International Biodeterioration & Biodegradation 95 (2014) 338e345

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International Biodeterioration & Biodegradation

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

Comparison of bioremediation strategies for soil impacted with

petrochemical oily sludge

vio A.O. Camargo c,
Vanessa S. Cerqueira a, *, Maria do Carmo R. Peralba b, Fla

tima M. Bento a, *
Fa
a
b
c

Department of Microbiology, Federal University of Rio Grande do Sul, Sarmento Leite, 500, CEP 90050-170, Porto Alegre, RS, Brazil
Department of Inorganic Chemistry, Federal University of Rio Grande do Sul, Bento Gonalves, 9500, CEP 91500-970, Porto Alegre, RS, Brazil
Department of Soils, Federal University of Rio Grande do Sul, Bento Gonalves, 7712, CEP 91540-000, Porto Alegre, RS, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 December 2013
Received in revised form
7 June 2014
Accepted 24 August 2014
Available online 3 October 2014

Different bioremediation techniques (natural attenuation, biostimulation and bioaugmentation) in

contaminated soils with two oily sludge concentrations (1.5% and 6.0%) in open and closed microcosms
systems were assessed during 90 days. The results showed that the highest biodegradation rates were
obtained in contaminated soils with 6% in closed microcosms. Addition of microbial consortium and
nutrients in different concentrations demonstrated higher biodegradation rate of total petroleum hydrocarbons (TPH) than those of the natural attenuation treatment. Soils treated in closed microcosms
showed highest removal rate (84.1 0.9%) when contaminated at 6% and bacterial consortium and
nutrients in low amounts were added. In open microcosms, the soil contaminated at 6% using biostimulation with the highest amounts of nutrients (C:N:P of 100:10:1) presented the highest degradation
rate (78.7 1.3%). These results demonstrate that the application of microbial consortium and nutrients
favored biodegradation of TPH present in oily sludge, indicating their potential applications for treatment
of the soils impacted with this important hazardous waste.

Keywords:
Bioaugmentation
Biostimulation
Bioremediation
Oily sludge
Soil landfarming

2014 Elsevier Ltd. All rights reserved.

1. Introduction
Petroleum processing generates considerable amounts of a
residue named oily sludge. One of the main issues faced by reneries and petrochemical industries is related to the safe disposal
of this residue, since its destination and/or inappropriate treatment
can cause serious impact to the environment and potential risk to
human health (Cameotra and Singh, 2008; Xu and Lu, 2010). Oil
sludge has been classied by the United States Environmental
Protection Agency (US EPA) as a hazardous organic complex (Ubani
et al., 2013). Usually, the oily sludge contains water, sand, oils,
grease, organic compounds, chemical elements, and metals. Among
the organic compounds present, the most common are alkenes,
cyclic alkenes, benzene, toluene, ethyl-benzene, xylenes and polycyclic aromatic hydrocarbons (PAHs) and phenols (Kriipsalu et al.,
2008). Polycyclic aromatic hydrocarbons (PAHs) consist in a

* Corresponding authors. Tel.: 55 51 33084497; fax: 55 51 3308 3665.

E-mail addresses: vanescerqueira@yahoo.com.br (V.S. Cerqueira), fatimabento@
yahoo.com.br (F.M. Bento).
http://dx.doi.org/10.1016/j.ibiod.2014.08.015
0964-8305/ 2014 Elsevier Ltd. All rights reserved.

serious environmental concern, as many of them are cytotoxic,

mutagenic and potentially carcinogenic (Ubani et al., 2013).
Bioremediation of hydrocarbon-contaminated soils, which exploits the ability of microorganisms to degrade and/or detoxify
organic contaminants, has been established as an efcient, economic, versatile and environmentally safe treatment (Singh and
Lin, 2008). In petroleum processing plants, when treating residues, mainly oily sludge, the landfarming process is a frequently
chosen method, due to its operational simplicity, low cost (when
compared to other techniques), high potential for contaminant
removal and high rate of residue application, varying from 83 to
252 m3/ha/year (Marn et al., 2005; Jacques et al., 2007). However,
the method still shows several limitations especially to what
regards to appropriate management and control, large areas requirements, the environmental unfavorable conditions of microbial
growth and inefciency in the reduction of inorganic contaminants
level (Khan et al., 2004).
Bioremediation process efciency of contaminated areas depends on several factors; the most important ones are the presence
of microorganisms with appropriate catabolic capacity on the
contaminated site, favorable environmental and nutritional

