VIEWS: 1 PAGES: 11 POSTED ON: 8/25/2012
Aerobic and Anaerobic Transformations of Pentachlorophenol in Wetland Soils Elisa M. D'Angelo* and K. R. Reddy ABSTRACT its degradation of chlorinated toxic organic chemicals Strategies to enhance biotransformation of pentachlorophenol (Renner, 1998). (PCP) in a spectrum of wetland soils were investigated under labora- Historically, environmental persistence of PCP and tory conditions, which included manipulations of electron acceptors less chlorinated phenols has been attributed to the ab- and donors, and PCP concentrations. Maximum transformation rates sence of degrading populations of microorganisms. were found at PCP concentrations <10 jxA/ (methanogenic condi- However, increasing numbers of observations in diverse tions) and >6 \iM to >23 \s,M (aerobic conditions). Differences in habitats indicate that transformation potential is wide- PCP toxicity and sorption among soils and treatments were largely spread, but is manifested only under favorable environ- governed by the activities of microbial groups. Within this concentra- mental conditions. Important variables include temper- tion range, transformation was observed in soils under aerobic and methanogenic conditions, but was inhibited under denitrifying and ature (Kohring et al., 1989), availability of electron SOa -reducing conditions. Aerobic PCP transformation initially pro- acceptors (Haggblom et al., 1993), electron donors (Ku- duced small amounts of pentachloroanisole (PCA). However >75% watsuka and Igarashi, 1975; Chang et al., 1996), nutrients of both chemicals disappeared in 30 d from five soils. Measured soil (Mileski et al., 1988; Schmidt, 1996), and toxic metals properties were not significantly correlated to aerobic transformation (Kuo and Genther, 1996). In wetland and aquatic sys- rates. Under methanogenic conditions, PCP was reductively dechlori- tems, these properties are often present as gradients nated to yield a mixture of tetra-, tri-, and dichlorophenols in eight resulting in a continuum of microbial activities. soils, with rates strongly correlated to measures of electron donor In aerobic soils, transformation of PCP by Flavobacte- supply (total C, N, organic C mineralization rates) and microbial rium and Rhodococcus spp., among others, proceeds biomass. Addition of protein-based electron donors enhanced reduc- though sequential hydroxylation and reductive removal tive dechlorination in a soil low in organic matter and microbial of chlorine substituents yielding poly-hydroxybenzene biomass. Results demonstrated the widespread occurrence of PCP transforming microorganisms in soils, which may be promoted by compounds that are eventually mineralized to CO2 (Uo- manipulating environmental conditions. tila et al., 1995; Xun et al., 1992). Several Rhodococcus spp. also methylate PCP resulting in production of vola- tile PCA (Middelorp et al., 1990). Several species of fungi mineralize PCP via ligninase enzymes (Mileski C ONTAMINATION of the environment with polychlori- nated phenols (CPs) is of global concern because of their widespread distribution and universal toxicity et al., 1988) and produce extracellular enzymes that polymerize CPs with humic substances (Bhandari et al., 1996; Ruttimann-Johnson and Lamar, 1997). to life (Escher et al., 1996; ATSDR, 1998). The most In anaerobic soils, the pathway for anaerobic transfor- common usage of CPs is treatment of wood against fungi mation of PCP is sequential replacement of chlorines by and insects, but other sources include production from hydrogen (reductive dechlorination), leading to phenol, chlorine bleaching of pulp (Kringstad and Lindstrom, benzoate, acetate, CO2 and CH4 (Kuwatsuka and Igara- 1984), combustion of organic matter and municipal solid shi, 1975; Zhang and Wiegel, 1990). To date, a few waste (Kanters et al., 1996), and partial transformation anaerobic isolates having the capacity for reductive de- of phenoxy pesticides such as 2,4-D and 2,4,5-T (Mike- chlorination of PCP have been discovered, and these sell and Boyd, 1985). often gain energy by coupling this process to oxidative Chlorophenols that enter nontarget upland, wetland, phosphorylation (Mohn and Tiedje, 1991; Loffler et al., and aquatic environments associate with colloidal and 1996). The resultant lesser chlorinated products from particulate matter and, if not photodegraded, eventually reductive dechlorination likely function as carbon and settle onto surface soils (Shiu et al., 1994). There they energy sources for those aerobic and anaerobic microor- may be biodegraded, depending on whether degrading ganisms involved (Haggblom and Young, 1990). microorganisms are present and whether appropriate Predicting the persistence of microbially transformed conditions exist for expression of this activity. There is toxic organic contaminants is currently hindered by lim- still much controversy about whether the presence of ited knowledge of the influence of environmental vari- microbial populations or environmental conditions lim- ables on degradation rates (Hart, 1996). Previous exper- iments in numerous wetland soils, however, have E.M. D'Angelo, Soil & Water Biochemistry Lab., Dep. of Agronomy, demonstrated that heterotrophic microbial activities Univ. of Kentucky, N-122 Agricultural Sci. Bldg. North, Lexington, were related to soil properties (D'Angelo and Reddy, KY 40546-0091; and K.R. Reddy, Univ. of Florida Wetland Bio- 1999), suggesting that similar relationships may also ex- geochemistry Lab., Soil and Water Sci. Dep., 106 Newell Hall, P.O. ist for transformation of toxic organic chemicals. The Box 110510, Gainesville, FL 32611-0510. Florida Agric. Exp. Stn. Journal Ser. no. R-07269. Mention of a specific product or trade name does not constitute endorsement of the University of Florida to the Abbreviations: A, aqueous concentration; CPs, polychlorinated phe- exclusion of others. Received 11 Mar. 1999. ""Corresponding author nols; DCP, dichlorophenol; EC;0, effective concentration; Ka, acid (email@example.com). dissociation constant; Kp, linear sorption coefficient; PCA pentachlor- oanisole; PCP, pentachlorophenol; T, total soil concentration; TCP, Published in Soil Sci. Soc. Am. J. 64:933-943 (2000). trichlorophenol; TeCP, tetrachlorophenol. 