effects of ca(oh)2 treatments (“overliming”) on the composition and toxicity of bagasse...

Effects of Ca(OH)2 Treatments(“Overliming”) on the Composition andToxicity of BagasseHemicellulose Hydrolysates

Alfredo Martinez,1,2 Maria E. Rodriguez,1,2 Sean W. York,1

James F. Preston,1 Lonnie O. Ingram1

1Institute of Food and Agricultural Sciences, Department of Microbiologyand Cell Science, University of Florida, PO Box 110700, Gainesville, Florida32611, USA; telephone: 352-392-8176; fax: 352-392-5922; e-mail:[email protected] de Biotecnologıa, Universidad Nacional Autonoma de Mexico,Cuernavaca, Mexico

Received 3 October 1999; accepted 23 April 2000

Abstract: Hemicellulose syrups from dilute sulfuric acidhydrolysates of hemicellulose contain inhibitors that pre-vent efficient fermentation by yeast or bacteria. It is wellknown that the toxicity of these hydrolysate syrups canbe ameliorated by optimized “overliming” with Ca(OH)2.We have investigated the optimization of overlimingtreatments for sugar cane bagasse hydrolysates (primar-ily pentose sugars) using recombinant Escherichia coliLY01 as the biocatalyst. A comparison of compositionbefore and after optimal overliming revealed a substan-tial reduction in furfural, hydroxymethylfurfural, andthree unidentified high-performance liquid chromatogra-phy (HPLC) peaks. Organic acids (acetic, formic, levulinic)were not affected. Similar changes have been reportedafter overliming of spruce hemicellulose hydrolysates(Larsson et al., 1999). Our studies further demonstratedthat the extent of furan reduction correlated with increas-ing fermentability. However, furan reduction was not thesole cause for reduced toxicity. After optimal overliming,bagasse hydrolysate was rapidly and efficiently fer-mented (>90% yield) by LY01. During these studies, titra-tion, and conductivity were found to be in excellentagreement as methods to estimate sulfuric acid content.Titration was also found to provide an estimate of totalorganic acids in hydrolysate, which agreed well with thesum of acetic, levulinic, and formic acids obtained byHPLC. Titration of acids, measurement of pH before andafter treatment, and furan analyses are proposed as rela-tively simple methods to monitor the reproducibility ofhydrolysate preparations and the effectiveness of over-liming treatments. © 2000 John Wiley & Sons, Inc. BiotechnolBioeng 69: 526–536, 2000.Keywords: ethanol; fermentation; lignin; phenolic com-pounds; furfural; hydroxymethylfurfural; lignocellulose;

biomass; hemicellulose; Escherichia coli; xylose; bag-asse; hemicellulose hydrolysate; calcium hydroxide;Ca(OH)2 treatment; overliming

INTRODUCTION

The production of renewable fuels and chemicals from lig-nocellulose using microbial catalysts offers the opportunityto reduce global dependence on fossil energy and improvethe environment (Himmel et al., 1997; Lynd, 1996; Lynd etal., 1991). Approximately 70% of the dry weight of ligno-cellulose is carbohydrate, of which 20% to 60% is hemicel-lulose and 20% to 50% is cellulose. Unlike cellulose, hemi-cellulose can be readily hydrolyzed to monomeric sugarswith dilute mineral acids at modest temperatures (Groh-mann et al., 1985). However, a complex mixture of micro-bial toxins is generated during dilute acid hydrolysis, whichincludes acetate from the deacetylation of xylan, furan de-hydration products (furfural and hydroxymethylfurfural)and aliphatic acids (formic and levulinic) from sugars, andassorted phenolic compounds from lignin (Clark andMackie, 1984; du Preez, 1994; Larsson et al., 1999a,b; Mc-Millan, 1994; Olsson and Hahn-Hagerdal, 1996; Perego etal., 1990).

The toxicities of many of the compounds in hemicellu-lose hydrolysates have been examined individually (Larssonet al., 1999b; McMillan, 1994; Nishikawa et al., 1988; Ra-natunga et al., 1997; Zaldivar and Ingram, 1999; Zaldivar etal., 1999, 2000; Zemek et al., 1979). No single compoundhas emerged as the dominant toxin, although furfural wasfound to increase the toxicity of other compounds for yeast(Boyer et al., 1992; Palmqvist et al., 1998, 1999a,b) andethanologenicE. coli (Zaldivar and Ingram, 1999; Zaldivaret al., 1999). The toxicity of these compounds for ethanolo-genicE. coli is directly related to hydrophobicity (Zaldivaret al., 1999, 2000).

Over 50 years ago, Sjolander et al. (1938), Leonard and

Correspondence to:L. O. IngramContract grant sponsors: Florida Agricultural Experiment Station; Con-

tract grant number: R-07137; Contract grant sponsor: U.S. Department ofAgriculture (National Research Initiative); Contract grant numbers: 98-35504-6177, 98-35505-6976; Contract grant sponsor: U.S. Department ofEnergy; Contract grant number: DE-FG02-96ER20222; Contract grantsponsor: CONACyT Me´xico; Contract grant number: XXL-9-29034-01

© 2000 John Wiley & Sons, Inc.

Hajny (1945), and Perlman (1944) reported that the toxicityof acid hydrolysates of lignocellulose could be mitigated bythe addition of Ca(OH)2 (“overliming”). The resulting sugarsyrup, containing gypsum and other residues, was ferment-able by yeast and bacteria. This approach continues to beused to improve the fermentability of hemicellulose hydro-lysates (Amarty and Jeffries, 1996; Beck, 1986; Ingram etal., 1998; Larsson et al., 1999a; McMillan, 1994).

