Continuous culture studies on the synthesis of capsular polysaccharide by Klebsiella pneumoniae K1

The synthesis of capsular polysaccharide by Klebsiella pneumoniae K1 was investigated in a minimal salts medium by continuous culture. The organism produced larger amounts of polysaccharide under nitrogen-limited conditions than under carbon-limited conditions. The synthesis of polysaccharide was dependent not only on the availability of excess carbon, but also on growth rate. The rate of polysaccharide synthesis was greatest at low dilutions, low temperature (30°C) and at neutral pH. Prolonged growth in nitrogen-limited culture resulted in the development of non-mucoid variants, possibly due to a selective growth advantage over mucoid cells. The non-mucoid isolate was more susceptible to some bacteriophages, possibly due to the reduction or absence of capsular polysaccharide.     

Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures

Summary Cryptococcus albidus var. Albidus CBS 4517 was grown in nitrogen-limited and in carbon-limited chemostat cultures. The effect of growth rate and limiting nutrient on lipid accumulation and fatty acid composition was investigated.The maximum lipid content in the biomass was, in both cultivation systems, observed at the lowest dilution rate (growth rate) tested. At this dilution rate, D=0.31 h^-1, cells from the nitrogen-limited culture contained 41% (w/w) lipid and cells from the carbon-limited culture 37%. These results indicate the ability of C. albidus, unlike other oleaginous yeasts, to accumulate lipid also in carbon-limited chemostats.The yield of lipid from carbon source was about the same at D=0.031 h^-1 in nitrogen-limited (Y _L/S=0.16 g/g) as in carbon-limited (Y _L/S=0.17 g/g) cultures and decreased with increasing growth rates. In the nitrogen-limited culture, the lipid productivity was about constant at low growth rates (0.031–0.056 h^-1) and a slight decrease was observed at D=0.08 h^-1, while the specific lipid productivity, q _L, increased to 27.5 mg/g per hour. In the carbon-limited culture, however, lipid productivity increased with increasing growth rates and reached its maximum value near _max, whereas q _L was about constant at 20 mg/g per hour.The fatty acid composition was influenced by the specific growth rate in nitrogen-limited as well as in carbon-limited cultures, although the changes were more pronounced during carbonlimitation. A decrease in the degree of unsaturation (/mole) was also observed with increasing lipid content in the cells.     

Immunological and biological relationship among flagellin of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Partial degradation of p-aminoazobenzene by a defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia

A defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia was used to accomplish the partial biodegradation of the azo-dye p-aminoazobenzene (pAAB). Kinetic experiments were conducted, under aerobic conditions, to study the mineralization of p-aminoazobenzene by the above-defined mixed culture, under aerobic conditions. The combination of two previously developed models, (Zissi et al., 1997), which describes pAAB biodegradation by Bacillus subtilis into aniline and p-phenylenediamine, and (Zissi and Lyberatos, 1999), which describes aniline biodegradation by Stenotrophomonas maltophilia, is shown to predict well the anticipated mixed culture growth and partial biodegradation of pAAB. In previous work (Zissi et al., 1997) it was observed that pphenylenediamine was unstable during the experiments therefore the fate of p-phenylenediamine was not studied. The overall kinetic model of the defined mixed culture was then used to study the behavior of the mixed culture system in a range of operating conditions in the chemostat. The partial degradation of pAAB (regarding one of the two products, aniline) was described by an interaction between the two bacteria with competitive and commensalistic elements. The two bacteria are shown to coexist in a CSTR for some ranges of the operating variables.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

The kinetics and physiology of stipitatic acid and gluconate production by carbon sufficient cultures of Penicillium stipitatum growing in continuous culture

The yield from glucose of ammonia-grown carbon-limited continuous cultures of Penicillium stipitatum was ca. 20% higher than that of nitrate-grown cultures at all growth rates examined. However, the yield from oxygen was similar during growth on both nitrogen sources. Under phosphate limitation the specific rate of gluconic acid and stipitatic acid production increased with growth rate, but the former product accounted for virtually 100% of the excreted carbon. Stipitatic acid was not produced under nitrogen limitation, and glucose supplied to the culture in excess of that required for growth was virtually quantatively converted into gluconic acid. Productivities of 11.4 g gluconic acid/L/h were stably maintained in continuous culture. Under conditions of glucose excess the enzyme glucose oxidase was excreted into the culture. The specific activity of this extracellular enzyme increased when the input glucose concentration to the culture was progressively increased. The excretion of a protein under nitrogen limitation suggests that this enzyme plays an important role under these conditions. Indeed, it was demonstrated that nitrogen-limited cultures did not overmetabolize gluconate at either pH 6.5 or 3.5, although up to 29 g/L gluconate was present in the culture. The Y(gluconate) and YO(2) of C- and N-limited gluconate-grown cultures were similar indicating that the rapid conversion of glucose to gluconate probably affords a means of regulating carbon flow in this organism. Nitrogen-limited cultures of P. stipitatum overmetabolized glucose to a much greater extent than acetate, fructose, or gluconate.     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas maltophilia PB1.