933 934 SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000 Table 1. Origin, classification, and description of organic and mineral wetland soils used in the study. State Soil name (symbol) Taxonomic class Description (domination vegetation) Organic Michigan Houghton Lake Peat (HLPI) euic, mesic Typic Haplosaprists Impacted by domestic waste discharge (Typha latifolia) Michigan Houghton Lake Peat (HLPU) euic, mesic Typic Haplosaprists Not impacted (Carex spp.) Florida Everglades (W2) euic, hyperthermic Typic Medihemists Impacted by P-enriched agricultural discharge (Typha spp.) Florida Everglades (W8) euic, hyperthermic Typic Medihemists Not impacted (Cladium spp.) Louisiana Salt marsh (LSM) euic, hyperthermic Typic Haplosaprists (Spartina spp.) North Carolina Belhaven muck (NCB) loamy, mixed, dysic, thermic Terric Haplosaprists Subsided organic agricultural soil Florida Lake Apopka muck (LAAF) euic, hyperthermic Typic Medifibrists Subsided organic agricultural soil Mineral Alabama Talladega (TAL) loamy-skeletal, mixed, mesic Rupta-Lithic-Entic, Freshwater sediment (Juncus spp.) Haplaudult North Dakota Parnell (PPP) fine, smectitic, frigid Vertic Argiaquolls Prairie pothole Louisiana Crowley (CR) fine, smectitic, hyperthermic Typic Albaqualfs Paddy soil (Oryza saliva) objectives of this study were to (i) determine whether 8-wk period. Samples of W8 (5 mL) and TAL (10 mL) were PCP transformers are commonly found in wetland soils, transferred to serum bottles (60 mL) and sealed with teflon- and (ii) identify chemical and biological conditions that lined butyl stoppers and aluminum crimps (Wheaton, Millville, promote this activity. This investigation attempts to NJ). Anaerobic bottles were purged with O2-free N2. Bottles were incubated in the dark at 28°C on a rotary shaker at 180 quantify the boundary conditions for microbial transfor- rpm. All experiments were conducted in triplicate. Separate mations of PCP in soils, including effects of concentra- EC50 values, defined as the effective concentration of PCP tion, sorption, electron acceptors and donors, and micro- that inhibited microbial activity by 50%, were calculated for bial biomass. the activities of CO2 production, methanogenesis, and PCP transformation in both soils. Effective concentrations were MATERIALS AND METHODS expressed on both PCP concentration dissolved in soil solution (EC50(diss0|Vf!(i), |jJW) and PCP concentration dissolved plus Soil Collection and Incubation sorbed to the soil (EC5o(lotai), (jimol kg"1). Three mineral and seven organic soils were collected from various wetlands in the continental USA, including soils from Influence of Chemical Amendments freshwater and estuarine, eutrophic and oligotrophic, organic on Pentachlorophenol Transformation and mineral, and natural and constructed wetlands (Table 1). Soils were previously shown to possess a wide range of Before initiation of transformation studies, soil slurries were biogeochemical properties (D'Angelo and Reddy, 1999), and pre-incubated for 14 d to obtain either aerobic or anaerobic selected characteristics are summarized in Table 2. These soils conditions (denitrifying, sulfate-reducing, or methanogenic). had no known previous exposure to PCP. For aerobic treatments, slurry (100 mL) was incubated in glass Samples of the surface soil were collected with a polyvinyl media bottles (500 mL) fitted with teflon-lined caps (Wheaton, chloride (PVC) corer (7.5 cm i.d. 15 cm), transferred to a 4-L Millville, NJ) and opened twice a week to re-aerate the head- plastic bottle, and returned in an ice chest to the laboratory space. For anaerobic treatments, slurry (80 mL) was incubated by overnight mail. When present, surface water samples from in serum bottles (160 mL) fitted with teflon-lined rubber stop- each wetland were also collected. Soils were passed through pers and aluminum crimps (Wheaton) and purged with O2- a 0.5 cm2 mesh sieve to remove large plant debris, shells, and free N2. Different anaerobic electron acceptor treatments were stones. Soils and water were stored in the dark at 4°C for a imposed by amending soils with a 30-d supply of a given maximum of 3 mo before being used in experiments. electron acceptor, calculated from consumption rates deter- Soils were collected either under drained or flooded condi- mined previously (D'Angelo and Reddy, 1999). Appropriate tions and, hence, were initially at different water contents and electron acceptor reducing conditions were confirmed by mea- redox potentials. To avoid diffusion constraints and develop- suring pore water for loss of NO3~ in denitrifying treatments, ment of microsites during transformation experiments, some loss of SOI" in SO4~-reducing treatments, loss of NH/ and soils were prepared as slurries with site water. The amount production of NO^ and SOJ" in aerobic treatments, and heads- of water used to prepare slurries was arbitrary except to avoid pace production of CH4 in the methanogenic treatments. Soils overdilution of solids and microbial biomass. The dry bulk with high amounts of bioavailable Fe (PPP, CR, TAL) were densities of slurries were 0.06 to 0.4 kg L~' for organic soils evaluated for PCP transformation under Fe(III)-reducing con- and 0.1 to 0.7 kg L"1 for mineral soils. Although there was a ditions instead of SOl'-reducing conditions. Iron(III) solution wide range in bulk densities, these were largely attributed to was prepared as described by Ghiorse (1994). All bottles were differences in organic matter content. Results are presented incubated horizontally with shaking. Preliminary experiments on a soil dry weight basis unless otherwise indicated. showed that this shaking protocol did not influence methano- genic activity, and previous work found it to be optimal for Pentachlorophenol Toxicity to Aerobic aerobic assays (Stark, 1996). All experiments were conducted