Many studies have investigated the components of hy-drolysates (Fenske et al., 1998; Larsson et al., 1999a; Nishi-kawa et al., 1988; Palmqvist, 1998; Ranatunga et al., 1997)and alternative methods to reduce toxicity (Buchert et al.,1990; Frazer and McCaskey, 1989; Jonsson et al., 1998). Arecent comparison of six different methods confirmed thatthe addition of Ca(OH)2 was the most effective detoxifica-tion treatment for spruce (softwood) hydrolysate using yeastas the biocatalyst (Larsson et al., 1999a). Although the hy-drolysate investigated contained only low levels of sugars(32 g/L, primarily hexose sugars) and presumably lowerlevels of toxins than may be expected in more concentratedhemicellulose syrups, excellent analytical studies were re-ported that examined the changes in chemical compositionafter detoxification. Overliming to pH 10 with Ca(OH)2 atambient temperature for 1 h reduced the concentrations offurfural, hydroxymethylfurfural (HMF), and phenolics byapproximately 20%, but did not alter the concentrations oforganic acids. No similar studies have been reported forpentose-rich hemicellulose hydrolysates (hardwoods, rota-tional crop residues) or using the pentose-fermenting re-combinantEscherichia coli.

In this article, we have systematically investigated theeffects of different Ca(OH)2 concentrations at 25°C and60°C on the fermentability of a bagasse hemicellulose hy-drolysate that contains high levels of sugars (100 g/L, pri-marily pentose sugars) usingE. coli LY01 as the biocata-lyst. In contrast to the experiments with spruce hydrolysate(Larsson et al., 1999a), which used a large inoculum (2.0 gcell dry wt/L) and measured ethanol production from hex-ose sugars after 6 h of incubation, our experiments em-ployed a low inoculum (165 mg cell dry weight/L) and 24-hincubations. In our experiments, effective ethanol produc-tion requires both the growth of the biocatalyst and themetabolism of pentose sugars. Changes in hydrolysate prop-erties and composition were correlated with differences inethanol production byE. coli LY01. Our results demon-strated a dose-dependent reduction of furans (furfural andHMF) by Ca(OH)2 for incremental additions above pH 7.5,and a reduction in lignin products. Treatments at 60°C re-quired less Ca(OH)2 and were more effective in reducingthe toxicity and furan concentrations than treatments at25°C. However, supplements that restored the furan contentin treated hydrolysate to original levels did not restore tox-icity. Furans were estimated to contribute one third of thetoxicity in bagasse hemicellulose hydrolysate, the balancebeing attributed to unidentified compounds.

MATERIALS AND METHODS

Microorganisms and Media

E. coli strain LY01 (Yomano et al., 1998) was used in allexperiments. This organism contains chromosomally inte-grated genes encoding the ethanol pathway fromZymomo-nas mobilisand produces ethanol as the dominant fermen-tation product. Cultures were grown in Luria broth (LB)containing 5 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl,and 50 to 110 g/L xylose. Stock cultures were maintained onsolid medium. For solid medium, xylose was reduced to 20g/L; agar (15 g/L) and chloramphenicol (600 mg/L) werealso added.

Hemicellulose Hydrolysate

Dilute acid hydrolysate of sugar cane bagasse was providedby a commercial supplier. This syrup was prepared usingdilute sulfuric acid essentially as previously described (As-ghari et al., 1996; Beall et al., 1992) based on the optimi-zation studies of Grohmann et al. (1985). Hydrolysate wasstored at ambient temperature in 55-gallon drums. Hydro-lysate contained 75.7 g/L of xylose, 13.5 g/L arabinose plusmannose, and 13.2 g/L of glucose.

Fermentation

The bioconversion of sugars to ethanol by LY01 was usedas a measure of toxicity. Samples of treated and untreatedhydrolysate were adjusted to pH 7.5 (unless indicated oth-erwise) with solid Ca(OH)2 or concentrated (36N) H2SO4 tominimize dilution. Neutralized hydrolysate was not steril-ized. Sterile corn steep liquor was added at a final concen-tration of 25 g/L on a dry-solids basis. Corn steep liquor(50% w/w dry solids) was adjusted to pH 7.5 and dilutedwith water to a volume equivalent to twice its weight (10×stock; 25% w/v) prior to steam sterilization. Addition ofcorn steep liquor stock (10% v/v) and 5% v/v inocula re-sulted in a 15% v/v dilution of hydrolysate.

For investigation of hydrolysate toxicity by dilution withLB containing xylose, inocula were centrifuged and the pel-lets resuspended directly in fermentation broth to avoid di-lution. Two different nutrients were tested. Corn steep li-quor (as noted previously) and laboratory nutrients (10 g/Ltryptone and 5 g/L yeast extract). Laboratory nutrients wereadded as a dry powder without sterilization.

Stock inocula of LY01 were prepared by growing cul-tures to 10 OD550 nm(3.3 g cell dry weight/L) in unbaffled2-L flasks (720 mL LB containing 50 g/L xylose) at 35°C(120 rpm). This stock was diluted 20-fold as an inoculum(165 mg cell dry weight/L after dilution). Fermentationswere conducted in unbaffled 250-mL flasks containing 100mL of broth and incubated for 48 h at 35°C (120 rpm).Ethanol was measured after 24 h and 48 h using gas chro-matography (Asghari et al., 1996).

MARTINEZ ET AL.: EFFECTS OF “OVERLIMING” ON HYDROLYSATES 527

Treatment of Hydrolysate with Ca(OH)2

Hydrolysate (3 kg) was held at either 25°C or 60°C in atemperature-controlled water bath and mixed vigorouslyduring the rapid addition of 55 to 91 g of anhydrousCa(OH)2. After 30 min of incubation, hydrolysate wascooled to ambient temperature in a second water bath andused immediately for analyses or in fermentations withoutfurther storage. For maximum accuracy, all Ca(OH)2 treat-ments were done on a weight basis. Approximately 80% ofthe treated hydrolysate was decanted for use in fermentationexperiments. Residual suspended particulate was not re-moved.

