Wood hydrolyzate treatments for improved fermentation of wood sugars to 2,3-butanediol

Biomass 18 (1989) 31-42

Wood Hydrolyzate Treatments for Improved Fermentation of Wood Sugars to 2,3-Butanediol F. R. Frazer* & T. A. McCaskey-~ Department of Animal & Dairy Sciences, Alabama Agricultural Experiment Station, Auburn University, Alabama 36849, USA (Received 28 September 1987; revised version received 28 October 1988. accepted 2 November 1988) ABSTRA CT Acid-hydrolyzed hardwood contains compounds inhibitory to microorganisrm" that convert wood sugars to fermentation products such as fuels and chemicals. Several methods of treating acid-hydrolyzed hardwood (hydrolyzate) to reduce the levels of potential microbial inhibitors (acetate, furfural, sulfate, and phenolics) were evaluated. The methods evaluated were precipitation with calcium hydroxide, extraction with organic solvents, treatment with ion-exchange resins, adsorption resins, and activated charcoal. Treatment of the hydrolyzate with an anion exchange resin (Amberlite IRA-400) was the most effective method for removing potential inhibitors. Non-treated hydrolyzate adjusted to p H 6 inhibited growth of a 2,3-butanediol-producing culture of Klebsiella pneumoniae. However, hydrolyzate treated with Amberlite IRA-400 was not inhibitory and resulted in yieMs of 2, 3-butanediol that were greater than 90% of theoretical. Key words: acid-hydrolyzed hardwood, wood hydrolyzate treatments, microbial inhibitors, 2,3-butanediol. INTRODUCTION Most micro-organisms that have been evaluated for production of fuels and chemicals f r o m acid-hydrolyzed h a r d w o o d (hydrolyzate) show lower productivity in hydrolyzate than in refined c a r b o h y d r a t e substrates. T h e apparent reason is inhibitory c o m p o u n d s in the hydrolyzate that are released or f o r m e d during w o o d hydrolysis, z Four *Present address: Southern Research Institute, PO Box 55305, Birmingham, Alabama 35255-5305, USA. +To whom correspondence should be addressed. 31 Biomass 0144-4565/89/S03.50 - © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain

32

t~ R. Frazer, T. A. McCaskev

groups of microbial inhibitors have been identified in wood hydrolyzate. 2 One group consists of inhibitors derived from the metals or minerals in wood, soil, or hydrolysis equipment. Another consists of carbohydrate decomposition products including furfural, hydroxymethylfurfural, and acetic acid. Other potential inhibitors are lignin degradation products and compounds derived from wood extractives, which are composed primarily of phenolic compounds. Decomposition of the hemicellulose fraction of wood results in production of furfural and acetic acid. During acid hydrolysis, a portion of the xylose in the hemicellulose molecule is degraded to furfural. The release of acetyl groups that are attached to the molecule results in formation of acetic acid. Although the lignin fraction of wood is resistant to acid hydrolysis, some decomposition does occur, releasing a variety of monomeric and polymeric phenolic compounds. Phenolic compounds that have been identified in hardwood hydrolyzate include vanillin, coniferyl aldehyde, syringaldehyde, vanillic acid, and syringic acid? The extractive portion of wood is degraded during acid hydrolysis to a variety of compounds including terpenes, alcohols, and aromatic compounds such as tannins. The residual sulfate in wood hydrolyzate is derived from the sulfuric acid used for hydrolysis. Treatment of wood hydrolyzate to remove compounds inhibitory to micro-organisms has been shown to improve the bioconversion of wood sugars in acid-hydrolyzed hardwood to ethanol and other chemicals. 4~5 First stage sulfuric-acid hydrolyzed hardwood (0"5% H2SO a, 190°C for 3 rain) contains furfural, phenolics and acetic acid at concentrations approximating 2.1, 4.6 and 12.0 mg ml-~, respectively.6 More rigorous hydrolysis conditions can result in higher levels of inhibitors. Studies with Pachysolen tannophilus grown in a minimal medium broth supplemented with various levels of these compounds showed that the yeast was killed by 2"0 mg ml -~ furfural, 2.5 mg ml -t of syringaldehyde-vanillin (2:1 mix), or by 5 mg ml-~ acetic acid. ~ Treatment of first stage acid-hydrolyzed hardwood with calcium hydroxide or with a strong anionic exchange resin reduced the concentration of these compounds and improved growth and ethanol production by P. tannophilus, v Phenolics such as isoeugenol, syringaldehyde and furulic acid are inhibitory to Saccharornyces cerev&iae at concentrations of 0.4 mg ml-1 or more. 8 In addition to yeasts, wood-derived inhibitors also suppress the activities of bacteria. The theoretical yield of 2,3-butanediol (BDO) by Klebsiella pneurnoniae l-D3 was reduced by approximately 25%, and the fermentation time was more than doubled with 0.19 mg ml-~ vanillin or 0-09 mg ml-J syringaldehyde in the fermentation sub-

