Eur J AppI Microbiol Biotechnol (1983) l 8 : 186- 188

Short Communication

European Apphed Microbiology and Biotechnology �9 Springer-Verlag 1983

Conversion of Sucrose to Ethanol by a Flocculent Zymomonas sp. Strain in a Continuous Upflow Floc Reactor

Emilio Rodriguez I and Danley A. S. Callieri=

Ptanta Piloto de Procesos Industriales Microbiologicos (PROIMI), Avda. Belgrano y Pie. Caseros (4000) S. M. de Tucumfin; 2 Career Researcher from Consejo Nacional de Investigaciones Cientfficas y Tdcnicas (CONICET), Argentina

Summary . A floccu]ent strain of Zymomonas sp. isolated from sugar cane juice was used to produce ethanol (EtOH) from sucrose in a 50 ml upf]ow f]oc continuous reactor. A maximun productivity of 92 g EtOH/l per hour (estimated on a total volume basis) with 10% sucrose feed and 60% conversion to ethanol was achieved at a dilution rate (D) of 3 h -I. At D = 2 h -I the productivity was 80 g EtOH/] per hour and the yield 0.4 g EtOH/g sucrose supplied. Dilution rates up to 7 h -1 were assayed and even when no wash out occurred, productivity dropped to 63 g EtOH/I per hour and conversion was very low (16%). These results are compared with those obtained using Zymomonas mobilis ATCC 10988 under the same conditions.

I n t r o d u c t i o n

Strains of Zymomonas sp. were isolated in this laboratory from sugar cane juice, and tested for ethanol production using sucrose as the carbon source. Several of the isolates showed a flocculent growth and produced higher yields of ethanol than those of the non-flocculent type s t r a i n Zymomonas mobilis ATCC 10988. The f l o c c u l e n t growth c h a r a c t e r i s t i c has been mentioned as a means of ma in ta in ing high ce l l concentration within the continuous fermentor without adding material to retain the cells (Strandberg eta]. 1982). However, the reactors already described were not of the same design as the one used in this work, nor were they employed to convert sucrose to ethanol. One of the reasons for isolating Zymomonas strains from sugar cane was to find a microorganism capable of converting sucrose to ethanol at a high yield in order to use sugar cane molasses for the industrial production of ethanol.

Hater ia l and Hethods

Hicroorqanisms. a) St ra ins of Zymomonas sp. i so la ted from sugar cane j u i ce , b) Zymomonas mobilis ATCC 10988 (See i n t r o d u c t i o n ) .

Fermentation medium. In grams per litre: sucrose, 100; (-NHq-T2SOq, I; KH2PO 4 I; HgSOq.7H20 , I; yeast extract, 5. The resulting pH was 5.5.

Culture maintainance and inocula. The micro- organisms were maintained by monthly transfer to fermentation medium. After 24-48 h incuba- tion at 30~ the cultures were stored at 4~ Inocula for the reactor were 24 h cultures in 10 ml fermentation medium grown at 30~

Assays. Sucrose was measured using the oravi- metric method of Fehling-Soxlet (AOAC Methods ]980) and ethanol was determined by means of a Carl Zeiss Jena immersion refractometer.

Reactor. The reactor used is simple in design and was constructed from glass tubing accord- ing to the scheme in Fig I. The narrower part of the cylindrical body took the main stream of gas out through an inverted funnel which partia]ly avoided the loss of floccu]es along with the effluent. The total working volume was 50 ml and the temperature was regulated at 30~ by means of a water jacket.

Operation of the reactor. The assembled reactor was sterilized by autoclaving at 121~ for 15 min. After cooling, 30 m] of sterilized fermentation medium was pumped into the reactor, inoculated (10 ml) and incubated batchwise at 30~ to allow growth. A slow feed of medium was then started until the reactor working volume was reached. Afterwards, the inflow of medium was regu]ated according to the desired dilution rate and once the steady

Offprint requests to: Danley A. S. Callieri

E. Rodriguez and Danley A. S. Callieri: Continuous Conversion of Sucrose to Ethanol 187

state was established, sucrose and ethanol were determined in the effluent.

