Patent Application: US-201113809003-A

Abstract:
the present invention relates to a cell retaining matrix for producing solvents and alcohols and an apparatus comprising said bio - column . the invention also relates to the method for producing solvents and alcohols by using said cell retaining matrix . said cell retaining biomatrix .

Description:
some embodiments of the invention will be explained next in more detail in examples . clostridia acetobutylicum b 5313 ( dsm 792 , atcc 824 ) was obtained from russian national collection of industrial microorganisms at the institute of genetics and selection of industrial microorganisms ( moscow , russia ). frozen stock cultures containing 20 % ( w / v ) glycerol were stored in 2 ml ampoules at − 70 ° c . inoculum for fermentation was prepared in 125 - ml air - tight , anaerobic glass flasks and grown overnight on mss - medium ( berezina et al ., 2008 ) at 37 ° c . mss - medium contained 5 g / l of yeast extract powder ( scharlau ), 60 g / l of glucose ( vwr ), 0 . 8 g / l k 2 hpo 42 hpo 4 ( j . t . baker ), 0 . 01 g / l p - aminobenzoic acid ( fluka ), 0 . 5 g / l cysteine - hydrochloride ( aldrich ), 3 g / l ch 3 coonh 4 ( merck ), 1 g / l kh 2 po 4 ( j . t . baker ), 1 g / l mgso 4 . 7h 2 o ( j . t . baker ), and 0 . 05 g / l feso 4 . 7h 2 o ( merck ). two - stage chemostat cultivations were carried out in two 1 - liter fermenters ( braun biostat q ) f1 , f2 on mss medium ( 20 g / l of glucose concentration ) or sol medium at 37 ° c . with a stirrer speed 50 rpm . sol medium was prepared according to liubimova et al . ( 1993 ) containing : 1 g / l tryptone ( lab m ), 1 g / l yeast extract powder ( scharlau ), 60 g / l glucose ( vwr ), 0 . 7 g / l k 2 hpo 4 ( j . t . baker ), 0 . 5 g / l cysteine - hydrochloride ( aldrich ), 0 . 01 g / l nacl ( j . t . baker ), 3 g / l ch 3 coonh 4 ( merck ), 0 . 7 g / l kh 2 po 4 ( j . t . baker ), 0 . 1 g / l mgso 4 . 7h 2 o ( j . t . baker ), 0 . 02 g / l mnso 4 . h 2 o ( merck ), and 0 . 015 g / l feso 4 . 7h 2 o ( merck ). alternatively the sugar mixture of glucose 6 . 3 g / l ; arabinose 2 . 23 g / l ; galactose 6 . 43 g / l ; mannose 22 . 85 g / l ; xylose 7 . 51 g / l were used . the medium was autoclaved for 20 min at 121 ° c . the culture ph was set at 4 . 6 and 5 . 1 in the f1 and f2 unit respectively and it was controlled with 4 m naoh . the dilution rate was adjusted to 0 . 05 h − 1 and 0 . 1 h − 1 consecutively . the working volume of 500 ml was kept constant by removing the effluent with peristaltic pumps from both fermentation units ( peristaltic pump p - 3 , pharmacia fine chemicals ). samples for biomass and hplc were taken on a two consecutive days to confirm the steady state conditions . culture samples ( 40 ml ) were centrifuged ( eppendorf , centrifuge 5804r ) at 5000 rpm for 10 minutes , washed with milli - q water , and then dried in an oven at 80 ° c . for 24 - hours ( heraeus ) in glass petri plates , which were weighed before adding the sample . once dry , the plates were weighed . pharmacia column ( xk26 ) was filled with water saturated cellulosic fibers supported by a plastic net . 50 g w / w spruce chips fibers were rolled together into a tubular form with a plastic net and inserted into column . the bed height was 20 . 7 cm and the corresponding void volume was 102 cm 3 . the column was sterilized overnight with ethanol and the column volume of 4 . 6 cm 3 was determined by flushing the column with a growth medium . actively growing and producing clostridia cell mass was loaded into the bio - column by pumping cell suspension with a high flow rate through the matrix . the out flowing cell mass was returned to the f2 bioreactor unit . cell mass retention was monitored by the decreasing optical density value at 600 nm . after the bio - matrix was saturated with cells the loading was stopped . the substrate solution feeding was initiated from the separate substrate bottle placed in the water bath at 37 ° c . from the bottom direction . the abe - solution product was collected from the top of the column . when productivity was decreased the cell loading was repeated . industrial spruce chips screened to 2 - 4 mm thickness and then air - dried were used as a raw material for pulping . chips were fractionated by the so 2 - ethanol - water ( sew ) pulping process , also termed avap ™ process by american process inc . ( api ). pulping was done in a silicon oil bath using 6 bombs of 220 ml and 25 grams of oven dried of chips were placed in each bomb . the fresh fractionation liquor was prepared by injecting gaseous sulfur dioxide into a 55 % ( by volume ) ethanol - water solution . deionized water and ethanol etax a ( 96 . 