Physiology of yeasts in relation to biomass yields

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<ul><li><p>Antonie van Leeuwenhoek 60: 325-353, 1991. 9 1991 Kluwer Academic Publishers. Printed in the Netherlands. </p><p>Physiology of yeasts in relation to biomass yields </p><p>Cornelis Verduyn Department of Microbiology and Enzymology, Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands </p><p>Key words: yeasts, cell yield, bioenergetics, YATP, P/O-ratio, uncoupling </p><p>Abst ract </p><p>The stoichiometric limit to the biomass yield (maximal assimilation of the carbon source) is determined by the amount of CO2 lost in anabolism and the amount of carbon source required for generation of NADPH. This stoichiometric limit may be reached when yeasts utilize formate as an additional energy source. Factors affecting the biomass yield on single substrates are discussed under the following headings: - Energy requirement for biomass formation (YAle)" YATP depends strongly on the nature of the carbon </p><p>source. - Cell composition. The macroscopic composition of the biomass, and in particular the protein content, has </p><p>a considerable effect on the ATP requirement for biomass formation. Hence, determination of for instance the protein content of biomass is relevant in studies on bioenergetics. </p><p>- Transport of the carbon source. Active (i.e. energy-requiring) transport, which occurs for a number of sugars and polyols, may contribute significantly to the calculated theoretical ATP requirement for biomass formation. </p><p>- P/O-ratio. The efficiency of mitochondrial energy generation has a strong effect on the cell yield. The P/O-ratio is determined to a major extent by the number of proton-translocating sites in the mitochondrial respiratory chain. </p><p>- Maintenance and environmental factors. Factors such as osmotic stress, heavy metals, oxygen and carbon dioxide pressures, temperature and pH affect the yield of yeasts. Various mechanisms may be involved, often affecting the maintenance energy requirement. </p><p>- Metabolites such as ethanol and weak acids. Ethanol increases the permeability of the plasma membrane, whereas weak acids can act as proton conductors. </p><p>- Energy content of the growth substrate. It has often been attempted in the literature to predict the biomass yield by correlating the energy content of the carbon source (represented by the degree of reduction) to the biomass yield or the percentage assimilation of the carbon source. An analysis of biomass yields of Candida utilis on a large number of carbon sources indicates that the biomass yield is mainly determined by the biochemical pathways leading to biomass formation, rather than by the energy content of the substrate. </p><p>I n t roduct ion </p><p>Biomass yield (amount of biomass produced per amount of substrate) may be an important factor in industrial processes. A high biomass yield is espe- </p><p>cially important when the price of the raw material (e.g. the carbon source) makes up a large fraction of the costs of the final product. This, for example, holds for the cultivation of baker's yeast on molas- ses. Furthermore, any improvement in yield will </p></li><li><p>326 </p><p>decrease the oxygen demand and reduce heat pro- duction. This reduces the product price since sup- ply of oxygen and cooling are also significant costs in large-scale processes. </p><p>Alternatively, in some processes it is necessary to keep biomass formation to a minimum in order to achieve a maximal conversion of the carbon source into extracellular products like ethanol or citric acid. It is evident, therefore, that more funda- mental knowledge on the factors that govern the biomass yield is required for the optimization of large-scale fermentation processes. </p><p>In this work, the effect of various parameters on the biomass yield will be discussed in relation to the physiology. Although the data and discussions mainly concern yeasts, they in our opinion are ap- plicable to heterotrophic organisms in general. </p><p>Practical aspects </p><p>In order to obtain reliable data on the relation between the physiology of an organism and its bio- mass yield, a number of practical aspects are of importance. These are summarized below. </p><p>Batch versus chemostat cultivation Results on biomass yields of batch cultures are difficult to interpret. This is due to the fact that in batch cultures cells are exposed to a continuously changing environment, that exerts profound ef- fects on the physiology. Therefore, in this contribu- tion, mainly data obtained with chemostat culti- vation are considered. In addition, biomass yields are nearly always expressed as weight of dry bio- mass obtained per amount of substrate utilized. However, the biomass composition (notably the protein content) needs consideration since com- parison of growth yields of different yeasts requires knowledge on the biomass composition. Where possible, the protein and/or nitrogen contents of biomass will therefore be considered. </p><p>Medium composition The importance of a correct medium composition