in triplicate. and Anaerobic Soil Microorganisms After appropriate reducing conditions were attained, slur- Toxicity was evaluated under aerobic and methanogenic ries were spiked with PCP from a stock solution prepared in conditions in a selected organic soil (W8) and a mineral (TAL) 0.05 M NaOH to give an average final PCP concentration of soil using a dose-response approach. Five PCP concentrations 0.66 mmol kg"1 dry soil. This PCP concentration was chosen between 0 and 3.8 mmol kg~' were tested for effects on rates because it is the median level measured at contaminated sites of CO2 and CH4 production and PCP transformations over an in the USA (ATSDR, 1998). Both aerobic and methanogenic D'ANGELO & REDDY: TRANSFORMATIONS OF PENTACHLOROPHENOL IN WETLAND SOILS 935 sterile controls were included for the most biologically active rH rH soil (HLPI), and these were prepared by adding HgCl2 to a s n .s o ooo ooo O OO final concentration of 2% (soil dry weight basis) alone or with | +| +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 oo f> in M ^t in -^ autoclaving at 121°C at 0.1 MPa for 1 h on three consecutive Ids •8 ^ y o- TT t' tfi •<* '"' * pi 313 days. Sample sterility was confirmed by monitoring CO2 and methane production during the experiment. On Days 1, 3, 6,10,15,20,25, and 30, PCP and transforma- "3 tion intermediates were extracted from slurries (1-3 mL) with OB § f> « t»> rH in p»« m r< 6 mL acetonitrile containing 0.36 M H2SO4 for 16 h on a u.2 B ooooo eo 0 O 0 •~ ? +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 reciprocal shaker. After centrifugation (700 X g), crude ex- "o "^ in f*} f*j QO «\ ^ t-; 1*- in o tracts were stored in amber glass vials with teflon-lined caps f*l rH r*5 O CO f> ^ opi-i c ^ & f> rH rH rH at 4°C before derivitization and analysis. Preliminary spike _e recovery experiments showed this procedure yielded >95% recovery of mixed CPs from peat and mineral soils. Similar 01 •a I approaches for extraction of pesticides from soil matrices have a rH rH rH O O rH O rH |H rH been described (Chang et al., 1996). tn a .a jr o ooooeo 0 O 0 2 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 Additional experiments were conducted to determine e \o oo N o \o oo in whether additions of nutrients, vitamins, and electron donors S I 5SS2 could promote the methanogenic transformation of PCP in a B e soil that previously lacked this capacity. The protocol used was +1 similar to that employed to examine transformations under ; replications Aerobic pH rH O rH P4 rH rH rH different electron acceptor reducing conditions, except a lower O O0 PCP concentration was used (0.13 mmol PCP kg"1 equivalent + 1 +1 +1 +1 +1 +1 +1 +1 +1 +1 to 5.8 fxA/ in the dissolved phase) to avoid PCP toxicity. The vo -t in w ON rj p; rH P4 ^ in >n vo vo fj i/i ("* TT in K following deoxygenated solutions (1 mL) were added to sepa- rate glass tubes (27 mL) containing PPP soil slurry (3 mL) to provide the following 16 treatments: water (control), inorganic 1 O nutrients (Owens et al., 1979), inorganic nutrients + vitamins CM O -J p) m in oo N »t >n VO rH rH (Owens et al., 1979), catechol, benzoate, casein, yeast extract, 2 peptone, glucose, sucrose, maleic acid, fructose, maltose, acetic i E + 1 +1 +1 +1 +1 +1 +1 SS8S3S8 + 1 +1 +1 rH rH fl fi P< rH acid, ethanol, and propionate. Hydrogen (5 mL) was added a 1 to separate tubes (final concentration of 25 kPa). All carbon- based electron donor treatments were added at concentrations 'n VI of 88 mmol C kg"1 soil and included amendments with inor- o -H- ganic nutrients + vitamins. All treatments were conducted in a a, 3 2o oooeo O rH f*> O O 00 0 rH 0 0 OO triplicate and, at the end of 5 wk, PCP and transformation 2 .3 +1 +1 +1 +1 +1 +1 +1 + 1 +1 +1 *S PI in pi intermediates were extracted from soils as described above. V 9 J3 o 3 5 P! S. 2 o in o o O 0 9 O O0 1 b Analytical Methods •s n H Dissolved SOJ" and NO3~ concentrations were determined •H- » by analyzing pore water with a Dionex 4500i ion chromato- 'S z — n ^i ^n 0 OO V o „ f*) O O fl rH ^ ^ graph (Sunnyvale, CA). Headspace gases were measured us- B "8 1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 ing gas chromatographs equipped with detectors employing flame ionization for CH4 and thermal conductivity for O2 and o a. X r ^ rH O I/I O\ GO ^$ *** rH rH r'l C^ rH r-, |fj rH S«i3 V CO2. Carbon in living, nonresting microbial biomass was esti- mated using the substrate-induced respiration technique, as .s •a 1 described previously (D'Angelo and Reddy, 1999). 1 B. ^ ^ rH O CS I/I M rH rH W 4) •c Pentachlorophenol and transformation intermediates pres- £ 3 +1 +1 +1 +1 +1 +! +1 + 1 +1 +1 B ent in extracts were prepared as acetylated derivatives and '3 jS ffi M M rH M W rH O rH "fl 1/1 analyzed using a gas chromatograph equipped with a 63Ni t/3 S •a a B electron capture detector (Nicholsen et al., 1992). The identity 2 B and concentration of PCP and transformation intermediates V ^ O\ ("- C\ f> I-- ^ ^* •** fl fl M rH ^ G\ 00 PI rH rH f) £ u E were determined by comparing retention times of derivitized tM + 1 +1 +1 +1 +1 +1 +1 + 1 +1 +1 e 3 authentic standards of the highest purity available (>98%) o 9\ co in o i/) f} (Supelco, Bellefonte, PA, and Ultra Scientific, Hope, RI). a rH ("^ M l/> f'l G^ H GO t- rH O 00 ^f m rH rH fM M •° + •S "I Data Analysis rH rH O a u OJD OOOOOHrH 0 O 0 1 1 B b Transformation rates of PCP and intermediate metabolites qj ^ -H +1 +1 +1 +1 -H +1 +1 +1 +1 were described by zero-order kinetics (p-mol kg"1 d"1) deter- 1 I - 1 ^ 00 'S eg mined by linear regression analysis. Since experiments were rH-e initiated with high concentrations of PCP under well mixed ~ C« V g conditions, these rates may be considered maximum velocity . .3 ^ •B ' = rates, or potentials. As experiments progressed, PCP concen- _a •a ^s. H| trations decreased, and transformation rates became a func- jt ES l «4 rJ ?J ^ ^5 y ^ Sii S ffi 1? ? J Z »J | H§;S tion of PCP concentrations. Hence, first order (d"1) rates 1 5 6 s II 936 SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000 