Detoxification of Dilute Acid Hydrolysates of Lignocellulose withLime

Alfredo Martinez,†,‡ Maria E. Rodriguez,†,‡ Melissa L.Wells,† Sean W. York,†James F. Preston,† and Lonnie O. Ingram*,†

Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science, University of Florida,Gainesville, Florida 32611, and Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico,Cuernavaca, Mor. 62250, Mexico

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis isaccompanied by the production of inhibitors that retard microbial fermentation.Treatment of hot hydrolysate with Ca(OH)2 (overliming) is an effective method fordetoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our resultsindicate that the optimal lime addition for detoxification varies and depends on theconcentration of mineral acids and organic acids in each hydrolysate. This optimumwas shown to be readily predicted on the basis of the titration of hydrolysate with 2N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average compositionof 15 hydrolysates prior to treatment was as follows (per L): 95.24 ( 7.29 g sugar, 5.3( 2.99 g acetic acid, 1.305 ( 0.288 g total furans (furfural and hydroxymethylfurfural),and 2.86 ( 0.34 g phenolic compounds. Optimal overliming resulted in a 51 ( 9%reduction of total furans, a 41 ( 6% reduction in phenolic compounds, and a 8.7 (4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarityof microorganisms, it is possible that the titration method described here may alsoprove useful for detoxification and fermentation processes using other microbialbiocatalysts.

Introduction

Hemicellulose is a renewable resource that could serveas a carbohydrate feedstock for the production of fuelethanol and other chemicals (1-5). Unlike cellulose, itis readily hydrolyzed at high yields by dilute mineralacids (1, 6, 7). During this hydrolysis, other compoundsare also produced at sufficient levels to inhibit microbialgrowth and fermentation (8-13). These inhibitors includefurfural, hydroxymethylfurfural (HMF), soluble phenoliccompounds, acetic acid, and other organic acids (14-17).No single compound or group of compounds has emergedas the dominant toxin, although furan concentrations inhydrolysates appear to correlate with toxicity (18-20).In general, the toxicity of each of these compounds forbacteria and yeast is directly related to hydrophobicity(4, 20-22).

The destruction of soluble lignin products using laccaseenzymes (23) and the chemical alteration of furfural andHMF (and other compounds) by overliming treatments(pH 9-10) with Ca(OH)2 (24-27) have each been shownto reduce the toxicity and improve the fermentability ofhydrolysates (8, 15-17, 28, 29). Overliming is generallyregarded as the best available technology for detoxifica-tion (1, 4, 7, 25). Neutralization with lime has the addedbenefit of forming a relatively insoluble salt with sulfuricacid (gypsum) and thus reduces the concentration ofsoluble salts during fermentation. In the United States,

this gypsum has many low value uses such as concrete,wallboard, soil amendment, capping of landfills, etc.Further research is also being pursued to develop etha-nologenic biocatalysts with improved resistance to thetoxins in hemicellulose hydrolysate (28, 30, 31).

Previous studies in our laboratory have focused on theoverliming process using recombinant, ethanologenicEscherichia coli as the biocatalyst (27, 32-34). For thisorganism, overliming at 60 °C was found to be moreeffective than overliming at 25 °C (27). Overliming atelevated temperatures has several advantages. Hydroly-sate would be expected to emerge from a dilute acidreactor at or near 100 °C (6). By overliming at a highertemperature, the requirement for immediate coolingwould be reduced. Less lime is required for detoxificationat 60 °C in comparison to 25 °C, and thus less acid isneeded to reduce the pH for fermentation. Treatinghydrolysate at an elevated temperature would have theadded benefit of reducing problems associated withsterility. However, optimization of overliming at 60° ismore difficult than at 25 °C. At 25 °C, overliming to pH10 is often adequate (25, 27) although somewhat tediousas a result of the need to minimize dilution of thehemicellulose syrup, the limited solubility of Ca(OH)2,mixing requirements for uniform dispersion, and aneutralization reaction that forms an insoluble product(gypsum). Adjustments of pH must be made slowly toallow stabilization of readings. At 60 °C, these problemsare further compounded by an increase in chemicalreactivity and the generation of new acidic products(4, 27).

Previous studies with hemicellulose hydrolysates in ourlaboratory have been conducted after initial fermentation

* Ph: (352) 392-8176. FAX: (352) 846-0969. E-mail:ingram@ufl.edu.

