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Bioremediation of petroleum hydrocarbon-contaminated soil by

composting in biopiles
K.S. Jrgensen*, J. Puustinen
1
, A.-M. Suortti
Finnish Environment Institute, Research Laboratory, Hakuninmaantie 4-6, FIN-00430 Helsinki, Finland
Received 29 August 1998; accepted 22 May 1999
``Capsule'': Composting is an ecient method that relies on added matrix material and on mixing/aeration, but not on addition of
microbial inoculum.
Abstract
Composting of contaminated soil in biopiles is an ex situ technology, where organic matter such as bark chips are added to
contaminated soil as a bulking agent. Composting of lubricating oil-contaminated soil was performed in eld scale (5 40 m
3
)
using bark chips as the bulking agent, and two commercially available mixed microbial inocula as well as the eect of the level of
added nutrients (N,P,K) were tested. Composting of diesel oil-contaminated soil was also performed at one level of nutrient addi-
tion and with no inoculum. The mineral oil degradation rate was most rapid during the rst months, and it followed a typical rst
order degradation curve. During 5 months, composting of the mineral oil decreased in all piles with lubrication oil from approxi-
mately 2400 to 700 mg (kg dry w)
1
, which was about 70% of the mineral oil content. Correspondingly, the mineral oil content in
the pile with diesel oil-contaminated soil decreased with 71% from 700 to 200 mg (kg dry w)
1
. In this type of treatment with
addition of a large amount of organic matter, the general microbial activity as measured by soil respiration was enhanced and no
particular eect of added inocula was observed. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Bioremediation; Oil-contaminated soil; Composting; Biopiles
1. Introduction
Bioremediation technologies are today well estab-
lished for the clean-up of chemically contaminated land,
and many technologies are applied commercially in
large scale. The rst bioremediation technologies that
developed were ex situ technologies, i.e. the treatment of
excavated soil in contrast to in situ technologies which
aim at treatment without excavation and often is taking
care of both groundwater and soil pollution (Skladany
and Metting, 1993).
Ex situ bioremediation technologies include slurry-
phase remediation, where a water phase is added to
enhance the physical mixing; treatment-bed remediation,
where usually only nutrients are added and the bed of
usually 01 m height is agitated mechanically at inter-
vals by a mixing device. Biopiles refer to the piling of the
material to be biotreated by adding nutrients and air
into piles or windrows usually to a height of 24 m.
Biopiles may be static with installed aeration piping or
they may be turned or mixed by special devices for this
purpose. Biopiles may be amended with a bulking
agent, usually with straw, saw dust, bark or wood chips
or some other organic material. If organic material is
added the technology is termed composting. A more
advanced type of composting is a drum compostor,
which is closed and has a continuous feeding and output.
Landfarming is the term for the older practice of treating
oil wastes by adding, for example, oil sludge and nutrients
to agricultural land and mixing by agricultural practices.
Basically, it is possible to add microbial inocula, i.e.
bioaugmentation, to all these types of technologies.
Many organic contaminants have successfully been
bioremediated in biopiles. This technology has been
demonstrated to function in eld pilot or full scale
especially for petroleum hydrocarbons (Samson et al.,
0269-7491/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PI I : S0269- 7491( 99) 00144- X
Environmental Pollution 107 (2000) 245254
www.elsevier.com/locate/envpol
* Corresponding author. Tel.: +358-9-403-00851; fax: +358-9-403-
00890.
E-mail address: kirsten.jorgensen@vyh. (K.S. Jrgensen).
1
Present address: Mikrobiologitoimisto Puustinen and Rahkonen,
Helsinki Science Park, Koetilantie 3, FIN-00710 Helsinki, Finland.
1994; Puustinen et al., 1995; Filauro et al., 1998; Koning
et al., 1998), polyaromatic hydrocarbons (PAHs)
(Dubourguier et al., 1995; Porta et al., 1997), chlor-
ophenols (Valo and Salkinoja-Salonen, 1986; Ha ggblom
and Valo, 1995; Laine and Jrgensen, 1997, 1998; Laine
et al., 1997a,b) and for nitro aromatics.
The important questions for full-scale bioremediation
applications are: (1) how low a concentration of the
contaminant can you obtain (bioavailability, microbial
activity)?; (2) what is the fate of the contaminant
(mineralization, biotransformation, evaporation, build-
up of microbial biomass, incorporation to the bound
residue, etc.)?; (3) how much time is needed to obtain the
set goal (degradation rate)?; and (4) what are the costs?
A large range of microbial genera have been reported
to degrade hydrocarbons (Atlas, 1981; Rosenberg,
1992), and a broad spectrum of genera can be isolated
from hydrocarbon- contaminated soil and ground water
(Ka mpfer et al., 1991). This is despite the fact that cyclic
hydrocarbons, especially, are toxic to bacterial mem-
branes (Sikkema et al., 1995). Contaminated soil is
often poor in organic matter and has a general low
microbial activity. Usually the bacterial community is
adapted to the presence of the contaminant, but other
environmental conditions such as nutrient availability
and oxygen concentration may be unfavourable, and
thus the microbial degradation of the contaminant is
slow in situ. Composting technologies can be applied to
clean-up contaminated soil ex situ. By adding an
organic matrix to contaminated soil the general micro-
bial activity is enhanced and also the activity of specic
degraders, which may be found in the contaminated soil
or introduced along with the organic material.
We have tested the application of this technology in eld
scale using bark chips as the introduced organic matter for
the clean-up of petroleumhydrocarbon-contaminated soil.
Here we report on the results of composting two dierent
kinds of hydrocarbon-contaminated soil: lubricating oil-
and diesel oil-contaminated soil. The main emphasis was
on the composting of lubricating oil-contaminated soil,
where we also tested the ecacy of commercially available
microbial inocula as well as the level of added fertilizers.
2. Materials and methods
2.1. Pretreatment of the soil
Soil contaminated with lube oil (motor lubricating oil)
and diesel oil was excavated from three service stations
in the Helsinki area. Approximately 400 m
3
of soil (600
tonnes) consisting of approximately equal volumes of
lube oil- or diesel oil-contaminated soils was trans-
ported to an asphalted composting area at a waste
treatment station 35 km north of Helsinki and treated
separately throughout the process. In order to avoid
stones and other rough material causing mechanical
problems during mixing, the soils were sieved with a
screen shaker (65-mm grid). The reject consisted of dif-
ferent coarse material such as stones, lumps of concrete,
oil containers and asphalt, etc. It was deposited on the
neighbouring landll area.
2.2. Piling
The sieved soils were stacked with the bulking agent
in separate piles of approximately 1.51.7 m height and
3.5 m width. Five piles of 1520 m length were con-
structed with the lube oil-contaminated soil. The diesel
oil-contaminated soil was rst placed in two piles of 45
m length. These two piles were combined to one after 1
month of composting due to space limitation at the site.
The ambient temperature during the preparation work
was 1520

