the exclusive precursor for tetrapyrrole synthesis.     

Expression of BHRF1 improves survival of murine hybridoma cultures in batch and continuous modes

Cell death by apoptosis limits growth and productivity in most animal cell cultures. It is therefore desirable to define genetic interventions to generate robust cell lines with superior performance in bioreactors, either by increasing specific productivity, life-span of the cultures or both. In this context, forced expression of BHRF1, an Epstein–Barr virus-encoded early protein with structural and functional homology with the anti-apoptotic protein Bcl-2, effectively protected hybridomas in culture and delayed cell death under conditions of glutamine starvation. In the present study, we explored the potential application of BHRF1 expression in hybridomas for long-term apoptosis protection under different biotechnological process designs (batch and continuous) and compared it to strategies based on Bcl-2 overexpression. Our results confirmed that long-term maintenance of the anti-apoptotic effect of BHRF1 can be obtained using bicistronic configurations conferring enhanced protection compared to Bcl-2, even in the absence of selective pressure. Such protective effect of BHRF1 is demonstrated both in batch and continuous culture. Moreover, a further analysis at high cell densities in semi-continuous perfusion cultures indicated that the mechanism of action of BHRF1 involves cell cycle arrest in G0–G1 state and this is translated in lower numbers of dead cells.     

Stability of plasmid-bearing microorganisms in batch and continuous cultures

The stability of five microbial strains bearing a domestic and/or exotic plasmid was investigated in continuous culture to obtain basic information on the fate of genetically engineered microorganisms released in the natural environment.The three strains with an exotic plasmid were constructed by the conjugal or mobilized transfer of conjugative plasmid R100-1 and non-conjugative plasmid RSF2124. Plasmid loss occurred only at the declining growth phase of batch culture of the transconjugants; the ratio of plasmid-free cells was 40–50% at the end of the culture, independent of the strains, whereas the plasmid in the native host cells was maintained at almost 100% of stability.In continuous culture of the transconjugant cells, the population ratio of plasmid-free cells at the pseudo-steady state was between 5–80% depending on the strain. The plasmid-bearing cells were not washed out of the continuous fermentor for 43 generations but maintained their quasi-stable concentration with some degree of oscillation. Simultaneous loss and retransfer of the plasmid from and to its host cells is suggested for the explanation.     

Isolation of ethanol-tolerant mutants of yeast by continuous selection

Summary Mutants of Saccharomyces uvarum, 5D-cyc with increased tolerance to ethanol have been isolated by a continuous selection technique which allows the culture itself to determine the intensity of selection via a feedback control circuit. The output of CO₂ from a continuous culture of the yeast was monitored using an infrared analyser and the signal from that analyser fed to a potentiometric controller which regulated the introduction of a concentrated ethanol solution into the culture vessel. The frequency of ethanol addition to the culture thus increased as the tolerance of the organisms improved.The use of this system permitted the selection of mutants of yeast which were viable in the presence of 12% w/v ethanol and which showed higher fermentation rates (as measured by CO₂ production) than the wild-type in the presence of 10% w/v ethanol and above. The technique of continuous selection with feedback should be generally applicable to the isolation of mutants of any microorganism to improved tolerance to any inhibitory condition of either its physical or chemical environment.     

Fermentation kinetics of recombinant yeast in batch and fed-batch cultures

Fed-batch cultures of recombinant microorganisms have attracted attention as they can separate cell growth stage from cloned-gene expression phase during fermentations. In this work, the effect of different glucose feeding strategies on cell growth and cloned gene expression was studied during aerobic fed-batch fermentations of recombinant yeast, containing the plasmid pRB58. The plasmid contains the yeast SUC2 gene, which codes for the enzyme invertase. Some feeding policies resulted in a constant glucose concentration inside the fermentor, while others deliberately introduced a cyclic variation. The cell mass yield was found to be higher at low glucose concentrations, thus indicating a shift to the more energy-efficient respiratory pathway. The SUC2 gene expression was derepressed at glucose levels below 2 g/L. The response of specific invertase activity to changes in the medium glucose concentration was found to be almost immediate.     

Cometabolic degradation of chloroallyl alcohols in batch and continuous cultures

The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis- and trans-3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal.     

Kinetics of protein release from yeast sonicated in batch and flow systems at 20 kHz

The release constant, k, of brewers yeast sonicated at powers up to 200 W at 20 kHz has been shown to be independent of cell concentration up to values of 60 g made up to 100 ml. It is inversely proportional to the volume of the treatment vessel in the range 75 to 450 ml, and almost proportional to the input acoustic power from 60 to 195 acoustic watts. A flow system is described and a relationship linking protein release, flow rate, and the protein release constant, determined from batch experiments, is derived. Good agreement between the theoretical prediction of protein release and experimental results with the flow system was obtained.     