Wood hydrolyzate treatments

33

strate. 9 A 10% decline in the production of BDO and acetylmethylcarbinol was reported for K. pneumoniae grown in medium containing 0"5 mg ml-~ hydroxymethylfurfural, 0.1 mg ml-~ furfural, 0"1 mg mlsyringaldehyde or 0.3 mg ml- ~of vanillin. ~0 Studies reported earlier showed that treatment with sodium hydroxide, calcium hydroxide or with a strong anionic exchange resin (Amberlite IRA-400) removed some of the microbial inhibitory compounds from acid-hydrolyzed hardwood. 7 This study investigated several other treatments of hydrolyzate including calcium hydroxide, three types of ionic or adsorption resins, five organic solvents, activated charcoal, and combinations of a few of the treatments. The hydrolyzate was analyzed before and after treatment to determine the effects of the treatment on levels of selected potential microbial inhibitors. For this study acetate, furfural, and phenolic compounds were used as representatives of wood-derived inhibitors. The effect of the treatments on sulfate levels in the hydrolyzate was monitored also, since its presence in sulfuric acid-hydrolyzed wood might be excessive and therefore inhibitory to microbes. Samples of untreated and treated hydrolyzate were inoculated and fermented to determine the effect of the treatments on culture growth and on 2,3-butanediol production.

MATERIALS AND METHODS Hardwood hydrolyzate used in this study was prepared by Stage I digestion of oakwood chips by the Cederquist method. ~l Dried oakwood chips were impregnated with 0.5% sulfuric acid and heated to 190°C with 12 kg cm-2 steam pressure for 3 min. Solubles were leached from the chips with a 10:1 (water to solids) wash with distilled water. The hydrolyzate contained 4"0% (v/w) total carbohydrate, which was greater than 85% xylose. Treatment with calcium hydroxide Powdered calcium hydroxide was added to the hydrolyzate until the pH reached 10-11. The hydrolyzate was then centrifuged at 17 000 g for 10 min to remove insolubles. Extraction with organic solvents Solvent extraction of the hydrolyzate was evaluated alone and in combination with calcium hydroxide precipitation. The solvents evaluated

34

1=. R. brazer, T. A. McCaskey

were trichloroethylene, benzene, ethyl acetate, chloroform, and hexane. Prior to extraction, raw hydrolyzate (pH 1-2) was adjusted to pH 5"0-5"5 with sodium hydroxide pellets or a saturated sodium hydroxide solution. The hydrolyzate was then centrifuged at 17 000 g for 10 min to remove insolubles. Calcium hydroxide-treated hydrolyzate (pH 10-11) was adjusted to pH 5.0-5.5 with concentrated hydrochloric acid. A pH of 5.0-5"5 was selected because it has been reported that solvent extraction of hydrolyzate is most effective when the pH is in the acid range. 5 The hydrolyzate was combined with solvent (3: 1, volume basis) and extracted by agitation for 5 min. The mixture was then transferred to a separatory funnel and after the organic and aqueous phases separated, the aqueous phase (hydrolyzate) was recovered. The pH of the treated hydrolyzate was then determined.