C.
Spruce bark was used as the bulking agent. The total
amount of bark used in the piles was approximately 150
m
3
and the ratio of soil to bulking agent was approxi-
mately 1:3 on a volume basis. Nutrients and inocula
were added to the piles according to Table 1.
2.3. Nutrient amendments
Nutrients were added as dry nitrogen-rich compound
fertilizer (Typpirikas Y-lannos 1, Kemira, Finland). The
product contained 26% N, 3% P, 3% K, 1.5% S, 0.5%
Mg, 0.03% B and 0.0006% Se. Nitrogen compounds
consisted of NH
4
-N (54%) and NO
3
-N (46%). In the
four lube oil-contaminated soil piles the addition was
approximately 1 kg of fertilizer per cubic metre of
compost matrix. In the control pile no nutrients were
added. In one of the piles (pile 5) the dosage was initi-
ally two times higher than in the other piles. Further-
more, half of the original dosage was applied to pile 5
after the 60 and 95 days mixings. Nutrients were added
manually on the top of the piles before the mixing.
2.4. pH adjustment
The original pH of the contaminated soils varied in
the range of 6.06.5. The pH of the bulking agent
(spruce bark) was 4.9. The pH of the composts was
Table 1
Nutrient and inocula amendments in the lube oil-contaminated soil
piles
Pile No. Nutrients Inocula
1 + Oilbac
2 +
3 + PRC 107 DTX
4 (control)
5 ++
246 K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254
adjusted by ne granular lime (Kalkki 2, Nordkalk Oy,
Sipoo, Finland). The addition was done according to
the measured pH values and the known buering capa-
city of the contaminated soil types. The target value was
neutral or slightly basic. Adjustment resulted in initial
pH values of 7.17.4.
2.5. Inocula
For the composting of lube oil-contaminated soil two
commercial inocula were used. One was a dry powder con-
sisting of a mixed culture of oil-degrading bacteria, trade
name ``PRC 107 DTX'' (Interbio Inc., USA, distributed in
Finland by Biocora Oy) and the other one, ``Oilbac''
(Ecolution Oy, Helsinki, Finland) was a matrix of sur-
face soil from a landfarming site used for the disposal of
oil wastes. The inocula were added manually on the top
of the piles just before the rst mixing.
2.6. Mixing and aeration of the piles
The piles were mixed and aerated by turning with a wheel
loader-drawn or tractor-drawn screw-type mixer (Anon.,
RTA Virtanen Oy, Hyvinka a , Finland and Allu 24, Idea-
chip Oy, Hollola, Finland). For the rst two months (June
July) the piles were turned twice a month and after this
once a month. The last mixing was done in September.
After mixing the piles were covered with tarps. Covering
was to prevent drying and wetting of the piles and also to
diminish the release of volatile compounds fromthe system.
The total volume of the completed piles was about 400 m
3
.
2.7. Sampling
Compost samples for chemical and microbiological
analyses were taken every time the piles were turned
(mixed). One composite sample was taken from each pile
by sampling from 1012 random locations at 020 cm
depth, combining and mixing in a big jar. Because of the
vigorous mixing of the piles prior to sampling it was con-
sidered unnecessary to sample at higher depths. The com-
posite samples (approximately 1520 l per pile) were sieved
(8-mm grid), collected in glass jars and stored at +4