1 v / v %) were used . the liquor - to - wood ratio used was 6 l / kg . pulping was carried out in two different conditions . fractionation conditions including the concentration of so 2 in the liquor by weight , temperature and fractionation time including the heating - up period are shown in table 1 . conditions were chosen so that the pulp obtained in the fractionation 2 has notably lower viscosity and cellulose degree of polymerization . in the further context , unfermented reference pulp samples are referred to as ref135 and ref150 according to the pulping temperatures , whereas the fermented pulps are referred to as fer135 and fer150 . after pulping , the bombs were rapidly removed from the oil bath and cooled in cold water . the pulp suspension from each bomb was poured in a washing sock and spent liquor was separated from pulp by squeezing . pulp was then washed 2 times with 300 ml of 40 % ethanol - water solution at 60 ° c . and finally 2 times with 3 l of deionized water at room temperature . a portion of the washed pulp was placed into the fermentation column whereas some pulp was kept as a reference sample with no further treatment . homogenization of the untreated reference pulps and fermented pulps was carried out by disintegration at 1 % consistency for 30 , 000 revolutions ( apparatus according to iso 5263 ). disintegrated pulp was contained in a washing sock and excess water was filtered out . filtrate was re - filtered through the fiber mat to diminish the loss of fines . pulp was then air dried prior to further experiments . the chemical composition of the pulps was determined after milling air dried pulps in a wiley mill using a 30 mesh screen . extractive content was determined according to scan - cm 49 : 03 , using acetone as an extracting solvent . for the determination of lignin and structural carbohydrates , the procedure by nrel ( nrel / tp - 510 - 42618 ) was followed . exception to the cited procedure was the determination of the acid soluble lignin ( asl ) with an uv - vis spectrophotometer at 205 nm wavelength , according to equation : where odw pulp - oven dry weight of the pulp sample . the absortivity value used was 128 l g − 1 cm − 1 , which is common for softwood species . the intrinsic viscosity of pulp solutions in ced was analyzed according to scan - cm 15 : 99 . prior to the determination , the pulps ref150 and fer150 prepared with lower so 2 charge ( 3 %) and thereby having higher lignin content were exposed to chlorite delignification according to t230 om - 66 ( 5 g pulp in 200 ml water + 5 g naclo 2 + 2 ml acetic acid at 70 ° c . for 5 min ). the cellulose degree of polymerization ( dp ) was calculated from the intrinsic viscosity according to the following equation ( da silva perez and van heiningen , 2002 ). where η — intrinsic viscosity of pulps in ced , ml / g ; [ hemi ] pulp — hemicelluloses content of pulp ( unit fraction ) and [ cel ] pulp — cellulose content of pulp ( unit fraction ). the cellulose content of the pulp was calculated using the equation ( janson , 1974 ). where [ glu ] tot — total glucan content of the pulp and [ man ]— mannan content of the pulp . glucan in hemicelluloses was calculated as the difference of the total glucan content and the cellulose content of the pulp . samples of 2 ml were centrifuged at 14 000 rpm for 5 min ( eppendorf , centrifuge 5424 ) and the supernatant was recovered for analysis . glucose , acetic acid , butyric acid , acetone , butanol , and ethanol concentrations were analysed by hplc ( alliance , waters 2690 and 2695 separations modules ). two stage chemostat set up was constructed by connecting two bioreactor units with peristaltic pumps . the division between acidogenetic and solventogenetic phase in f1 and f2 bioreactor units was based on the ph difference ( table 1 ) according to mutschlechner et al . ( 2000 ). the chemostat was running with two different dilution rates with glucose and sugar mixture substrates . the lower dilution rate was set to 0 . 