John Dudley Linton

Interactions in a Methane-utilizing Mixed Bacterial Culture in a Chemostat

Summary: The interactions between the component species of a mixed bacterial culture grown on methane were investigated. The culture contained the methane-utilizing bacterium Methylococcus sp. and four heterotrophic bacteria which were unable to grow on methane but utilized products released by lysis of the Methylococcus sp. Products of methane oxidation such as methanol, formaldehyde and formate did not constitute a significant amount of the organic carbon found in the culture supernatant of the pure methane-utilizing bacterium grown in chemostat culture. The organic carbon was accounted for mainly as protein and nucleic acid and originated from a growth-dependent lysis of the methane-utilizing bacterium. In the mixed culture the level of the organic carbon was reduced and it is suggested that extracellular enzymes produced by the heterotrophic bacteria play a role in the degradation and utilization of these compounds. The major heterotrophic bacterium Pseudomonas sp. ncib11310 produced an extracellular neutral protease that degraded the proteins present in the culture supernatant of the methane-utilizing bacterium.

The Effect of Mixtures of Glucose and Formate on, the Yield and Respiration of a Chemostat Culture of Beneckea natriegens

Beneckea natriegens oxidizes sodium formate constitutively when grown on glucose or glycerol in chemostat culture, but cannot utilize formate as the sole source of carbon and energy for growth. However, when grown on a mixture of glucose and formate (D=0.37 h-1, pH 7.6) the yield is higher than on glucose alone.The yield, expressed in terms of g bacterial dry weight g-1 glucose plus formate carbon utilized, gave a linear relationship when plotted against the total heat of combustion of glucose plus formate utilized. Extrapolation of the plot cut the abscissa at a value equivalent to the heat of combustion of formate, which suggests that formate is not utilised as a source of carbon but only energy.In cultures with nitrate as the sole source of nitrogen the yield from glucose was lower than that observed with ammonia but the addition of formate to the culture utilizing nitrate resulted in an increase in the yield from glucose to a value similar to that observed with ammonia.At a culture pH value of 7.65 unused formate (<0.15–227 mM) in the culture supernatant had no effect on respiration spiration or yield, but at a culture pH of 6.7 excess formate caused a marked increase in respiration rate and a large decrease in the yield from glucose; further decrease in the pH value caused washout of the culture. This may be explained by undissociated formic acid causing uncoupling of oxidative phosphorylation.

The use of response surface analysis to study the growth of Bacillus acidocaldarius throughout the growth range of temperature and pH

A minimal-salts medium has been optimised to support the growth of the acidophilic thermophilic bacterium Bacillus acidocaldarius. This medium was used during a study of the effect of temperature and pH on the growth rate and growth yield of this organism in batch cultures; a statistical method was used to design the experimental points, and the data were subjected to a response surface analysis which allowed the growth rate and growth yield to be predicted over the entire temperature and pH range from a minimum number of experimental points. The results indicate different responses for growth rate (optimum, 60°C, pH 4.1) and growth yield (optimum tending towards low temperature and neutral pH).

The occurrence and identification of intracellular polyglucose storage granules inMethylococcus NCIB 11083 grown in chemostat culture on methane

The accumulation of intracellular storage granules (0.03–0.5 μm) byMethylococcus NCIB 11083 when grown under conditions of ammonia limitation with methane as the sole source of carbon and energy was inversely proportional to the dilution rate. The isolated material was composed entirely of glucose residues and the infra-red spectrum exhibited characteristic absorption bands at 925 cm-1, 845 cm-1 and 745±4 cm-1, indicating the presence of α (1→4) glycosidic linkages. The polymer dissolved in hot water to give an opalescent solution that formed a violet iodine complex with an absorption maximum at 550 nm, identical to that observed with reference amylopectin. The percentage of the polysaccharide released as maltose by the action of β- and α-amylases was 55–64% and 80–90% respectively, values very similar to those obtained by the action of these enzymes on reference amylopectin and glycogen. Methylation analysis indicated that the average interior and exterior chain lengths of the polymer were 2.7 and 10.0 glucose units respectively and confirmed that theMethylococcus polyglucose is a branched polymer composed of units joined by 1→4 and 1→6 linkages. The number average molecular weight of the polymer is 2–4.5×105. The stored polymer was metabolised by the organism and its metabolism resulted in the synthesis of protein.

