In-line toxic product removal during solvent production by continuous fermentation using immobilized Clostridium a cetobutylicum B. M. Ennis*, N. Qureshi and I. S. Maddox Biotechnology Department, Massey University Palmerston North, New Zealand (Received 10 April 1987; revised 15 June 1987) Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used in a two-stage continuous reactor for solvent production from whey permeate. Gas-stripping (N 2 gas), an adsorbent resin ( XAD-16 ) and a molecular sieve (silicalite ) were evaluated for their use in between-stages solvent removal. All three techniques removed significant quantities of solvents, but not lactose, and allowed increases in sugar utilization and solvent productivity in the second stage. Gas stripping was the most successful technique, possibly because it removed only volatile solvents and not essential nutrients. Keywords: Clostridium acetobutylicum; continuous solvent production; in-line product removal;whey permeate Introduction The attainable products concentration in the acetone/ butanol/ethanol (ABE) fermentation is limited by product inhibition such that concentrations exceeding 20 g 1- * are rarely achieved. L2 For this reason, intensified ferment- ation technologies are being developed in attempts to minimize the effects of product inhibition and thus improve the reactor productivity. Continuous fermenta- tion proceses based on free cells, 3'4 immobilized cells, 5 - 8 and external cell-recycle using cross-flow microfiltration have been reported. 9- a0 Novel, alternative solvent recovery techniques have been developed, principally for the ethanol fermentation, some of which may be suitable for integration (in line or in situ) with the ABE fermentation process, thus minimiz- ing accumulation of toxic products, a~ Recently reported examples of integrated ABE fermentation/product recovery processes include continuous fermentation using alginate-immobilized cells/pervaporation ~2 and contin- uous fermentation using cross-flow microfiltration/ adsorbent resin. ~ 3 In this laboratory, integration of batch fermentation/gas stripping resulted in significant increases in the sugar utilization rate and the reactor solvents productivity. 14 The purpose of the present work was to investigate the use of a two-stage continuous fermentation process in- corporating between-stages solvent removal for solvent production from whey permeate. The reactors consisted *Present address: New Zealand Dairy Research Institute, Palmerston North, New Zealand of cells immobilized by adsorption onto bonechar, 8 while three different techniques of product removal were evaluated, i.e., gas-stripping, ~4 an adsorbent resin and a molecular sieve. Synthetic, polymeric resin adsorbents have been evaluated for ethanol recovery from ferment- ation broths, either in integrated processes or during downstream processing, 15'16 but there are few data describing their application to the ABE fermentationJ 7 Molecular sieves, e.g., silicalite, are porous, solid materials that can be used to preferentially adsorb alcohol from alcohol water mixtures.~ ~- 2o Thus, the objective was to develop an integrated process whereby the extent of lactose utilization, and hence the solvents productivity, were increased compared with a process without in-line solvents removal. Materials and methods Organism Clostridium acetobutylicum P262 was obtained from D. R. Woods (University of Cape Town, South Africa), and was stored as a spore suspension in sterile distilled water at 4°C. Mater&ls Spray-dried cheese whey permeate, obtained from the New Zealand Dairy Research Institute (Palmerston North, New Zealand), was reconstituted to 60 g 1- ~ using distilled water, supplemented with yeast extract (5 g 1-1 Difco Laboratories, Detroit, MI, USA), and sterilized in an autoclave at 121°C for 20min. The adsorbent resin, XAD-16, was obtained from Rohm and Hass (Philadel- phia, USA); on receipt it was washed in 50% (v/v) 0141-0229/87/110672-04 $03.00 672 Enzyme Microb. Technol., 1987, vol. 9, November ~ 1987 Butterworth Publishers