Growth of a methanol-utilizing yeast

Growth of a methanol-utilizing yeast H. S. Pal and I. Y. Hamdan Kuwait Institute for Scientific Research, P.O. B o x 24885, Kuwait

(Received 23 October 1978; revised 21 December 1978)

A halophilic thermotolerant yeast species, identified as Hansenula polymorphaMorais etMaia, was isolated from a mixed culture obtained from sea-water from the Arabian Gulf. The species grew on methanol at 25-42°C, pH 3 . 5 - 6 . 7, and in a medium compounded with 75% sea-water. Either thiamin HCl and biotin or yeast extract proved essential for growth. In shake flask studies a depression o f the yield was observed when methanol concentration increased; at concentrations in excess o f 0.1%, v/v, inhibition o f growth also occurred. In a batch culture grown in a 14 l fermenter, the values o f T d , It and Ys were f o u n d to be 3 h, 0.23h -1 and 0.38, respectively.

The utilization of C l compounds by yeasts is less widespread than among procaryotic microorganisms, l Whilst the growth of bacteria on methanol has been well documented since the early work of Bassalik, 2 the utilization of methanol by a yeast was first reported only nine years ago by Ogata. 3 Since then, a variety of methanol-utilizing yeasts have been described by several investigators and summarized by Minami et al.,4 van Dijken 6 and Papoutsakis et al. 7

For single cell protein (SCP) production, yeasts have some distinct advantages, despite their generally low specific growth rate and protein content. For economic considerations, thermophilic or thermotolerant cultures are of the utmost importance for SCP processing in tropical countries, where the efficiency of removing metabolic heat is greatly hampered by the elevated temperature of the cooling water. Most of the yeasts reported in the literature to date have shown optimum growth temperatures within the range 25-30°C. However, other investigators 4,6,a have isolated thermotolerant yeast species which are capable of growing in the temperature range 37-42°C. Pal et al. 9 reported preliminary data for a methanol-utilizing thermotolerant mixed culture, designated SC, which was obtained from water samples taken from the Arabian Gulf. This article describes some of the growth characteristics of a yeast species isolated from the above mentioned mixed culture.

Materials and methods The salt medium used for the growth studies contained 2.5 g NaH2PO 4 .2H20, 0.25 g K2HPOn, 2 g (NHn)zSOn, 0.05 g NaC1, 0.2 g MgSOn.7H20, 2 mg FeSO4.7H20, 1 mg ZnSO 4.7H20 , 0.1 mg MnSO 4-2H20 , 0.1 mg CuSO 4. 5 H20 , 0.1 mg Co(NO3) 2 •6H20, 0.02 mg NazMoO 4 -2H20, and 50 ml of tap water. The pH was adjusted to 4.0 with 1 N-HC1 and the volume was made up to 11 with distilled water. The medium was steam-sterilized at 12 I°C for 30 rain, then cooled; 5 0 - 7 0 ml portions were added to 250 ml Erlenmeyer flasks.

Membrane-sterilized yeast extract (5%) and methanol were added to give final concentrations of 100-200 mg/l and 0.1-0.2%, v/v, respectively. The medium was solidified by adding 1.8% Difco Bacto agar to the above medium prior to autoclaving. After cooling to about 45°C, yeast extract and methanol were added. In some experiments the medium was compounded with 75% sea-water, obtained from the Arabian Gulf. The water was filtered through a sintered funnel to eliminate suspended particles. Its natural pH was 8.1 ; upon adjustment of the medium to pH 4.2 and autoclaving, there was no precipitation of the salts. Aliquots of 3 - 6 ml were removed aseptically from the cultures and the absorbance was measured in the range 0.05-0.5 at 620 nm using a Spectronic 20. Where necessary, the cultures were diluted with distilled water. An absorbance-dry weight relationship, described in an earlier work, 9 was established and was shown to be linear in the above range. Cultures were centrifuged at 6375g at 4°C; 10 ml portions of the supernatant were immediately frozen at -18°C. Prior to methanol determination, the samples were thawed, spiked with pentanol and mixed thoroughly. Methanol was determined quantitatively in a microprocessor-controlled Hewlett-Packard 5840 gas chromatograph. A 3 m long column, packed with 80/100 mesh Carbowax 20M, was maintained at 150°C. The temperature of both the injection port and flame ionization detector was 250°C. The carrier gas was nitrogen. With regard to fermentation, all shake flask studies were undertaken with a New Brunswick G-20 Gyrotary shaker, maintained at a temperature of 42°C, a 1 in. stroke and 300 rev/min. In some experiments a 14 1 New Brunswick MF-114 fermenter with a condenser (8°C) at the air exit was used. Throughout the course of fermentation, no antifoam agent was added and the pH and temperature were maintained at 4.0 and 39°C, respectively. The dissolved oxygen of the culture was maintained at 60% by automatic regulation of the sparge and agitation rate. The total nitrogen was determined by Kjeldahl's method,

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Enzyme Microb. Technol., 1979, vol. 1, October 265

Papers and the crude protein content calculated by multiplying nitrogen content by 6.25. Values for the specific growth rate (/2) were related to the biomass doubling time (Ta) according to the following equation: In 2 0.693 /2