05 and the higher 0 . 1 1 / h , respectively . above 0 . 1 1 / h dilutions rates biomass started to slowly wash out indicating that the dilution rate maximum was surpassed . the analysed glucose substrate concentrations were 18 . 7 and 56 . 2 g / l . the sugar mixture substrate concentrations were glucose 6 . 3 , arabinose 2 . 23 , galactose 6 . 43 , mannose 22 . 85 and xylose 7 . 51 g / l . this sugar composition was based on the average results obtained from spruce chips wood hydrolysis by water - ethanol - so 2 cooking by rakkolainen et al . ( 2010 ). the results indicated that different ph value cannot completely separate the c . acetobutylicum metabolism into acidogenetic and solventogenetic phases . acids and solvents were found from both f1 and f2 bioreactor units . biomass increases with the increasing dilution rate on 18 . 7 g / l of glucose feed concentration . this indicated that cells cannot have their maintenance energy demand completely fulfilled at the dilution rate of 0 . 06 1 / h . the metabolite production indicated that acetone and butanol were the main products from glucose and they can be found from both f1 and f2 units in these conditions . at the 18 . 7 g / l of glucose concentration both units f1 and f2 had approximately the same metabolite levels . this is due to the fact that cell suspension was continuously pumped from the f1 to f2 unit . however , only after the glucose concentration was increased to 56 . 2 g / l the butanol and acetone concentration increased correspondingly in the f2 unit indicating that solventogenetic phase required sufficient excess substrate concentration ( table 3 ). mannose and glucose were consumed with the yields of 0 . 67 and 0 . 90 g / g respectively being the most preferred substrates . consequently , arabinose and xylose were the least preferred substrates with the yields 0 . 04 and 0 . 11 g / g respectively ( table 4a - b ). all mixed substrates were consumed simultaneously by the c . acetobutylicum , but no solvent production was detected in the chemostat conditions . the figures are presented as yields of g of consumed sugar / g of sugar concentration in the medium ( y s / s ) and f1 and f2 refers to the fermentation units and f1 + f2 is the total consumption of sugars . in order to achieve high volumetric productivity of abe products the c . acetobutylicum cells from the f2 bioreactor were loaded into the column . cellulosic fibers were used as a cell retaining matrix supported by a plastic net . the results indicate that the highest volumetric butanol production from glucose was 4 . 38 g / l h with the final concentration of 4 . 14 g / l ( data not shown ). the highest dilution rate ( 1 . 06 1 / h ) allowed rapid removal of butanol alleviating the inhibition effect on cell metabolism . the acetic and butyric acid concentrations were 1 . 19 g / l and 1 . 63 g / l respectively indicating that cells were actively producing acids ( data not shown ). the volumetric productivity and final concentration for the sugar mixture feed at the dilution rate of 0 . 2 1 / h was 0 . 82 g / l h and 4 . 02 g / l ( data not shown ). the results on the viscosity and dp of cellulose show that c . acetobutylicum can degrade cellulosic fibers by producing extracellular cellulases in the column conditions . dp of cellulose was decreased by 9 . 6 % in case of the pulp with higher viscosity , whereas the effect on pulp with lower viscosity was negligible ( 1 . 