Fed-Batch Cultivation of Cellulornonas on Sugarcane Bagasse Pith

Hilda Rodriguez and Antonio Enriquez Department of Industrial Microbiology, National Center for Scientific Research, Aptdo. 6990, Havana, Cuba

Accepted for Publication April 10, 1984

A high biomass concentration (19.9 gll) was obtained with the fed-batch cultivation of Cellulomonas on pre treated sugarcane bagasse pith. Similar results in biomass concentration, yield, and substrate consump- tion were obtained with the discontinuous feed of bagasse as with discontinuous feed supplemented with a partial continuous addition of salts. Two or more growth phases were detected, probably caused by the differential utilization of bagasse components. An ac- ceptably low content of bagasse components remained in the biomass after separation.

INTRODUCTION

In a previous article,' we reported on the production of cell concentrations of 13 g/L with Cellulomonas growing on pretreated sugarcane bagasse, for the production of single-cell protein (SCP). However, higher cell concentra- tions could not be obtained by normal batch fermentation, mainly due to the necessity of using high concentrations of an insoluble substrate. Nevertheless, the attainment of high cell densities is most desirable to make the process economically feasible.

Srinivassan and co-workers2 obtained cell densities of 21 g/L with Cellulomonas growing on glucose, by using a gradient-feed method of cultivation. On the other hand, different authors have reported an increase in cellulolytic activity of fungi in fed-batch fermentations on cellul0se.3~~ This method appears to be useful in systems where high cell concentrations are to be attained and/or when the in- soluble nature of the substrate makes it difficult to run a continuous culture.

In this article, we report on the application of a method of fed-batch fermentation for obtaining high cell densities of Ceiiuiomonas grown on sugarcane bagasse pith.

MATERIALS AND METHODS

Microorganism and Cultivation Conditions

Cellulomonas sp. IIbG was grown in a culture medium (MF medium) consisting of (g/L): KH2P04, 0.6; NH4CI, 0.25; MgS04. 7H20, 0.25; and thiamine, 0.01. The culti-

vations were carried out in a 5-L (effective volume) fer- mentor, at approximately optimal growth conditions7 (pH 6.5, 32"C, and 2 v/v /min air flow rate). The stirring fre- quency was increased to 1.33 Hz.

Analysis

Growth was analyzed by measuring the absorbance at 600 nm after filtration of the culture through a sintered- glass crucible of No. 1 porosity. At the end of.growth, the culture was filtered through a nylon wool of 200-mesh and the retained bagasse was washed and dried at 90°C to con- stant weight. The biomass concentration was determined by centrifugation of the filtered cell suspension at 1@g for 15 min. The pellet was washed twice with distilled water and dried at 80°C to constant weight. Protein in the cell suspension was estimated by the biuret method,8 with lipids measured according to Folch and co-w~rkers.~ Hemicellulose determination was carried out according to Jones.Io Cellulose was determined by the method of Crampton and Maynare" and lignin by that of Ritter and co-workers.12 Ash content was obtained by 4 h combustion at 650°C. The bagasse pith was pretreated with 10% NaOH.13

Fed-Ba tch Cultivation

Three variants of cultivation were assayed: (1) Discontinuous feed of bagasse in sixfold concen-

trated MF medium (except for NaCl which was used in the basal concentration).

(2) Discontinuous feed of bagasse, together with contin- uous feed of salts was also tried. In this case, the initial medium in the fermentor contained simple salt concentra- tions and sixfold concentrated thiamine. One liter of con- centrated salt solution (to achieve a sixfold concentrated MF medium) was pumped at a flow of 0.11 L/h during the first nine hours of cultivation. During this time, the optical density values are affected by the continuous addition of salts so these are taken as approximate values. The final increase in volume was 25%.