Gas f

4 ,Drain

Inverted funnel

I . ~ ~ ~ -------Water jacked

Feed

Fig. I Schematic configuration of the reactor, constructed from 3.3 cm ID glass tubing, with a total working volume of 50 ml

Results and Discussion After 2-3 days of incubation in batch, small congTomerates of cells were observed in the medium. These floccules (about I mm diameter or less) appeared under the microscope as associations of short chains which entangled pairs and single cells. When the con%inuous flow of medium started, gas production rose,

thus stirring the flocs which were kept in suspension. These conditions produced a kind of "peTleting effect" on the small flocs which grew, reaching a diameter of 2-4 mm. Flocs of different sizes resulted and the gas produced was no longer able to maintain a homogeneous distribution of them. The bigger flocs, which very often entrapped smaller ones, sank to the bottom of the reactor and were only slightly stirred by the oas, while the smaller ones in the upper part were vigorously agitated. In order to see if the big flocs maintained their fermentative capacity, a sample was drawn from the bottom of the reactor. The flocs were washed with water to eliminate free cells attached to the surface, placed in fresh medium and incubated. A normal and vigorous fermenta- tion took place within 24 h. About a week after starting the continuous in- flow of medium, the floc bed volume remained apparently constant (40 ml), small flocs and free cells bein 9 eliminated along with the effluent. The increase of inflow medium posit.ively influenced productivity until a dilution rate of 3 h -l was reached. At this point the volu- metric productivity estimated on the total basis (50 ml) was 92 g EtOH/l per hour and the yield was 0.4 g EtOH/g sucrose supplied. Higher dilution rates impaired productivity. The dilution rate also influenced the ethanol yield, which rose from 0.4 to 0.45 g EtOH/g sucrose supplied when D was increased from 0.3 to i.O h -I (Fig. 2). Above this value, these parameters showed an inverse correlation, probably due to a steady decrease in cell population (which was not measured) and/or changes in the physiological state of the microorganisms affecting the fermentative per- formance of the strain (M~lek 1958). Dilution rates up to 7 h -I were tested and even if no wash out occurred, obviously due to the flocculent growth characteristics of the strain, the conversion of sucrose to ethanol was so low that no further increase of D was assayed.

The above results also indicate that the high- est overall efficiency of the system occurred when the dilution rate was about 2 h -I with a yield of 0.4 g Etnrl/O sucrose and 80 g

EtOH/l per hour productivity. The performance of the non-flocculent type strain under the same experi,mental conditions was substant'ially lower than that of the flocculent strain (Fig. 2). From the beginning, both yield and productivity were negatively affected by the increase in dilution rate. No washing out occurred at a dilution rate as high as 7 h -l, probably due to the cells tendency to attach to the walls of the reactor.

It was also observed that after several days under continuous cu|ture, small powder-like cells associations appeared which were quite

188 E. Rodrfguez and Danley A. S. Callieri: Continuous Conversion of Sucrose to Ethanol

0.5

0.4

0,3

0.2

0.1

0.5

0.4

0.3

0.2

0.1

4---ETOH yield -o -oIg ETOH/g sucrose)

-ETOH in the effluent -i-A-(g/I) D-

-Sucrose in the effluent -A-t - (g/ I ) ~.

-ETOH volumetric productivity -*- ,-(g ETOH/I per hour---~

o~ '~ ~ ~

m

! : ; I I I O :

100

80

60

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�9 ~ ..,. 0 , , .

= ,, O

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100

80

60

40

20

-Fig. 2. Continuous etffanol production by a flocculent strain of Zymomonas sp. isolated from sugar cane juice (--). Results are compared with those obtained with Zymomonas mobilis ATCC 10988 (---)

different from the flocs previously described. When a sample of this culture was transferred to fresh medium and incubated in batch the growth was normal with no conglomerations of cells being produced.

Acknowledgements. This work was partially supported by grants from Subsecretarra de Cien- ciay TecnologTa and DirecciSn Nacional del Az~ car, Argentina.

References

AOAC ~1ethods 13th ed. (1980) 31.034 p 513 Mglek I (1958) The physiological state of

microorganisms during continuous cultiva- tion. In: Continuous cultivation of micro- organisms, Publishing House of the Czechos- lovak Academy of Sciences, Prague, p II

Strandberg G~4, Donalson TL, Arcuri EJ (1982) Continuous ethanol production by a floccu- l e n t s t r a i n o f Zymomonas mobilis. B i o t e c h L e t t e r s 4 ( 6 ) : 347 -352 .

Received May 13, 1983