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Remediation of pentachlorophenol-contaminated soil by composting with immobilized

World Journal of Microbiology & Biotechnology (2006) 22:909–913 DOI 10.1007/s11274-006-9134-4

? Springer 2006

Remediation of pentachlorophenol-contaminated soil by composting with immobilized Phanerochaete chrysosporium
Xiao-yun Jiang1, Guang-ming Zeng1,*, Dan-lian Huang1, Yang Chen1, Fang Liu2, Guo-he Huang1,3, Jian-bing Li1,4, Bei-dou Xi5 and Hong-liang Liu1,5 1 College of Environmental Science and Engineering, Hunan University, 410082 Changsha, Hunan, China 2 Institute of Mineral and Waste Processing and Dumping Technology, Clausthal University of Technology, D-38678 Clausthal-Zellerfeld, Germany 3 Faculty of Engineering, University of Regina, S4S 0A2 Regina, Canada 4 Environmental Engineering Program, University of Northern British Columbia, V2N 4Z9 Prince George, Canada 5 Chinese Research Academy of Environmental Science, 100012 Beijing, P.R. China *Author for correspondence: Tel.: +86-731-8822754, Fax: +86-731-8823701, E-mail: zgming@hnu.cn
Received 7 November 2005; accepted 19 January 2006

Keywords: Composting, degradation, immobilization, pentachlorophenol (PCP), Phanerochaete chrysosporium, soil remediation

Summary To reduce and eliminate the hazards of pentachlorophenol (PCP) to the soil, the method of inoculating free and immobilized white rot fungi, Phanerochaete chrysosporium to PCP-polluted soils was investigated. Three parallel beakers A, B, C are adopted with the same components of soil, yard waste, straw and bran for aerated composting to degrade the PCP in soil. A was with no inoculants as control, B was added with the inoculants of immobilized P. chrysosporium, C was inoculated with non-immobilized P. chrysosporium, and additionally D contained only PCP-contaminated soils also as control. By contrastive analyses, the feasibility of applying composting to the bioremediation of the PCP-polluted soil was discussed. From the experimental results, it could be seen that the degradation rate of PCP by the immobilized fungi exceeded 50% at day 9, while that of the non-immobilized fungi achieved the same rate at day 16. However, the ?nal degradation rates of PCP for both of them were beyond 90% at day 60 and that the rate of A was much lower than the others. The above data have shown that the degradation e?ect of inoculating P. chrysosporium was better than that of no inoculation, and that of the immobilized fungi was better than that of non-immobilized ones. Meanwhile, shown by all the indicators the composts of A, B and C were mature and stabilized at the end of the experiment. Therefore, the method of composting with immobilized P. chrysosporium is e?ective for the bioremediation of PCP-contaminated soil.

Introduction Pentachlorophenol (PCP) have often been used as herbicides, algicides, bactericides, insecticides, biocides, disinfectants and wood preservatives (Becaert et al. 2000; Cortes et al. 2002) all over the world. Therefore large areas of soils and sediments in lakes or waters have been polluted by PCP which can then enter the food chain and is thought to be teratogenetic, carcinogenic and mutant to humans (Yu & Ward 1996). Moreover, its degradation is di?cult because of its stable aromatic ring system and high chlorine content. Bioremediation techniques have become a very popular approach for the treatment of soil or sediment contaminated with PCP, among which composting is of advantage over other technologies, because of relatively

low capital and operating costs, simplicity of operation and design, and relatively high treatment e?ciency (Namkoong et al. 2002). In composting, organic amendments including manure, yard wastes, food processing wastes and inoculation of fungi are often added to supplement the amount of nutrients and readily degradable organic contaminants in soil (USEPA 1996, 1998). White rot fungi, as specialized ?lamentous fungi, have been often concerned due to their high e?ciency, complete and non-speci?c ability to degrade a variety of environmental pollutants (Zouari et al. 2002; Walter et al. 2005). Phanerochatete chrysosporium is the most extensively characterized white rot fungus. It has been the subject of extensive investigation and many biodegradation studies (Chung & Aust 1995; Shim & Kawamoto 2002). In technical operations, immobilized