120 TAL 120i -» Initial NO., • Final NO, - 392i6 mmol kg'1 232±18 mmol kg' •4- aerobic -B- methanogenic 'en ————————B ^ "3 0 EC SO(lotal) 0.75 1.5 2.25 *-SO(total) 0 1.25 2.5 3.75 5.0 10 20 10 20 E 120 E. 0.8- C. 0.8 • D. -•-PCP *———————————— £ -*- 2,3,4,5 TeCP 0.6- 1 Initial SO, - Final SO, *• 254±17 mmol kg-1 86±14 mmol kg-1 „ , -•-3,4,5 TCP 0.4- 0.2- 0.4 • 0.2- [\ A -«-3,5DCP 0 10 20 30 0 10 20 30 0.61 E. 0.4 ^k= 0.2- 0.0 20 Time (d) Time (d) l.S 2.25 0 1.25 2.5 3.75 5.0 Fig. 2. Microbial transformation of pentachlorophenol (PCP) in Initial PCP concentration (mmol kg"1) Houghton Lake constructed marsh soil (HLPI) under four electron acceptor reducing conditions, and in aerobic and methanogenic Fig. 1. Influence of pentachlorophenol (PCP) concentration on activ- sterile controls: (A) O2; (B) NO3~; (C) S O S ; (D) CO2; (E) O2 + ities of aerobic and methanogenic microorganisms in mineral TAL 2% HgCl2; and (F) CO2 + 2% HgCl2. Dotted lines in (E) and (F) (A-C) and organic W8 (D-F) wetland soils: (A) and (D) CO2 represent autoclave + HgCl, treatments. Initial and final NO.f and production; (B) and (E) CM, production; and (C) and (F) PCP S(>4~ refer to concentrations at the beginning and end of the experi- degradation. Each value represents the mean of three ment. Each value represents the mean of three replications ± one replications ± one standard deviation. EC5l)(M,i) represents the total standard deviation. PCP concentration in the soil (dry weight basis) that inhibits micro- bial activity by 50%. RESULTS were also determined from nonlinear regression analysis. Rate Pentachlorophenol Toxicity to Aerobic constants were calculated from data generated after any lag and Anaerobic Soil Microorganisms period. The length of the lag period was defined as the period before a significant (P < 0.05) decrease in concentration was Aerobic and methanogenic activities (CO2 and CH4 observed between successive time increments, as determined production and PCP transformation) were typically in- by t statistic. hibited when soils were amended with increasing con- In separate batch isotherm experiments, linear sorption co- centrations of PCP (Fig. 1). However, specific effects efficients (Kf, L kg"1) for PCP were determined for all soils differed between aerobic and anaerobic treatments and examined (D'Angelo, 1998). These were then used to convert total PCP concentration (T, mmol kg"1) to aqueous concentra- between mineral and organic soils. In the mineral TAL tions (A, mmol L"1): soil, CO2 and methane production, and PCP transforma- tion (under methanogenic conditions only), were signifi- A = T • (Kf + 9 • pb"1)-1  cantly reduced at >0.38 mmol PCP kg"1 soil. In contrast, where 9 is the volumetric water content (L water L"1 soil) CO2 production and PCP transformation were relatively and pb is dry bulk density (kg L"1) of soil slurry. unaffected at any PCP concentrations tested under aero- Table 3. Inhibition of aerobic and methanogenic microbial activities by pentachlorophenol (PCP) in mineral TAL and organic W8 wetland soils. TAL W8 Aerobic Methanogenic Aerobic Methanogenic Parameter Units C02 PCP CO2 CH4 PCP C02 PCP CO2 CH4 PCP 1 ECso (total)t mmol 1kg" >1.9 >1.9 0.48 0.68 0.56 >3.8 >3.8 3.0 >3.8 1.2 Kpt Lkg" 1 307 307 32 32 32 147 147 99 99 99 <e • P-')§ Lkg- 6 6 7 7 7 17 17 18 18 18 ECso (dittolved)H (unol L ' >6.0 >6.0 12 17 14 >23 >23 26 >32 10 t PCP concentration on a soil dry weight basis that inhibited microbial activity by 50%; EQoitoui) is effective concentration. £ Soil:water partition coefficient determined in separate experiments (D'Angelo, 1998). Values are summarized in Tables 4 and 6. § Ratio of volumetric water content (6) and bulk density (p) of soil slurries used in the study. H PCP concentration dissolved in the soil solution that inhibited microbial activity by 50%, calculated from Eq.  in text. Overall loss 75 ± 11 Chlorophenol concentration (mmol kg'1) 96 ± 1 100 ± 0 87 ± 1 in 30 d 95 ± 0 O % 0 0 0 0 0 0 0 OC O\ *• K» O Table 4. Aerobic transformations of pentachlorophenol (PCP) in wetland soils. Each value represents the mean of three replications ± one standard deviation. 0.009 0.042 0.023 0.006 0.161 ± 0.011 First-order rate d-' 0.139 ± 0.338 ± 0.224 ± 0.139 ± 0 0 0 0 0 0 0 00 o O\ 4*. s» PCP loss H-mol kg"1 d"1 O JB 0a B - f & J g° t Maximum 45 ± 1 32 ± 3 77 ± 7 44 ± 6 44 + 8 *||ti, „ rate 0 0 0 0 0 0 0 o c/5 c 13 H [a re a I i-s§ III;- §g§- S5 a. S " a O §^ I «9 3 * %£ a' g- B> e g t &9 </) ^o o a o B ^xI l s 3 s re •- « o •^ re 81 o. a E? B B -t r? s re s &a 1 So re re z *T re VI O o' ^», O. I re i a I. 3 B S E VI & \ a- 0 a a a a •f re re i I S =" 82, I: - 1 u W A<» 10 ± 0 If a 3 % re 16 ± 0 20 ± 0 4± 0 S 4 ± 2 •9 VI NAfl time Lag NA NA NA NA NA ND a. S' 1+ ^_ a o 0 re fD VI d bic conditions. Therefore, EC50 for aerobic samples could not be calculated. However, based on the maxi- 0.0002 0.0003 0.0002 0.0027 0.0034 0.0006 0.0012 ± 0.0005 0.0030 First-order rate mum concentration tested, the EC50(totai) (PCP concentra- tion on a dry soil basis) was >1.9 mmol kg"1 soil for aerobic activities compared to between 0.5 to 0.7 mmol d-1 0 0 0 0 0.0017 0.0029 0.0025 0.0099 0.0009 0.0056 0.0030 kg ' soil for methanogenic populations (Table 3). For the organic W8 soil, similar trends in toxicity were observed as for TAL, except that the threshold Methylation concentrations of PCP for microbial inhibition were sig- nificantly higher (Fig. 1 and Table 3). For example, the Maximum rate nmol kg"1 d"1 1.1 ± 0.1 1.1 ± 0.1 1.8 ± 0.2 1.2 ± 0.3 3.5 ± 1.9 2.4 ± 1.2 0.9 ± 0.3 EC5o(totai) for aerobic activities and methanogenesis were 0+ 0 >3.75 mmol kg"1, and between 1.2 and 3.0 mmol kg"1 II Lag time longer than 30-d experimental period; NA, not applicable; ND, not determined. 