Steady-state Continuous Cultures Research Articles

The Production of Alginate by Pseudomonas mendocina in Batch and Continuous Culture

The effect of growth conditions on the production of alginate by highly mucoid strains of Pseudomonas mendocina was investigated, using pH-controlled batch and continuous cultures. In batch culture, alginate was produced both during the exponential and stationary phases of growth. A polysaccharide concentration of 20 g 1−1 was obtained from medium containing glucose (50 g l−1). In steady-state continuous culture under nitrogen limitation (dilution rate, D = 0·05 h−1), a similar alginate concentration was attained; more than 60% of the utilized glucose was converted to alginate and the polysaccharide concentration was ten-fold the biomass concentration. Increasing the dilution rate above 0·1 h−1 decreased the proportion of glucose converted to alginate although the biomass was unchanged. The optimum dissolved oxygen tension (d.o.t.) for alginate production in nitrogen-limited continuous culture was approximately 40 mm Hg (1 mm Hg is approximately 133 Pa). The respiration rate and bacterial density were independent of d.o.t. above 10 mm Hg. Under oxygen limitation, no alginate was produced but acetate was excreted into the culture fluid. Variants producing little alginate arose during growth in continuous culture. Under carbon limitation, alginate production was substantially decreased but not eliminated.

Effects of copper concentration in continuous culture on the aa 3-type cytochrome oxidase and respiratory chains of Paracoccus denitrificans

The role(s) of copper in a bacterial cytochrome oxidase of the aa3-type was investigated by growth of Paracoccus denitrificans NCIB 8944, in batch and steady state continuous culture, in a medium from which the bulk of the copper had been extracted. In a medium containing approximately 0.02 μM copper, cellular copper content, cytochromes a+a3 and cytochrome a3 were reduced to 55%, 58% and 33% respectively of control values and there were also less marked decreases in cytochromes c+c1 (to 85%) and a CO-binding b-type cytochrome, possibly cytochrome o (to 71%). Copper deficiency elicited in reduced minus oxidized difference spectra a shift to shorter wavelengths and narrowing of the band width of the α-band of the oxidase, and loss of a (negative) band near 830 nm attributable to CuA (the copper functionally associated with haem a in the oxidase complex). The oxidase in copper-deficient cells reacted with oxygen to form the oxy “Compound A” at rates similar to that in control cells but CO recombination to ferrous haem a3 was slowed 4-fold in the copper deficient case. The results are interpreted as indicating loss of CuA and changes in the proportions of haems a and a3 with retention of catalytic activity. Titrations of respiration rates with antimycin suggested that copper deficiency did not result in diversion of electron flux through an antimycin A-insensitive, cytochrome o-terminated branch of the respiratory chain.

Continuous and biomass recycle fermentations of Clostridium acetobutylicum

The availability and demand of biosynthetic energy (ATP) is an important factor in the regulation of solvent production in steady state continuous cultures of Clostridium acetobutylicum. The effect of biomass recycle at a variety of dilution rates and recycle ratios under both glucose and non-glucose limited conditions on product yields and selectivities has been investigated. Under conditions of non-glucose limitation, when the ATP supply is not growth-limiting, a lower growth rate imposed by biomass recycle leads to a reduced demand for ATP and substantially higher acetone and butanol yields. When the culture is glucose limited, however, biomass recycle results in lower solvent yields and higher acid yields.

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (me) rather than the max growth yield coefficient (Yxsάx). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.

Oxidation of an inhibitory substrate by washed cells (oxidation of phenol by Pseudomonas putida)

The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. Then the equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.

Determination of the Monod substrate saturation constant for microbial growth

Methods used to determine the Monod substrate saturation constant for microbial growth are surveyed. The preferred and most accurate method is to assay the concentrations of growth rate-limiting nutrients in steady-state continuous cultures. But, this is not always possible due to the lack of sufficiently sensitive assay methods or due to high nutrient fluxes in rapidly growing cultures. It is suggested that an acceptable and simple alternative method for aerobic microorganisms is to measure initial oxygen uptake rates during growth in the presence of different initial concentrations of growth rate-limiting nutrient. It is important in this method that the microbial cells are taken from rapidly growing cultures and are suspended in a medium permitting growth.

