End product inhibition can be reduced by in situ removal of inhibitory fermentation products as they form. The first experiments were performed by Bekhtereva who studied the effect of BuOH on the ABE fermenting process and on the development of Clostridium acetobutylicum in concentrated mash. Experimental removal of neutral products from the substrate during fermentation was tested by continuous extraction with castor oil. This oil could extract acetone 13-60, EtOH 5-20 and BuOH 50-88% from the wort. By adding the oil to the medium in varying amounts depending on the carbohydrate content, it was possible to ferment corn mash of 3-5 times the usual concentrations. The yield of acetone was 20-37 g L-1 of wort, that of all neutral products 60-100 g L-1. Their concentrations in the wort under the oil layer was usually lower than in control vessels, e. g., total products 1.4-2.3%, BuOH 0.4% against 1.2-1.3% in usual fermentation. The extraction was beneficial to the development of the bacteria [151]. Other starch-containing materials could also be fermented in the usual manner, and BuOH and Me2CO were continuously removed by means of a solvent immiscible with H2O, e. g. castor oil [152]. The extraction processes were coupled to batch, fed-batch, and continuous BuOH fermentation to affirm the applicability of recovery techniques in the actual process. In batch and fed-batch fermentation, a 3-fold increase in the substrate consumption, in continuous fermentation ~30% increase could be achieved. [142].

Toxicity and selectivity of 13 organic compounds were tested in extractive batch fermentations performed with C. acetobutylicum. Among them, oleyl alcohol and mixed alcohol (the mixture of oleyl alcohol and C18 alcohol) were the best for acetone-BuOH fermentation. The orthogonal — cross-test method with 3 elements and 3 levels was used to evaluate effects of fermentation temperature, inital glucose concentration, and solvent/water ratio on extractive batch ABE fermentation. Extractive batch ABE fermentation in a stirred fermentor was studied at different initial glucose concentrations at 41/35° and at solvent/water ratio 1:2. When initial glucose concentration was 110 g L-1, at the end of extractive fermentation the BuOH concentration in the broth and in the solvent was 5.12 and 22.3 g L-1, respectively. The total BuOH and ABE concentrations based on the broth volume were ~16.27 and 33.63 g L-1, the conversion ratio of glucose was 98% and the total ABE yield was 0.312. In situ extractive fermentation could eliminate the inhibition of BuOH on microbial growth, increased the initial glucose concen­tration and reduced the wastewater amount, thus the consumption of energy could be reduced for the separation and purification of the products [153]. Roefler et al [154] studied the effect of six extractants in batch extractive fermentation: kerosene, 30 wt.% tetradecanol in kerosene, 50 wt.% dodecanol in kerosene, oleyl alcohol, 50 wt.% oleyl alcohol in a decane fraction, and 50 wt.% oleyl alcohol in benzyl benzoate. Best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of C. acetobutyli- cum, glucose consumption is limited to ~80 kg m-3 due to accumulation of BuOH in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, >100 kg m-3 of glucose can be fermented. Maximal volumetric BuOH productivity was increased by ~60% in extractive fermentation compared to batch fermentation. BuOH produc­tivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations [154].

A medium for ABE fermentation by C. acetobutylicum was mixed with 0.2-5.0% 1-octanol or 2- ethylhexanol and various parameters of fermentation were studied. Glucose consumption, cell growth, ABE formation, and acetate and butyrate formation were inhibited, especially at higher solvent concentrations. Octanol was more toxic than 2-ethylhexanol [155]. A mathematical model for simultaneous fermentation and extraction of the products was derived for ABE pro­duction by immobilized C. acetobutylicum cells in a microporous hollow fiber based tubular fermentor-extractor. The solvent, 2-ethyl-1-hexanol, is used for in situ dispersion-free extrac­tion of products. Both predicted and experimental data follow the same trend. The experimen­tally observed value of total solvent productivity increased by >40% as a result of in situ solvent extraction [156]. Unfortunately, good extractants for BuOH, such as decanol, are toxic to C. ace — tobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextrac­tants, was tested to circumvent this toxicity. Decanol appeared to inhibit BuOH formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol, however, maintenance of the pH at 4.5 alleviated the inhibition of BuOH production and the consump­tion of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced BuOH formation by 72% under pH-controlled conditions. A mechanism for the effects of decanol on product formation is proposed [157]. The same mixed extractant that contained 20% decanol in oleyl alcohol were combined by Wang et al. to carry out in-situ extrac­tive acetone-butanol fermentation, resulting 19.21 g/L of butanol concentration. Butanol pro­ductivity could be 62.8% higher than that of control; meanwhile, total organic solvent productivity increases by 42.3% as compared to the control [145].