0 0 0 0 for anaerobic PCP transformation and CO2 production. § Initial PCP concentration in the dissolved phase calculated from Eq. , see text. Influence of Chemical Amendments on PCP Transformation 6±0 6±0 5±2 time NDfl Lag <1 <1 <1 <1 NA NA NA NA d The presence or absence of specific types of electron acceptors had significant effects on rates and mecha- nisms of PCP transformation in wetland soils (Fig. 2 and 3; Tables 4-6). Transformation was attributed to nmol L-' 2.82 2.28 0.83 0.36 2.34 Initial biological activity since HgCl2 + autoclaved samples PCP§ 1.25 1.29 10.4 12.8 238 showed no changes in PCP concentration during the study (Fig. 2). Except for two soils (PPP and CR), PCP I Determined in a separate study (D'Angelo, 1998). transformation occurred in each soil under at least one electron acceptor-reducing condition. coefficient (A'p)i Linear sorption Under aerobic conditions, eight soils transformed PCP, including all organic soils and one mineral soil Lkg- 1 1.3 t See Table 1 for soil name symbols. 931 463 147 549 225 307 50 49 2017 (TAL) (Table 4). Pentachlorophenol transformation proceeded by at least two different mechanisms. Within the first week after PCP treatment, pentachloroanisole (PCA) was detected in seven soils as a methylation product of PCP, with maximum rates of between 0.9 and 3.4 (xmol kg"1 d"1 and first order rates between HLPI + Hg + 0.0009 and 0.01 d"1. Between 4 and 20 d, four of the autoclave Sterile controls HLPI + Hg organic soils (HLPI, W2, W8, and LAAF) and the min- HLPU LAAF HLPI eral soil TAL additionally exhibited losses of PCA. NCB Organic LSM TAL Mineral PPP W8 W2 CR ! 9. o Soilt 3 C/3 *1 0) P= X fa D. O. h+i 3 P5 o> a P 938 SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000 Table 5. Anaerobic transformation rates of pentachlorophenol (PCP) and reductive dechlorination intermediates (tetra-, tri-, and di- chlorophenols) in wetland soils in the presence of inorganic electron acceptors. The sequential dechlorination pathway was PCP—>2,3,4,5 tetrachlorophenol (TeCP)—»3,4,5 trichlorophenol (TCP)—'3,5 dichlorophenol (DCP) unless otherwise indicated. Each value represents the mean of three replications ± one standard deviation. Values in parentheses are first-order rates (d '). Siilfate Overall loss Soilt Nitrate Lag time PCP — —— > TeCP ————> TCP —— > DCP — in 30 d p.mol kg"1 d~' d ——————— (xmol kg"1 d"1 ———————— Organic HLPI 0 NAt 0 ND 0 HLPU 0 NA 0 ND 0 W2 0 15 ±0 39 ± 2 (0.29) 54 ± 12 (0.20) 21 ±S (0.027) ND 33 ± 26 W8 0 NA 0 ND 0 LSM 8 ± 3 (0.009) <1 23 ± 3 (0.065) 2±S (0.005) trfl 13 ± 19 NCB 0 NA 0 ND 0 LAAF 0 12 ± 0 40 ± 10 (0.22)# 27 ± 3 (0.25) 23 ± 7 (0.10) 22 ± 9 (0.09) 69 ± 15 Mineral TALft 0 NA 0 ND 0 PPPtt 0 NA 0 ND 0 CRtt 0 NA 0 ND 0 t See Table 1 for soil name symbols. t Lag phase longer than 30 d experimental period. § Not detected. II Trace amounts of 3,4,5-TCP were detected. # Degradation pathway was PCP-»2,3,4,5-, 2,3,5,6,-, 2,3,4,6-TeCP—23,4,5-, 3,4,5-TCP. ft These soils were evaluated for PCP reduction under Fe(III)-reducing conditions instead of sulfate-reducing conditions. order rates between 0.139 and 0.338 d"1. No other chlori- transformation of PCP (Table 5). Under SO^-reducing nated organics were detected. This indicated the pres- conditions, three organic soils (W2, LSM, and LAAF) ence of either mineralization or chemical binding- showed loss of PCP. After lag periods of between 1 and attachment to soil. In these five soils, total loss of PCP 15 d, PCP was transformed at maximum rates between and PCA ranged between 75 and 100% in 30 d. The 23 and 40 (xmol kg"1 d"1 and first-order rates between remaining five soils showed no loss of PCP during the 0.065 and 0.29 d"1. The dominant mechanism of PCP incubation period. transformation was sequential reductive dechlorination Under denitrifying conditions, only LSM transformed to tetrachlorophenol (TeCP), trichlorophenol (TCP), PCP, as indicated by accumulation of trace amounts of and dichlorophenol (DCP), which occurred primarily the reductive dechlorination product 2,3,4,5-TeCP (Fig. through the pathway PCP-»2,3,4,5-TeCP-^3,4,5-TCP-> 2 and 3; Table 5). Of those soils tested under Fe (III)- 3,5-DCP, demonstrating a preference for removal of reducing conditions (PPP, TAL, and CR), none showed chlorines ortho- and para- to the hydroxyl group. The Table 6. Transformations of pentachlorophenol (PCP) and reductive dechlorination intermediates (tetra-, tri-, and di-chlorophenols) in wetland soils under methanogenic conditions. The sequential dechlorination pathway was PCP—>2,3,4,5 tetrachlorophenol (TeCP)—>3,4,5 trichlorophenol (TCP)—>3,5 dichlorophenol (DCP) unless otherwise indicated. Each value represents the mean of three replications ± one standard deviation. Values in parentheses are first-order rates (d~'). Linear sorption Initial Lag Overall loss Soilt coefficient (K,)$ PCP§ time PCP - -*• TeCP — -> TCP — *• DCP in30d j _ M ,___, j_, 0/ (jimol L"1 d Organic HLPI 116 5.52 6±0 70 ± 9 (0.26) 70 ± 7 (0.24) 65 ± 5 (0.23) 65 ± 5 (0.25) 97 ± 0.6 HLPU 314 2.32 13 ± 4 64 ± 9 (0.18) 77 4 (0.23) ± 59 ± 9 (0.10) 59 ± 9 (0.10) 63 ± 14 W2 88 5.58 6±0 60 ± 4 (0.39) 66 ± 7 (0.50) 45 ± 2 (0.24) 45 ± 2 (0.24) 99 ± 0.2 W8 99 5.51 9 ± 2 63 ± 4 (0.38) 57 ± 7 (0.44) 53 ± 9 (0.41) 49 ± 16 (0.39) 99 ± 0.2 LSM 66 11.0 25 ± 2 (0.082) 0 Irll ND# 0 NCB 286 2.89 NAtt 0 ND 0 LAAF 48 6.50 4± 0 38 ± 2 (0.21)ft 39 ± 13 (0.23) 38 ± 3 (0.16) 22 ± 8 (0.17) 93 ± 1.4 Mineral TAL 32 19.4 NA 0 ND 0 PPP 13 50.0 NA 0 ND 0 CR 1.8 159 NA 0 ND 0 Sterile controls HLPI + Hg ND 25 ± 2 (0.15) (0.15) (0.018) ND 0 HLPI + Hg + NA 0 ND 0 autoclave t See Table 1 for soil name symbols. t Determined in D'Angelo (1998). § Initial PCP concentration in the dissolved phase calculated from Eq. , see text. H Trace levels detected. # Not detected. ft Lag phase longer than 30 d experimental period. D'ANGELO & REDDY: TRANSFORMATIONS OF PENTACHLOROPHENOL IN WETLAND SOILS 939 Table 7. Relationships between soil properties and maximum rates of pentachlorophenol (PCP) transformation ((unol kg'1 d ') in wetland soils. These relationships apply to soils with PCP concentrations <10 |JtM in the dissolved phase. Aerobic transformation rate Methanogenic transformation ratef 2 Soil property (*) Units Equation r Equation r2 First order rate d-i 230 A: 0.89*** 196 x 0.80** 10 Total C mmol g ' NS| 1.7 x - 2.7 0.58* 7 Total N ixmol N g" NS 35 In x - 200 0.77** 7 Total P |unol N g~ NS 30 Injr - 43 0.49* 7 Microbial C (jimol C g~ NS 35 In x - 80 0.94*** 7 Aerobic CO2 + CH, (jimol C g" d'1 NS 29 In x - 20 0.66* 7 Anaerobic CO2 + CH4 ixmol C g~ d^1 NA§ 22 In x + 17 0.83** 7 Inorganic Nil ixmol N g~ NS 19 In x - 17 0.71* 7 Organic N (jimol N g~ NS 35 In x - 200 0.77** 7 Unavailable P1[ ixmol P g~ NS NS 7 *, **, *** Significance at the P =s 0.05, 0.01, and 0.001 levels, respectively. t Nitrate and sulfate inhibited degradation under anaerobic conditions. t Not significant at P £ 0.05. § Not applicable. 