International Biodeterioration & Biodegradation 63 (2009) 316–321

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Biological treatment of oilfield-produced water: A field pilot study

Mang Lu a, *, Zhongzhi Zhang a, Weiyu Yu a, Wei Zhu b
a
College of Chemical Engineering, China University of Petroleum, Beijing 102249, China
b
Petroleum Survey, Design & Research Institute, Shengli Oilfield, Dongying 257000, Shandong Province, China

a r t i c l e i n f o a b s t r a c t

Article history: A field test was conducted on a hydrolysis acidification/bio-contact oxidation system (HA/BCO) to treat
Received 25 May 2008 oilfield-produced water with high salinity. By operating the biodegradation system for three months
Received in revised form with a hydraulic retention time (HRT) of 32 h and a volumetric load of 0.28 kg COD m3 d1, the treatment
31 August 2008
process achieved mean removal efficiencies of 63.5% for chemical oxygen demand (COD), 45% for NH3-N,
Accepted 29 September 2008
Available online 4 February 2009
79.5% for total suspended solid (TSS), and 68.0% for total petroleum hydrocarbon (TPH). GC/MS was used
to analyze relative changes of components of main organic waste in a process indicating that the influent
wastewater contained organic compounds from C12H26 to C35H72, which could be degraded effectively
Keywords:
Oilfield-produced water with the coordinated action of hydrolysis acidification and aerobic treatment. The use of maize powder
Biological treatment can enhance environmental adaptability of microorganisms and biodegradation ability and is recom-
Total petroleum hydrocarbon mended as a nutrient supplement to maintain good treatment performance.
Biodegradation Ó 2009 Elsevier Ltd. All rights reserved.
GC/MS

1. Introduction Generally speaking, oilfield-produced water has a high flow

Produced water is the main source of oily water as many Chinese
oilfields are in their mid- or final stage of production and the oil
produced contains a very large volume of waterdup to 90 percent
(Yang and Wang, 2002). Until now, most of the oilfield-produced
water in China was re-injected into the stratum without being
treated properly, which could cause a variety of damages to the
stratum (Moghadasi et al., 2004; Bader, 2005). In addition, the
impact of untreated produced water discharged from world oil and
gas operations on shallow water coastal wetlands has recently
become an issue of significant environmental concern (Rayle and
Mulino, 1992).
Biological treatment is an effective and economical approach
that can be used in oil de-emulsification and wastewater treatment
(Campos et al., 2002; Eusébio et al., 2007; Chavan and Mukherji,
2008). Petroleum hydrocarbon-degrading microorganisms, namely
bacteria, yeast, and fungi, which can grow using crude oil as
a source of carbon and energy, have been reported as oil degraders
(Atlas, 1981; Wammer and Peters, 2005; Verma et al., 2006; Man-
cera-López et al., 2008).
* Corresponding author. Tel.: þ86 10 89734284; fax: þ86 10 69744636.
E-mail address: bjlumang@hotmail.com (M. Lu).
rate and a high suspended solid (SS) content, is composed of
complex components, and is heavily polluted and difficult to
degrade. In addition, as oilfield wastewater is low in N and
P compounds, the conventional activated sludge (CAS) process
cannot operate efficiently over the long term, as bulking and
foaming tend to occur (Stringfellow and Alvarez-Cohen, 1999;
Tellez et al., 2002). Moreover, the high salinity of oilfield waste-
water affects the metabolism of the microorganisms in activated
sludge, causing plasmolysis and attenuating endogenous respira-
tion (Zhao et al., 2006). More importantly, the COD of oilfield
effluent in China is generally lower than 300–400 mg l1 that is the
usual load of the CAS process. Therefore, it is difficult to meet the
discharge criterion using simple biological treatment processes
like CAS, while physical and chemical treatment processes incur
high operation expenses.
Researchers have recently developed new immobilized microbe
treatment processes, in one of which the wastewater is first
hydrolyzed under anoxic conditions, and then treated under
aerobic conditions (Wu et al., 2007). Anoxic hydrolysis–aerobic
treatment of industrial wastewater has been considered to have
some advantages over the conventional processes. For anoxic
hydrolysis, the hydraulic retention time (HRT) is short, and non-
degradable organic compounds of wastewater can be transformed
into degradable substances, i.e., the degradable performance of the