Synchronous protein cycling in batch cultures of the yeast Saccharomyces cerevisiae at log growth phase

The assumption that cells are temporally organized systems, i.e. showing relevant dynamics of their state variables such as gene expression or protein and metabolite concentration, while tacitly given for granted at the molecular level, is not explicitly taken into account when interpreting biological experimental data. This conundrum stems from the (undemonstrated) assumption that a cell culture, the actual object of biological experimentation, is a population of billions of independent oscillators (cells) randomly experiencing different phases of their cycles and thus not producing relevant coordinated dynamics at the population level. Moreover the fact of considering reproductive cycle as by far the most important cyclic process in a cell resulted in lower attention given to other rhythmic processes. Here we demonstrate that growing yeast cells show a very repeatable and robust cyclic variation of the concentration of proteins with different cellular functions. We also report experimental evidence that the mechanism governing this basic oscillator and the cellular entrainment is resistant to external chemical constraints. Finally, cell growth is accompanied by cyclic dynamics of medium pH. These cycles are observed in batch cultures, different from the usual continuous cultures in which yeast metabolic cycles are known to occur, and suggest the existence of basic, spontaneous, collective and synchronous behaviors of the cell population as a whole.     

Analysis of protein and cell volume distribution in glucose-limited continuous cultures of budding yeast

Method of flow cytometric analysis have recently been developed that make it possible to obtain segregated data on a single cell basis. In particular, it has been previously demonstrated that protein distributions obtained by flow cytometry give information about the law of growth of the cell population and the law of growth of the single cell; thus these distribution show how the microbial population is actually growing at the moment of the analysis and may yield more accurate and predictive information. We have extended the analysis of protein distribution and cell volume distribution to continuous cultures of Saccharomyces cerevisiae growing in a glucose-limited chemostat. We have found that: (1) to each dilution rate corresponds a given protein and volume distribution that does not change with time in steady state cultures; (2) there is a good proportionality between the average cell volume and the average protein content; (3) the protein distribution obtained can be easily analyzed with the model of growth of yeast previously developed in our laboratory; (4) the analysis of perturbed states shows that both protein distribution and volume distribution change very quickly; thus they are very sensitive parameters and can be used for monitoring and controlling industrial fermentation.     

Studies on exopolysaccharide release by diazotrophic batch cultures of Cyanospira capsulata

Summary Diazotrophic batch cultures of Cyanospira capsulata producing large amounts of a soluble exopolysaccharide (EPS) were studied over a period of about 30 days under continuous illumination. The thickness of the capsule surrounding the trichomes remained almost the same throughout the growth phases and the EPS was continuously released into the medium at a rate which was roughly constant throughout the culture period. A mean EPS productivity of about 6 g m^–2 day^–1 was attained. Purified EPS samples exhibited a saccharidic composition consisting of four neutral sugars (glucose, mannose, fucose and arabinose) and galacturonic acid in a molar ratio of 1:1:1:1:2, respectively. The EPS was also characterized by the presence of pyruvic residues and by a protein content of about 2%. O-Acetyl groups and sulphate residues were not detected. The massive release of this polysaccharidic material into the liquid medium made the cultures progressively more viscous. Offprint requests to: M. Vincenzini     

Effect of ammonium on chloramphenicol production by Streptomyces venezuelae in batch and continuous cultures

Cultures of Streptomyces venezuelae presented with a mixture of ammonium and an amino acid as nitrogen sources used both compounds together. Absence of ammonium repression of alternative nitrogen assimilation pathways was also observed when ammonium was added to cultures already growing on proline. The presence of ammonium in the medium ab initio depressed the yield of chloramphenicol. However, its addition to a culture growing on proline caused only a temporary inhibition of antibiotic synthesis, even when sufficient ammonium was added to create an excess. Continuous cultures supplied with ammonium as the growth-limiting nutrient showed no significant change in specific antibiotic production at different specific growth rates. The overall results indicate that in S. venezuelae neither nitrogen utilization pathways nor chloramphenicol biosynthesis is controlled by nitrogen repression.     

Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of phenol under denitrifying conditions by a bacterial consortium was studied both in batch and continuous cultures. Anaerobic degradation was dependent on NO_f3 ^p– and concentrations up to 4 mm phenol were degraded within 2–5 days. During continuous growth in a fermenter, steady states could be maintained at eight dilution rates (D) corresponding to residence times between 12.5 and 50 h. Culture wash-out occurred at D=0.084 h^–1. The kinetic parameters obtained for anaerobic degradation of phenol under denitrifying conditions by the consortium were: maximam specific growth rate = 0.091 h^–1; saturation constant = 4.91 mg phenol/l; true growth yield = 0.57 mg dry wt/mg phenol; maintenance coefficient = 0.013 mg phenol/mg dry wt per hour. The Haldane model inhibition constant was estimated from batch culture data giving a value of 101 mg/l. The requirement of CO₂ for the anaerobic degradation of phenol with NO_f3 ^p– indicates that phenol carboxylation to 4-hydroxybenzoate was the first step of phenol degradation by this culture. 4-Hydroxybenzoate, proposed as an intermediate of phenol carboxylation under these conditions, was detected only in continuous cultures at very low growth rates (D=0.02 h^–1), but was never detected as a free intermediary metabolite either in batch or in continuous cultures. Correspondence to: N. Khoury     

Reduction of hexavalent chromium by Pseudomonas fluorescens LB300 in batch and continuous cultures

Batch and continuous cultures of Pseudomonas fluorescens LB300 were shown to reduce hexavalent chromium, Cr(VI), aerobically at neutral pH (pH 7.0) with citrate as carbon and energy source. The product of Cr(VI) reduction was previously shown and confirmed in this work to be trivalent chromium, Cr(III), by quantitative reoxidation to Cr(VI) with KMnO₄. In separate batch cultures (100 ml) containing initial Cr(VI) concentrations of 314.0, 200.0 and 112.5 mg Cr(VI) L^–1, the organism reduced 61%, 69% and 99.7% of the Cr(VI), respectively. In a comparison of stationary and shaken cultures, the organism reduced 81% of Cr(VI) in 147 h in stationary culture and 80% in 122 h in shaken culture. In continuous culture, the organism lowered the influent Cr(VI) concentration by 28% with an 11.7-h residence time, by 39% with a 20.8-h residence time and by 57% with a 38.5-h residence time. A mass balance of chromium in a continuous culture at steady state showed an insignificant uptake of chromium by cells of P. fluorescens LB300. Correspondence to: P. C. DeLeo     

Anaerobic degradation of p-cresol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of p-cresol under denitrifying conditions by a bacterial consortium was studied in batch and continuous cultures. Concentrations up to 3 mm were degraded within 5–6 days with 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde and 4-hydroxybenzoate as intermediates. Steady states could be maintained at only one dilution rate, D=0.04 h^–1. A further increase in the dilution rate to 0.0 8 h^–1 resulted in culture wash-out. An estimation of the Saturation constant was made (<1 mg/l), taking the maximum specific growth rate as 0.045 h^–1, thus yielding a value of 0.125 mg p-cresol/l. Correspondence to: N. Khoury     

Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5

We find that overexpression in yeast of the yeast MCK1 gene, which encodes a meiosis and centromere regulatory kinase, suppresses the temperature-sensitive phenotype of certain mutations in essential centromere binding protein genes CBF2 and CBF5. Since Mck1p is a known serine/threonine protein kinase, this suppression is postulated to be due to Mck1p-catalyzed in vivo phosphorylation of centromere binding proteins. Evidence in support of this model was provided by the finding that purified Mck1p phosphorylates in vitro the 110 kDa subunit (Cbf2p) of the multimeric centromere binding factor CBF3. This phosphorylation occurs on both serine and threonine residues in Cbf2p.     

Extracellular xylanase production by two thermophilic alkali-tolerant Bacillus strains in batch and continuous cultures

Xylanase production of newly isolated thermophilic alkali-tolerant Bacillus sp. strain SP and strain BC was investigated in batch and continuous cultures. Enzyme synthesis was inducible with both strains and was observed only in xylan-containing media. Xylan from oat spelt is a better inducer than xylan from birch for strain Bacillus sp. BC while such difference was not observed for strain SP. Compared with batch cultures xylanase production of both strains increased about two times and its rate became more than four times faster in continuous cultures at a dilution rate of 0.2 h(-1).     

Analysis of metabolic fluxes in batch and continuous cultures of Bacillus subtilis

It is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose-limited continuous cultures of Bacillus subtilis by combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by-products, CO(2) evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) used in biosynthesis. (c) 1993 John Wiley & Sons, Inc.