910 microbial cell systems could also provide additional advantages over freely suspended cells, such as being stable, less in?uenced by the external environment and more easily separated etc. Natural polymers, such as alginate, chitosan, chitin etc., have been mostly used as the matrix for the immobilization of microbial cells via the entrapment technique, which can also enhance microbial cell performance and adsorptive capacity (Arica et al. 2001). In previous studies, either composting or fungi were applied to degrade the PCP in soil but few researches have been conducted on the combination of composting and immobilized inoculants for PCP-contaminated soil remediation. Normally, physicochemical, chemical and biological analyses are used in the assessment of contaminated soil and in monitoring the e?ciency of soil remediation processes. Therefore, the above analysed parameters and PCP degradation are discussed respectively to investigate if the method of composting with free and immobilized P. chrysosporium inoculants is e?ective for the bioremediation of PCP-contaminated soil. Composting establishment

X. Jiang et al.

Materials and methods Preparations of free and immobilized inoculants The white-rot basidiomycete, P. chrysosporium strain BKM-F-1767 purchased from China Center for Type Culture Collection (CCTCC) was used. Stock cultures were maintained on malt extract agar slants at 4 °C. Mycelial suspensions were prepared in sterile distilled water. The fungal concentration was measured and adjusted to 2.0?106 c.f.u. ml)1. The immobilized fungi bead is prepared as follows (Arica et al. 2001): A total of 5 ml free cell suspension were mixed with 20 ml sterile alginate solution, and then dropped into 100 ml of a sterile CaCl2 solution, where the Ca–alginate beads were formed by ionotropic gelation. After 24 h, the beads were rinsed ?ve times and were ready for inoculation.

PCP was purchased from American ADL Co. with a purity >98%. The raw soil was obtained from Yuelu Mountain Changsha, China. The soil was air-dried and ground to pass through a 2-mm mesh, and then stored at 4 °C in amber-colored jars. The main physicochemical characteristics were measured as follows: 39% of clay, an organic C content of 0.83%, total N of 0.059%, a pH value of 4.9. The non-sterile soil, wheat straw, kitchen waste, wood litter and bran were prepared as compost materials to which were added PCP solution to achieve a concentration of 100 mg PCP kg)1of dry soil. The organic matter content of this mixture reached 57.8%, and the carbon-to-nitrogen ratio (C/N) was about 25:1. The above simulated wastes were controlled to 70% water content. After one night they were evenly distributed into A, B, C reactors. A was with no inoculant; B was added with 0.15 ml/g dry soil plus immobilized fungal beads; C was inoculated with the same amount of the above free mycelial suspensions; and D only contained soil contaminated with 100 mg PCP kg)1of dry soil. Experimental apparatus used for this research consisted of a composting reactor, a CO2 removal trap, a humidi?er, and a trap for collecting CO2 evolved through biodegradation as shown in Figure 1. The temperature of the composting environment was maintained at 30 °C by a temperature controller. A blower fan was used for aeration with the air ?ow controlled at 0.1 m3 h)1 by a ?ow meter. Analytical methods All composting lasted 60 days. Triplicate samples were collected from each pile at days 0,3,6,9,12,15, 18,21,24,27,30,42 and 60. All the following parameters were analysed, such as temperature, volatile solids (VS), coarse ?ber content, lignin content, nitrogen content, pH, water content, germination index (GI), microbial biomass carbon (MBC), PCP degradation etc. Each was considered time-zero and performed in triplicate for each sample taken from di?erent depths of compost.












1.timer controller 2.air blower 3.NaOH 4. H2O 5. airflow meter 6. attemperator 7.reactor 8. aeration head 9. thermometer

Figure 1. Schematic diagram of experimental apparatus A (B and C are the same as A).

Biodegradation of PCP by composting with fungi The aqueous compost extracts were obtained by mechanically shaking the samples with distilled water at a solid:liquid ratio of 1:10 (w/v, dry weight basis) for 1 h. The suspensions were centrifuged at 12,000 rev min)1 for 20 min and ?ltered through 0.45 lm membrane ?lters. The ?ltrates were used for the following analyses. pH was determined using a 716 DMS Titrino pH meter (Metrohm Ltd. CH.-9101 Herisau, Switzerland) ?tted with a glass electrode. The moisture content (ovendried at 105 °C for 24 h), total organic matter (weight loss on ignition at 550 °C for 72 h) and total nitrogen (Kjeldahl method, by Buechi Distillation Unit B-324 and Metrohm T19S Titrino) were determined. Cress seed germination index test (Ahtiainen et al. 2002) Seed germination and root length tests were carried out on water extracts by mechanically shaking the fresh samples for an hour with a solid:liquid ratio of 1:10 (w/v, dry weight basis). About 5.0 ml of each extract was pipetted into a sterilized plastic petri dish lined with a ?lter paper. Ten cress seeds (Lepidium sativum L.) were evenly placed on the ?lter paper and incubated at 25 °C in the dark for 48 h. Triplicates were analysed for each pile sample. Treatments were evaluated by counting the number of germinated seeds, and measuring the length of roots. The responses were calculated by a germination index (GI) that was determined according to the following formula:

