Methanol-limited Cultures Research Articles

The Regulation of Exopolysaccharide Production and of Enzymes Involved in C1 Assimilation in Methylophilus methylotrophus

Summary: Methylophilus methylotrophus produced viscous and non-viscous exopolysaccharides (EPS) when grown in batch culture. Both types contained glucose, galactose, mannose and an unidentified 6-desoxyhexose, and were substituted with pyruvate and acetate residues. When the organism was grown in continuous culture only the non-viscous EPS was synthesized; the rate of production was 18·5 mg h−1 (g biomass)−1 in methanol-limited cultures and increased by approximately 3- and 4-fold when growth was limited by oxygen or nitrogen respectively. The specific activity of methanol dehydrogenase in cell extracts was relatively low when bacteria were grown under conditions of methanol excess and increased 2-fold in carbon-limited cells, reflecting the need to scavenge the small amounts of available methanol. In contrast, the specific activities of several key enzymes of the ribulose monophosphate (RuMP) pathway were greater in cells grown under conditions of nitrogen or oxygen limitation than when growth was Limited by the availability of carbon, indicating the potential for increased carbon flux round the cycle when excess methanol was present in the growth medium. When methylotrophs are grown under conditions of methanol excess it is important that there is a mechanism to prevent the overproduction of formaldehyde, and we suggest that these changes in EPS production and in the specific activities of the key enzymes of the RuMP cycle are necessary for the efficient removal of this toxic metabolite of methanol.

Environmental Regulation of the Methanol Oxidase System of Methylophilus methylotrophus

Summary: Methylophilus methylotrophus was grown in continuous culture at different dilution rates under either methanol or oxygen limitation. Methanol dehydrogenase and cytochrome oxidase aa 3 were maximally repressed in oxygen-limited cultures and also in methanol-limited cultures at dilution rates close to mUmax, whereas cytochrome oxidase co was maximally repressed in methanol-limited cultures and also in oxygen-limited cultures at dilution rates close to μmax. The observed changes in the activity and concentration of methanol dehydrogenase were accompanied by approximately parallel changes in the whole cell respiration rate with ethanol, formaldehyde or acetaldehyde, but not with methanol. These and other results suggest that whole cell methanol oxidase activity reflects respiration from both methanol and its oxidation product, formaldehyde, and is regulated via repression–derepression of methanol dehydrogenase and the cytochrome oxidases. The methanol oxidase system is thus precisely tuned to the concentration of methanol or oxygen in the culture such that the latter is able to maintain a rate of respiration that satisfies the energy demands of the imposed growth rate.

A continuous culture study of methanol and formate utilization by the yeast Pichia pastoris

Quantitative aspects of C1 metabolism by chemostat cultures of Pichia pastoris have been studied. Under methanol limitation the maximum growth yield was 0.41 (g/g), the maintenance energy was 9.1 mg CH3OH/g.h and the μmax was estimated to be 0.18 h−1. Formate was utilized by methanol-limited cultures, and increased the cell yield approx. 20% as compared to that on methanol alone.

Energy conservation in the terminal region of the respiratory chain of the methylotrophic bacterium Methylophilus methylotrophus.

Reduced + CO minus reduced difference spectra of respiratory membranes prepared from methanol-limited cultures of Methylophilus methylotrophus confirm the presence of three CO-binding cytochromes i.e. cytochromes aa3, o and cco. The kinetics of cyanide inhibition indicate that the respiratory chain of this organism is branched at the level of cytochrome c to two major terminal oxidases, viz. cytochromes aa3 and o; cytochrome cco is probably not a physiologically significant oxidase. Determination of proton and charge translocation stoichiometries (leads to H+/O and leads to K+/O quotients) during oxidation of ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine shows that the terminal oxidase system of this organism exhibits a net inward translocation of 2e-, but no net proton translocation, when a pair of electrons are passed from cytochrome c to oxygen. The use of appropriate concentrations of cyanide to selectively inhibit cytochrome o indicates that the overall translocation stoichiometries are achieved by the two oxidases, aa3 and o, functioning similarly. These and other results suggest that methanol oxidase is organised as a simple redox arm with the methanol oxidation site and oxygen consumption site(s) on the periplasmic and cytoplasmic faces of the inner membrane respectively.

