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Biomass 15 (1988) 201-207

Ethanol Fermentation of Cassava Starch by Zymomonas mobilis NRRL B-4286


H. Nellaiah, T. Karunakaran & E Gunasekaran*
Department of Microbiology, School of Biological Sciences, Madurai Kamarai University, Madurai 625 021, India (Received 11 October 1987; revised version received 8 January 1988; accepted 12 January 1988)

ABSTRACT Four strains o f Z y m o m o n a s mobilis were compared for ethanol production from enzymatically hydrolysed cassava starch. Strain NRRL B-4286 performed efficiently, producing 80 g/litre ethanol from 171 g/litre initial sugar concentration. Addition of yeast extract, calcium pantothenate, ammonium sulphate or magnesium sulphate did not significantly increase ethanol production by this strain. Key words: Ethanol fermentation, cassava starch, Zymomonas mobilis.

NOTATION

E P qp q~ Qp Q~ Su T X /~

Yp/~ Yx/~

Percentage of theoretical yield (%) of ethanol Final ethanol concentration (g/litre) Specific ethanol productivity (g/g/h) Specific sugar uptake (g/g/h) Volumetric ethanol productivity (g/litre/h) Volumetric sugar uptake (g/litre/h) Sugar utilized (g/litre) Period of study (hours) Final biomass (g/litre) Ethanol yield Cell yield Specific growth rate (h- ~)

201 *To whom all offprint requests should be addressed.

Biomass 0144-4565/88/S03.50 -- 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain

202

H. Nellaiah, T. Karunakaran, P. Gunasekaran

INTRODUCTION Production of fuel alcohol from renewable raw materials such as wheat starch, corn starch, sweet sorghum, cane molasses, bagasse, etc. is well k n o w n , 1-4 but there are fewer reports concerning the use of c a s s a v a . 5-7 Recently, Poosaran et al 8 reported ethanol production from cassava starch using Z. mobilis ZM4. This paper extends our previous studies on fermentation of glucose, fructose and sucrose by another strain of Z. mobilis ( N R R L B-4286) to aspects of cassava starch fermentation.

MATERIALS AND METHODS


Z y m o m o n a s mobilis strains NRRL B-14023 (ZM4), NRRL B-14022 (ZM3), NRRL B-4286 and NRRL B-806 (ZM1) were obtained from the Northern Regional Research Laboratory, Illinois, USA and maintained on RM (Glucose 2%; Yeast extract 1%; KH2PO 4 0"2%, pH 7.0) slants. The liquid seed culture for all fermentation experiments had the same composition as the above growth medium with the exception of that used with the adapted cultures, where cassava starch hydrolysate (CSH) was used. Fermentations were inoculated using a 10% (v/v) culture in the exponential phase of growth. Starch hydrolysate was prepared by mixing cassava flour with distilled water (1:5 w/w) and digesting with alpha amylase (Termamyl, 0.6% v/v) at 90C for 1.5 h. Amyloglucosidase was added at 0.6% (v/v) and incubated at 37C for 20 h (alpha amylase and amyloglucosidases were from Novo Industries, Denmark). The CSH was boiled and the precipitate removed by filtration. The solution was then sterilized. The CSH, as used, contained 168-171 g/litre sugar (approx. 86% conversion). Batch fermentations were carried out at pH 7.0. Ethanol was estimated using the method of Caputi et al. 9 Biomass was determined as absorbance at 580 nm while the corresponding dry weight was obtained from a standard curve of absorbance versus dry weight as described by Doelle and Greenfield. 1 Residual sugar was estimated by the DNS method.11

RESULTS AND DISCUSSION Initially ethanol fermentation of four Z. mobilis strains (non-adapted) (NRRL B-14023, NRRL B-14022, NRRL B-4286 and NRRL B-806) on CSH (initial sugar concentration 168 g/litre) were compared (Table 1). Strains NRRL B-4286, NRRL B-14023 and NRRL B-14022 performed efficiently with ethanol yields of over 86% and final biomass

Ethanol from cassava using Zymomonas

203

TABLE l Comparison of Ethanol Production by Z. mobilis Strains on CSH (Initial Sugar Concentration - - 168 g/litre)