Effect of High pH on Putative Toxins

The stabilities of selected compounds were tested duringCa(OH)2 treatment. Solutions containing 0.25 g/L of eachwere prepared in synthetic hydrolysate (14.5 g/L H2SO4,11.4 g/L acetic acid, 70 g/L xylose, and 30 g/L glucose).Each was treated with 18.8 g of Ca(OH)2/kg at either 25°Cor 60°C (approximately pH 10) using a temperature-controlled water bath. Controls were held at the respectivetemperature without base addition. After 30 min of incuba-tion, samples were cooled to ambient temperature in a sec-ond water bath. For some compounds, analogous tests wereconducted using NaOH instead of Ca(OH)2.

Chemical modification of individual components wasmonitored by measuring changes in the UV-visible spec-trum using a Beckman DU 640 spectrophotometer (Fuller-ton, CA). A decline in absorbance of the principle peak wasinterpreted as evidence of chemical modification or destruc-tion. Due to complex spectral changes that occurred uponheating to 60°C prior to the addition of Ca(OH)2, base-dependent destruction or modification of syringic acid wasquantified only at 25°C. Results are expressed as a percent-age of absorbance prior to treatment. Linearity of responsefor dilutions was established for all chemicals tested.Samples were diluted 25-fold with synthetic hydrolysatebefore analysis to minimize differences in pH.

HPLC Analyses

Sugar composition was analyzed by high-performance liq-uid chromatography (HPLC) using a Waters HPLC system(Milford, MA) and refractive index monitor. Separationswere performed on a Biorad (Hercules, CA) Aminex HPX-87P column (300 × 7.8 mm i.d.) at 85°C using pure water asthe mobile phase (0.5 mL/min). Prior to sugar analysis,samples were neutralized with CaCO3 or Ca(OH)2 and clari-fied by centrifugation and filtration.

Acetic acid was determined using an Aminex HPX-87Hcolumn (60°C) with 0.01N sulfuric acid as the mobile phase(0.5 mL/min). Furfural, hydroxymethylfurfural (HMF),phenolic compounds (in order of elution: 4-methylcatechol,vanillyl alcohol, hydroquinone,p-hydroxybenzoic acid, va-nillic acid, syringic acid, guaiacol,p-hydroxybenzaldehyde,

vanillin, syringaldehyde, and ferulic acid) were analyzed at55°C using the same column (Yuan and Chen, 1999). Totalfurans is defined as the arithmetic sum of furfural and HMF.Total soluble phenolics is defined as the sum of peaks elut-ing in the regions corresponding to the aforementionedcompounds and does not include phenolics that may remainbound to the column. These compounds were separated us-ing a mobile phase consisting of 0.01N sulfuric acid (84%v/v) and acetonitrile (16% v/v) at an elution rate of 0.35mL/min. Levulinic and formic acids were analyzed at 65°Cwith 0.018N sulfuric acid as the mobile phase at an elutionrate of 0.7 mL/min. All peaks were quantified using a vari-able-wavelength UV detector.

Identifications were made by comparing retention timesto standards and by comparing changes in peak heights atdifferent wavelengths (210 to 300 nm). Changes in peakheights were compared with UV spectra of pure compoundsto aid in identification.

Estimation of Acid Content by Titration

The amount of total mineral acids and total organic acids inhydrolysate was estimated by titration with 2N NaOH usinga Corning Model 350 pH meter. Total mineral acid (inmilliequivalents [mEq]) was estimated as the base requiredto increase the pH from the initial value to pH 2.5; totalorganic acid (mEq) was estimated as the base required toincrease from pH 2.5 to pH 7.

Other Analytical Methods

Conductivity was measured using an Oakton ModelWD35607-10 portable meter (distributed by Fisher Scien-tific, Norcross, GA). Refractive index was measured as Brixusing a digital palette refractometer (NSG Precision Cells,Farmingdale, NY) and converted to refractive index usingthe chart provided by the manufacturer. Viscosity was mea-sured with a Digital LVTD Wells/Brookfield Cone/PlateViscometer (Middleboro, MA) using cone no. CP-40 (60rpm, 25°C). The viscometer was calibrated with water andaqueous mixtures of glycerol.

Chemicals

Fine chemicals, laboratory media components, and otherchemicals were obtained from either Fisher Scientific orSigma. Corn steep liquor (50% dry solids) was obtainedfrom Grain Processing Corp. (Muscatine, IA).

RESULTS AND DISCUSSION

Toxicity of Bagasse Hemicellulose Hydrolysate

Hemicellulose syrup from dilute acid hydrolysis contains acomplex mixture of compounds that are toxic to bacteriaand yeasts (du Preez, 1994; Olsson and Hahn-Hagerdal,

528 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 5, SEPTEMBER 5, 2000

1996). The toxicity of toxins in bagasse hydrolysate (102g/L sugar) was examined in batch fermentations usingE.coli LY01 by diluting neutralized hydrolysate [adjusted topH 6.5 with Ca(OH)2 and supplemented with nutrients] withreagent-grade xylose (110 g/L) containing the correspond-ing nutrients (Fig. 1). A 50% (v/v) dilution was sufficient toreduce toxicity and permit partial fermentation. The highestvolumetric productivities, 1.9 g/L per hour for laboratorynutrients and 1.2 g/L per hour for corn steep liquor, wereachieved in 20% hydrolysate (fivefold dilution by volume).Higher levels of ethanol were produced using laboratorynutrients (yeast extract and tryptone) than with corn steepliquor during 24-h incubation, although similar dilutionswere required for fermentation with both nutrients. At lowconcentrations (20% v/v hydrolysate), the rate of ethanolproduction was stimulated by components in the treatedhydrolysate. Ethanol concentrations in 20% (v/v) hydroly-sate exceeded those with laboratory nutrients and xylose.This apparent stimulation by low levels of hydrolysate maybe due in part to differences in sugar composition in theabsence of toxic levels of other compounds. LY01 is knownto ferment glucose more rapidly than xylose (Yomano et al.,1998; Zaldivar et al., 1999). Glucose was present in treatedhydrolysate (12 g/L), but only xylose was present in themedia used for dilution. Broth containing 20% hydrolysateby volume would contain approximately 2.2 g glucose/L.

