SOIL COMPOSITION AND SUPPRESSION OF PYRENE MINERALIZATION BY PHANEROCHAETE CHRYSOSPORIUM B. Tucker1, J.E. McLean2, J.S. Brar2 and A.J. Anderson1, 1Department of Biology, Center for Environmental Toxicology, Utah State University, Logan, UT 84322-5305; and 2Utah Water Research Laboratory, Utah State University, Logan, UT 84322-5305 ABSTRACT Soils from four different geographic locations with different compositions all reduced mineralization of pyrene by P. chrysosporium when compared to a non-soil system. Two clays, bentonite and kaolinite, differentially decreased the mineralization of pyrene. Bentonite sorbed pyrene to a greater extent and was more inhibitory to mineralization than kaolinite. Sand had little effect on mineralization. Four artificial soils, made with different proportions of sand, silt and clay, free of organic matter and microbes, inhibited mineralization by P. chrysosporium. Inhibition increased with greater proportions of silts. These findings suggest that soil type may influence the bioremediation potential of P. chrysosporium. KEY WORDS pyrene, Phanerochaete chrysosporium, bentonite, kaolinite, mineralization. INTRODUCTION P. chrysosporium were isolated from the native soil [11]. However, mineralization The wood decay fungus Phanerochaete with the sterilized soil and P. chrysospo- chrysosporium utilizes an extracellular rium was still below that of the inoculum complex of lignin peroxidase enzymes to placed directly onto the pyrene-agar su r- degrade the lignin content of the wood cell face [11]. Consequently, in this paper we walls [1, 2]. This same complex is impl i- searched for abiotic features of the soil that cated in the degradation of pollutants of could explain the suppression in the steri l- diverse structures [3, 4]. Such degradation ized soil. is readily demonstrated in the laboratory in defined media conditions [3, 5]. Cons e- It seemed possible that the reduction in p y- quently, the potential of P. chrysosporium rene mineralization by sterilized soil could to degrade pollutants in the environment is be both physical and chemical in nature. being investigated [3, 6-9]. Inhibition by physical barriers was studied using the individual soil components, sand, Both abiotic and biotic factors may infl u- silt and clays, alone and in combination to ence the ability of P. chrysosporium to de- provide soils with different textures. Two grade pollutants in soil [10, 11]. We o b- different types of clays were used, the e x- served that a native soil suppressed the panding clay bentonite and the non- mineralization of pyrene by P. chrysospo- expanding clay kaolinite. These clays were rium [11]. Because suppression of mine r- examined for their relative abilities to sorb alization of the native soil was in part r e- pyrene and to determine whether sorption lieved by sterilization of the soil, antag o- of the pollutant was a significant feature. nism of the bioremediation potential of the Soils from diverse locations in the United fungus by soil microbes seemed likely [11]. States of America were also used to see Microbes that in vitro reduced the growth of whether inhibition was a generalized or Proceedings of the 10th Annual Conference on Hazardous Waste Research 325 variable trait or was related to such fe a- State University Analytical Laboratories Soil tures as soil texture and pH. Testing Laboratory, Logan, Utah. Sterilized soils were prepared by autoclaving for 4 h PROCEDURES on two consecutive days. Maintenance of P. chrysosporium The microcosms P. chrysosporium BKM-F-1767 ATCC Microcosms to examine mineralization of #24725 was maintained at 26°C by subcu l- pyrene by P. chrysosporium were estab- turing at two weekly intervals on potato lished in Corning polystyrene tissue culture dextrose agar (PDA) (Difco, Detroit, Mich i- flasks (Corning Co. 25110-75, Corning, gan) amended with 1% agar. Stocks were NY). The flasks were laid flat and contained stored as plugs of mycelia frozen at -80°C 40 ml of 1% water agar with no added n u- in 15% glycerol. trients. Pyrene (14C-labeled [500,000 dpm]) from Sigma Chemical Company, St. Louis, P. chrysosporium was grown on sterilized MO, in 5 µl benzene was applied to the ground corn cobs for one month to provide agar surface. The benzene was removed an inoculum for mineralization studies as by passing air through the flasks for 18 h described by Tucker et al. [11]. Both spores using the sterile gas exchange system fi t- and mycelia were present on the corn cobs ted to the flasks as described by Tucker et at the time of inoculation into the micr o- al. [11]. The agar layer was overlaid with a cosms. 