911 min, and UV detector at 254 nm. Under such conditions, the retention time of PCP was 8.1 min. PCP concentrations were calculated by reference to appropriate standard PCP solutions.

Results and discussion Physicochemical and chemical change during composting The change of VS contents in Reactor A, B, and C is shown in Table 1. There was a continuous decrease in the VS percentage for all the tested samples during the composting. It reached 19.71%, 18.82%, 20.49%, 4.24% in Reactor A, B, C and D at day 60, respectively. Usually VS tended to reduce during composting due to the decrease of the substrate carbon resulting from CO2 loss (William et al. 1992). It was also observed that the VS content retained in Reactor A was lower than that in Reactor B and C. This was probably because of the weakening microorganism activity and the slight growth of microorganism. The pH values of all samples ranged from 6 to 9, as in Table 1, which was within the optimum range for composting. The whole tendency of pH change was increasing from weak acid to weak alkali, but at day 12 there was a slight decrease, because at the beginning of the process of the composting, organic acid is produced and later on some NH3 generated from nitrogen consumption.

Germination index ?%? ?

Seed germination ?%? ? Root length of treatment ? 100 Root length of control The C/N ratio is often used as the parameter for evaluation of maturity of compost. At the beginning of composting, it should be adjusted to between 25:1 and 30:1, which will facilitate the growth of the microbes and degradation of the organic matters. The C/N ratio then decreased with the progress of the composting. Compost is thought to be mature when the C/N has dropped to lower that 20:1 (Garcia et al. 1992). From Table 1 it is shown that the composts of A, B and C achieved maturity at the ?nal stage. Biological parameter and phytotoxicity test The Germination index (GI), which combines the measurement of the relative seed germination and relative root length of cress seed, is an integrated biological indicator, which is regarded as the most sensitive parameter used to evaluate the toxicity and degree of maturity of compost (Zucconi et al. 1981). As shown in Figure 2, at the beginning of the composting, GI in all reactors increased slowly due to phytotoxicity of PCP but all except D achieved more than 150% ?nally. It was also apparent that GI in Reactor A remained above 80% after 12 days of composting, whereas 30 days were

Microbial biomass carbon content Fumigation extraction was performed according to the method described by Vance et al. (1987) and Anderson et al. (1997). The aqueous sample was divided into two portions equivalent to 2.5 g dry soil. One portion was fumigated for 24 h at 25 °C with ethanol-free CHCl3 containing 20 ll 2-methyl-2-butene l)1. Following fumigant removal, the soil was extracted with 100 ml 0.5 M K2SO4 by 30 min horizontal shaking (200 rev min)1) and ?ltered. The non-fumigated portion was extracted similarly at the time fumigation concerned. Original C in K2SO4 soil extraction was measured by American OI 1010 TOC instrument. Soil microbial biomass C (FEbiomass C) was calculated by Ec/kEC, where Ec=original C extracted from fumigated soil-organic C-extracted from non-fumigated soil, and KEC is 0.45. PCP analysis The PCP in samples was extracted with hexane and then determined by HPLC (Agilent 1100) analysis using UVD with a column temperature at 25 °C, mobile phase of methanol and water (80:20, v/v), ?ow-rate at 1 ml/


X. Jiang et al.

Table 1. Change of VS content, pH, C/N ratio in the composting (A: without inoculants, B: with immobilized P. chrysosporium, C: with free P. chrysosporium, D: soil only). Time (days) 0 3 6 9 12 15 18 21 24 27 30 42 60

Volatile solids (%) A 30.7 B 31.2 C 31.1 D 6.0 pH A B C D C/N ratio A B C 6.0 6.0 6.0 4.6 30.62 30.11 30.60