0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2008.09.009
 M. Lu et al. / International Biodeterioration & Biodegradation 63 (2009) 316–321 317

wastewater is improved greatly. Simultaneously, a portion of COD temperatures (15  C to 5  C). Considering that the temperature
can be removed. of injection water was about 55  C, which was nearly an unlimited
heat source, a series of serpentine pipes were laid on the bottom of
2. Materials and methods the tanks to allow injection water flow to heat the wastewater. The
temperature of the system was controlled between 25 and 30  C
2.1. Site description through a valve.
The pH of influent was maintained at 7.0  0.25 with a pH-
The treatment system for this study was constructed in the controller (type FC660, Shanghai) coupled to a peristaltic pump,
South Water Injection Station of the Qinghe Oil Production Plant, which added buffered water containing NaOH and K2HPO4.
Shengli Oilfield, China. The location provided an unlimited source According to titration trials, 350 g NaOH m3 wastewater was
of relatively constant-strength produced water, as well as facilities needed to adjust the pH of influent to 7.0. In addition, with
to house the entire system. The inherent variability of the waste- 300 mg l1 of the influent COD, 16.8 g K2HPO4 m3 wastewater was
water composition is illustrated in Table 1. needed to satisfy the desired C: P ratio of 100:1, and the content of
K2HPO4 in the buffer was only 4.8 percent of NaOH, so the effect of
2.2. Hydrolysis acidification/bio-contact oxidation system K2HPO4 on pH was negligible.
(HA/BCO)
2.4. Source of inoculum
Fig. 1 is the schematic diagram of the pilot-scale arrangement,
which was constructed of armored concrete with a glass fiber Two distinct inocula were utilized in this study. A mixed culture
reinforced plastic (GRP) lining applied to prevent leakage. The of microorganisms isolated from crude oil-contaminated soil was
water flow was driven by gravity, from the bottom of the tank to the used to feed the aerobic tanks. The prominent role of the micro-
top, by means of gravitational potential energy. organism mixture in degrading crude oil, at flask level, was
While in operation, air used to produce oxygen-saturated reported on in our former study (Wei et al., 2007). The second
conditions was supplied to the two aerobic tanks through air inoculum, composed of anaerobic sludge obtained from a local
diffusers, located at the bottom of the tanks, to supply sufficient treatment plant of refinery wastewater, was used to start up the
oxygen to the biomass and to stir the liquid as well as to exfoliate hydrolytic tank.
the aged biofilm.
One kind of combined semi-soft medium constructed by plastic 2.5. Startup and operation of the system
ring and synthetic fiber string, provided by Yixing Strong Ltd., was
chosen as the support for biofilm in both the hydrolytic and the Microorganisms of the aerobic inoculum were first acclimated
aerobic tanks, whose principal physical characteristics are pre- to produce wastewater in a digester operating with a 24-h cycle.
sented in Table 2. The media had a theoretical specific surface area The first six cycles of the aerobic tanks’ operation marked the
of 1520 m2 m3. These semi-soft media were not clogged due to the startup phase during which the tanks operated with 22 h of aera-
special configuration and large specific surface area, and microor- tion, and 2 h of quiescent condition (to allow the biomass to settle
ganisms that were attached onto the media would be able to out of solution). At the start of each cycle, diesel oil and
accumulate faster. The carrier was hung from 25 cm below the top the fermentation broth of human feces were added to the tanks.