C for
a maximum of 7 days if not analysed immediately. On one
occasion (November 1993) the composite samples were
taken in duplicate to see the variation between the samples.
In order to monitor volatile degradation products in
the piles and to estimate the loss of oil hydrocarbons
into the atmosphere compost gases were also analysed.
The samples were taken after a 12-days equilibration
period after each mixing. Gases were vacuum pumped
(Aircheck Sampler Model 224-PCXRT; SKC Inc.,
USA) from perforated tubes (12 tubes per pile) which
were set at approximately 1-m depth in the piles. Vola-
tile hydrocarbons were absorbed onto activated carbon
ampoules (SKC Inc., USA) for later analysis. CO
2
and
moisture samples were taken by the Dra ger gas detector
system (Dra ger, Dra gerwerk AG Lu beck, Germany).
2.8. Microbial respiration in the soil compost piles
Basic and substrate-induced respiration were mea-
sured with a method modied from DECHEMA (1992).
Field-moist soil corresponding to 10 g dry soil was
incubated in triplicate at 22

C in 126-ml glass asks


sealed with teon-lined rubber stoppers. The evolved
CO
2
in the headspace was then measured using an Easy-
Quant IR carbon analyser. For the basic respiration the
bottles were incubated for up to 2 days. For the sub-
strate-induced respiration samples were incubated for 2-
h. This short incubation time does not allow time for
exponential microbial growth but, according to Ander-
son and Domsch (1978), this immediate activity is a
direct measure of the microbial biomass size. In the
substrate-induced respiration, carbon, nitrogen and
phosphorus were added as powder as follows: 100 mg of
glucose, 15 mg of NH
4
Cl and 2 mg of KH
2
PO
4
per 10 g
dry soil.
2.9. Enumeration of bacteria
The number of colony-forming heterotrophic bacteria
in compost samples was determined by plating a dilu-
tion series on tryptoneglucoseyeast extract medium
(TGY) diluted to 1:5 (American Public Health Associa-
tion, 1989). Each dilution was plated in duplicate. Also,
enumeration of hydrocarbon-degrading bacteria was
attempted on a mineral medium containing motor oil or
diesel as the sole carbon source. The mineral medium
contained (in mg l
1
) the following: KH
2
PO
4
, 8.5;
K
2
HPO
4
, 21.7; Na
2
HPO
4
2H
2
O, 33.4; NH
4
Cl, 0.5;
CaCl
2
2H
2
O, 39.4; MgSO
4
7H
2
O, 22.5; FeCl
3
6H
2
O,
0.2; cycloheximidine, 150; noble agar, 1500. Dierent
methods for addition of motor oil were tested: sonica-
tion of hot agar solution after autoclaving, application
of hexaneacetone solubilized oil to solidied plates
(200 ml of oil per plate [1520 ml of medium]). Diesel
(200 ml) was added to a small piece of lter paper which
was placed in the lid of the petri dish. These plates were
incubated in a closed glass jar. The nal amount of the
carbon source available in the solidied medium was
approximately 1% (w/w). Control plates were incubated
without any carbon source.
2.10. Isolation and identication of bacteria from
inocula and compost piles
Colonies were isolated from TGY plates with the
highest dilutions. These were anticipated to represent the
most numerous culturable bacteria. Approximately 10
colonies were isolated from each of the following sam-
ples: inoculum PRC 107 DTX, inoculum Oilbac, original
K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254 247
lube oil-contaminated soil, compost pile 2 (no inoculum)
and compost pile 3 (inoculated with PRC 107 DTX).
The isolates were puried by restreaking single colo-
nies three times on TGY (1:5) solid medium. The iden-
tication of isolates was done by API NE and API 20E
(bioMerieux sa, France) and Biolog (Biolog, Inc., CA,
USA) identication systems.
2.11. Denitrication in compost piles
Denitrication in the compost piles was followed
during the course of the composting season. The rate of
denitrication was measured using the acetylene block-
age method (Tiedje, 1982). In this method the accumu-
lation of the intermediate nitrous oxide (N
2
O) was
measured in the presence of acetylene (C
2
H
2
), which
inhibits the further reduction to N
2
.
Natural moist soil samples corresponding to 10 g of
dry soil were incubated in 126-ml asks sealed with
teon-lined rubber stoppers. Half of the bottles were
ushed with nitrogen, N
2
, to create anaerobic condi-
tions before the addition of acetylene. Acetylene was
added with a syringe through the rubber stopper to a
nal concentration of 9% (vol). The asks were incu-
bated at 22

