COMPARISON OF POLISH INDUSTRIAL DISTILLERY YEAST WITH ETHANOL PRODUCING BACTERIA ZYMOMONAS MOBILIS

Jacek Nowak

ABSTRACT

Comparison between ethanol producing bacteria Zymomonas mobilis (strain CCM 3881 and CCM 3883) and commercial yeast (D₂, Bc-16a) was made using glucose medium. While the differences between bacteria and yeasts in low glucose medium (85.5 g/dcm³) were not of statistical importance, ethanol yield and sugars utilization were higher for Z. mobilis on medium with high concentration of glucose (150-250 g/dcm³). The bacteria growth was less inhibited by alcohol in the medium than yeasts. The bacteria biomass needed for 1 dcm³ ethanol production was from 2 times (glucose medium 85.5 g/dcm³) to 3-4 times (241 g glucose in 1 dcm³ medium) less than yeasts.

Key words: yeast, Zymomonas mobilis, ethanol yield, ethanol productivity, continuous fermentation.

INTRODUCTION

From other microorganisms than Saccharomyces cerevisiae tested as a potential ethanol producers, Zymomonas mobilis is probably the most suitable organism. It converts glucose almost stoichiometrically to ethanol and CO₂, grows more rapidly than the yeasts and demonstrates highest productivity during continuous fermentation [2, 20, 22]. Significantly higher specific rates of sugar uptake and ethanol production compared to those found for yeasts [11, 15, 22]. Zymomonas cultures grow anaerobically and unlike yeasts do not require the controlled addition of oxygen to maintain viability at high cell concentrations [10, 22]. Ethanol tolerance of some strains of Zymomonas is comparable and somethimes higher than strains of Saccharomyces cerevisiae [3, 21]. Zymomonas mobilis produces less by-products, especially fusels [7, 14]. The genetic manipulation of Zymomonas is simpler than for yeasts which give the opportunity to widen the spectrum of raw materials for ethanol production to lignocellulosic materials and direct digestion of starch [5, 14, 24].

In this work the fermentation capabilities of two Zymomonas mobilis (CCM 3881 and CCM 3883) strains from Czech Culture Collection were compared with two stains of industrial Polish yeasts D₂ and Bc-16a. Both batch and continuous techniques were used as well as immobilization of cells in alginate in continuous process. Advantages of bacteria on glucose medium were demonstrated.

MATERIALS AND METHODS

- Dried Polish commercial yeast Saccharomyces cerevisiae Bc-16a and D₂ as well as bacterial culture Zymomonas mobilis CCM 3881 and CCM 3883 from Czech Culture Collection were used.

- Dried yeast were rehydrated 20 min in water (1:10) and acidified with H₂SO₄ (5 cm³ in 1 dcm³ water) for decontamination.

- Z. mobilis was cultured on glucose medium (glucose 80 g/dcm³, yeast extract 10 g/dcm³, KH₂PO₄ 1 g/dcm³, (NH₄)₂SO₄ 1 g/dcm³, and MgSO₄ × 7 H₂O 0.5 g/dcm³).

- Glucose medium with different glucose level was used in the experiments.

- Glucose medium (glucose 80 to 250 g/dcm³, yeast extract 10 g/ dcm³, KH₂PO₄ 1 g/dcm³, (NH₄)₂SO₄ 1 g/dcm³, and MgSO₄ × 7 H₂O 0.5 g/dcm³).

- Batch fermentation were done in 0.5 dcm³ Erlenmeyer flask containing 0.2 dcm³ medium and 10% of inoculum (80 g/dcm³ glucose medium after 24 hours fermentation).

- For continuous fermentation BioFlo C 30 fermentor (New Brunswick) was used with 1 dcm³ reactor (0.38 dcm³ working volume) and 10 % of inoculum (80 g/dcm³ glucose) was added and after 24 hours culturing the continuous process was started.

