Wolfgang Babel

Correlation between cell composition and carbon conversion efficiency in microbial growth: a theoretical study

SummaryAs the macromolecular composition of microorganisms varies during their life cycle it was asked whether, and to what extent such changes exert any influence on substrate consumption, i.e. growth yield and carbon conversion efficiency, respectively. This question was dealt with in a theoretical study by use of the YAPTmax-concept. The growth substrates considered were methanol, acetate and glucose; the latter was assumed to be assimilated via both the glycolytic and the oxidative hexosemonophosphate pathway. Five fictitious biomasses were used which were altered in their proportion of polysaccharides, proteins, lipids, RNA and DNA. As a result, only small variations in the individual “biomass formulae” were obtained. On the basis of the energy balances for the syntheses of all cell constituents it was found that variations in the macromolecular composition of microbial biomass have only a slight effect on carbon conversion efficiency, amounting to maximally 3%. From the material balances it could be calculated that the upper, solely metabolism-determined limit of carbon conversion efficiency is 85% for substrates assimilated glycolytically via phosphoglycerate; for gluconeogenetic substrates, the upper limit was 75%. These limits are essentially determined by carbon loss on the way to amino acid syntheses.

Glucose as an energy donor in acetate growing Acinetobacter calcoaceticus

Since glucose can be oxidized but not assimilated by Acinetobacter calcoaceticus 69-V the question arose whether energy generated by glucose oxidation can help incorporate carbon from heterotrophic substrates and, if so, what the efficiency of ATP production is like. For this reason this species was grown in the chemostat on acetate. After having reached steady state conditions an increasing concentration of glucose was added. This led to an increase in the biomass level from about 0.4 g/g for growth on acetate alone to 0.6–0.65 g/g in the presence of glucose, independently of either the growth rate or the steepness of the glucose gradient used. This upper value approximates about the limit of the carbon conversion efficiency calculated for non-glycolytic substrates. Glucose was almost exclusively oxidized to gluconic acid, 2- and 5-ketogluconates, and pentose 5-phosphates were found only in traces. These results demonstrate that glucose functions as an additional energy source in Acinetobacter calcoaceticus 69-V. From the transient behaviour of biomass increase and the mixing proportion at which the maximum growth yield on acetate in the presence of glucose was obtained it followed that two mol of ATP must have been generated per mol of glucose oxidized. This property is discussed in terms of coupling glucose dehydrogenase with the respiratory chain.

Improvement of growth yield of yeast on glucose to the maximum by using an additional energy source

The experimentally determined growth yield on glucose under aerobic conditions is approximately 0.5 g/g, but on the basis of the carbon content a value of 0.71 g/g should be the upper limit if carbon conversion is improved by the use of an additional energy source. This assumption was investigated with the methylotrophic yeast Hansenula polymorpha MH 20. Formate served as an additional energy source. The growth yield experiments were performed with a transient-state fermentation technique in which formate was fed via an increasing concentration gradient to a culture growing continuously on glucose. As a result the growth yield on glucose was improve, the extent was dependent on the formate feeding rate, i.e. the slope of this formate gradient. The predicted maximum growth yield of 0.7 g/g was obtained at a slope of the formate gradient of 0.21 g/l·h at a glucose concentration of about 1 g/l. Steeper gradients did not further improve this value, but rather impaired the growth yield due to the appearence of a high residual formate concentration in the fermentation medium. The yield patterns are influenced by the culture pH, a value of at least 4.8 is necessary to achieve the maximum growth yield on glucose. At lower pH formate became increasingly toxic. The ratio of formate to glucose necessary to obtain the maximum yield coefficient was 1...1.6:1 (in grams). On the basis of the energy content of formate a ratio of 1.2...1 (P/O=2) was calculated to substitute the part of glucose which is endoxidized for energy generation. Deviations from this value are explained in terms of the manner of uptake and uncoupling property of formic acid/formate and the existence of a second, formate-“wasting” enzyme.

Mixed Substrate Utilization in Micro-organisms: Biochemical Aspects and Energetics

SUMMARY: The energy-based classification of heterotrophic substrates requires biochemical evaluation because some substrates can be assimilated by a variety of different metabolic pathways. By using the YnATP-concept it was shown that the classification depends on the yield of ATP and reducing equivalents already generated on the way to the precursor (phosphoglycerate). With carbon-excess substrates a part of the total substrate consumed must be oxidized to completion merely for energy production, whereas with energy-excess substrates more energy is provided on the route to the precursor than is needed for assimilation of the precursor carbon. By means of this approach it was possible to assess experimental growth yields obtained on mixed substrates and to predict the optimum mixing proportion in order to attain the maximum carbon conversion efficiency. The validity of this method was shown for some examples.