Treatment with ion-exchange and adsorption resins These treatments were evaluated alone and in combination with calcium hydroxide precipitation. The resins evaluated were strongly acidic cation-exchange resin (Amberlite IR-120 Plus), strongly basic anionexchange resin (Amberlite IRA-400), and non-ionic polymeric-adsorption resin (Amberlite XAD-4). All resins were purchased from Mallinckrodt, Inc., Paris, KY. Before treatment with strongly basic anion-exchange resin, the hydrolyzate was adjusted with sodium hydroxide pellets to pH 10, while the hydrolyzates to be treated with strongly acidic cation-exchange resin or adsorption resin were respectively adjusted to pH 6 and 5. The hydrolyzate and resin were combined (4 : 1, v/w) and stirred for 1 h. The resin was then removed by filtration.

Treatment with activated charcoal Non-treated and calcium hydroxide-treated hydrolyzate was treated with activated charcoal (6/14 mesh, Fisher Scientific Co., Fair Lawn, N J) in a manner similar to that used to treat the hydrolyzate with resins. Prior to treatment the hydrolyzate was adjusted to pH 5-6 with sodium hydroxide pellets.

Analyses Samples of non-treated and treated hydrolyzate were analyzed to determine the effect of the treatments on the levels of acetate, sulfate, furfural, and phenolic compounds. The level of acetate was determined using a Varian Model 1400 gas chromatograph equipped with a Model 8000

Wood hydrolyzate treatments

35

auto sampler, Model CSD III integrator, and Model 9176 recorder. The column packing material was Chromosorb 101, 80/100 mesh. The level of phenolic compounds was determined with a colorimetric procedure based on the reaction of tannin or lignin with tungstophosphoric and molybdophosphoric acids to produce a blue color. ~2 The results obtained depend on the compound used as the standard. Two compounds suspected to be present in hardwood hydrolyzate were used: syringaldehyde (monomeric standard) and tannic acid (polymeric standard). The total phenolic concentrations are reported as 'syringaldehydelike' or 'tannin-like' compounds. The sulfate concentration was determined using a turbidimetric method based on the reaction of sulfate with barium chloride in an acid (HCI) medium to form barium sulfate. ~ Furfural concentrations were determined using a colorimetric procedure based on the reaction of aniline and acetic acid with furfural. ~3 Sugar levels were determined by the reducing sugar method of Shaffer and Somogyi. ~4

2,3-Butanediol (BDO) fermentation Fermentation of sugars in acid-hydrolyzed hardwood to BDO was accomplished with a culture of Klebsiella pneumoniae designated culture l-D3 that was isolated from rotting sawdust. In a synthetic (minimal) medium, the culture produced BDO yields greater than 0.4 mg ml-~ of D-xylose. In addition to BDO the bacterium produced small amounts of ethanol, acetic acid, and acetoin. BDO production by the culture has been reported previouslyY.~-~ Prior to fermentation the culture was serially transferred in minimal medium (MM) broth which contained the following ingredients (g liter-~): 10 g D-xylose, 4 g ammonium phosphate dibasic, 1 g potassium phosphate monobasic, 1 g sodium chloride, 1 g sodium phosphate dibasic, 0.2 g magnesium sulfate, and 1-5 g yeast extract. Before the wood hydrolyzate was fermented, it was supplemented with the MM ingredients in the proportions listed above. The supplemented hydrolyzate was adjusted to pH 6"0-6-3 and centrifuged at 17 000 g for 10 min to remove insolubles. Sterilization was accomplished by passing the hydrolyzate through a Selas porcelain filter (Osmonics, Inc. Minnetonka, MN) with a nominal pore size of 0-3 #m. The culture was acclimated to wood hydrolyzate by adding small amounts of hydrolyzate to the MM broth used to grow inoculum for the wood hydrolyzate fermentation studies. To quantitate improvements made through culture acclimation, MM broth + 1% D-xylose with 5 or 10% (v/v) wood hydrolyzate was inoculated with low levels of an unacclimated culture. They were incubated until each contained about

