Microb Ecol (1989) 17:201-206

MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1989

Effects and Fate of Phenol in Simulated Landfill Sites Brian Jonathan Tibbles and Albin Alexander Wladyslaw Baecker Department of Microbiology, Universityof the Witwatersrand, PO WITS 2050, Johannesburg, South Africa

Abstract. Phenol was administered to landfill waste in concentrations from 150 to 1,000 ppm via the feed-liquor oflysimeter systems over an 18-week incubation period. Biotic contributions to phenol removal in the landfill waste were of greater significance than abiotic removal. The addition of phenol did not cause the isolation ofthermophilic phenol degraders. Plates inoculated from the test lysimeter receiving phenol were eventually predominated by mesophilic phenol-degrading Micrococcus, Nocardia, and Arthrobacter spp.; plates inoculated from the control lysimeter, receiving water, were predominated by species incapable of utilizing phenol.

Introduction The escalating production of synthetic organic chemicals has had an environmental consequence in the increasing dissemination of xenobiotics. Controlled landfill has proved to be the cheapest way to treat urban waste and constitutes the most commonly used waste disposal method [13]. Landfills are ecologically heterogeneous environments varying in physical, chemical, and biological parameters. The incorporation of hazardous waste with municipal refuse in landfills is commonly practiced in a technique called "codisposal." In such situations hazardous wastes may affect the landfill ecology directly by suppressing or inhibiting the growth of sensitive strains, or may promote the growth of those microorganisms capable of utilizing the compound as a carbon or energy source. Furthermore, leachates from such sites may contain toxic compounds which may adversely affect the environment. Phenol is a common constituent of effluents from polymeric resin production plants, oil refining factories, paper pulp processing, and coal liquefaction industries [9]. Young and Rivera [24] quote the statistics of phenol production in 1983 to have been some 1.2 • 109 kg, placing it among the 50 chemicals produced in the greatest quantities in the United States. The environmental and health risks of phenol are well documented [3]. Although considerable attention has been paid to pollution effects on the ecosystems of soils [I 7] and aqueous [ 10] communities, very little attention has been paid to similar effects on the microbial communities of landfills. Indeed, Grainger et al. [8] have COmmented, " . . . it is not easy to imagine a more difficult habitat to study microbiologically." The present study was designed to determine the influence of phenol on the microbial succession patterns in landfill waste, and the contributions of the indigenous microflora to phenol depletion.

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Fig. 1. Concentrations of phenol detected in leachates from lysimeters. [] lysimeter A (sterile, receiving phenol); A lysimeter B (unsterile, receiving phenol); (> lysimeter C (unsterile, receiving water).

Methods Three lysimeters, A, B, and C, were constructed using 1,001 openhead polyethylene drums (Kohler Plastics, SA) filled with 8-year-old landfill waste collected from 1 m below the landfill surface. The control lysimeter A was sterilized by gamma irradiation (Isoster, SA) whereas lysimeters B and C remained unsterilized. A Minipuls peristaltic pump (Gilson, France), was used to administer the feed-liquors, via fitted irrigation systems, to the lysimeters at a flow rate of 500 ml/lysimeter/dayLysimeters A and B received phenol-supplemented feed-liquors and the second control lysimeter C received only distilled water. The concentration of phenol in the feed-liquor oflysimeters A and B was increased every 3 weeks (Fig. 1) to determine the effects of increasing phenol concentrations on the microbiology of the waste of B. The lysimeters were incubated in a temperature-controlled chamber at 30~ Leachate samples were taken concurrent with phenol concentration increments in feed-liquors at 3-week intervals over 18 weeks. Residual phenol in filtered leachates was determined by reversedphase high pressure liquid chromatography (HPLC) and ultraviolet (UV) detection (278 nm). The liquid chromatograph comprised an LKB 2150 pump system, LKB Uvicord 2138S UV detector, LKB 2220 integrating recorder (Bromma, Sweden), and a Rheodyne 7125 injection valve (Cotati, USA). All isocratic separalions of prefiltered samples were performed on an LKB Ultropac column 2134-215 packed with Lichrisorb RPI8 5 um C18 packing material. The mobile phase was a degassed mixture of analytical grade methanol (Merck) and glass-distilled water (1:1) with 2 g 1-' sulphuric acid. Concurrent with leachate sampling, five 3 g waste samples were extracted from lysimeters B and C at depths of 0, 5, 10, 15, and 20 cm to monitor the microbiological populations. A calibrated

