Effective and Safe Composting of Chlorophenol-Contaminated Soil in

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Environ. Sci. Technol. 1997, 31, 371-378

Effective and Safe Composting of Chlorophenol-Contaminated Soil in Pilot Scale M. MINNA LAINE* AND KIRSTEN S. JØRGENSEN Finnish Environment Institute, Hakuninmaantie 4-6, FIN-00430 Helsinki, Finland

A pilot-scale composting of chlorophenol-contaminated soil was performed to compare chlorophenol degradation by two different inoculants, straw compost and remediated soil, with that by indigenous soil microbes. Four compost piles with a size of 13 m3 each were constructed. Chlorophenol and chloroanisole concentrations as well as numerous physical and microbiological parameters were monitored during 6 months of composting. Over 90% of the chlorophenols were removed during the composting period. The biodegradation of chlorophenols was efficient and fast despite the inocula. Frequent mixing and control of the nutrient level enhanced the chlorophenol degradation activity of the indigenous microbes in the contaminated soil. In a parallel bench-scale experiment, an average of 60% mineralization of radiolabeled pentachlorophenol ([14C]PCP) was obtained in 4 weeks in 1-kg piles with or without inocula. This result showed that a major part of chlorophenols was completely mineralized.

Introduction In Finland, chlorophenols (CPs) have been widely used at sawmill sites to preserve wood. Because of the large amounts of wood preservatives used and their persistence in the environment, there are now around 800 chlorophenolcontaminated old sawmill sites that need to be remediated. In most of the already performed remediation cases in Finland, composting in biopiles has been the predominating remediation technology. The cold climate gives an extra challenge for bioremediation of contaminated soil in Finland. Many chlorophenol-degrading microbes have been isolated and their properties have been carefully investigated in the laboratory (see refs 1-5 for reviews). However, very few experiments have included outdoor field tests (6-8; for a review, see ref 3). Microbiological processes occurring in soil during bioremediation are nonetheless not fully understood. While some bacteria may mineralize chlorophenols completely, some microorganisms may O-methylate or polymerize chlorophenols under certain conditions. These biotransformation products may cause ecotoxicological risks and effects on the soil environment. A complete mineralization of chlorophenols should always be the goal of the bioremediation process. Yet, rapid, more effective, and safer purification methods with no harmful side reactions need to be developed. Straw compost is the unspawned growth substrate made for the cultivation of edible mushrooms, and it is produced in large scale in most countries. In our previous laboratory experiments (9), the biotransformation of pentachlorophenol * Corresponding author phone: (358) 9 40300884; fax: (358) 9 40300880; e-mail: [email protected].

S0013-936X(96)00176-9 CCC: $14.00

 1997 American Chemical Society

(PCP) and the mineralization of radiolabeled [U-14C]PCP by straw compost and remediated soil were studied under fieldsimulating conditions before and after 3 months of adaptation with pentachlorophenol in a percolator. After PCP adaptation, the straw compost mineralized up to 56% of the [14C]PCP. No partial dechlorination of PCP was found. The native straw compost did not mineralize PCP, but partial dechlorination of PCP occurred. Enrichment in the percolator enhanced the mineralization rate of remediated soil to 56% as compared to the native remediated soil, which mineralized 24% of [14C]PCP added. No harmful side reactions, such as extensive methylation, were observed. Based on these results, straw compost and remediated soil were chosen as inocula for a pilot-scale field test to bioremediate chlorophenol-contaminated soil. The aim of this study was to develop a fast and controlled system to bioremediate chlorophenol-contaminated soil with the complete mineralization of chlorophenols. We compared chlorophenol degradation by two different inoculants, straw compost and remediated soil, with that by indigenous soil microbes and studied the biodegradability of heavily contaminated wooden parts from the bottom of the former wood preservative dip basin. The present study was made to produce a well-documented field experiment, with as many parameters detected as possible.