Dilution alone may not represent an optimal solution tothe problem of toxins in hemicellulose hydrolysate. Dilutionwithout a source of concentrated supplemental sugar wouldlimit final ethanol concentrations (0.51 g ethanol per gramof sugar) and increase the cost of product recovery.

Effects of Ca(OH)2 Additions on EthanolProduction from Hemicellulose Hydrolysate

The addition of Ca(OH)2 has been previously reported todecrease the toxicity of hemicellulose hydrolysates from

many different lignocellulosic materials (Asghari et al.,1996; Larsson et al., 1999a; Olsson and Hahn-Hagerdal,1996). Figure 2A,B confirms that this is also true for thefermentation of bagasse hydrolysate to ethanol byE. coliLY01. Treatments with Ca(OH)2 for 20 to 60 min producedsimilar results (data not shown). A treatment time of 30 minwas selected to minimize the influence of small variations inheating and cooling times. Treatments at 60°C were moreeffective than treatments at 25°C in reducing the toxicityand resulted in higher initial rates of ethanol production. At60°C (Fig. 2B), slightly smaller amounts of Ca(OH)2 wererequired for detoxification than were needed at 25°C (Fig.2A). At the lowest concentration of Ca(OH)2 (18.5 g/kg ofhydrolysate), ethanol production in hydrolysate treated at60°C was approximately twice that of hydrolysate treated at25°C. Based on ethanol production during the initial 24-hperiod, the addition of 25.9 to 30.4 g Ca(OH)2/kg of hydro-lysate was optimal for treatments at 25°C, whereas 24.4 to28.9 g Ca(OH)2/kg of hydrolysate was optimal for treat-ments at 60°C.

Ethanol concentrations after 48 h were higher than thoseobserved after 24 h. Surprisingly, substantial amounts ofethanol were produced during the second 24 h of incubationin hydrolysates treated with 18.5 to 21.5 g of Ca(OH)2,hydrolysates in which sugars were essentially unmetabo-lized during the initial 24-h period. A similar lag in ethanolproduction has been observed with LY01 in response to theaddition of furans (Zaldivar et al., 1999) and several com-pounds found in untreated hemicellulose hydrolysates(Zaldivar and Ingram, 1999; Zaldivar et al., 1999, 2000).Furfural additions have been shown to cause a dose-dependent lag in the growth and fermentation ofSaccharo-myces, which was correlated with the metabolism of thiscompound (Larsson et al., 1999b; Palmqvist et al., 1999a,b).Both organisms are known to metabolize this compound.

The highest concentrations of ethanol were produced af-ter 48 h in hydrolysates treated with Ca(OH)2 at 23 g/kg(60°C) and 24.5 g/kg (25°C). The higher ethanol valuesobtained after treatment at 25°C as compared to 60°C maybe attributed to a combination of factors. Fermentations aretypically more vigorous after optimal Ca(OH)2 treatment at60°C and are completed sooner than 48 h, potentially al-lowing higher losses from both evaporation (7.8% of re-maining ethanol lost per day based on control experiments;data not shown) or the aerobic catabolism of ethanol afterthe exhaustion of sugar. Also, HPLC analyses have shownthat more sugar may be destroyed at the higher temperature(Fig. 3B).

Effects of Ca(OH)2 Additions on pH, SugarContent, and Ethanol Yield

The pH of treated hydrolysate (measured at ambient tem-perature) increased with the amount of added Ca(OH)2 asexpected (Fig. 3A). However, the measured value differedwith the temperature of treatment. Final pH values aftertreatments at 25°C were up to 1.0 pH unit higher than valuesafter treatments at 60°C. This difference in pH may be

Figure 1. Fermentation of neutralized hydrolysate after dilution usingcorn steep liquor (CSL) and Luria broth as nutrients. Ethanol was measuredafter 24 h of incubation.


attributed to increased chemical reactivity and the genera-tion of additional acidic products at the higher temperature.An increase in Ca(OH)2 was accompanied by a decline insugar content as measured by HPLC, which was particularlysignificant at 60°C (Fig. 3B). Pentose sugars are less stablethan hexose sugars and the observed decline resulted pri-marily from a reduction in xylose, the most abundant sugar.The decline in ethanol production (48 h; Fig. 2B) in hydro-lysates treated with increasing concentrations of Ca(OH)2

appears to result in part from sugar destruction as observedpreviously (Amarty and Jeffries, 1996; McMillan, 1994).

Fermentation yields were calculated after 24 h and 48 hbased on HPLC analysis of total sugars at the start of fer-mentation (Fig. 2C). The highest ethanol concentrations(39.3 and 38.1 g/L) were obtained after 48 h in hydrolysatestreated with Ca(OH)2 at 25°C and 60°C, respectively.Greater than 90% of the theoretical yield (0.51 g of ethanolper gram of sugar) was obtained after treatments at 25°Cwith 24.4 to 30.4 g of Ca(OH)2/kg of hydrolysate, and at60°C with 23.0 to 28.9 g of Ca(OH)2/kg of hydrolysate. Thealkalinity of these fermentable hydrolysates ranged from pH9.4 to pH 10.7 after treatments at 25°C, and from pH 7.5 topH 9.8 after treatments at 60°C (measured after cooling to25°C).

