## 2 Results and Discussion
### Operational Stability  
For operational stability, the conditions previously optimized for immobilization and curdlan production were used. The curdlan production by immobilized cells was evaluated by seven repeated cycles of 10 days each. The glucose consumption was 99.37% in the first five cycles and dropped to 87.36% and 85.40% in the sixth and seventh cycles, respectively, and the increased consumption led to increased production levels. In the first cycle, the curdlan production was 20.62 g/L, 31.62% higher than that obtained by free cells (14.10 g/L) and higher than those obtained by the immobilized cells in the other operational cycles (Figure 5). Until the fourth cycle, the immobilized cells maintained an average of 87.40% of the curdlan production obtained in the first cycle and showed a reduction of 29.43%, 40.10% and 49.32% only in the fifth, sixth and seventh cycles, respectively. Considering that the production of curdlan occurred for several consecutive cycles, it is possible to suppose that the immobilization matrix did not negatively affect the mass transfer. Considering that the curdlan serves as an energy reserve, its production by free cells in the second cycle was null, due to the presence of curdlan in the medium since the beginning of the second cycle, when the contents of curdlan present in the first cycle were transferred with the cells for the second cycle. The glucose consumption was 99% in the first cycle and 79% in the second cycle. Curdlan is used as both an energy reserve and a means for cellular adherence to solid surfaces and to improve cell survival [34]. Other authors have also obtained good operational stability through the use of loofa sponge as a matrix for immobilization. Ethanol production was obtained by _Saccharomyces cerevisiae_ in the  
Figure 4: Curdlan production rate by _Agrobacteriumium_ sp. IFO 13140. Predicted rate (solid line) and observed rate (open circles) for immobilized cells and predicted rate (sectioned line) and observed rate (open squares) for free cells, at 30 \({}^{\circ}\)C and 150 rpm. course of four cycles with less than a 5% reduction in production [25]. The decreased production of \(\varepsilon\)-poly-l-lysine by _Kitasatospora_ sp. MY 5-36 was detected only after the fourth production cycle [32]. In Figure 5, it is also observed that the highest volumetric productivity of curdlan achieved was 0.09 g/L/h, which was obtained in the first cycle; thereafter, a decrease occurred throughout the cycle, reaching 0.04 g/L/h in the last cycle. The volumetric productivity obtained by free cells was 0.06 g/L/h of curdlan in the first cycle. Other authors working only with free cells, large volumes of culture medium and incubation times of approximately 120 h obtained different productivity values. The use of the _Agrobacteriumum_ sp. ATCC 31750 mutant afforded a volumetric productivity of 0.63 g/L/h of curdlan [15]. When a parental strain was used, 0.55 g/L/h of curdlan was obtained with the addition of uracil 46 h after ammonia depletion [18] and 0.20 g/L/h of curdlan when sucrose was utilized as a carbon source [19]. A volumetric productivity of 0.36 g/L/h of curdlan by _Agrobacteriumum_ sp. ATCC 31749 was reached when a rate of dissolved oxygen equal to 60% was utilized [23]. The determination of intracellular protein confirmed the cell desorption from the immobilization matrix. These cells were not transferred to the subsequent production cycle. The average protein content in the first four cycles was 0.24 mg/mL, and in the last three cycles, there was an average reduction of 53.53% (Figure 5). The formation of free biomass in all cycles evaluated contributed to glucose consumption and curdlan formation. The increased number of dead cells immobilized due to its reuse may also have contributed to the decreased production of curdlan after the fourth cycle, which was described previously for the biosynthesis of \(\varepsilon\)-poly-l-lysine [32] and gibberell acid [27], by microorganisms immobilized on loofa sponge.