Patent Application: US-87900597-A

Abstract:
this invention presents a method for the production of ethanol that utilizes a soy hydrolysate - based nutrient medium or a yeast autolysate - based medium nutrient medium in conjunction with ethanologenic bacteria and a fermentable sugar for the cost - effective production of ethanol from lignocellulosic biomass . the invention offers several advantages over presently available media for use in ethanol production , including consistent quality , lack of toxins and wide availability .

Description:
raw soybeans and soybean products , such as soy flour , soy meal , and ground soybeans , are widely available from such sources as grain mills and food processors . alternatively , a hyrolyzed soybean product can be obtained directly from food processors . a non - hydrolyzed soybean product can be hydrolyzed through the addition of such commercial proteases as spezyme ™ fan ( genencor , south san francisco , calif .). the conditions for protease hydrolysis are typically selected in consideration of the conditions suitable for the specific protease source . in general , typical conditions include a temperature between about 35 ° c . and 60 ° c . and a ph between about 6 and 10 . vitamins , macronutrients , and micronutrients are available through a variety of chemical supply companies , such as sigma chemical company ( st . louis , mo .). an ethanologenic microorganism is one which has the ability to convert a sugar or oligosaccharide to ethanol . ethanologenic microorganisms are known in the art and include ethanologenic bacteria . the microorganisms are ethanologenic by virtue of their ability to express one or more enzymes which , individually or together , convert a sugar to ethanol . preferred examples of ethanologenic microorganisms include ethanologenic bacteria expressing alcohol dehydrogenase and pyruvate decarboxylase , such as can be obtained with or from zymomonas mobilis ( see u . s . pat . no . 5 , 028 , 539 to ingram et al ., u . s . pat . no . 5 , 000 , 000 to ingram et al ., u . s . pat . no . 5 , 424 , 202 to ingram et al ., u . s . pat . no . 5 , 487 , 989 to fowler et al ., u . s . pat . no . 5 , 482 , 846 to ingram et al ., u . s . pat . no . 5 , 554 , 520 to fowler et al ., u . s . pat . no . 5 , 514 , 583 to picataggio , et al ., and copending applications having u . s . ser . no . 08 / 363 , 868 filed on dec . 27 , 1994 , u . s . ser . no . 08 / 475 , 925 filed on jun . 7 , 1995 , u . s . ser . no . 08 / 218 , 914 filed on mar . 28 , 1994 , attorney docket no . uf96 - 01 filed on apr . 7 , 1997 , attorney docket no . uf97 - 01 filed on apr . 7 , 1997 , attorney docket no . uf97 - 02 , filed on apr . 7 , 1997 , the teachings of all of which are hereby incorporated by reference , in their entirety ). in another embodiment , the ethanologenic microorganism can express xylose reductase and xylitol dehydrogenase , which convert xylose to xylulose . xylose isomerase converts xylose to xylulose , as well . the ethanologenic microorganism can further express xylulokinase , which catalyzes the conversion of xylulose to xylulose - 5 - phosphate . additional enzymes to complete the pathway can include transaldolase and transketolase . these enzymes can be obtained or derived from escherichia coli , klebsiella oxytoca and erwinia species . for example , see u . s . pat . no . no . 5 , 514 , 583 . it is particularly preferred to employ a microorganism which is capable of fermenting both pentoses and hexoses to ethanol , such as are obtained from preparing a recombinant organism which inherently possesses one set of enzymes and which is genetically engineered to contain a complementing set of enzymes . examples of such microorganisms include those described in u . s . pat . nos . 