Although yields based on sugar analyses remained highover these broad ranges, sugar losses due to base destructionreduced the final ethanol concentrations in treatments abovepH 10.0 at 25°C or pH 9.0 at 60°C.

Ethanol yields with hydrolysates were higher than ex-pected based on our prior experience using flask fermenta-tions and laboratory sugars. Ethanol losses due to evapora-tion and aerobic metabolism of ethanol after sugars are ex-hausted typically prevent this high level of productaccumulation. Control experiments were conducted usingmixtures of xylose, glucose, and arabinose at concentrationspresent in hydrolysate after treatment with 25.9 g ofCa(OH)2/kg at 25°C (treated hydrolysate4; 92.8 g of sugarper liter before addition of nutrients and inocula) and at60°C (treated hydrolysate4; 85.6 g of sugar per liter beforeaddition of nutrients and inocula). These fermentations pro-

duced 33.3 ± 0.5 g and 31.3 ± 0.6 g of ethanol per liter after48 h (averages of four replicates ± 1 standard deviation),respectively, equivalent to 80.2% and 81.5% of the theoret-ical yield. Thus, HPLC measurements of sugar compositionin Ca(OH)2-treated hydrolysates appear to underestimatethe total carbohydrate available for bioconversion by ap-proximately 10%. This may be due to the fermentation ofnew products formed by the exposure of monomer sugars tohigh pH.

Effects of Ca(OH)2 on Properties and Compositionof Hemicellulose Hydrolysate

We compared the properties and composition of untreatedhydrolysate to fermentable hydrolysate that was treated at60°C (30 min) with 25.9 g of Ca(OH)2/L (Table I). Both aretan solutions with a pleasantly aromatic odor. Treatmentwith Ca(OH)2 increased the pH and viscosity, decreased theconductivity (salt content), and had no measurable effect onthe density or refractive index of the clarified hydrolysate.The increase in pH and reduction in conductivity (formationof insoluble CaSO4) were as expected. However, the basisfor the increase in viscosity is unknown. The conductivity oftreated hydrolysate (10.6 mS) was approximately equal tothat of LB (10.4 mS). The viscosity of treated hydrolysatewas approximately equal to that of 20% w/w aqueous glyc-erol at 25°C.

Total mineral acid was estimated in untreated hydrolysateby titration with 2N NaOH. The 0.25 mol of NaOH /L ofhydrolysate required to reach pH 2.5 is equivalent to a sul-furic acid concentration of 12.5 g/L. Because sulfuric acidwould be expected to represent the dominant ionized com-ponent in hydrolysate, a second estimate was made usingconductivity by diluting sulfuric acid into a solution con-taining 100 g/L sugar and 13 g/L acetic acid. Using thisapproach, the conductivity of untreated hydrolysate wasequivalent to a sulfuric acid concentration of 11.6 g/L, simi-lar to the value obtained by titration.

Total organic acids were estimated in untreated hydroly-sate by titration between pH 2.5 and 7.0 using 2N NaOH

Figure 2. Effect of Ca(OH)2 treatment on the fermentation of hemicellulose hydrolysate. (A) Treatments for 30 min at 25°C. (B) Treatments for 30 minat 60°C. (C) Ethanol yield as a percentage of the theoretical maximum based on initial sugar content. The maximum theoretical yield for monomeric sugarsis 0.51 g ethanol per gram of sugar. Error bars for (A) and (B) represent one standard deviation (three replicates).


(Table I). Previous studies have shown that acetic acid is themost abundant organic acid in hemicellulose hydrolysates(du Preez, 1994; Olsson and Hahn-Hagerdal, 1996). The0.30 mol of NaOH /L hydrolysate required to reach pH 7.0is equivalent to an acetic acid concentration of 18 g/L, con-siderably higher than that measured by HPLC (Table II).

More detailed analyses were made using HPLC (Fig. 4,Tables I and II). Total sugars in hydrolysates declined froman initial concentration of 102 g/L to 85.6 g/L after treat-ment with 25.9 g Ca(OH)2/kg at 60°C. Untreated hydroly-sate contained 75.7 g/L xylose, 13.5 g/L arabinose plusmannose, and 13.2 g/L glucose. After treatment at 60°C,hydrolysate contained 61.4 g/L xylose, 12.9 g/L arabinoseplus mannose, and 12.0 g/L glucose.

HPLC analyses confirmed that acetic acid (12.5 g/L) wasthe most abundant organic acid in bagasse hemicellulosehydrolysate, followed by levulinic acid (5.5 g/L) and formicacid (1.5 g/L). The sum of these three acids (200, 47, and 33

mEq/L, respectively) was in good agreement with the esti-mate for total organic acids by titration (300 mEq/L). Con-centrations of acetic acid, formic acid, and levulinic acidremained unchanged by treatments with Ca(OH)2 (Fig. 3B,Table II).

The concentrations of furfural and HMF were dramati-cally reduced by treatment with Ca(OH)2 (Fig. 3C). Theoptimal Ca(OH)2 treatment for fermentation reduced totalfuran (furfural + HMF) content by more than half, from1333 mg/L to 590 mg/L (Table I). A substantial reduction infurans was observed in all Ca(OH)2-treated hydrolysates,which were fermented to 90% of the theoretical yield after48 h (Figs. 2C and 3C). It is of interest to note that littlefuran destruction occurred during Ca(OH)2 treatments be-low pH 7.5 (Fig. 3C,D). Above this pH, furfural and HMFdeclined rapidly with further increases in base and pH.