Determination of the Monod substrate saturation constant for microbial growth

Methods used to determine the Monod substrate saturation constant for microbial growth are surveyed. The preferred and most accurate method is to assay the concentrations of growth rate-limiting nutrients in steady-state continuous cultures. But, this is not always possible due to the lack of sufficiently sensitive assay methods or due to high nutrient fluxes in rapidly growing cultures. It is suggested that an acceptable and simple alternative method for aerobic microorganisms is to measure initial oxygen uptake rates during growth in the presence of different initial concentrations of growth rate-limiting nutrient. It is important in this method that the microbial cells are taken from rapidly growing cultures and are suspended in a medium permitting growth.

Catabolite repression of amylase synthesis in yeast

Amylase synthesis by the yeasts Saccharomycopsis fibuligera and Schwanniomyces castellii and alluvius is repressed by glucose. Steady state continuous culture data for amylase activity, E, biomass concentration, X, and reducing sugar concentration, S, were fitted to the three-parameter catabolite repression model E/X = [1 + a(S/X)]/[1 + b(S/X)], and biomass productivity, DX, and amylase productivity, DE, were determined for S. castellii and S. alluvius.

Growth and enzyme synthesis during continuous culture of Trichosporon cutaneum on phenol

The soil yeast Trichosporon cutaneum was grown in continuous culture with phenol as the sole carbon source. The cultures were operated as carbon-limited chemostats or as steady-state continuous cultures without carbon limitation. Selected comparative runs were also conducted on glucose or acetate as carbon source. In addition to growth parameters, the activities of several intracellular enzymes were determined, comprising those directly involved in the degradation of phenol as well as auxiliary enzymes required for the generation of reducing power. All enzymes were assayed in detergent-permea-bilized cells. Phenol was found to serve as an excellent carbon source, comparable to glucose or acetate. The utilization of phenol in T. cutaneum is very efficient as indicated by a low maintenance requirement (0.01 g phenol/g cells.h). The cell yields obtained were on the order of 0.8 g cells/g phenol. Although the phenol-limited chemostats were run with fully phenol-induced cells, a further increase in the activities of isocitrate DH(NADP(+)), maleate DH and the phenol-degrading enzymes occurred after transition to nonlimiting condition. Enzyme activities increased in parallel with increasing phenol levels in the effluent, as well as with increasing toxicity. The significance of this phenomenon is discussed. The significance of this phenomenon is discussed. This elevation in enzyme activities in not related to an increase in specific growth rate.

Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK).

Steady-state chemostat cultures of Azotobacter vinelandii strain CA11, carrying a deletion of genes encoding the structural polypeptides of nitrogenase nifHDK, were established in a simple defined medium chemically purified to minimize contamination by Mo. The medium contained no utilizable N source. Growth was dependent on N2 (1.1 X 10(8) viable cells X ml-1 at D = 0.176 h-1), and was inhibited by Mo (20 nM). DNA hybridization showed the deletion to be stable during prolonged (55 days) growth in the chemostat (132 doublings). Since batch cultures, using unsupplemented 'spent' chemostat medium, showed good growth (1.9 X 10(8) cells X ml-1), no requirement for subnanomolar concentrations of Mo was found. The biomass yield, as the dilution rate (D) was varied, showed that the N content of the culture, protein and dry wt. increased as D was decreased, indicating that neither N2 nor O2 were limiting growth. The limiting nutrient was not identified. Substantial amounts of H2 were evolved by the chemostat cultures, probably as the result of inhibition of O2-dependent hydrogenase activity by nitrilotriacetic acid present in the medium. Over a range of D values approx. 50% of the electron flux through the alternative system was allocated to H+ reduction. C2H2 was a poor substrate, being reduced at 0.14-0.1 times the rate of N2 fixation, calculated from the N content of the cells. SO4(2-)-limited steady-state continuous cultures of strain UW136 (wild-type for nifHDK) had a 2-fold greater biomass in the presence of MoO4(2-) (1 microM). The significance of this finding for 'Mo-limited' continuous cultures [Eady & Robson (1984) Biochem. J. 224, 853-862] is discussed.