- For immobilization sodium alginate solution (30 g/dcm³) was mixed with bacterial or yeast cells resuspended in sterile water, added dropwise to 0.1 mol/dcm³ CaCl₂ and allow to solidify for 1 hour. Gel beads (180 g) were used in the bioreactor. Bacterial and yeast cells were centrifuged after 24 hours fermentation on 80 g/dcm³ glucose medium (3000 min^-1, K70, 10 min). In 180 g of alginate beads 4.5-4.75 g d.m. of yeast cells and 3.3-3.5 g d.m. of bacterial cells were immobilized.

- All the fermentations were carried out in 30°C for 24-96 hours (batch fermentation) or 15 days (continuous fermentation).

- Sugars were estimated spectrophotometrically using 3,5-dinitrosalicylic acid and glucose as a standard [13]

- Ethanol was measured by density measurement after distillation.

- Cell biomass was estimated gravimetrically after drying at 60 ° C with ethanol and then at 105°C to a constant weight.

- Ethanol yield was calculated for used sugars and the amount of sugars in the medium after sterilization and after fermentation was estimated for each sample. The theoretical ethanol production was calculated for the sugars used and taken as 100%. The productivity was expressed as the amount of ethanol in gram formed in bioreactor within one hour counting for one dcm³ of working volume of fermentor.

RESULTS AND DISCUSSION

The most popular medium used for testing Z. mobilis ethanol production was glucose medium enriched in yeast extract, ammonium sulphate and magnesium sulphate [1, 6, 10, 17, 21, 23].

Ethanol yield of bacteria and yeast was very close when a glucose medium with lower level of glucose (85.5 g/dcm³) was used (Tab. 1).Using medium with the amount of sugars normally present (136 g/dcm³) in industrial mashes the yield for both types of microorganisms start to differentiate and the use of glucose in the amount close to 250 g in 1 dcm³ medium proved that in high osmotic pressure circumstances bacteria producing ethanol with significantly higher yield (Fig. 1). The physiological basis of the exceptionally high sugar tolerance of Zymomonas was investigated by Struch et al. [23]. Z. mobilis has a facilitated diffusion system which enables a rapid equilibration between internal and external glucose concentration.

This is to stress that the amount of bacteria biomass produced during fermentation was less than yeast biomass.

The comparison of biomass needed to produce 1 dcm³ of 100% ethanol proved, that with growing concentration of glucose in the medium the amount of biomass needed to form 1 dcm³ ethanol decreased both for yeasts and bacteria. However to produce the same volume of pure ethanol we need significantly less bacterial biomass than yeast biomass (especially for the media with high glucose level). The bacteria biomass needed for 1 dcm³ ethanol production was from 2 times (glucose medium 85.5 g/l) to 3-4 times (241 g glucose in 1 l medium) less than yeasts (Fig.2). Close to the theoretical ethanol yield for bacteria for media with high glucose concentration is likely to be connected with both less metabolic energy used for growth of population and less maintenance energy used by bacteria biomass in that situation.

Accumulation of alcohol during fermentation is accompanied by a progressive decrease of rate of sugar conversion to ethanol and repress microorganisms growth. The mechanism of this is not fully recognized there is evidence that inhibition of fermentation can be attributed to an indirect effect of ethanol and by repression of the enzymes of glycolysis involving the plasma membrane which allow leakage of essential cofactors and coenzymes (Doelle et al.1993, Osman and Ingram 1985, Hobley and Pammet 1994).

In this work the attempt just to estimate the growing possibilities of used strains of bacteria and yeast in the present of ethanol in the medium was made. The test was planned to compare the amount of biomass formed in standard medium (100 g/dcm³ glucose) connected 0, 5, 10 and 15% (v/v) ethanol in the beginning of fermentation. The addition of ethanol to the medium influenced yeast growth more than bacteria (Fig. 3). Still 5% ethanol concentration in the medium partly inhibited growth of both types of microorganisms and 15% concentration nearly fully inhibit the growth of used ethanologens. From the four strains tested ( two strains of Zymomonas and two strains of yeasts), Z.mobilis CCM 3883 was the best to adopt to the ethanol presence in the medium.

