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 Liu1,51College of Environmental Science and Engineering, Hunan University, 410082 Changsha, Hunan, China2Institute of Mineral and Waste Processing and Dumping Technology, Clausthal University of Technology, D-38678Clausthal-Zellerfeld, Germany3Faculty of Engineering, University of Regina, S4S 0A2 Regina, Canada4Environmental Engineering Program, University of Northern British Columbia, V2N 4Z9 Prince George, Canada5Chinese 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, soilremediation

Summary

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

Introduction

Pentachlorophenol (PCP) have often been used as her-bicides, algicides, bactericides, insecticides, biocides,disinfectants and wood preservatives (Becaert et al.2000; Cortes et al. 2002) all over the world. Thereforelarge areas of soils and sediments in lakes or waters havebeen polluted by PCP which can then enter the foodchain and is thought to be teratogenetic, carcinogenicand mutant to humans (Yu & Ward 1996). Moreover,its degradation is difficult because of its stable aromaticring system and high chlorine content.Bioremediation techniques have become a very pop-

ular approach for the treatment of soil or sedimentcontaminated with PCP, among which composting is ofadvantage over other technologies, because of relatively

low capital and operating costs, simplicity of operationand design, and relatively high treatment efficiency(Namkoong et al. 2002). In composting, organicamendments including manure, yard wastes, food pro-cessing wastes and inoculation of fungi are often addedto supplement the amount of nutrients and readilydegradable organic contaminants in soil (USEPA 1996,1998). White rot fungi, as specialized filamentousfungi, have been often concerned due to their high effi-ciency, complete and non-specific ability to degrade avariety of environmental pollutants (Zouari et al. 2002;Walter et al. 2005). Phanerochatete chrysosporium is themost extensively characterized white rot fungus. It hasbeen the subject of extensive investigation and manybiodegradation studies (Chung & Aust 1995; Shim &Kawamoto 2002). In technical operations, immobilized

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

microbial cell systems could also provide additionaladvantages over freely suspended cells, such as beingstable, less influenced by the external environment andmore easily separated etc. Natural polymers, such asalginate, chitosan, chitin etc., have been mostly used asthe matrix for the immobilization of microbial cells viathe entrapment technique, which can also enhancemicrobial 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 researcheshave been conducted on the combination of compostingand immobilized inoculants for PCP-contaminated soilremediation.Normally, physicochemical, chemical and biological

analyses are used in the assessment of contaminated soiland in monitoring the efficiency of soil remediationprocesses. Therefore, the above analysed parametersand PCP degradation are discussed respectively toinvestigate if the method of composting with free andimmobilized P. chrysosporium inoculants is effective forthe bioremediation of PCP-contaminated soil.

Materials and methods

Preparations of free and immobilized inoculants

The white-rot basidiomycete, P. chrysosporium strainBKM-F-1767 purchased from China Center for TypeCulture Collection (CCTCC) was used. Stock cultureswere maintained on malt extract agar slants at 4 �C.Mycelial suspensions were prepared in sterile distilledwater. The fungal concentration was measured andadjusted 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 suspensionwere mixed with 20 ml sterile alginate solution, and thendropped into 100 ml of a sterile CaCl2 solution, wherethe Ca–alginate beads were formed by ionotropic gela-tion. After 24 h, the beads were rinsed five times andwere ready for inoculation.

Composting establishment

PCP was purchased from American ADL Co. with apurity >98%. The raw soil was obtained from YueluMountain Changsha, China. The soil was air-dried andground to pass through a 2-mm mesh, and then storedat 4 �C in amber-colored jars. The main physicochemi-cal characteristics were measured as follows: 39% ofclay, 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 ascompost materials to which were added PCP solution toachieve a concentration of 100 mg PCP kg)1of dry soil.The organic matter content of this mixture reached57.8%, and the carbon-to-nitrogen ratio (C/N) wasabout 25:1. The above simulated wastes were controlledto 70% water content. After one night they were evenlydistributed into A, B, C reactors. A was with no inoc-ulant; B was added with 0.15 ml/g dry soil plus immo-bilized fungal beads; C was inoculated with the sameamount of the above free mycelial suspensions; andD only contained soil contaminated with 100 mgPCP kg)1of dry soil.Experimental apparatus used for this research con-

sisted of a composting reactor, a CO2 removal trap, ahumidifier, and a trap for collecting CO2 evolvedthrough biodegradation as shown in Figure 1. Thetemperature of the composting environment was main-tained at 30 �C by a temperature controller. A blowerfan was used for aeration with the air flow controlled at0.1 m3 h)1 by a flow meter.

Analytical methods

All composting lasted 60 days. Triplicate samples werecollected from each pile at days 0,3,6,9,12,15,18,21,24,27,30,42 and 60. All the following parameterswere analysed, such as temperature, volatile solids (VS),coarse fiber content, lignin content, nitrogen content,pH, water content, germination index (GI), microbialbiomass carbon (MBC), PCP degradation etc. Each wasconsidered time-zero and performed in triplicate foreach sample taken from different depths of compost.