9 %) ( table 6 ). the weight fraction of the total carbohydrates was decreased in the fermented pulp compared to the reference , whereas the proportional share of lignin was increased ( table 7 ). mainly the sugars mannan and xylan , originated from hemicelluloses were consumed rather than glucan originating from cellulose . continuous bio - catalytic conversion of sugar mixture to acetone - butanol - ethanol by immobilized c . acetobutylicum dsm 792 glucose and d - xylose was purchased from vwr international , finland , yeast extract , tryptone were purchased from lab m ltd , uk . mannose , d - galactose , l - arabinose were purchased from danisco , finland . p - amino benzoic acid , mgso 4 , fecl 3 , namoo 4 and cacl 2 were obtained from fluka , switzerland . l - cysteine hydrochloride and biotin were purchased from sigma aldrich , usa . k 2 hpo 4 , sodium sulphate , znso 4 , znso 4 , cuso 4 and reinforced clostridia medium ( rcm ) were obtained from merck , germany . naoh , hcl and h 2 so 4 were obtained from j . t . baker , holland . all the chemicals were analytical grade . c . acetobutylicum dsm 792 was obtained from dsmz , germany ( german collection of microorganisms and cell cultures ). initially sporulated cells were activated by heat shock at 80 ° c . for 10 min . the activated spore culture ( 2 . 5 ml ) was inoculated in 100 ml sterile rcm in 125 ml air tight , anaerobic glass bottles and grown for 20 h at 37 ° c . after 20 h , the inoculum was used for batch experiments ( 5 % v / v ) as well as for immobilization of matrix for continuous experiments . the inoculum medium ( rcm ) contained meat extract 10 g / l , peptone 5 g / l , yeast extract 3 g / l , d (+) glucose 5 g / l , starch 1 g / l , sodium chloride 5 g / l , sodium acetate 3 g / l and l - cysteine hydrochloride 0 . 5 g / l ( final ph 6 . 8 ± 0 . 2 ). the production medium contained ( in g / l ) glucose 60 , magnesium sulphate 0 . 2 , sodium chloride 0 . 01 , manganese sulphate 0 . 01 , iron sulphate 0 . 01 , potassium dihydrogen phosphate 0 . 5 , potassium hydrogen phosphate 0 . 5 , ammonium acetate 2 . 2 , biotin 0 . 01 , thiamin 0 . 1 and p - aminobenzoic acid 0 . 1 . modified production medium contained sugar mixture ( 50 g / l ) of glucose , mannose , arabinose , galactose and xylose instead of a single carbon source . it contained ( in g / l ) glucose 8 . 5 , mannose 22 . 0 , arabinose 2 . 3 , galactose 4 . 5 and xylose 10 . 50 . the medium was adjusted to ph 6 . 5 with hcl . after preparation , the medium was purged with oxygen free nitrogen and autoclaved at 10 5 pa ( 121 ° c .) for 20 min and cooled . in the present study , we chose support materials like wood pulp , loofa sponge , coconut fibers , wood chips and sugarcane baggase . the wood pulp fibers and wood chips were obtained from department of forest products technology , aalto university school of science and technology , espoo , finland . the matrices were cut into 3 - 5 mm from their raw sources . they were washed with water for three times and dried in oven at 70 ° c . all the immobilization materials were evaluated for maximum solvent production in batch mode . processed matrices were added to production medium at ratio of 1 : 4 v / v in 125 ml air tight bottles . it was purged with nitrogen and autoclaved at 10 5 pa ( 121 ° c .) for 20 min and cooled . it was inoculated ( 5 % v / v ) with 20 h actively growing seed culture and incubated for 110 h at 37 ° c . the wood pulp soaked in excess of water was distributed evenly on plastic mesh and rolled to remove excess of water . the plastic mesh was used for holding the wood pulp . the roll was put into a jacketed glass column and used for immobilization of cells . the column was filled with 70 % ethanol and kept for 24 h for sterilization . the ethanol was replaced with deoxygenated production medium after 24 h . the inoculum was pumped into the column and re - circulated for 24 h for cell adsorption and growth . after immobilizing cells , the production medium was continuously fed to the immobilized cell reactor at different dilution rates . the dilution rate was altered whenever a steady state was reached in terms of production of solvents