5 , 000 , 000 ; 5 , 028 , 539 ; 5 , 424 , 202 ; 5 , 482 , 846 ; 5 , 514 , 583 ; and ho et al ., wo 95 / 13362 , all of which are incorporated herein by reference . particularly preferred microorganisms include klebsiella oxytoca p2 and escherichia coli ko11 . fermentable sugars can be obtained from a wide variety of sources , including lignocellulosic material . lignocellulose material can be obtained from lignocellulosic waste products , such as plant residues and waste paper . examples of suitable plant residues include stems , leaves , hulls , husks , cobs and the like , as well as wood , wood chips , wood pulp , and sawdust . examples of paper waste include discard photocopy paper , computer printer paper , notebook paper , notepad paper , typewriter paper , and the like , as well as newspapers , magazines , cardboard , and paper - based packaging materials . a cellulase enzyme can be added to the lignocellulosic material . the cellulase can be provided as a purified enzyme or can be provided by a cellulase - producing microorganism in said aqueous mixture . cellulases , as that term is used herein , includes any enzyme that effects the hydrolysis or otherwise solubilizes cellulase ( including insoluble cellulose and soluble products of cellulose ). cellulase enzymes , including purified enzyme preparations , organisms expressing the same , are known in the art . suitable sources of cellulase include such commercial cellulase products as spezyme ™ cp , cytolase ™ m104 , and multifect ™ cl ( genencor , south san francisco , calif . ), and such organisms expressing cellulase as the recombinant bacterium of u . s . pat . no . 5 , 424 , 202 , which is incorporated herein by reference . the conditions for cellulase hydrolysis are typically selected in consideration of the conditions suitable for the specific cellulase source , e . g , bacterial or fungal . for example , cellulase from fungal sources typically works best at temperatures between about 30 ° c . and 48 ° c . and a ph between about 4 . 0 and 6 . 0 . in general , typical conditions include a temperature between about 30 ° c . and 60 ° c . and a ph between about 4 . 0 and 8 . 0 . the conditions for converting sugars to ethanol are typically those described in the above referenced u . s . patents . generally , the temperature is between about 30 ° c . and 40 ° c . and the ph is between about 5 . 0 and 7 . 0 . it is generally advantageous to add supplements to the nutrient medium , such as vitamins , macronutrients , and micronutrients . vitamins include choline chloride , nicotinic acid , thiamine hcl , cyanocobalamin , p - aminobenzoic acid , biotin , calcium pantothenate , folic acid , pyridoxine . hcl , and riboflavin . macronutrients include ( nh 4 ) 2 so 4 , k 2 hpo 4 , nacl , and mgso 4 . 7h 2 o . micronutrients include fecl 3 . 6h 2 o , zncl 2 . 4h 2 o , cocl 2 . 6h 2 o , molybdic acid ( tech ), cucl 3 . 2h 2 o , cacl 2 . 2h 2 o , and h 3 bo 3 . the methods and materials described below were used in carrying out the work described in the examples which follow . materials and methods for a soy hydrolysate - based nutrient medium are described first , followed by a yeast autolysate - based nutrient medium . tryptone ™, soytone ™ and yeast extract were obtained from difco ( detroit , mich ., usa ). soybeans , soy flour , and soy meal were purchased locally ( approximately 8 % moisture ). the particular size of soybeans and soy meal was reduced with a commercial coffee grinder prior to digestion . spezyme ™ fan protease was generously provided by genencor international ( south san francisco , calif ., usa ). soy hydrolysates prepared by different methods were tested in shaken , 250 - ml flasks ( 50 ml broth , 35 ° c .). media for flask - fermentations ( and for initial studies in ph - stats ) contained per liter : 50 ml of test hydrolysate , 100 g glucose , 2 g ( nh 4 ) 2 so 4 , 1 g k 2 hpo 4 , 2 g nacl , 0 . 25 g mgso 4 . 7h 2 o , 5 . 4 mg fecl 3 . 6h 2 o , 0 . 4 mg zncl 2 . 4h 2 o , 0 . 4 mg cocl 2 . 6h 2 o , 0 . 4 mg molybdic acid ( tech ), 0 . 2 mg cucl 3 . 2h 2 o , 0 . 2 mg cacl 2 . 2h 2 o , 0 . 1 mg h 3 bo 3 , 2 mg choline chloride , 2 mg nicotinic acid , 1 mg thiamine . hcl , 0 . 1 μg cyanocobalamin , 0 . 2 μg p - aminobenzoic acid , 0 . 2 μg biotin , 0 . 4 μg calcium pantothenate , 0 . 2 μg folic acid , 0 . 2 μg pyridoxine . hcl , and 0 . 