A mixed microbial culture capable of metabolizing the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A bacterium, Stenotrophomonas maltophilia PB1, isolated from the culture used RDX as a sole source of nitrogen for growth. Three moles of nitrogen was used per mole of RDX, yielding a metabolite identified by mass spectroscopy and 1H nuclear magnetic resonance analysis as methylene-N-(hydroxymethyl)-hydroxylamine-N'-(hydroxymethyl)nitroamin e. The bacterium also used s-triazine as a sole source of nitrogen but not the structurally similar compounds octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, cyanuric acid, and melamine. An inducible RDX-degrading activity was present in crude cell extracts.     

Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture.

Lipid accumulation of Candida 107, grown at dilution rates from 0.03 to the maximum of 0.21/h, with carbon, nitrogen, phosphate, and magnesium limitations in a chemostat, was maximal at about 40% (wt/wt) with nitrogen-limited medium at a dilution rate of 0.06/h, giving an efficiency of substrate conversion of 22 g of lipid per g of glucose consumed. At higher dilution rates the lipid content decreased. With carbon-limited growth, the highest lipid content (14%, wt/wt) was at the maximum dilution rate. High lipid contents also occurred with phosphate + nitrogen as double limitations of growth, with the lipid content of the yeast (about 35%, wt/wt) continuing to be near maximum at dilution rates also near maximum (0.17/h), thus giving the highest specific rate of lipid formation of any growth conditions (0.59 g of lipid/g of yeast per h). However, the efficiency of substrate utilization was only 5.2 g of lipid formed per 100 g of glucose consumed. The composition of the fatty acyl residues within the lipid remained constant over many weeks if the steady-state conditions remained unchanged. With carbon-limited growth, the degree of unsaturation of the fatty acids markedly decreased as the dilution rate was increased, but with nitrogen limitation the reverse trend was seen. In all cases, linoleic and oleic acids were the principal fatty acyl residues affected, and their relative proportions always varied in opposite directions. When magnesium was a limiting nutrient, there was a considerable increase in the proportion of myristic acid produced within the lipid. Neutral lipids (predominantly triglycerides) varied from 66 to 92% of the total lipid from carbon- and nitrogen-limited growth; phospholipids (varying from 2 to 25%) were highest in nitrogen-limited growth. The fatty acyl residues within each lipid fraction showed the same variations with changing growth rates.     

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Influence of environmental factors on endo-β-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring

The production of endo-β-1,4-glucanase by a Bacillus strain isolated from a hot spring in Zimbabwe was studied in batch culture, chemostat culture, and carbon dioxide-regulated auxostat (CO₂-auxostat). The bacteria produced the enzyme in the presence of excess glucose or sucroso, but not under carbon-limited conditions in a chemostat using mineral medium. There was a specific growth rate dependent linear increase in enzyme production in glucose excess, nitrogen-limited chemostat cultures. A high specific growth rate of 2.2 h^-1 and a high rate of enzyme production of 362 nkat (mg dry mass h)^-1 were attained under nutrient rich conditions in the CO₂-auxostat. The bacteria had the highest specific growth rate and endo-β-1,4-glucanase enzyme production at 50° C. The maximum specific growth rate and the rate of enzyme production increased when yeast extract and tryptone were added in increasing amounts to the mineral medium used for cultivation in separate experiments. Increasing the glucose concentration in the CO₂-auxostat cultures increased the rate of enzyme production but did not affect the specific growth rate.     

Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae

For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast.     

Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa.

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.     