11 Determined as water soluble + KCl-exchangeable nutrients. agricultural LAAF soil exhibited more complex dechlo- peptone (37%), and acetate (5.1%) treatments (data not rination pathways, in which 2,3,5,6-TeCP, 2,3,4,6-TeCP, shown). The dominant pathway was through reductive and 2,3,5-TCP were also detected, indicating sequential dechlorination yielding 2,3,4,5-TeCP and 3,4,5-TCP; para-, ortho-, as well as meta- dechlorination pathways. however, no net loss of CPs was observed. After 30 d, losses of PCP and breakdown intermediates were between 13 and 69% for these three soils (Table Relationships between PCP Transformation 5). Monochlorophenols were not detected in any of Kinetics and Soil Properties the samples. Under methanogenic conditions, six organic soils Regression analysis showed that none of the mea- transformed PCP, but none of the mineral soils did so sured soil properties was significantly (P < 0.05) corre- (Fig. 2 and 3; Table 6). For the transforming soils, there lated to the duration of the lag periods before detection was a lag phase of between 1 and 13 d before loss of PCP transformation. Moreover, none of the mea- of PCP, after which maximum rates were found to be sured soil properties was significantly correlated to aero- between 25 and 70 (jimol kg"1 d"1 and first-order rates bic PCP transformation rates (Table 7). between 0.082 and 0.39 d"1. As observed under SOi~ Pentachlorophenol transformation under anaerobic -reducing conditions, the dominant mechanism for conditions was typically inhibited by the presence of transformation was reductive dechlorination, which usu- NO3~, Fe(III), SO?- (Table 5) and dissolved PCP con- ally followed the sequence PCP->2,3,4,5-TeCP—3,4,5- centrations >10 to 14 (jiM (Table 3, see discussion). TCP—>3,5-DCP. Again, LAAF exhibited more complex Hence, to predict anaerobic PCP degradation rates in dechlorination pathways than other soils, including soils, one must ensure that conditions are methanogenic PCP-»2,3,5,6-TeCP—2,3,5-TCP, 2,3,6-TCP->3,5-DCP and that the dissolved PCP concentration is below the and PCP—2,3,4,6-TeCP—2,3,6-TCP. The sum of losses toxicity threshold level. After taking these considera- of PCP and intermediates ranged between none and tions into account, several soil properties were signifi- 99% in 30 d (Table 6). cantly correlated to PCP transformation, including total Rate constants for PCP transformation intermediates organic C, N, and P content, microbial biomass, aerobic were determined by summing the concentrations for a and anaerobic carbon mineralization rates, and bioavai- given intermediate and all preceding products at each lable N. Microbial C accounted for more than 90% of time step, and calculating the least squares fit through the points (Tables 5 and 6). For soils capable of PCP transformation under SOij'-reducing conditions, the rate-limiting steps in overall CP loss were reductive dechlorination of TeCP, TCP, and DCP, as indicated O HLPI by accumulation of these intermediates compared to the parent compound PCP. For example in LSM soil, y = 35 Ln(x) - 80 PCP was almost completely transformed to 2,3,4,5- r2 = 0.94 TeCP, which was not reduced further (Table 5). Under methanogenic conditions, most soils (except LSM) dem- onstrated similar dechlorination rates for CPs con- 0 100 150 taining high and low numbers of chlorine substituents (Table 6). Microbial C (mmol C kg-') In PPP soil maintained under methanogenic condi- Fig. 4. Relationship between microbial biomass C and maximum pentachlorophenol (PCP) degradation rate in seven wetland soils tions and amended with nutrients, vitamins, and elec- under methanogenic conditions. This relationship applies to soils tron donors, PCP transformation was only observed in where PCP concentration in the dissolved phase was less than the the water-alone control (8.1%), yeast extract (34%), toxic level of 10 (J.M (see discussion). 940 SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000 the variability in rates of reductive dechlorination of plains why these soils did not transform PCP. In compar- PCP (Fig. 4). ison, the TAL and PPP soils transformed PCP in the toxicity and electron donor studies when concentrations were below threshold levels. DISCUSSION Transformation under both aerobic and anaerobic con- Our results indicate that most wetland soils contain ditions was also restricted at concentrations of PCP < microorganisms with the capacity to transform PCP, 0.3 u,M, at which PCP transformation rates became first despite no known history of contamination. However, order (Fig. 2d, 3a, and 3d). At low concentrations, slow expression of this capacity was regulated by chemical desorption kinetics plays a major role in regulating and biological conditions, including contamination transformation rates (Schlebaum et al., 1998). More- level, type of electron acceptors and donors, microbial over, in soils contaminated for long periods, PCP and biomass, and the co-contaminant (Hg (II)). intermediates may become less bioavailable as they dif- High (>10 (jiAf) and low (<0.3 jjJVf) PCP concentra- fuse into microbially inaccessible soil zones. Therefore tions typically restricted transformation in soils. This rates in the present study, in which experiments were inhibition was attributed to toxicity and limited bioavail- conducted in freshly contaminated soils, may overesti- ability, respectively. Pentachlorophenol toxicity results mate actual rates. Our results and those of others (Apa- from its influence on energy