2 μg riboflavin . flasks were inoculated by transferring a small colony from solid media . ethanol produced after 24 h was used as the endpoint . ethanol was determined by gas chromatography ( beall et al ., 1991 ). free amino nitrogen was determined spectrophotometrically using glycine as a standard ( european brewery convention et al ., 1987 ). combinations of mineral and vitamin - supplements were tested in ph - stats ( 350 ml working volume , ph 6 . 0 , 35 ° c ., 100 rpm ) ( beall et al ., 1991 ). these were inoculated from broth cultures to an initial density of 165 mg cell dry weight l - 1 . optimized soy - based medium contained per liter : 50 ml crude soy hydrolysate ( 9 . 2 g soy solids l - 1 ) or 5 g tryptone ™, 100 g glucose , 2 g ( nh 4 ) 2 so 4 , 1 g k 2 hpo 4 , 2 g nacl , 0 . 25 g mgso 4 . 7h 2 o , 10 . 8 mg fecl 3 . 6h 2 o , 0 . 25 mg thiamine . hcl , 0 . 1 μg calcium pantothenate , 0 . 05 μg pyridoxine . hcl , and 0 . 02 μg cyanocobalamin . ethanol production by e . coli ko11 ( flask cultures ) was used as a bioassay to optimize the denaturation and hydrolysis of soy with spezyme ™ fan . the resulting procedure for the production of a 20 - fold nutrient concentrate ( 300 ml ) was : 1 ) combine 60 g soy flour , ground soybeans or soy meal ( approximately 8 % moisture ) with 220 ml tap water in a 500 - ml screw - capped erlenmeyer flask ( approximately ph 8 ); 2 ) denature by heating to 95 ° c . for 2 h with reciprocal shaking ; 3 ) cool to room temperature and adjust to ph 9 with 10 n sodium hydroxide ; 4 ) add 20 ml of 95 % ethanol and 1 . 2 ml of spezyme ™ fan ; 5 ) mix thoroughly and incubate for 18 h at 50 ° c . with reciprocal shaking . hydrolysates were stored frozen until needed and pasteurized for 15 min at 90 ° c . immediately before use . inclusion of ethanol in the hydrolysate appeared to increase the efficacy of pasteurization . approximately 50 % of soy dry weight was solubilized with a free amino nitrogen content of 1 . 0 g l - 1 . e . coli ko11 was used in these studies . unless noted otherwise , media for ph - stats contained per liter : macronutrient mineral salts ( 2 g ( nh 4 ) 2 so 4 , 1 g k 2 hpo 4 , and 2 g nacl ); 0 . 5 g mgso ( 4 ). 7h 2 o , 11 mg fecl 3 . 6h 2 o ; vitamins ( 25 μg cyanocobalamin , 100 μg calcium pantothenate , 50 μg pyridoxine . hcl , and 500 μg thiamine . hcl ), complex nutrient ( 5 g difco product of 10 g crude yeast autolysate ), 100 g glucose , and 40 mg chloramphenicol . a chloramphenicol stock ( 1000 ×) was prepared in 70 % ethanol . inocula for ph - stats were grown for 18 h at 30 ° c . in lbg medium ( luria and delbruck , 1943 ) ( per liter : 50 g glucose , 5 g difco yeast extract , 10 g difco tryptone , 5 g sodium chloride ) and 40 mg chloramphenicol . cells were harvested by centrifugation ( 5 , 000 × g , 5 min ) for use as inocula ( 158 mg dry weight l - 1 ) pressed yeast cakes were purchased from a local bakery . inorganic salts , except molybdic acid ( technical ), were reagent grade . salts , difco tryptone , difco soytone and difco yeast extract were purchased from the fisher scientific company ( norcross , ga .). chloramphenicol was purchased from the sigma chemical company ( st . louis , mo .). a crude yeast autolysate was prepared in batches by a modification of the method described by kollar et al ., ( 1992 ). sufficient water was added to 200 g of yeast cake ( 70 % moisture ; 200 g dry weight l - 1 ), 3 g nacl , and 16 g ethanol ( 20 ml ) to produce a total volume of 300 ml . a glass marble was added to aid agitation ( 60 cycles min - 1 ) and the mixture incubated in a sealed 500 - ml flask for 24 h at 50 ° c . this serves as a 20 - fold concentrate and was stored frozen at - 20 ° c . autolysate was pasteurized ( 15 min , 90 ° c .) immediately prior to use ( final concentration of 10 g autolyzed yeast l - 1 ). modified corning fleakers ™ ( 500 ml total volume , 350 ml working volume ) were used as vessels for ph - stats ( beall et al ., 1991 ). these were immersed in a 35 ° c . water bath and agitated with a magnetic stir bar ( 100 rpm ). broth ph was controlled during fermentation by the automatic addition of 2n koh and was not allowed to fall below ph 6 . 