26.4 28.9 29.8 5.8 6.3 6.6 7.2 4.8 30.52 28.03 29.58

26.6 30.4 29.0 5.8 7.1 6.7 7.3 5.0 25.36 24.28 27.15

24.7 28.8 29.3 5.6 7.1 7.4 6.9 5.0 22.71 23.29 25.69

22.6 27.8 25.2 5.3 6.9 6.8 6.5 5.0 22.27 19.07 18.68

22.8 27.0 26.0 5.1 7.4 7.9 7.1 5.0 21.09 18.00 19.49

23.4 26.6 27.8 5.0 7.2 8.3 6.8 4.9 20.99 18.30 20.20

22.9 26.3 26.6 4.6 7.8 8.1 7.6 4.9 20.93 19.37 18.48

20.6 24.7 26.8 4.5 8.1 8.1 6.5 5.0 18.50 17.42 17.67

21.0 22.2 25.1 4.4 8.2 8.2 8.1 4.6 18.72 13.83 17.01

22.6 23.8 22.2 4.6 7.8 8.4 8.3 5.1 19.86 16.51 15.97

20.2 20.3 21.4 3.9 8.2 8.3 8.7 4.7 16.54 14.02 12.85

19.7 18.3 20.5 4.2 8.1 8.1 8.3 5.1 14.98 12.35 11.14

needed by Reactor B and C. All showed that the phytoxicity of compost in Reactor C was lower than that in Reactor B and higher than that in Reactor A. Microbial biomass re?ects the growth of the microbes (Wang et al. 2003). From Figure 3, it can be seen that there were two peaks, at day 6 B achieved the highest while A and C achieved at day 9. That is to say at this stage the activity of microbes was the highest. Another peak arrived at day 24 to day 27, but earlier for B than that for A and C. The reason might be that at the beginning of composting, the oxygen, nutrients and carbon source were su?cient and the microbes could also make use of PCP as nutrients, so the microbes grew very quickly, but after that composting went into the high temperature stage, and some of the compost was in an anaerobic stage with the oxygen de?cient, and then in the middle stage of composting, aerobic composting again predominated and the toxicity of PCP was much less than before, so another peak appeared. While with the protection of the immobilized support, the P. chrysosporium could be less in?uenced by the PCP, so its activity increased earlier than A and C.

Degradation of PCP From Figure 4 the degradation of PCP of A, B, C and D all decreased, but the e?ect of B was the best, which might be due to the protection of the immobilization support from the high load of pollutants and the absorption capability of the polymer–alginate which facilitated the su?cient contacts between pollutants and fungi. And in a whole, A without inoculants was not as good as B and C, which showed the addition of inoculants was helpful for the degradation and the PCP concentration in D also decreased due to the phototransformations (Piccinini et al. 1998). Interactions of parameters At the beginning of the composting (0–12 days), the PCP was degraded very quickly. At day 12 the degradation of PCP for B had reached 71.56%, and meanwhile GI had increased to almost 85%, which showed the toxicity of the compost decreased to a great amount,


250 200 150 100 50 0 0 6 12 18 21 30 A C B

MBC(ugC g–1)


800 600 400 200 0 0 3 6 9 12 15 18 21








composting time(days)
Figure 2. Cress Germination index in the composting (A: without inoculants, B: with immobilized P. chrysosporium, C: with free P. chrysosporium, D: soil only).

composting time(days)

Figure 3. Microbial biomass carbon (MBC) in the composting (A: without inoculants, B: with immobilized P. chrysosporium, C: with free P. chrysosporium, D: soil only).