V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

conditions to microbial growth and metabolism, contaminant

composition and concentration and the pollutant bioavailability to
microbial attack.
Two techniques have been proposed to increase the efciency of
bioremediation process: biostimulation and/or bioaugmentation.
Biostimulation process consists in the introduction of nutrients,
mainly nitrogen and phosphorus under the form of organic and/or
inorganic fertilizers, in the contaminated system to stimulate the
present microbial population growth and activity. The bioaugmentation process consists in the introduction of microorganisms, previously characterized as potential degraders to target
contaminant, in the contaminated environment aiming an increase
in the microbial population and, as a consequence, an increase in
the biodegradation rate (Das and Chandran, 2011; Tyagi et al., 2011;
Taccari et al., 2012).
The objective of the present work was to evaluate bioremediation potential in contaminated soils with different oily sludge
concentrations in open and closed microcosms using biostimulation and bioaugmentation techniques.
2. Material and methods
2.1. Sampling
Dystrophic Red Argisoil soil samples (depth of 0e30 cm) were
collected randomly in 5 points in landfarming cells, which have
received petrochemical oily sludge for three decades. The sampling
was made at the end of the treatment period and before applying
more oily sludge. The samples were sent to the laboratory, homogenized and the soils sieved to 2 mm. Representative samples of
soil and oily sludge were submitted to physical chemical characterization (Table 1).
2.2. Bioremediation experiments
For soil bioremediation, two contamination levels (1.5% and 6%)
of oily sludge were tested in open and closed microcosms. The soil
contamination was executed in the laboratory at the experiment
start. For each different combination of contamination level and
microcosm condition, the following treatments were carried out in
triplicate:
1) Natural attenuation (NA): soil oily sludge
2) Biostimulation BS(NP): soil oily sludge nutrients with
high concentration
3) Bioaugmentation BA(NP): soil oily sludge nutrients with
high concentration bacterial consortium
Table 1
Physical chemical analyses of Landfarming soil and oily sludge.
Parameters

Landfarming
soil

Oily
sludge

Methodology

Humidity
pH
SMP index
Oil and
greases
Organic
carbon
Nitrogen
Total
Phosphorus
Total
Potassium

15%
6.4
6.8
1.75%

ND
ND
ND
48%

Gravimetry/105  C
pH in water 1:1
e
Soxhlet extraction

9.5%

88%

Humid combustion/Walkey Black

0.14%
0.08%

0.06%
0.01%

0.08%

<0.01%

Kjeldahl
Humid digestion nitric-perchloric/ICP;
OES
Humid digestion nitric-perchloric/ICP;
OES

ND: not determined.

339

4) Biostimulation BS(NeP): soil oily sludge nutrients with

low concentration
5) Bioaugmentation BA(NP): soil oily sludge nutrients with
low concentration bacterial consortium
2.3. Bacterial consortium
On the bioaugmentation experiments a bacterial consortium of
5 bacteria with biodegradation potential for oily sludge and production of biosurfactants isolated directly from the oily sludge
(Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus
cibi) and soil (Pseudomonas aeruginosa and Bacillus cereus) was
used. The isolates were identied based on gene partial sequencing
16S rRNA (Cerqueira et al., 2011).
The bacterial inoculum was prepared through the transference
with a platinum wire from the isolate (stock culture) to asks
containing the sterile nutritive broth, which were kept in an orbital
shaker at 100 rpm, 30  C for 24 h. Thereafter the cells were
centrifuged at 4  C, 9000 rpm during 15 min, washed with sterile
saline solution (0.85%) and the processes was repeated 3 times. The
cells were suspended in sterile saline solution (0.85%). The inoculum of each isolate was standardized and the consortium added to
the soils of each experimental unit in order to inoculate 108 cells/g
of soil.
2.4. Experimental design
Experiments were carried out during 90 days in 1.0 L microscoms (glass asks). In experiments with closed microcosm the
asks were kept tightly closed and in open microcosm experiments
the asks were kept without lids. Experiments in open microcosms
were performed to represent eld conditions, where the humidity
adjustment was more frequent. The amount of soil in each ask was
calculated in dry basis (200 g). The soils were contaminated with
1.5% and 6% w/w of petrochemical oily sludge.
In the biostimulation and bioaugmentation experiments, nitrogen and phosphorus were added using solutions of (NH4)2SO4
and KH2PO4 respectively. The C:N:P ratio was adjusted to 100:10:1
and 100:0.4:0.2 for experiments that corresponds to high and low
amounts of nutrients. Control experiments were carried out with
soil without applying contaminants and treatments.
Soil humidity was periodically adjusted by adding sterile
distilled water, after the weighing of asks, in order to keep the
eld capacity in 70%. The eld capacity was determined according Silva et al. (2007). The soils were periodically revolved in
order to increase the aeration rate. The asks were maintained
indoor and exposed to temperature variations to represent eld
conditions. Room temperature was monitored as a function of
time.
2.5. Soil analyses
Soils were sampled at 0 and 90 days for quantication of total
petroleum hydrocarbons (TPH) and after 7, 14, 30, 60 and 90 days
for microbiological analyses.
2.5.1. Counting of hydrocarbon degrading microorganisms and total
heterotrophics
The counting of microorganisms in soils was performed in each
of the triplicate samples for every treatment at the corresponding
sampling time by the Most Probable Number Method (MPN)
(Braddock and Catterall, 1999). Initially, the soil sample was submitted to decimal dilution with a saline solution (0.85%) in test tube
and homogenized in a vortex for 30 s. Dilutions in 24-well microtitre plates were then made.