Experimental Section Straw Compost. Straw compost was obtained from a mushroom farm in Finland and had been prepared as follows: After prewetting, rye or wheat straw was stacked for 10-12 days in piles with chicken litter. Windrows (2 m × 2 m × 70 m) amended with gypsum (1800 kg) and lime (320 kg/windrow) were mixed, and the compost was allowed to maturate for 10 more days. This compost is so-called phase I compost (10). The straw compost contained (in g/kg dry weight): NH4+-N, 0.14; total N, 18; total P, 6.3; and soluble P, 0.9. The ignition loss was 83% (wt/dry wt), water content was 74% (wt/wt), and the pH was 8.0. Remediated Soil. Soil was obtained from successfully remediated, full-scale 3-year composting of chlorophenolcontaminated soil in biopiles where bark chips and nutrients had been added, but no inoculum. The remediated soil contained (in g/kg dry weight): NH4+-N, 0.004; total N, 1.4; total P, 0.59; and soluble P, 0.022. The ignition loss was 17% (wt/dry wt), water content was 32% (wt/wt), and the pH was 6.4. The chlorophenol concentration was 1.5 mg (kg dry wt)-1. Contaminated Soil. The contaminated soil was from a Finnish sawmill where a chlorophenol-containing wood preservative (Ky-5) had been used from 1955 to 1977 to impregnate wood. Ky-5 consisted mainly of 2,4,6-trichlorophenol (2,4,6-TCP, 7-15%), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP, about 80%), and pentachlorophenol (PCP, 6-10%) (11). Ky-5 contained also trace amounts of polychlorinated phenoxyphenols, dibenzo-p-dioxins, and dibenzofurans as impurities (12). The contaminated soil contained (in g/kg dry weight): NH4+-N, 0.004; total N, 0.4; total P, 0.23; and soluble P, 0.003. The ignition loss was 5% (wt/dry wt), water content was 16% (wt/wt), and the pH was 3.4. The ability of the native soil to mineralize [U-14C]PCP was determined as described later under Mineralization Assay of [14C]PCP. The [14C]PCP was, however, added in a phosphate buffer, pH 7, instead of mineral salts medium. Construction of Pilot Piles. The pilot-scale composting was started at the end of May 1994. Four cone-shaped compost piles (3 m × 3 m × 1.5 m) were built in the field (Table 1). The total volume of each pile was about 13 m3 (7500 kg, Table 1). Each inoculant was mixed with contami-

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TABLE 1. Mass of Materials in Pilesa 1000 kg (m3)b pile no.

contaminated soil

bark chips

1 2 3 4

5.6 (7) 5.6 (7) 5.6 (7) 5.6 (7)

1.2 (6.5) 0.54 (3) 0.54 (5) 0.9 (5)

remediated soil

straw compost

contaminated wood chips

total

0.42 (2)

6.8 (13.5) 6.8 (13.0) 7.4 (13.5) 8.2 (15.5)

0.66 (3) 1.3 (1.5) 1.3 (1.5)

a

The mass of the materials is calculated from their densities. After 2 months, 1 m3 of heavily contaminated soil was further added to piles 1-3; and 1 m3 of fresh bark was added to piles 1 and 3. b The volume of the materials in m3 is shown in parentheses.

TABLE 2. Measured Nutrient Concentrations (per Dry Weight) and pH Values in Piles during the Pilot Compostinga pile no.b

time (weeks)

NH4-Nc (mg kg-1)

NO3-Nd (mg kg-1)

Ntote (g kg-1)

Psolf (mg kg-1)

Ptotg (mg kg-1)

pH

1

1 7 25 1 7 25 1 7 25 1 7 25

279 15 8 269 19 7 236 12 3 238 131 8

199 1 52 148 152 389 211 22 62 211 121 128

1.13 nmh 1.34 1.79 nm 2.26 1.35 nm 1.27 1.35 nm 1.16

2.0 3.7 3.0 14.9 24 28.1 4.3 4.9 4.6 1.8 4.0 3.0

415 423 464 801 801 792 471 468 662 436 521 518

7.0 6.9 6.5 6.9 6.7 6.5 6.9 7.1 6.6 6.5 6.3 6.5

2

3

4

a On week 9, nutrients were added to piles 1-3, and bark was added to piles 1 and 3. b Pile numbers: 1, reference pile; 2, straw compost pile; 3, remediated soil pile; and 4, wood chips pile. c Nitrogen in form of ammonium. d Total nitrogen. e Nitrogen in form of nitrate. f Soluble phosphorus. g Total phosphorus. h nm, not determined.