† University of Florida.‡ Universidad Nacional Autonoma de Mexico.

287Biotechnol. Prog. 2001, 17, 287−293

10.1021/bp0001720 CCC: $20.00 © 2001 American Chemical Society and American Institute of Chemical EngineersPublished on Web 02/08/2001

trials that empirically determined the optimal additionof Ca(OH)2 for each batch of hydrolysate (27, 32-34).Although this gravimetric optimization has proven quiteeffective for university work, the effort and time delayrequired for these biological assays may be prohibitivefor commercial production. Recent studies by Moniruz-zaman and Ingram (27) have shown that optimal over-liming for rice hull hemicellulose hydrolysate at 60 °Cwas approximately equal (in milliequivalents) to theamount of NaOH required to adjust hydrolysate topH 11.

In this paper, we have investigated titration with 2 NNaOH as a potential method to predict the optimalamount of Ca(OH)2 for overliming at 60 °C using 15different batches of bagasse hemicellulose hydrolysate.Near identical predictions were obtained on the basis oftitration to pH 11.0 and to pH 7.0. After overlimingtreatments based on these predictions, all hydrolysateswere readily fermented by ethanologenic E. coli LY01.Changes in hydrolysate compositions caused by overlim-ing were also examined.

Materials and Methods

Microorganisms and Media. Ethanologenic E. colistrain LY01 (31) was used in these experiments. Thisorganism can efficiently ferment all pentose and hexosesugar constituents of hemicellulose hydrolysate (4). Stockcultures were maintained at 30 °C under argon on LBmedium (per L: 5 g yeast extract, 10 g tryptone, 5 gNaCl) supplemented with 20 g/L xylose, 0.6 g/L chloram-phenicol, and 15 g/L agar. Inocula were grown for 16 hin 2-L flasks containing 720 mL of modified LB medium(50 g/L xylose, no antibiotics) at 35 °C with gyratoryagitation (120 rpm). Fermentation tests with hydroly-sates were conducted without antibiotics using corn steepliquor (CSL) as a nutrient. A 10× stock of CSL (25 g/Lon a dry solids basis) was prepared by adjusting to pH7.2 with NaOH and sterilized by autoclaving for 20 min.

Treatment of Hydrolysate with Ca(OH)2. Hydroly-sates (3 kg) were heated to 60 °C in a water bath andmixed vigorously during the rapid addition of 50-100 gof anhydrous Ca(OH)2. After 30 min of incubation,hydrolysates were cooled to ambient temperature in asecond water bath and used immediately for analyses orin fermentations. As an “untreated control”, hydrolysateswere adjusted to pH 6.5-6.7 with Ca(OH)2 at roomtemperature and supplemented with CSL immediatelybefore inoculation. (Note that Ca(OH)2 was used forneutralization of the control to prevent an increase insoluble salts that are potentially inhibitory.) After in-oculation, little ethanol was produced in these controlhydrolysates.

Fermentation. Inhibition of ethanol production dur-ing a 24-h incubation at 35 °C was used as a measure oftoxicity. Samples of treated and untreated hydrolysatewere adjusted to pH 6.5-6.7 with solid Ca(OH)2 orconcentrated H2SO4 as necessary and supplemented withsterile CSL as a nutrient. Fermentations were conductedin unbaffled 250-mL flasks containing 100 mL of brothand incubated for 24 h at 35 °C (120 rpm). Flasks wereinoculated to provide an initial cell density of 0.16 g/Lcell dry weight.

Analyses. Sugar composition was analyzed by HPLCusing a Waters HPLC system (Milford, MA) equippedwith a Biorad (Hercules, CA) Aminex HPX-87P columnand refractive index monitor (25). Mannitol was addedas an internal standard. Furfural, HMF, and total furans(furfural + HMF) were determined by HPLC using a

Biorad HPX-87H column and a variable wavelengthdetector at 278 nm (35). Total furans were also estimatedby a spectrophotometric method based on the differencein absorbance at 284 and 320 nm (36) using a BeckmanDU 640 spectrophotometer (Fullerton, CA). Acetic acidwas measured using an Aminex HPX-87H column andrefractive index monitor (27). Total soluble phenoliccompounds were estimated by a modification of thePrussian blue method (24, 25, 37) using ferulic acid as astandard and 1.6 mM K3Fe(CN)6 as the color reagent.Conductivity was measured using an Oakton modelWD35607-10 portable meter (distributed by Fisher Sci-entific, Norcross GA). Ethanol was measured using aVarian (Sugarland, TX) Star 3400 gas chromatographand a Supelco (Bellefonte, PA) column (0.2% Carbowax1500 on 80/100 Carbopack C) with normal propanol asan internal standard.