BuOH fermentation was carried out by contact with solvent containing C10-14 alcohols, as well. A seed culture of C. acetobutylicum IAM19013 was inoculated and mixed with tridecanol. The broth was anaerobically fermented, with stirring, at 37 °C for 60 h. The solvent layer at the top of the fermentor was circulated to the bottom. The concentrations of BuOH in the solvent and vapor were 41.6 g L-1 and 66%, respectively [158]. Higher alcohols, e. g. C16-18 unsaturated alcohols and C16-20 branched alcohols were also tested for continuous extraction of BuOH from the medium during the fermentation period. Extraction of the BuOH from the medium by using unsaturated or branched alcohols innoxious to the microorganism markedly increased BuOH yield. Thus, C. acetobutylicum was anaerobically cultivated at 37°C on a medium containing 10% glucose and, after 30 h, 40% oleyl alcohol was added to the broth to remove the BuOH from the aqueous phase and thereby reactivate the fermentation. This increased the total BuOH concentration to 2.5-fold in an additional 70 h [159]. Oleyl alcohol was found to be one of the best solvents for in-situ extractive ABE fermentation. Its butanol partition coefficents value was varied between 3.0 and 3.7 depending on the composition of the broth, nontoxic, nonmiscible and its boiling point is high as compared to ABE solvents. Batch and fed-batch extractive fermentation by C. acetobutylicum was studied with oleyl alcohol as extractant. Extractive fermentation could reduce the product inhibition, increase the initial glucose concentration and increase the fermentation rate. A mathematical model was suggested to describe batch fermentation processes. The proposed model could simulate the experimental data fairly well [160].

In situ removal of inhibitory products from C. acetobutylicum resulted in increased reactor productivity; volumetric butanol productivity increased from 0.58 kg m-3 h-1 in batch fermen­tation to 1.5 kg m-3 h-1 in fed-batch extractive fermentation using oleyl alcohol as the extraction solvent. The use of fed-batch operation allowed glucose solutions of up to 500 kg m-3 to be fermented, resulting in a 3.5-5-fold decrease in waste water vol. Butanol reached a concentra­tion of 30-35 kg m-3 in the oleyl alcohol extractant at the end of fermentation, a concentration that is 2-3 times higher than is possible in regular batch or fed-batch fermentation. Butanol productivity and glucose conversion in fed-batch extractive fermentation was compared with continuous fermentation and in situ product removal fermentation [161].

In ABE fermentation using C. acetobutylicum IAM 19012, it was necessary not only to keep BuOH concentration below the toxic level (2 g L-1), but also to control glucose concentration at <80 g L-1 and pH between 4.5 and 5.5. The amount of glucose consumed could approximatly be estimated as 4 times the volume of gas evolved, and BuOH was produced from glucose with an average yield of 0.173. It was thus possible to estimate the concentration of glucose and BuOH at any fermentation time using the volume of gas evolved as an indicator. As oleyl alcohol was an excellent extracting solvent for BuOH, a fed-batch culture system for the microorganism was developed, where withdrawing and feeding operations of the solvent were done automatically based on gas evolution [162]. Ohno combined the fermentation by C. beijerinckii ATCC 25752 which perfectly inhibited the process at the BuOH concentration of 12 kg m-3 with the extraction with oleyl alcohol and removing the butanol from its mixture with oleyl alcohol which was car­ried out by prevaporation with hollow fiber membrane. When the BuOH concentration in oleyl alcohol was 22 kg m-3, the BuOH flux was 3.6’10-4 kg m-2 h-1 at 35 °C [163].