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Results For our yeast isolation programme, methanol-utilizing organisms obtained from various samples of soil, water and plants served as culture sources. The yeast was isolated from a mixed culture designated 'SC'. The SC culture was derived from a mixture of water and weeds from the Arabian Gulf using enrichment culture techniques conducted at 42°C. 9 A combination of low pH (4.0) and an acid-stable antibiotic, cephalosporin,* was employed to enrich the culture with the yeast prior to its separation on agar plates. The yeast was subsequently identified as Hansenula polymorpha Morais et Maia.t In order to optimize the various growth parameters for the yeast, the following studies were undertaken. Unless indicated otherwise, the incubation temperature was 42°C, the initial pH was 4.0, and the concentrations of methanol and yeast extract in the medium were 2 ml/1 and 50 mg/1, respectively. No corrections were made for the evaporative losses of methanol encountered in the shake flask or 14 1 fermenter studies. The effect of temperature on growth is shown in Figure 1. The results indicate that the culture was able to grow better at 35 42°C than at 25°C. Moreover, both the growth rate and yield coefficient for the yeast at 42°C were not substantially lower than the values at 35°C, thereby showing the thermotolerant nature of the yeast. With regard to the effect of pH, the initial pH values of the medium were adjusted within the range of 3.5 to 6.7. After sterilization, the pH was re-checked in duplicate flasks. After 23 and 42 h incubation, the absorbance of the cultures was measured. The results, shown in Figure 2, indicate that the optimum pH is 4.0. However, at pH 3.5 and 6.7, the biomass was only 10% lower than at pH 4.0. The pH of the cultures declined by 0.2 to 0.5 units at the termination of incubation (42 h).

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* Cephradine (Velosef~), a semisynthetic cephalosporin of E. R. Squibb and Sons, Athens, New York. t CBS, personal communication, Delft, The Netherlands.

266 Enzyme Microb. Technol., 1979, vol. 1, October

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Time (h) F i g u r e 3 E f f e c t of d i f f e r e n t c o n c e n t r a t i o n s of v i t a m i n s a n d y e a s t e x t r a c t o n g r o w t h in s h a k e flasks at 4 2 ° C . (a) Biotin + t h i a m i n HCI: = c o n t r o l ; e , 5 / ~ g b i o t i n + 3 0 0 ~ g t h i a m i n hydrochloride; o, 15/~g b i o t i n + 9 0 0 g g t h i a m i n h y d r o c h l o r i d e . (b) Y e a s t e x t r a c t : =,control;,% lOmg/I;o 25mg/I;A 50mg/I;e, lOOmg/I

To observe the effect of growth factors, a combination of biotin and thiamin HC1 was tested in one case ; yeast extract was added at four concentrations in the second case. The results shown in Figure 3 show that the growth curve obtained with 5/ag biotin plus 300 ~tg thiamin HC1 was almost identical to that with three times the concentration of the two vitamins. The incorporation of 100 mg yeast extract/1 of medium increased the biomass by 12% over and above the increase obtained with the biotin and thiamin HCI. When all the growth factors were deleted (control) from the medium, there was no increase in the biomass. Growth curves obtained with the initial methanol concentration in the range 0.01-0.8%, v/v, are plotted in Figure 4, which shows the effect of methanol concentration on growth. It was noted that above 0.1% methanol, the growth rate is inhibited and the yield of the biomass is depressed. The culture was grown at 39°C in a 14 1 fermenter under the conditions described above. The concentrations of methanol and yeast extract in the medium were 1.5 and 100 my/l, respectively, and the pH of the medium was 4.0. The increase in the biomass and the corresponding decline in the concentration of methanol, observed during the course of fermentation, are shown in Figure 5. A brief lag

Growth of a methanol-utilizing yeast: H. S. Pal and I. Y. Hamdan

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Incubation period at 42°C (h) Figure 4 Effect of different methanol concentrations on growth in shake flasks at 42°C: =, zero control; e, 0.01%; x, 0.05%; ~, 0.1%; ,~, 0.2%; A, 0.3%; o, 0.6%; +, 0.8%

phase was followed by a progressive increase in the biomass during the exponential phase. This lasted until the eighteenth hour. At that point, the residual methanol concentration was only 2 ~ % of the initial level, and the growth curve began to level off. The values for Ta, ~t and Ys were found to be 3 h, 0.23 h - I and 0.38, respectively. At the termination of fermentation, the yeast cells were harvested after centrifuging at 4°C, washed twice with an equal volume of chilled distilled water, and lyophilized. The final product was a free flowing white powder containing 40.3% crude protein. Finally, a trial experiment was conducted to note the effect of sea-water on growth. The salt medium described above was compounded with 75% sea-water and included 200 mg/1 of yeast extract and 0.2%, v/v, methanol. The growth of the yeast in this medium was similar to that obtained with the salt medium. The results indicate that the high salinity and the constituents of such a medium did not have a deleterious effect on the growth of the yeast.

