W. Alexander Scheffers

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: Current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.

The role of redox balances in the anaerobic fermentation of xylose by yeasts

SummaryThe kinetics of glucose and xylose utilization by batch cultures of Candida utilis were studied under aerobic and anaerobic conditions during growth in complex media. Rapid ethanol formation occurred during growth on glucose when aerobic cultures were shifted to anaerobic conditions. However, with xylose as a substrate, transfer to anaerobiosis resulted in an immediate cessation of metabolic activity, as evidenced by the absence of both ethanol formation and xylose utilization. The inability of the yeast to ferment xylose anaerobically was not due to the absence of key enzymes of the fermentation pathway, since the addition of glucose to such cultures resulted in the immediate conversion of glucose to ethanol. Furthermore, when the enzyme xylose isomerase was added to an anaerobic xylose culture, immediate conversion of xylose to ethanol was observed. This indicates that the inability of the yeast to form ethanol from xylose under anaerobic conditions is caused by metabolic events associated with the conversion of xylose to xylulose. A hypothesis is put forward which explains that ethanol production from xylose by yeast under anaerobic conditions is negligible. It is suggested that the failure to ferment xylose anaerobically is due to a discrepancy between production and consumption of NADH in the overall conversion of xylose to ethanol. When a hydrogen acceptor (i.e. acetoin) was added to anaerobic cultures of C. utilis, xylose utilization resumed, and ethanol and acetate were produced with the concomitant stoicheiometric reduction of acetoin to 2,3-butanediol.

Colorimetric alcohol assays with alcohol oxidase

Abstract A method is described for manufacturing crude alcohol oxidase (EC 1.1.3.13) preparations which are suitable for application in colorimetric alcohol assays. The procedure involves a one-step removal of catalase activity from a partially purified preparation of alcohol oxidase from the yeast Hansenula polymorpha via dialysis against 3-amino-1,2,4-triazole and hydrogen peroxide. Thus, the irreversible inactivation of more than 90% of the catalase present was achieved, which is prerequisite for the use of alcohol oxidase preparations in colorimetric alcohol assays via peroxidase-mediated oxidation of dyes. This type of assay was shown to be rapid, accurate and sensitive. The influence of the relative concentrations of the various assay constituents such as alcohol oxidase, catalase and peroxidase is discussed. It is concluded that this colorimetric alcohol assay is particularly suitable for the determination of ethanol in fermentation broths, both in qualitative and in quantitative tests.

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

SummaryAn alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial bakers yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l-1 glucose.Similar experiments with batch cultures of certain ‘non-fermentative’ yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells-1·h-1.

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

Candida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066 were grown in glucose-limited chemostat cultures with formate as an additional energy source. In both yeasts formate was oxidized via a cytoplasmic NAD+-linked formate dehydrogenase. Other formate-oxidizing enzymes could not be detected.WithCandida utilis the steady-state cell yield on glucose increased with increasing amounts of formate in the medium until growth became carbon-limited. The maximum growth yield on glucose in the presence of excess formate was dependent on the nitrogen source used for growth. With ammonium and nitrate the maximum yields were 0.69 and 0.56 g cells/g glucose, respectively. Calculations showed that this difference correlates with the NADPH requirement for biomass formation with these two nitrogen sources. This implies that the NADH produced from formate oxidation cannot replace the NADPH needed for biomass formation. It therefore is concluded that inCandida utilis transhydrogenase activity is absent.AlsoSaccharomyces cerevisiae was capable of oxidizing formate in glucose-limited chemostat cultures. However, in contrast toCandida utilis utilization of formate by this yeast did not enhance the cell yield on glucose.

Adaptation of the Kinetics of Glucose Transport to Environmental Conditions in the Yeast Candida utilis CBS 621: a Continuous-culture Study

SUMMARY: The relation between the kinetic parameters of glucose transport and the physiology of Candida utilis CBS 621 was studied in chemostat cultures. In glucose-limited cultures the transport parameters were dependent on the growth rate of the yeast. Three different transport systems were found which differed by an order of magnitude in their affinity constants, namely a high-affinity (K m 25 μM), a medium-affinity (K m 190 μM), and a low-affinity uptake system (K m 2000 μM). Cells growing at a dilution rate of 0.45 h-1 or less had the high- and medium-affinity uptake systems. At a dilution rate of 0.52 h-1 the high-affinity system was absent and both the medium-and low-affinity systems were present. At a dilution rate close to μmax (0.57 h-1) only the low-affinity system was detected. The in situ contribution of each of the transport systems to glucose consumption in glucose-limited cultures was estimated on the basis of their kinetic parameters (K m and V max) and the residual glucose concentration in these cultures. The sum of the calculated rates of transport corresponded to the in situ rate of glucose consumption by the cultures as determined from the yield constant and the dilution rate. The dependence of the transport parameters on the growth rate and hence on the environmental sugar concentration was also evident in cells grown under nitrogen limitation. In contrast to carbon-limited cells, nitrogen-limited cultures growing at D = 0.15 h-1 did not exhibit the high-affinity glucose uptake system, whereas the medium- and low-affinity systems were present.

Is the Kluyver effect in yeasts caused by product inhibition

Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0.1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the cultures respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

DETERMINATION OF PROTEIN CONCENTRATION BY TOTAL ORGANIC CARBON ANALYSIS

Determination of the carbon concentration in protein solutions by total organic carbon analysis was found to be a sensitive and reliable method for the estimation of protein concentrations. Using a carbon content of 0.53 g/g in protein and of 0.44 g/g in carbohydrate, the concentrations of normal proteins, proteins containing chromophoric groups, and proteins containing carbohydrate could be established. The method appeared to be independent of the nature of the protein and showed complete linearity between 25 and 1000 mg/l (0.5-20 micrograms per assay) when protein was serially diluted. Determination of specific absorption coefficients by measuring both the absorbance of protein solutions at 280 nm and their carbon concentrations gave values which, on the average, coincided within 12% with values reported in the literature. The method may have special applicability in protein purification studies, as it does not require knowledge of molar extinction coefficients beforehand, and also monitors the disappearance of carbon compounds other than protein.

In Delft: a personal account.

The author looks back on his development in microbiology and yeast research, and on the establishment in Delft of the FEMS Central Office, FEMS Publications Office and the birth of FEMS Yeast Research.