1

8764 4

A

3

9

5

23

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).

910 X. Jiang et al.

The aqueous compost extracts were obtained bymechanically shaking the samples with distilled waterat a solid:liquid ratio of 1:10 (w/v, dry weight basis) for1 h. The suspensions were centrifuged at 12,000 rev min)1

for 20 min and filtered through 0.45 lm membrane filters.The filtrates were used for the following analyses. pH wasdetermined using a 716 DMS Titrino pH meter(Metrohm Ltd. CH.-9101 Herisau, Switzerland) fittedwith a glass electrode. The moisture content (oven-dried at 105 �C for 24 h), total organic matter (weightloss on ignition at 550 �C for 72 h) and total nitrogen(Kjeldahl method, by Buechi Distillation Unit B-324and Metrohm T19S Titrino) were determined.

Cress seed germination index test (Ahtiainen et al. 2002)

Seed germination and root length tests were carried outon water extracts by mechanically shaking the freshsamples for an hour with a solid:liquid ratio of 1:10 (w/v,dry weight basis). About 5.0 ml of each extract waspipetted into a sterilized plastic petri dish lined with afilter paper. Ten cress seeds (Lepidium sativum L.) wereevenly placed on the filter paper and incubated at 25 �Cin the dark for 48 h. Triplicates were analysed for eachpile sample. Treatments were evaluated by counting thenumber of germinated seeds, and measuring the lengthof roots. The responses were calculated by a germina-tion index (GI) that was determined according to thefollowing formula:

Microbial biomass carbon content

Fumigation extraction was performed according to themethod described by Vance et al. (1987) and Andersonet al. (1997). The aqueous sample was divided into twoportions equivalent to 2.5 g dry soil. One portionwas fumigated for 24 h at 25 �C with ethanol-freeCHCl3 containing 20 ll 2-methyl-2-butene l)1. Follow-ing fumigant removal, the soil was extracted with 100 ml0.5 MK2SO4 by 30 min horizontal shaking (200 rev min)1)and filtered. The non-fumigated portion was extractedsimilarly at the time fumigation concerned. Original C inK2SO4 soil extraction was measured by American OI1010 TOC instrument. Soil microbial biomass C (FE-biomass C) was calculated by Ec/kEC, where Ec=ori-ginal C extracted from fumigated soil-organic C-ex-tracted from non-fumigated soil, and KEC is 0.45.

PCP analysis

The PCP in samples was extracted with hexane and thendetermined by HPLC (Agilent 1100) analysis usingUVD with a column temperature at 25 �C, mobile phaseof methanol and water (80:20, v/v), flow-rate at 1 ml/

min, and UV detector at 254 nm. Under such condi-tions, the retention time of PCP was 8.1 min. PCPconcentrations were calculated by reference to appro-priate standard PCP solutions.

Results and discussion

Physicochemical and chemical change during composting

The change of VS contents in Reactor A, B, and C isshown in Table 1. There was a continuous decrease inthe VS percentage for all the tested samples duringthe 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 tothe decrease of the substrate carbon resulting from CO2

loss (William et al. 1992). It was also observed that theVS content retained in Reactor A was lower than that inReactor B and C. This was probably because of theweakening microorganism activity and the slight growthof microorganism.The pH values of all samples ranged from 6 to 9, as in

Table 1, which was within the optimum range for com-posting. The whole tendency of pH changewas increasingfrom weak acid to weak alkali, but at day 12 there was aslight decrease, because at the beginning of the process ofthe composting, organic acid is produced and later onsome NH3 generated from nitrogen consumption.

The C/N ratio is often used as the parameter forevaluation of maturity of compost. At the beginning ofcomposting, it should be adjusted to between 25:1 and30:1, which will facilitate the growth of the microbes anddegradation of the organic matters. The C/N ratio thendecreased with the progress of the composting. Compostis thought to be mature when the C/N has dropped tolower that 20:1 (Garcia et al. 1992). From Table 1 it isshown that the composts of A, B and C achievedmaturity at the final stage.

Biological parameter and phytotoxicity test

The Germination index (GI), which combines the mea-surement of the relative seed germination and relativeroot length of cress seed, is an integrated biologicalindicator, which is regarded as the most sensitiveparameter used to evaluate the toxicity and degree ofmaturity of compost (Zucconi et al. 1981). As shown inFigure 2, at the beginning of the composting, GI in allreactors increased slowly due to phytotoxicity of PCPbut all except D achieved more than 150% finally. It wasalso apparent that GI in Reactor A remained above80% after 12 days of composting, whereas 30 days were

Germination index ð%Þ ¼ Seed germination ð%Þ �Root length of treatment

Root length of control� 100

Biodegradation of PCP by composting with fungi 911

needed by Reactor B and C. All showed that the phy-toxicity of compost in Reactor C was lower than that inReactor B and higher than that in Reactor A.Microbial biomass reflects the growth of the microbes