C in the dark. Each sample was prepared in


triplicate. The accumulation of N
2
O was measured by
withdrawing a 0.5-ml gas sample and injecting it into a
gas chromatograph equipped with an electron capture
detector. Separation was done on a 1.8-m long porapak
Q column using a carrier gas ow of 30 ml min
1
(ArCH
4
[5%]). Samples with atmospheric composition
in the headspace were incubated for up to 1 day and
samples with a nitrogen atmosphere were incubated up
to 8 h.
2.12. Chemical and physical analysis
Compost matrices were analysed for the total oil
hydrocarbon (oil and grease) content (i.e. compounds
soluble in carbon tetrachloride, CCl
4
) and the mineral
oil content with an in-house method based on the Fin-
nish standard, SFS 3010 (1980). The procedure included
an extraction of 2030 g of the eld-moist or air-dried
samples with 50 ml of CCl
4
in an ultrasonic bath for 30
min. The extract was allowed to settle and was ltered
through prewashed lter paper (589
1
or 589
3
; Schleicher
and Schu ll). The extraction was repeated twice with 25
ml of CCl
4
. The extracts were combined and the
volume was made up to 100 ml with CCl
4
. The extract
was measured with infrared spectrometry by calculating
the sum of absorbances at wavelengths of approxi-
mately 2960 and 2930 cm
1
. The results for total oil
hydrocarbons and for the mineral oil content were
obtained before and after the removal of polar material
by aluminium oxide (Al
2
O
3
) column separation, respec-
tively.
To visualize the hydrocarbon composition of the
samples, some of the ltered extracts were also analysed
by a gas chromatograph equipped with a ame ioniza-
tion detector (GCFID). This was performed on sam-
ples of the original contaminated soil, bark chips and
the compost mixture during the composting time. A gas
chromatograph (HP 5890 II) tted with a SE-54 (length
50 m; i.d. 0.25 mm) column was used. Asplitless injection
of 1 ml at 250

C was performed and the oven pro-


gramme was as follows: 40

C for 4 min, ramps from 40


to 280

C at 10

C/min and 280

C for 32 min. The chro-


matograms were examined only visually and were only
used for qualitative interpretation. Quantitation or
fractionation on the silica gel column into aliphatic and
aromatic components was not performed. The total
volatile organic compounds (TVOCs) in the air pores of
the compost piles were measured from the activated
carbon ampoules. The trapped TVOCs were desorbed
with carbon disulde and analysed by GCFID.
Separation was done on a HP-5 (length, 50 m; i.d. 0.3
mm) column using the oven programme as follows:
45