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Table 1. Diversity of microorganisms isolated (+) from landfill waste in lysimeters during 18Week incubation period

Phenol-treated lysimeter B (weeks) Genus Aspergillus Bacillus Aerobacter Beijerinkia Cellulomonas Pseudomonas Corynebacterium Arthrobacter Flavobacteriurn A cinetobacter Noeardia~ Arthrobacter, Micrococcus~ Cladosporium~ Aci net o b a c t er~

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sterile auger designed to remove 3 g samples from given depths in the waste was used to take these samples. The external diameter of the auger was 14 m m and it was inserted vertically. When the auger was withdrawn, the moist waste automatically filled the space left, and sampling by this method did not appear to jeopardize the physical integrity and water relations of the system. Samples were serially diluted and used to inoculate tryptose soy agar (BBL Laboratories), tryptose soy agar plus soil extract [ l ], and phenol agar [9]. Replicate spread-plates were incubated aerobically and anaerobically at 30 and 55~ for 2-7 days until growth on each was complete. The isolates were subcultured and purified on tryptose soy agar plates for identification. Determinations of Gram, catalase, and oxidase reactions; determination of motility, cell morphology, colony morPhology, pigmentation, the production of spores, and the mode of glucose utilization were conducted for all isolates named in Table 1. Furthermore, physiological profile assessments of all isolates Were obtained using the API 20B system (API Systems, France). The isolates were then identified Using the keys of Waksman [20], Skerman [l 8], Raper and Fennel [ 15], Cross and Goodfellow [7], Barksdale [4], Weeks [23], Becking [5], Stolp and Gadkari [19], Keddie and Jones [l 1], Norris et al. [14], Schleifer et al. [16], Bovre and Hagen [6], and Krieg and Holt [12]. The identities of the fungal isolates were established in consultations with Dr. D. Knox, Department of Botany, UniVersity of the Witwatersrand. Three waste samples were extracted at depths 0, 10, and 20 cm from all lysimeters at week 18 and diluted in 50 ml distilled water by shaking (150 rpm) overnight at 4~ Solutions were analyzed for phenol as above and the diluted sample from lysimeter A was also plated out to confirm that Sterility had been maintained throughout the test period.

Results Abiotic contributions to phenol depletion were determined by analyses ofleachates from the sterile lysimeter A, and biotic contributions were subsequently C a l c u l a t e d f r o m a n a l y s e s o f l e a c h a t e s A a n d B. E f f e c t s o f p h e n o l a d d i t i o n o n

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the landfill waste microflora were determined from analyses of waste samples from the unsterilized lysimeters B and C. Leachate analyses showed that biotic contributions were significant in the depletion of phenol in landfill waste (Fig. 1). Whereas phenol was detected in the leachate from the control lysimeter A when the administered feed-liquor contained 150 ppm phenol, it was not detected in the leachate from lysimeter B until feed-liquor with 650 ppm phenol was applied. When the latter feedliquor was administered, the results indicated phenol depletion of 77% by lysimeter A and of 98.5% by lysimeter B. Furthermore, HPLC analyses of extracts at 18 weeks showed that the sterile waste in lysimeter A contained 713 ppm phenol, whereas lysimeter B contained no detectable phenol. The disadvantages associated with the use of dilution plates to quantify microbial populations in heterogeneous ecosystems are widely recognized, therefore the present study qualitatively recorded genus diversity on plates. Furthermore, no anaerobic phenol degraders were detected. Therefore, in this work, this process was concluded to have no significance in the biotic degradation of phenol. Only data derived from aerobic populations are presented here. The addition of phenol-supplemented feed-liquors to lysimeter B did not appear to affect the diversities of the thermophilic general or phenol-degrading populations (Table 1). Thermophilic phenol degraders did not occupy, nor were stimulated to occupy, the dominant populations on plates from unsterilized lysimeters; these thermophilic populations were predominated by a nonphenol utilizing actinomycete throughout the duration of the experiment. However, isolate patterns indicated that the mesophilic succession pattern which developed within lysimeter B differed from that in the control lysimeter (Table 1) as the extent of genus diversity among the general mesophiles was reduced by addition of phenol to lysimeter B. Furthermore, after 18 weeks of treatment, the dominant genera on plates from lysimeter B were phenol degraders, whereas those strains that dominated on plates from the control lysimeter C did not grow on phenol agar and were incapable of phenol degradation.