0 . ethanol , base consumption and ph were measured at 24 h intervals . ethanol was measured by gas liquid chromatography using isopropanol as an internal standard ( ohta et al ., 1991 ). ethanol yields were calculated after correcting for dilution by added base and for ethanol present at the time of inoculation ( yeast autolysate and chloramphenicol stock ). yields are expressed as a percentage of the maximum theoretical yield ( 51 g ethanol per 100 g glucose ) and were not corrected for unmetabolized sugar . moisture content was measured after drying for 48 h at 70 ° c . free amino nitrogen ( fan ) was measured using glycine as a standard ( european brewery convention , 1987 ). kollar et al ., ( 1992 ) developed an optimal batch procedure for yeast autolysis ( 100 g autolyzed yeast l - 1 ) based on the solubilization of protein . this procedure included freshly prepared autolysate ( prior batch ), 1 % nacl and 5 % ( w / v ) ethanol . initial experiments , using ethanol production by e . coli ko11 ( 48 h ) as a bioassay , indicated that the yeast concentration can be doubled to produce a nutrient which was equivalent on a dry weight basis . using this higher yeast concentration eliminated any benefit from the addition of autolysate from a prior batch . approximately 58 % of the yeast dry weight was solubilized during the preparation of 20 % yeast autolysate . using the materials and procedures outlined above , vitamin and mineral supplements were optimized for soy hydrolysate - based medium ( 9 . 2 g l - 1 total soy , 4 . 6 g l - 1 soy solubles ), using ethanol production by e . coli ko11 as a measure . fig1 shows the beneficial effects of supplements on ethanol production . subsequent experiments indicated that fecl 3 can fully replace the mixture of trace metals . only 4 of the 10 vitamins tested were beneficial : thiamine . hcl , cyanocobalamin , calcium pantothenate and pyridoxine . hcl . when compared on the basis of solubles , soy hydrolysate prepared as described was equivalent to commercial soytone ™ as a nutrient for ethanol production ( 44 - 45 g ethanol l - 1 ). neither fermentation rate nor ethanol yield was increased significantly by increasing the levels of vitamins or minerals above that in the optimized soy medium . fermentations were also conducted in modified lb medium ( luria and delbruck , 1943 ) ( per liter : 10 g tryptone ™, 5 g yeast extract , 5 g nacl , and 100 g glucose ) for comparison . no combination of vitamins and minerals was found which allowed fermentations with soy hydrolysate ( 4 . 6 g soy solubles l - 1 ) to reach completion as rapidly as fermentations with lb medium ( 15 g soluble hydrolysate l - 1 ) the completion of fermentations in soy medium can be slower due to the increased demand for amino acid biosynthesis . final ethanol concentrations achieved with both media were nearly equivalent ( fig2 ). a series of experiments was conducted with 20 %- yeast autolysate to determine how to optimize the supplements ( macronutrient salts , magnesium , trace metals , and vitamins ) for ethanol production . an initial mixture of 7 trace metals was completely replaced by fecl 3 ( 11 mg l - 1 ) similarly , a mixture of 10 vitamins was reduced to 4 vitamins ( per liter : 25 μg cyanocobalamin , 100 μg calcium pantothenate , 50 μg pyridoxine . hcl , and 500 μg thiamine . hcl ). fig3 and table 1 illustrate the effect of omitting either of these supplements . inorganic sources of nitrogen ( 2 g ( nh 4 ) 2 so 4 l - 1 ) and phosphorous ( 1 g k 2 hpo 4 l - 1 ) were also beneficial ( fig3 ; table 1 ). a further increase in ethanol production was obtained with a 50 % increase in the level of ammonia , phosphate , or both . reducing either macronutrient or magnesium resulted in lower ethanol concentrations and lower yields . as a complex nutrient , 20 %- yeast autolysate was clearly superior to 10 %- yeast autolysate ( table 1 ). the more concentrated nutrient also has the advantage of reducing the volumes which must