nated soil, the pH was adjusted with fine granular lime (dolomite, 5 kg m-3), nutrients (1 kg m-3) were added as a commercial fertilizer, and bark chips (fresh pine) as supporting and aerating material were added to enhance the conditions for microbial degradation of chlorophenolic waste in soil. The ratio of the materials was 2 parts contaminated soil and 1 part inoculant and/or bark by volume. The commercial fertilizer contained 26% N (14% ammonium-N and 12% nitrate-N), 3% P (2.1% water soluble P), 3% K, 1.5% S, 0.5% Mg. 0.03% B, and 0.0006% Se. The concentrations of the nutrients after addition are shown in Table 2. The contaminated wooden parts (logs) were chipped beforehand to a size of about 2 cm × 3 cm × 0.5 cm, making them easier to mix thoroughly with the soil. The contaminated wood chips were added to one pile of contaminated soil with remediated soil as inoculum (Table 1). The adjustment of pH to near neutral in the compost piles was based on laboratory tests where 100 mL of contaminated soil (59 g wet wt) was mixed with 10 mL of water and 0.5, 3, or 7 g of fine granular lime (dolomite). After incubation overnight at room temperature, water was added to 100 mL, and the pH was measured. According to the results obtained, 5 kg of lime was added/m3 pile constituents. The chlorophenol-contaminated soil was excavated from 0 to 1 m outside the wood preservation shed, where it had been exposed to rainfall for many years. The soil was homogenized with a crushing machine. The piles were constructed on plastic beds each covered with a 3-m3 insulation layer of bark. After all the materials were piled, the piles were mixed, sampled, and covered with tarps. The piles were mixed with a mechanical clamshell every second week for the first 2 months and then once a month for the following 4 months. The moisture content of the compost piles was about 26% and was maintained by watering the piles once during the composting period. Since the chlorophenol concentrations decreased to less than 10 mg (kg dry wt)-1 already after 9 weeks, the chlorophenol concentration was raised by a second addition of heavily contaminated soil. The soil was excavated from 0 to 1 m depth right beside the wood-preserving basin,

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under the roof of the wood-preserving building. A total of 1 m3 of contaminated soil as well as nutrients (0.5 kg m-3) were added to piles 1-3, and 1 m3 of fresh bark was added to piles 1 and 3. Sampling. After the piles were mixed, they were sampled. A combination sample of 10-20 different points from each pile was mixed by shaking and sieved through an 8-mm sieve. The pieces of bark and contaminated wood chips from the sample remained on the sieve. A glass jar was filled with the sieved sample, and aluminium foil was placed between the jar and the cap. The samples were stored at 4 °C before microbiological measurements, and a subsample was frozen from each combination sample for chemical analysis. Bench-Scale Piles. Bench-scale piles were constructed to imitate conditions and material ratios in the pilot-scale composting. Nutrients, lime, and bark as well as inocula were added to contaminated soil in the same proportions as to the compost piles (see Table 1). The mini-piles (1 kg mass) were stacked in trays, covered with aluminium foil, and kept at room temperature in the hood for 6 months. The piles were mixed twice a month, and the moisture was maintained by adding distilled water. Mineralization Assay of [14C]PCP. After 6 months of incubation, the mineralization potential was examined from the bench-scale compost piles. One-sixth volume of a 100mL infusion bottle was filled with compost (10 g wet wt). A sample of 20 µg of PCP with or without [U-14C]PCP (specific activity 10.6 mCi mmol-1, 14 000 dpm per bottle) was added in 5 mL of mineral salts medium (containing, in g/L, the following: K2HPO4, 2.9; KH2PO4, 1.04; NH4Cl, 2.0; MgSO4‚ 7H2O, 0.4; NaCl, 0.03; CaCl2, 0.003; and FeSO4‚7H2O, 0.001) to make a slurry of 50-60% (wt/vol). The exact radioactivity of the mineral solution with [U-14C]PCP was determined by liquid scintillation counting. Labeled and unlabeled bottles were studied in duplicate. The mineralization test was performed at room temperature by shaking the bottles on a platform shaker (135 rpm). Every third or fourth day, the amount of 14CO2 absorbed to 1 M NaOH from the labeled samples was measured using a liquid scintillation counter

