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Research ArticleReceived: 28 November 2008 Revised: 6 February 2009 Accepted: 6 February 2009 Published online in Wiley Interscience: 30 March 2009

(www.interscience.wiley.com) DOI 10.1002/jctb.2167

Development of a fungal pre-treatmentprocess for reduction of organic matter incontaminated soilErika Winquist,a∗ Lara Valentin,b Ulla Moilanen,a Matti Leisola,a

Annele Hatakka,b Marja Tuomelab and Kari T. Steffenb

Abstract

BACKGROUND: Combustion at high temperature is a common treatment method for heavily contaminated soils. The capacityof the combustion process is negatively correlated with organic matter content of the soil. Thus, by reducing the amount of soilorganic matter, batch size could be increased and the combustion process improved. In this study, the possibility to pre-treatsoil containing high levels of organic matter with white-rot and litter-decomposing fungi was examined and scaled up.

RESULTS: Calculations based on the CO2 production in laboratory experiments indicated that 20% of the soil organic carbonwould have been degraded in 6 months when treated with Sphaerobolus stellatus and 10% when treated with Strophariarugosoannulata. In a pilot-scale experiment with S. rugosoannulata mass loss due to degradation of soil organic matteraccounted for 10% of the total weight of the soil in 6 months.

CONCLUSION: A fungal pre-treatment process for contaminated soils with high organic matter content was developed. Goodresults were obtained with S. stellatus and S. rugosoannulata and the process was successfully scaled up to 300 kg scale.c© 2009 Society of Chemical Industry

Keywords: bioremediation; bioaugmentation; litter-decomposing fungi; Stropharia rugosoannulata; Sphaerobolus stellatus

INTRODUCTIONThe natural environment for white-rot fungi (WRF) is wood andfor litter-decomposing fungi (LDF) plant litter material in the topsoil layer. Many wood-degrading species, e.g. members of thegenera Phanerochaete, Pleurotus and Trametes, may also survivein soil if suitable substrates are available.1 For the degradationof cellulose and hemicellulose from wood or plant litter, WRFand LDF utilize various extracellular hydrolytic enzymes such asendoglucanase, cellobiohydrolase, β-glucosidase, endoxylanaseand endomannanase.2 WRF and LDF are also efficient lignindegraders in nature.3 They produce extracellular oxidizingenzymes, such as laccase and manganese peroxidase (MnP), whichare involved in lignin degradation of wood.4 These enzymesare non-specific, and in addition to lignin they can oxidize awide range of organic compounds with structural similarities tolignin including soil humic substances and organic contaminants,especially aromatic compounds.5 – 7 WRF and LDF are the mostimportant organisms in the degradation of recalcitrant humicsubstances.8 Various studies have proved the capability of lignin-degrading fungi to degrade polycyclic aromatic hydrocarbons(PAH) and chlorinated organic compounds (pentachlorophenol,PCP, and polychlorinated biphenyls, PCB).9 – 12

The starting point for this study was the development of afungal pre-treatment process for contaminated soils with highorganic matter content. In experiments, soil from an old sawmillsite was used. Sawmill soil has typically high organic mattercontent due to small wood particles in the soil and is often

contaminated with chlorophenols, polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F). Chlorophenols were used asactive ingredients in wood preservatives for several decades untilthe 1980s and PCDD/F were found in these products as impurities.PCDD/F may also have been formed from chlorophenols incontaminated soil through microbial activity.13 The currenttreatment method used for this type of soil is combustion athigh temperature (over 1300 ◦C), which however is expensiveand energy-intensive. The capacity of the combustion process isnegatively correlated with the organic matter content of the soil.Thus, this process could be improved and batch size increased byreducing the amount of soil organic matter (Rantsi R, Niska andNyyssonen, personal communication).