of the tanks to the part 25 cm above the rectangular bottom of the The addition of human feces supplied abundant nutrients to the
tanks. When in operation, the tanks were filled with wastewater to microorganisms. During the acclimation stage, exhausted waste-
a depth of 2 m, which resulted in the submergence of all packing. water and excess sludge were removed daily. Afterwards, fresh
wastewater was fed into the tanks. On cycle seven, crude oil was
2.3. Temperature and pH adjustment introduced into the tanks at the start of the aeration period. This
addition further selected for microorganisms in the population that
During the preparative trial, it was found that the water could degrade oil. The aerobic tanks were cultivated over a 24-h fill-
temperature of the first aerobic tank was below 15  C even at the and-draw cycle to stimulate the formation of biofilm on the
HRT of 10 h, because these facilities were operated at ambient packing. Continuous-flow experiments were initiated until the
formation of biofilm on the packing had become evident (verified
by visual inspection). The formation of biofilm on the aerobic
Table 1 packing media was evident 15 days after startup.
Qinghe oil production plant wastewater characterization.
As to anoxic operation, after seeding, the anaerobic sludge was
Parameter Average valve Date range kept under anaerobic conditions without any feeding for 48 h to
Temperature (centigrade) 52 50–55 eliminate any residual dissolved oxygen prior to the operation. The
pH 5.8 5.6–6.0 tank was seeded two times over a week. Each time, 150 l of the
Total solids (mg l1) 46,700 42,300–54,000 anaerobic sludge sample were screened with a 2-mm sieve to
Total dissolved solids (mg l1) 46,530 42,130–53,740
Total suspended solids (mg l1) 155 90–180
remove coarse particles. The pH of the sludge was adjusted to 7.0
TOC (mg l1) 84 76–105 before it was pumped into the tank. The anoxic tank was stabilized
Total phosphorus (mg l1) 0.15 – after one month by re-circulating the waste stream. Biofilms were
Total hydrocarbon (mg l1) 23 14–32 observed on the surface of the packing media. After that the system
CODcr (mg l1) 345 228–481
was fed with constant wastewater and the day was denoted as
CODcr filtered (mg l1)a 302 200–421
BOD5 (mg l1) 72 65–80 day 1.
BOD5/CODcr 0.24 – Operation of the engineered biofilter began in January 2005, and
NH3-N (mg l1) 82 70–90 data collection was started after steady-state conditions were
Mg2þ (mg l1) 143 – obtained. Operational parameters such as gas flow rate and liquid
Ca2þ (mg l1) 1600 –
Cl (mg l1) 24,350 –
rate were monitored if necessary, depending on the test needs. The
a
test, carried out for three months, comprised four stages; the
Millipore membrane – 0.45 mm.
operating conditions of the system are shown in Table 3.
318 M. Lu et al. / International Biodeterioration & Biodegradation 63 (2009) 316–321
biofilm carrier
1 2 3 4
influent
sludge air diffusers
heating ducts sludge sludge
Fig. 1. Schematic diagram of the HA/BCO setup. 1. Settling tank a; 2. Anoxic tank; 3. Settling tank b; 4. Aerobic tank a; 5. Aerobic tank b; 6. Settling tank c.
2.6. Nutrient
As shown in Table 1, the concentration of NH3-N in the waste-
water was sufficient to support the COD removal. However, the
phosphorus source was far below the conventional treatment
standard of 100:5:1. Therefore the desired C:P ratio was maintained
by changing the concentration of K2HPO4 in pH buffer water with
no exogenous nitrogen added.
2.7. Sampling frequency and water quality detection
Water sampling for COD was carried out every day between
9:30 and 10:30 a.m. Sampling for other measurements was carried
out at irregular intervals. The wastewater quality parameters were
monitored according to standard methods (State Environmental
Protection Administration of China, 2002). COD was determined by