The dependence of primary production in the mixed layer of the eastern tropical Pacific on the vertical transport of nitrate

Primary production measured in the mixed layer of the eastern tropical Pacific around 10°N ranged from 0.24 to 3.49 μg C l −1 h −1. Regression of chlorophyll on productivity indicates that 89% of the variation in primary production can be attributed to variations in phytoplankton standing stock, suggesting that algal growth rates are relatively invariant. A calculated carbon:chlorophyll ratio of 86.6, when combined with a mean productivity index of 7.5 mg C mg Chl −1 h −1, results in an average growth rate of 1.0 d −1 (0.083 h −1), despite concentrations of nitrate and ammonium frequently below limits of analytical detection. Positive variation of algal growth rates with zooplankton standing stocks and algal standing stocks with nitrate supply rates are discussed in relation to analogous features of the steady-state continuous culture. It is concluded that primary production in the eastern tropical Pacific is not controlled by inorganic nitrogen concentrations, but rather by rates of nitrate supply by vertical mixing.

Invertase activity of intact cells of Saccharomyces cerevisiae growing on sugar cane molasses. I. Steady‐state continuous culture tests

During the steady-state continuous culture of Saccharomyces cerevisiae on sugar cane blackstrap molasses under different experimental conditions, oscillatory variations of the invertase activity of the intact yeast cells were observed. The continuous morphological changes of the cells wall and of the periplasmic space affecting the interaction between invertase and sucrose molecules could be responsible by the observed oscillatory phenomena. The average invertase activity at the steady state is linearly correlated to the cell's growth rate.

The fate of planktonic primary production1

The carbon dynamics of phytoplankton communities in six different lakes were investigated. In each lake carbon‐specific rates of production (b) and loss (d) were highly correlated and closely balanced through most of the year. As a result carbon‐specific growth rates were usually very low and carbon standing crop was relatively stable between sampling dates. Major fluctuations in algal biomass occurred only when the production‐loss balance was significantly perturbed. The frequency of these perturbations and the seasonal variability of b and d differed between lakes. The most consistent pattern was found in the low altitude lakes of the temperate zone, where b and d were highest in summer and lowest in winter. Values were also low during the spring and fall mixing periods when perturbations in the production‐loss balance caused major peaks in carbon standing crop. The fate of primary production varied significantly between lakes. In most of the lakes considered the majority of carbon fixation was consumed in metabolic processes. However, in Lake Wingra a large part of the production was apparently lost through grazing and sedimentation. Differences in the average values of b, d, and % metabolic loss between lakes appeared to depend on the combined loss rate due to sinking and grazing. These results suggest a general pattern of carbon dynamics similar to that found in steady state continuous cultures.

Nitrogen metabolism in Xanthobacter H4-14

N2-fixation was investigated in the chemoautotrophic hydrogen bacterium Xanthobacter H4-14. N2-fixing batch cultures of this organism could only be grown at pO2 values of around 0.02 bar, and in continuous culture dissolved oxygen tensions above 16 μM were found to inhibit N2-fixation. Xanthobacter H4-14 utilized a variety of amino acids, nitrate and ammonia as nitrogen sources. Cell-free extracts from steady-state continuous cultures of ammonia grown, nitrate grown and N2-fixing Xanthobacter were assayed for the presence of ammonia assimilation enzymes. No alanine dehydrogenase or glutamate dehydrogenase activity was detected. Ammonia was assimilated exclusively via the glutamine synthetase/glutamate synthase pathway, irrespective of the extracellular concentration of ammonia.

