Birch (Betula papyrifera Marsh) condensate collected from a veneer plant in Michigan was analyzed for its major chemical constituents. This condensate contained carbohydrates and lipids. In the lipid fractions, triglycerides were the most abundant component (35-40%), followed by phenolic compounds (30%) and waxes (25-30%). Gas Chromatography-Mass Spectrometry (GC-MS) and Fast Atom Bombardment (FAB) MS were used to identify 14 lipid compounds. A white rot fungus, Phanerochaete chrysosporium [Burdsall-lombard, 5176, HHB-6251], was tested as a means for the bio-remediation of the condensate. P. chrysosporium reduced the total organic content (TOC) of the condensate from 350 ppm to 22 ppm and the color intensity from 0.614 to 0.355 absorbance units, after 2 weeks incubation in a liquid medium containing yeast and peptonc at pH 5

Dawson-Andoh, Benjamin E.

Gage, Douglas

Kamdem, D. Pascal

Loconto, Paul

Ma, Wanli

Pan, Yanlyang

English

Wood and Fiber Science (E-Journal)

CHARACTERIZATION AND BIOREMEDIATION OF BIRCH CONDENSATE Wanli Ma Graduate Assistant D. Pascal Kamdemt Associate Professor Department of Forestry Paul Loconto Rcscarch Assistant Yanlyang Pan Rescarch Assistant Department of Civil and Environmental Engineering Douglas Gage Assistant Professor Department of Biochemistry Michigan Statc University East Lansing, MI 48824 and Benjamin E. Dawson-Andoh Assistant Professor West Virginia University Morgantown, WV 26505-6 125 (Received September 1998) ABSTRACT Birch (Betula papyrifera Marsh) condensate collected from a veneer plant in Michigan was analyzed for its major chemical constituents. This condensate contained carbohydrates and lipids. In the lipid fractions, triglycerides were the most abundant component (35-40%), followed by phenolic com- pounds (30%) and waxes (25-30%). Gas Chromatography-Mass Spectrometry (GC-MS) and Fast Atom Bombardment (FAB) MS were used to identify 14 lipid compounds. A white rot fungus, Pha- nerochaete chrysosporium [Burdsall-lombard, 5176, HHB-62511, was tested as a means for the bio- remediation of the condensate. P. chrysosporium reduced the total organic content (TOC) of the condensate from 350 ppm to 22 ppm and the color intensity from 0.614 to 0.355 absorbance units, after 2 weeks incubation in a liquid medium containing yeast and pcptonc at pH 5.  Keywords: Birch (Betula papyrifera Marsh), condensate, extractives, fungi trcatmcnt, Phanerochaete chrysosporium, GC-MS, FAB-MS, lipids, triglycerides. i Member of SWST. Wood und hiher Science. 3 1(4), 1999, pp. 370-175 0 1999 by the Society of Wood Sclence and Technology Mu rr 01.-CHARACTERIZATION AND BIOREMEDIATION OF BIRCH CONDENSATE INTRODUCTION The forest products industry is currently confronting several environmental issues. Le- gal mandates emanating from the 1990 amendments to the "Clean Air and Water Act" and other legislation focus not only on air quality but also on condensate emissions from wood processing operations (Rice et al. 1994). It is imperative to find alternative ways to dispose of condensates emanating from wood processing operations other than dispos- al in land fills, which is a common current practice. The availability of landfill space is rapidly diminishing and the cost is increasing at an exponential rate (Stalker 1993). In this study, birch condensate is used as a model because birch is one of the Michigan hardwood species widely used for veneer pro- duction. To facilitate peeling of veneer from logs, green birch logs are preconditioned by treatment with live steam (170°F to 200°F) or heating with hot water in vats for 12 to 36 h depending upon log species, size, and the har- vesting season. Consequently, the resulting condensate or leachate is a complex mixture of hot-water extractable material. The conden- sate presents potential disposal problems be- cause of its bio-toxicity, high biological oxy- gen demand, high total organic carbon, and low pH (Servizi et al. 1986; Leach and Tha- kore 1975). A significant fraction of birch con- densate is composed of carbohydrates and lip- ids (Edhborg 1956) and carbohydrates are used as food source by microorganisms (Theander et al. 1993; Ballard et al. 1982). In this study, we focused on the chemical iden- tification of the lipophilic compounds, which are considered as one of the main causes of the pollution. Bioremediation of birch conden- sates was evaluated by inoculating conden- sates with the ligninolytic fungus, P. chryso- sporium, which is reported to be capable of degrading a wide variety of aromatic