In this study, a method to pre-treat soil containing high organicmatter with lignin-degrading fungi was examined and scaled up.In addition to sawmill soil, fungal treatment was tested also for twoother types of contaminated soil, one from a shooting range andanother from a landfill area. White-rot fungi have been studied

∗ Correspondence to: Erika Winquist, Helsinki University of Technology, Depart-ment of Biotechnology and Chemical Technology, PO Box 6100, 02015 TKK,Finland. E-mail: erika.winquist@tkk.fi

a Helsinki University of Technology, Department of Biotechnology and ChemicalTechnology, P.O. Box 6100, 02015 TKK, Finland

b University of Helsinki, Department of Applied Chemistry and Microbiology, P.O.Box 56 (Viikki Biocenter), 00014 University of Helsinki, Finland

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Table 1. Properties of soils used in the experiments

Soil Contamination pH

Drymatter

(%)

Organicmatter(% dm)

C(% dm)

N(% dm) Organic matter : Cc

Sawmill Aa chlorophenols, PCDD/F 4.3 ± 0.0d 34 ± 2 82 ± 2 46 ± 14 0.41 ± 0.00 1.77

Sawmill Bb chlorophenols, PCDD/F 4.3 ± 0.0 29 ± 1 82 ± 3 42 ± 3.0 0.43 ± 0.08 1.94

Shooting range heavy metals (Pb), PAH 3.9 ± 0.0 64 ± 2 28 ± 2 16 ± 1.0 0.72 ± 0.03 1.78

Landfill petroleum hydrocarbons 7.1 ± 0.0 78 ± 1 14 ± 1 8.3 ± 0.7 0.13 ± 0.01 1.70

a Used in the laboratory experiments with S. rugosoannulata, G. luteofolius and P. velutina.b Used in the laboratory experiments with S. stellatus and in the pilot-scale experiment with S. rugosoannulata.c Organic matter : organic carbon ∼ 1.72 in forest soil, van Bemmelen factor.14

d Average value of three replicates ± standard deviation.

extensively for bioremediation applications.1 LDF was includedbecause these fungi occur naturally on the soil surface and theyare capable of growing in the soil.

EXPERIMENTALProperties of soil used in the experimentsThree soils from different origins were used in the experiments(Table 1). Moisture content of soil was determined by dryingthe fresh soil at 105 ◦C overnight and organic matter by losson combustion at 440 ◦C for 5 h. Total carbon and nitrogencontent were analysed using a Vario Max CN elemental analyser(ELEMENTAR Analysensysteme, Germany). Soil pH was measuredin 0.01 mol L−1 CaCl2 solution with suspension ratio 1 : 2.5 (w/v).

Sawmill soil was slightly contaminated with chlorophenolsand PCDD/F (60–70 ng kg−1 I-TEQ) and contained over 40%organic carbon (Table 1). Both sawmill soils, sawmill A andsawmill B, originated from the same locality. Shooting rangesoil was contaminated mainly with lead (700–1200 mg kg−1) andPAH compounds (mainly fluoranthene and pyrene, sum of 16PAH compounds was 150 mg kg−1) and contained 16% organiccarbon. Landfill soil was contaminated mainly with petroleumhydrocarbons (C10 –C40: 10–34 g kg−1) and contained only 8%organic carbon.

For this study, it can be assumed that the analysed totalcarbon content represented well the organic carbon contentof the soil, because the soil was acidic and thus contained verylittle carbonates. From these values estimates were made of theorganic matter degradation. According to Smolander14 there aremany factors used to determine organic matter from organiccarbon. One commonly used for forest soils is the van Bemmelenfactor (the ratio of organic matter to organic carbon = 1.72). Thisratio was calculated for soils used in this study and the values werein the same range except for the sawmill B soil used in the pilot-scale experiments, which contained less carbon in proportion toorganic matter. This is probably because the soil contained a lotof small wood particles and wood contains more oxygen than soilhumic substances. However, the van Bemmelen factor for this soilwas close to that (1.9) presented by Nelson and Sommers.15

Fungi and culture conditionsFungal strains were obtained from the Fungal BiotechnologyCulture Collection (FBCC) of the Department of Applied Chem-istry and Microbiology, University of Helsinki. The strains weremaintained on potato dextrose agar (PDA, Difco laboratories,USA). Four fungal strains (with old strain number in parenthesis),

Stropharia rugosoannulata FBCC475 (11 372), Gymnopilus luteo-folius FBCC466 (X9), Phanerochaete velutina FBCC941 (T244) andSphaerobolus stellatus FBCC253 (PO203), were selected for labo-ratory experiments after extensive screening of more than 100strains of wood-degrading and litter-decomposing fungi (ValentinL et al., unpublished). S. rugosoannulata belongs to the ecologicalgroup LDF, P. velutina to WRF and G. luteofolius and S. stellatus to agroup whose habitat overlaps WRF and LDF.

