Effect of copper on the degradation of phenanthreneby soil micro-organisms

J. Sokhn, F.A.A.M. De Leij, T.D. Hart and J.M. LynchSchool of Biomedical and Life Sciences, University of Surrey, Guildford, UK

2001/73: received 16 May 2001 and accepted 31 May 2001

J . SOKHN, F .A .A .M . DE LEIJ , T .D . HART AND J.M. LYNCH. 2001.

Aims: The effect of copper on the degradation by soil micro-organisms of phenanthrene,

a polycyclic aromatic hydrocarbon, was investigated.

Methods and Results: Inert nylon ®lters were incubated in the soil for 28 days at 25°C. Each

®lter was inoculated with a soil suspension, phenanthrene (400 ppm), copper (0, 70, 700 or

7000 ppm) and nitrogen/phosphorus sources. The ®lters were assessed for phenanthrene

degradation, microbial respiration and colonization. Phenanthrene degradation proceeded even

at toxic copper levels (700/7000 ppm), indicating the presence of phenanthrene-degrading,

copper-resistant and/or -tolerant microbes. However, copper at these high levels reduced

microbial activity (CO2 evolution).

Conclusions: High levels of copper caused an incomplete mineralization of phenanthrene and

possible accumulation of its metabolites.

Signi®cance and Impact of the Study: The presence of heavy metals in soils could seriously

affect the bioremediation of PAH-polluted environments.

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in

the environment and mainly produced by combustion

processes (Mueller et al. 1996). They are known or suspected

to be genotoxic or carcinogenic and have been classi®ed as

priority pollutants. The study of their fate in nature is

therefore of great environmental concern (Mueller et al.1996; Cuny et al. 1999; Wilcke 2000). While concentrations

of individual PAHs in soil produced by natural processes are

estimated to be around 1±10 lg kg±1, recently measured

lowest concentrations are frequently 10 times higher (Wilcke

2000). Organic horizons of forest soils and urban soils may

contain individual PAH concentrations of several 100 lg kg±1

(Wilcke 2000), whereas concentrations of PAHs in highly

polluted soils vary from 10 mg kg±1 to 10 g kg±1 dry weight

(Stieber et al. 1994). Persistence of PAHs in the environment

is linked to their general recalcitrance, binding to the soil

matrix and low water solubility, making them non-bioavail-

able to PAH-degrading organisms (Cuny et al. 1999).

Heavy metal exposure has, since the last century, been

known to affect microbial growth and survival (BaÊaÊth

1989). An extensive literature is available on the effects of

heavy metals on microbial populations and microbial

processes, such as litter decomposition and carbon miner-

alization (Tyler 1974; BaÊaÊth 1989; Angle and Chaney 1991;

Hattori 1992). However, little is known about the effect of

heavy metals on the degradation of recalcitrant hydrocar-

bons, such as PAHs. Whereas some metals, such as copper,

are essential for bacteria and fungi in trace amounts, high

concentrations are known to be toxic (Yamamoto et al.19851 ). The addition of copper to soil signi®cantly inhibits

soil respiration, nitrogen mineralization and nitri®cation

(BaÊaÊth 1989; Hattori 1992; McGrath 1994). However,

tolerance and adaptation of micro-organisms to heavy

metals are common phenomena, and the presence of

tolerant fungi and bacteria in polluted environments has

frequently been observed (Arnebrant et al. 1987; Deighton

and Goodman 1995). The negative effects of heavy metals

on soil microbes and soil microbial processes means that

their presence in contaminated soils can potentially limit

the bioremediation of organic pollutants. The in¯uence of

heavy metals on PAH degradation in polluted soils has

only recently been emphasized (Baldrian et al. 2000). This

study examined the effect of different copper concentra-

tions on the indigenous microbial communities, and its

effect on the biodegradation of phenanthrene.Correspondence to: Prof. J.M. Lynch, School of Biomedical and Life Sciences,

University of Surrey, Guildford, Surrey, GU2 7XH, UK

(e-mail: j.lynch@surrey.ac.uk).