be processed . omission of yeast autolysate reduced fermentation rates by over 50 % ( fig3 & amp ; 4 ). reductions in the level of yeast autolysate ( 25 % and 50 %) also reduced the rates of ethanol production and ethanol yields . the addition of spezyme ™ protease ( 4 ml l - 1 ) during autolysis did not appear to improve the nutritional value of autolysate when assayed at a 1 : 20 dilution ( table 1 ). when tested at one - half strength , however , autolysate containing protease supported higher rates of ethanol production and a higher product yield than control autolysate . the addition of 50 % higher levels of ammonia and phosphate also improved fermentations with half - strength hydrolysate but yields remained below that obtained with full strength autolysate . ethanol yield with crude yeast autolysate and optimal supplements was equivalent to that obtained with lbg ( fig4 ; table 1 ) albeit with a slightly slower rate of ethanol production . the nutritional value of crude yeast autolysate was compared to difco products ( table 1 ). when compared using the same supplements , 20 %- yeast autolysate diluted to 10 g autolyzed yeast l - 1 was equivalent to 5 gl - 1 yeast extract or soytone but less effective than tryptone . the fan contents of these fermentation media were examined as a possible bias for the nutritional differences ( jones and ingledew , 1994b ; thomas and ingledew , 1990 ). lbg ( 475 mg l - 1 ) contained the highest level of fan , followed by protease - supplemented yeast autolysate ( 225 mg l - 1 ), yeast extract ( 177 mg l - 1 ), yeast autolysate ( 168 mg l - 1 ), tryptone ( 149 mg l - 1 ) and soytone ( 83 mg l - 1 ). although it is clear that complex nutrients containing amino acids stimulate fermentation , factors other than fan content must also contribute to the effectiveness . table 1______________________________________effects of nutrients on fermentation ( 100 g glucose / l ) ethanol base . sup . e yield (% nutrients . sup . a , b , c n . sup . d ( g / l ) ( ml / l ) theoretical ) ______________________________________1 . lbg medium ( difco ) 23 44 . 7 ± 1 . 9 62 . 9 92difco nutrient ( g / l ) + salts + mg + fe + vitamins2 . yeast extract ( 5 ) 4 43 . 7 ± 0 . 9 62 . 9 903 . tryptone ( 5 ) 3 47 . 9 ± 0 . 5 51 . 4 974 . soytone ( 5 ) 6 44 . 8 ± 1 . 7 54 . 3 9110 %- yeast autolysate ca yeast / l ) + salts + mg + fe + vitamins5 . yeast autolysate ( 10 ) 3 44 . 3 ± 0 . 5 77 . 1 8320 - yeast autolysate ( g yeast / l ) + salts + mg + fe + vitamins6 . yeast autolysate ( 10 ) 21 46 . 0 ± 1 . 7 68 . 6 907 . # 6 minus fe 3 44 . 7 ± 1 . 2 62 . 9 878 . # 6 minus salts 3 29 . 7 ± 5 . 4 97 . 1 589 . # 6 minus vitamins 6 41 . 3 ± 3 . 2 91 . 4 8310 . # 6 minus yeast 4 26 . 7 ± 1 . 9 71 . 4 50 autolysate11 . # 6 plus protease 3 44 . 2 ± 1 . 5 85 . 7 8812 . # 6 with 1 . 5 × mg 2 43 . 5 71 . 4 8613 . # 6 with 1 . 5 × nh . sub . 4 2 47 . 6 54 . 3 9314 . # 6 with 1 . 5 × po . sub . 4 2 48 . 7 57 . 1 9515 . # 6 with 1 . 5 × [ nh . sub . 4 + po . sub . 4 ] 4 47 . 0 ± . 1 62 . 9 9216 . # 6 with 0 . 5 × mg 2 42 . 5 94 . 3 8517 . # 6 with 0 . 5 × nh . sub . 4 2 42 . 7 114 . 3 8718 . # 6 with 0 . 5 × po . sub . 4 2 44 . 5 80 . 0 8819 . # 6 with 0 . 75 × yeast 4 42 . 9 ± 4 . 6 62 . 9 85 autolysate20 . # 6 with 0 . 5 × yeast 5 35 . 8 ± 2 . 3 77 . 1 72 autolysate21 . # 20 with protease . sup . d 3 39 . 9 ± 0 . 5 94 . 3 8222 . # 20 with 1 . 5 × nh . sub . 4 2 41 . 4 65 . 7 8323 . # 20 with 5 .× [ nh . sub . 4 + po . sub . 4 ] 2 38 . 9 62 . 9 77______________________________________ . sup . a abbreviations : mg , mgso . sub . 4 . 7h . sub . 2 o ; fe , fecl . sub . 3 . 6h . sub . 2 o ; nh . sub . 4 ; and po . sub . 4 , k . sub . 2 hpo . sub . 4 . . sup . b average initial ethanol concentrations in fermentation broth were as follows : 0 . 7 g / l for lbg and difco products ; 5 . 3 g / l for 10 % yeast autolysate ; 3 . 0 g / l for 20 % yeast autolysate ( 2 . 4 g / l for 0 . 75 × and 1 . 9 g / l for 0 . 