Extraction with the non-toxic immiscible solvent, oleyl alcohol was combined with fermenta­tion performed with immobilized C. acetobutylicum to ferment glucose to ABE solvents in a fluidized-bed bioreactor. The extracting solvent had a distribution coefficient of near 3 for butanol. Nonfermenting system tests indicated that equilibrium between the phases could be reached in one pass through the column. Steady-state results are presented for the fermentation with and without extractive solvent addn. One run, with a continuous aqueous feedstream containing 40 g L-1 glucose, was operated for 23 d. Steady state was established with just the aqueous feedstream. About half of the glucose was consumed, and the pH fell from 6.5 to 4.5. Then, during multiple intervals, the flow of organic extractive solvent (oleyl alc.) began into the fermenting columnar reactor. A new apparent steady state was reached in about 4 h. The final aqueous butanol concentration was lowered by more than half. The total butanol production rate increased by 50-90% during the solvent extraction as the organic-to-aqueous ratio increased from 1 to 4, respectively. A maximal volumetric productivity of 1.8 g butanol h-1 L-1 was observed in this nonoptimized system. The butanol yield apparently improved because of the removal of the inhibition. More substrate is going to the desired product, butanol, and less to maintenance or acid production, resulting in a 10-20% increase in the ratio of butanol relative to all products [164].

Whole broth containing viable cells of C. acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter-currently through the column. A concentrated solution containing 300 g L-1 glucose was fermented at an overall butanol productivity of 1.0 g L-1 h-1, 70% higher than productivity of normal batch fermentations. The continuous extraction process provides flexible operation and lends itself to process scale-up [165].

A new type of bioreactor containing a porous permeable wall to recover the biobutanol pro­duced in anaerobic ABE fermentation processes was developed [166, 176]. The ferment liquor is contacted with a non-toxic organic solvent as oleyl alcohol and the butanol in the fermentation liquor distributes between the organic phase and the ferment liquor. The butanol containing solvent located at one side of the permeable wall is in diffusion equilibrium with a same kind of auxiliary solvent with lower butanol concentration located at the other side of the permeable wall. Due to concentration difference, butanol diffuses from one side of the wall to the other side. The concentration difference is kept to be constant by continuous removal of the butanol form the auxiliary solvent phase in which the butanol concentration is always lower than in the extractant phase but much higher than the butanol concentration in the ferment liquor phase. In this way, the primary extractant solvent contacting the ferment liquor is only a transmitting me­dia between the ferment liquor and a small volume of the auxiliary solvent separated with the permeable wall. Energy demand of the distillation to remove the butanol from the auxiliary sol­
vent is less than energy demand of the direct butanol recovery from the ferment liquor or from the extractant phase [166]. The porous composite membranes used as permeable walls for ABE production can be prepared by the method of Tamics et al. [167].

Not only simple alcohols but polyols can also be used in extractive fermentation systems for ABE production. Mattiasson et al. [168] produced acetone and BuOH by C. acetobutylicum in an aqueous two-phase system using 25 % polyethylene glycol 8000. Bacteria remained in the lower phase, and the partition coefficients of acetone and BuOH favoring the upper phase were 2.0 and 1.9, resp. Mean productivity was estimated at 0.24 g BuOH L-1 h-1, producing 13 g BuOH L-1 in 50 h. Poly(propylene)glycol 1200 is the highest partition coefficient reported to date for a biocompatible ABE extracting solvents. Extractive fermentations using concentrated feeds produced ~58.6 g L-1 acetone and BuOH in 202 h, the equivalent of 3 control fermentations in a single run. Product yields (based on total solvent products and glucose consumed) of 0.234-0.311 g g-1 and within-run solvent productivities of 0.174-0.290 g L-1 h-1 were consistent with conventional fermentation reported in the literature. The extended duration of fermen­tation resulted in an overall improvement in productivity by reducing the fraction of between — run down-time for fermentor cleaning and sterilization [141].