36

F. R. Frazer, T. A. McCaskev

the same number of viable cells. The cells were harvested by centrifugation and resuspended in calcium hydroxide-treated wood hydrolyzate. These hydrolyzates were fermented for 120 h and monitored daily for culture growth, BDO production, and sugar utilization. All fermentation studies were conducted with 100-ml quantities of hydrolyzate or MM broth in 250-ml Erlenmeyer flasks capped with 38-mm Morton closures. The flasks were incubated in a water shaker bath at 100 rpm and 32°C. The fermentation medium was maintained at pH 6"0-6"3 by adjusting twice daily with concentrated hydrochloric acid or saturated sodium hydroxide solution. Inoculation levels were calculated on a per cent v/v basis, i.e. inoculation of 100 ml of minimal medium or hydrolyzate with cells harvested from 50 ml of minimal medium constituted a 50% inoculum. The amount of BDO produced was quantitated by gas chromatography. The column packing material was Porapak Q, 50/80 mesh. During fermentation culture growth was measured by the pour plate technique using plate count agar (Difco) as the plating medium. Sugar utilization was monitored by the reducing-sugar method of Shaffer and Somogyi. ~4 The stoichiometric yield of BDO achievable from xylose is 0.5 mg mg-~ of xylose consumed. Total reducing sugar concentrations were reported as xylose, which was the only sugar used in the MM broth and which accounted for greater than 85% of the total sugar in wood hydrolyzate.

RESULTS AND DISCUSSION Treatments

The effects of the treatments on inhibitor concentrations in wood hydrolyzate are summarized in Table 1. None of the treatments except activated charcoal, alone and in combination with calcium hydroxide, had an effect on the sugar level in the hydrolyzate. Both treatments reduced the sugar level from 40 to 33 mg ml -~. A number of treatments removed acetate from the hydrolyzate. However, the most effective treatments removed only about 27% of the acetate present initially. During extraction with organic solvents the hydrolyzate pH changed only slightly; the hydrolyzates were at pH 5"0-5"5 after extraction. The removal of acetate or any of the other inhibitors by organic solvent extraction is dependent on their preferential solubility in the organic

37

Wood hydrolyzate treatments

TABLE 1 Effects of Hydrolyzate Treatments on Levels of Selected Microbial lnhibitors Treatment

Inhibitor Ong ml ~) Phenolics"

Ca(OH:) + IR- 120 Plus resin IR-120 Plus resin Ca(OH)e+IRA-400 resin IRA-400 resin Ca(OH)2 + adsorption resin Adsorption resin Ca(OH)2 + act. charcoal Act. charcoal Ca(OH2) + ethyl acetate Ethylacetate Ca(OH)2 + chloroform Chloroform Ca(OH)2+TCE' TCE Ca(OH)2+benzene Benzene Ca(OH)z+hexane Hexane Ca(OH)2 Non-treated

A cetate

Furjhral

SulJate

Monorneric

Polymeric

11.(I 11.0 8.(/(27) 8.0(27) 11.0 11.0 2.{)(27) 8.{)(27) 11.11 11.1) 8.0(27) 9.1)(18) 8.0(27) 8.0(27) 8.{)(27) 8'/)(27) 2'0(27) 10-0 (9) 11.0 114)

1.2 1.2 (/.7(42) (/.7(42) 1'2 1.2 1.2 1-1 (9) /).4(67) 0.2(83) 1.2 1.2 /).8(33) /)-9(75) 0.8(33) 1/'8(33) 0.2(83) t).9(75) 1.2 1-2

l.(1 1.(I 0.2(80) 0.2(80) 1.0 1.0 /).1(9{)) 0.1(90) 1.0 1.1) 1.1) 1.1) 0.8(20) 0.8(20) 1.1) 0-8(20) /).8(20) //.9(10) 1.0 1.1)

2.5(44)/' 4.2 (7) 1.8(60) 2'1(53) 0"6(87) 1.3(71 ) {).7(84) 1.2(73) 2.2(38) 3"5(22) 2"9(35) 4.1 (9) 3.5(22) 4.1 (9) 3.1(31) 4-2 (7) 3.4(25) 4.5 3.4(25) 4.5

1.4(46) 2.(1(23) 1.0161) 1'2(54) 0.4! 85) (/.7(73) t).4(85) {).8(69) 1.6(38) 2.0(23) 1.7(35) 2.3( 11 ) 2./)(23) 2.4 (8) 1,8(31) 2.4 (8) 2.0(23) 2.6 1.9(27) 2.6