Liquid inoculumLiquid inoculum was prepared by cutting between two and four(5 mm × 5 mm) agar plugs from the PDA-plates. These wereextruded through a syringe to the liquid medium in 500 mLErlenmeyer flasks containing 200 mL of malt extract (2% w/v).Fungi were cultivated for 7–10 days at 25 ◦C with continuousagitation (100–150 rpm). After cultivation, mycelial pellets werehomogenized with an Ultra-Turrax (IKA Werke GmbH, Germany)for 10 s at 17 500 rpm.

Fungal bark inoculumPine bark, used as a co-substrate in soil experiments, was firstsoaked in water overnight and then drained. The fungi werecultivated on bark in high density polyethene (HDPE) plastic bags(Suominen Oy, Kauhava, Finland). Wet bark, 1 kg in each plasticbag, was autoclaved (30 min, 121 ◦C) and the cooled bark wasinoculated with 100 mL of homogenized liquid inoculum. Thebags were incubated at room temperature (21 ◦C) and aeratedcontinuously with moisturized air (1.5 L min−1). The incubationtime varied: S. rugosoannulata and G. luteofolius were cultivated for4 weeks while P. velutina and S. stellatus for 6 weeks for laboratoryexperiments. For pilot-scale experiments S. rugosoannulata wascultivated for 6 weeks.

Soil experiments in laboratory2 L glass bottles (Fig. 1) were filled with layers of soil (700 g)and fungal bark inoculum (100 g). The intensity of the myceliumgrowth could be followed through the glass wall of the bottles.

All bottles were aerated with moist air (0.5 L min−1) and CO2

was analysed online from the exhaust air by mass spectrometer(OmniStar GSD 301, Pfeiffer Vacuum GmbH, Germany). Due to thelimited amount of air channels in the mass spectrometer only tworeplicate bottles were used. However, the fairly large scale of 700 gfor laboratory experiments diminished the variations betweenparallel bottles.16 CO2 production from fungal bark inoculum wasestimated by using gravel (Maxit crushed stone for sanding, grain

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150 g expanded clay

350 g soil

100 g fungal inoculum

350 g soil

Air in Air outAir in Air out

Figure 1. Experimental set-up of the laboratory experiments.

0

5

10

15

20

25

0 15 30 45 60 75 90

Time (d)

CO

2 (g

)

S. rugosoannulataP. velutina

G. luteofoliusS. stellatus

Figure 2. Average cumulative CO2 production from soil organic matterwith four fungi in the laboratory experiments with sawmill soil.

Aeration 30 l/min

Fungal inoculum tube,d = 9 cm

10 cm, intermediate floor

100 cm of soil

Plexi-glass

75 cm75 cm

5 cm of expanded clay

75 cm75 cm

Figure 3. Experimental set-up of the pilot-scale experiment.

size 3–6 mm, Optiroc Oy Ab, Finland) instead of soil in controlbottles. Furthermore, CO2 from soil organic matter (om) wasestimated by subtracting the amount of CO2 of the gravel bottles(CO2 from fungal bark inoculum) from that of the soil bottles (CO2

from soil and fungal bark inoculum):

CO2 prod from soil om (g) =Av soil CO2 prod (g) − Av bark CO2 prod (g) (1)

Figure 4. Stropharia rugosoannulata mycelia through Plexi-glass window(49 d) in the pilot-scale experiment.