Total inorganic acids in untreated hydrolysates (pri-marily sulfuric) were estimated by titration of 100-gsamples to pH 2.5 with 2 N NaOH. Total organic acidsin untreated hydrolysates (acetic plus others) wereestimated by titration from pH 2.5 to 7.0 with 2 N NaOH.Titrations were carried out at 25 °C using a Corningmodel 540 pH meter and a conventional buret. Above pH8, 3-5 min of mixing was required for stabilization afterbase additions. For consistency, a time increment of 5min was maintained for all additions throughout eachtitration.

Materials. Fifteen different samples of bagasse hemi-cellulose hydrolysate were provided by a commercialsupplier. Small samples were provided for 13 of these (<1kg); two were provided in large amounts (nos. 13 and 15in Table 1). Hydrolysates were produced using a continu-ous reactor and dilute sulfuric acid (32-34, 27). Moredetailed information regarding processing conditions wasnot provided. On the basis of previous studies (6),conditions are assumed to be between 1% and 2% sulfuricacid at 100-160 °C for 5-30 min. Furfural, HMF, xylose,and other chemicals were obtained from the FisherScientific Company (Norcross, GA). CSL was purchasedfrom the Grain Processing Corporation (Muscatine, IA).

Results and Discussion

Composition of Bagasse Hydrolysates. Table 1summarizes the major components of 15 bagasse hy-drolysates prior to treatment with Ca(OH)2. Total sugarcontent was the least variable constituent on a percent-age basis, ranging from 80.31 g/L in no. 14 to 109.75 g/Lin no.12, and averaged 95.24 ( 7.29 g/L. All hydrolysatescontained approximately the same proportions of xylose(76.9 ( 7.6%), glucose (13.3 ( 1.4%), and arabinose (9.8( 4.5%) with small amounts of galactose and mannose(data not shown). Large variations (percentage basis)were observed for acetic acid (3.65 g/L in no. 4 to 12.68g/L in no.12), furfural (0.299 g/L in no.14 to 1.442 g/L inno. 8), HMF (0.180 g/L in no.15 to 0.698 g/L in no.13),total furans (0.656 g/L in no. 14 to 1.711 g/L in no. 8),and total organic acids (169.6 mequiv/L in no. 4 to 322.2mequiv/L in no.12). Soluble phenolic compounds averaged2.86 ( 0.34 g/L. Inorganic acid content varied from 201.0mequiv/L in no.15 to 354.0 mequiv/L in no. 9 with anaverage value of 306.0 ( 35.6 mequiv/L. Conductivity wasmeasured in these samples and varied from 38.0 mS inno. 14 to 56.8 mS in no. 9 with an average value of 50.05( 5.37 mS. The density of all samples was essentiallyconstant at 1.057 ( 0.003 g/mL.

Empirical Optimization of Overliming for theFermentation of Hydrolysate No. 13. Large amounts

288 Biotechnol. Prog., 2001, Vol. 17, No. 2

of hydrolysate no. 13 were available for testing. Todetermine the optimal overliming for this hydrolysate,samples were treated with different concentrations of Ca-(OH)2 at 60 °C (Figure 1A). After cooling, nutrientaddition, and adjustment to pH 6.7, each treated hy-drolysate was inoculated and assayed for ethanol after24 h of incubation. Excellent detoxification was achievedin samples of no.13 that had been treated with 660-780mequiv of Ca(OH)2 per kg hydrolysate as evidenced bythe production of 31.19 g/L ethanol (average volumetricproductivity of 1.3 g/Lh). However, all lime additionscaused a progressive decline in sugar content, whichcorrelated with an increase in pH after overliming(Figure 1B). A pH of 8.5 and above (after treatment andcooling) was sufficient to eliminate toxicity. Above pH 9.0,the rate of sugar loss increased dramatically. On thebasis of these results, a Ca(OH)2 treatment with 700mequiv/kg hydrolysate was selected as optimal for hy-drolysate no. 13.