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V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

In order to determine total heterotrophic microorganisms

250 mL of Tryptic Soy Broth medium (TSB) was dispensed into 96well microtitre plates, added with 10 mL of each dilution. The
plates were incubated at 30  C during 14 days and the microbial
growth was determined by turbidimetry. For the determination of
hydrocarbon degrading microorganisms the minimum mineral
medium (MM1) was used (composition in g/L: KCl, 0.7; KH2PO4,
3.0; NH4NO3, 1.0; micronutrients solution, 1 mL/L (MgSO4, 4.0;
FeSO4, 0.2; MnCl2, 0.2; CaCl2, 0.2)) (Richard and Vogel, 1999) and a
solution 2.3.5-tripheniltetrazolium chloride (TTC) as indicator
(Bento et al., 2005; Cerqueira and Costa, 2009). After inoculation of
each dilution the plates were inoculated with 1% of the oily sludge.
The plates were incubated at 30  C during 14 days and the microbial
growth was determined by the development of a violet color
(signaling reduction of the indicator via microbial respiration). The
estimative microbial population was then determined using MPN
tables (APHA, 1995).
2.5.2. Respiratory activity
The microbial respiratory activity in closed microcosm containing oily sludge contaminated soil was evaluated by the cumulative liberation of CO2 (Stotzky, 1965). A capturing system for CO2
was assembled in each microcosm. The capturing system consisted
in a plastic cup containing 20 mL of NaOH 0.5 mol/L suspended in a
metal frame. Flasks without soil were used as blank in order to
quantify the CO2. The asks were kept tightly closed and only
periodically opened for basal respiration determination. To perform
the analysis, 1 mL of BaCl2 30% and 3 drops of phenolphthalein 1%
were added to the NaOH. The residual amount of NaOH was titrated
with HCl 0.5 mol/L. The carbonic gas produced was calculated by
the equation:

CeCO2 generated mg=Kg soil

VB  VA $MC =2$ MHCl $FC=m

where: VB is volume in mL of HCl 0.5 mol/L used to titrate the blank

proof; VA is volume in mL of HCl 0.5 mol/L used to titrate the
treatment; MC is the carbon molar mass in g/mol; MHCl is the
concentration in mol/L of the standardized HCl solution; FC is the
correction factor for the molarity (MHCl/MNaOH) and m is the mass in
kg of dry soil in the ask.
2.5.3. Total petroleum hydrocarbons analyses
The Total Petroleum Hydrocarbons were determined by the EPA
8015B/3540 method. Soil samples were extracted with hexane and
dichloromethane in 1:1.5 proportion and an aliquot of the extract
(2 mL) was injected in a gas chromatograph (Dani GC1000) with
ame ionization detector (FID) and capillary column VF-5MS
(30 m  0.53 mm  1.5 mm). Nitrogen was used as carrier gas
and the hydrocarbon concentration was quantied using external
standardization. The method detection limit was 0.05 mg/kg.
2.5.4. Statistic analysis
The obtained results were analyzed by the Tukey test (comparison between mean values) to a condence level of 95% using
the program Statistica 7.1.
3. Results and discussion
3.1. Microbial growth evaluation
The population of total heterotrophic microorganisms (TH) and
hydrocarbon degrading microorganisms (HD) were quantied as a
function of time in the biodegradation experiments of