as Hansenula polymorpha Morais et Maia. (Van Dijken 6 has undertaken intensive biochemical studies on a similar species of yeast.) Marine yeasts are known to grow in a wide range of saline media as well as media prepared with fresh water, is Our preliminary data show that the yeast can grow on methanol in a medium compounded with 75% fresh, filtered sea-water taken from the Arabian Gulf. Higher concentrations of sea-water in the medium were not tested, Since this yeast was originally isolated from sea-water, its halophilic property was not unexpected. From the standpoint of the economics of SCP production, the yeast's ability to withstand high salinity may prove to be advantageous in countries like Kuwait, where the availability of groundwater is extremely limited while sea-water is abundant. Moreover, some organic compounds present in sea-water could possibly serve as cosubstrates for the heterotrophic yeast mentioned above, thereby enhancing the production of the biomass. Our results in Figure 3 show that, like all methanolutilizing yeasts, our culture has an absolute requirement for vitamins, which could also be provided by yeast extract. Generally, the data derived from batch fermentations on the biomass yield and specific growth rates do not reflect precisely the corresponding figures at steady-state in the continuous system. However, batch cultures may furnish significant preliminary information on the kinetics of growth and substrate inhibition of growth. In the 14 1 batch fermentation at 39°C (Figure 5) our observed value of/2 was 0.23 h - l . In methanol-limited continuous cultures, the values of ~ reported by other investigators were 0.18-0.2 h -1 at 37°C, 6 0.22 h -1 at 3 7 - 4 2 ° C 8 and 0.28h -1 at 37-43°C. 5 We observed a Ys 1.2

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Discussion Since the first report, by Ogata, 3 on a methanol-utilizing yeast, several such yeasts have been isolated from natural sources. However, not all of the species are thermotolerant. The original source for our culture was a mixture of bacteria and yeasts. In order to reduce the number of bacterial cells, we employed a combination of low pH (4.5), high incubation temperature (42°(2) and a high concentration of the antibiotic cephalosporin (500 mg/1). We avoided the use of cycloserine and penicillin G, which according to van Dijken 6 are unstable at low pH. The cephalosporin which we used proved stable in the pH range 2 . 5 - 8 . 0 ) o The resulting yeast isolate has been identified by CBS of Delft, The Netherlands,

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Enzyme Microb. Technol., 1979, vol. 1, October

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Papers value o f 0.38 in our batch cultures compared to 0.36, 8 0.386 and 0.3864 reported b y others for the continuous cultures. Specific growth rate and the biomass yield are known to be affected significantly by different methanol concentrations in the culture. 7,8,n-13 Our results in Figure 4 are in agreement with these findings and suggest, moreover, that even at methanol concentrations below the lower limit for growth inhibition (0.1%, v/v) depression o f the yield coefficient occurs. 16 Our observed protein content o f 40% fails within the reported range ( 3 5 - 5 0 % ) for methanol-grown yeasts. 14

Acknowledgements The authors are grateful to Miss Lewa Khosrawi for her technical assistance, Dr Farid Shunbo and Mr Rouhi Abu Tabanja for the g.c. analysis and Dr Geoffrey Hamer for his comments.

References 1 Quayle, J. R. Adv. Microb. Physiol. 1972, 7, 119-203 2 Bassalik, K. Jahrb. Wiss. Bot. 1914, 53,255-298

268 Enzyme Microb. Technol., 1979, vol. 1, October

3 Ogata, K., Nishikawa, H. and Ohsugi, M. Agric. BioL Chem. 1969, 33, 1519-1520 4 Minami, K., Yamamura, M., Shimuzu, S., Ogawa, K. and Sekine, N. J. Ferm. TechnoL 1978, 56, 1-7 5 Minami, K., Yamamura, M., Shimuzu, S., Ogawa, K. and Sekine, N. J. Gen. AppL Microbiol. 1978, 24, 155-164 6 Van Dijken, J. P. PhD Thesis, Groningen University, The Netherlands (1976) 7 Papoutsakis, E., Lim, H. and Tsao, G. AIChE J. 1978, 24, 406-417 8 Levine, D.W. andCooney, C.L. AppLMicrobioL 1973,26, 982-990 9 Pal, H., Hamdan, I. and A1-Mouthen, F. J. Univ. Kuwait (Science) in press 10 Windholz, M. The Merck lndex o f Chemicals and Drugs 9th ed., Merck & Co., Rahway, New Jersey, 1976 11 Reuss, M., Gnieser, J., Reng, H. and Wagner, F. Eur. J. AppL MicrobioL 1975, 1,295-305 12 Wagner, F. Experientia 1977, 33, 110-112 13 Wilken, T., Hazeu, W., van Leengoed, L. and de Snoo, T. Alcohol, Industry and Research 1977, pp. 220-226 14 Cooney, C., Levine, D. and Snedecor, B. Food TechnoL February 1975, 34-43 15 MacLeod, R. A. Tolerance in Marine Ecology (Kinne, O., ed.), Wiley- Interscience, New York, 1971, ch. 4, pp. 689- 703 16 Hamer, G., Pal, H. S. and Hamdan, I. Y. BiotechnoL Lett. 1979, 1, 9-14