FIGURE 1. Mineralization of [14C]PCP to 14CO2 by the PCP-adapted straw compost and the remediated soil after 6 months bench-scale enrichment with bark, nutrients, and contaminated soil. Reference soil ) soil were no inoculum was added. Wood chips were contaminated with chlorophenols. (Wallac). NaOH was kept in a cup anchored by the rubber stopper of the bottle (15). After sampling, the NaOH in the cup was replaced by 1 mL of fresh NaOH. The mineralization test was terminated after 4 weeks. Chlorophenol and Chloroanisole Analyses. Chlorophenols were extracted from the solid samples (2-5 g wet wt) by adding 50 mL of acidic acetone (pH 2 with HCl), sonicating in a water bath for 15 min, and shaking on a platform shaker overnight. A 10-mL aliquot of acetone extract (supernatant) was transferred to a test tube, 5 mL of water was added, and the extract was washed with 5 mL of hexane. The hexane phase was recovered and dried with Na2SO4 for chloroanisole analysis. The residue was neutralized with 2 M NaOH and washed with another 5 mL of hexane. The purified extract was derivatized for chlorophenol analysis with 15 mL of 0.2 M K2CO3 and 295 µL of acetic anhydride. The extract was allowed to acetylate for 10 min before the chlorophenols were transferred to a hexane phase, let stand for another 10 min, and then dried with Na2SO4. Chlorinated compounds were analyzed by a Hewlett Packard (HP) 5890 gas chromatograph (GC) with two HP electron capture detectors connected to HP-1 and HP-5 fused-silica capillary columns, respectively. The internal standards used were 2,4,6-tribromophenol and 2,4,6-tribromoanisole. The GC was calibrated with 16 different chlorophenol compounds (di-, tri-, and tetrachlorophenols and pentachlorophenol) and seven different chloroanisole compounds. Nutrient, Ignition Loss, and pH Determinations. The total phosphorus concentration was measured by plasma emission spectroscopy after dry combustion and HCl extraction. Total nitrogen was measured with the Kjeldahl method using a Kjeltec Auto 1030 analyzer. Soluble nitrogen and soluble phosphorus were extracted by shaking for 1 h with 0.1 M K2SO4 solution before analyzing with Kjeltec Auto 1030 analyzer or plasma emission spectroscopy, respectively. The pH was measured in a 1 M KCl solution. The ignition loss was measured by combusting an oven-dried sample (105 °C) at 850 °C for 2.5 h. Soil Respiration Rates. Basic and substrate-induced respiration rates were measured with a method modified from

FIGURE 2. Relative chlorophenol removal (in %) in compost piles 1-3. (A) Low contamination level from 45 to below 10 mg (kg dry wt)-1 during weeks 0-9. (B) High contamination level from approximately 850-50 mg (kg dry wt)-1 during weeks 9-25. (O) ) pile 1, reference pile; (9) ) pile 2, straw compost pile; and (b) ) pile 3, remediated soil pile. the one described by Anderson and Domsch (13). Two replicate 125-mL closed glass bottles were filled with the amount of natural moist soil equivalent to 10 g by dry weight and incubated for 2 h (substrate-induced respiration) and for 20 h (basic respiration), at a constant temperature of 22 ( 1 °C in a water bath. The production of CO2 was measured by injecting a 0.5-mL gas sample from the headspace into an Easy-Quant IR carbon analyzer. For measurements of substrate-induced respiration rate, glucose was added to the test bottles as a powder, mixed, and let stand stabilize for 30 min. The bottles were flushed with air before the zero point measurement and after each sampling. The carbon analyzer was calibrated with a standard gas mixture containing CO2 (AGA, Sweden) of known concentration. Determination of Soil Gas Components and Temperature. The soil air composition in the compost piles was measured before each mixing in perforated gas collection tubes installed inside the piles. Oxygen concentrations were measured using a Crowcon Triple 84TR (Crowcon Instruments, U.K.) gas meter. For CO2 and humidity measurements, gas detector tubes (Dra¨ger, Germany) were used. The temperature inside the piles was measured using a 2-m-long temperature probe. The temperature gradient was measured at 0.25-m depth intervals, and the average temperature was calculated. Enumeration of Bacteria. Bacteria were extracted from the compost by blending 10 g wet wt of compost for 2 min with 90 mL of 0.9% NaCl, 1 mL of 10.4% Na5P3O10, and 100

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TABLE 3. Total Chlorophenol Concentrations and Relative Amount of PCP Compared to 2,3,4,6-TeCP as Mass % During Composting total chlorophenols (mg (kg dry wt)-1) and ratio of PCP/2,3,4,6-TeCP as % (av ( SD) pile 1

pile 2

pile 3

time (weeks)

total CPs

PCP/TeCP

total CPs

PCP/TeCP

0 1 3 5 7 9

43 ( 2 19 ( 0.6 16 ( 2 10 ( 0.5 9 ( 0.3 7 ( 0.4

70 ( 6 100 ( 5 102 ( 17 90 ( 1 78 ( 12 63 ( 0.5

45 ( 4 23 ( 0.5 21 ( 0.5 13 ( 0.1 10 ( 0.04 10 ( 0.4

80 ( 5 111 ( 4 97 ( 4 102 ( 5 78 ( 3 70 ( 0.5

9 13 17 21 25 51

771 ( 48 203 ( 30 35 ( 5 33 ( 5 34 ( 2 29 ( 7

37 ( 1 89 ( 2 121 ( 44 123 ( 51 70 ( 3 90 ( 27

Addition of Heavily Contaminated Soil 683 ( 9 42 ( 2 1108 ( 26 233 ( 29 156 ( 25 585 ( 82 42 ( 4 99 ( 8 103 ( 16 44 ( 5 83 ( 4 53 ( 5 42 ( 1 88 ( 8 67 ( 19 38 ( 3 89 ( 6 49 ( 3