contaminated soils with 1.5% and 6% of oily sludge conducted in

open microcosms (Fig. 1) and in closed microcosms (Fig. 2).
The biostimulation (BS) and bioaugmentation (BA) experiments
in open systems with contamination of 1.5% and 6% showed that
the maximum population of microorganisms TH and HD was achieved in up to 30 days of processing. The maximum population of
HT (10.3 0.0 log MPN/g) was achieved in 30 days in the experiment BS(NP) in contaminated soils with 6%. The maximum
population of HD (8.6 0.0 log MPN/g) was achieved in the experiments: BS(NP) and BA(NP) in contaminated soils with
1.5% in 14 days and in the experiment BA(NP) in contaminated
soils with 6% also in 14 days.
The biostimulation and bioaugmentation experiments in closed
microcosms with a contamination of 1.5% showed that the
maximum population of microorganisms TH and HD was achieved
in up to 30 days of processing. In contaminated soils at 6%, the
addition of nutrients in higher quantities showed higher populations of TH in 60 and 90 days and of HD in 30 and 90 days. On the
other hand, the lower application of nutrients showed maximum of
TH and HD in up to 30 days. The experiment BA(NP) at 6%
contamination was the one who presented the higher population of
TH microorganisms (10.54 0.0 log MPN/g) in 30 days of processing. The maximum population of HD microorganisms
(9.38 0.0 log MPN/g) was achieved in experiments BS(NP) in
soils at 6% contamination in 7 days of processing.
In all applied treatments the microorganisms showed capacity
of tolerance and growth in oily sludge. In hydrocarbons contaminated environments, the microorganisms survival after inoculation,
the content and diversity of autochthones microbial communities
and the depletion of nutrients are important factors that affect the
biodegrading contaminant rate.
The addition of nutrients can affect both the rate and the
extension of organic pollutant biodegradation since they alter the
community dynamics of heterotrophic microbial populations (Das
and Mukherjee, 2007; Machn-Ramrez et al., 2008).
Other important factor that affects the microbial metabolism is
the temperature. Unfavorable temperature conditions may delay or
inhibit the growth and metabolism of the local microbiota resulting
in reduced efciency of the biodegradation process and consequently the permanence of this residue for longer periods. In this
work, the room temperature was monitored as a function of time
and presented an average of 27.5  C with a minimum of 19.1  C
between 17 and 21 days and a maximum of 37.3  C between 42 and
46 days (Fig. 3). These values are within the considered ideal range
for the effectiveness of Landfarming that is 10e45  C (EPA, 2004).
3.2. Respiratory microbial activity
The microorganism metabolic activity was monitored by
measuring the microbial respiration in contaminated soils at 1.5%
and 6% with oily sludge treated in closed microcosms (Fig. 4).
All the applied treatments in contaminated soils at 1.5% showed
higher production of CeCO2 at the rst 30 day processing. The
highest daily mineralization rates occurred in the treatments
BA(NP) (282.4 8.8 mg/kg) and BS(NP) (305.9 8.5 mg/kg)
in 10 days. In treatments with lower of nutrients rate, the highest
production was achieved in 3 day processing. Probably, in the initial
process period, the more labile fractions present in the oily sludge
can have been readily used as a source of carbon and energy for the
microbiota, since the maximum HD microbial populations and
highest mineralization rates were observed in that period. It has
been reported that organic compounds with low molecular mass
and simpler molecular structures are degraded preferably by many
microorganisms while the ones with more complex structures like
aromatic polycyclic hydrocarbons with more than ve benzene

V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

341

Fig. 1. Estimate of the number of total heterotrophic microorganisms (log MPN/g of soil) in soils contaminated with (a) 1.5% and (b) 6% and of total hydrocarbon degrading microorganisms (log MPN/g of soil) in soils contaminated with (c) 1.5% and (d) 6% during 90 days in open system.

Fig. 2. Estimate of the number of total heterotrophic microorganisms (log MPN/g of soil) in soils contaminated with (a) 1.5% and (b) 6% and of total hydrocarbon degrading
microorganisms (log MPN/g of soil) in soils contaminated with (c) 1.5% and (d) 6% during 90 days in closed system.