com- pounds that are reported as environmental pol- lutants, including certain polycyclic aromatic hydrocarbons (PAHs) (Hammel et al. 1992). MATERIALS AND METHODS Birch condensate Two liters of birch condensate, provided by a veneer company based in Escanaba, Michi- gan, were stored at 4OC in a laboratory refrig- erator. Aliquots were pipetted into 10-ml glass vials with rubber stoppers and lyophilized. Lyophilized samples were kept in a freezer at - 10°C until further use. The pH of condensates was measured with a Brinkmann Instruments 716 DMS Titrino pH meter. The pH meter was calibrated using buffers with pHs 3, 7, and 10 before measur- ing the pH of condensate in triplicate. Elemental analysis/(carbon/hydrogen/ nitrogen) of birch condensates Lyophilized birch condensate was analyzed for carbon, hydrogen, and nitrogen content us- ing a C/H/N 2400 Elemental Analyzer (Perkin Elmer). Lyophilized samples were dried in an oven at 105OC for 2 h to remove any residual water before the elemental analysis. In order to get the total organic carbon content, 10% phosphoric acid was used to acidify samples, followed by purging with N, to remove inor- ganic carbon by conversion of carbonate ion C 0 , 2  to gaseous CO,. UV-Vis spectra and color measurements of birch condensate Ultraviolet and visible spectrophotometric analysis was performed on a Beckman DU 640B spectrometer using a quartz cuvet with light path of 10 mm. The sample solutions ex- hibited a maximum peak absorbance at 465 nm, and this peak was used for measuring col- or intensity (Marton et al. 1969). All solutions were prefiltered through a 0.45-pm filter mil- lipore to minimize the particulate interference before the UV measurements. Conjnnation of presence of reducing sugars in birch condensates The presence of reducing sugars in birch condensates was determined by using the pro-- 372 WOOD AND FIBER SCIENCE, OCTOBER 1999, V. 31(4) tocol of Morrow and Sandstrom (1935). After the addition of a solution of copper sulfate and alkaline tartrate, the condensate-a precipitate of orange cuprous oxide in test solution, con- firmed the presence of reducing sugars. The colorimetric method to quantify sugars (Dubois et al. 1956) was also used to estimate the content of glucose equivalents in the birch condensate using a standard curve made with D-glucose at concentrations of 0, 5, 10, 15, 20, 25, and 30 mglml and UV-Vis absorbance reading at 490 nrn. Although this method is simple, rapid, and relatively mexpensive, the presence of colored substances such as qui- nones can interfere with the analysis. Determination of gross lipophilic contents of birch condensate About 100 mg of freeze-dned lyophilized birch condensate sample was dissolved in 5 ml CHCl,, filtered through anhydrous sodium sul- fate cartridge column to remove trace amounts of water and other solid particulate and was then washed twice with 5 ml of CHCI,. The eluant was collected in a clean, preweighed glass vial and then evaporated to dryness under a nitrogen purge, and the glass vial was then reweighed to determine the total lipid content. Solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) Different classes of lipids in the birch con- densate were fractionated by SPE using a pro- tocol developed by Chen et al. (1994). An SPE cartridge containing 200 mg of aminopropyl silica (Isolute International Sorbent Technol- ogy) was loaded with 15-20 mg of total lipid extract from the lyophilized condensate sam- ple. The fractionation scheme using various elution solvents was the same as reported by Chen et al. (1994). Each fraction was collected in a prepared clean bottle. After elution, the fractions were dried under a stream of N, and weighed accurately using an analytical bal- ance. The weight percentage of each class was then calculated. Using this technique, we iso- lated and quantified the following fractions: (1) fatty and resin acids; (2) steryl esters and waxes; (3) triglycerides; (4) sterols, fatty al- cohols, and diglycerides; (5) monoglycerides; and (6) others. An autosystem Gas Chromatograph (Perkin Elmer) interfaced to a Series 800 ion trap mass spectrometer (Finnigan) equipped with a 30- m X 0.25-mm DB-5 fused-silica capillary col- umn (J & W scientific) was used to analyze the different fractions. The following temper- ature program was used: 160°C for 2 min, ramp at 7OCImin to 310°C and hold for 3.5 minutes. The m/z range scanned was from 0 to 650. Fatty and resin acids were methylated with fresh diazomethane in ether before GC injection (Chen et al. 