Lignin-derived phenolic compounds were estimated us-ing HPLC with a 210-nm detector (Table II; Fig. 4A,B). The

Figure 3. Effect of Ca(OH)2 on pH and composition of hemicellulose hydrolysate. (A) pH of hydrolysate measured at ambient temperature after treatmentwith Ca(OH)2 at 25°C and 60°C. (B) Effect on sugar and acetic acid content. (C) Effect on furan content. Relationship between furan content and pH afteroverliming. Error bars for (B), (C) and (D) represent one standard deviation (three replicates).


complexity of this material makes specific identificationsdifficult and some of these compounds may remain boundto the analytical column. It is likely that most peaks ob-served at 210 nm represent combinations of compounds.However, differences in elution profiles do offer useful in-dications of changes caused by treatments with Ca(OH)2.

Total eluted absorbance at 210 nm declined by 17% aftertreatment of hydrolysate at 60°C with 25.9 g of Ca(OH)2/kg. No peaks were increased by treatment with Ca(OH)2

and only a few peaks declined. Absorbance at the front(unknown 1), unknown 4, and unknown 5 declined by ap-proximately 40%, 25%, and 50%, respectively. Unknown 5eluted just before HMF and at the same time as furoic acid.However, control experiments indicated that, unlike un-known 5, furoic acid was resistant to the Ca(OH)2 treat-ment. Based on the retention times of standards, three phe-nolic regions were identified and quantified using an aver-age absorbance. These included region 1, region 2, and afused peak consisting of ferulic acid and unknown 6. Thecomponents at the front (unknown 1) and unknown 5 maybe phenolic, but were not estimated. Using the sum of threephenolic regions, untreated and treated bagasse hydrolysatewas estimated to contain 700 mg of total soluble phenolicsper liter. When compared on a soluble-sugar basis (to nor-malize for different biomass concentrations during reac-tion), this value is similar to results reported by Fenske et al.(1998).

Table II. HPLC analysis of bagasse hemicellulose hydrolysates.

Compound

Retentiontimes(min)

Untreated(mg/L)

Ca(OH)2-treated

(mg/L or %change)

Unknown 1 (front) 11.3 40% declineUnknown 2 14.6 UnchangedGallic acid 16.5 Trace UnchangedUnknown 3 18.5 UnchangedLevulinic acid 21.8 ∼5500 UnchangedFormic acid 23.1 ∼1500 UnchangedAcetic acid 23.8 12,500 12,500Unknown 4 25.6 25% declineUnknown 5 29.2 50% declineHMF 33.6 749 340Region 1a (4-methylcatechol,

vanillyl alcohol, hydroquinone,p-hydroxybenzoic acid, vanillicacid, and syringic acid) 34–43 ∼70 ∼70

Furfural 46.62 584 250Region 2b (guaiacol,

p-hydroxybenzaldehyde,vanillin, syringaldehyde) 49–59 ∼45 ∼45

Ferulic acid 60.1 290 290Unknown 6 61.5 290 UnchangedTotal soluble phenolicsc 695 Unchanged

aCompounds eluting in region 1, quantified using average absorbance at210 nm.

bCompounds eluting in region 2, quantified using average absorbance at210 nm.

cSum of region 1, region 2, ferulic acid, and unknown 6.

Table I. Summary analysis of hemicellulose hydrolysate before and afterthe amelioration of toxins by treatment with Ca(OH)2.

Properties andcomposition Untreated

Ca(OH)2-treated(25.9 g/kg; 30 min, 60°C)

PropertiespH 0.98 9.28Density (g/L)a 1.06 1.06Viscosity (cP)a 1.57 2.12Conductivity (mS)a 43.3 10.6Refractive indexa 1.357 1.323

Composition (units)Total mineral acids (mEq/L) 250 Not determinedTotal organic acids (mEq/L) 300 Not determinedTotal sugar (g/L)b 102 86Acetic acid (g/L) 12.5 12.5Total furans (mg/L) 1300 590Total soluble phenolics (mg/L) 700 700

aMeasured after centrifugation to remove suspended particulate material.bTotal sugar is the arithmetic sum of xylose, glucose, arabinose, and

mannose.

Figure 4. HPLC elution profile for hemicellulose hydrolysate. (A) Be-fore treatment. (B) After treatment with Ca(OH)2 (25.9 g/kg) for 30 min at60°C. Unknown peaks are labeled from 1 (front) to 6. Letters indicateprovisional identifications: G, gallic acid; L, levulinic acid; F, formic acid;A, acetic acid; H, hydroxymethylfurfural; R1, phenolic region (see TableII); Fur, furfural; R2, phenolic region (see Table II); and Fer, ferulic acid.


Effect of pH 10 on Selected Compounds

Based on comparisons to standards, two peaks that werereduced by Ca(OH)2 treatments were provisionally identi-fied as HMF and furfural. To confirm these results andidentifications, we examined the effect of Ca(OH)2 treat-ment on individual compounds in synthetic hydrolysate(Table III). Spectra were compared immediately after 30-min incubation at 25°C and 60°C with and without theaddition of Ca(OH)2 (pH 10). Spectra were measured aftercooling to ambient temperature. All compounds tested werestable during incubation without Ca(OH)2. Consistent withthe HPLC results, most of the phenolic compounds wereunchanged by this treatment. Of the phenolic compoundstested, only one (syringic acid) was destroyed or modified.Both furfural and HMF were also destroyed or modified,consistent with results observed in Ca(OH)2-treated hydro-lysate and reported previously (Larsson et al., 1999a,b). Theextent of destruction (or modification) was higher at 60°Cthan at 25°C. It is of interest to note that destruction (ormodification) continued more slowly even at ambient tem-perature (23°C) for at least a 24-h period (data not shown).

