Continuous fermentation is a preferred operational mode to decrease cost of production and increase efficiency. It can easily be performed with using cascade reactors with suppressing butanol concentration below the inhibition limit. The butanol concentration supressing can be performed by dilution or with various methods of recovery with adsorption, extraction, stripping, membrane techniques or with combination of these methods. Increase of the active biomass amount in the mash by cell recycling plays key role in continuous ABE fermentation processes, as well.

Dyr et al. [100] observed formation of neutral solvents in continuous ABE fermentation process by means of C. acetobutylicum without morphological adaptation due to the altered way of cul­tivation. The results obtained leave no doubt as to the possibility of employing the continuous method for acetone-butanol fermentation [101]. A cascade type continuous ABE fermentation method was developed from soluble starch by building an equipment consistsing a battery of 11 fermenting tanks [102]. The first tank is used as an incubator and an activator for the culture. In the remaining tanks, the actual fermentation is carried out. The feed liquor is continuously sup­plied. The continuous fermentation process for Me2CO-BuOH production is a lst-order reac­tion. A continuous ABE fermentation process was developed and adopted in plants using starch raw materials by Yarovenko [103]. The basis for a continuous process is knowledge of laws of continuous mixing of liquids in batteries of connected vessels which are discussed by Yarovenko [104]. The length of fermentation considerably influences the acidity of the ferment­ed mixture at the end of the process. Owing to differences in mash composition and duration of process, acid level is mostly higher in continuous fermentation than in a discontinuous one. With continuous acetone-butanol process fermentation, speed could be raised 1.58 times com­pared to the semicontinuous method. In the continuous fermentation, it is useful to operate with 2-5 parallel batteries and to cultivate bacteria in separated vessels. The carbohydrates produced by saccharification under different conditions were studied as they were of great importance on length and course of fermentation. Operation of the battery’s head fermentor has a great influ­ence on the whole process, the amount of inoculum, acid production, and fermentation speed. To provide an adequate microorganism concentration and to reduce the risk of infection in the battery’s head fermentor, mash from the 2nd vessel is recycled. The acidity increase was evident primarily in the last tank. Optimum concentration of the cells to be inoculated at the start of the fermentation 7*109/ml for C. acetobutylicum and physiologically mature cells should comprise about 80% of the total inoculum. The flow rate into the main fermentor should be harmonized with the utilization rate of carbohydrate in the battery. Bacteria in the main vessel must be main­tained at their respective stationary phase of growth. The continuous ABE fermentation in­creased productivity efficiency 20%. The carbohydrate utilization was improved by 2.4%, along with the characteristics of the beer [103,104]. The Japanese K. F. Engineering [105] described an apparatus for production of Me2CO and BuOH by immobilized ABE-producing microorgan­isms, where the immobilized microorganisms are first exposed to a batch process until active gas formation is observed, and then, a continuous production process was performed.