Estimated CO2 production from soil organic matter in 6 monthswas calculated by dividing the CO2 production by the duration ofthe experiment and multiplying by 180:

Est CO2 prod in 6 months (g) =CO2 prod from soil om (g)/t(d) × 180d (2)

Estimated carbon loss from soil was calculated by comparingthe CO2 production from soil organic matter with the theoreticalmaximum CO2 production from soil (Equation (3)) which wascalculated based on the analysed carbon content (Equation (4)):

Est carbon loss from soil (%) =CO2 prod (g)/Theor max CO2 prod from soil (g) × 100% (3)

Theor max CO2 prod from soil (g) =m(dry soil) × c(carbon)/12 g/mol × 44 g mol−1 (4)

Soil experiments in pilot-scaleA box with a volume of 0.56 m3 (Fig. 3) was filled with soil (approx.300 kg) and six netlike tubes containing fungal bark inoculum(1.5 kg in each tube). During the experiment, soil was aerated withmoist air (30 l min−1) from the bottom of the box. The box wascovered with a tarpaulin to prevent drying of the soil. Mycelialgrowth of the fungus could be followed through a Plexi-glass wall

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Table 2. Cumulative CO2 production with four fungi in different soils

Fungus SoilTime

(d)

Averagecumulative

soil CO2production (g)

Averagecumulative

bark CO2production (g)

CumulativeCO2

productionfrom soil om (g)

Estimated carbonloss from soilin 6 months(% max CO2)

Gymnopilus luteofolius Sawmill A 84 22.2 ± 0.2a 7.0 ± 1.6 15.2 ± 1.8 8.0

Phanerochaete velutina Sawmill A 70 25.9 ± 0.7 17.6 ± 1.8 8.3 ± 2.4 5.3

Sphaerobolus stellatus Sawmill B 70 35.0 ± 1.7 11.2 ± 0.3 23.8 ± 2.0 19.6

Sphaerobolus stellatus Shooting range 53 16.2 ± 0.3 4.6 ± 0.2 11.5 ± 0.4 15.2

Stropharia rugosoannulata Sawmill A 90 23.5 ± 0.8 4.2 ± 0.8 19.3 ± 1.5 9.5

Stropharia rugosoannulata Landfill 60 15.5 ± 0.5 6.7 ± 0.5 8.7 ± 0.9 13.9

a Average value of two replicates ± range of variation.

(Fig. 4). The radial mycelium growth was measured with a ruler.The box was placed on top of scales and mass loss was followedduring the experiment. A CO2 sensor (Priva, the Netherlands) wasplaced above the soil surface and CO2 production was monitored.After 3 and 6 months samples were taken and dry matter analysed.

Enzyme activity assaysA sample of fungal-bark inoculum (20 g) from each tube wasextracted with 100 mL of 25 mmol L−1 sodium phosphate buffer(pH 7.0) for 1.5 h on a rotary shaker (160 rpm) at 28 ◦C. Dry matter ofthe bark was 39% before extraction. Extracts were filtered throughMiracloth (Calbiochem, USA). After centrifugation (9.3×g, 10 min),enzyme activities were analysed. MnP (EC 1.11.1.13) activity wasmeasured by monitoring formation of Mn3+-malonate complexesat 270 nm, and laccase (EC 1.10.3.2) activity by the oxidation ofABTS at 420 nm.17,18 Activities are expressed in units (U), definedas 1 µmol of substrate reacted in 1 min at 25 ◦C. Activities of endo-1,4-β-xylanase (EC 3.2.1.8), endo-1,4-β-mannanase (EC 3.2.1.78)and endo-1,4-β-glucanase (EC 3.2.1.4) were measured with azo-dyed substrates of birchwood xylan, carob-galactomannan andcarboxymethyl cellulose, respectively. The assays were performedaccording to the protocol of the azo-dyed-substrate supplier(Megazyme, Ireland) with some modifications.19 One unit (U) ofenzyme activity is defined as 1 µmol of reducing sugars released in1 min at 40 ◦C. All the enzymes were measured with a Shimadzu UV-1700 Pharma spectrophotometer (Shimadzu Corporation, Japan).