filtering with a 0.45-mm filter and then oxidation with potassium
dichromate under strongly acidic conditions and at an elevated
temperature for 2 h. The oil content in the wastewater was deter-
mined by using an infrared spectrophotometry oil-measuring
instrument (H3-OCMA-350, Japan). NH3-N was determined by
Nessler’s reagent colorimetry. The content of phosphorus was
determined by the ascorbic acid method. Additionally, tempera-
ture, dissolved oxygen, and pH were routinely monitored with
probes. Generally, samples were drawn at the end of each tank.
2.8. Gas chromatography/mass spectrometric (GC/MS) analysis
The GC–MS analysis was conducted on day 26. A 50-ml sample
of wastewater was extracted by 5 ml dichloromethane three times
for pH 2, 12, and 7. The three extract layers were combined and
dried using nitrogen, and the residue was dissolved in a 1-ml
solution of n-hexane. Samples were analyzed by gas chromatog-
raphy, using a Thermo-Finnigan SSQ710 GC/MS with HP-5MS
elastic silica capillary columns (60 m  0.25 mm  0.25 mm). The
injection volume was 1 ml. The carrier gas was helium at 37 kPa.
Table 2
Principal physical characteristic of the biofilm anoxic and aerobic tanks.
Physical characteristics* Value
Settling tank a Anoxic tank
Total volume (m3) 3.50 8.75
Working volume (m3) 2.10 5.25
Effective volume (m3) – 4.75
Biofilm carrier density (g cm3) – 0.90
Total volume of the carrier (m3) – 3.75
Total surface areas of the carrier (m3) – 5700
*Determined before the beginning of the biological treatment process.
5 6
effluent
air diffusers
heating ducts
sludge sludge sludge
Flow velocity was 1 ml min1. The analytical conditions were:
initial temperature of 50  C, with isothermal operation for 1 min;
heating to 120  C at a constant rate of 20  C min1; and heating to
a final temperature of 310  C at a constant rate of 4  C min1, with
a 30-min isothermal.
Mass spectrometer conditions were: ionization mode, El; elec-
tron energy, 70 eV; filament current, 100 mA; multiplier voltage,
1200 V; full scan.
3. Results and discussion
3.1. Overall COD removal in the pilot-scale HA/BCO
Generally, if the BOD/COD ratio for untreated wastewater is 0.5
or greater, the waste is easily biodegradable. Below 0.3, the
wastewater contains some toxic components, and special kinds of
microorganisms are required for biodegradation. In this study,
however, the ratio of BOD5 to COD (filtered) (Table 1) was only 0.24,
which meant the remaining organic substance in the wastewater
was refractory for biological treatment. The wastewater was highly
saline, but was low in organic substances and contained refractory
compounds such as cyclic alkanes and PAHs. For all that, as shown
in Fig. 2, after 60 days of operation, the average COD removal effi-
ciency of the system was about 65%. As a general trend, the COD
removal efficiency did not decrease as HRT was reduced from 230
to 32 h, which demonstrated the capability of high resistance for
the COD loading. During the first three days, the effluent COD was
more than 200 mg l1, due to fermentation liquid accumulating in
the wastewater. After that, COD of the effluent decreased sharply to
about 120 mg l1.
From day 12, COD concentration in the effluent climbed slowly;
this was considered to be caused by reduced biological vitality in
the highly salty environment. High salt content in wastewater is
known to significantly reduce COD and nitrogen removal efficiency
(Panswad and Anan, 1999). It has been well demonstrated that
environmental adaptability of microorganisms and biodegradation
Settling tank b Each aerobic tank Settling tank c
1.75 7.00 1.75
1.05 4.20 1.20
– 3.80 –
– 0.90 –
– 3.00 –
– 4560 –
 M. Lu et al. / International Biodeterioration & Biodegradation 63 (2009) 316–321 319