5 ×). . sup . c omitted salts were replaced by an equivalent weight of nacl . . sup . d number of fermentation trials . . sup . e 2n naoh added to maintain ph during fermentation . . sup . f yeast autolysate prepared with spezyme ™ fan protease ( 4 m / l ) a a supplement . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , many equivalents to the specific embodiments of the invention described herein . such equivalents are intended to be encompassed by the following claims . amartey , s . and t . w . jeffries , &# 34 ; comparison of corn steep liquor with other nutrients in the fermentation of d - xylose by pichia stipitis cbs 6054 &# 34 ; biotechnol . lett ., 16 : 211 - 214 ( 1994 ). asghari , a ., r . j . bothast , j . b . doran , l . o . ingram , &# 34 ; ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered escherichia coli strain ko11 &# 34 ; j ind . microbiol ., 16 : 42 - 47 ( 1996 ). atkinson , b . and f . mavituna , biochemical engineering and biotechnology handbook , 2nd edn . stockton press , new york , n . y . ( 1991 ). beall , d . s ., l . o . ingram , a . ben - bassat , j . b . doran , d . e . fowler , r . g . hall and b . e . wood &# 34 ; conversion of hydrolysates of corn cobs and hulls into ethanol by recombinant escherichia coli b containing integrated genes for ethanol production &# 34 ;, biotechnol . lett ., 14 : 857 - 862 ( 1992 ). beall , d . s ., k . ohta and l . o . ingram , &# 34 ; parametric studies of ethanol production from xylose and other sugars by recombinant escherichia coli &# 34 ; biotechnol bioeng ., 38 : 296 - 303 ( 1991 ). brooks , t . a . and l . o . ingram , &# 34 ; conversion of mixed office paper to ethanol by genetically engineered klebsiella oxytoca strain p2 &# 34 ; biotechnol . progr ., 11 : 619 - 625 ( 1995 ). european brewery convention , 1987 . &# 34 ; free amino nitrogen &# 34 ; in analytica - ebc , 4th edn ., pp e141 - e142 , european brewery convention , zurich , switzerland . guimaraes , w . v ., g . l . dudey and l . o . ingram , &# 34 ; fermentation of sweet whey by ethanologenic escherichia coli &# 34 ; biotechnol bioeng ., 40 : 41 - 45 ( 1992 ). hohmann , n . and c . m . rendleman , &# 34 ; emerging technologies in ethanol production &# 34 ; us department of agriculture information bulletin number 663 , pp . 1 - 17 ( 1993 ). jones , a . m . and w . m . ingledew , &# 34 ; fermentation of very high gravity wheat mash prepared using fresh yeast autolysate &# 34 ; biores . technol ., 50 : 97 - 101 ( 1994a ). jones , a . m . and w . m . ingledew , &# 34 ; fuel alcohol production : appraisal of nitrogenous yeast foods for very high gravity wheat mash fermentation &# 34 ; process biochem ., 29 : 483 - 488 ( 1994b ). lawford , h . g . and j . d . rousseau , &# 34 ; ethanol production by recombinant escherichia coli carrying genes from zymomonas mobilis &# 34 ; appl biochem biotechnol ., 28 / 29 : 221 - 236 ( 1991 ). katzen , r . and d . e . fowler , appl . biochem . biotechnol ., 45 / 46 : 697 - 707 ( 1994 ). kollar , r ., e . sturdik and j . sajbidor , &# 34 ; complete fractionation of saccharomyces cerevisiae biomass &# 34 ;, food biotechnol ., 6 : 225 - 237 ( 1992 ). luria , s . e . and m . delbruck , &# 34 ; mutations of bacteria from virus sensitivity to virus resistances &# 34 ; genetics 28 : 491 - 511 ( 1943 ). ohta , k ., d . s . beall , j . p . mejia , k . t . shanmugam and l . o . ingram , &# 34 ; genetic improvement of escherichia coli for ethanol production : chromosomal integration of zymomonas mobilis encoding pyruvate decarboxylase and alcohol dehydrogenase ii &# 34 ; appl environ microbiol ., 57 : 893 - 900 ( 1991 ). shah , m . m . and m . cheryan , &# 34 ; acetate production by clostridium thermoaceticum in corn steep liquor media &# 34 ;, j . indust . microbiol ., 15 : 424 - 428 ( 1995 ). sheehan , j . j ., &# 34 ; bioconversion for production of renewable transportation fuels in the united states : a strategic perspective &# 34 ; in enzymatic conversion of biomass for fuels production , acs symposium series 566 ( himmel m . e ., j . o . baker and r . p . overend , eds . ), pp 1 - 53 , american chemical society , washington , d . c . 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