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Letters in Applied Microbiology 2001, 33, 164168

Effect of copper on the degradation of phenanthrene


by soil micro-organisms
J. Sokhn, F.A.A.M. De Leij, T.D. Hart and J.M. Lynch
School of Biomedical and Life Sciences, University of Surrey, Guildford, UK
2001/73: received 16 May 2001 and accepted 31 May 2001

J . S O K H N , F . A . A . M . D E L E I J , T . D . H A R T A N D J . M . L Y N C H . 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 25C. 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.
Signicance 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 classied 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 110 lg kg1, 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 kg1
(Wilcke 2000), whereas concentrations of PAHs in highly
polluted soils vary from 10 mg kg1 to 10 g kg1 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-bioavailable to PAH-degrading organisms (Cuny et al. 1999).
Heavy metal exposure has, since the last century, been
known to affect microbial growth and survival (Baath
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).

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


heavy metals on microbial populations and microbial
processes, such as litter decomposition and carbon mineralization (Tyler 1974; Baath 1989; Angle and Chaney 1991;
Hattori 1992). However, little is known about the effect of
heavy metals on the degradation of recalcitrant hydrocarbons, 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.
1 1985). The addition of copper to soil signicantly inhibits
soil respiration, nitrogen mineralization and nitrication
(Baath 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 inuence 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 concentrations on the indigenous microbial communities, and its
effect on the biodegradation of phenanthrene.
2001 The Society for Applied Microbiology

Cu AND PHENANTHRENE DEGRADATION

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 01 ml1) and a solution
containing 50 g l1 KH2PO4, 35 g l1 NaNO3, 07 g l1
Tween 80, phenanthrene in acetic acid (400 ppm), and
CuSO4 (0, 70, 700 or 7000 ppm) were added to each
moistened lter. The solution (02 ml) and soil suspension
(01 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 25C, 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 gas
chromatography (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 min1,
25C). The hexane fraction was recovered after a 24 h
extraction period and evaporated in the fume hood overnight. The dried phenanthrene extracts were re-dissolved
in 09 ml hexane and 01 ml of a 2000 ppm hexamethylbenzene (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,
2 Bracknell, UK) and auto-sampler. The carrier gas was
helium. The column was a fused silica, non-polar BP1 column
(25 m, 022 mm). The oven temperature was kept at 200C
for 12 min. All injectors and detectors were set at 250C.
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 25C, a 60 ml sample was collected from each
ask using a disposable syringe. Each sample was manually

165

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


3 (Analytical Development Co. Ltd, Hoddesdon, UK). Nitrogen 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 107 were prepared and 01 ml aliquots of
each dilution were inoculated onto 10% Tryptone Soya
Agar (TSA, Oxoid). The plates were incubated at 25C for 6
days, after which the numbers of visible bacterial colonies
were counted. The colony-forming unit (cfu) counts were
quantied 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 ml1, respectively. The
plates were incubated at 25C for 6 days. The fungal species
dominating the MEA plates were identied.
Statistical analysis
Statistical analyses were carried out using SPSS 100 for
4 Windows 98 (SPSS Inc., Chicago, IL, USA). All data were
analysed using one-way analysis of variance (ANOVA), at 005
signicance level, using three replicates (n 3). Where
more than two means were compared, signicant differences
between treatments were analysed using a test for least
signicant difference (LSD).
RESULTS
Increasing concentrations of Cu (7000 and 700 ppm)
resulted in decreasing degradation of phenanthrene
(P < 001 and P < 005, respectively). No signicant difference 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, 1015% 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 < 0001) and 700 ppm
(P < 001). Cu (70 ppm) also signicantly reduced microbial

2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164168

166 J . S O K H N E T A L .

100
% Phenanthrene remaining

90
80
70
60
50
40
30
20
10
0
0

28

21

14
Days of incubation

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 25C over a 28 day incubation period.
n 3, S.E. bars are shown

3000

CO2 evolution (ppm)

2500
2000
1500
1000

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 25C over a 28-day incubation period.
n 3, S.E. bars are shown

500
0
0

14

21

28

Days of incubation

12.00

log10 (cfu)

10.00
8.00
6.00
4.00
2.00
0.00
0

14
Days of incubation

activity throughout the incubation period (P < 005)


(Fig. 2).
In general, the presence of Cu decreased the microbial
colonization of the lters (Fig. 3). Cu at 7000 ppm signicantly decreased (P < 0001) microbial numbers at all

21

28

Fig. 3 Microbial colonization of lters incubated with different concentrations of Cu:


(d), 0 ppm; (h), 70 ppm; (n), 700 ppm;
(s), 7000 ppm. Filters were incubated in the
soil at 25C over a 28 day incubation period.
n 3, S.E. bars are shown

times. A similar trend was observed with the 700 ppm


(P < 0001), 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 < 005).

2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164168

Cu AND PHENANTHRENE DEGRADATION

The species of fungi identied 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 (25C, 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 prole was probably
a reection of the time period needed for the phenanthrenedegrading 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 environmental 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

167

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 inuencing 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 proles, as the
microbial communities needed to adapt to the acidic environment. 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 phenanthrene 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 phenanthrene degradation observed.
The most important abiotic factor that inuenced 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 highlyadapted, 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 detoxication of
PAH-polluted environments. The degradation of the parent
PAH molecules is indeed a critical step towards detoxication, but the accumulation of possibly toxic metabolites in

2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164168

168 J . S O K H N E T A L .

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 Scientic Research for partial nancial
funding of this work.
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2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 164168