ã 2001 The Society for Applied Microbiology

Letters in Applied Microbiology 2001, 33, 164±168

MATERIALS AND METHODS

Treatments

Inert ®lters (4 cm2) consisting of nylon capillary matting

were treated in the following way. An 8% w/v soil

suspension (approximately 104 cfu 0á1 ml±1) and a solution

containing 5á0 g l±1 KH2PO4, 3á5 g l±1 NaNO3, 0á7 g l±1

Tween 80, phenanthrene in acetic acid (400 ppm), and

CuSO4 (0, 70, 700 or 7000 ppm) were added to each

moistened ®lter. The solution (0á2 ml) and soil suspension

(0á1 ml) were added to the centre of the ®lters and assumed

to have spread uniformly by capillarity. The treated ®lters

were transferred to Petri dishes (all with a diameter of

14 cm) ®lled with sieved sandy soil (Holiday Hills series). In

total, 18 Petri dishes were prepared for each treatment, each

containing 20 ®lters. After 0, 2, 7, 14, 21 and 28 days

incubation at 25°C, three Petri dishes for each treatment and

each time point were destructively sampled to assess

phenanthrene degradation, microbial activity and microbial

populations associated with the different Cu treatments. For

this purpose, at each sampling day, ®lters were removed

from the soil, cleaned from any adhering soil and extracted

as described below.

Measurements

Phenanthrene degradation: solvent extraction and gaschromatography (GC). Eight ®lters taken from each Petri

dish were placed in a 250 ml ¯ask containing 60 ml hexane

(BDH). The ¯asks were placed on a shaker (250 rev min±1,

25°C). The hexane fraction was recovered after a 24 h

extraction period and evaporated in the fume hood over-

night. The dried phenanthrene extracts were re-dissolved

in 0á9 ml hexane and 0á1 ml of a 2000 ppm hexamethyl-

benzene (HMB) stock solution and analysed by GC.

Phenanthrene recovered from the ®lters (re-dissolved in a

1 ml volume) was calculated using the standard curve

equation. Hence, the level of phenanthrene remaining

per ®lter was calculated for every treatment. The GC used

for this experiment was a Hewlett-Packard 5890 A, with

a Hewlett-Packard integrator 3396 A (Hewlett-Packard,

Bracknell, UK) and auto-sampler2 . The carrier gas was

helium. The column was a fused silica, non-polar BP1 column

(25 m, 0á22 mm). The oven temperature was kept at 200°C

for 12 min. All injectors and detectors were set at 250°C.

Microbial respiration. Ten ®lters taken from each Petri

dish were placed in a 250 ml Erlenmeyer ¯ask. Flasks were

sealed with a rubber bung and CO2 evolving from the ®lters

was allowed to accumulate in the ¯ask. After a 32 h

incubation at 25°C, a 60 ml sample was collected from each

¯ask using a disposable syringe. Each sample was manually

injected into an infrared (IR) CO2 analyser, type 225 MK3

(Analytical Development Co. Ltd, Hoddesdon, UK3 ). Nitro-

gen was used to dilute the CO2 samples where appropriate.

Bacteria. From each Petri dish, one ®lter was taken, soaked

in 10 ml sterile Ringers-Agar solution (quarter strength

Ringers and 0.05% Bacteriological Agar (Oxoid)) and

allowed to stand for 15 min. The tubes were then mixed

on a Vortex mixer for 30 s, after which the ®lter was

removed from the tube using sterile metal forceps. Ten-fold

dilutions down to 10±7 were prepared and 0á1 ml aliquots of

each dilution were inoculated onto 1á0% Tryptone Soya

Agar (TSA, Oxoid). The plates were incubated at 25°C for 6

days, after which the numbers of visible bacterial colonies

were counted. The colony-forming unit (cfu) counts were

quanti®ed on plates containing between 20 and 350 colonies.

From the total cfu present on the dilution plate, the original

number of bacteria in each ®lter was calculated.

Fungi. The ®lters for fungal assessment (one per Petri dish)

were aseptically cut into 2 mm2 pieces and washed with

1 ml RA solution to remove most of the conidia. Each

2 mm2 piece was placed in the middle of a Malt Extract

Agar (MEA, Oxoid) plate containing streptomycin and

penicillin (Sigma) at 100 and 60 lg ml±1, respectively. The

plates were incubated at 25°C for 6 days. The fungal species

dominating the MEA plates were identi®ed.

