3.1. Immobilization

Immobilization of C. Acetobutylicum strains prevents bacteria from existing in the ferment mash and is a very essential facility in a variety of integrated solvent recovery methods. Haeggstroem and Molin [87] concluded that immobilized vegetative cells of C. Acetobutyli — cum have a similar product formation pattern when incubated in a simple glucose-salts solution as ordinary growing cells. If vegetative cells of the organism are immobilized in the solvent production phase, solvents are continuously produced on extended incubation. By immobi1izing spores of the organism, the disturbance of the cells metabolic activity during the immobilization procedure was avoided. After the outgrowth of viable cells within the gel, the washed gel preparation retained at a high production capacity in the non-growth stage

and the results indicate that continuous production might be fully possible. The butanol productivity was also found to be higher with immobilized cells than in a normal batch process. Haeggstroem [88] used immobilized spores of Clostridium acetobutylicum in a calcium alginate gel. The productivity of the system was 67 g BuOH/L-day and with immobilized cells it was possible to achieve continuous BuOH production for 1000 h. Foerberg et al. [89] developed a technique for maintaining constant activity during continuous production with immobilized, non-growing cells. A single stage continuous system with alginate-immobilized C. Acetobutylicum, was mainly fed with a glucose medium that supported fermentation of acetone-BuOH but did not permit microbial growth. The inactivation that occurred during these conditions was prevented by pulse-wise addition of nutrients to the reactor. By using this technique, the ratio of biomass to BuOH was reduced to 2% compared to 34% in a traditional batch culture. At steady state conditions BuOH was the major end product with yield coefficients of 0.20 (g/g glucose). The productivity of BuOH was 16.8 g L-1 d-1 during these conditions. In a corresponding system with immobilized growing cells the ratio of biomass to BuOH was 52-76% and the formation of butyric and acetic acid increased thereby reducing the yield coefficients for BuOH to 0.11 (g g-1). With the intermittent nutrient dosing technique, const. activity from immobilized non-growing cells has been achieved for 8 weeks.

Several carriers have been tested for production of ABE solvents by immobilized local strain of C. acetobutylicum. Thus, both batch and continuous fermentations were performed by using sodium alginate, polyacrylamide, activated carbon, and silica gel carriers. Calcium alginate was found to be the most suitable with batch culture techniques where the total solvent production was 19.55 g L-1 after 4 days. On the other hand, higher solvent yields with continuous fermenta­tion was noticed with silica gel G-60 (0.063-0.2 mm) with 13.06 g L-1 solvent production. In all cases, the tested solid supports were of inferior effect for solvent production under the exptl. conditions used as compared with Ca-alginate [90]. High-strength carriers were also tested for C. acetobutylicum ATCC 824 in batch fermentation. Coke, kaolinite and montmorillonite clay appeared to have a beneficial effect on the fermentation, although the effectiveness appeared to be dependent on the medium used. One of the least expensive materials, coke, was suitable for use in continuous culture. Steady state conditions could be maintained for more than 30 days with total solvent productivity and a yield of 12 g L-1, 1.12 g L-1 h-1 and 0.3 g total solvent/g glu­cose used, respectively [91]. Entrapment of C. acetobutylicum AS 1.70 with PVA as the base and by means of absorption in the corncob as the carrier is recommended. Experiments have been done to produce acetone and butanol in a statical way in batches and by changing the corn as medium circulatingly [92]. The vegetative cells of C. acetobutylicum AS 1.70 were also immobi­lized onto CR (ceramic ring) carriers by adsorption. The continuous production of acetone- BuOH from 8% corn mash concentration was carried out for 90 days in a system of 3-stage packed column reactor (total vol. 5.18 L). The maximal concentration of solvent (acetone, BuOH, and EtOH) was 21.9 g L-1 and the productivity of the column was 24.73 g L-1 d-1. The resid­ual starch concentration was 0.43% and the conversion efficiency of starch was 40.5% [93]. ABE solvent production was also carried out with C. acetobutylicum DSM 792 (ATCC 824) in a two — stage stirred tank cascade using free and immobilized cells. The cells were immobilized by algi­nate, к-carrageenan or chitosan. The cell-containing pellets were dried or chemically treated to improve their long-term stability. Dried calcium alginate yielded the best matrix system. It re­mained stable after a fermentation time of 727 h in stirred tank reactors. The solvent (sum of ace­tone, butanol and ethanol) productivity of 1.93 g L-1 h-1 at a solvent concentration of 15.4 g L-1 with free cells was increased to 4.02 g L-1 h-1 at a solvent concentration of 4.0 g L-1 h-1 with calcium alginate-immobilized cells (25% cell loading, 12 g L-1 pellet concentration, 3 g L-1 wet cell mass concentration). With pellet diameter of 0.5 mm, the biocatalyst efficiency was <50% [94]. Immo­bilized cells of C. saccharoperbutylacetonicum N1-4 (ATCC 13564) were tested in an anaerobic batch culture system. Two different methods of immobilization, active immobilization in algi­nate and passive immobilization by employing stainless steel scrubber, nylon scrubber, polyur­ethane with uniform pore’s size, polyurethane with different pore’s size and palm oil empty fruit bunch fiber were studied. Immobilization in alginate was carried out on the effect of cell’s age, initial culture pH and temperature on the production of ABE. Immobilized solventogenic cells (18 h) produced the highest total solvents concentration as compared to other phases with productivity of 0.325 g L-1 h-1. The highest solvents production by active immobilization of cells was obtained at pH 6.0 with 30 °C with productivity of 0.336 g L-1 h-1. Polyurethane with differ­ent pore’s size is significantly better than other materials tested for solvents productivity and YP/S at 3.2 times and 1.9 times, respectively, compared to free cells after 24 h fermentation. We concluded that passive immobilization technique increases the productivity (215.12 %) and