"Syringaldehyde was used as the monomeric phenolic standard; tannic acid was used as the polymeric phenolic standard. J'Values in parentheses represent percent removal as compared to nontreated hydrolyzate. ' Trichloroethylene.

rather than aqueous (hydrolyzate) phase. In the case of acetate, the solubility is greatly affected by the medium pH. If the hydrolyzate pH had been lower, pH 2 - 3 instead of 5.0-5.5, more of the acetate might have been extracted because the protonated form is probably more soluble in an organic medium than the non-protonated form. The hydrolyzate treated with the IRA-400 resin was at pH 10 prior to treatment where the non-protonated (negatively charged) form of acetate predominates and can be bound by the resin. Extraction of hydrolyzate with ethyl acetate alone or with hexane in combination with calcium hydroxide were the most effective treatments

38

F. R. Frazer, T. A. McCaskey

for removing furfural. Both treatments reduced the fuffural level by 83%. It should be noted that calcium hydroxide treatment alone did not reduce the furfural level. It is possible that the fuffural content of the ethyl acetate- and hexane-treated hydrolyzates is about the same. The discrepancies could be due to the accuracy of the furfural assay. Several treatments reduced the sulfate level. The most effective were calcium hydroxide and activated charcoal treatment or activated charcoal alone. Both treatments decreased the sulfate level by 90%. Syringaldehyde and tannic acid are representatives of lignin and wood extractive decomposition products. All of the treatments in Table 1 except hexane removed a portion of the phenolics. Calcium hydroxide was the simplest and most economical of the treatments evaluated. This treatment resulted in a precipitation of 25-27% of the phenolics but the levels of the other inhibitors remained unchanged. Treatment of the hydrolyzate with XAD-4 (adsorption) resin or activated charcoal alone or in combination with calcium hydroxide were the most effective methods for removing phenolics. When used alone, both materials reduced the level of phenolics by 69-73%, and when the treatments were coupled with calcium hydroxide precipitation, the concentration was reduced by 84-87%. The XAD-4 resin has been reported previously to remove phenolic compounds from solutions. ~' Cation and anion exchange resins also have been reported to effectively remove phenolics from solutions. ~7 The removal is thought to be due to ionic bonding to charged chemical groups or by physical adsorption to non-polar regions of the molecule. Phenolic compounds were the only inhibitors removed by the cation exchange resin. The hydrolyzate was at pH 6 before treatment with this resin. While the resin is reported to be effective at this pH, it might have removed more inhibitors if the medium pH had been lower where cationic species are more predominant.

Fermentation

A preliminary fermentation was conducted to evaluate culture growth and BDO production by culture l-D3 in wood hydrolyzate. Non-treated hydrolyzate was inoculated at the 100% level (approximately 1.3 x 109 viable cells ml -~) with an unacclimated culture. The culture did not survive for more than 24 h and produced only 7% of theoretical yield of BDO. A similar fermentation was conducted with hydrolyzate treated with calcium hydroxide. Initially, cell number declined, but by the end of the fermentation (72 h), viable cells were approximately the same level as

3'3 3"4 3"5

0~

9"5 9"5 9"5

48 9-4 9"4 9"3

Ok 8'6 9"3 9"2

24

Log cell number relative to time (h)

8-7 9"4 9'3

72 7"3 9'4 9"1

120 11 31 31

Carbohydrate utilized' (mg m l - 1)

5-9 10"3 11"0

Conc (mg ml i)

0"54d(0"15) e 0"33 (0'26) 0"35 (0"28)

Yield (mg rag- 1 6"110)

BDO produced

"Minimal medium containing 0, 5, or 10% hydrolyzatc was inoculated and fermented for 48 h in a rotary-shaker bath ( 100 rpm, 32°C). bCells were harvested from minimal medium and resuspended in calcium hydroxide-treated hydrolyzate. 'Initial carbohydrate concentration (85% xylose) was 40-0 mg m l - 1. dyield based on the amount of carbohydrate consumed. eYield based on total carbohydrate in the substrate.