C for 4 min, ramps from 45 to 175

C at 10

C/ min,
175

C for 5 min. Injection and detector temperatures


were 220 and 250

C, respectively. The TVOC analyses


were performed at the Tampere Regional Institute for
Occupational Health.
Nutrients (N, P) were analyzed by the Kjeldahl
method (N) and plasma emission spectrophotometry
(P). NH
4
-N and NO
3
-N assay included sample extrac-
tion by 0.1 M K
2
SO
4
followed by distillation (NO
3
-N
with Devarda reagent) and titrimetric analysis. The
nutrient as well as pH measurements were done by
Novalab Oy (Karkkila, Finland).
Soil moisture was measured by drying (at 105

C) to
constant weight. All results throughout the paper are
calculated per dry weight.
Temperature measurements in the composts were
done at three to ve locations per pile at approximately
50 cm depth. The pile temperatures recorded are the
averages of the measured values. The thermometer
(Testo 700, Testoterm GmbH and Co, Lenzkirch, Ger-
many) included a measuring instrument and a rigid
probe up to 3 m long.
3. Results
3.1. Oil hydrocarbon degradation
The mineral oil content in the piles was monitored
after each mixing of the piles except for the last sam-
pling in November, when no mixing was performed.
The degradation in the piles containing lubricating oil
and diesel oil are shown in Fig. 1.
The presented data is obtained from extraction of
eld-moist samples. Control experiments with air-dried
248 K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254
samples during the last two samplings showed that the
mineral oil content in the eld-moist samples was
underestimated up to 2673% depending on the con-
centration level. The average relative standard deviation
for the mineral oil content of the duplicate composite
samples [n 7 (ve duplicates from the lube oil piles
and two duplicate samples from the diesel pile)] was 16
% (431%) in the eld-moist samples.
There was some variation (approximately 300 mg
kg
1
) in the initial concentrations (time [t] = 0) of the
parallel ve lube oil piles due to the heterogenous nature
of the piles. The pattern of the decline was very clear.
The degradation was most rapid during the rst month
and the rate declined with time. Lube oil compounds
were degraded at the same rate in all piles, but the con-
trol pile (pile 4) showed a clear lag period. The residual
concentrations of mineral oil after 5 months composting
were about one third of the initial ones, which were on
average 2400 and 700 mg kg
1
for the lube oil and diesel
oil piles, respectively.
The chromatograms gave qualitative information on
the composition of the hydrocarbons in the samples.
Some of the peaks seen at t 0 were originating from
the bark chips, based on the chromatograms of the ori-
ginal soil and the bark chips separately (data not
shown). They disappeared however for the most part in
1 month. They were not seen in the compost matrix
extracts puried with Al
2
O
3
column before GC run.
After 2 months the GC trace of the lube oil compost
matrix revealed a signicant reduction in the unre-
solved `envelope'. Also many of the resolved peaks (not
alkanes) had decreased (Fig. 2).
3.2. Volatile hydrocarbons in the compost gases
The gas phases in the piles were analysed for TVOCs.
The summary of the measurements (Table 2) shows that
volatilization of the oil hydrocarbons happened during
the rst few weeks of processing. Volatile losses in the
control pile (pile 4) were about one fourth lower than in
all the other piles.
3.3. Composting temperatures
The lube oil pile temperatures clearly showed a dis-
tinct increase of pile temperature during the rst month
of composting (Fig. 3). Only in the control pile was the
temperature rise was somewhat slower, reaching the
maximum after 2 months of processing. The control pile
also remained remarkably warmer for about 30 days
longer. The maximum temperatures were about the
same for all of the piles, the highest slightly exceeding
40