µL of 2% Tween 80 before serial dilution. The total number of bacteria was determined by acridine orange staining as follows: The bacterial extract was diluted with formaldehyde and saline and filtered through a 5-µm membrane prefilter that was washed with KCl. The extract was then filtered on to a polycarbonate filter, and acridine orange was added. After 5 min of incubation, the filter was dried by suction. The stained cells were counted using a Leitz SM Lux epifluorescence microscope by counting 20 randomly chosen fields. For determining the number of culturable bacteria in the compost, a rich medium specific for actinomycetes, R8 medium with cycloheximide (14), was used. Bacteria able to grow on mineral salts medium with PCP as a sole carbon source were also enumerated in samples from the compost piles. The PCP medium contained, in g/L, the following: K2HPO4, 2.1; KH2PO4, 0.4; NH4NO3, 0.5; MgSO4‚7H2O, 0.2; CaCl2‚ 6H2O, 0.023; and FeCl3‚6H2O, 0.002; and 1 mM PCP. Bacteria were grown on R8 and PCP plates at 30 °C for 5 days.

Results and Discussion Mineralization of PCP in Bench Scale. According to our previous work, the two inoculants studied, straw compost and bioremediated soil, showed effective mineralization of PCP after 3 months adaptation time (9). The 1-kg mini-piles containing these inoculants together with contaminated soil were incubated at room temperature for 6 months before the mineralization test of [14C]PCP was performed. The mineralization of [14C]PCP by the native contaminated soil was also determined. The contaminated soil alone (chlorophenols 850 mg (kg dry wt)-1) showed no (0.4% in 1 week) in situ mineralization activity for [14C]PCP when studied in phosphate buffer (pH 7). The incubation of the contaminated soil with straw compost and nutrients resulted in 60 ( 0.3% mineralization of [14C]PCP in 4 weeks. The initial mineralization rate was lower than in other treatments (Figure 1). The contaminated soil with bark (reference bench pile) and without inoculum mineralized 63 ( 0.4% of the [14C]PCP added in 4 weeks (Figure 1). The 6-month incubation enhanced the initial mineralization rate of the chlorophenols to be even higher than that with the inoculants, although the mineralization reached the same level (60-63%) in all treatments. This indicated that the conditions in the bench-scale piles (nutrients, pH) favored bacteria in contaminated soil that were able to mineralize PCP efficiently despite the augmentation. The degree of mineralization obtained can be considered large, since the remaining 40% have probably been incorporated to cell biomass, although part of the chlorophenols may have absorbed to the soil organic matter. Chlorophenol Removal during Pilot-Scale Composting. Piles 1-3 removed 80% of the chlorophenols and reached

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total CPs 43 ( 0.5 21 ( 2 18 ( 3 11 ( 0.6 9 ( 0.8 7 ( 0.9