0 5 19

Hydrolyzate in minimal medium % (v/v)

TABLE 2 Effect of Culture Acclimation on Growth and 2,3-Butanediol Production by Culture l - D 3

40

F. R. Frazer, T. A. McCaskey TABLE 3

Effects of Treatments on Fermentation of Wood Hydrolyzate to 2,3-Butanediol" Hydrolyzate treatment

Ca(OH) 2+ 1R- 120 Plus resin IR-120 Plus resin Ca(OH)2 + IRA-400 resin IRA-400 resin Ca(OH)2 + adsorption resin Adsorption resin Ca(OH) 2+ act. charcoal Act. charcoal Ca(OH)2 + ethyl acetate Ethyl acetate Ca(OH), + chloroform Chloroform Ca(OH) 2+ TCE" TCE Ca(OH)2 + benzene Benzene Ca(OH)2 + hexane Hexane Ca(OH) 2 Non-treated

Carbohydrate utilized b (mg ml- t)

40.0 8.8 40.0 40-0 40.0 32.2 33.0 33'0 28.4 20.2 39.3 15.8 40.0 35.3 31.1 24'0 25' 1 15.5 27.7 8.4

BDO produced Conc. (mg ml - 9

Yield (mg mg- i CHO)

19' 1 4.3 18.7 18.7 15.6 5.7 10.9 8.7 12.0 2.9 13.7 3-6 11-9 2.3 9"8 1-3 10"5 3.4 9.2 4-0

0.48 '(0.48) '/ 0.49 (0.11 ) 0.47 (0.47) 0.47 (0.47) 0'39 (0'39) 0' 14 (0' 18) 0'33 (0'33) 0.26 (0.26) 0.30 (0.42) 0'07 (0' 14) 0.34 (0.35) 0'09 (0.23) 0.30 (0.30) 0.06 (0-07) 0.25 (0.32) 0'03 (0"04) 0.26 (0-42 ) 0-09 (0.22) 0.33 (0.23) 0.48 (0.10)

aHydrolyzate was inoculated at 50% level and fermented 120 h in rotary-shaker bath (100 rpm, 32°C). qnitial carbohydrate concentration (85% xylose) was 40.0 mg ml-t except for activated charcoal samples where the concentration was 33.0 mg ml- ~. 'Yield expressed on the basis of carbohydrate consumed. aYield expressed on the basis of total carbohydrate in the substrate. eTrichloroethylene.

the original i n o c u l u m . T h e yield o f B D O i m p r o v e d to 2 1 % o f t h e o r e t i c a l . I m p r o v e d g r o w t h a n d B D O p r o d u c t i o n in w o o d h y d r o l y z a t e was a c h i e v e d b y a c c l i m a t i n g c u l t u r e l - D 3 first to a dilute h y d r o l y z a t e substrate. Mixtures of M M broth and h a r d w o o d hydrolyzate were i n o c u l a t e d with a n a v e r a g e o f 2-5 x 103 viable cells ml-~. T h e results o f the a c c l i m a t i o n s t u d y a r e s h o w n in T a b l e 2. A f t e r 4 8 - h i n c u b a t i o n , cell n u m b e r i n c r e a s e d to 3-2 x 109 cells m l - ~ . In 1 0 0 % h y d r o l y z a t e , the cells a c c l i m a t e d in M M b r o t h with 5 o r 1 0 % a d d e d h y d r o l y z a t e s u r v i v e d b e t t e r t h a n cells g r o w n in M M b r o t h with n o h y d r o l y z a t e . P r o d u c t i o n o f B D O was also i m p r o v e d . T h e c o n c e n t r a t i o n o f B D O a c h i e v e d in

Wood hydrolyzate treatments"