Statistical analysis

Statistical analyses were carried out using SPSS 10á0 for

Windows 98 (SPSS Inc., Chicago, IL, USA4 ). All data were

analysed using one-way analysis of variance (ANOVAANOVA), at 0á05

signi®cance level, using three replicates (n � 3). Where

more than two means were compared, signi®cant differences

between treatments were analysed using a test for least

signi®cant difference (LSD).

RESULTS

Increasing concentrations of Cu (7000 and 700 ppm)

resulted in decreasing degradation of phenanthrene

(P < 0á01 and P < 0á05, respectively). No signi®cant dif-

ference in phenanthrene degradation was observed at

70 ppm Cu during the course of the experiment. Whereas

almost no phenanthrene was recovered at the end of the

incubation from the ®lters containing 0 ppm and 70 ppm

Cu, 10±15% of the compound could still be recovered from

the 700 and 7000 ppm Cu treatments (Fig. 1).

Cu reduced microbial respiration at all times during the

28 day incubation period. This was especially evident in the

®lters containing Cu at 7000 ppm (P < 0á001) and 700 ppm

(P < 0á01). Cu (70 ppm) also signi®cantly reduced microbial

Cu AND PHENANTHRENE DEGRADATION 165

ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164±168

activity throughout the incubation period (P < 0á05)

(Fig. 2).

In general, the presence of Cu decreased the microbial

colonization of the ®lters (Fig. 3). Cu at 7000 ppm signi-

®cantly decreased (P < 0á001) microbial numbers at all

times. A similar trend was observed with the 700 ppm

(P < 0á001), apart from the last day of incubation where

microbial colonization seemed to have increased, but at

70 ppm Cu microbial counts were only slightly lower

(P < 0á05).

100

90

80

70

60

50

40

30

20

10

0

0 7 14 21 28

Days of incubation

% P

hena

nthr

ene

rem

aini

ng

Fig. 1 Phenanthrene (%) recovered from

®lters treated with different concentrations of

Cu: (d), 0 ppm; (h), 70 ppm; (n), 700 ppm;

(s), 7000 ppm. Filters were incubated in the

soil at 25°C over a 28 day incubation period.

n � 3, S.E. bars are shown

0 2 7 14 21 28

3000

2500

2000

1500

1000

500

0

Days of incubation

CO

2 ev

olut

ion

(ppm

)

Fig. 2 Microbial activity measured in the

form of CO2 (ppm) produced over 32 h from

®lters treated with different concentrations of

Cu: (h), 0 ppm; (j), 70 ppm; ( ), 700 ppm

( ), 7000 ppm. Filters were incubated in the

soil at 25°C over a 28-day incubation period.

n � 3, S.E. bars are shown

12.00

10.00

8.00

6.00

4.00

2.00

0.000 7 14 21 28

Days of incubation

log 10

(cf

u)

Fig. 3 Microbial colonization of ®lters incu-

bated with different concentrations of Cu:

(d), 0 ppm; (h), 70 ppm; (n), 700 ppm;

(s), 7000 ppm. Filters were incubated in the

soil at 25°C over a 28 day incubation period.

n � 3, S.E. bars are shown

166 J . SOKHN ET AL.

ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164±168

The species of fungi identi®ed included species of

Penicillium and Trichoderma, but by far the most dominant

fungus up to day 28 of incubation was a Zygorrhynchus sp.

This fungus was tolerant to the presence of Cu (7000 ppm);

its growth characteristics (25°C, medium humidity, acidic

environment), and zygospores shape resembled those of

Zygorrhynchus moelleri (Domsch and Gams 1980). No

conclusive evidence regarding the phenanthrene degradation

potential was obtained. It is, however, evident that the

fungus is both phenanthrene- and Cu-tolerant.

DISCUSSION

The static phase in the bacterial growth pro®le was probably

a re¯ection of the time period needed for the phenanthrene-

degrading bacteria to multiply several fold before any

appreciable loss of the chemical caused by bacterial activity

could be detected. It is known that the percentage of the

substrate that is either mineralized or incorporated depends

on the species carrying out the transformation, the identity of

the substrate, its concentration and, probably, other envi-

ronmental factors (Alexander 1994). Before this principle can

be applied to phenanthrene degradation, the disappearance

of phenanthrene should be considered carefully.