'soil' layer that was inoculated with the P. chrysosporium-colonized corn cobs. The Soils microcosms were incubated at 26°C and The soils used in these studies were from sampled for CO2 evolution using the sterile gardens in College Park (Maryland), and gas exchange system as described [11]. Logan (Utah), a semi-industrial site from Chicago (Illinois) and from agricultural fields Amendments used in the in Parma (Idaho) and Kaysville (Utah). ‘soil’ layer The soils were sieved through a 2 mm The ‘soil’ layer consisted of native and sieve and stored at room temperature for sterilized soils, or matrixes of defined te x- less than one year. Soil characteristics ture. Sand (particle size < 355 µm) was (Table 1) were determined by the Utah purchased and autoclaved at 121°C for one Table 1. Soil characteristics. 326 Proceedings of the 10th Annual Conference on Hazardous Waste Research study performed with the Kaysville soil. Soil and corn cobs (15 ml) were removed sep a- rately from the microcosms and mixed with 20 ml sterile distilled water, and the pH of the suspension was measured after 15 min. Sorption of pyrene The soil-water sorption coefficient, k d, was measured for Kidman soil and compared with the kd value for kaolinite and bentonite. The EPA protocol for sediment and soil isotherm (CFR SEC 796.2750) was fo l- lowed. Clay (4 g) was mixed with 40 ml Figure 1. Suppression of mineralization of pyrene 0.01 M Ca (N03)2. A mixture of pyrene and by P. chrysosporium by native soils. Microcosms 14C-pyrene (0.1 to 7 mg/L) was added to 50 were established without soils as a control (-- --) or with soils from College Park (-- --), Chicago (-- --), ml Teflon tubes filled with 0.01 M Ca(NO 3)2 Parma (-- --) and Logan (-- --). Mineralization of to capacity. The tubes were sealed and 14 14 pyrene was measured as CO2 evolution from C- weighed. The solutions were shaken at 20 labeled pyrene as described in Procedures. Data are ± 1°C in a rotary mixer at 30 rpm for 24 h. from duplicate flasks of a single experiment. Error Samples were centrifuged at 7000 g for 30 bars represent standard deviation. Each study was replicated twice. min. A 1 ml aliquot of supernatant was counted using a Beckman 1400 Liquid Scintillation Counter in Ready Safe® coc k- h on two consecutive days before use. Silt tail. The sorbant were dried overnight and (particle size < 45 mm) was obtained by two 1 g portions each for the soil and two sieving and sedimenting Timpanogos soil clays were combusted in a Harvey Biolog i- that has a high silt content [11]. To remove 14 cal Oxidizer. The resulting CO2 was organic materials, a suspension of silt (50 trapped in 10% monoethanolamine, 40% g) in 20 ml 30% hydrogen peroxide was methanol and 50% Ready Gel (Beckman) heated to boiling for five min and cooled, prior to counting. The study was repeated and the process was repeated after add i- in triplicate. Kd values were calculated by tion of another aliquot of hydrogen peroxide plotting S mg/kg versus C e mg/L where S is [12]. The hydrogen peroxide was removed the amount of pyrene sorbed by the clay by extensive washing with sterile distilled (mg/kg) and Ce is the equilibrium solution water. Kaolinite and bentonite clays were concentration (mg/L). The slopes of the line obtained commercially and were sterilized were calculated from three separate stu d- by autoclaving at 121°C for one h on two ies for the bentonite and kaolinite and the consecutive days. The sodium-saturated means calculated. bentonite clay was suspended in 10 M MgCl2 and rinsed in sterile distilled water to RESULTS partially exchange the cations to minimize swelling from water. Artificial soils were Suppression of pyrene produced by mixing the sand, silt and mineralization by native soils commercial kaolinite clay in known propo r- tions. Mineralization of pyrene by P. chrysospo- rium was suppressed by the inclusion of a The pH of the soil and corn cob layers were layer of native soil from four different ge o- determined throughout a time course in a graphic locations into the microcosms Proceedings of the 10th Annual Conference on Hazardous Waste Research 327 Figure 2. Suppression of mineralization of P. chrysosporium by sterilized Kaysville soil. Mineralization of pyrene 14 14 was measured as CO2 evolution from C-labeled pyrene as described in Procedures. Microcosms were esta b- lished with no soil layers (-- --) or with sterilized Kaysville soil (-- --). Data are from duplicate flasks of a single experiment. Error bars represent standard deviation. Each study was repl icated twice. (Figure 1). Two of the soils, a garden soil Effects of artificial soils on from Logan and an agricultural soil from mineralization Parma, were more inhibitory than the soils from College Park and Chicago. A sterilized Soils of different textures were achieved by sandy-loam soil, Kaysville, also inhibited mixing fractions of purified sand, silt and mineralization (Figure 2). kaolinite clay. These systems were free of organic material and microbes. The soil The native soils were of different textures mixtures all decreased mineralization of (Table 1).