The discontinuous addition of substrate in both vari- ants was calculated for the desired increase in biomass

Biotechnology and Bioengineering, Vol. XXVII, Pp. 121-123 (1985) 0 1985 John Wiley &Sons, Inc. CCC 0006-359218510201 21 -03$04.00

concentration at intervals according to the expression AS = A X / A Y , where AS is the substrate concentration to be added, AX is the increase in biomass concentration, and A Y is the yield. For the calculation, a yield of 0.35 was as- sumed, based on previous re~u1ts.l~

(3) The final cultivation variation was batch fermenta- tion using sixfold concentrated medium and 5.7% bag- asse pith.

RESULTS AND DISCUSSION

Figure 1 shows the growth curve obtained in the cultiva- tion with discontinuous feed of bagasse pith in a concen- trated medium (variant 1). A high final value of absor- bance was reached. At least three exponential growth phases are distinguished, with specific growth rates of 0.075, 0.073, and 0.056 h-l, respectively. Srinivassan and co-workers2 pointed out that in some cases, high concen- trations of mineral salts could decrease the specific growth rate. Therefore, we decided to try a double fed-batch fer- mentation, with a partial continuous addition of medium salts, besides a discontinuous feed of bagasse pith. The corresponding growth curve is shown in Figure 2. Similar final absorbance values were reached as with variant 1, and specific growth rates of 0.082 and 0.059 h-' were ob- tained in the two distinguishable exponential growth phases.

The observed first diauxie in both cases (Figs. 1 and 2 ) does not seem to be due to a limitation in substrate concen- tration since this is still high enough at the point where the first growth phase stops. Furthermore, in variant 2 (Fig. 2) growth starts again before the addition of fresh substrate at 25 h. The delayed induction of cellulase synthesis and the utilization of hemicelluloses at the beginning of growth ap-

7 1 I

Figure 1. Growth of Cellulomonus sp. IIbc with discontinuous feed of bagasse pith in MF X 6 medium. Bagasse additions included (0) 20.6 g, (1) 27.4 g, (2) 54.9 g, (3) 45.7 g, (4) 45.7 g, (5) 51.4 g, and (6) 51.4 g each.

60 h @ J 0 20 40

Figure 2. Growth of Cellufomonas sp. IIbc with discontinuous feed of bagasse pith and continuous feed of salts. The medium was MF X 6. Bagasse additions included (0) 17.9 g, (1) 22.8 g, (2) 45.7 g, (3) 45.7 g, (4) 45.7 g, (5) 51.4 g, and ( 6 ) 51.4 g. Salts were fed continuously during the first 9 h of growth.

pears to be the most probable explanation of this diauxie, taking into account previous results obtained in batch fermentations where a differential development of hemi- cellulases and cellulases was observed, in correspondence with a diauxic pattern of g r o ~ t h . ' ~ , ~ ~

Figure 3 shows the growth curve obtained on 5.7% substrate in a sixfold concentrated medium. The specific growth rates were 0.068,0.030, and 0.039 h-I for the three

5.0

F ~ r e 3. asse pith and MF X 6 medium.

Batch fermentation of Cellulomonus sp. IIbc on 5.7% bag-

122 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 27, FEBRUARY 1985

Table I. Results from three variants of cultivation of Cellulomonas on bagasse pith in a MF X 6 medium.

Percent Y Total fed Bagasse Y (g biomass/

Biomass bagasse consumption (g biomass/ g metabolized Variant (g/L) (g) ( 7 0 ) g fed bagasse) bagasse)

Discontinuous

Discontinuous feed of bagasse 19.1 297 76.7 0.297 0.388

feed of bagasse plus continuous feed of salts 19.9 280 75.8 0.299 0.394

Batch cultivationa 3.8 285 20.0 0.03 0.137 ~~

aBagasse pith (5.7%) and salts (MF X 6 medium) were present from the beginning.