Predicting the Optimal Addition of Ca(OH)2 forOverliming by Titration to pH 11.0 with 2 N NaOH.

The use of bioassays to empirically determine the opti-mum Ca(OH)2 addition for each batch of hydrolysate maynot be practical during commercial ethanol production.Faster and less labor-intensive methods would be desir-able. Titration with 2 N NaOH at ambient temperaturerepresents a potential method to estimate the optimaladdition of Ca(OH)2 for overliming at 60 °C provided areliable correlation can be established (Figure 2A). Theaddition of 700 mequiv NaOH/kg hydrolysate no. 13resulted in a pH of 10.91( 0.14. This value was roundedto pH 11.0 for convenience.

Hydrolysate no.15 was used to test the hypothesis thatthe mequiv of NaOH required to adjust the pH ofhydrolysate to pH 11.0 at ambient temperature is nu-merically equal (mequiv/kg hydrolysate) to the optimaladdition of Ca(OH)2 during overliming at 60 °C. Hydroly-sate no. 15 required 620 mequiv NaOH/kg hydrolysatefor adjustment to pH 11.0 (Table 2). Using 620 mequiv/kg as a central point, overliming treatments were con-ducted with Ca(OH)2 concentrations from 540 mequiv/kg to 700 mequiv/kg. After cooling, each was bioassayed

Table 1. Composition of Hemicellulose Hydrolysates

hydr.no.

total sugarsa

(g/L)acetic acidb

(g/L)furfuralc

(g/L)cHMF(g/L)

total furans(g/L)

phenolicsc

(g/L)inorg. acids(mequiv/L)

organic acids(mequiv/L)

bcond.(mS)

density(g/mL)

1 90.60 3.87 1.003 0.191 1.194 3.03 343.4 184.4 54.9 1.0552 93.00 3.80 1.076 0.224 1.300 2.84 292.6 188.7 50.1 1.0573 92.40 4.40 1.032 0.194 1.226 2.75 318.0 178.1 52.8 1.0554 94.00 3.65 1.037 0.159 1.196 2.84 279.8 169.6 45.9 1.0555 96.10 3.86 1.314 0.216 1.530 3.05 320.1 192.9 51.6 1.0556 93.30 3.88 1.432 0.232 1.664 2.90 330.7 190.8 53.8 1.0567 99.10 4.14 1.398 0.220 1.618 2.97 311.6 195.0 50.8 1.0578 100.90 4.26 1.442 0.269 1.711 3.06 349.8 199.3 56.0 1.0569 97.70 4.23 1.163 0.228 1.391 2.95 354.0 188.7 56.8 1.058

10 96.00 4.26 1.143 0.235 1.378 3.09 326.5 188.7 52.9 1.05711 98.55 5.25 0.796 0.209 1.005 2.89 271.4 209.9 43.8 1.05912 109.75 12.68 0.777 0.665 1.442 2.71 303.2 322.2 47.3 1.06413 103.45 12.50 0.630 0.698 1.328 3.01 265.0 315.9 43.1 1.06414 80.31 4.80 0.299 0.357 0.656 1.70 222.6 288.3 38.0 1.05615 83.50 3.96 0.749 0.180 0.929 3.12 201.0 195.0 53.0 1.053

av. 95.24 5.30 1.019 0.285 1.305 2.86 306.0 213.8 50.05 1.057SD 7.29 2.99 0.324 0.167 0.288 0.34 35.6 50.4 5.37 0.003

%SD 7.65 56.33 31.80 58.65 22.04 12.03 11.64 23.59 10.72 0.29a Average of two independent determinations. Total sugar analysis includes xylose, glucose, galactose, arabinose, and mannose. b Average

of two independent determinations. c Average of three independent determinations. In all cases standard deviation was lower than 7%.Abbreviations: HMF, hydroxymethylfurfural; cond., conductivity; hydr. no., hydrolysate number.

Figure 1. Empirical optimization of overliming at 60 °C (hydrolysate no. 13). (A) Effect of Ca(OH)2 additions on ethanol productionand sugar content. (B) Relationship between pH after overliming (measured at ambient temperature), ethanol production, and sugarcontent. Data are an average of three replicates. Error bars are shown for a single standard deviation.