Table 3
Operation conditions of Hydrolysis acidification/bio-contact oxidation system.
Items Stage 1 Stage 2
Duration (days) 1–3 4–11
HRT (h) 230 95
Volume load 0.03–0.04 0.07–0.10
(kg CODm3 d1)
Carrier volume load 0.02–0.03 0.05–0.07
(kg CODm3 d1)
ability can be enhanced by the addition of conventional nutrients,
such as yeast extract and glucose (Armenante et al., 1995; Yuan
et al., 2000; Yuan et al., 2004). In view of this, the proper addition of
appropriate nutrient was believed to be necessary in an attempt to
maintain good performance of the treatment system. From day 13,
a kind of commercially available maize powder was used for the
nutrient supplement for the exogenous hydrocarbon degrader,
with a dosing quantity of 3 kg/day. The cheap maize powder, with
abundant nutrition, was added directly to the anoxic tank. The
powder was fermented by the anaerobic microorganisms and the
nutrients released permitted the aerobic microorganisms to pros-
per. The result showed that with the supplement of maize powder,
biofilter performance improved quickly as COD removal efficiency
increased from 43 to 67%. When the nutrient was added, the COD
content of the system seemed to be increased. The performance of
the system would have declined if the COD brought by the nutrient
entered the effluent. However, the removal efficiency increased
considerably. In fact, the COD brought by the nutrient was mostly
consumed by the microorganisms. The improvement in biotreat-
ment performance indicated that microbial activity was enhanced
with the additional nutrient.
From day 19, cyanobacteria began to be observed in the upper
carrier of the aerobic tanks and gradually flourished. From day 28,
a continuous increase was observed in the effluent COD at a good
operation rate, which ranged from 87 mg l1 on day 28 to
234 mg l1 on day 36. The disruption was deemed caused by cya-
nobacteria toxicity to the microorganisms. Some publications
suggest that cyanobacteria may produce substances toxic to
bacteria (Mezrioui et al., 1994; Oufdou et al., 1998; Vasconcelos and
Pereira, 2001).
In order to block cyanobacteria photosynthesis and therefore
their growth, the two aerobic tanks were covered with two pieces
of black tarpaulin from day 30. This procedure achieved good
results as the effluent COD decreased to 100 mg l1 on day 38 and
remained stable thereafter.
It is obvious that the anoxic section was the major contributor to
COD removal during steady state. In a strictly anaerobic process, in
Stage 3 Stage 4 general, the influent COD content can be transformed into volatile
12–20 21–60 fatty acids (VFA), alcohol, hydrogen, and biomass. However, the
54 32 overall COD content should remain unchanged. In this study, the
0.12–0.17 0.20–0.28 hydrolysis acidification tank operated under anoxic conditions, and
upper biofilms were exposed to dissolved oxygen to a certain
0.09–0.12 0.15–0.20
extent. Therefore, the influent COD was partially decomposed and
another part was transformed into VFA, alcohol, and hydrogen, as
well as biomass.
3.2. Ammonia nitrogen (NH3-N) removal
It can be observed from Table 4 that the anoxic section
contributed little to NH3-N removal although it shouldered the
major part of COD removal in the system. This is because nitrate and
nitrite in the wastewater were reduced to ammonia by fermenta-
tive bacteria under anaerobic conditions. It was believed that
ammonia nitrogen removal obtained under our experimental
conditions was due not only to the nitrogen assimilation process.
Obviously, there was also an overall loss of nitrogen, which would
logically be assumed to be ammonia volatilization, adsorption on
the biofilm due to different electric charges or through simulta-
neous nitrification–denitrification in the aerobic biofilm (Gálvez
et al., 2003). Neither assimilation, volatilization, adsorption, nor
aerobic denitrification were directly measured or individually
calculated for this study.
3.3. Total suspended solid (TSS) removal
Normally, suspended solids contain clay, silt, sand, algae,
microbes, fractions of hydrocarbon in suspension, and other
substances. Total suspended solid (TSS) values for all the treatment
units are compared in Table 5. Obviously, the settling tank (a) and
anoxic tank showed a significant removal of TSS. In this study, the
biological filter was proven to be very efficient in the removal of
non-biological particles. These results can be explained by the
combined structure of the support material, the low flow velocity of
water, and the roughness of the biofilm surface, which facilitates
suspended solid attachment. Other dominant forces that can
control the attachment of particles are electrical interactions
between charged particles and the charged media surface (Gálvez
et al., 2003).
3.4. Total petroleum hydrocarbons(TPH) degradation