Continuous fermentation of glucose medium (136 g/dcm³) was confronted using 0.38 dcm³ working capacity bioreactor with gentle stirring (50 min^-1). Two dilution rates was used, one D=0.12 h^-1 which was thought to be preferable for slower growing yeast cells and the second dilution rate D=0.2 h^-1 which was close to the maximum growing rate expecting for yeasts. Theoretically the minimum retention time for medium containing glucose (150 g/dcm³) should not be less than 4-4.5 h [12]. Practically longer retention times are used. Using dilution rate D=0.2 h^-1 glucose medium is kept in the fermentor for 5 hours. Mixing and slight aeration for yeasts in such a dilution rates are preferable.

The comparison of ethanol content, glucose utilization and ethanol productivity (Fig. 4 and 5) showed that in both dilution rates used tested, Zymomonas was significantly better than the used commercial strains of yeasts. Even for the process with lower dilution rate (the retention time 8.33 h) both ethanol yield, and sugar utilization were higher for bacteria and the productivity, which seems to be one of the most important parameter in continuous fermentations, of bacteria was almost twice of that of yeasts (Fig. 4).

Better results was achieved in D=0.2 h^-1 when the productivities of yeast (5.68-7.73 g/dcm^3. h) were much closer to that of bacteria strains (8.84-11.05 g/dcm^3. h). Still the sugar utilization and concentration of produced ethanol was not acceptable. The best results gave Z.mobilis 3883. It produced 5.5 g/dcm³ ethanol, consuming 95.1% of total glucose and achieved the productivity exceeded 11 g/dcm^3. h.

The relatively low parameters (but comparable with those cited in the literature - Ishizaki et al. [9]) we linked with high wash out of biomass from bioreactor during fermentation. The amount of yeast biomass coming out with the effluent was as high as 6-7.5 g d.m. in 1 dcm³ and 3-6 g of dry bacterial bomass in 1 dcm³. Because of that it was decided to use the immobilization procedure to enhance the biomass concentration in the bioreactor and enhance the productivity.

The same bioreactor BioFlo C30 (New Brunswick) was used, filled with 180 g of calcium alginate beads containing immobilized bacteria or yeasts. Gentle stirring (50 rpm) was employed which did not destroy the structure of gel.

Using the immobilized system higher dilution rates could be applied which markedly increased the productivities of the used bioreactor. When dilution rate D=0.5 h^-1 employed the ethanol productivities of bacteria settled on the level of 23.8-25.5 g/dcm^3. h (yeasts 14.5-15.8 g/dcm^3. h) (Fig. 6). Applying D=1.0 h^-1, dilution rate relatively for that kind of immobilized fermentor, the ethanol productivity up to 37.0 g/dcm^3. h for Z.mobilis CCM 3881 and 32.3 g/dcm³*h for Z.mobilis CCM 3883 was achieved. The productivity of yeast was in this fermentation increased much less, reaching 16.8 g/dcm^3. h (D₂) and 19.1 g/dcm³*h for Bc-16a (Fig. 7).

CONCLUSIONS

Comparison of two strains of ethanol producing bacteria Zymomonas mobilis and two commercially used in Polish distillery industry yeasts proved that the bacteria represents a high potential possibilities as ethanol producer. The two used strains of bacteria have some advantages against the industrially used yeast as: higher yield when media with high glucose concentration is used, less inhibition by ethanol and higher productivities in continuous fermentation and especially in continuous fermentation when cells were immobilized in alginate beads.

The above mentioned advantages, from the other hand, were demonstrated on rich glucose medium. There is evidence that the situation might be different when industrial media as rye mashes are used (almost 90% of ethanol is produced in Poland from rye). The yield of alcohol produced by tested bacteria are the same as industrial yeasts [15].

However there is to emphasise that bacteria are more suitable for ethanol production, when more dynamic, continuous fermentation techniques are used, especially with immobilized cells.

As it seems that a serious challenge will concern bioethanol producing industry of Central Europe when fuel bioethanol government programs will be opened, applying of more productive techniques and microorganisms must be taken into account. Zymomonas mobilis appears to have considerable potential for industrial alcohol production especially for high productivity fermentation systems.

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