Exocellular Succinoglucan Production by Agrobacterium radiobacter NCIB 11883

SUMMARY: The efficiency of growth and exopolysacharide production by Agrobacterium radiobacter NCIB 11883 was examined in both carbon- and nitrogen-limited chemostat cultures. Under carbon limitation this organism exhibited two distinct Y maxO2 values, one below D = 0·15 h-1 (40 g mol-1) and the other above this dilution rate (84 g mol-1). Under nitrogen limitation optimum exopolysaccharide production occurred at low dilution rates and under these conditions accounted for virtually all the product carbon excreted. The maximum observed yield of exopolysaccharide was 3·5 g (g O2)-1 and 0·65 g (g glucose)-1. These observed yields when corrected for the cellular requirement for glucose and oxygen gave values very similar to the theoretical value if the ATP/O quotient of carbon-limited cultures grown at corresponding dilution rates was used. Thus, the efficiency of growth of both carbon- and nitrogen-limited cultures was similar once an allowance for exopolysaccharide production was made. Under conditions optimum for polysaccharide production virtually all the respiratory activity occurring over and above that required for growth was utilized in polysaccharide production. Exopolysaccharide production is a major event in energetic terms and the rate of ATP utilization for its synthesis can be equivalent to 90% of that required for cell production. Nevertheless, because of the relationship between the structure of the polysaccharide and the ATP/O quotient extant in A. radiobacter succinoglucan production supplies up to approximately 56% of its own ATP demand during the synthesis of the acid moieties that comprise this polymer.

Molar growth yields, respiration and cytochrome patterns of Beneckea natriegens when grown at different medium dissolved-oxygen tensions.

The effect of medium dissolved-oxygen tension on the molar growth yield, respiration and cytochrome content of Beneckea natriegens in chemostat culture (D 0-37 H-1) was examined. The molar growth yield (Y), the specific rate of oxygen (qo2) and glucose consumption, and the specific rate of carbon dioxide evolution were independent of the dissolved-oxygen tension above a critical value (greatest than 2 mmHg). However, the potential respiration rate increased with reduction in the dissolved-oxygen tension at values of the dissolved-oxygen tension well above the critical value. Changes in the cytochrome content occurred at dissolved-oxygen tensions well above the critical value. An increase in cytochrome c relative to cytochrome b was observed as the dissolved-oxygen tension was decreased. Reduction of the dissolved-oxygen tension to less than I mmHg caused a switch to fermentative metabolism shown by the apparent rise in YO2 and decrease in the molar growth yield from glucose. At this point the potential respiration rate (qO2) increased to its highest value, while the cytochrome pattern reverted to that observed at dissolved-oxygen tensions above 96 mmHg. There appeared to be no correlation between cytochrome content, potential qO2, in situ qO2, and cyanide sensitivity of the organism at various dissolved-oxygen tensions.

Growth Efficiency of Xanthomonas campestris in Continuous Culture

SUMMARY: Xanthomonas campestris NCIB 11854 was grown glucose-limited in continuous culture at 28 °C, pH 6.8, in a defined minimal salts medium. Whole cells contained cytochromes b, c, aa3, o-type cytochromes and possibly a1, and yielded →H+/O quotients of up to approximately 6 for the oxidation of endogenous substrates. These data, by analogy with results obtained previously with other species of bacteria, suggest the presence of up to three sites of respiratory chain energy conservation. However, molar growth yields on glucose [Y = 53.6 g dry wt bacteria (mol glucose)-1] and oxygen [Y = 26.4 g dry wt bacteria (mol O2)-1] were extremely low and indicated an ATP/O quotient of approximately 1 which was only marginally increased when corrected for polymer production. A relatively rapid decay of the pH gradient generated by in vitro respiration was observed, probably indicating either an enhanced permeability of the cytoplasmic membrane to H+ or a rapid rate of ATP turnover, either of which could in part account for the observed low growth efficiency of the organisms.