The potential for adsorption to gypsum rather than de-struction or modification was considered. However, similarchanges in furans and syringic acid were also observed intests using NaOH to increase pH instead of Ca(OH)2. Theseresults indicate that the loss of furans and syringic acidbased on spectra is a consequence of high pH rather thanadsorption to gypsum or a specific feature of calcium. Thedecrease in absorbance could result from polymerization athigh pH or a variety of other chemical transformations.

Correlation Between Furan Contentand Fermentability

The treatment of hydrolysate with Ca(OH)2 resulted in adecline in furans and an increase in ethanol production after

24 h. The correlation between these results is summarized inFigure 5A. Very similar correlations were obtained for treat-ments at both 25°C and 60°C. Ethanol production during theinitial 24-h incubation was strongly inhibited in treated hy-drolysates containing furan concentrations of >900 mg/L.To further explore the relationship between toxicity andfuran content, we also plotted the results from the hydroly-sate dilution experiment (Fig. 1) as a function of furan con-tent (Fig. 5A). Again, ethanol production was strongly in-hibited in diluted, untreated hydrolysate containing over900 mg of furan per liter. However, 900 mg of furan perliter is lower than concentrations (3.5 to 4.0 g/L) previouslyreported to inhibit the growth and fermentation of ethanolo-genicE. coli (Zaldivar et al., 1999).

Further experiments were conducted to test the hypoth-esis that total furan content was the dominant factor thatinhibited ethanol production (Fig. 5B). Hydrolysate wastreated with 25.9 g of Ca(OH)2/kg at 60°C to mitigate toxinsand then subdivided. This treated hydrolysate contained 450mg of total furans per liter. One portion served as a positivecontrol for fermentation. Sufficient furfural, HMF, and acombination of both were added to samples of treated hy-drolysate to restore the furans destroyed or modified duringthe Ca(OH)2 treatment at 60°C. Hydrolysate neutralizedwith Ca(OH)2 at room temperature (1290 mg furan per liter)served as a control for the toxicity of untreated hydrolysate.After the addition of corn steep liquor and pH adjustment,fermentation flasks were inoculated and incubated for 48 h.After 24 h, little ethanol was produced in the untreatedhydrolysate. Treated hydrolysate supplemented with furfu-ral or HMF alone produced equivalent levels of ethanol, 26g/L. Full restoration of furan content did not restore toxicity.Treated hydrolysate supplemented with both furans pro-duced 23 g of ethanol per liter, 90% of that produced with-out added furans. After 48 h, all treated hydrolysates were

Table III. Effect of high pH on the destruction or modification (% of total) of individual selected compounds in synthetic hydrolysate.a

Compound lmax (nm)

Treatment temp. 25°C Treatment temp. 60°C

Posttreatment 0 h Posttreatment 24 h Posttreatment 0 h Posttreatment 24 h

(−) (+) (−) (+) (−) (+) (−) (+)

Furfural 278 0 6 3 39 0 55 4 66Furoic acid 254 0 0 0 0 0 0 0 05-Hydroxymethylfurfural 284 0 11 2 36 0 58 0 724-Hydroxybenzaldehyde 284 0 0 0 0 0 0 0 04-Hydroxybenzoic acid 256 0 0 0 0 0 0 0 0Syringaldehyde 305 0 0 0 0 0 0 0 0Syringic acid 272 0 74 0 82 0 NDb 0 NDb

Ferulic acid 320 0 0 2 5 0 0 0 3

aCa(OH)2 (18.8 g/L) was added to synthetic hydrolysate containing individual compounds (0.25 g/L) to raise the alkalinity to approximately pH 10 andthen the solution was incubated at 25°C or 60°C for 30 min. Control samples were also incubated without Ca(OH)2. Each solution was diluted 25-fold intosynthetic hydrolysate prior to spectral analysis. Spectra were determined immediately after treatment (0 h) and after 24-h storage at ambient temperature(24 h). Loss in absorbance at the indicated wavelength was used to calculate the extent of destruction (loss, removal, polymerization, or chemicalmodification) as a percentage of initial value. During treatments at 60°C, some color was also developed in unmodified synthetic hydrolysate treated withCa(OH)2. This absorbance was subtracted from that of treated hydrolysates during analysis: (−), incubated without addition of Ca(OH)2; (+), treated with18.8 g Ca(OH)2/L.

bND, not determined. Complex changes in the spectra of synthetic hydrolysate at 60°C during incubation without Ca(OH)2 that prevented an estimationof destruction or modification at this temperature (increased color development in the region of the initial maximum).


equivalent, regardless of furan addition, whereas the un-treated sample (neutralized) contained less than 15 g ofethanol per liter. Production of ethanol after an initial 24-hlag may be due in part to the metabolism of furans by LY01.E. coli (Zaldivar et al., 1999), andSaccharomyces(Larssonet al., 1999b; Palmqvist et al., 1999a,b) have been shownpreviously to metabolize this compound. With yeast, theduration of growth lag was directly related to the initialfuran concentration (Palmqvist et al., 1999a,b).

Higher levels of furfural and HMF were also added toadditional samples of treated hydrolysate in fermentationtests (Fig. 5A). A total furan content of approximately 3600mg/L was required to cause inhibition equivalent to theuntreated control. This value is in good agreement withminimal inhibitory concentrations determined for furfural(3.5 g/L) and HMF (4.0 g/L) in LB with LY01 (Zaldivar etal., 1999). Together, these results indicate that furans aloneare not the sole or dominant factor that determines the tox-icity of hemicellulose hydrolysates for ethanol productionby LY01, in agreement with previous studies withSaccha-romyces(Larsson et al., 1999a; Palmqvist et al., 1999a,b).Based on the relationship between furan content in treatedhydrolysate and ethanol production, the furans present inuntreated hydrolysate (1300 mg/L) are estimated to contrib-ute one third of the toxicity. Additional amelioration oftoxicity by Ca(OH)2 treatment is presumed to involve de-struction or modification of unknown compounds and mayinclude those present in unknown peaks 1, 4, and 5 (Fig.4A). With reduced levels of these unknown compounds andfurans, the organic acids (acetic, levulinic, and formic) thatwere unchanged by Ca(OH)2 treatment did not inhibit fer-mentation.