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis in Methylobacterium rhodesianum

The enzymes β-ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The β-ketothiolase had a relatively highKm for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (Ki 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (Km 0.07 mM) and was significantly inhibited by NADH (Ki 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)-1 during growth to the detection limit when poly(β-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6–0.7, and 1.0 nmol (mg dry mass)-1, respectively, were determined during growth and PHB synthesis. During growth, the β-ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the β-ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (KmNADPH 18 μM). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.

Simultaneous utilization of heterotrophic substrates by Hansenula polymorpha MH30 results in enhanced growth rates

SummaryThe maximum specific growth rate (μmax) of Hansenula polymorpha MH30 on xylose as the sole source of carbon and energy is 0.175 h−1, on methanol 0.21 h−1, on glycerol 0.27 h−1 and on glucose 0.61 h−1. On mixtures of xylose plus methanol, xylose plus glycerol, xylose plus glucose and glycerol plus glucose H. polymorpha MH30 grows faster: 0.36 h−1, 0.37 h−1, 0.47 h−1 and 0.52 h−1, respectively. Attempts have been made to explain these somewhat surprising results, especially the fact that the growth rates on xylose plus methanol and xylose plus glycerol exceed the specific growth rates of these on even the “faster” partner in the mixture.

Flow of 14C-methanol via assimilatory and dissimilatory sequences with yeast in presence of glucose

Experiments were performed to reveal the extent to which individual heterotrophic substrates of a mixture contribute to the overall carbon and energy metabolism. For this reason Hansenula polymorpha MH 20 was chemostatically (C-limited) cultivated at different growth rates on mixtures of methanol and glucose fed at proportions of 3:1 and 1:3 (in weight units), respectively. The distributions of 14C-carbon from methanol in biomass as well as carbon dioxide (and supernatant) fractions were determined. From these results it followed, firstly, that energy derived from methanol dissimilation was used in part for the incorporation of glucose carbon, resulting in carbon conversion efficiencies for this substrate equivalent to yield coefficients of 0.61–0.69 g/g. Secondly, the growth yield data revealed that the efficiency of methanol conversion had to be increased in order to account for the experimentally determined yield figures. This was further confirmed by theoretical treatment of the growth yield data which showed that these could only be obtained if P/O-quotients for methanol conversion similar to those for glucose, i.e. 2.0–2.5, were considered. The latter property was regarded as the main reason for the observed improvement of growth yield accompanying the simultaneous utilization of methanol and glucose in this yeast.

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)-β-hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)-β-hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent Km values of 15 μM and 30 μM for AcAc-CoA, 18 μM for NADPH and 30 μM for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 μM and 50 μM, respectively. The contribution of the two reductases to poly-β-hydroxybutyrate synthesis is discussed.

Use of formate gradients for improving biomass yield of Pichia pinus growing continuously on methanol

SummaryInvestigations were made into the improvement of growth yield (Y) of Pichia pinus MH 4 growing continuously on methanol by feeding formate so as to create an increasing concentration gradient (transient state). Under particular formate supply conditions, Y could be increased from 0.37 g·g-1 on methanol alone to 0.55 and 0.47 g·g-1 in the presence of formate at dilution rates (D) of 0.045 and 0.075 h-1, respectively. These differences could be explained as being due to a limiting formate consumption rate of 50–60 nmol·min-1·g-1 dry wt., coupled to a net-energy generation independent of D. Any further formate oxidation proceeded without energy gain. Deviations from optimum conditions of biomass increase are discussed in terms of different formate oxidizing systems and uncoupling properties of formate itself. These results are compared to and confirmed by steady-state considerations.

Glucose as an auxiliary substrate

SummaryA theoretical consideration is presented of the comparative efficiency of carbon conversion of glucose by the Embden-Meyerhof-Parnas (EMP) and the oxidative hexosemonophosphate (HMP) pathways. As a result it is shown that maximum carbon conversion, that is 89%, is possible when glucose is assimilated via the EMP pathway. This value is diminished in proportion to the participation of the HMP pathway in carbon assimilation and is halved when glucose is incorporated entirely via this pathway. If NADPH is included as a source of energy, glucose may behave both as an excess carbon and an excess energy substrate, the latter being the case when greater portions of the HMP pathway operate, and the extent of this is in turn dependent on the P/O quotient. If NADPH cannot be used for ATP synthesis, glucose remains an excess carbon substrate throughout, although when the HMP pathway accounts for more than 26% of glucose assimilation an increasing excess of reduction equivalents is produced. These results are interpreted in terms of mixed-substrate utilization for improving growth yield when glucose is to be used as the excess carbon component.