Discussion Phenol depletion from the percolating feed-liquor was shown to be greater in the presence of viable microflora than in their absence (Fig. 1). Moreover, HPLC analyses of extracts of waste retrieved from the lysimeters revealed that phenol removed by abiotic processes had accumulated on the soil particles or in the interstitial fluids of the waste, whereas that removed by biotic processes in soil had been mineralized by the indiginous microflora. These findings from landfill waste studies support the contentions of Alexander [2] that abiotic processes rarely bring about significant changes in chemical structure and that biotransformations are more important in the removal of toxic xenobiotics. The general populations of nonphenol degraders isolated from lysimeters B and C were largely similar in diversity throughout the experiment, although

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Aspergillus and Bacillus spp. were not isolated from phenol-treated waste samPles (Table 1). In contrast, the addition of phenol to lysimeter B coincided with the isolation of phenol-degrading Nocardia, Arthrobacter, Micrococcus, Cladosporium, and Acinetobacter spp. These isolates predominated, apparently at the expense of the general microflora, on both tryptose soy agar and phenol agar. Most of these were never isolated from lysimeter C. The effects of phenol, therefore, altered microbial diversity and caused increased isolation of aerobic, mesophilic phenol-degrading populations from landfill waste. These findings are supported by those of Watanabe [21, 22] who reported that the number of the indigenous pentachlorophenol (PCP)-degrading bacteria in soil increased 1,000-fold over a 3-year period of continual PCP applications. Phenol added to the sterile waste remained associated with it and also emerged in leachate, whereas the fate of phenol added to unsterile waste appears to have been mineralization, presumably by the phenol-degrading microflora.

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14. Norris JR, Berkeley RCW, Logan NA, O'Donnel AG (1981) The genera Bacillus and Sporobacillus. In: Starr MP, Stolp H, Truper HG, Balows A, Schlegel H G (eds) The prokaryotes: A handbook on habitats, isolation, and identification of bacteria. Springer-Verlag, Berlin, PP 1711-1742 15. Raper KB, Fenneli DI (1965) The genus Aspergillus. Williams and Wilkins, Baltimore 16. Schleifer KH, Kloos WE, Koeur M (1981) The genus Micrococcus. In: Start MP, Stolp H, Truper HG, Balows A, Schlegel H G (eds) The prokaryotes: A handbook on habitats, isolation, and identification of bacteria. Springer-Verlag, Berlin, pp 1539-1547 17. Scow KM, Simkins S, Alexander M (1986) Kinetics of mineralization of organic compounds at low concentrations in soil. Appl Environ Microbiol 51:1028-1035 18. Skerman VBD (1959) A guide to the identification of the genera of bacteria with methods and digests of genetic characteristics. Williams and Wilkins, Baltimore 19. Stolp H, Gadkari D (1981) Non-pathogenic members of the genus Pseudomonas. In: Staff MP, Stolp H, Truper HG, Balows A, Schlegel H G (eds) The prokaryotes: A handbook on habitats, isolation, and identification of bacteria. Springer-Verlag, Berlin, pp 719-741 20. Waksman SA (1950) Distribution ofactinomycetes in nature. In: The actinomycetes. Chronica Botanica Co, Watham, Mass, USA, pp 133-148 21. Watanabe I (1977) Pentachlorophenol-decomposing and PCP-tolerant bacteria in field soil treated with PCP. Soil Biol Biochem 9:99-103 22. Watanabe 1 (1978) Pentachlorophenol (PCP) decomposing activity in field soils treated annually with PCP. Soil Biol Biochem 10:71-75 23. Weeks OB (1981) The genus Flavobacterium. In: Starr MP, Stolp H, Truper HG, Balows A, Schlegel HG (eds) The prokaryotes: A handbook on habitats, isolation, and identification of bacteria. Springer-Verlag, Berlin, pp 1365-1370 24. Young LY, Rivera MD (1985) Methanogenic degradation of four phenolic compounds. Water Res 19:1325-1332