These results indicate that the furan content may serve asan excellent indicator of relative toxicity after treatment

with Ca(OH)2. Although not the dominant cause of thisinhibition, furan measurements may provide a useful way tomonitor the effectiveness and reproducibility of toxin ame-lioration for fermentations using LY01.

CONCLUSIONS

Many of our results with bagasse hemicellulose hydrolysateand ethanologenicE. coli are quite similar to those reportedby Larsson et al. (1999a) for spruce hemicellulose hydro-lysate and yeast. Although both used dilute sulfuric acid,there were large differences in feedstock composition, sugarcomposition (pentose sugars vs. hexose sugars), and experi-mental design. Considering these differences, the excellentagreement of results suggests that they may be of generalapplication.

Bagasse hydrolysate contained over 100 g/L sugar (pri-marily xylose) and spruce hydrolysate contained 32 g/Lsugar (primarily hexoses). Using a gravimetric approachrather than target pH and a low inoculum, we determinedthat pH 10 was optimal for overliming at 25°C for LY01,confirming the value reported previously for yeast fermen-tations (Larsson et al., 1999a). Furfural and hydroxymeth-ylfurfural were reduced by this treatment but not eliminated.Organic acid content was not affected.

Surprisingly, few compositional changes were observedthat correlated with the amelioration of toxins by treatmentswith Ca(OH)2. HPLC analyses revealed that Ca(OH)2 ad-dition caused a dose-dependent reduction in the levels ofboth furans (furfural and HMF) and three unknown peaks(unknowns 1, 4, and 5), whereas regions corresponding tophenolic compounds remained essentially unchanged. Al-though many phenolic compounds appear resistant to

Figure 5. Summary plot illustrating the correlation between furan content and ethanol production. (A) Summary plot of the data from the hydrolysatedilution experiment (Fig. 1), with hydrolysates treated with Ca(OH)2/kg at 25°C (Fig. 2A) and 60°C (Fig. 2B), and hydrolysate treated with 25.9 gCa(OH)2/kg at 60°C to which supplemental furans were added (after cooling). (B) Effect of replacing furans destroyed or modified by treating with 25.9g Ca(OH)2/kg at 60°C (detoxed hydrolysate) on ethanol production. Error bars represent one standard deviation (three replicates). Abbreviations: Fur,furfural; HMF, hydroxymethylfurfural. Hydrolysate that had been neutralized at ambient temperature with Ca(OH)2 served as a control to demonstratetoxicity without overliming treatment.


Ca(OH)2 treatments, some, such as syringic acid, were de-stroyed or chemically modified. Also, treatment withCa(OH)2 caused a 17% decrease in 210-nm absorbing ma-terial, which eluted from our HPLC column, indicating de-struction or modification of additional unresolved com-pounds. The reduction in furans was more extensive at 60°Cthan at 25°C. No measurable loss of furfural or HMF oc-curred in controls incubated at either temperature withoutthe addition of Ca(OH)2. Concentrations of acetic, formic,and levulinic acids were unchanged by Ca(OH)2 treatments,although partial sugar destruction was noted, particularly at60°C and with high levels of this base. The presence ofrelatively high concentrations of these organic acids in hy-drolysates after Ca(OH)2 treatment did not appear detrimen-tal to ethanol production by LY01, in agreement with pre-vious studies of individual organic acids in LB (Zaldivarand Ingram, 1999). However, the toxicity of organic acidscould be synergistic with other compounds that were ini-tially present in untreated hydrolysate.

Total furan content has been proposed as a predictivemeasure of hydrolysate fermentability by yeasts (Mande-nius et al., 1999). For ethanologenicE. coli, the decline infuran content after Ca(OH)2 treatments did not appear to bethe principal change responsible for the improvement infermentation. The addition of three times the original furanconcentration was needed to restore inhibition in Ca(OH)2-treated hydrolysate. Total furans were estimated to contrib-ute one third of the toxicity in hydrolysate with ethanolo-genicE. coli LY01 as the biocatalyst. Optimal toxin ame-lioration with Ca(OH)2 also caused a 17% reduction inelution of material with an absorbance at 210 nm, most ofwhich remains unidentified, such as unknown peaks 1, 4and 5. Remaining toxicity must be attributed to these un-knowns, unresolved compounds, and perhaps other com-pounds that were retained on the column.

The decline in total furan content, although not causallyinvolved, may serve as a useful marker to monitor toxinamelioration during the Ca(OH)2 treatment process. A de-cline in furans to approximately 0.6 g/L in treated hydroly-sate was accompanied by a progressive increase in the rateof fermentation and ethanol production. Our results alsoindicate that relatively simple methods can be used to moni-tor hemicellulose hydrolysates. With proper controls, con-ductivity and titration can be used to estimate mineral andorganic acid concentrations. After toxin amelioration bytreatment with Ca(OH)2, conductivity declined by 75% andviscosity increased. As expected, the pH was dramaticallyincreased by this treatment. The pH after treatment couldserve as a useful indicator of process reproducibility. Thus,with convenient quantitative measures, optimal titrationconditions can be employed to ameliorate toxins in hemi-cellulose hydrolysates, in a manner sufficient to allow theefficient conversion of sugars to ethanol byE. coli LY01.

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effects of ca(oh)2 treatments (“overliming”) on the composition and toxicity of bagasse...

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