Biotechnology Letters Vol.4 4 253-256 (1982)

PRODUCTION OF ETHANOL FROM SUGAR CANE MOLASSES

BY Zymomonas mobilis

H.J.J. van Vuuren*and Lourine Meyer

Sentrachem Research Unit, Department of Microbiology University of the O.F.S. BLOEMFONTEIN R. SOUTH AFRICA

SUMMARY

Zymomonas mobilis strains were compared with each other and with a

Saccharomyces cerevisiae strain for the production of ethanol from sugar

cane molasses in batch fermentations. The effect of pH and temperature on

ethanol production by Zymomonas was studied. The ability of 2. 'mobiZis

to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae.

However, at higher sugar concentrations the yeast produced considerably

more ethanol than Zymomonas.

INTRODUCTION

Recently a number of papers have been published dealing with the

ability of different Zymomonas strains to produce ethanol mainly in complex

laboratory media (Rogers et al,., 1979; 'Lee et al., 1980 and Rogers et al.,

1980). These results indicated that Z. mobiZis seem to have considerable

potential for large scale alcohol fermentations. However, the main cost component for the industrial production of ethanol is the substrate. It

is therefore essential that ethanol production by Zymomonas from cheap

substrates, available in large quantities, be investigated to evaluate Zymomonas as micro-organism for the industrial production of ethanol. In

this study blackstrap sugar cane molasses, normally used for the industrial production of ethanol in South Africa and elsewhere, was used as substrate.

MATERIALS AND METHODS

Strains and media. Zymomonas mobiLis strain 10988 was obtained from ATCC. Other Zymomonas strains were isolated from palm wine according to the

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methods described by Swings (1974>, The Zymomonas strains were adapted to

grow in molasses media according to the following procedure:

Stock cultures 1

1,6% glucose + 0,4% molasses + 0,5% yeast extract (YE)

1 l,O% glucose + 1% molasses + 0,4% YE

I 0,5% glucose + 1,5% molasses + 0,2% YE (2x)

1 2% molasses + 0,2% YE

1 4% molasses + 0,2% YE (2x)

I 4% molasses

The adapted Zymomonas strains were maintained on agar slants containing 4%

molasses at a pH of 5,0 at 4°C. Saccharombces cerevisiae Y102 was obtained

from National Chemical Products (Pty) Ltd and maintained according to their

procedures.

EXPERIMENTAL PROCEDURES

The effect of pH on ethanol production. Molasses media containing 10%

sugars at the following pH levels (after sterilization) were used: 4,20;

5,0; 5,60; 5,80; 6,20; 6,40 and 6,60. Concentrated H2S04 or 10 N NaOH were

used to adjust the pH. Fermentation tubes containing 15 me/tube were ino-

culated with 1 m& of a 16 hr old culture grown in molasses media contain-

ing 5% reducing sugars. Incubation took place at 30°C for 72 hrs.

The effect of temperature on ethanol production. The same medium and pro-

cedures used for pH studies were employed. The pH was set at 5,60. Incu-

bation took place at 28; 30; 32; 34 and 38°C respectively.

Comparison of Z. mobilis ATCC 10988 and s". cerevisiae Y102. Fermentations

were carried out under sterile conditions in a 1,5 .JZ Multigen fermenter.

Diluted molasses (1 2) containing 5; 10; 15 and 20% reducing sugars were

used respectively. The pH of the molasses wort was adjusted to S,60 and

4,80 for ,zly~o~oncr:; and ?. cerevisias respectively. Sixteen hour old Zymo-

mow,; and BJ~. cercvisiati cultures were standardized to contain approximate-

ly 1 x lo8 cells/me. A 10% cv/v> inoculum was used. Ethanol production

was monitored continuously by gas chromatography.

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RESULTS AND DISCUSSION

The effect of pH and temperature on ethanol production

by Zymomonas are indicated in Figs. 1 and 2 respectively.

The set pH of the molasses wort had a marked effect on ethanol production

by all Zymomonas strains studied. The optimum pH for strains P156, P159,

P160 and P163 were approximately between 5,0 and 5,60 compared with an

optimum pH of ca. 5,70 for strain 10988 (Fig. 1). 2. mobilis 10988 pro-

duced considerably more ethanol than the other strains studied and was

therefore the only strain used in further studies.

The effect of temperature on ethanol production by Z. mobilis 10988 is

shown in Fig. 2. The optimum fermentation temperature for this strain is

approximately 32'C. Temperatures above 34°C severely inhibited ethanol

production.

Final amounts of ethanol produced by Z. mobilis 10988 and S. cerevisiae

Y102 from different sugar concentrations in molasses media are indicated in

Table 1. At a 5% sugar level Zymomonas produced more ethanol than Saccharo-

myces. However, at the higher sugar concentrations the yeast produced con-

siderably more ethanol than Zymomonas. The ability of Z. mobiZis 10988 to

grow and produce ethanol at high sugar concentrations in synthetic media

has been demonstrated (Rogers et al., 1979). The low efficiencies obtained

in this study can probably be ascribed to the presence of certain compounds

in the molasses which is inhibitory to growth and ethanol production by

Zymomonas. This phenomenon has also been observed by Australian workers

(Dr. D.E. Tribe, personal communication); Zymomonas is therefore not a

suitable micro-organism to produce ethanol on a large scale from sugar cane

blackstrap molasses. However , promising results have been achieved using

maize as substrate (Faber and van Vuuren, unpublished data).

REFERENCES

Lee, K.J., Lefebvre, M., Tribe, D.E. and Rogers, P.L. (1980) Biotechnol.

Lett. 2, 487 - 492.

Rogers, P.L., Lee, K.J. and Tribe, D.E. (1979) Biotechnol. Lett, 1, 165 .- - 170.

Rogers, P.L., Lee, K.J., Skotnicki, M.L. and Tribe, D.E. (1980) Proceedings

of the 6th IFS Symposium London Ontario Canada.

Swings, J.G. (1974) Ph.D. thesis, State University of Gent Belgium.

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5,0

4, O 10988

P160

3,0 ,j.

o z,O 1,4,1

P 163 1,0 P156

P159

4,2 4,6 4,8 5,2 5,6 6,0 6,4 6,6

pH

Fig. 1 The effect of pH on ethanol production by. five Zymomonas strains

5,0

4,0 ) ,

: ' 3,0 Z O *" 2,0 m

1,0

I 1 ! I I t

28 30 32 34 36 38

TEMPERATURE °C

Fig. 2 The effect of temvera-~re on etha~ol production by Z. r~obilis 10988

TABLE 1 Final amounts of ethanol produced by g. mobilis 10988 and

,;. ocretoisiae YI02 from different sugar concentrations in

molasses media

Sugar concentration

(~ m/v)

5

I 0

15

20

% Ethanol (V/v)

7. mobilis S. ccrevisiae

2 , 5 0

3,68

5,0

2,0

2,30

5,20

7,66

I0,50

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