RESULTSSoil experiments in laboratoryCO2 production with four different fungi was first studied insawmill soil (Table 1). All tested strains were able to colonizethe soil and G. luteofolius even produced some fruiting bodiesduring the experiment. Cumulative CO2 production of all fungalstrains varied remarkably (Fig. 2). Even though CO2 production waspresented cumulatively the range of variation was small (Table 2).Because the duration of the experiments varied, estimated CO2

production for 6 months was calculated in order compare theresults (Table 2). It was possible to extrapolate the CO2 productionfor 6 months, because the cumulative CO2 production was linear.Of these fungi S. stellatus and S. rugosoannulata were the mostefficient to degrade soil organic matter. In 6 months 19.6% of thesoil organic matter would have been degraded when treated withS. stellatus and 9.5% when treated with S. rugosoannulata.

Organic matter degradation by S. stellatus and S. rugosoannulatawas further studied in soil from a shooting range and in soil from a

Table 3. Enzyme activities in the fungal bark inoculum (Strophariarugosoannulata) used in the pilot-scale experiment

EnzymeActivity

(mU g−1 dm−1)

Manganese peroxidase 1670 ± 1049a

Laccase 16 ± 28

Endo-1,4-β-glucanase 69 ± 36

Endo-1,4-β-xylanase 39 ± 18

Endo-1,4-β-mannanase 27 ± 14

a Average value of six replicates ± standard deviation.

landfill area, respectively. These soils varied in their organic mattercontent as well as type of contamination (Table 1). S. stellatusproduced more CO2 than S. rugosoannulata in both soils studied(Table 2), although S. stellatus did not grow as well in shootingrange soil as in sawmill soil.

Soil experiments in pilot-scaleS. rugosoannulata was chosen for a pilot-scale experiment becauseit performed well in laboratory experiments and because wehad more experience with S. rugosoannulata than with theother fungi. As S. rugosoannulata belongs to LDF, it was alsoexpected to have the best potential to grow in the soil. Thefungal bark inoculum used had high initial MnP activity, and alsosome laccase, glucanase, xylanase and mannanase activities werepresent (Table 3). Extensive fungal growth from inoculum tubeswas detected visually at the beginning of the experiment throughthe Plexi-glass window (Fig. 4). Radial hyphal growth was constantup to 53 days of incubation (Fig. 5). After 53 days the radial growthof fungus could not be followed. However, when the soil wasremoved from the box, strong and thick mycelia was observedeverywhere in the soil. In particular, the mycelium was attached towood pieces in the soil.

Respiration activity correlated with radial hyphal growth upto 53 days (Fig. 6). After 70 days the respiration activity startedto cease and after 100 days it remained constant (100 mg kg−1).Mass loss was constant during the whole experiment (Fig. 6). Drymass of the soil did not change during the experiment indicatingthat evaporation was not the reason for mass loss. Mass loss frombark in inoculum tubes was 2.2 kg, and 0.8 kg of mass was lostwhen samples were taken from the soil. Thus, the corrected massloss due to degradation of soil organic matter was 30.5 kg, whichrepresents 10% of the original soil mass (308 kg).

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0

5

10

15

20

25

30

0 10 20 30 40 50 60

Time (d)

Myc

eliu

m (

cm)

0

2

4

6

8

10

Gro

wth

rat

e (c

m /

wee

k)

Figure 5. Radial mycelium length (�) and growth rate (×) of Strophariarugosoannulata in the pilot-scale experiment.

270

280

290

300

310

0 30 60 90 120 150 180

Time (d)

Mas

s (k

g)

0

200

400

600

800

CO

2 (m

g/kg

)

Figure 6. Total mass of the soil and fungal bark inoculum tubes (�) andconcentration of CO2 measured from exhaust air (◦) in the pilot-scaleexperiment.