C. The latest measurements were done in November.


At that time the outdoor temperature was 2

C and
the compost temperatures had dropped to around 5

C.
3.4. Microbial respiration
Microbial activity in the piles was monitored by soil
respiration (CO
2
production) tests and bacteria enu-
meration. Basic soil respiration, which reects the actual
degradation of organic matter, and substrate-induced
respiration (so called potential bioactivity) measure-
ments, where glucose, nitrogen and phosphorus were
added to all sample bottles, are shown in Fig. 4.
Respiration in the lube oil piles followed the same
pattern as the temperatures. The control pile showed a
lag phase, but the activity was higher than in the others
after 2 months of composting. Substrate induced
respiration (SIR) in the lube oil piles showed the same
chronological pattern as the basic respiration. SIR was,
however, about three times higher than the basic
respiration. A distinct exception was the SIR activity in
the control pile. It increased slowly during the season as
did the basic respiration.
Fig. 1. Degradation of mineral oil in lube oil- and diesel oil-con-
taminated soil during composting in biopiles.
Fig. 2. Chromatograms (GCFID) of mineral oil content of lube oil-
contaminated soil compost matrix (pile 5) at time zero and after 2
months.
K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254 249
3.5. Enumeration of bacteria
The numbers of culturable heterotrophic bacteria in
the lube oil compost piles varied from 0.8 to 8:2 10
8
colony-forming units (CFU) g
1
(dry soil) (data not
shown). The counts did not change signicantly during
the composting period. The number of bacteria in the
sieved soil before the preparation of the piles was 1:8
10
7
CFU g
1
(dry soil). The number thus increased
about 10-fold immediately after the preparation of the
compost piles.
The plate counts of oil-degrading bacteria did not
succeed very well. The addition of lubricating oil to the
mineral medium created an irregular surface and the
counts were unreliable. Plates incubated with diesel as
the carbon source in the headspace were possible to
count. The control plates without any carbon source,
however, gave about the same count or even higher than
the ones incubated in the presence of diesel or gasoline.
That might be due to toxic eects of diesel. Counts on
mineral medium ranged from 3:0 10
7
to 1:8 10
8
CFU g
1
(dry soil) and mineral medium plus diesel as
the carbon source counts ranged from 5:3 10
7
to 1:5
10
8
CFU g
1
(dry soil). There was no distinct pattern in
the counts (data not shown).
3.6. Identication of bacteria isolated from inocula and
compost matrix
Four dierent bacterial species were identied in the
PRC 107 DTX inoculum: Enterobacter sakazakii,
Bacillus mycoides, Klebsiella oxytaca and Acinetobacter
calcoaceticus/Gen 13. In the Oilbac inoculum, bacteria
were identied as Bacillus megaterium, Pseudomonas
diminuta, Gluconobacter cerenius, Pasteurella caballi and
one species belonging to the genus CDC plus several
unidentiable species.
In pile 2, which was not inoculated, we found Sphin-
gomonas paucimobilis, Sphingobacterium multivorum
and several other bacteria that did not match any of the
data bases of the identication systems. In pile 3, which
was inoculated with PRC 107 DTX, we identied iso-
lates as Pseudomonas vesicularis, Sphingomonas pauci-
mobilis, Sphingobacterium mizutaii, Sphingobacterium
multivorum, CDC group DF-3 and several unidenti-
able bacteria.
None of the species present in the PRC 107 DTX
inoculum were found among the 10 colonies isolated
from the most dilute plate of the inoculated compost
pile (pile 3).
The API NE system was only able to identify ve
strains out the 54 tested strains to a reliable level
(>98%). The API 20E system identied ve strains of
19 tested and the Biolog system gave identication to 18
out of 23 tested strains. There was good correlation
between the identication of the API and the Biolog
system.
3.7. Denitrication
The rates of denitrication in the compost matrices
are shown in Fig. 5. The rates under aerobic conditions
were ranging from 0 to 18 ng N
2
O-N g
1
(dry soil) h
1
Table 2
Total volatile organic compounds (TVOCs, mg m
3
) in gas samples from lube oil-contaminated soil piles
a
Windrow No. Treatment 5.7.93 16.7.93 21.7.93 11.8.93 24.8.93
1 Oilbac inoculum 261 79 nm 2.8 0
Nutrients
2 No inoculum 381 235 32 13 0
Nutrients
3 PRC 107 DTX inoculum 325 214 46 29 0
Nutrients
4 Control 89 43 nm 15 3.7
No nutrients
5 No inoculum 313 308 nm 12 <0.5
Extra nutrients
a
Not measured.
Fig. 3. Temperatures in lube oil-contaminated soil piles during com-
posting.
250 K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254
and under anaerobic conditions from 0 to 2285 ng N
2
O-
N g
1
(dry soil) h
1
. The rate under anaerobic condi-
tions was thus more than 100 times higher than under
aerobic conditions. The true oxygen conditions in the
compost piles were not measured but CO
2
concentra-
tions up to 35% in the soil gas air indicated that the
oxygen concentrations would have been fairly low
between the mixing of the piles. Under both aerobic and
anaerobic conditions the rate of denitrication
decreased during the composting time.
4. Discussion
4.1. Oil hydrocarbon analyses
The measurement of mineral oil hydrocarbon ana-
lyses for soils were performed on eld-moist samples.
Air drying of the samples before extraction was avoided
because of the possible loss of volatile compounds.
Drying can, however, be done chemically by using
sodium sulphate (Na
2
SO
4
) and this appears to be
important when using a non-polar solvent for extrac-
tion, as in this case. Sodium sulphate absorbs water
which interferes with the extraction of oil hydrocarbon
compounds. The yield can thus be improved. The ana-
lytical data presented here are therefore to some extend