Two aqueous two-phased systems involving polyol-type extractants were investigated to determine their ability to reduce product inhibition in the acetone-BuOH-EtOH fermentation. An industrial-grade dextran (DEX) and a hydroxylpropyl starch polymer (Aquaphase PPT (APPT)) were tested as a copolymer with polyethylene glycol (PEG) to form a two-phased fermentation broth. Two-phase fermentation performances in the DEX-PEG and APPT-PEG 2-phase systems were compared to a single-phase conventional fermentation through a series of batch runs. Effects of the phase-forming polymers on C. acetobutylicum also were investi­gated. With a BuOH partition coefficient of 1.3, the BuOH yield with the two-phase system was increased by 27% over conventional fermentation [169].

Dibutyl phthalate is one of the ester-type extractants used in extractive fermentation of glucose, glucose-xylose mixtures and hydrolyzates of lignocellulosics to acetone-butanol solvents. Dibutyl phthalate has satisfactory physical properties, nontoxic and mildly stimulates the growth of the organism used, C. acetobutylicum P262. Sugar concentrations mainly in the range of 80-100 g L-1 resulted in solvent concentrations of 28-30 g L-1 in 24 h extractive fermen­tation compared to 18-20 g L-1 for nonextractive control fermentation. Conversion factors of 0.33-0.37 g solvents g-1 sugar consumed were obtained. Rapid fermentation was achieved by high cell concentrations and cell recycling from every 24 h fermentation to succeeding similar 24 h fermentation. Somewhat higher nutrients were also helpful. By this means, 255 L of acetone-butanol solvents were obtained per ton of aspen wood, 298 L per ton of pine, and 283 L per ton of corn stover. Such high product yields from inexpensive substrates offer the prospect of economic viability for the process [170].

Induction of flocculation of Clostridia led to a reduction of the specific solvent production rate. Cells adhering to sintered glass are better than flocculating cells for continuous BuOH-acetone fermentation. Due to low toxicity, in-situ application of paraffin, oleic alcohol or stearic acid butyl ester with the cells in the fermenter is possible. Solvent production by Clostridia can be considerably enhanced by the extractive process. Extraction may be directly integrated into a continuous fermentation. Separation of BuOH from oleic acid is easy due to the high boiling point of the extractant (260 °C) being far above the boiling point of BuOH (117 °C). Thus, BuOH can be obtained by normal distillation and the extractant can be recycled [171].

BuOH could be manufactured by cultivating BuOH-producing microorganisms such as C. ace — tobutylicum in a medium containing a fluorocarbon extractant. The generation time, the mean BuOH production rate, and the mean final BuOH concentration in the C. acetobutylicum cul­ture medium containing Freon-11 (1 g L-1) were increased by 29, 19, and 12%, respectively. Pro­duction of acetone and EtOH was not affected [172]. Continuous fermentation of a carbohydrate substrate with continuous extraction of the product by CFCl3 took place in a cylin­drical fermentor, with an inlet at the center and a filter membrane concentric with the outer wall, allowing the medium to diffuse outward and to retain microorganisms. The collected medium is pumped to an extractor, where it contacts CFCl3 or another material with a high solvency for BuOH and a low solvency for H2O and then separated into two phases. The extracted medium is recycled to a feed tank. The solvent is removed from BuOH in an evaporator, where BuOH is collected and the solvent pumped to a compressor and re-utilized [173].