and acids and use of substrate . after changing the dilution rate , sufficient time was allowed to pass in order to reach a new steady state before samples were taken from the top of the column and centrifuged at 15000 rpm for 5 min and supernatants were used for the substrate and product analysis . the column temperature was maintained at 37 ° c . by continuously circulating water through the jacket . the solvents and acids were quantified by using gas chromatography . the gas chromatograph ( hewlett packard series 6890 ) equipped with a flame ionization detector was used . separation took place in db - waxetr capillary column ( 30 m × 0 . 32 mm × 1 μm ) from agilent technologies , finland . the injector temperature was 200 ° c . and detector temperature was 250 ° c . the injector volume was 10 μl . glucose , mannose , arabinose , galactose and xylose were determined by high - performance liquid chromatography ( biorad laboratories , richmond , calif . ), equipped with an inores s 259 - h column ( inovex , vienna , austria ) packed with inores cation exchanger ( particle size , 9 mm ). the column was heated at 70 ° c ., and the eluent ( 0 . 01m h 2 so 4 ) was circulated with a flow rate of 0 . 60 ml min − 1 . a cellobiose ( roth , karlsruhe , germany ) solution was added to the samples as an internal standard . a refractive index detector ( model 1755 ; bio - rad ) was used for quantification . dilution rate in h − 1 was calculated as flow rate divided by the working volume of the column . the overall solvent productivity in g /( i · h ) during continuous cultivation of solvent - producing clostridia was expressed as g / l of total solvents multiplied by dilution rate ( h − 1 ). solvent yield was calculated by dividing total solvents in g / l by utilized substrate in g / l . cell immobilization is often used to improve the performance of traditional continuous fermentation process by increasing the amount of cells per bioreactor volume , and cell deposition on support matrix . cell immobilization through adsorption provides a direct contact between nutrients and the immobilized cells . this technique involves the transport of the cells from the bulk phase to the surface of support , followed by the adhesion of cells , and subsequent colonization of the support surface . both electrostatic and hydrophobic interactions govern the cell - support adhesion , which is the key step in controlling the cell immobilization on the support it was found that addition of support matrix helps in improving substrate consumption and conversion to solvents . coconut fibers and wood pulp were found to be the most promising support matrices . the maximum solvent concentration of 18 . 88 g / l and 18 . 60 g / l was obtained with wood pulp and coconut fiber respectively , after 110 h of fermentation as compared to 8 . 18 g / l with control . among all the support matrices , wood pulp and coconut fibers containing bottle complete glucose consumption was observed . in control experiment i . e . without adding any support matrix maximum glucose consumption was approximately 65 %. in summary : continuous production of acetone , n - butanol and ethanol ( abe ) was successfully carried out using immobilized clostridium acetobutylicum dsm 792 . wood pulp fibers were used to immobilize c . acetobutylicum cells . initially , different lignocellulosic materials were evaluated as an immobilization matrix for maximum production of abe . coconut fibers and wood pulp fibers were found to be promising . further , wood pulp was used as cell holding material in column reactor for continuous production of abe mixture . glucose as well as sugar mixture ( glucose , mannose , galactose , arabinose and xylose ) identical to lignocellulosic hydrolysate was used as substrate for the production of solvents . the effect of dilution rate on solvent production was studied during continuous ( nearly 25 days ) operation . the maximum total solvent concentration of 14 . 32 g / l was obtained at a dilution rate of 0 . 22 h − 1 with glucose as substrate as compared to 12 . 