Influence of medium composition on sporulation byStreptomyces coelicolor A3(2) grown on defined solid media

Summary Reproducible sporulation using solid-plate cultures ofStreptomyces coelicolor A3(2) was obtained on a defined medium with glucose as the primary source of carbon and sodium nitrate as the sole source of nitrogen. The type of agar used as a solidifying agent, and the inclusion of trace salts to the medium, significantly affected sporulation. An interrelationship between the medium carbon to nitrogen ratio and sporulation was observed, with clear promotion of spore formation under nitrogen-limited conditions, whilst carbon-limited conditions suppressed sporulation. The influence of medium phosphate upon sporulation is also reported, with high concentrations exerting an inhibitory effect.     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Lipid Accumulation in an Oleaginous Yeast (Candida 107) Growing on Glucose Under Various Conditions in a One- and Two-Stage Continuous Culture

Lipid accumulation and fatty acid composition in Candida 107 have been studied using a two-stage continuous culture system in which the first vessel was run under carbon-limited conditions and then the entire output was passed into a second vessel, where lipid accumulation was stimulated by adding only glucose. Maximum lipid accumulation (28% of yeast [dry weight]) occurred for a volume ratio of vessel 1 to vessel 2 of 3:5, with 30 g of glucose per liter being added to vessel 2 operated at 25°C with an aeration rate of between 0.1 and 1.0 volume of air/volume of medium per min. Although the maximum specific rate of lipid formation (0.05 g of lipid/g of yeast per h) was higher than in a nitrogen-limited, single-stage system, the efficiency of lipid formation was much less and never exceeded 14 g of lipid produced per 100 g of glucose consumed. The fatty acid composition was not significantly altered in either the two-stage or single-stage culture (nitrogen-limited) systems by changes in growth temperature (from 19 to 33°C) or aeration rates (0.05 to 1.0 volume of air/volume of medium per min); or, in the two-stage system, by changes in the residence time of the yeast in the second vessel (from 3.2 to 24.4 h), or, in the single-stage system, by changes in pH (from 3.5 to 7.5). Only when the concentration of glucose entering vessel 2 of the two-stage system was less than 30 g/liter did significant changes in the fatty acids occur. Thus, although a two-stage continuous culture system allows lipid accumulation to be separated from the growth phase, it offers no practical advantages over a single-stage system as a means of producing microbial oils and fats.     

Starvation response of Saccharomyces cerevisiae grown in anaerobic nitrogen- or carbon-limited chemostat cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(-1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Studies on the microflora associated with xenic cultures of Entamoeba gingivalis

The microflora associated with xenic stock cultures (ATCC 30927) of Entamoeba gingivalis, the major protozoan of the human oral cavity, were isolated and identified as Citrobacter diversus, Yersinia enterocolitica, Acinetobacter anitratus and Pseudomonas maltophilia. In studies to determine whether the bacterial isolates were able to utilize rice starch as a sole carbon source, Y. enterocolitica exhibited excellent growth in rice starch minimal medium and TYSGM-9 medium (with rice starch), but growth was weak in TYSGM-9 medium (without rice starch). C. diversus, A. anitratus and P. maltophilia exhibited poor growth in rice starch minimal medium, but they produced excellent growth in TYSGM-9 medium with or without rice starch. In order to determine the effect of the rice starch hydrolysis on Entamoeba growth, the filtrate from each isolate grown in rice starch minimal medium was added to an E. gingivalis culture grown in TYSGM-9 medium. The filtrate from a Y. enterocolitica culture grown in rice starch minimal medium enhanced E. gingivalis growth, but the filtrates from cultures of C. diversus, A. anitratus and P. maltophilia suppressed E. gingivalis growth. This supported the concept that Y. enterocolitica is capable of metabolizing rice starch into intermediate products, which in turn can be utilized by the amoeba.     

Behaviour of Nif− mutants of Rhodobacter capsulatus in photosynthetic, ammonia-limited chemostat cultures

Abstract Nif⁻ mutants of Rhodobacter capsulatus carrying mutations either in the nifR4 regulatory gene or in the nifH structural gene both outgrew the wild-type strain B10 in mixed chemostat cultures under conditions favouring nitrogenase-mediated H₂ production by the wild-type (ammonia as limiting nutrient, inert argon atmosphere, light as energy source), whereas under aerobic conditions in the dark, or in batch culture, the growth of Nif⁻ mutants was not favoured. Nitrogenase-mediated H₂ production therefore appears to be detrimental to the growth of R. capsulatus in nitrogen-limited continuous culture, as may also be the case for other nitrogen fixers.