Comparison of ethanol feed-type on yeast growth at various po2 levels

The growth characteristics of the yeastCandida utilis in the individual stages of a multistage tower fermentor obtained with single- and multistream ethanol feeding were compared. In addition, various types of pure oxygen supply were tested for each type of ethanol feed. The results, obtained from steady-state continuous cultures, provided evidence that the two types of ethanol and oxygen supply significantly affect the cell growth rate, ethanol dissimilation rate, acetate excretion in the medium, biomass yield and productivity.

Kinetics of ethanol production by yeast in continuous culture

The rate of fermentation of glucose by Saccharomyces uvarum in steadystate continuous culture in excess of substrates showed non-competitive inhibition kinetics with respect to ethanol. A model is presented which predicts that growth stops at a finite ethanol concentration, which was calculated to be 95 gl-1 for the system used here. The observed maximum ethanol concentration in a single stage continuous culture was 92 gl-1.

Continuous Cultivation for Apparent Optimization of Defined Media for Cellulomonas sp. and Bacillus cereus

Steady-state continuous culture was used to optimize lean chemically defined media for a Cellulomonas sp. and Bacillus cereus strain T. Both organisms were extremely sensitive to variations in trace-metal concentrations. However, medium optimization by this technique proved rapid, and multifactor screening was easily conducted by using a minimum of instrumentation. The optimized media supported critical dilution rates of 0.571 and 0.467 h for Cellulomonas and Bacillus, respectively. These values approximated maximum growth rate values observed in batch culture.

The Continuous Culture of Phytoplankton: Mathematical Equivalence Among Three Steady-State Models

Within a framework of three-state variables-and the corresponding differential conservation equations-three alternative models of phytoplankton growth in a steady-state continuous culture are formulated, based on commonly accepted uptake and growth functions. Simple algebraic analysis of the three models proves them strictly equivalent, making them merely three (of many) minimal representations of one underlying metamodel. Correspondingly, and as sometimes implied (Droop 1973a, 1973b; Chisholm 1976), each of the three models is an equally valid (or equally invalid) formal description of algal growth at steady state. Experimental results from chemostats are not in principle applicable to dynamic conditions. Neither do they permit the distinction between internal and external nutrient pools as the determinant of growth.

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia ( Rhizobium sp.), newly fixed NH 4 + is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg 2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg 2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH 4 +, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH 4 + produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.

Production of a yeast killer factor in the chemostat and the effects of killer yeasts in mixed continuous culture with a sensitive strain.

In batch culture in a complex medium the killer yeasts NCYC 738 and NCYC 235 gave maximal killer activity when grown in the pH ranges 4·2–4·4 and 4·6–4·8 respectively. Incubation of culture filtrates of NCYC 738 for 10 h at 25 °C or 2 h at 29 °C resulted in a 50% reduction in activity. The addition of bovine serum albumin or gelatine to a complex medium stabilized killer activity. In a defined medium the addition of yeast extract stimulated the production of killer activity. When killer yeast NCYC 738 was grown in a chemostat, killer activity was influenced by temperature, pH and the rate at which the culture was stirred. The production of killer activity was growth‐linked and increased as dilution rate was raised to a maximum of 0·15 h”1. Steady state continuous cultures of the sensitive strain, NCYC 1006, were contaminated deliberately with either killer or killer‐cured strains. During the first 30 h cultivation, the cell concentration of both strains increased. Subsequently the sensitive strain was displaced from the culture. When killer‐cured NCYC 738 was added, the rate of displacement was proportional to the culture temperature. However, with killer NCYC 738 increase of temperature reduced the rate of displacement. When killer NCYC 235 was employed, a lowering of pH decreased the rate of displacement but had no effect when killer‐cured NCYC 235 was used.