YP/S (88.37 %) of solvents by C. saccharoperbutyl-acetonicum N1-4 [95]. C. beijerinckii was im­mobilized in calcium alginate to produce BuOH continuously from glucose. Two different algi­nate geometries (beads and coated wire-netting) were used for continuous experiments and two mathematical models (sphere and flat plate) were developed. Calculations revealed that no glucose limitation was present in both cases. Furthermore, the biomass build-up in the alginate was probably a surface process [96].

Cells of C. acetobutylicum immobilized on bonechar were used for the production of ABE sol­vents from whey permeate. When the process was performed in packed bed reactors operated in a vertical or inclined mode, solvent productivities up to 6 kg m-3 h-1 were obtained. However, the systems suffered from blockage due to excess biomass production and gas hold-up. These problems were less apparent when a partially-packed bed reactor was operated in horizontal mode. A fluidized bed reactor was the most stable of the systems investigated, and a productivi­ty of 4.8 kg m-3 h-1 was maintained for 2000 h of operation. The results demonstrate that this type of reactor may have a useful future role in the ABE fermentation [97]. Schoutens determined the optimal conditions necessary for the continuous BuOH production from whey permeate with C. beyerinckii LMD 27.6 immobilized in calcium alginate beads. The influence of three parame­ters on the BuOH production was investigated: fermentation temperature, dilution rate (during start-up and at steady state) and concentration of Ca2+ in the fermentation broth. Both a fermen­tation temperature of 30 °C and a dilution rate of <0.1 h-1 during the start-up phase are required to achieve continuous BuOH production from whey permeate. BuOH can be produced continu­ously from whey permeate in reactor productivities 16-fold higher than those found in batch cultures with free C. beyerinckii cells on whey media [98]. Fermentation of cane sugar molasses by immobilized C. acetobutylicum cells was greatly affected by inoculum size, calcium alginate concentration and molar ammonium nitrogen to molasses ratios. The pH value of the medium and incubation temperature both influenced the ABE production. The maximum total solvent content reached 22.54 g L-1 at inoculum size 6% (w/w), molasses concentration 140 g/l, sodium alginate amount 3 %, and molar ammonium nitrogen to molasses ratios 0.48, pH 5.5. Attempts to recyclize the fermentation process by using immobilized spores of C. acetobutylicum afford­ed total solvent contents of 22.54, 20.64, 19.31 g L-1 during the first 3 runs, respectively [99].