pile 4 PCP/TeCP

total CPs

PCP/TeCP

66 ( 0.02 102 ( 3 106 ( 13 109 ( 17 78 ( 5 61 ( 8

1842 ( 64 3756 ( 1635 2374 ( 1156 1814 ( 45 1132 ( 224 437 ( 36

21 ( 1 25 ( 5 51 ( 19 69 ( 6 60 ( 1 76 ( 0.3

54 ( 0.01 106 ( 5 183 ( 13 168 ( 37 138 ( 59 110 ( 18

437 ( 36 506 ( 24 91 ( 1 127 ( 33 200 ( 10 106 ( 37

76 ( 0.3 52 ( 0.2 82 ( 22 76 ( 27 62 ( 6 92 ( 7

acceptable (below 10 mg (kg dry wt)-1) concentrations of contaminants in 2 months (low contamination level, Figure 2A, Table 3). After this period, 1 m3 of heavily contaminated soil as well as nutrients were added to piles 1-3. The chlorophenol concentrations increased to about 850 mg (kg dry wt)-1, and more than 90% disappeared during the following 3 months (high contamination level, Figure 2B, Table 3). No differences were found between the piles with or without augmentation. The proper and often mixing of the piles as well as improved nutrient and pH conditions apparently enhanced chlorophenol degradation activity of the indigenous microbes in the contaminated soil. The chlorophenols from the second addition of contaminated soil were degraded very fast, indicating that the first 2 months of composting had increased the chlorophenol degradation activity in the compost piles. The chlorophenol concentration in the forth pile, where the contaminated wood chips had been added, remained high for the first 2 months. After 2 months, the chlorophenol concentration in pile 4 also started to decrease (Table 3). In the source of chlorophenol contamination at Finnish sawmills, the technical wood preservative Ky-5, the relative concentration of PCP compared to 2,3,4,6-TeCP was 8-13% (11). After the contaminant had resided in soil for decades, the relative amount of PCP compared to 2,3,4,6-TeCP was about 66-80%, indicating that PCP had been more persistent or less mobile than 2,3,4,6-TeCP in the surface soil used in compost piles (Table 3). During the composting, the relative concentration of PCP increased after 1 week, indicating that 2,3,4,6-TeCP was degraded faster than PCP but then decreased, showing that PCP was also degraded (Table 3). When the heavily contaminated soil was added to the pilot composts, this pattern was repeated. The results indicated that tetrachlorinated phenols were degraded more readily than PCP, but PCP did not accumulate. In pile 4, where the contaminated wood chips had been added, the relative concentration of PCP compared to 2,3,4,6TeCP of 21% was nearest the original PCP/TeCP ratio in Ky-5 (8-13%), indicating that little or no degradation had taken place in the wood. PCP may also have been more tightly bound to the wood chips than TeCP. The ratio increased during the composting due to release of chlorophenols from degrading wooden parts together with faster removal of TeCP. Biotransformation of Chlorophenols during Composting. The chloroanisole concentrations detected were from 0.1 to 0.3 mg (kg dry wt)-1 after 3 weeks of composting (low contamination level). After the second addition of contaminated soil, the chloroanisole concentration increased to between 0.7 and 2.4 mg (kg dry wt)-1, because the heavily chlorophenol-contaminated soil added also contained more chloroanisoles. However, the chloroanisole concentration

FIGURE 3. Temperatures in pilot piles during the composting. The temperature is the average of the temperature profiles measured in the piles. The ambient temperature was measured in the air at the sampling time. decreased to less than 0.1 mg (kg dry wt)-1 during the composting at high contamination level in all four piles. Thus, no methylation of chlorophenols to the corresponding chloroanisoles was found. On the contrary, removal of chloroanisoles present in the contaminated soil was observed. Another potential biotransformation of chlorophenols, e.g., polymerization or dimerization to polychlorinated dibenzop-dioxins or dibenzofurans, was not observed during the composting (Laine et al., in preparation.). The result of the mineralization experiment in bench scale showed that a major part of chlorophenols was completely mineralized and that indigenous soil microbes were responsible for chlorophenol degradation. As a conclusion, chlorophenol degradation was complete and effective without any harmful side reactions. Physicochemical Conditions for Biodegradation in Compost Piles. The average temperature in the compost piles was 36 °C at its highest, and it was well above the ambient day temperature (Figure 3). The reference pile (pile 1) had the highest temperature during the pilot experiment. The degradation of chlorophenols was efficient when the temperature of the piles was maintained above 10-15°C (between May and September, weeks 0-17). During the winter (between weeks 25 and 51), the degradation of chlorophenols stopped since the piles were completely frozen, probably because they were too small to keep the temperature above zero. The pH of the piles decreased from near neutral (6.9-7.0) with about 0.5 pH unit to 6.5-6.6 (Table 2) during the composting due to the bacterial catabolism of chlorophenols and other carbon sources to organic acids, CO2, and HCl. The average ignition loss in compost piles 1-3 was 9.8 ( 1.1% of dry weight, and it increased sligthly after the addition of contaminated soil, nutrients, and bark to 11.0 ( 0.9%. In pile 4, the ignition loss was higher (13.0 ( 1.4%) because of the added wood chips. Nutrients were added to the compost piles in the form of a commercial field fertilizer. The measured nutrient concentrations during the composting are given in Table 2. The amounts of total phosphorus and nitrogen remained at the same level during the composting. A small increase could be

FIGURE 4. (A) Basic and (B) substrate-induced respiration rates in the piles during the composting. Notice the different scale in respiration rates. seen in nutrient concentrations due to the second addition of fertilizer to piles 1-3 on week 9 (Table 2). The highest amount of total and soluble phosphorus was found in pile 2 where it originated from the straw compost added. Ammonium was used up in piles 1-3 already during the first 7 weeks, when chlorophenol degradation and microbial activity were high, but in pile 4 only when the efficient chlorophenol degradation started after 7 weeks of composting (Table 2). The amount of soluble phosphorus that was added in the form of fertilizer (calculated value 58-90 mg (kg dry wt)-1) was not seen in the compost samples after 1 week of composting (measured value 2-15 mg (kg dry wt)-1, Table 2), since available phosphorus was taken for microbial use instantly. The increase in the nitrate concentration in pile 2 (Table 2) indicated that ammonium was nitrified to nitrate by nitrifying bacteria. This phenomenon did not, however, affect the chlorophenol degradation. As a conclusion, the amounts of total phosphorus and total nitrogen remained the same during the composting, although the amounts of available form of nutrients, soluble phosphorus, and ammonium (and nitrate) were rapidly used by the microbes. Based on this observation, the analysis of total nutrient concentrations is not an informative parameter to followup the composting process. On the contrary, measurements of available nutrients gave a tool to evaluate the microbial status during the composting.