Biodegradation of PCP by composting with fungi
120 100

composting of creosote-contaminated soil. Ecotoxicology and Environmental Safety 53, 323–329. Anderson, T.H. & Joergensen, R.G. 1997 Relationship between SIR and FE estimates of microbial biomass C in deciduous forest soils at di?erent pH. Soil Biology and Biochemistry 29, 1033–1042. Arica, M.Y., Kacar, Y. & Genc, O. 2001 Entrapment of white rot fungi Trametes versicolor in Ca–alginate beads: preparation and biosorption kinetic analysis for cadmium removal from an aqueous solution. Bioresource Technology 80, 121–129. Becaert, V., Deschenes, L. & Samson, R. 2000 A simple method to evaluate the concentration of pentachlorophenol degraders in contaminated soils. FEMS Microbiology Letters 184, 261–264. Chung, N. & Aust, S.D. 1995 Degradation of pentachlorophenol in soil by Phanerochaete chrysosporium. Journal of Hazardous Materials 41, 177–183. Cortes, D., Barrios-Gonzalez, J. & Tomasini, A. 2002 Pentachlorophenol tolerance and removal by Rhizopus nigricans in solid-state culture. Process Biochemistry 37, 881–884. Garcia, C., Costa, H.F. & Ayuso, M. 1992 Evaluation of the maturity of municipal waste compost using simple chemical parameters. Communications in Soil Science Plant Analysis 23, 1501–1512. Lasaridi, K.E. & Stentiford, E.I. 1998 A simple respirometric technique for assessing compost stability. Water Research 32, 3717– 3723. Namkoong, W., Hwang, E.Y., Park, J.S. & Choic, J.Y. 2002 Bioremediation of diesel-contaminated soil with composting. Environmental Pollution 119, 23–31. Piccinini, P., Picha, P. & Guillard, C. 1998 Phototransformations of solid pentachlorophenol. Journal of Photochemistry and Photobiology A: Chemistry 119, 137–142. Shim, S.S. & Kawamoto, K. 2002 Enzyme production activity of Phanerochaete chrysosporium and degradation of pentachlorophenol in a bioreactor. Water Research 36, 445–4454. Tuomela, M., Lyytikainen, M., Oivanen, P. & Hatakka, A. 1999 Mineralization and conversion of pentachlorophenol (PCP) in soil inoculated with the white-rot fungus Trametes versicolor. Soil Biology and Biochemistry 31, 65–74. USEPA, 1996 Engineering Bulletin: Composting (EPA/540/S-96/502). USEPA, 1998 An Analysis of Composting as an Environmental Remediation Technology (EPA530-R-98-008). Vance, E.D., Brooks, P.C. & Jenkinson, D.S. 1987 An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. Walter, M., Kirsty, S.H., Boyd, W., McNaughton, D. & Northcott, G. 2005 Laboratory trials on the bioremediation of aged pentachlorophenol residues. International Biodeterioration and Biodegradation 55, 121–130. Wang, W.J., Dalal, R.C., Moody, P.W. & Smith, C.J. 2003 Relationships of soil respiration to microbial biomass, substrate availability and clay content. Soil Biology and Biochemistry 35, 273–284. William, H.S., Margolis, Z.P. & Janonis, B.A. 1992 High altitude sludge composting. Biocycle 8, 68–71. Yu, J. & Ward, O. 1996 Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed cultures. International Biodeterioration and Biodegradation, 181– 187. Zouari, H., Labat, M. & Sayadi, S. 2002 Degradation of 4-chlorophenol by the white rot fungus Phanerochaete chrysosporium in free and immobilized cultures. Bioresource Technology 84, 145– 150. Zucconi, F., Forte, M., Monaco, A. & Bertoldi, M.D. 1981 Biological evaluation of compost maturity. Biocycle 22, 27–29.


concentration of PCP (mg kg–1)

80 60 40 20 0 0 3 6 9 12 15 18



21 24




composting time(days)
Figure 4. Degradation of PCP in the composting (A: without inoculants, B: with immobilized P. chrysosporium, C: with free P. chrysosporium, D: soil only).

the microbial activity also reached its ?rst peak. From the physical character, the color of the compost turned dark and the odor became stronger. All criteria indicated that composting was proceeding well, together with degradation of the pollutants. In the middle stage of composting, the degradation of PCP became slower and the microbial activity decreased, but the GI increased. This suggests that the PCP had almost no toxic e?ect on seeds and at the same time the compost was almost mature, so pH, C/N and VS were all are in a steady phase. At the ?nal stage of the composting, PCP was almost consumed and the compost was then mature (Tuomela et al. 1999). This maturity was compatible with the safe application of the compost product (Lasaridi & Stentiford 1998).

Acknowledgements The study was ?nancially supported by the National 863 High Technologies Research Foundation of China (No. 2004AA649370), the National Basic Research Program (973 Program) (No. 2005CB724203), the Natural Foundation for Distinguished Young Scholars (No. 50425927, No. 50225926), the Doctoral Foundation of Ministry of Education of China, the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of MOE, P.R.C. (TRAPOYT) in 2000.

Ahtiainen, J., Valo, R., JaKrvinen, M. & Joutti, A. 2002 Microbial toxicity tests and chemical analysis as monitoring parameters at

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