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V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

Fig. 3. Maximum and minimum temperatures along the 90 days.

pez et al.,
rings present higher resistance to microbial attack (Lo
2008). Marn et al. (2005) showed that the CO2 emissions gradually decrease while the most labile hydrocarbon fractions disappear, remaining only the more recalcitrant fraction.
It was veried that after the thirtieth day the accumulated
CeCO2 production had started to stabilize for all evaluated treatments. This indicates that the mineralization of the oily sludge
occurred markedly in a period of 30 days. Beyond 70 day processing
a reduction in the production of CeCO2 was observed in all tested
treatments, indicating a possible inhibition of the catabolic activity
of the local microorganisms.
Biostimulation and bioaugmentation treatments in contaminated soils at 1.5% showed higher mineralization values than the
natural attenuation treatment at the end of the period. Higher
accumulation productions of CeCO2 after 90 day treatment
occurred in the experiments BA(NP) (3010.1 63.9 mg/kg) and
BS(NP) (2825.9 55.7 mg/kg), which values differ statistically
from the natural attenuation treatment (2014.5 36.7 mg/kg).
The period of high CeCO2 production in experiments with
contaminated soils at 6% oily sludge was higher than that in
contaminated soils at 1.5%, probably due to the higher amount of
hydrocarbons to be metabolized and microbial competence in
metabolizing hydrocarbons.
The natural attenuation, biostimulation and bioaugmentation
treatments with lower amounts of nutrients showed high

production during 90 days. The maximum daily CeCO2 production

occurred after a 10 day experiment BA(NP) (332.9 6.9 mg/kg)
and in 7 day experiment BS(NP) (285.9 14.9 mg/kg). The
maximum population of HD microorganisms (9.38 0.0 log MPN/
g) in contaminated soils at 6% was achieved after 7 day processing
in experiment BS(NP). Addition of nutrients in lower quantity
favored the mineralization during the entire degrading process
showing higher daily production in the process initial steps. Probably, the initial step of CeCO2 liberation is related to the aliphatic
hydrocarbon fraction which corresponds to the most labile fraction
presented in the oily sludge where the maximum mineralization
was veried. In these experiments, is probable that the present
microbiota was able to degrade in larger amount the aromatic hydrocarbon fraction which is characterized as recalcitrant. As
pointed out by Tahhan et al. (2011), in spite of the alkane degradation starting almost immediately to the soil incubation the aromatic compounds are actively degraded in a later stage in a
scenario of mixed hydrocarbon degradation, probably because alkanes are preferential carbon sources until the microorganisms are
able to use both alkanes and aromatics. Furthermore, aromatic
compounds are more strongly adsorbed by the soil than alkane,
which makes their degradation slower. It must be considered that
along with the sorption, the low solubility and hence the low
bioavailability constitutes a limiting step in the biodegradation of
PAHs (Tahhan et al., 2011).
In treatments where higher amounts of nutrients were added, a
reduction in the production of CeCO2 was observed after 54 days
when compared to natural attenuation. Maximum daily productions for the treatments BS(NP) (271.2 36.7 mg/kg) e
BA(NP) (266.5 14.0 mg/kg) occurred in 38 and 33 days,
respectively. Furthermore it was observed that the natural attenuation showed higher mineralization rate than treatments where
higher nutrient amounts were added (p < 0.05). Similar behavior
was observed for the rate of mineralization in work realized by
Tahhan and Abu-Ateih (2009), where was observed inhibitory effect of the added nutrients (NH4NO3 and KH2PO4) in the mineralization of TPH from the oily sludge. Since nutrients were added as a
ratio of the added carbon, inhibition was the greatest with the
highest TPH treatment. This could be attributed to a possibility of
biodegradation inhibition by high concentrations of nutrients,
where nutrients may inhibit microorganisms that are adapted to an
originally oligotrophic soil environment.
The addition of bacterial consortium showed higher mineralization rates when compared to biostimulation treatments. This
shows the capacity of added microorganisms in contributing to the
increase of mineralization rate in different hydrocarbons fractions
present in the oily sludge. The bacteria on the consortium were

Fig. 4. Accumulated production of CeCO2 (mg/kg of soil) during 90 days in closed system in soils contaminated with (a) 1.5% and (b) 6%.

V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

isolated from the landfarming soil and from the studied oily sludge
and selected in liquid medium according to the hydrocarbon
degradation capacity and biosurfactant production. The isolates
showed capacity of growing and mineralization in contaminated
soils at 1.5% and 6%. According to Nasseri et al. (2010) the use of
native microbiota for bioaugmentation is preferred since these
microorganisms have higher adaptation capacity to the pollutant
than exogenous microorganisms and genetically modied microorganisms which can lead to gene transference, which is undesirable (Nasseri et al., 2010). The use of microbial consortiums can be
interesting when an increase of biodegradation rate and extension
of the hydrocarbons that constitute the oily sludge is required, in
view of their complex composition of alkanes, aromatic compounds, resin fractions and asphaltenes. Mixed populations with a
large range of enzymatic capacities and co-metabolism are required
for mineralization of oily sludge as opposed to individual microorganisms, which are able to mineralize only a limited range of