underestimated. However, they are sucient to follow
the trend of degradation and eciency of biodegradation
and to make other conclusions. Recent method devel-
opment is focussed on extraction of eld-moist samples
with a mixture of non-polar and polar solvents.
4.2. Biodegradation
The results of the eld experiment showed that
there was a distinct decline in the mineral oil con-
centrations during the composting. Two thirds of the
original mineral oil was degraded. The maximum
degradation intensity was highest during the rst 2
months. There was about the same degradation pattern
for the hydrocarbons in the original lube- and diesel-
contaminated soils. The degradation pattern followed a
typical rst-order degradation. A similar pattern is
reported by most authors independent on the starting
concentration.
Fig. 4. Rates of basic respiration (upper panel) and substrate-induced
respiration (lower panel) in lube oil-contaminated soil piles during
composting.
Fig. 5. Rates of denitrication in lube oil-contaminated soil pile
matrices during composting. Aerobic assay (upper panel); Anaerobic
assay (lower panel).
K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254 251
Filauro et al. (1998) and Porta et al. (1998) reported
48% degradation from an initial total petroleum
hydrocarbon (TPH) concentration of 10,000 mg kg
1
and 60% degradation from a starting concentration of
TPH of 16,000 mg kg
1
. These were eld-scale biopiles
with wood chips as bulking agent. Bench-scale experi-
ments from the same site showed a slightly more eective
degradation of 80% (Tamburini, 1997), but still the
same pattern.
Mixed aqueous cultures of hydrocarbon-degrading
bacteria also show a rst-order degradation pattern.
Dott et al. (1989) reported degradation percentages (1%
fuel oil) from 24 to 64% from commercially available
inocula for oil degradation. The degradation of n-
alkanes in these cultures were from 50 to 90%.
Experiments with dierent types of hydrocarbon-
spiked soil show the same pattern of degradation (Song
et al., 1990; Zhou and Crawford, 1995). However, it
seems that it is possible to obtain lower end point con-
centrations with freshly gasoline-spiked soil than with
aged contaminants at concentrations of TPH below
1800 mg kg
1
(Zhou and Crawford, 1995). Further-
more, it was shown that the residual amount is higher in
heavier distillates (gasoline<jet fuel<heating oil<die-
sel oil<bunker oil; Song et al., 1990). The level of the
residual amount, which is degraded very slowly, is very
important for the applicability of the bioremediation
process. The exact nature of the residual amount is not
known, but it is likely that it consists of branched-
chain alkanes, multi-ring saturates (naphtenes) and
aromatics, each of which may have alkyl side chains
attached to their core ring structure. For old con-
taminations a poor bioavailability of the residual
fraction, which is `locked up' in particle pores, seem to
be the reason for this phenomenon even though it
cannot be excluded that the residual fraction is inher-
ently recalcitrant (Heusemann, 1997). Another expla-
nation may be changes in the microbial community. At
the end of a bioremediation event, the stimulated
microbial community is aged and the benets of
degrading the contaminant may be lost.
4.3. Microbial activity
Respiration rates seem to reect the unspecic degra-
dation of the added bulking material, and did not
directly correlate with the mineral oil degradation rates.
Especially, the respiration rates of the control pile did
not correlate with the degradation pattern. This was
also observed by Samson et al. (1994), who used saw
dust as bulking agent in composting of oil-con-
taminated soil. These authors found good correlation
with the actual degradation using
14
C-radiorespirometry
for specic hydrocarbons as well as gene probes for
alkanes (alkB) and xylene (xylE) degradation. The same
phenomenon was observed by Laine et al. (1997a) when
composting chlorophenol-contaminated soil in biopiles
using bark chips as the bulking agent. In this case,
plating on selective media for chlorophenol degraders
correlated better with the degradation rate than did the
respiration rate, which reects the unspecic microbial
activity. The plating on selective media in the present
study did not give reliable results, but other techniques
such as most probable number, gene probes or radio-
respirometry may describe the actual hydrocarbon-
degradation activity better than respiration or the total
number of heterotrophic micro-organisms.
As judged from the gas chromatograms the hydro-
carbons present in the soil did not resemble the
chromatograms of fresh products (data not shown).
Apparently, evaporation of volatile compounds has
taken place during the initial excavation, transport and
sieving of the soil. The hydrocarbons left in the soil were
degraded during the composting. TVOCs were tran-
siently produced in the piles (Table 2) and mainly in
piles with high microbial activity. If all these TVOCs
were lost to the atmosphere during mixing they would,
however, only constitute a very small fraction of the
total oil content. Due to the health and environmental
risks associated with the loss of volatile compounds,
composting of gasoline-contaminated soil, which con-
tain more than 50% of TVOCs, should not be recom-
mended.
4.4. Denitrication
The degradation of hydrocarbons is mainly thought
to be an aerobic process, at least the initial oxidation of
the terminal end methyl group. Degradation products
of hydrocarbons and monoaromatic hydrocarbons
(BTEX) can also be degraded under anaerobic condi-
tions in the presence of nitrate through the denitrica-
tion process (Schocher et al., 1991). This may happen in
anaerobic micro sites in the otherwise aerobic compost
matrix.
Denitrication is the reduction of nitrate (NO
3