64 g / l at 0 . 5 h − 1 dilution rate with sugar mixture . the maximum solvent productivity ( 13 . 66 g / l · h ) was obtained at dilution rate of 1 . 9 h − 1 with glucose as substrate whereas solvent productivity ( 12 . 14 g / l · h ) was obtained at dilution rate of 1 . 5 h − 1 with sugar mixture . the immobilized column reactor was found to be suitable for continuous production of abe using sugar mixture . continuous acetone - butanol - ethanol fermentation using so 2 - ethanol - water spent liquor from spruce the cost of biomass is an important parameter for the economical production of butanol . lignocellulose biomass is considered as the cheapest as well as sustainable feedstock . continuous production of acetone , butanol and ethanol ( abe ) was studied using so 2 - ethanol - water ( sew ) spent liquor . initially , batch experiments were performed using spent liquor to check the suitability for production of abe . the overall study will serve the concept of forest biorefinery by using forest biomass for production of valuable compounds . the spent liquor from sew fractionation process was successfully used for abe solvent production . a continuous abe solvent production process using an efficient column reactor with wood pulp fibers as an immobilization material and sew spent liquor as substrate is developed . the bioreactor was operated for nearly 20 days in continuous flow mode . the use of cheap substrate along with continuous mode production makes the process industrially attractive . further , we developed method for efficient utilization of spent broth to make process more economical . glucose was purchased from vwr international , finland . p - amino benzoic acid , mgso 4 , feso 4 , nacl were obtained from fluka , switzerland . l - cysteine hydrochloride and biotin were purchased from sigma aldrich , usa . k 2 hpo 4 , kh 2 po 4 , mnso 4 , ammonium acetate , and reinforced clostridia medium ( rcm ) were obtained from merck , germany . naoh and hcl were obtained from j . t . baker , holland . all the chemicals were analytical grade . amberlite xad - 4 resin was a kind gift from rohm and haas , france . c . acetobutylicum dsm 792 was obtained from dsmz , germany ( german collection of microorganisms and cell cultures ). initially sporulated cells were activated by heat shock at 80 ° c . for 10 min . the activated spore culture ( 2 . 5 ml ) was inoculated in 100 ml sterile rcm in 125 ml air tight , anaerobic glass bottles and grown for 20 h at 37 ° c . after 20 h , the inoculum was used for batch experiments ( 5 % v / v ) as well as for immobilization of matrix for continuous experiments . the production of solvents was studied using sew spent liquor . the liquor was supplemented with the medium components reported by tripathi et al . ( 2010 ). the supplement contained ( in g / l ) magnesium sulphate 0 . 2 , sodium chloride 0 . 01 , manganese sulphate 0 . 01 , iron sulphate 0 . 01 , potassium dihydrogen phosphate 0 . 5 , potassium hydrogen phosphate 0 . 5 , ammonium acetate 2 . 2 , biotin 0 . 01 , thiamin 0 . 1 and p - aminobenzoic acid 0 . 1 . the glucose was added as and when mentioned . the ph was adjusted to 6 . 5 with hcl . after preparation , the medium was purged with oxygen free nitrogen and autoclaved at 10 5 pa ( 121 ° c .) for 20 min and cooled . rcm was used for inoculum preparation . the rcm contained meat extract 10 g / l , peptone 5 g / l , yeast extract 3 g / l , d (+) glucose 30 g / l , starch 1 g / l , sodium chloride 5 g / l , sodium acetate 3 g / l and l - cysteine hydrochloride 0 . 5 g / l ( final ph 6 . 8 ± 0 . 2 ). the sew spent liquor was produced and conditioned as reported by sklavounos et al . ( 2011 ). the fractionation of spruce wood chips was carried out using sew liquor with liquor - to - wood ratio of 6 : 1 kg . the spent sew liquor was processed with a sequence of conditioning steps including evaporation , steam stripping , liming and catalytic oxidation for making it fermentable . in sew liquor , the acetic acid concentration was 1 . 5 g / l ( 1 . 