pez et al., 2008).
hydrocarbon substrates (Gallego et al., 2007; Lo
The contamination level and the treatments, as well as the
interaction of these factors, showed inuence on the CeCO2 production rate. In average, higher mineralization rates were obtained
in contaminated soils at 6%. It can be observed that the best condition to obtain the highest oily sludge mineralization rates in
closed microscosms corresponds to bioaugmentation and biostimulation treatments using lower nutrient concentration in
contaminated soils with 6% of oily sludge.
3.3. Total petroleum hydrocarbons degradation
Table 2 shows the biodegradation rates achieved in different
treatments in open and closed microcosms in soils contaminated
with 1.5% and 6% of oily sludge.
Analyzing the systems, it can be observed that higher biodegradation rates were obtained using the closed microcosms
(p < 0.05). The closed microcosms presents the better control of
abiotic losses as well as a better soil humidity control as some
advantages. During the experimental period it was veried that the
open microcosms presented a higher rate of water evaporation,
requiring more frequent periods of humidity correction in order to

Table 2
TPH degradation (%) obtained in different treatments applied to soils contaminated
with 1.5 and 6.0% in open (O) and closed (C) system.
Experiment

System

Contamination
level

Treatment

TPH degradation (%)*

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

O
O
O
O
O
O
O
O
O
O
C
C
C
C
C
C
C
C
C
C

1.5%
1.5%
1.5%
1.5%
1.5%
6.0%
6.0%
6.0%
6.0%
6.0%
1.5%
1.5%
1.5%
1.5%
1.5%
6.0%
6.0%
6.0%
6.0%
6.0%

NA
BS(NP)
BA(NP)
BS(NP)
BA(NP)
NA
BS(NP)
BA(NP)
BS(NP)
BA(NP)
NA
BS(NP)
BA(NP)
BS(NP)
BA(NP)
NA
BS(NP)
BA(NP)
BS(NP)
BA(NP)

52.7
60.4
57.0
67.2
58.7
70.3
78.7
74.6
76.7
72.8
59.7
74.9
72.4
72.2
74.3
74.9
78.8
79.6
84.1
81.3

0.5a
3.5ab
0.2a
4.2bdi
0.3ab
2.4cd
1.3cjk
1.4 cdk
3.2cjk
0.0cdk
1.5ai
2.5eghcd
3.9 egcd
4.4egcd
0.0egcd
2.3 fgcd
0.4fgc
2.8fgc
0.9 fhj
0.5fgk

*
Equal letters indicate that the experiments do not presented signicant differences (p > 0.05) for the studied response.

343

keep the soil with 70% of the eld capacity. In a bioremediation

process the soil humidity must be kept between 50% and 80% of the
eld capacity in order to obtain optimal degrading rates (Rizzo
et al., 2006). The bioremediation in open microcosms, as in the
case of landfarming, has as main disadvantage the inuence of
environmental conditions such as temperature and humidity,
which are limiting conditions to microbial activity. Considering that
in south of Brazil the seasons are characterized by large climatic
variations, showing extremes of temperature and periods with
predominance of rainfall or droughts, it is possible to say that the
open system does not show regularity in degrading efciency
throughout the year. It must be considered that reduced humidity
negatively affects the microbial metabolism, the microorganism
movement and the nutrient transport in soil. On the other hand, an
excess of humidity limits the oxygen transport in soil (Rizzo et al.,
2006).
The applied contamination level showed inuence in the oily
sludge biodegradation rate (p < 0.05) with the 6% contamination
showing in average a higher biodegradation rate when compared to
15% contamination. Considering both systems and two contamination levels, in average, the highest degradation occurred in
treatments BS (NP) and BA(NP) followed by treatments BA
(NP) and BS (NP) in closed system and 6% contamination.
It was observed that nutrient ratios of C:N:P of 100:0.4:0.2
favored, in average, the oily sludge TPH degrading rate. The ratio
C:N:P of 100:10:1 also showed to be effective for hydrocarbon
degrading. According to Adoki and Orugbani (2007), the bacterium
requires about 10 parts of carbon and 1 part of nitrogen for an
efcient growth. If this ratio becomes higher, for instance, 100:1 or
1000:1 as a result of environment contamination by petroleum
products, the growth and utilization of carbon source will be
retarded. This fact was not evidenced in the present study.
The introduction of microorganisms showed, in average, lower
biodegradation rates than the treatments containing only the
nutrient addition, however there was no signicant difference between treatments. In studies by Mariano (2006), the bioaugmentation with the bacterial consortium was the variable
presenting the smaller positive effect on biodegradation rates. According to this author, the bioaugmentation might not present
positive effects or present slightly marginal effects in hydrocarbon
biodegradation since inhibition can occur among the used strains in
the articially built consortium. According to Hamed et al. (2010),
inhibition of strains inoculated in natural environments can occur
because they are susceptible to competitors present in the environment, besides toxic effects caused by intermediate metabolites
produced by some strains. Furthermore, the microorganisms can
metabolize other organic compounds in detriment to the pollutant
or being incapable of percolating the soil until reaching the
contamination points (Mariano, 2006). The success to microbial
inoculums use requires that the microorganisms reach the contaminants, however, the physical adsorption to the soil particles or
the ltration through small pores can limit the microorganism
transport. In studies conducted by Machn-Ramrez et al. (2008),
where different biodegradation treatments of oily sludge contaminated soils were studied, the removal of TPH was higher when
applied to biostimulation than natural attenuation and bioaugmentation with endogenous and exogenous consortiums, being
the initial TPH concentration reduced from 133,000 to 93,000 mg/
kg of soil in 15 days of biostimulation treatment.
The biodegradation percentage varied from 52.7 0.5% (NA in
open microcosms contaminated at 1.5%) to 84.1 0.9% (BS(NP)
in closed microcosms contaminated at 6%). Addition of nutrients
and microorganisms to contaminated soils at 1.5% of oily sludge, in
open and closed microcosms showed to favor biodegradation when
compared to natural attenuation treatments. This evidences that