should not be overlooked. Rigorous testing must be performed to determine the medium compo- </p><p>nent that limits the growth yield. Rieger et al. (1983), for example, showed that the physiology (and cell yields) of S. cerevisiae H1022 were strong- ly affected by a hidden manganese deficiency in the medium. </p><p>A number of theoretical studies have been pub- lished on the nutritional demands of yeasts (Egli 1980; Jones &amp; Greenfield 1984; Jones &amp; Gadd 1990). In anaerobic cultures, the provision of an adequate amount of sterols and unsaturated fatty acids is of primary importance. Low anaerobic growth yields and YAxP values (g cells.mol ATP formed -a) as reported by Dekkers et al. (1981) can be explained (Verduyn et al. 1990a) by the fact that no ergosterol and oleic acid, which are required as vitamins during anaerobic growth (Andreasen &amp; Stier 1953, 1954), had been added to the growth medium. All data discussed in this contribution refer to carbon- and energy-limited growth in che- mostat cultures, unless mentioned otherwise. </p><p>Oscillations Some yeasts, notably Saccharomyces and Schizo- saccharomyces strains may exhibit oscillations in metabolic activity during sugar-limited growth due to spontaneous synchronization of the cell cycle (Parulekar et al. 1986; Novak &amp; Mitchison 1990). These oscillations can result in variations in for instance dry weight (Rouwenhorst et al. 1991) and make yield studies with S. cerevisiae a difficult en- terprise. Our data for S. cerevisiae presented here- in were obtained with cultures that were not oscil- lating, as shown by measurements of 02 consump- tion and CO2 production. </p><p>Effluent removal Selective effluent removal in chemostat cultures, especially when culture fluid is removed upwards from the culture surface by suction, can result in accumulation of biomass in the fermenter. This can be checked by measuring the biomass concentra- tions both in samples taken directly from the fer- menter via a sample port and in samples taken from the effluent line. These data should be the same. We have found that, although a steady-state bio- mass concentration may be established, the differ- ence between fermenter and outlet biomass con- </p></li><li><p>centration may in some cases be more than 10% (H. Noorman, pers. comm.). These problems can often be solved by placing the outlet tube in the centre of the fermenter fluid (rather than on the surface) and controlling the fluid removal rate by means of an electrical contact sensor at the surface of the fluid. The sensor then steers the effluent pump to maintain the required working volume. </p><p>Metabolite analysis and sampling Assays of metabolites in culture supernatants may supply useful information on the presence of po- tentially inhibitory compounds, such as weak acids (to be discussed later). Simple HPLC methods can detect a wide range of short-chain acids via UV- detection at 210 nm with a high sensitivity (Favre et al. 1989; Verduyn et al, 1990b). Furthermore, by placing a refraction index meter in series with this equipment, (poly)alcohols and sugars can also be assayed. The detection limit for these !atter com- pounds is, however, insufficient to assay residual substrate concentrations. </p><p>Sampling of culture fluid also deserves special attention when it concerns measurements of low residual substrate concentrations like, for exam- ple, glucose in glucose-limited chemostat cultures. Uptake of glucose will continue during sampling and thus leads to an underestimation of the residual concentration in the fermenter. Therefore, fast sampling methods have to be applied which either separate cells immediately from the medium or, alternatively, instantaneously stop metabolic activ- ity. Methods to achieve this include rapid filtration (e.g. Rutgers et al. 1987), and the direct transfer of cells into liquid nitrogen (Postma et al. 1988). </p><p>Whatever the method applied, it should be checked that, as prescribed by chemostat mathe- matics, the residual concentration of the growth- limiting substrate is independent of the biomass density in the culture. To this end, the residual substrate concentration should be determined at different reservoir concentrations of the growth- limiting nutrient. </p><p>Carbon recovery Finally, studies on biomass yields must include con- struction of carbon balances in order to check </p><p>327 </p><p>whether unexpected byproduct formation does oc- cur. This should also be verified with a Total Orga- nic Carbon (TOC) analysis of culture supernatants. The yield data reported herein refer to situations in which the carbon recovery was 100 __ 5%. </p><p>Basic flows in metabolism: assimilation and dissimilation </p><p>The biomass yield is determined by two factors: the energy requirement of biomass formation and the efficiency of energy transduction in dissimilation. It is therefore convenient to make a general divi- sion of metabolism into two main processes: assi- milation and dissimilation. Assimilatory processes involve the biosynthetic reactions leading to forma- tion of biomass. Dissimilatory processes