41

hydrolyzate fermented with inoculum grown in MM broth with 10% hydrolyzate was almost twice the concentration achieved with inoculum grown in MM broth containing no hydrolyzate. Fermentation of treated hydrolyzates The acclimated culture grew well and used all of the available sugar in hydrolyzates treated with calcium hydroxide and ion-exchange resins (Table 3). The same response was seen with hydrolyzate treated with strongly basic anion-exchange resin only. The highest concentrations of BDO were achieved in hydrolyzate treated with calcium hydroxide and Amberlite IR-120 Plus resin, calcium hydroxide and Amberlite IRA400 resin, and Amberlite IR--120 Plus resin alone. In each of these hydrolyzates, the highest concentrations of BDO were achieved after incubation for 96 h. Highest production of BDO occurred in hydrolyzate treated with calcium hydroxide and IR-120 Plus resin where a concentration of 19-1 mg ml -~ (96% of theoretical yield) was attained. Fermentation of hydrolyzate treated with calcium hydroxide and IRA400 resin or IRA-400 resin alone produced 18,7 mg ml-~ of BDO (94% of theoretical yield). These concentrations were more than twice the concentration achieved in hydrolyzate treated with calcium hydroxide alone. The concentrations reached in hydrolyzate treated with ion-exchange resins probably represent the maximum achievable by culture l-D3 and demonstrate that the sugar in the hydrolyzate can be readily fermented to 2,3-butanediol.

REFERENCES 1. Johnson, M. C. & Harris, E. E., Acclimation of various yeasts to wood sugar. J. Am. Chem. Soc., 70 (1948) 2961-3. 2. Leonard, R. H. & Hajny, J. G., Fermentation of wood sugars to ethyl alcohol. Ind. Eng. Chem., 37 (1945) 390-95. 3. Stanek, D. A., A study of the low-molecular weight phenols formed upon the hydrolysis of Aspenwood. TAPPI, 41 (1958) 601-9. 4. Pearlman, D., Production of 2,3-butylene glycol from wood hydrolyzates. Ind. Eng. Chem., 39 (1944) 803-4. 5. Sjolander, N. D., Langlykke, A. E & Peterson, W. H., Butyl alcohol fermentation of wood sugar. Ind. Eng. Chem., 30 (1938) 1251-5. 6. McCaskey, T. A. & Martin, C. P., Components of hardwood hydrolyzate inhibitory to Pachysolen tannophilus. Proceedings TAPP1 Research and Development Conf., 1984, pp. 293-9. 7. McCaskey, T. A., Rice, M. D. & Smith, R. C., Improved fermentation of wood hydrolyzate to ethanol by removal of compounds inhibitory to

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8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

F. R. Frazer, T. A. McCaskey Pachysolen tannophilus. In Biomass Energy Development, ed. W. H. Smith. Plenum, New York, 1986, pp. 573-85. Zemek, J., Kosikova, B., Augustin, J. & Joniak, D., Antibiotic properties of lignin components. Folia Microbiol., 24 (1979)483-6. Tran, A. V. & Chambers, R. P., Production of 2,3-butanediol and methyl ethyl ketone from mannose. Proceedings TAPPI Research and Development Conf., 1984, pp. 199-209. Nishikawa, N. K., Sutcliffe, R. & Saddler, J. N., The effect of wood-derived inhibitors on 2,3-butanediol production by Klebsiella pneumoniae. Biotech. Bioeng., 31 (1988) 624-7. Cederquist, K. N., Seminar on production and use of power alcohol in Asia and the Far East, Lucknow, India, 23 October-6 November, 1952. American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 16th edn. American Public Health Association, Washington, DC, 1985. Dinsmore, H. L. & Nagy, S., Improved colorimetric determination for furfural in fruit juices. J. Assoc. Off. Analyt. Chem., 57 (1974) 332-5. Shaffer, P. A. & Somogyi, M., Copper-iodometric reagents for sugar determination. J. Biol. Chem., 100 (1932) 695-713. Veeraraghaven, S., Lee, Y. Y., Chambers, R. P. & McCaskey, T. A., Ethanolbutanediol fermentation of xylose. Enzyme Eng., 5 (1980) 171-3. Gresier, M. D. & Pietrzyk, D. J., Liquid chromatography on a porous polystyrene-divinyl benzene support -- separation of nitro- and chlorophenyls. Analyt. Chem., 48 (1973) 1348-53. How, J. S. L. & Moor, C. V., Removal of phenolic compounds from soy protein extracts using activated carbon. J. Food Sci., 47 ( 1982 ) 933-40.