Biotechnology Techniques Vol 4 No 6 385-390 (1990) Received as revised 11th September

ETHANOL PRODUCTION FROM MIXTURES OF DRIED CELLS OF SACCHAROMYCES CEREVISIAE AND SOLID GLUCOSE EXPOSED TO

RELATIVE HUMIDITIES LESS THAN 100%'

Henry Schneider*, Rick Little and Neil W. Ross Division of Biological Sciences

National Research Council of Canada Ottawa, Canada KlA OR6

SUMMARY

Dried cells of Saccharomyces cerevisi= mixed with solid glucose can produce ethanol when incubated over a range of relative humidities less than 100%. Solid glucose can be added repeatedly during the fermentation. The results suggest that vapour-solid methodology can be used with whole yeast cells to generate volatile products that require multi-enzymic bioconversions, as well as cofactor regeneration or conversion of ADP to ATP.

INTRODUCTION

Several enzymes function when dried and then suspended at relative humidities less than 100%. The approach works when solid substrates are mixed with dry enzyme (Skujins

and McLaren, 1967, Stevens and Stevens, 1979) and when volatile substrates are presented to the enzyme as vapour (Barzana et al, 1987). The approach is of biotechnological interest, in part, because of possible increases in mass transfer rates with volatile substrates and products, and facilitated downstream processing. The relatively small amounts of water involved could also aid in waste disposal.

Vapour-solid bioconversions studied previously have involved either one (Yagi et al, 1969) or two enzymatic steps (Barzana et al, 1987), and a single bioconversion coupled with regeneration of NADH (de Bont et al, 1983, Hou, 1984). It was of interest to determine if the approach could be used to carry out complex bioconversions which

' Issued as NRCC Publication Number 31876

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involve several enzymic reactions in sequence, including cofactor regeneration, and in particular if such multiple enzymic processes could be carried out with whole yeast cells in a vapour-solid mode. The question was approached through an investigation of ethanol production from mixtures of solid glucose and dried cells of Saccharomvces cerevisiae exposed to relative humidities less than 100%. The present paper delineates some of the factors involved in ethanol production under xeric conditions and discusses some of the implications.

MATERIALS AND METHODS

The organism employed was a commercial dried yeast preparation, Fleischmann's "rapid rise" yeast, which consisted of granules with a diameter of about 0.1 mm and lengths ranging up to about 3 mm. D-glucose was supplied by BDH Chemicals and D-[6,6'- 'Hz]-glucose by MSD Isotopes, Canada.

Two hundred milligrams of cells and either 25 or 50 mg of powdered glucose were placed in a capped 1.5 ml centri- fuge tube and mixed by shaking. The mixture was transferred to a cone made of Whatman No. 1 filter paper which was then fitted into a 1.5 ml microcentrifuge tube which had several 5 mm holes in the body. The centrifuge tube was placed in a 30 ml serum vial containing 5 ml of a saturated salt solution used to control relative humidity. The vial was sealed with a rubber septum, and incubated at 37OC. Samples of the aqueous phase for ethanol analysis by GC or GC-MS were removed through the septum. In re-feeding experiments, the vials were opened, a weighed quantity of powdered glucose was added to the filter paper cone, the contents of the cone mixed gently with a spatula and the vial resealed. Water uptake of cells, or cells plus sugar, after exposure to chosen relative humidities was determined by the difference in weight between wet and dry cells. Drying was accomplished by exposure to a vacuum of 30-40 millitorr at 45OC for 18 hours.

Relative humidity was controlled with saturated KNO, (90%) , KC1 (83%), NaCl (75%) and NaBr (54%) (Greenspan, 1977).

RESULTS

Mixtures of dried S. cerevisiae cells and solid

glucose produced ethanol when exposed to a relative humidity of 90% (Fig. 1). The ethanol came from two sources, endogenous reserves and the exogenously added glucose. Production from reserves was indicated by ethanol

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accumulation in the absence of added glucose, and ethanol from the added glucose was taken to be the amount in excess

of the endogenous values. Ethanol production with no added glucose was 14.2 mg. The yield of ethanol due to the addition of 50 mg glucose at day 10 was 22 mg, or 86% of theoretical (0.51 g/g). The corresponding yield with 25 mg of glucose was 73% of theoretical.

40

W

0 0 0 2 2 4 4 6 6 6 6 10 10

Day Figure 1 Time course of ethanol production at 90% relative humidity with different glucose loads: no glucose 0; 25 mg glucose 0; 50 mg glucose A.

50 /

0 3 6 9 12 15

Day

Figure 2 Effect of re-feeding glucose on ethanol production at 90% relative humidity: 25 mg glucose added initial1 D-[6,6

y o--O; 25 mg glucose added initially then 25 mg I- H,]-glucose added on day 3 O......O; 25 mg glucose

added initially then 25 mg D-[6,6'-2H2]-glucose added on days 3 and 8 A--A.