Table II. fermentations on pretreated bagasse pith.a

Chemical composition (70) of the biomass from Cellulomonas sp. IIbc obtained from fed-batch

Protein Free lipids Ash Cellulose Hemicellulose Lignin ~

45 * 1 10.5 k 0.8 15.5 31 0.1 4.8 k 0.4 0.17 k 0.01 0.25 k 0.02

aOther components were reported previously (see ref. 1).

exponential growth phases. The first probably took place at the expense of reducing sugars brought in by the high bagasse concentration in the medium.14 Afterwards, a dif- ferential utilization of hemicellulose and cellulose appears to take place. However, low final values of absorbance were reached in comparison with variants 1 and 2. Besides this, difficulties in manipulation of the samples and the culture were encountered, due to the high bagasse concen- tration in the fermentor. In fact, growth may be limited by slow enzyme and/or cell diffusion caused by this high ma- terial concentration. Repression due to the high substrate concentration may also take place.

Table I summarizes the results obtained in the three variants of cultivation. High biomass concentrations were obtained in both variants of fed-batch fermentation. The substrate consumption and yields were also higher in these cultures in comparison with batch fermentation using a high substrate concentration (variant 3). In our case, no appreciable differences were detected between the two forms of fed-batch cultures assayed.

For the application of this system to SCP production, the question to be answered concerns the possible effect of remaining substrate particles in the biomass, since the separation step is probably less efficient at a high cell and bagasse concentration. Therefore, we carried out a partial characterization of the biomass produced in the fed-batch cultures, as shown in Table 11.

The total remaining lignocellulose material in the biomass after separation does not exceed 5’70, which is an acceptable value. A relatively high lipid content was also found. These data complement a previous characteriza- tion of Cellulomonas sp. IIbc biomass,’ and corroborate the good perspectives of this strain from the point of view of its composition for SCP production. The results ob- tained show the feasibility of the fed-batch method for

SCP production from cellulosic substrates. Even better yields could be expected by improving the method of discontinuous addition, seeking optimum intervals of ad- dition and optimum bagasse concentration in the fermen- tor at any point in time. Thus, the method appears to be a profitable technique for the practical application of this process on an industrial scale.

References

1. A. Enriquez and H. Rodriguez, Biotechnol. Bioeng., 25,877 (1983). 2. V. R. Srinivassan, M. B. Fleenor, and R. J. Summers, Biotechnol.

Bioeng., 19, 153 (1977). 3. A. L. Allen and R. E. Mortensen, Biotechnol. Bioeng., 23, 2641

(1981). 4. M. Gottwaldovi, J. Kurera, and V. Podrazki, Biolechnof. Lett., 4,

229 (1982). 5. A. Enriquez, “The preparation of SCP from cellulosic wastes by the

fermentation process,” Ph.D. thesis, Institute of Microbiology, Prague, Czechoslovakia, 1978.

6. H. Rodriguez, A. Enriquez, and 0. Volfovi, Foliu Microbiol., 28, 163 (1983).

7. A Enriquez, Biotechnol. Bioeng., 23, 1425 (1981). 8. H. W. Robinson and C. G. Hogden,J. Biol. Chem., 135,727 (1940). 9. J. Folch, M. Lees, and G. H. Sloane, J. Bid . Chem., 226, 497

(1957). 10. H. A. Jones, Tappi, 44, 745 (1961). 11. E. W. Crampton and L. A. Maynard, J. Nutr., 15,385 (1938). 12. G. J. Ritter, R. M. Seborg, and R. L. Mitchell, Iizd. Eng. Chem.

Anal., 4,202 (1932). 13. C. E. Dunlap, “Protein from waste cellulose by chemical-microbial

processing,” Ph.D. thesis, Louisiana State University, Baton Rouge, LA, 1969.

14. H. Rodriguez, ‘‘Growth of cellulolytic bacteria on sugarcane wastes,” Ph.D. thesis, Institute of Microbiology, Prague, Czechoslovakia, 1983.

15. H. Rodriguez and 0. Volfovl, “Localization of Cellulomonas cellulolytic enzyme complex during the growth on bagasse,” Third Symposium of the Socialist Countries on Biotechnology, Bratislava, 1983.

123 RODRIGUEZ AND ENRIQUEZ: CULTIVATION OF CELLULOMONAS