Growth yields and the efficiency of oxidative phosphorylation during autotrophic growth of Paracoccus denitrificans on methanol and formate

Paracoccus denitrificans was grown aerobically in chemostat or pH-auxostat cultures with methanol or formate as the limiting carbon source. The maximum growth yields (expressed in g biomass/mole), determined in methanol-limited cultures, on methanol, oxygen and carbon-dioxide were 13.4, 15.5 and 24.8 respectively. The maximum growth yields, determined in formate-limited cultures, on formate and oxygen were 2.9 and 10.5 respectively. Elemental analysis of these cells yielded a cell formula of C6H10.8N1.5O2.9. With the help of the known elemental cell composition an equation for material balances could be drawn up. The calculated yields on oxygen and CO2 (Y O 2 and Y CO 2) were in good agreement with the experimental values. Autotrophically grown cells on methanol possess three active energy conserving sites in the electron-transport chain. The overall P/O ratio (the amount of adenosine triphosphate (ATP) synthesized per atom oxygen consumed) is calculated to be 2.05 with the matching Y ATP max value of 3.8, which is lower than the theoretical value of 6.5. With only 2 phosphorylation sites impossible Y ATP max values would result. Autotrophic growth on formate yielded cells in the possesion of only two active energy conserving sites, most probably sites 1 and 2. Formate crosses the cytoplasmic membrane as free acid, which is equivalent to co-transport with one proton. Therefore transport of formate is energy requiring. With →H+/site ratios of 2 or 3 respectively 0.5 or 0.33 moles of ATP are necessary to transport 1 mole of formate. Consequently the P/O ratio in formate-grown cells is respectively 1.5 or 1.67 with the matching Y ATP max values of 3.1 or 3.5. Therefore during autotrophic growth on methanol and formate the same amount of adenosine triphosphate is necessary to synthesize 1 g of biomass. The 95% confidence intervals of the Y ATP max values overlap completely. The found Y ATP max is 50–60% of the theoretical value as was found for heterotrophic growth of Paracoccus denitrificans. We propose that metabolism in Paracoccus denitrificans is regulated on the level of phosphorylation in the site 1 and the site 3 region of the electron-transport chain.

The Effect of Discontinuous Methanol Addition on the Growth of a Carbon-limited Culture of Pseudomonas

Summary: As the interval between methanol additions to methanol-limited cultures of Pseudomonas methylotropha was increased beyond 20 s the yield of bacterial dry wt/g of methanol fell significantly. Discontinuous methanol additions also caused cycling in the values of a number of parameters, including pH, dissolved oxygen tension, CO2 production and amino acid pool concentration. It is suggested that after each addition of methanol a burst of growth occurred, followed by a period of starvation. These observations are discussed in the light of continuous culture practice and theory.

The influence of dissolved oxygen on Pseudomonas AM1 grown on methanol in continuous culture.

SUMMARY: Methanol-limited cultures of Pseudomonas ami were grown in chemostats (D, 0·1 h.−1; methanol 10 g./l.) at dissolved oxygen tensions between 1 and 690 mm. Hg. Decreasing the dissolved oxygen tension from no to 28·5 mm. Hg did not affect either respiration or carbon balance, but lowering it further caused a progressive increase in respiration which was maximal at 3 mm. Hg (the lowest dissolved oxygen tension at which a steady state could be maintained at the fixed dilution rate). Increasing the dissolved oxygen tension from 97 to 560 mm. Hg increased production of CO2 and decreased yield; this effect was particularly evident between 420 and 560 mm. Hg. Yield varied little between 560 and 690 mm. Hg but CO2 output decreased and exocellular products increased in concentration indicating a metabolic change. The possible mechanisms involved in metabolic regulation are discussed; it is concluded that dissolved oxygen tensions between 3 mm. and 690 mm. Hg exert a continuous quantitative control over respiration and metabolism of growing organisms. A stoichiometric equation is derived for the growth of Pseudomonas am i on methanol.