Organic solvents having relatively high distribution coefficients for BuOH against water, often higher alcohols, esters, and organic acids, are very toxic to the microorganisms for BuOH fermentation. Most fermentation inhibition caused by solvent toxicity was eliminated by re­extracting the primary extractant solvent from the residual phase, to be recycled from the product extraction column to the fermentor by paraffin as an extractive fermentation process applied externally to product extraction. After selecting 2-octanol as the extractant from the standpoint of energy consumption in BuOH recovery, a two-stage-extraction BuOH extractive fermentation process having the possibility of reducing the production cost of BuOH was proposed [174]. Heptanal shows strong toxic effect towards C. Acetobutylicum R1 and T5 strains [175] but it has extremely high distribution coefficent (11.5) for butanol [175,176]. Ex — situ extraction with heptanal and recycling the residual broth into a new fermentation cycle proved to be unsuccesfull because the broth contained approx. two times higher heptanal concentration than the toxic limit. Diluting the recycled broth or extracting it with a secondary non-toxic apolar solvent such as hexane to remove the residual dissolved heptanal, inoculate the recycled broth with fresh bacteria in each cycle showed that 4-5 cycles of fermentation could be obtained without important decreasing in the ABE yields and productivity [175]. A multiple solvent extraction is described by Shi et al [177].

Mathematical formulation was made for the performance evaluation considering two types of solvent-supplying strategies. One is to add multiple solvents simultaneously and the product is removed at one time. Another is to add them one by one consecutively. Computer simula­tions were made for batch, fed-batch, and repeated fed-batch operation of acetone-BuOH fermentation to show the power of the approach. Significant improvement in terms of productivity and product concentration is expected when two extractants such as oleyl alcohol and benzyl benzoate are used, as compared to using only one solvent [177]. A two-stage — extraction butanol extractive fermentation process was developed and studied using a bench — scale extractive fermentation plant with a butanol production capacity of ~10 g h-1. The production rate equation for extractive fermentation was simply expressed by a previously reported equation multiplied by an equation for the extraction raffinate recycling effect. A butanol production-cost calculation program for the two-stage-extraction process determined the optimum operational conditions to be when butanol concentration, residual sugar concentration and recycling ratio were 6 kg m-3, 15 kg m-3 and 3, respectively. These optimal conditions were achieved in the bench-scale plant when it was operated with total sugar concentration, dilution rate and recycling ratio of 113 kg m-3, 0.158 h-1 and 3, respectively [178].

A special kind of in-situ extractive fermentation is the so-called perstraction, where a selective membrane is located between the broth and the extractant phase. Both sides of the membrane contact with each phase and ensures a medium betwen two immiscible phases to exchange butanol content. Due to lack of direct contact betwen two phases, toxicity or other problems can be eliminated and a dispersion-free extraction is possible, leading to an easy operation of the equipment, but the mass transfer in the membrane becomes important. This extraction processes were coupled to batch, fed-batch, and continuous BuOH fermentation to affirm the applicability of the recovery techniques in the actual process. In batch and fed batch fermen­tation a 3-fold increase in the substrate consumption could be achieved, while in the continuous fermentation it increases by~30% [142]. Jeon and Lee [179] described a fed-batch operation for enhanced separation with a semipermeable silicon membrane which showed high specific permeability to BuOH and acetone. Among various solvents examined, oleyl alcohol and polypropylene glycol were the most suitable as extractants. In fed-batch operation of the membrane-assisted extractive BuOH fermentation system, significant improvements were found in comparison to a straight batch fermn. The total glucose uptake per run was raised to 10 times of the value normally found in batch fermentation. The solvent productivity increased by a factor of 2. The total solvent yield increased by 23% due to reduction of acid production and reuse of cells in the fed-batch operation [179]. A continuously operated membrane bioreactor was connected to a 4-stage mixer-settler cascade and Clostridium acetobutylicum was cultivated in this reactor. BuOH was selectively extracted with butyric acid-saturated decanol from the cell-free cultivation medium, and the BuOH-free medium was refed into the reactor. Due to high boiling point of decanol, recovery of BuOH from the decanol solution is easy. Both partition coefficient and selectivity of BuOH in the cultivation medium-decanol system are sufficiently high for removing it from the medium. Direct contact of cells with the decanol phase causes cell damage. However, decanol is practically insoluble in the fermenta­tion medium, thus the contact of the cell-free medium with the solvent phase does not influence cell growth neither product formation. At a dilution rate of D=0.1 h-1, BuOH productivity was increased by a factor of 4 by removing BuOH from the medium [180].