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V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

deciency of nutrients and/or low degrading activity from the local

microbiota were in part responsible for the oil persistency in the
contaminated soil.
Considering TPH reduction rate of each biostimulation and
bioaugmentation treatment in relation to the natural attenuation
treatment inside each experimental block, the higher degrading
rates are observed for contaminated soils with the lowest tested
concentration (1.5%) in closed microscosms, being the maximum
achieved of 15.2% in BS(NP). Similar behavior was observed by
Tahhan and Abu-Ateih (2009), who veried that the efciency in
TPH removal from oily sludge in different concentrations (44.2,
22.2, 11.1 g/kg of TPH) in soils during 120 days was higher for the
lowest rate of applied oily sludge. On the other hand, contaminated
soils at 6% of oily sludge in closed system showed higher reduction
rate (9.3%) in concentration of TPH compared to natural attenuation
occurred in the BS(NP). The BS(NP) and BA(NP) treatments in contaminated soils at 6% in closed systems showed lower
reduction rate (3.9% and 4.7%, respectively) in the concentration of
TPH as well as lower mineralization rate.
3.4. Behavior of microbial activity  degrading rates
In contaminated soils at 1.5% treated in open microcosms, the
addition of nutrients in lower amounts favored the growing of HD
microorganisms in both experiments BS and BA after 2 weeks,
remaining in higher number up to the end of the cultivation when
compared to the experiments which showed to favored the TH
population. The treatments BS(NP) and BA(NP) showed
higher degrading rates, reaching 67.2 4.2% and 58.7 0.3% of TPH
reduction, respectively. In this case, the degradation occurred
probably during all the experimental period as indicated by the
predominance of HD after 30 days. On the other hand for BS(NP)
and BA(NP), the degradation of TPH might have occurred in the
initial steps were the population of HD was higher. The condition of
larger amounts of nutrients favored the development of TH which
most probably competed for the carbon source and nutrients
available with the HD, ending up by predominating in the environment. As a consequence, a lower TPH biodegradation rate was
obtained. One of the main issues on the majority of the isolates
used in bioremediation of contaminated soils with oil is their incapacity of competing with the autochthon soil population when
applied in eld (Adoki and Orugbani, 2007).
In contaminated soil at 1.5% treated in closed systems, the
addition of nutrients in lower amounts favored the growing of HD in
treatment with the consortium (BA(NP) remaining with higher
number after 30 days. Addition of larger amounts of nutrients promoted a fall along the time of BA treatment and favored the growing
in the initial steps with BS. The highest degradation rates were obtained in BS(NP) (74.9 2.5%) and BA(NP) (74.3 0.0%) which
showed no signicant difference between the treatments, but
showed signicant difference with natural attenuation. Treatment
with variations on amount of nutrients showed similar mineralization rates (p > 0.05). According to the analyses of CeCO2 liberation it
was observed that with the tested nutrient amounts this study did
not show statistical difference on the mineralization rate.
In contaminated soils at 6% treated in open microcosms, the
addition of nutrients in lower amounts favored the growing of HD
hydrocarbons in the initial period. However, for the BA(NP) a
sharp growth decrease was veried, especially after 8 weeks,
achieving the same number of HD microorganisms present in the NA
treatment. In this treatment, the smallest TPH biodegradation rate
was observed, however there was no signicant difference
compared to the other. Nutrients such as nitrogen and phosphorus
were probably limiting due to the presence of a larger amount of
degrading microorganisms and added carbon. The addition of larger