Biotechnol. Prog., 2001, Vol. 17, No. 2 289

for the production of ethanol during a 24-h incubation(Figure 3). Little ethanol (<1 g/L) was produced bycontrol samples that had been adjusted with Ca(OH)2 topH 6.7 at ambient temperature. The highest level ofethanol (32.7 g/L) was produced in the hydrolysatesample that was treated at 60 °C with the predictedamount of Ca(OH)2, 620 mequiv/kg of hydrolysate. ThepH of this hydrolysate (pH 9.03) was very similar tothat of hydrolysate no. 13 treated with 700 mequiv/kg(pH 9.14).

All 15 samples of hydrolysate were used to furtherevaluate the utility of NaOH titration as a guide foroverliming (Figure 2B). Each sample was titrated todetermine the milliequivalents of 2 N NaOH required toreach pH 11.0 and then overlimed with the corresponding(mequiv) amount of Ca(OH)2 at 60 °C (Table 2). Theresulting pH range after overliming (and cooling) wasvery narrow, averaging pH 8.92 ( 0.15. Excellent ethanolproduction was observed for all overlimed samples av-eraging 32.26 ( 1.14 g/L (Table 2). Again, controls that

had been neutralized with Ca(OH)2 to pH 6.7 at ambienttemperature produced little ethanol (<1.0 g/L) (data notincluded).

Effects of Overliming at 60 °C on HydrolysateComposition. Compositional changes after overlimingoffers a potential method to evaluate the reliability ofCa(OH)2 additions based on titration. All 15 overlimedhydrolysates exhibited the same trends despite differ-ences in the amount of added Ca(OH)2 (Table 3). Totalfurans were reduced by 51 ( 9%. Soluble phenoliccompounds were reduced by 41 ( 6%. Presumably, thesefurans and phenolic compounds are converted to less toxicproducts by overliming. Total sugars were reduced by8.74 ( 4.46%. Conductivity was reduced by 84.94 (3.63%. As previously observed (27), acetic acid contentwas not affected by overliming at 60 °C.

It is interesting to note that excellent ethanol produc-tion was observed in all hydrolysates after overliming,even though significant levels of furfural and phenoliccompounds remained. In general, the highest residual

Figure 2. Titration of hydrolysates. (A) Hydrolysate no. 13 titrated with 2 N NaOH and with solid Ca(OH)2. (B) Summary of 15hydrolysates titrated with 2 N NaOH.

Table 2. Prediction and Testing of Optimal Ca(OH)2 Additions Based on Titration of Hydrolysate with 2 N NaOHa

titration with NaOH (milliequivalents/kg hydrolysate) treatment and fermentation

hdst.no.

initialpH

inorg. acids(init. 2.5)

total acids(init. 7)

total titration(init. 11)

constant(7-11)

total acids + K(init. 7 + K)

Ca(OH)2 added(mequiv/kg)

pH aftertreatment

ethanol producedafter 24 h (g/L)

0.94 324 498 648 150 649 640 8.92 33.311 0.97 276 454 598 144 605 600 9.16 31.843 0.94 300 468 608 140 619 600 9.07 30.794 0.99 264 424 568 144 575 560 8.76 31.685 0.94 302 484 634 150 635 640 8.87 32.916 0.93 312 492 642 150 643 640 8.65 32.877 0.95 294 478 634 156 629 640 9.00 31.948 0.91 330 518 678 160 669 680 8.67 31.979 0.90 334 512 656 144 663 660 8.92 33.84

10 0.92 308 486 630 144 637 640 8.81 32.4211 0.97 256 454 600 146 605 600 8.98 33.9612 0.94 286 590 740 150 741 740 8.91 32.6613 1.00 250 548 706 158 699 700 9.14 31.1914 1.12 210 482 652 170 633 660 8.92 29.7115 1.00 284 468 628 160 619 620 9.03 32.74

av. 0.96 288.67 490.40 641.47 151.07 641.47 641.33 8.92 32.26SD 0.05 33.61 40.42 43.20 8.21 40.42 44.37 0.15 1.14

%SD 5.57 11.64 8.24 6.73 5.43 6.30 6.92 1.71 3.53a Abbreviations: hdst. no., hydrolysate number; init., initial pH; K, constant ) 151 mequiv/kg hydrolysate.