transduction processes car- jalahti and Salkinoja-Salonen, 1984; Bellin et al., 1990 ried out by the cell (Escher et al., 1996). These effects Mileski et al., 1988; Mohn and Kennedy, 1992), clearly were reflected as decreased rates of CO2 production, show the influence of contaminant concentration on methanogenesis, and PCP transformation. transformation of toxic organics in soils, and the role Comparing EC50(totai) values, PCP was apparently more of sorption in regulating contaminant bioavailability. toxic to methanogenic activities compared to aerobic Sorption of PCP may be controlled by manipulating pH, activities and in mineral soils compared to organic soils. types of microbial activity (e.g., aerobic and anaerobic), These results can at least be partially explained by differ- and organic C content in contaminated soils. ences in sorption, which was greater under aerobic con- Availability of electron acceptors was also a key regu- ditions and in organic soils (Tables 3, 4, and 6). For lator of both rates and pathways of PCP transformation example, sorption is known to provide a protective in soils. In the presence of O2, most soils produced mechanism by removing PCP from the soluble (bioavai- pentachloroanisole (PCA) within 1 d after PCP treat- lable) pool (Apajalahti and Salkinoja-Salonen, 1984). ment. Methylation is mediated by common aerobic bac- Sorption of chlorophenols is increased in soils with high teria and fungi such as Rhodococcus rhodochrous, Pha- concentrations of H + (pKa of PCP = 4.74), dissolved nerochaete chrysosporium, and P. sordida (Middelorp cations, and soil organic matter (Westall et al., 1985; et al., 1990; Lamar et al., 1990). Subsequently, PCP and D'Angelo, 1998). Aerobic processes such as nitrification PCA were lost from many soils without the appearance and sulfide oxidation tended to acidify soils, while an- of chlorinated intermediates, indicating either mineral- aerobic activities produced higher pH values (Table 2). ization to CO2 or chemical binding-attachment with Hence, it was expected that PCP transformations would other pesticide moieties or humic substances. Assuming proceed at higher concentrations in aerobic and organic degradation was the dominant mechanism, the maxi- soils which matched with experimental results (Table 3). mum PCP loss rates of up to 77 (xmol kg"1 d""1 observed Using Eq. , average concentrations of PCP dis- in this study are typically higher than those measured solved in soil solution ranged between 0.4 and 238 jxM previously (Briglia et al., 1994; Haggblom and Valo, (Tables 4 and 6). The EC50(dissoived) for PCP transformation 1995; Laine and Jorgensen, 1997). Higher rates in the (i.e., concentration of dissolved PCP that decreased PCP present study perhaps reflected an exclusion of diffusion transformation by 50%) was 10 to 14 u.M for methano- constraints by constant shaking. genic treatments (Table 2). The EC50(diSsoived) for aerobic Among the soil properties measured, none was signif- treatments could not be calculated because inhibition icantly correlated to aerobic transformation rates, indi- was not observed at any concentration tested (up to cating that availability of C, inorganic N (ranging be- 6 |xM for the mineral soil and 23 u.M for the organic tween 1330 and 9770 u,M), and soluble P (ranging soil). These results suggest that aerobic microorganisms between 3 and 488 u,M) were not the primary regulators were less affected by PCP level than methanogenic con- in the soils tested (Table 7). Schmidt (1996), however, sortia. These results generally agree with others, with found a highly significant correlation between aerobic reported toxicity values ranging from 15 to 1900 jjuM transformation rates in PCP-contaminated groundwater for aerobes (Mileski et al., 1988; Stanlake and Finn, and soluble P between 8 and 90 \iM. As observed in 1982) and 0.45 to 10 |o,M for anaerobes (Mohn and our study, Laine and Jorgensen (1997) also found no Kennedy, 1992; Wu et al., 1993; Uberoi and Bhatta- correlation between bacterial biomass and aerobic PCP charya, 1997). transformation rates in pilot-scale bioremediation ef- Knowledge of toxicity threshold concentrations are forts in Finland. key to predicting the transformation potential of PCP Under anaerobic conditions, but not under aerobic in soils. For example, in the electron acceptor study, conditions, NO3", Fe(III), and SO^~ inhibited PCP trans- PCP concentrations in the methanogenic mineral soils formation. For most soils, lack of transformation under PPP (50 p,Af), CR (159 |xM), and TAL (19 p,Af) were intermediate reducing conditions was probably not due higher than the threshold EC50(dissoived) (l>«M), which ex- to toxicity, since both NO3~ and SOi" were consumed D'ANGELO & REDDY: TRANSFORMATIONS OF PENTACHLOROPHENOL IN WETLAND SOILS 941 in anaerobic treatments (Fig. 2 and 3). These results Recent discovery of toxaphene-, dieldrin-, and DDT- are in accordance with the paradigm that denitrifiers, contaminated soils in this former agricultural field Fe(III)-reducers, and SO4~-reducers outcompeted de- (SJRWMD, 1999) suggests the occurrence of cross-accli- halogenators for common electron-donating substrates mation by PCP dechlorinators. (Chang et al., 1996; Fennel and Gossett, 1998). The co- Several soil properties were highly correlated to PCP occurrence of reductive dechlorination and SC^" reduc- transformation rates under methanogenic conditions, tion in LAAF, W2, and LSM soils, however, may reflect including total C, N, and P, microbial C, aerobic and the ability of some dehalogenators to compete effec- anaerobic C mineralization rates, and bioavailable N tively with SOI'-reducers for electron equivalents. The (Table 7). Kuwatsuka and Igarashi (1975) also observed specific identity of electron donors required for reduc- high correlations with soil organic matter. Microbial tive dechlorination of PCP, and comparisons of affinity C showed the highest correlation probably because it constants between anaerobic microbial groups, have yet integrated many of the regulators of