The availability and demand of biosynthetic energy (ATP) is an important factor in the regula­tion of solvent production in steady state continuous cultures of C. acetobutylicum. The effect of biomass recycle at a variety of dilution rates and recycle ratios on product yields and selectivi — ties was determined. Under conditions of non-glucose limitation, when the ATP supply is not growth-limiting, a lower growth rate imposed by biomass recycle leads to a reduced demand for ATP and substantially higher acetone and butanol yields. When the culture is glucose limit­ed, however, biomass recycle results in lower solvent and higher acid yields [106]. Wijjeswara — pu et al. studied continuous BuOH fermentation by C. acetobutylicum in a stirred tank reactor. The results of glucose fermentation with cell recycling revealed the formation of small amounts of EtOH, moderate amounts of Me2CO and BuOH, and large amounts of AcOH and butyric acid. Without cell recycling overall BuOH production was decreased by a factor of 3.5 [107]. Af — shar et al. used a cascade system and cell recycling. At a dry cell mass concentration of 8 g/L and a dilution rate of D=0.64 h-1, a solvent productivity of 5.4 g/L-1 h-1 could be attained. To avoid de­generation of the culture which occurs with high concentrations of ABE solvents a 2-stage cas­cade with cell recycling and turbidostatic cell concentration control was used as optimal solution, the 1st stage of which was kept at relatively low cell and product concentrations. A sol­vent productivity of 3 and 2.3 g L-1 h-1, respectively, was achieved at solvent concentrations of 12 and 15 g L-1 [108]. Huang and Ramey [109] determined the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum in a fibrous bed bioreactor with high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as co­substrates, the fermentation was maintained in the solventogenesis phase, and the optimal bu­tanol productivity of 4.6 g L-1 h-1 and a yield of 0.42 g g-1 were obtained at a dilution rate of 0.9 h-1 and pH 4.3. Eight Clostridium acetobutylicum strains were examined for a-amylase and strains B-591, B-594 and P-262 had the highest activities. Defibered-sweet-potato-slurry containing starch supplemented with potassium phosphate, cysteine-HCl, and polypropylene glycol was used as continuous feedstock to a multistage bioreactor system. The system consisted of four columns (three vertical and one near horizontal) packed with beads containing immobilized cells of C. acetobutylicum P-262. The effluent contained 7.73 g solvents L-1 (1.56 g acetone; 0.65 ethanol; 5.52 g butanol) and no starch. Productivity of total solvents synthesized during contin­uous operation was 1.0 g L-1 h-1 and 19.5% yield compared to 0.12 g L-1 h-1 with 29% yield in the batch system [110]. Pierrot et al. introduced a hollow-fiber ultrafiltration to separate and recycle cells in continuous ABE fermentation. Under partial cell recycling and at a dilution rate of 0.5 h-1, a cellular concentration of 20 g L-1 and a solvent productivity of 6.5 g L-1 h-1 is maintained for sev­eral days at a total solvent concentration of 13 g L-1 [111]. The device developed was sterilizable by steam and permitted drastic cleaning of the ultrafiltration membrane without interrupting continuous fermentation. With total recycle of biomass, a dry weight concentration of 125 g L-1 was attained, which greatly enhanced the volumetric solvent productivity averaging 4.5 g L-1 h-1 for significant periods of time (>70 h) and maintaining solvent concentration and yield at accept­able levels [112].

A stable continuous production system with nongrowing cells of C. acetobutylicum adsorbed to beechwood shavings was obtained by different types of adsorption procedures for produc­tion of ABE solvents by Foerberg and Haegsstroem [113]. The system was started with continuous flow of a complete nutrient medium. A thick cell layer was formed on the wood shavings during the 1st day but it disappeared rapidly. Under glucose limitation, a new cell layer developed during the following period (2-5 days). After this phase, a continuous flow of nongrowth medium with nutrient dosing (8 h dosing interval) was started. This led to a washout of most adsorbed cells and ~85% of suspended cells. Another cell layer was formed during this period and the system was controlled by the nutrient dosing technique. The system was stable with no cell leakage for weeks. The maximal productivity of butanol, acetone, and EtOH was 36 g L-1 d-1 with a product ratio of 6:3:1 [113].

A continuous ABE production system with high cell density obtained by cell-recycling of Clos­tridium Saccharoperbutylacetonicum N1-4 was also studied. In a conventional continuous ABE culture without cell-recycling, the cell concentration was below 5.2 g L-1 and the maximal ABE productivity was only 1.85 g L-1 h-1 at a dilution rate of 0.20 h-1. To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtra­tion and were able to obtain ~20 g L-1 of active cells after only 12 h of cultivation. Continuous cul­ture with cell recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g L-1. The maximum ABE productivity of 11.0 g L-1 h-1 was obtained at a dilution rate of 0.85 h-1. However, a cell concentration >100 g L-1 resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. There­fore, to maintain a stable cell concentration, cell bleeding and cell recycling were performed. At dilution rates of 0.11 h-1 and above for cell bleeding, continuous culture with cell recycling could be operated for more than 200 h without strain degeneration and an overall volumetric ABE productivity of 7.55 g L-1 h-1 was achieved at an ABE concentration of 8.58 g L-1 [114].