290 Biotechnol. Prog., 2001, Vol. 17, No. 2

furans and phenolic compounds were found in treatedhydrolysates that contained the highest levels prior tooverliming. The amount of residual furans and phenoliccompounds in many overlimed (fermentable) hydroly-sates was higher than the initial furan and phenoliclevels in hydrolysate no. 14. Since all 15 treated hydroly-sates were fermented well during the 24-h incubationperiod, the average concentration of each toxic componentcan be regarded as a permissible level (Table 3): furans(e0.640 g/L), phenolic compounds (e1.70 g/L), and aceticacid (e5.33 g/L). From our data, neither component canbe identified as being the dominant toxin.

Together, these results confirm the efficacy of titrationwith 2 N NaOH as a method to predict optimal Ca(OH)2additions for the overliming of bagasse hemicellulosehydrolysates. Titration to pH 11.0 and bioassays predictequal amounts of Ca(OH)2 for optimal overliming. Using15 different hydrolysates, overliming with the predictedadditions of Ca(OH)2 based on titration provided excellentdetoxification as measured by ethanol production. Alltreatments resulted in a similar final pH after treatmentand an equivalent level of chemical modification ofseveral important components (furans, soluble phenoliccompounds, and sugars).

Titration to pH 7.0 with 2 N NaOH as a Predictorof Optimal Ca(OH)2 Additions for Overliming. Ti-tration curves for all hydrolysates were extremely similarin shape, particularly above pH 7.0 (Figure 2B). This wasfurther confirmed by manual alignment of individualcurves, suggesting the possibility that titration to pH 7.0may be sufficient to predict the optimal addition of Ca-(OH)2. Titration to pH 7.0 would be particularly attractiveas a result of the steepness of response in this region ascompared to pH 11.0, and the accuracy with which pH7.0 can be determined using routine calibration methods.Unlike titration to pH 11.0, titration to pH 7.0 can beperformed rapidly with 2 N NaOH and requires minimaltime (<1 min) for pH stabilization between additions.Table 2 summarizes the amounts of NaOH required toadjust the 15 hydrolysates to various pH values. Inspec-tion of these values suggested that almost all of thevariations in the base requirements were contained inthe region below pH 7.0. This was confirmed by plottingtitration data as a function of an unrelated variable,conductivity (Figure 4). The scatter in the plot fortitration to pH 11.0 was almost identical to that fortitration to pH 7.0. A plot of the difference between these

Table 3. Composition of Hemicellulose Hydrolysates after Optimal Overliminga

hdst.no.

total sugars(g/L)

% sugarreduction

total furans(g/L)

% furanreduction

total phenolics(g/L)

% phenolicreduction

cond.(mS)

% cond.reduction

1 88.61 2.20 0.591 50.52 1.64 45.97 6.98 87.292 84.72 8.90 0.480 63.09 1.73 39.28 6.94 86.153 85.00 8.00 0.504 58.89 1.63 40.56 6.71 87.294 86.01 8.50 0.711 40.59 1.64 42.25 6.80 85.195 88.99 7.50 0.832 45.59 1.95 35.99 7.00 86.436 85.93 7.90 1.076 35.35 1.86 35.75 6.92 87.147 90.18 9.00 0.780 51.78 1.64 44.86 6.95 86.328 91.11 9.70 0.994 41.89 1.68 45.14 6.98 87.549 97.41 0.30 0.760 45.36 1.87 36.49 6.81 88.01

10 86.50 9.90 0.703 48.98 1.84 40.43 6.86 87.0311 91.80 6.8 0.469 53.33 1.65 42.68 7.10 83.7912 91.20 16.9 0.515 64.31 1.98 26.81 9.91 79.0513 87.73 15.2 0.415 68.73 1.96 34.90 9.54 77.8714 75.89 5.50 0.295 55.11 0.87 48.91 8.44 77.7915 71.14 14.8 0.469 49.50 1.59 49.05 6.75 87.26

av. 86.81 8.74 0.640 51.54 1.70 40.60 7.38 84.94SD 6.37 4.46 0.221 9.34 0.27 5.98 1.04 3.63

%SD 7.34 51.0 34.61 18.13 15.78 14.74 14.07 4.28a Abbreviations: hydr. no., hydrolysate number; cond., conductivity.