1994). Triglycerides, steryl esters, and waxes were saponified ac- cording to the procedure of Saranpaa and Ny- berg (1987). These fractions were heated with 0.5M KOH in 90% ethanol for 3 hours at 70°C (water bath). The solutions were then diluted with one volume distilled water and acidified to pH 2.0 with 1M HC1. The solutions were subsequently extracted with hexane-diethyl ether (1 : 1, vlv), dried under a stream of nitro- gen, and methylated with ethereal diazometh- ane. The derivatization and GC analysis of other fractions were performed as reported by Chen et al. (1994). Fast Atom Bombardment Mass Spectrometry (FAB-MS) FAB analysis was performed on a JEOL HX- 1 10 double-focusing mass spectrometer operating in the positive mode with ions pro- duced by bombardment with a beam of Xe atoms (6 keV). The accelerating voltage was 10 kV, and the resolution was set at 1,000. Aliquots of the sample solution were mixed with the glycerol matrix on the end of the di- rect insertion probe. The matrix-sample solu- tion was then introduced into the mass spec- trometer via a vacuum interlock. The instru- ment was scanned from m/z 0 to 1,500, and data were collected in the range 50 to 1,500. Mo ut 01.-CHARACTERIZATION AND BIOREMEDIATION OF BIRCH CONDENSATE 3-73 Total organic carbon (TOC) content in birch condensate Total organic carbon content of aqueous samples was determined by direct aqueous in- jection using model 700 TOC Analyzer (0-1 Analytical). Only prefiltered solutions of birch condensate were used for TOC analysis. Fungal bioremediation treatment of birch condensate A strain of P. chrysosporium [Burdsall-lom- bard, 5176, HHB-62511 was plated on a 2% malt-agar media and incubated at 25OC for 2 weeks. About 0.1% peptone hydrolysate I from Sigma Chemicals Co. and 0.5% yeast ex- tract from Difco Chemical Co. were added to 200 ml of birch condensate in a 500-ml Er- lenmeyer flask. A parallel set of controls was carried without peptone or yeast to determine their effects. The pH of the solutions was ad- justed to 5, 7, or 10 using sodium acetate and/ or sodium hydroxide. With a sterile hollow tube, five discs (5-mm diameter) were punched out of the periphery of the mycelia of P. chrysosporium growing on the 2% malt- agar plate and added aseptically to each flask. Six replicas were made for each condition. The flasks were inoculated and kept at 25°C and 90% relative humidity without shaking for 2 weeks. The temperature level used was sug- gested by the American Type Culture Collec- tion (1991). At the end of the incubation pe- riod, the pH, the absorbance at 465 nm, and the TOC of the solutions were measured. RESlJLTS AND DISCUSSION Chemical characterization of birch condensates Color and pH.-The birch condensate has a brown color and a pH of 3.90. When it was left exposed to air at room temperature for a minimum of 3 days, the color became darker and the pH increased to about 5. The low pH of condensate is attributed to formic, acetic, and other organic acids produced during the hot-water or steam treatment of green wood TARI F I. Types of lipids and their relative proportions. Proportiun o i  thc ltpld content Free fatty and rcsin acid 1.6% Steryl esters and waxes 27% Triglyceridcs 38% Sterols, fatty alcohols and diglycerides 1.6% Monoglyccridcs 1.6% Others (mainly Phenolic compounds) 30% (Edhborg 1956). The color of the condensate may be due to the presence of hot-water-sol- uble lignin and breakdown of some phenolic glycosides. At lower pH (3.9-6), the color of birch condensate was lighter than at higher pH (7- 14). Elemental analysis and sugar content.-- The lyophilized birch condensate contains 50.2% carbon, 6.05% hydrogen, and 2.37% nitrogen by weight, corresponding to the mole ra.tio of CIWN to be 4.1816.0510.17 (average of 10 samples). Samples pretreated by the phosphoric purge method gave the same TOC results as those that were not pretreated. This indicates that the carbon was entirely in or- ganic form. A carbon to hydrogen molar ratlio of 4 to 6 was an indication of the presence of unsaturated hydrocarbons. The colorimetric method measurements in- dicated that the sugar (D-glucose equivalent) content of the birch condensate was about 8.9 mglml. Gross lipid content of lyophilized birch con- densate.-Gross lipid content of lyophilized birch condensate was 9.1%. This is equivalent to 91 mg per gram of lyophilized birch con- densate. Lipids include fatty and resin acids, sterols, waxes, triglycerides, phenolic com- pounds, etc. Those different fractions were separated and analyzed by solid phase extrac- tion and GC-MS. Separation and identification of chemicals in lipophilic fractions.