Growth-linked biodegradation, in which the degrading

organisms convert the substrate to CO2, as well as cell

components and products typical of the usual catabolic

pathways (Alexander 1994), is observed at all Cu levels.

However, although phenanthrene was disappearing in all the

treatments, the rate of CO2 production did not correspond

to the decrease in phenanthrene at high Cu concentrations.

This suggests that at high Cu levels, the parent compound

was not being fully mineralized to CO2. Hence, the

metabolites of phenanthrene were probably accumulating

at high Cu levels.

There are fundamental differences in the mechanisms of

PAH metabolism used by micro-organisms. In complex

environments such as soil, C assimilated is generally

estimated as Cassimilated � Csubstrate ± Cmineralized. The C

assimilated is further mineralized as the cells metabolizing

the parent compound are themselves decomposed

(Alexander 1994). This could further explain the delayed

mineralization, marked by the high CO2 levels observed

towards the end of the experiment, when almost all the

parent compound had disappeared. Therefore, it could be

concluded, that the activity of the soil inoculum was spread

into three phases. These phases have previously been

reported in the degradation of PAHs in contaminated soils

(Stieber et al. 1994) and proceed as follows: (i) metabolism

of the initial compound phenanthrene; (ii) degradation of

the dissolved metabolites, accumulated in the aqueous

phase; (iii) degradation of the biomass itself. It should be

noted that the three phases occur simultaneously in this

experiment. The soil inoculum provided the organisms that

degraded phenanthrene and its metabolites. In addition, the

use of the non-ionic surfactant, Tween 80, meant that the

phenanthrene was partitioned into the water phase. Hence,

it was more accessible to the inoculum.

Apart from the supply of nutrients and factors that

controlled the bioavailability of phenanthrene, the chief

abiotic factors in¯uencing its microbial transformation

included temperature, pH, moisture level and presence of

toxins (Cu). The acidity of the ®lters, a result of the addition

of acetic acid, could also explain the short lag phase in both

the bacterial colonization and degradation pro®les, as the

microbial communities needed to adapt to the acidic environ-

ment. It has been demonstrated that, at more moderate pH

values, degradation tends to be faster (Alexander 1994).

Hence, it remains to be investigated whether a higher pH

would have increased the rate of biodegradation of phe-

nanthrene in the ®lters. On the other hand, acetate could

have been used by the soil microbes as an additional carbon

source. Therefore, it could be argued that the presence of

acetic acid could have contributed to the rate of phenanth-

rene degradation observed.

The most important abiotic factor that in¯uenced the

degradation of phenanthrene was the presence of Cu. It has

been established that in a soil environment contaminated

with Cu, growth conditions favour Cu-resistant/tolerant

strains within the community. Over time, an increase in

Cu-tolerant organisms, which were already present at low

density in the non-polluted environment, might have

occurred. Only organisms able to withstand or adapt to

the toxicity of Cu probably persisted. The toxicity of Cu at

700 and 7000 ppm was evident as bacterial populations and

microbial activity were low in these treatments. The

reduction in microbial activity could be due to the direct

toxicity of Cu at the cellular level, or to the inhibition of

enzymes involved in the degradation of the intermediates in

the phenanthrene degradation pathways. However, the

degradation of phenanthrene occurred even at toxic Cu

levels, implying that the degradation of the parent

phenanthrene molecule was being performed by highly-

adapted, Cu-resistant species.

In summary, the degradation of phenanthrene was

retarded by the presence of Cu. Most likely, high Cu levels

were directly toxic to organisms metabolizing intermediates

formed from the degradation pathway of phenanthrene, or

inhibited the enzymes involved in the degradation of these

intermediates (or both). The outcome in both cases was

the incomplete mineralization of phenanthrene and the

presumed accumulation of its metabolites. This implies that

heavy metals could seriously affect the detoxi®cation of

PAH-polluted environments. The degradation of the parent

PAH molecules is indeed a critical step towards detoxi®ca-

tion, but the accumulation of possibly toxic metabolites in

Cu AND PHENANTHRENE DEGRADATION 167

ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164±168

the presence of metals could prove to be increasingly

important issues, especially relating to their toxicity and

persistence in the environment.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the Lebanese

National Council for Scienti®c Research for partial ®nancial

funding of this work.

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