Figure 3. Evaluation of the predicted optimal addition of limeat 60 °C (hydrolysate no. 15). On the basis of titration to pH11.0 with 2 N NaOH, 620 mequiv Ca(OH)2 per kg of hydrolysatewas predicted as the optimal addition (hatched bar) for over-liming. Hydrolysate was treated with a series of Ca(OH)2concentrations above and below this predicted value. Eachtreated hydrolysate was evaluated for toxicity by measuring theproduction of ethanol after 24 h.

Figure 4. Relationship between initial conductivity and theamount of 2 N NaOH required to increase the pH of 15hydrolysates to the indicated values. Titration of hydrolysateto pH 2.5 provides a rough measure of mineral acid content.Titration to pH 7 provides an estimate of total acid content.Titration to pH 11.0 provides an estimate of the optimal Ca-(OH)2 addition for overliming. Note that the amount of NaOHrequired to increase the alkalinity of hydrolysates from pH 7.0to 11.0 is essentially constant, 151 ( 8 mequiv/kg hydrolysate.

Biotechnol. Prog., 2001, Vol. 17, No. 2 291

curves was nearly constant for all hydrolysates, 151 ( 8mequiv/kg hydrolysate. The arithmetic sum of the NaOH(mequiv/kg) required to neutralize hydrolysates to pH 7.0plus this constant (151 mequiv/kg) is within 10 mequiv/kg of the base required to titrate to pH 11.0 for allhydrolysates except no. 14 (19 mequiv/kg) (Table 2).These results indicate that titration to pH 7.0 with 2 NNaOH (plus a constant, 151 mequiv/kg) is essentiallyequivalent to titration to pH 11.0. Either approach couldbe used to predict optimal additions of Ca(OH)2 foroverliming at 60 °C.

Relationship between Conductivity and MineralAcid Content. Upon examination of the data in Table2, an excellent correlation was observed between titrationto pH 2.5 and initial conductivity (Figure 4). Titration topH 2.5 is essentially a measure of mineral acid content,primarily sulfuric acid in this case. At the low initial pHof hydrolysates (pH =1.0), sulfuric acid is the mostabundant ionized species in hemicellulose hydrolysatesmade from bagasse residues. At this low pH, the con-ductivity of a 15 g/L sulfuric acid solution is reduced bythe addition of organic acids such as acetic acid (3.45%reduction for 10 g/L acetic acid) or sugars (16.04%reduction for 100 g/L xylose). For hydrolysates of a fairlyuniform composition (sugars, acetic acid, phenolic com-pounds), sulfuric acid content can be calibrated bytitration and subsequently estimated by conductivity.

Conclusions

Titration to pH 7.0 plus a constant (151 mequiv/kghydrolysate) or titration to pH 11.0 can be used toaccurately predict the optimal addition of Ca(OH)2 mequiv/kg hydrolysate for the detoxification of bagasse hemicel-lulose hydrolysate at 60 °C. Overliming at elevatedtemperatures reduces the amount of lime required, theamount of acid required for the readjustment of pH, andminimizes problems associated with sterility.

Predicted optima based on titration and ethanologenicE. coli LY01 may be similar for other microorganisms.With some exceptions, most bacteria (16, 17, 20-22, 29)and yeast (15, 17, 19, 24) are approximately equal in theirresistance to the individual compounds present in hemi-cellulose hydrolysates. Overliming conditions at ambienttemperature (adjusted to pH 10.0 with Ca(OH)2) havebeen reported effective for both yeast (8, 11, 25, 28) andE. coli LY01 (32-34, 27). Thus the titration method fordetoxification may prove generally useful for the com-mercial production of many renewable chemicals. Titra-tion and conductivity may also be useful to monitorsulfuric acid concentrations in effluent streams fromcontinuous biomass hydrolyzers.

Acknowledgment

This study was supported by the Florida AgriculturalExperiment Station (publication no. R-07372), the U.S.Department of Agriculture National Research Initiative(98-35504-6177 and 98-35505-6976), and the U.S. De-partment of Energy (DE-FG02-96ER20222 and DE-FC36-97GO10224/A005). A.M. was partially supportedby CONACyT-Mexico.

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Accepted for publication December 22, 2000.

BP0001720

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