transformation to be determined. (e.g., types and amounts of electron donors and enzyme Under methanogenic conditions, PCP transformation systems) into one measurement. Also, since bacteria are in eight soils proceeded via reductive dechlorination, in largely composed of protein, it is plausible that dead which electrons derived from decomposition of organic microbial cells served as electron donors for reductive matter replaced Cl~ atoms of PCP. One can only specu- dechlorination by the degrading populations. While ad- late about the identity of microbial species and enzymes dition of nutrients and vitamins did not enhance PCP responsible for anaerobic PCP transformation in this transformation in PPP soil under methanogenic condi- study. However, enhanced transformation in PPP soil tions, protein-based donors did, demonstrating the pri- in response to the addition of the protein-based electron mary role that electron donors and microorganisms play donors yeast extract and peptone indicated the involve- in regulating reductive dechlorination. ment of proteolytic and amino-acid fermenting bacteria. Methanogenic soils treated with 2% HgCl2 plus auto- Clostridium-like species have previously been impli- claving did not show reductive dechlorination, indicat- cated in dechlorination of CPs (Zhang and Wiegel, 1990; ing a requirement for biological activity. In the absence Madsen and Licht, 1992). These results indicate that of autoclaving, however, reductive dechlorination pro- addition of proteinaceous compounds may be a viable ceeded, albeit at reduced rates, suggesting that dechlori- strategy to enrich for PCP-transforming microorganisms nation activity can be highly resistant to Hg (II) (Fig. 2). and to bioremediate chlorophenol-contaminated soils. In contrast to methanogenic soils, PCP transformation The lag time of 1 to 6 d observed prior to initiation under aerobic conditions was completely inhibited in of dehalogenation was not significantly correlated to Hg (II) treatments with and without autoclaving, indi- any of the soil properties. Lag times have been observed cating a higher microbial sensitivity to Hg (II). Thus co- for other microbial activities, including denitrification, contamination with Hg and possibly other heavy metals SO4~-reduction, and methanogenesis (D'Angelo and may be an important consideration when formulating Reddy, 1999), and reductive dechlorination (Linkfield remediation protocols. et al., 1989). Lag times may be explained by limiting In conclusion, this study has demonstrated the wide- environmental (electron donors and redox potential) or spread geographic distribution of microorganisms capa- biological conditions (populations and induction and ble of PCP transformation, even in systems with no synthesis of enzyme systems) (Linkfield et al., 1989). In known history of contamination. In addition, this study our study, however, the latter likely predominated, in has shown the extent to which PCP transformations are view of the fact that soils were preincubated under de- regulated by selected factors, including PCP concentra- sired reducing conditions well before PCP amendment tion, types of electron acceptors and donors, and micro- to the soils. bial biomass. The relationships between these proper- Maximum PCP transformation rates under methano- ties and transformation processes provided in this study genic conditions approached 70 jjimol kg~' d"1, which may be useful in predicting environmental persistence is higher than rates reported for many other soils (Ku- as a function of site specific conditions, as well as provide watsuka and Igarashi, 1975; Chang et al., 1996; Mikesell insight about potential impediments to in situ transfor- and Boyd, 1988). However, these reported rates are mation. Future studies should focus on identifying mi- lower than that observed for a methanogenic PCP-accli- crobial species involved in transformation, and de- mated consortia in bioreactors that transformed 10 ^M termining how chemical and biological factors influence PCP at 44000 ixmol kg^1 d~' (Wu et al., 1993). their transformation of PCP and other toxic organics Most soils that transformed PCP under SO;j~-re- in soils. ducing and methanogenic conditions showed preferen- tial ortho and para dechlorination, resulting in the pro- ACKNOWLEDGMENTS duction of 2,3,4,5-TeCP, 3,4,5-TCP and 3,5-DCP. This pathway has been shown to be common in unacclimated This research was partially financially supported by the U.S. Department of Agriculture National Research Initiative microbial communities (Nicholsen et al., 1992). In the Competitive Grants Program. We would like to gratefully agricultural LAAF soil, however, additional meta de- acknowledge the cooperation of several researchers who pro- chorination pathways were observed, which confirmed vided soils used in the study, Drs. E. Roden (Univ. of Ala- the pathways previously observed in anaerobic soils bama), C. Lindau and R. DeLaune (Louisiana State Univ.), (Kuwatsuka and Igarashi, 1975; Murthy et al., 1979). C. Crozier (North Carolina State Univ), J. Richardson (North 942 SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000 Dakota State Univ.), R. Kadlec (Wetland Management Ser- vices, MI), and Mr. J.R. White and M.M. Fisher (Univ. of Florida), and the statistical analysis advice of J.M. Harrison (Senior Statistician, Univ. of Florida). Florida Agricultural Experiment Station Journal Series no. R-07269. NAY & BORMANN: SOIL CARBON CHANGES 943 Wu, W.M., L. Bhatnagar, and J.G. Zeikus. 1993. Performance of Zhang, X., and J. Wiegel. 1990. Sequential anaerobic transformation anaerobic granules for transformation of pentachlorophenol. Appl. of 2,4-dichlorophenol in freshwater sediments. Appl. Environ. Mi- Environ. Microbiol. 59:389-397. crobiol. 56:1119-1127. Xun, L., E. Topp, and C.S. Orser. 1992. Purification and characteriza- tion of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp. J. Bacteriol. 174:8003-8007.
Pages to are hidden for
"_2000_ Aerobic and Anaerobic Transformations of "Please download to view full document