(Wang et al. 2003). From Figure 3, it can be seen thatthere were two peaks, at day 6 B achieved the highestwhile A and C achieved at day 9. That is to say at thisstage the activity of microbes was the highest. Anotherpeak arrived at day 24 to day 27, but earlier for B thanthat for A and C. The reason might be that at thebeginning of composting, the oxygen, nutrients andcarbon source were sufficient and the microbes couldalso make use of PCP as nutrients, so the microbes grewvery quickly, but after that composting went into thehigh temperature stage, and some of the compost was inan anaerobic stage with the oxygen deficient, and then inthe middle stage of composting, aerobic compostingagain predominated and the toxicity of PCP was muchless than before, so another peak appeared. While withthe protection of the immobilized support, the P. chry-sosporium could be less influenced by the PCP, so itsactivity increased earlier than A and C.

Degradation of PCP

From Figure 4 the degradation of PCP of A, B, C and Dall decreased, but the effect of B was the best, whichmight be due to the protection of the immobilizationsupport from the high load of pollutants and theabsorption capability of the polymer–alginate whichfacilitated the sufficient contacts between pollutants andfungi. And in a whole, A without inoculants was not asgood as B and C, which showed the addition of inocu-lants was helpful for the degradation and the PCPconcentration in D also decreased due to the photo-transformations (Piccinini et al. 1998).

Interactions of parameters

At the beginning of the composting (0–12 days), thePCP was degraded very quickly. At day 12 the degra-dation of PCP for B had reached 71.56%, and mean-while GI had increased to almost 85%, which showedthe toxicity of the compost decreased to a great amount,

0

50

100

150

200

250

0 6 12 18 21 30

composting time(days)

GI(

%)

A B

C D

Figure 2. Cress Germination index in the composting (A: without

inoculants, B: with immobilized P. chrysosporium, C: with free

P. chrysosporium, D: soil only).

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 26.4 26.6 24.7 22.6 22.8 23.4 22.9 20.6 21.0 22.6 20.2 19.7

B 31.2 28.9 30.4 28.8 27.8 27.0 26.6 26.3 24.7 22.2 23.8 20.3 18.3

C 31.1 29.8 29.0 29.3 25.2 26.0 27.8 26.6 26.8 25.1 22.2 21.4 20.5

D 6.0 5.8 5.8 5.6 5.3 5.1 5.0 4.6 4.5 4.4 4.6 3.9 4.2

pH

A 6.0 6.3 7.1 7.1 6.9 7.4 7.2 7.8 8.1 8.2 7.8 8.2 8.1

B 6.0 6.6 6.7 7.4 6.8 7.9 8.3 8.1 8.1 8.2 8.4 8.3 8.1

C 6.0 7.2 7.3 6.9 6.5 7.1 6.8 7.6 6.5 8.1 8.3 8.7 8.3

D 4.6 4.8 5.0 5.0 5.0 5.0 4.9 4.9 5.0 4.6 5.1 4.7 5.1

C/N ratio

A 30.62 30.52 25.36 22.71 22.27 21.09 20.99 20.93 18.50 18.72 19.86 16.54 14.98

B 30.11 28.03 24.28 23.29 19.07 18.00 18.30 19.37 17.42 13.83 16.51 14.02 12.35

C 30.60 29.58 27.15 25.69 18.68 19.49 20.20 18.48 17.67 17.01 15.97 12.85 11.14

0

200

400

600

800

1000

1200

0 3 6 9 12 15 18 21 24 27 30 42 60

composting time(days)

MB

C(u

gC g

–1)

A B

C D

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).

912 X. Jiang et al.

the microbial activity also reached its first peak. Fromthe physical character, the color of the compost turneddark and the odor became stronger. All criteria indi-cated that composting was proceeding well, togetherwith degradation of the pollutants. In the middle stageof composting, the degradation of PCP became slowerand the microbial activity decreased, but the GI in-creased. This suggests that the PCP had almost no toxiceffect on seeds and at the same time the compost wasalmost mature, so pH, C/N and VS were all are in asteady phase. At the final stage of the composting, PCPwas almost consumed and the compost was then mature(Tuomela et al. 1999). This maturity was compatiblewith the safe application of the compost product (Las-aridi & Stentiford 1998).

Acknowledgements

The study was financially supported by the National 863High Technologies Research Foundation of China (No.2004AA649370), the National Basic Research Program(973 Program) (No. 2005CB724203), the NaturalFoundation for Distinguished Young Scholars (No.50425927, No. 50225926), the Doctoral Foundation ofMinistry of Education of China, the Teaching andResearch Award Program for Outstanding YoungTeachers in Higher Education Institutions of MOE,P.R.C. (TRAPOYT) in 2000.

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conc

entr

atio

n of

PC

P(m

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porium, D: soil only).

Biodegradation of PCP by composting with fungi 913