Parameters
P
X Su

NRRL B-14023
75'8 1"66 161'50 0"47 0"01 86"7

NRRL B-14022
75"8 1"66 158'60 0'48 0"01 88'4

NRRL B-4286
75-8 1-17 160"70 0"47 0"0073 88'4

NRRL B-806
73"8 !'26 157'10 0'47 0"008 84"6

Yp/~ Y~/~
E

yields of over 1.0 g/litre. NRRL B-4286 gave the highest yield in 36 h while the other strains took 4 0 - 4 8 h. The lower yield of N R R L B-806 could be attributed to its poor volumetric sugar uptake and ethanol productivity rates. Since strain NRRL B-4286 was found to be efficient when grown on glucose, fructose, sucrose, ~2 cane juice or molasses, ~3 this strain and the already reported efficient strain Z M 4 j4 w e r e selected for further studies. A study was undertaken to compare the fermentation efficiency of adapted and non-adapted cultures of strains NRRL B-4286 and NRRL B-14023. Results are shown in Table 2 and Fig. 1. In all experiments fer-

TABLE 2 Comparison of Kinetics of Ethanol Production by Z. mobilis strains NRRL B-14023 and NRRL B-4286 on CSH (Initial Sugar Concentration - - 171 g/litre)

Parameters 14023
P X Su 77'9 1'35 160"7 0.48 1.95 5.02 16-28 4.41 7.72 0"084 0"01 89"3

Non -adapted 4286


78'5 1"38 160"8 0.49 1.96 5.03 16-28 6.71 12-62 0" 100 0"0086 90-0

Adapted 14023
78'4 1"44 157'0 0-5 2-4 4'9 16-28 7"14 13'80 0.060 0"0092 89"9

4286
80"1 2"88 160"2 0.5 2.8 5.72 16-28 1.97 3.38 0.193 0-018 91.9

Yp/s
Qp Q~ T qp q~ /~

Yx/~
E

204

H. Nellaiah, T. Karunakaran, P. Gunasekaran


A

80 ~ 60
~o

Z "r
"'

20

160
v

120
0

~n 80 z

~0

en

I
0 8 16 TIME 24 ( h )

1
32

I
40

Fig. 1. K i n e t i c s o f e t h a n o l p r o d u c t i o n o f Z. mobilis o n c a s s a v a starch h y d r o l y s a t e (CSH): o - - N R R L B-4286 (adapted); [ ] - N R R L B-14023 (adapted); - N R R L B-4286 (non-adapted); - - N R R L B-14023 ( n o n - a d a p t e d ) .

mentation was completed within 40 h. Z. mobilis NRRL B-4286 adapted to CSH, showed the fastest rate of fermentation, achieving a final ethanol concentration of 80.1 g/litre after 28 h.. Thus, the adaptation of the culture to a higher sugar concentration helped in achieving maximum yield in a short period. There was a considerable difference in the rates of growth and fermentation between adapted and non-adapted ceils of this strain. Parallel experiments were carried out using NRRL B-14023 (ZM4), but there was no significant difference in the rates of growth and fermentation between adapted and non-adapted cultures. The ethanol yield was 0.5 and 0.48-0.49 for adapted and non-adapted

TABLE 3 Effect of Nutrients on Ethanol Fermentation of Z. mobilis strain N R R L B-4286 on CSH (Initial Sugar Concentration - - 168 g/litre)

Parameters

CSH + Calcium pantothenate

CStt + A m m o n i u m sulphate

CSH + Magnesium sulphate

CSH + Yeast extract

Control ~

P X Su Yp/~ Qp Q~ T

qp

N
O O

q~

Yx/~ E

73"3 1'66 158"3 0"46 1"83 3"96 16-24 6"91 13"02 0"395 0-01 85-5

76.8 2"04 160"5 0"48 2"40 5'01 16-24 7"07 15"30 0'271 0"013 89"6

77"0 1"15 160.7 0"48 2"41 5"02 16-24 9'46 19-75 0"211 0"0072 89"8

75"2 2"04 160.5 0"47 2-35 5'01 16-24 8"10 14'40 0"124 0"0130 87.7

75"8 1"17 160-7 0"47 2"37 5"02 16-24 9-14 16"80 0"204 0"0073 88"4

a Cassava strarch hydrolysate only.