provide the energy (ATP) required for assimilation. Energy is also required for maintenance functions and transport of nutrients (Fig. 1). Finally a flow of carbon is directed towards the generation of NADPH, which is used as a reductant in anabo- lism. NADPH generation is considered here as an assimilatory process. The scheme shown in Fig. 1 is applicable to the carbon sources which support growth of yeasts. With very oxidized substrates, however, an additional dissimilatory flow is re- quired to reduce the carbon source to the level of biomass, i.e. to provide reducing equivalents. The carbon sources for growth need not be the same as the energy source. Many yeasts can be grown on mixed substrates in such a way that ATP for assimi- lation of the carbon source is derived almost exclu- sively from an energy source which itself cannot be assimilated, such as formate (Fig. 1). Under these conditions the yield on the carbon source is at its maximum (i.e. all carbon in the carbon source is directed towards cell synthesis). This is discussed below. </p><p>Stoichiometric limits to the cell yield </p><p>An important issue in the context of yield studies is the theoretical limit to the yield that can be achieved on a given carbon source. Frequently it is </p></li><li><p>328 </p><p>I ---,.- __. glucose / ~ C02 I ATP </p><p>~,~ NADH </p><p>~ CO 2 glucose d/ss/m//a~n or </p><p>formate </p><p>Fig. 1. Schematic representation of central metabolic processes. Formate can serve as an energy source during glucose-limited growth. That part of glucose which is normally dissimilated to provide ATP is then replaced by formate. </p><p>assumed that this is identical to complete incorpo- ration of the substrate carbon into biomass, i.e. a carbon conversion of 100%. However, during assi- milatory reactions, net production of CO: always occurs (Oura 1972). Therefore, the maximal car- bon conversion is always lower than 100%. The fraction of CO2 which is lost in assimilation de- pends on the carbon source and lies between ap- proximately 10% for growth on, among others, glucose and methanol, and 29% for carbon sources such as succinic acid or oxalic acid (Gommers et al. 1988). </p><p>Another factor that must be taken into account in assessing the limits of growth yields is the NADPH requirement of biomass formation. In mammalian mitochondria (Rydstr6m et al. 1970) and a number of prokaryotic organisms (Asano et al. 1967), NADPH can be generated from NADH by transhydrogenase (NADH + NADP NADPH + NAD This enzyme has not been detected in yeasts (Bruinenberg et al. 1983a). In yeasts, NADPH is generated via NAD(P)+-de - pendent isocitrate dehydrogenase and via the hex- ose monophosphate (HMP) pathway. It can be calculated (Bruinenberg et al. 1983b) that during growth on glucose, with ammonium as nitrogen source, insufficient NADPH can be produced via </p><p>the isocitrate dehydrogenase reaction alone. Therefore, the HMP route must be active and part of the glucose is oxidized to CO2 via this route to generate NADPH. This means that, even in cases where the assimilation of a carbon source would result in the production of sufficient NADH to provide the ATP necessary for biomass formation, some dissimilation of the carbon source would still be necessary for the generation of NADPH. This is nicely illustrated with studies in which yeasts were grown under carbon and energy limitation on mix- tures of glucose and formate. Formate can only be used as an energy but not as a carbon source by yeasts. Its oxidation is strictly NAD in yeasts (Veenhuis et al. 1983), hence formate can- not serve as a source of NADPH. Addition of formate to glucose-limited cultures of Candida uti- lis resulted in an increase in yield from 0.51 to 0.69 g.g glucose -1 (Bruinenberg et al. 1985). It can be calculated, however, that the theoretical maximum yield (not regarding the NADPH requirement) is 0.75 g.g glucose 1. Indeed radiorespirometric stud- ies on the contribution of the HMP pathway to glucose metabolism in C. utilis suggested that ca. 7% of the C-source is oxidized in the HMP pathway (Bruinenberg et al. 1986). Thus, during growth on mixtures of glucose and formate, formate oxida- tion can supply all NADH required for ATP forma- tion. However, since formate cannot supply NADPH required for biomass synthesis, NADPH must be generated by oxidation of the carbon source (Fig. 1). </p><p>The amount of NADPH required for assimila- tion is dependent on the nitrogen source for growth. When nitrate serves as a nitrogen source, additional NADPH is required for reduction of nitrate to NH4 (Bruinenberg et al. 1983). Indeed, it could be shown that during glucose-limited growth of C. utilis with nitrate, more glucose is directed to the HMP pathway (Bruinenberg et al. 1985). As a result, the maximum attainable yield on glucose in the presence of formate was lower dur- ing growth with nitrate than with NH4 + (Table 1). </p><p>Not only with glucose, but also with other carbon sources formate can serve as an energy source (Ta- ble 1). In most of these cases a considerable in- crease in yield (...</p></li></ul>