quantities of nutrients showed small variation in the HD population

in treatment BA(NP). However, with BS(NP) a sharp growing
in HD population occurred reaching a maximum in 30 days.
In contaminated soils at 6% treated in closed systems the addition of lower nutrient amounts favored the growing of HD reaching
maximum populations of 9.38 0.0 log MPN/g and 8.38 0.0 log
MPN/g in 7 and 14 days treatment BS(NP) and BA(NP),
respectively. Addition of larger doses of nutrients shows, in average,
smaller HD populations when compared to experiments with
lower doses of nutrients. In these treatments the maximum HD
populations were obtained in 90 days for BS(NP) (5.9 0.0 log
MPN/g) and in 30 days for BA(NP) (7.38 0.10 log MPN/g). This
inhibiting effect was also evidenced in the measurement of the
microbial activity where a lower rate of CeCO2 liberation occurred
when compared to NA, BS(NP) and BA(NP). However, according to chromatographic analyses, no signicant difference was
observed between biodegradation rates in treatments BS(NP)
and BA(NP) compared to BS(NP) and BA(NP) which presented biodegradation rates of 84.1% and 81.3% respectively.
Chaneau et al. (2005) veried that proper fertilization level
increased the biodegradation rates of crude oil in soils during 150
days, while excessive fertilization had a negative effect. The
maximum biodegradation achieved was 62% in soils with low
fertilization rate (850 mg N/kg of NH4NO3, 450 mg P/kg of Na2HPO4
and KH2PO4 and 1500 mg/kg of KH2PO4) while the natural attenuation contributed to 47% of degradation (Chaneau et al., 2005).
In this work, the use of nutrients and bacterial consortium
resulted in an increment of the biodegradation rate of oily sludge in
soils reaching a maximum of 84.1 0.9% and TPH degradation in 90
day processing in contaminated soils at 6% and 74.9 2.5% in
contaminated soils at 1.5%. Smaller values of biodegradation were
found in other works. In works conducted by Vasudevan and
Rajaram (2001), studying the effect of adding nutrients and the
microbial consortium formed by Acinetobacter, Pseudomonas, Bacillus, Flavobacterium, Corynebacterium and Aeromonas, on the
degradation of contaminated soils with 5% of oily sludge during 90
days a maximum of 65% of degradation was found in the treatment
containing inorganic nutrients and microbial consortium. Liu et al.
(2010) studied the biostimulation with endogenous microorganisms in contaminated soils with 241,000 mg/kg of oily sludge
during 360 days, reaching a maximum of 58.2% of degradation in
the TPH content of the oily sludge. Marn et al. (2005), studying the
bioremediation of renery oily sludge by landfarming, observed
that hydrocarbon degradation of 80% occurred in 11 months being
half of this reduction during the rst three months.
4. Conclusion
The bioaugmentation and biostimulation showed an increment
on the degradation of TPH of oily sludge. Higher biodegradation
rates were obtained using a closed microcosm and 6% of the
contaminant. The nutrients added did not show effect on biodegradation rate. However, the excess caused inhibition of hydrocarbon mineralization compared to other treatments.
Therefore, the study showed the possibility of treating larger
amounts of oily sludge using smaller amounts of nutrients and
bacterial pre-selected consortium. The possibility of treatment of
oily sludge in closed conditions can be considered, according to our
results, the one that showed higher TPH degradation and better
control of environmental conditions.
Acknowledgments
The authors acknowledge to CAPES (Coordena~
ao de Aperfeioamento de Pessoal de Nvel Superior) by the Doctoral

V.S. Cerqueira et al. / International Biodeterioration & Biodegradation 95 (2014) 338e345

Scholarship. This work was nanced with resources from the Laboratory of Biodeterioration and Biofuels and from the Post Graduation Programs in Agricultural and Environmental Microbiology
and Soil Sciences of the Universidade Federal of Rio Grande do Sul e
Brazil.

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