) to
gaseous nitrogen (N
2
) during the oxidation of organic
matter by bacteria (Tiedje, 1988). In other words,
nitrate is used as an alternative electron acceptor instead
of oxygen in bacterial respiration. Denitrication only
occurs when the oxygen concentration is very low or in
the absence of oxygen. Denitrication aects the
microbial activity in two dierent ways: (1) an ecient
respiration can continue in the compost piles if oxygen
is exhausted; and (2) the nitrate converted to nitrogen
gas is a loss of nitrogen source for growth.
According to the measured denitrication rates under
completely aerobic conditions (average 5 ng N g
1
h
1
)
a loss of nitrogen of 0.12 mg N kg
1
(dry soil) day
1
could have occured. Under completely anaerobic
conditions (average 1000 ng N g
1
h
1
) a loss of 24 mg
N kg
1
(soil) day
1
could occur. The actual oxygen
252 K.S. Jrgensen et al. / Environmental Pollution 107 (2000) 245254
concentration in the compost piles was probably in
between these two limits as was the actual denitrica-
tion rate. The true loss of nitrogen due to denitrication
was thus between 0.12 and 24 mg N kg
1
(dry soil)
day
1
. The nitrogen addition was 117 mg NO
3

-N kg
1
and 138 mg NH
4
+
-N kg
1
. The added nitrate could thus
be lost due to denitrication between 5 and 1000 days.
4.5. Inocula
Two commercial inocula were tested. No signicant
eect of this bioaugmentation on the process was
observed. The natural microbial community in soil is
usually able to degrade oil hydrocarbons, since many of
the bacterial species known to degrade hydrocarbons
are commonly found in soil. It is more important to
create suitable conditions for these indigenous bacteria
than to introduce new species. It is also important to
note that none of the introduced organisms were re-iso-
lated by traditional plating techniques. The Biolog sys-
tem was able to identify all the morphologicaly dierent
bacteria in the dried mixed culture (PRC 107 DTX) and
80% of the isolates from the Oilbac inoculum. How-
ever, from the compost piles around 40% of the isolates
did not match the database. During composting of
chlorophenol-contaminated soil straw compost with
induced chlorophenol-degradation activity as a mixed
inoculum was tested (Laine et al., 1997a, b). No eect
was observed in comparison to the addition of bark
chips and nutrients. Inocula may be benecial in certain
special cases such as single recalcitrant compounds or
bioremediation without bulking agent with soil with
poor microbial activity. Morrison et al. (1997) reported
from pilot-scale biopiles with no bulking agent 44%
degradation and showed up to 71% degradation with a
bacterial inoculum. On the other hand, Neralla and
Weaver (1997) did not nd any signicant impact of
commercial inocula on the degradation of crude oil in
marine microcosms. In most cases for petroleum
hydrocarbons inocula are only an extra cost and give no
benet.
Acknowledgements
This work was supported by the Finnish Petroleum
Federation and the Finnish Ministry of the Environ-
ment.
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