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Direct evidence that ethanol was produced from added solid sugar was provided by mixing solid, deuterium labelled glucose with the cells and following the accumulation of deuterium-labelled ethanol by GC and GC-MS (Fig. 2). These experiments also demonstrated that the cells could be re-fed with dry glucose. The experiments were carried out by initially providing three batches of cells with 25 mg of unlabelled glucose and adding 25 mg of D-[6,6V-2H,]-glucose to one batch on day 3 and on days 3 and 8 to another. The control had glucose added only at zero time and was mixed on days 3 and 8 like the samples to which deuterium-labelled glucose was added. Re-feedability by solid sugar was indicated by increased ethanol production in amounts related to the quantity of sugar refed. Production of deuterium- labelled ethanol demonstrated the utilisation of deuterium- labelled glucose. Twenty seven percent of the ethanol on day 3 and 43% on day 8 was derived from D-[6,6V-2H2]- glucose. Relative humidity influenced ethanol formation (Fig. 3).

Four replicate glucose/cell mixtures systems were incubated at 90% relative humidity for 24 hours, and three then switched to lower humidities. Decreased rates of ethanol production were obtained with decreased relative humidities. By returning low humidity samples to 90% relative humidity at day 8, ethanol production was

0 c a

40 r

Day Figure 3 Effect of relative humidity on ethanol production. Cells with 50 mg glucose were incubated at 90% relative humidity for 1 day then switched as follows: 90% 0; 83% A; 75% for 7 days then 90% v; 54% for 7 days then 90% 0.

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increased. The shift in ethanol production following changes in relative humidity suggest that it can be used to control ethanol formation to some extent.

The cells when suspended at 90% relative humidity, accumulated 35% water by weight over 8 days and about 38% water when glucose was added (Fig. 4). However, not all of the water taken up by the cells was necessary for ethanol formation since appreciable production occurred at 90% relative humidity during the first 3 days when water content had not reached maximal levels (compare Figures 1 and 4). Dried S. cerevisiae in a fluidised bed reactor at 100% relative humidity accumulates in excess of 45% water (Bauer and Rottenbacher, 1985).

40

30

20

10

0 ’ I / I I 0 2 4 6 6

Day Figure 4 Water uptake by cells + 50 mg glucose at different relative humidities. 90%, cells only M; 90%, cells + glucose O......U; 83%, cells only A-A; 83%, cells + glucose v...... v.

DISCUSSION The results show that in the gas-solid mode whole yeast

cells can carry out a complex series of concerted reactions when supplied with solid glucose. At least 11 enzymic reactions are required to produce ethanol from glucose via the glycolytic pathway. For production from internal carbohydrate reserves of 5. cerevisiae, glucan and trehalose, additional enzymes are needed.

The results indicate also that appreciable NADH

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regeneration occurs in the course of the xeric conversion of glucose to ethanol. The conclusion follows consideration of the weight of NADH that would have to be stored if regener- ation did not occur, and the weight of the dry cells. For the 36 mg of ethanol produced in the presence of 50 mg glucose, 260 mg of NADH are required if ethanol production involves only stored NADH. Regeneration of NADH has to occur in the system studied since the starting weight of the dry cells is only 200 mg. A comparable argument can be made for the occurrence of ADP-ATP interconversion.

The vapour-solid approach with whole yeast cells is of potential interest for the production of volatiles which require multi-enzymic synthesis, cofactor regeneration or ATP-ADP interconversion, using glucose or other inexpensive substrates. The results are also of interest in showing that the vapour-solid approach with whole yeast cells allows spatial and temporal separation of cell growth from bio- conversion, a factor of interest when it is desired to prepare cells for a particular bioconverion, but then transport or store them indefinitely for future use.

REFERENCES

Barzana, E., Klibanov, A.M. and Karel, M. (1987) Anal. Biochem. Biotechnol. 15, 25-34.

Bauer, W., Rottenbacher, L. (1985) Entronie 124, 18-23.

de Bont, J.A.M., van Ginkel, C.G., Tramper, J. and Luyben, K.Ch.A.M. (1983) Enzyme Microbial. Technol. 5, 55-59.

Greenspan, L. (1977) Nat. Bur. Std. J. Res. 87A, 89-96.

HOU, C.T. (1984) Appl. Microbial. Biotechnol. 19, l-4.

Skujins, J.J. and McLaren, A.D. (1967) Science 158, 1569- 1570.

Stevens, E. and Stevens, L. (1979) Biochem. J. 179, 161-167.

Yagi, T., Tsuda, M., Mori, Y. and Inokuchi, H. (1969) J. Am. Chem. Sot. 91, 2801.

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