W. de Koning

Experimental pulse technique for the study of microbial kinetics in continuous culture

A novel technique was developed for studying the growth kinetics of microorganisms in continuous culture. The method is based on following small perturbations of a chemostat culture by on-line measurement of the dynamic response in oxygen consumption rates. A mathematical model, incorporating microbial kinetics and mass transfer between gas and liquid phases, was applied to interpret the data. Facilitating the use of very small disturbances, the technique is non-disruptive as well as fast and accurate. The technique was used to study the growth kinetics of two cultures, Methylosinus trichosporium OB3b growing on methane, both in the presence and in the absence of copper, and Burkholderia (Pseudomonas) cepacia G4 growing on phenol. Using headspace flushes, gas blocks and liquid substrate pulse experiments, estimates for limiting substrate concentrations, maximum conversion rates Vmax and half saturation constants Ks could rapidly be obtained. For M. trichosporium OB3b it was found that it had a far higher affinity for methane when particulate methane monooxygenase (pMMO) was expressed than when the soluble form (sMMO) was expressed under copper limitation. While for B. cepacia G4 the oxygen consumption pattern during a phenol pulse in the chemostat indicated that phenol was transiently converted to an intermediate (4-hydroxy-2-oxovalerate), so that initially less oxygen was used per mole of phenol.

Regulation of methanol metabolism in the yeast Hansenula polymorpha

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DNA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DNA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.

Methanol-Utilizing Yeasts

The ability of yeasts to grow on methanol as a source of carbon and energy has been discovered relatively recently. Whereas bacterial utilization of methanol was found before the end of the previous century, it was not until 1969 that growth of eukaryotic microorganisms at the expense of this one-carbon compound was reported (Ogata et al. 1969). Independent studies by several other research groups soon followed this first report and established that a variety of yeasts (Lee and Komagata, 1980a) and some filamentous fungi (Goncharova et al., 1977) are able to utilize methanol as a source of carbon and energy for growth.

Trichloroethene degradation in a two‐step system by methylosinus trichosporium OB3b. Optimization of system performance: Use of formate and methane

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

Methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase

SummaryVarious factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C5 sugars gave the highest triose accumulation (ca. 20mM after 45h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resulted in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.

NADH-regulated metabolic model for growth of Methylosinus trichosporium OB3b. Cometabolic degradation of trichloroethene and optimization of bioreactor system performance

A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis‐Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth‐based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash‐out of suspended cultures at low TCE loads (below μM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash‐out. Pulsed addition of TCE (0.014–0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33–0.64 mM). This emphasizes the inefficient use of enzyme activity in growth‐based systems. A comparison of growth‐based and similar non‐growth‐based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two‐step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system.