and OutlookIn biotechnological processes, fundamental performances of microorganisms are used. The economy of these processes is essentially determined by the efficiency, velocity (productivity) and quality of thc products. Therefore it is a permanent task and challenge for basic and biotechnological research to seek out measures for improving the actually attained parameters. The auxiliary substrate concept supplics an approach. It is based on the fact that chemo-organo-heterotrophic substrates differ in the carbon:energy ratio, thus, growth yield is limited in energy and/or reducing power. I t SAYS that. by simultaneous utilization of physiologically similar substrates (mixed substrates), the growth yield increases. The substrates are to combine in such a way that with their simultaneous utilization a minimum of carbon is dissimilated merely for the purpose of the generation of biologically useful energy and/or reducing power. Since all chemo-organo-heterotrophic substrates are more or less energy-deficient, an increase in growth efficiency can be expected if the individual substrates of the mixture are assimilated more efficiently than the respective substrates alone. This may result, for instance, from an immediate assimilation of a substrate (according to the "manner of finished part construction"). An increased growth rate is rather the rule than the exception in mixed substrate utilization.In product syntheses the substrates are. depending on the concrete product and metabolic pathway. either energy-excess or energy-deficient, or, in other words, the processes are energy-generating or energy-consuming, respectively. If this is responsible for discrepancies between the possible yields determined by the carbon metabolism and the experimentally obtained yields. the discrepancies should be able to be decreased and the yields increased by mixing substrates. The substrates are to choose and combine so that, due to simultaneous utilization, the product formation process becomes energy neutral. As a rule, the enhanced efficiency is accompanied by an increased velocity. This does not only apply to syntheses, but also to degradation (and detoxification) reactions. Even supposedly inert compounds or persistent substances can be activated by simultaneous (co-)metabolization of another (an auxiliary substrate, victim substrate or co-substrate) and converted at a considerable rate. It is of interest for syntheses of products but in particular for degradation and decontamination of harmful and waste products in the environment that the residual concentrations of the substrates are smaller than those achieved if the compounds of a mixture are metabolized separately. The auxiliary substrate concept has proven to be fruitful, both for theoretical and practical questions. It was practically already being used before it was formulated (mixed substrate utilization, cometabolism). However, an abundance of regulatory and energetic aspects are waiting to be investigated in more detail. 212Acta Biotechnologica 13 (1993) 3

A bacterium classified as Rhodococcus opacus, which is able to use pyridine (a potentially growth-inhibiting substrate) as its sole source of carbon, energy and nitrogen, was isolated. In a carbon-limited chemostat culture, the kinetics was determined for growth on both pyridine and a mixture of pyridine and fructose (9 mM/22.15 mM). With growth on pyridine, stable steady states were achieved up to dilution rates of about 0.1 h-1. A further increase in the dilution rate resulted in the progressive accumulation of pyridine in the culture liquid and the cells were washed out. The maximum specific growth rate (mu max = 0.23 h-1) and the Ks value (0.22 mM) for growth on pyridine were determined from the residual pyridine concentrations measured within the range of stable steady states. With growth on the substrate mixture, the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone, and stable steady states were established at dilution rates of up to 0.13 h-1. The maximum pyridine degradation rate was enhanced to 270 mg pyridine l-1 h-1 compared to 210 mg pyridine l-1 h-1 with growth on pyridine as a single substrate. An external nitrogen source did not need to be added in the case of growth on the substrate mixture. Fructose was assimilated by means of ammonium released from pyridine. Analysis of the nitrogen balance furnished proof that pyridine is an energy-deficient substrate; pyridine was assimilated and dissimilated at a ratio of 1 mol/0.67 mol respectively. The resulting yield coefficient was about 0.55 g dry weight/g pyridine. Moreover, it was demonstrated that, in regard to the biologically usable energy, 1 mol pyridine corresponds to 0.43 mol fructose.

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The maximum specific growth rate Q..tmax) 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: 0.36h -1, 0.37h -a, 0.47h -~ and 0.52h -a, respectively. Attempts have been made to explain these somewhat surprising resuits, especially the fact that the growth rates on xylose plus methanol and xylose plus glycerol exceed the specific growth rates of those on even the "faster" partner in the mixture.

The maximum growth rate of methylotrophic yeasts during growth on methanol is about 0.2 h−1. Since they are able to grow faster on substrates such as glucose we tried to identify the putative limiting step in methanol metabolism within the assimilatory pathway, leading to the formation of a major precursor for biosyntheses, or within the linear dissimilatory sequence. Growth experiments with mixed substrates and determination of some kinetic parameters allowed us to restrict the number of possible pacemaker enzymes. The dissimilatory sequence does not seem to be growth‐rate limiting. This also applies to transketolase, transaldolase and fructose‐1,6‐bisphosphatase. Surprisingly, methanol oxidase appears to be the prime candidate.