Cultivation Medium Selection and Optimization Agitation Speed of Bacterial Cellulose Production by Acetobacter pasteurianum in Shaking Culture
Abstract
Bacterial cellulose is cellulose produced by microbe especially Acetobacter strain. So far, production process of bacterial cellulose which commonly use is static culture. At this culture, production mean per culture volume (rendemen) can be improved madely culture as shallow as possible. But, this matter require wide area to grow culture and impractical for the mass production scale (Okiyama et al., 1992 in Son et al., 2001). Therefore it needed an economic and practical mass production method to produce bacterial cellulose. Shaking Culture and agitated culture are potentials method for mass production method of bacterial cellulose due to not require wide place to grow the culture and fermentation time relative quickly. This research objectives are to obtain suitable cultivation medium (type of carbon and nitrogen source) and optimum agitation speed in producing bacterial cellulose by A. pasteurianum in shaking culture and also its kinetic aspect parameter. Optimization of agitation speert was carried out to obtain optimum agitation speed of cell propagation and production bacterial cellulose. This research was carried out in three steps. First step was selection of cultivation medium. Composition of cultivation medium, which were used in this step. was modification of CSL-Fru medium. This modification had done by substituted vitamin mix, mineral mix and CSL (Com Steep Liquor). Modification had also done to Cill"bon and nitrogen source of CSL-Fru medium. Meanwhile, the fixed material of CSL-Fru medium were 0.1 % w/v KHzP04.and 0.25 % WI" MgS04.7H20. Experiment design in this step was factorial complete random design with two treatment and two replications. First treatment factor was type of carbon source (C). which comprised four levels, that were 4.03 % w/v fructose (CI), 4.43 % WI" glucose (C2), 3.83 % WI" sucrose (C3), and 4.08 % WI" sorbitol (C4) . . Second treatment factor was type of nitrogen source (Y) comprised four level, that were 0.73 % WI. (NH.)zSO, (Yl ), 0.5 % WI. (NH.)zS04 + 0.5 % WI. yeast extract (Y2) and 1.57 % WI. yeast extract (Y3). Concentration of each level of treatment factors were made different in order to have equal weight of carbon atom and nitrogen percentage with CSL-Fru medium. that is 1.62 g weight of carbon atom and 0.1924 % nitrogen percentage. Analysis in this step was yield of dried pure bacterial cellulose. First research step was begun with refreshing isolate (slant agar medium. pH = 4.5, 4 day and 30 0C), then cell propagation (3 ose, statically, 100 ml working volume in 300 ml erleruneyer flask, pH = 5, 3 day, and room temperature) and production of bacterial cdlu!ose (5 % v/v inoculu..rru:, 100 rpm, 100 m1 working volume in 300 ml erlenmeyer flask, pH = 5. 7 day, and room temperature). Second research step was the assessment of optimum agitation speed. Cultivation medium in this step was the best medium that had obtained from first research step. The research was carried out by factorial complete random design with two treatments and two replications. First treatment factor was cell propagation agitation speed (A), which comprised four level, that were 0 rpm (AI), 80 rpm (A2), 160 rpm (AJ) and 240 rpm (A4). Meanwhile, second treatmellt factor was production agitation speed (B) which comprised 4 level, that were 100 rpm (Bl), 140 rpm (B2), 180 rpm (B3) and 220 rpm (B4). Analysis in this step was yield of dried pure bacterial cellulose. Research procedures in this step were same as first reseacrh step procedures, but incubation condition (agitation speed) was accordance to each treatment. Third research step was the assessment of kinetic parameter value of production bacterial cellulose in shaking culture. Cultivation medium, which was used in this step, was the best medium obtained from the first research step. Meanwhile, agitation speed, which was used in this step, was optimum agitation speed obtained from second research step. Analysis that carried out in' this step comprised of yield of dried pure bacterial cellulose, optical density of culture medium, weight of dried biomass and rest of sugar concentration. The analysis was carried out in ten day. First day was every 4 hour. Second until forth day was every 6 hour. Fifth day was every 8 hour and sixth until tenth day was every 12 day. Variance analysis result of first research step showed that yield of dried pure bacterial cellulose was influenced by both treatment factor (type of carbon source and type of nitrogen source) and interaction between treatment factors. The suitable cultivation medium for producing bacterial cellulose was YI C3 treatment with yield 2.27 gil. Composition medium of YIC3 treatment are coconut water as solvent, 0.1 % w/y KH2P04, 0.25 % w/y MgS04.7H2v, 3.83 % WI. sucrose and 0.73 % w/y (NH.hSO,. Variance analysis result of second research step showed that yield of dried pure bacterial cellulose was influenced by both treatment factor (agitation speed of cell propagation and agitation speed of production bacterial cellulose) and interaction between treatment factors. Optimum agitation was obtained from A I B2 treatment (0 rpm agitation speed of cell propagation and 140 rpm agitation speed of production bacterial cellulose). The yield of this treatment was 5.4995 g/l.