DISCUSSIONIn this study, a fungal pre-treatment process for reduction oforganic matter in contaminated soil was developed. At present,only big rocks are separated from contaminated soil by sievingprior to the combustion process (Rantsi R, Niska and Nyyssonen,personal communication). With the method proposed the amountof organic matter in the soil is reduced and the efficiency ofthe combustion process improved. The fungal pre-treatment isdesigned to work on site where the fungal bark inoculum tubescan be placed inside a soil pile. The process was tested in pilot-scale where a ratio of 3 : 100 (w : w) of fungal bark inoculum to soilwas used. The same inoculum to soil ratio is enough for full scaleapplication. For fungal treatment a daily average temperature ofat least 10 ◦C is required.1 In southern Finland possible treatmenttime would then be 6 months: from the beginning of May until theend of October. On the other hand, fungal activity also producesheat, e.g. the temperature of soil treated with P. chrysosporiumwas, during a winter in southern Finland, 10 ◦C higher than the airtemperature.20 This could allow even longer treatment times.

In this study, the degradation of soil organic matter in 6 monthswas estimated based on the results obtained in 53 to 90 days. Forthe sawmill soil this was 20% in 6 months when treated with S.stellatus and 10% when treated with S. rugosoannulata. In a furtherpilot-scale experiment with S. rugosoannulata, mass loss due todegradation of soil organic matter was 10% of the total mass of

the soil in 6 months supporting results from the laboratory scaleexperiments. S. rugosoannulata was selected for the pilot-scaleexperiment because it represents LDF and thus was expected tobe the most suitable for growth in the soil. WRF and LDF are bothcapable of degrading cellulose and hemicellulose from wood orplant litter as well as soil humic substances.2,8 Carney et al. noticedthat soils with higher abundance of fungi and higher activitiesof soil organic matter degrading enzymes compared to controls,gave more rapid organic matter degradation resulting in loweramounts of soil carbon.21

In addition to sawmill soils, two other types of soils differing intheir organic matter content and type of contamination werestudied. Soil from an old shooting range was treated withS. stellatus and another from an abandoned landfill area withS. rugosoannulata. Probably because of the lead contaminationS. stellatus did not grow as well in the shooting range soil as itdid in the sawmill soil. Fungi can tolerate higher concentrationsof toxic heavy metals than bacteria, but the tolerance of fungalspecies differ greatly and even tolerant species suffer in thepresence of high concentrations.22 Tuomela et al. showed thathigh concentrations of Pb inhibited the growth of LDF and theactivity of some lignin degrading enzymes, but the extent ofinhibition varied among different LDF species.23 According to thatstudy, one of the most tolerant species was S. rugosoannulata, thesame strain used in our study. Nevertheless, S. stellatus producedmore CO2 in Pb contaminated soil than S. rugosoannulata ineither of the studied soils. Unexpectedly, S. rugosoannulata wasable to degrade more organic matter in the landfill soil thanin the sawmill soil. This might be due to the high petroleumhydrocarbon content of the landfill soil. Oil hydrocarbons arerather easily degraded by soil indigenous microbes in addition tothe fungus.24 In the experimental conditions soil was aerated andindigenous microbial activity was enhanced.

At present the only efficient treatment method for soilscontaminated with PCDD/F is combustion. These compoundsare highly stable and lipophilic in nature. However, there arewhite-rot fungi capable of degrading PCDD/F.25 Chlorophenolson the other hand can be degraded by composting as well as withbioaugmentation technology using solid fungal inoculum.26 – 29 Itmight be possible to develop a fungal treatment process for soilscontaminated with PCDD/F and replace combustion, but moreresearch is needed on degradation of PCDD/F especially on solidmedia.

Although, promising results were obtained with both S. stellatusand S. rugosoannulata, economical calculations are needed tofind out whether the process would be commercially feasible.Also, it may not be possible to select only one fungal species fortreatment of all soil types. The tolerance of different contaminantsis species specific. Also the requirements of temperature, moisture,pH, oxygen and nutrients differ between species as well as thecrucial ability to compete against indigenous micro- or macro-organisms. However, many tolerant species have been foundand there is a possibility to choose between fungi for all typesof contamination and soil properties. Test methods could bedeveloped and standardized to find the most suitable fungiamong good candidates. This kind of fungal pre-treatment couldbe beneficial for all kinds of contaminated soils that have highorganic matter content and are intended to be combusted.

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ACKNOWLEDGEMENTSThis research was funded by the Finnish Funding Agency forTechnology and Innovation (TEKES) and the soil constructioncompany Niska & Nyyssonen Oy.

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