-Solid phase extraction ( S P E )  was used to separate the different clas- ses of lipid in the birch condensate. The type of lipid and relative amount are listed in Table 1. The quantitatively important components 374 WOOI) AND FIBER SCIENCE, OCTOBER 1999, V. 31(4) TABLE 2. Compounds idrrzrifified usinj: GC-MS Ketent~on Molecular mlr valuc of major peaks Nu .iarnplc name time weight (relative intcnslty. 9,) Pentadecanoic acid, 13-rnethylt 7,lO-Octadecadicnoic acidi. 8,1 1 -0ctadccadienoic acidt 9.15-Octadecadienoic acidt Heptadec:inoic acidi Eicosanoic acidi Nonadccanoic acid, 10-rncthylt Nonadccanol Bezenepropanol, 4-hydroxy, al- pha-mcthyl(R-) Phenol, 2,6-Bis(1,I-Dimethylc- thyl)-4-methyl Phcnol, 3,s-dimethoxy- Benzene, I -methyl-3-phenoxy- i The latt) ecldr wcrc analy.:ed as thell- cormeap~~ndit~p methyl esterc are triglycerides (38%), phenolic compounds (30%), and waxes (27%). Other components 0 such as free fatty and resin acids, monoglyc- erides, diglycerides, sterols, and fatty alcohols H ZC - 0-C- 17 31 I I add up to 4.8% of' the total weight. Table 2 lists the major chemical compounds identified by GC-MS. A total of 14 com- pounds was identified by comparing sample HC spectra with those in the literature and spectra of standards. The mass to charge ratio (mlz) values and the relative intensity of the pro- nounced peaks are provided in Table 2. All the 0 -0- I I C- 17 H 31 0 fatty acids listed were analyzed as their cor- H2C- 0-C- C 17 H 31 I I responding methyl esters. The GC-MS analy- sis of the triglyceride fraction following sa- ~ h ,  molecular weight is theoretically calcu- ponification showed that the dominant fatty lated to be 879.4, using accurate atomic acid was octadecadienoic acid (C,,H,,O,). weight for C: 12.01 1 15, H: 1.00797 and 0 :  This was confirmed by FAB-MS of the intact 15.9994 (Silverstein et al. 1981). triglyceride fraction. In the FZAB-MS spectra of the triglyceride fraction, a weak molecular Bioremediation of birch condensate ion peak, MH' at m/z 879.7 was observed for Flasks containing birch condensate with pH the triglyceride C ,,H,,O,: adjusted at 3.9, 5.0, 7.0, and 10.0 were inoc- Ma cr 01.-CHAKAC'I'ERIZATION AND BIOREMEDIATION OF BIRCH CONDENSATE 3;'s ulated with P. chrysosporium. No evidence of fungal growth was noticed. Another set of ex- periments was carried out using the same con- ditions as before except adding 0.1% peptone and 0.5% yeast to each of the flasks. This time the flasks with pH 3.9, 7.0, 10.0 showed no change, but vigorous growth of fungus and color change were observed in the flask with pH 5.0. The color appeared to be much lighter after inoculation; UV-Vis measurements showed color intensity decreased from 0.614 to 0.355 after the P. chrysosporium treatment. TOC measurements also showed that the total organic carbon content of the solution was re- duced from 350 ppm to 22 ppm after the treat- ment. This corresponds to a 42% reduction in color intensity and 94% reduction in total or- ganic carbon content. The birch condensate consists primarily of reducing sugars and lipids. In the lipid extract, triglycerides, waxes, and phenolics were the major compounds. The dominant fatty acid residue of triglycerides detected was octade- cadienoic acid (C lxH1202). The lab-scale bioremediation technology employed is simple and effective, the optimum pH for bioremediation treatment is near 5, and the first stage nutrient (peptone and yeast) is needed to accelerate the fungus growth. Fur- ther work is needed to determine the best ex- perimental parameters, such as temperature, fungus strain, fungus to solution ratio, incu- bation duration. ACKNOWLEDGMENTS We are grateful to Ismail Jusoh and Joseph Nguyen for their contribution. We wish to ac- knowledge the NIEHS Basic Superfund Re- search Center, Analytical Chemistry Core Grant P42ES04911, the MSU Mass Spectrom- etry Facility grant (DRR-00480) from the NIH, and the Michigan State University-East- ern Hardwood Utilization Program USDAI CSRS grant #95-34158- 1304 for providing funds. REFERENCES American Type Culture Collection (ATCC). 1991. Cala- loguc of filamentous fungi. 18th cd. Edited by S. C. 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Characterization and Bioremediation of Birch Condensate

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Characterization and Bioremediation of Birch Condensate

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