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FIGURE 5. Oxygen, carbon dioxide, and water concentrations in the gas phase of the compost pores during the composting. Microbial Status during Composting. The basic and substrate-induced respiration rates were measured during the pilot composting to follow the overall microbial activity in the compost piles. The basic respiration rates were similar in piles 1-3 (Figure 4). The basic respiration rates decreased during the first 2 months and then remained at the same level for the following 3 months (Figure 4A). The substrateinduced respiration rates followed the same pattern but were about three times higher in all piles except in pile 4 with wood chips (Figure 4B). The respiration rates in the compost piles were the highest at the beginning of the composting. This may be due to the addition of bark and nutrients, which provided easily available carbon sources for the microbes. The addition of nutrients and bark to piles 1-3 on week 9 increased however the respiration rates only slightly. This could be explained by selection of the microbes during the first 2 months of composting.

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FIGURE 6. Total bacterial number determined by acridine orange staining (AO), number of bacteria growing on R8 medium (R8), and number of bacteria growing on PCP medium (PCP) in compost samples during the composting. 1, reference pile; 2, straw compost pile; 3, remediated soil pile; and 4, wood chips pile. cfu, colony forming units. The high concentration of chlorophenols (about 1800 mg (kg dry wt)-1) seemed to inhibit both the basic and substrateinduced respiration rates in pile 4, indicating that it had a toxic effect on the microbial community (Table 3, Figure 4). The actual and potential respiratory activity were the same in pile 4 at high contamination level, since the addition of

glucose did not increase the substrate-induced respiration rate over the basic respiration rate. The high chlorophenol concentration may have inactivated other microbes than those that could tolerate chlorophenols and that were responsible for chlorophenol degradation. Gas measurements from the compost piles showed that oxygen was consumed, and CO2 and water were released during the most active chlorophenol degradation period of composting (Figure 5). When the microbial activity was low, as reflected by temperature and soil respiration results (Figures 3 and 4), the concentrations of water and CO2 were low and the concentration of O2 was high in the compost pores. In order to achieve more information on chlorophenoldegrading microbes in the compost piles, the total number of bacteria, and bacteria able to grow on plates with PCP as a sole carbon source, as well as on rich R8 medium, were monitored (Figure 6). The total number of bacteria, as determined by acridine orange staining and microbial counts on R8 medium, remained at the same level during the composting period. No clear correlation was seen between the bacterial counts and chlorophenol removal rates, although the lowest total number of bacteria was constantly found in pile 4 with the highest chlorophenol concentration. On the other hand, the highest number of bacteria growing on PCP medium was also seen in pile 4, showing that bacteria growing on PCP medium represented a higher proportion of the total bacteria. This result indicated the presence of selective environmental pressure in pile 4. The number of bacteria able to grow with 1 mM PCP as the sole carbon source increased right after the contaminated soil was added to the compost piles, indicating that those bacteria originated from contaminated soil (Figure 6). A small increase in bacterial counts on the PCP medium was seen in piles 1 and 3 three weeks after the addition of highly contaminated soil and nutrients to piles 1-3. To piles 1 and 3, bark was also added, so these two piles received fresh carbon sources together with additional nutrients. The number of bacteria growing on PCP medium decreased after 21 weeks of composting, when the temperature of the piles was less than 10 °C, but then increased again toward the end of the composting period. When the temperature in the compost piles decreased to less than 10 °C, the chlorophenol degradation stopped also (Figure 3, Table 3). As a conclusion, indigenous microbes that were chlorophenol degraders in the compost piles were not able to work under 10 °C, which is the average temperature in Finnish soils. Still, the addition of nutrients and bark and frequent aeration changed the conditions beneficial for chlorophenol degradation in the compost piles. Evaluation of Bioremediation. The degradation of chlorophenols was faster in the laboratory than in the field: the overall mineralization rate in the bench-scale experiment was about 2% per day, and chlorophenol degradation rate in pilotscale composting was 0.3 or 1.3% per day, depending on the starting concentration of the chlorophenols. The laboratoryscale experiments were done under more favorable and controlled conditions. For example, the constant room temperature above 20 °C improves chlorophenol degradation in the laboratory, but is difficult to maintain during the outdoor composting in Finland. A good review of the results from field tests for the bioremediation of chlorophenol-contaminated soil is presented by Ha¨ggblom and Valo (3). In order to compare the efficiency of chlorophenol degradation in different field studies, the removal rates of chlorophenols can be calculated. The removal rate expressed as concentration of chlorophenols depleted per time is dependent on the starting concentration of chlorophenols, and it holds an assumption that the degradation rate is constant throughout the bioremediation (following zero-order kinetics), which is hardly ever the case.