The effect of temperature and pH on the growth efficiency of the thermoacidophilic bacterium Bacillus acidocaldarius in continuous culture

The molar yield coefficients (Yglucose, YO2) of glucose-limited continuous cultures of the thermoacidophile Bacillus acidocaldarius have been measured as a function of dilution rate as well as over a range of temperature and pH (51°C to 64°C, pH 2.8–5.5) at a fixed dilution rate of approximately 0.1 h-1. The highest growth yields were observed at 51°C and pH>4.3 (Yglucose 54.8 g cells · mol glucose-1, YO2 15.0 g cells · mol O2-1), but were very much lower than those of mesophilic neutrophiles of similar respiratory chain composition to B. acidocaldarius. Even lower growth yields were observed when the temperature was raised or when the pH was lowered, lowest yields occurring at 64°C and pH 2.8 (Yglucose 23.4 g cells · mol glucose-1, YO2 5.9 g cells · mol O2-1).These decreases in growth yield could be correlated with increases in the permeability of the cytoplasmic membrane to protons, i.e. cells needed to catalyse enhanced rates of substrate oxidation in order to avoid a potentially lethal acidification of the cytoplasm. This strategy appears to be successful in that the specific death rates in situ were very low for all cultures except those growing under the most extreme conditions (64°C, pH 2.8).

Efficiency and Stability of Exopolysaccharide Production from Different Carbon Sources by Erwinia herbicola

Summary: Under conditions of glucose limitation the maximum yield of Erwinia herbicola from oxygen (Y max o2 ) and glucose (Y max gic) was 31 g mol−1 and 72 g mol−1 respectively. This corresponded to a relative ATP/O quotient between 1.1 and 1.5. The cytochrome profile indicated the absence of c-type cytochromes which is consistent with this low ATP/O quotient. During nitrogen-limited growth on glucose, the rate of polysaccharide production (0.34-0.37 g g−1 h−1) was independent of growth rate, whereas the rate of production of gluconate and 2-ketogluconate increased with growth rate. The ATP/O quotient of 1.25 derived from glucose-limited growth was used to calculate the theoretical yields of exopolysaccharide from fructose and oxygen. The values obtained were close to the observed yields once an allowance had been made for cell production. Thus the growth efficiency of E. herbicola appears to be unaltered during carbon- or nitrogen-limited growth on fructose. At an ATP/O quotient of 1.25 the synthesis of the acid moiety of the exopolysaccharide provides a significant proportion (51-65%) of the ATP needed for polymerization of the sugar backbone. Nevertheless, the rate of ATP turnover is large in relation to that required for cell production under these conditions. Polymer production may serve as a means of turning over ATP but it is not essential for growth as mutants that do not produce exopolysaccharide but turnover ATP by some other means are readily selected. The rate of ATP turnover rather than exopolysaccharide production appears to be physiologically important.

The effect of rate of respiration on sensitivity to cyanide and carbon monoxide in Beneckea natriegens grown in batch and continuous CULTURE

Respiration of cell-free extracts of the marine bacterium Beneckea natriegens was reported to be inhibited by 10 MM cyanide when ascorbate-TMPD* was the substrate but, with succinate as the electron donor, 1 mM KCN caused only 5% inhibition [l] . These results were explained in terms of a terminal branching of the electron transport system. Beneckea natriegens had been shown to possess 4 different potential terminal oxidases: cytochrome a 1, cytochrome d, cytochrome o and a CO-binding cytochrome c [2], and it was suggested by Weston and Knowles [2] that this may be further indication of a branching of the respiratory chain at the terminal oxidases, although they did not ascribe any particular cytochrome to the cyanide-insensitive pathway. Studies of cyanide sensitivity of whole cells of B. natriegens grown at different oxygen concentrations in chemostat culture failed to reveal changes in the sensitivity of the maximum potential respiration rate (i.e. respiration in the presence of excess substrate) to cyanide [3], although the relative content of cytochromes o and CO-binding c varied [4]. Further experiments showed that growth of glucose-limited cultures was resistant to inhibition by cyanide while harvested cells, supplied with excess substrate, were sensitive (Linton, Harrison and Bull, in preparation).