Total petroleum hydrocarbon (TPH) values estimate the amount

of petroleum hydrocarbon waste coming into the system. This was
500
influent anoxic outlet
450 Table 4
aerobic (a) outlet aerobic (b) outlet Effects of ammonium nitrogen removal for the HA/BCO during steady state.
400
350 Day Influent Anoxic outlet Effluent Total removal
COD (mg L-1)

(mg l1) (mg l1) (mg l1) efficiency (%)

300
39 72.6 73.4 38.6 46.8
250
41 69.3 71.2 36.9 46.8
200 43 79.6 78.6 41.3 48.1
150 45 73.1 71.3 42.5 41.8
47 80.5 78.1 45.1 44.0
100 49 72.3 73 39.7 45.1
50 51 75.2 76.6 43.6 42.0
53 77.1 77.2 46.2 40.1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 55 78.6 77.9 41.3 47.5
57 72.5 70.6 37.1 48.8
Time (d) 59 78.9 75.8 40.2 49.0
Average 75.4 74.9 41.1 45.5
Fig. 2. COD removal in the pilot-scale HA/BCO.
320 M. Lu et al. / International Biodeterioration & Biodegradation 63 (2009) 316–321

Table 5
a

C19
C20
C21
Total suspended solid removal.

C22
100

C17

C23
C18
1
TSS(mg l ) 90
Day Influent Settling tank a Settling tank b Effluent 80

C16

C24
C25
Relative Abundance
31 90 30 20 20 70

C15

C26
C27
33 140 40 30 10
60
35 180 60 40 20
37 160 70 50 10 50

C28
C31
C29
39 160 50 40 10

C14

C30
40
41 140 40 20 20

C32
30

C13
C12
43 180 70 40 20

C33
45 180 70 40 20 20

C34
C35
47 150 60 30 20
49 180 60 20 10
10
51 140 50 20 20 0
Average 154.5 54.5 31.8 16.4 0 5 10 15 20 25 30 35 40 45 50
Time (min)
b

C18
100
one of the important parameters that were measured to check the
90
removal efficiency of the treatment system. The system showed an
80
overall hydrocarbon waste removal efficiency of 68% (Fig. 3), which

Relative Abundance

C17
was similar to the total COD removal efficiency. Evaporation losses 70

C31
C25
of the lighter fraction of hydrocarbons and removal by the skimmer 60

C29
C16

C20
in the settling tank (a) account for the high percentage of hydro-

C24
50

C27
C26
C23
C19

C22
C21

C30
carbons removal.

C28
40

C33
C32
30

C34
C35
3.5. Biodegradability of n-alkanes

C15
20

C14
C13
C12
10
Gas chromatography/mass spectrometry analysis was per-
formed to identify the presence of the heavier petroleum TNA (total 0
0 5 10 15 20 25 30 35 40 45 50
n-alkanes) compounds in the influent and effluent streams of the
system, when the HRT was 32 h. The analysis is illustrated by Time (min)
chromatographs shown in Fig. 4. Influent produced water typically Fig. 4. Sequential GC/MS unit process analysis, with total n-alkane identification (C12–
consists mainly (>60%) of n-alkanes C12 through C35, with inter- C35) of an oilfield-produced water during hydrolysis acidification/bio-contact oxidation
mediate branched chain hydrocarbons and other petroleum-based system treatment: (a) treatment system influent; (b) treatment system effluent.
compounds. The highest peak response value occurs at 25.36 min,
corresponding to heptadecane, 2,6,10,14-tetramethyl-. The TNA resulting from the greater degradation of other compounds, when
contains eight kinds of branch-chain alkanes and 16 kinds of more easily degradable compounds were degraded and these
straight-chain alkanes that range from dodecane (C12H26) to pea- substances accumulated. However, further research is needed to
tatriacontane (C35H72). determine how this happened.
As can be seen from comparing Fig. 4(a and b), the GC–MS
abundance and area value of most substances (except for C18, C31,
4. Summary
C35) in the effluent decreased greatly, which means a significant
enhancement is achieved in the quality of the effluent, confirming
A field test was carried out to investigate the performance of an
the capability of the system in decomposing the organic
HA/BCO process using immobilized microorganisms on combined
compounds in the oilfield-produced water. The increase in the
plastic carriers. It was found that the HA/BCO was an efficient and
abundance of C18, C31, and C35 could be attributed to concentration
cost-effective biological treatment process not yet reported as
being able to purify oilfield-produced water with a high salinity of
50 90 46,530 mg l1 and low organic load of 302 mg l1 COD. During the
removal efficiency test period, the treatment process achieved mean removal effi-
45 80
influent ciencies of 63.5% for COD, 45% for NH3-N, 79.5% for TSS, and 68.0%
TPH concentration (mg L-1)

40 effluent for TPH. It is interesting to note that the whole domestication

70
Removal efficiency(%)

35 period was only 20 days with the addition of feces fermentation

60 broth, which was quite accessible and inexpensive. In addition,
30
50 the supplement of a kind of maize powder was found to be
25 effective in maintaining excellent performance of the treatment
40 process.
20
30
15
Acknowledgements
10 20

5 10 This work was funded by the National Natural Science Foun-

dation of China (40672211) and the Scientific Foundation of
0 0
18 23 28 33 38 43 48 53 58 Shengli Oil Field. The authors are grateful to State Key Laboratory
Time(d) of Heavy Oil Processing of China for their technical assistance and
to Shengli Oil Field for field and logistical assistance during this
Fig. 3. TPH degradation in the pilot-scale HA/BCO. work.
 M. Lu et al. / International Biodeterioration & Biodegradation 63 (2009) 316–321 321

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