Hence, the removal rate as such gives a very rough estimation of the efficiency of chlorophenol removal, but it does not take into account either the degree of mineralization or the contamination level reached after bioremediation. In our study, the chlorophenol removal rate was about 0.6 mg (kg dry wt)-1 d-1 at low contamination level (from 45 to below 10 mg of CPs (kg dry wt)-1), and 2.2-4.8 mg (kg dry wt)-1 d-1 at high contamination level (from about 1800 to 50 mg of CPs (kg dry wt)-1). In our laboratory test, the mineralization of radiolabeled PCP was 60% per month (2% per day). In pilot-scale composting studies by Valo and Salkinoja-Salonen (16) on an enrichment culture, the removal rate was 2.2 mg of CPs (kg dry wt)-1 d-1 at the level from 280 to 20 mg of CPs (kg dry wt)-1, and in studies by Mahaffey and Sanford (17) on a mixed culture in slurry bioreactors, the chlorophenol removal rate was 3.3 mg (kg dry wt)-1 d-1 at the contamination level from 100 to 0.5 mg of CPs (kg dry wt)-1. In both cases of Valo and Salkinoja-Salonen (16) and Mahaffey and Sanford (17), laboratory studies confirmed that the chlorophenol removal was due to mineralization. In the solid-phase treatment of soil in the field, Seech et al. (8) found the chlorophenol removal rate of 3.2 mg (kg dry wt)-1 d-1 (contamination level from 680 to 6 mg (kg dry wt)-1) in the soil inoculated with Pseudomonas resinovorans. The inoculated strain mineralized 12% of radiolabeled PCP in 48 h (6% per day) in the laboratory. The above-mentioned chlorophenol removal rates are in agreement with our studies with the consortium of chlorophenol degraders. Considerably higher removal rates could be found in field experiments on inoculation with white rot fungi Phanerochaete chrysosporium and Phanerochaete sordida: removal rates were from 4.3 to 9.3 mg (kg dry wt)-1 d-1 (contamination level from 1010 to 74 mg (kg dry wt)-1), but also up to 15% methylation of PCP and accumulation of pentachloroanisole were observed. The mineralization was not tested (6). According to earlier laboratory studies by Lamar and his co-workers (18), Phanerochaete spp. could mineralize PCP to a small extent (2-12% per month, 0.10.4% per day) in liquid culture, but in soil environment, most of the depletion of PCP by Phanerochaete spp. was due to methylation or conversion to non-extractable compounds. The augmentation with a chlorophenol-mineralizing strain Mycobacterium chlorophenolicum resulted in the chlorophenol removal rates of 0.8 and 9.5 mg (kg dry wt)-1 d-1 (the contamination levels from 790 to 10 mg (kg dry wt)-1 and from 8500 to 18 mg (kg dry wt)-1, respectively) (3). As a conclusion of these field tests, augmentation with chlorophenol-degrading pure cultures can significantly improve chlorophenol degradation in soil, provided that mineralization occurs. Relatively good degradation rates can also be achieved by amendment with mixed culture. However, when the contaminated soil itself has a sufficient number of chlorophenol-degrading (mineralizing) microbes, further inoculation will not enhance degradation, as was the case in our study.

Acknowledgments We thank Kirsi Sirkia¨ for skillful laboratory assistance and Rainer Peltola for his help in the field and the laboratory. The nutrients were analyzed by Novalab Oy, Finland. We thank also Antti Uusi-Rauva, head of Instrument Centre in University of Helsinki, for the use of radiorespirometry facilities and Kai Hurme for all his help with radioactive work. The straw compost was kindly provided by Mykora Oy, Kiukainen, Finland, and the remediated soil as well as the contaminated soil was provided by Metsa¨-Serla Oy, Finland. We appreciate the fruitful and fluent cooperation with Juha Hiltunen, head of Va¨a¨ksy Sawmill, and Assi Weber from Metsa¨-Serla Group, R&D Corporate. This work, which was part of EC Environment Programme (Contract EV5V-CT92-0240), was supported by the Finnish Ministry of the Environment.

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Received for review February 26, 1996. Revised manuscript received September 9, 1996. Accepted September 24, 1996.X ES960176U X

Abstract published in Advance ACS Abstracts, December 15, 1996.