0 g / 100 g o . d . wood ) and the formic acid concentration was close to zero . the furfural and hydroxy methyl furfural ( hmf ) concentrations in sew liquor were 0 . 7 and 0 . 2 g / l , respectively . the so 2 level was sufficiently minimized to 6 mg / l . the total sugar concentration in final liquor was approximately 111 . 0 g / l . the individual sugar concentration was ( in g / l ) glucose 18 . 8 , mannose 52 . 3 , galactose 9 . 7 , arabinose 5 . 4 and xylose 24 . 8 . the obtained liquor was further treated with anion exchange resin ( amberlite xad - 4 ). the resin was removed by filtration . the ph of spent liquor was finally adjusted to 6 . 5 with ca ( oh ) 2 before adding the medium components . the effect of dilution of sew liquor was studied on production of solvents . the sew spent liquor was diluted as 2 fold , 4 fold and 8 fold with water to make it suitable for growth and fermentation of clostridia . the diluted liquor ( 8 fold ) was also tried as an inoculum medium . all the production medium components except carbon source were supplemented to the spent liquor . the effect of supplementing the extra glucose ( 15 , 25 and 35 g / l ) to the 4 fold diluted sew liquor was also studied . the batch experiments were carried out in 125 ml screw cap bottles with 50 ml production medium . it was purged with nitrogen and autoclaved at 10 5 pa ( 121 ° c .) for 20 min and cooled . it was inoculated ( 5 % v / v ) with 20 h actively growing seed culture and incubated for 96 h at 37 ° c . the optimized medium composition was further tested for continuous operation . all the experiments were done at least in triplicates and values are given as means and standard deviation was established using microsoft excel software . the wood pulp was used as an immobilization material . the column was filled with 70 % ethanol and kept for 24 h for sterilization . the inoculum was pumped into the column and re - circulated for 24 h for cell adsorption and growth . after immobilizing cells , the sew spent liquor was continuously fed to the immobilized cell reactor at different dilution rates . the dilution rate was altered whenever a steady state was reached in terms of production of solvents and acids . after changing the dilution rate , 2 volume changes was allowed to pass in order to reach a new steady state before samples were taken from the top of the column and centrifuged at 15000 rpm for 5 min . supernatants were used for the substrate and product analysis . the column temperature was maintained at 37 ° c . by continuously circulating water through the jacket . the sb collected after continuous fermentation contained residual sugars and some medium components . the feasibility of sb as production medium was checked in batch experiments after removing the solvents produced . the solvents produced were removed by nitrogen gas purging . the sb was used as such or supplemented with production medium components . the batch experiments were carried out in 125 ml screw cap bottles with 50 ml production medium as reported in earlier section . the solvents and acids were quantified by using gas chromatography . glucose , mannose , arabinose , galactose and xylose were determined by high - performance liquid chromatography . in summary : maximum concentration of total abe was found to be 8 . 79 g / l using 4 fold diluted sew liquor supplemented with 35 g / l of glucose . the effect of dilution rate on solvent production , productivity and yield was studied in column reactor consisting of immobilized clostridium acetobutylicum dsm 792 on wood pulp . total solvent concentration of 12 g / l was obtained at a dilution rate of 0 . 21 h − 1 . the maximum solvent productivity ( 4 . 86 g / l · h ) with yield of 0 . 27 g / g was obtained at dilution rate of 0 . 64 h − 1 . further , to increase the solvent yield , the unutilized sugars were subjected to batch fermentation after taking out solvents produced . we successfully used sew liquor for batch and continuous production of abe solvents . bahl , h ., andersch , w . and gottschalk , g . 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