C~

206

H. Nellaiah, T. Karunakaran, P. Gunasekaran

cultures of both strains, respectively. However, strain NRRL B-4286 had slightly higher substrate consumption, ethanol productivity and percentage of theoretical yield (91.9%) values. These results are comparable to those of ZM4 obtained by Poosaran et aU The effect of addition of various nutrients (calcium pantothenate -- 5 mg/litre, ammonium s u l p h a t e - 1 g/litre, magnesium s u l p h a t e - 0.5 g/litre and yeast extract -- 10 g/litre, added separately) on fermentation was studied (Table 3) using a non-adapted culture of NRRL B-4286 on CSH (initial sugar concentration 168 g/litre). No significant increase in ethanol production was observed in comparison with the control. Similarly, there was no significant increase in the biomass and cell yield over the control in CSH supplemented with nutrients. This perhaps suggests the presence of growth factors in CSH. Z. mobilis strain NRRL B-4286 produced 94 g/litre, 76"9 g/litre and 66.5 g/litre ethanol at a 200 g/litre concentration of glucose, fructose and sucrose respectively. 12 It also produced 74 g/litre ethanol from cane juice (180 g/litre)and 26.3 g/litre from molasses (100 g/litre). These results, together with the performance of this strain on cassava starch clearly indicate its efficiency, suggesting its high ethanol tolerance and its suitability for genetic improvement.

ACKNOWLEDGEMENTS The authors acknowledge UGC/India and CSIR/India for financial support.

REFERENCES 1. Bringer, S., Sahm, H. & Swyzen, W. (1984). Ethanol production by Zymornonas mobilis and its application on an industrial scale. Biotechnol. Bioeng. Syrup., 14, pp. 311-19. 2. Chay, P. B. Chonvel, H., Cheftel, J. C. Ghomidh, C. & Navarro, J. M. (1984). Extrusion- hydrolysed starch and flours as fermentation substrates for ethanol production. Lebensm.-Wiss. Technol., 17, pp. 257-67. 3. Saddler, J. N., Chan, M. K. H., & Louis-Geize, G. (1981). A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture. Biotechnol. Lett., 3, pp. 321-6. 4. Tanaka, H., Kumosawa, H. & Murakami, H. (1986). Ethanol production from starch by a co-immobilized mixed culture of Aspergillus awamori and Zymornonas mobilis. Biotechnol. Bioeng., 28, pp. 1761-8.

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5. Fujio, Y., Ohta, M. & Ueda, S. (1985). Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas circulation type fermenter. Biotechnol. Bioeng., 27, pp. 1270-3. 6. Lee, S. Y. Shik, Y. C., Kim, S. V. & Byun, S. M. (1985). Ethanol fermentation of uncooked cassava starch. J. Ferm. Technol. 63, pp. 51-6. 7. Prema, P., Ramakrishna, S. V. & Madhusudhana Rao, J. (1986). Influence of composition of sugars in cassava starch hydrolysate on ethanol production. Biotechnol. Lett. 8, pp. 449-50. 8. Poosaran, N., Heyes, R. H. & Rogers, P. L. (1985) Ethanol production from cassava starch using a highly productive strain of Zymomonas mobilis and Saccharomyces uvarum ATCC 26602. Biomass, 7, pp. 171-83. 9. Caputi, A., Ueda, M. & Brown, T. (1968). Spectrophotometric determination of ethanol in wine. American J. Enol. Viticul., 19, pp. 160-8. 10. Doelle, H. W. & Greenfield, P. E (1985). The production of ethanol from sucrose using Zymomonas mobilis. Appl. Microbiol. Biotechnol., 22, pp. 405-10. 11. Miller, G. L. (1959). Use of DNS acid reagent for the determination of reducing sugars. Anal. Chem., 31, pp. 426-8. 12. Nellaiah, H., Karunakaran, T. & Gunasekaran, P. (1988). Ethanol fermentation by an efficient strain, NRRL B-4286, of Zymomonas mobilis. J. Ferm. Technol., 66 (2). 13. Gunasekaran, P., Karunakaran, T. & Kasthuribai, M. (1986). Fermentation pattern of Zymomonas mobilis strains on various substrates -- a comparative study. J. Biosci., 10, pp. 181-6. 14. Skotnicki, M. L., Warr, R. G., Goodman, A. E., Lee, K. J. & Rogers, P. L. (1983). High productivity alcohol fermentations using Zymomonas mobilis. Biochem. Soc. Symp., 48, pp. 53-86.

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