Journal of Environmental Management 157 (2015) 213e219

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Journal of Environmental Management

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

Use of Bacillus thuringiensis supernatant from a fermentation process

to improve bioremediation of chlorpyrifos in contaminated soils
~ eda b, Laura M. Castan
~ eda-Sandoval b
Angel E. Aceves-Diez a, *, Kelly J. Estrada-Castan
a
b

Research and Development Department, Minkab Laboratories, Av. 18 de Marzo No. 546, Col. La Nogalera, Guadalajara, Jalisco, P.O. Box 44470, Mexico
Microbiology School, University of Antioquia UdeA, Calle 70 No. 52-21, Medelln, Colombia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 6 May 2014
Received in revised form
9 March 2015
Accepted 14 April 2015
Available online 21 April 2015

The aim of this research was to investigate the potential of a nutrient-rich organic waste, namely the cellfree supernatant of Bacillus thuringiensis (BtS) gathered from fermentation, as a biostimulating agent to
improve and sustain microbial populations and their enzymatic activities, thereby assisting in the
bioremediation of chlorpyrifos-contaminated soil at a high dose (70 mg kg
1). Experiments were performed for up to 80 d. Chlorpyrifos degradation and its major metabolic product, 3,5,6-trichloro-2pyridinol (TCP), were quantied by high-performance liquid chromatography (HPLC); total microbial
populations were enumerated by direct counts in specic medium; and uorescein diacetate (FDA)
hydrolysis was measured as an index of soil microbial activity. Throughout the experiment, there was
higher chlorpyrifos degradation in soil supplemented with BtS (83.1%) as compared to nonsupplemented soil. TCP formation and degradation occurred in all soils, but the greatest degradation
(30.34%) was observed in soil supplemented with BtS. The total microbial populations were signicantly
improved by supplementation with BtS. The application of chlorpyrifos to soil inhibited the enzymatic
activity; however, this negative effect was counteracted by BtS, inducing an increase of approximately
16% in FDA hydrolysis. These results demonstrate the potential of B. thuringiensis supernatant as a
suitable biostimulation agent for enhancing chlorpyrifos and TCP biodegradation in chlorpyrifoscontaminated soils.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Chlorpyrifos
Bioremediation
Biostimulation
Organic waste (Bacillus thuringiensis
supernatant)
Soil microbial activity

1. Introduction
Pesticides play an important role in agriculture, helping to
reduce the economic losses caused by weeds, insects, and disease,
and their use has been increasing (Palanisami et al., 2009). Unfortunately, according to data from the World Health Organization,
only 2e3% of the pesticide applied to crops is utilized against pests.
The remaining 97e98% degrades slowly and remains in the environment, causing surface runoff, leaching, and percolation into the
soil-water environment and exerting toxic effects on biota and
humans through the food chain (Korade and Fulekar, 2009). In
addition to the natural processes mentioned above, accidental spills
and discharges from pesticide-manufacturing plants also
contribute to the presence of pesticides and their transformation
products and residues in the environment, which can then be

* Corresponding author.
E-mail addresses: aea@minkab.com (A.E. Aceves-Diez), laura.castaneda@udea.
~ eda-Sandoval).
edu.co (L.M. Castan
http://dx.doi.org/10.1016/j.jenvman.2015.04.026
0301-4797/ 2015 Elsevier Ltd. All rights reserved.

transported to other environmental compartments, as exemplied

by their frequent detection in surface- and ground-waters (Gilliom,
2007; Hildebrandt et al., 2008; Schipper et al., 2008). In soil, pesticides affect microbial populations and thereby alter their activities
(Pandey and Singh, 2006).
Chlorpyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) is a broad-spectrum organophosphorus insecticide
and acaricide that has been widely used since the 1960s (Cho et al.,
2002) in direct soil applications for the control of foliar insects in
cotton and paddy elds; grain, fruit, and vegetable crops; and
pastures. It is also used for the control of household pests and ectoparasites on sheep and cattle (Fang et al., 2006). Chlorpyrifos is
highly persistent in soil due to its low water solubility (1.4 mg L
1)
and high soil sorption coefcient (6,070e14,800 mL g
1; USDA, ARS
Pesticide Properties, 1995). This pesticide has a half-life in soil of
10e120 d, and its rate of decomposition depends on its initial
concentration and soil characteristics such as pH, temperature,
moisture and organic carbon content (Racke et al., 1990; Racke,
1993; Singh et al., 2003). The environmental fate of chlorpyrifos
has been studied extensively, with 3,5,6-trichloro-2-pyridinol (TCP)

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A.E. Aceves-Diez et al. / Journal of Environmental Management 157 (2015) 213e219

being identied as its main degradation product. TCP is a charged

molecule at neutral pH and is more mobile than chlorpyrifos due to
its greater water solubility, which causes the widespread contamination of soil and aquatic environments (Feng et al., 1997). This
molecule is highly persistent, has a half-life ranging from 65 to
360 d in soil (Armbrust, 2001), and exhibits broad-spectrum antimicrobial activity, preventing the proliferation of chlorpyrifosdegrading microorganisms in soil (Racke et al., 1990; Yang et al.,
2005).
The use of organic waste as a bioremediation strategy may
alleviate the inhibitory effects of residual pesticides on soil microbes due to its important role in enhancing the soil fertility and
soil microbial activity (Moreno et al., 1999). In general, organic
waste is rich in nutrients and microora and can therefore act as an
efcient biostimulating agent. Previous studies have demonstrated
the potential of various organic wastes, such as compost, farmyard
manure, and urban waste, to accelerate pesticide degradation in
different biobeds and soil (Vischetti et al., 2004; Coppola et al.,
2007; Tejada et al., 2011; Kadian et al., 2012; Tortella et al., 2012).
Additionally, the biodegradation of chlorpyrifos using different
types of agro-residues, including coconut husks, peat moss, peanut
shells, and rice husks, has recently been published (Romyen et al.,
2007); however, more research on this subject is required. Therefore, this study aimed to investigate the efcacy of the Bacillus
thuringiensis supernatant obtained from fermentation, a nutrientrich organic waste, as a biostimulating agent to improve and sustain microbial populations and its enzymatic activity in
chlorpyrifos-contaminated soil.
2. Materials and methods
2.1. Chemicals
Analytical-grade (99.5% purity) chlorpyrifos and TCP were purchased from Chem Service Inc. (West Chester, PA, USA) and stored
as recommended by the manufacturer. Water, glacial acetic acid,
acetone, acetonitrile, and ethyl acetate (HPLC grade) were obtained
from Merck, Inc. (Darmstadt, Germany). Fluorescein diacetate (FDA,
97% purity) was purchased from Janssen Pharmaceuticalaan (Geel,
Belgium). Chlorpyrifos and TCP solutions were prepared by dissolving the appropriate volumes of the respective substances in
water:acetonitrile:glacial acetic acid (50:50:1, v/v) to create
100 mg L
1 stock solutions, which were serially diluted in the same
phase (ranging from 2 to 100 mg L
1) to create standard solutions.
A commercial formulation of chlorpyrifos (Lorsban 4 EC manu, Colombia) was used to
factured by Dow Agrosciences, Bogota
achieve a nal concentration of 100 mg L
1 of the pesticide for the
biostimulation experiment on soils.
2.2. Bacterial strain
The B. thuringiensis var Kurstaki strain 4D4 used in the present
study was kindly donated by Dr. Daniel R. Zeigler (BGSC Director,
Ohio, USA). Sterile paper disks with containing 108 spores obtained
from growth in LB agar plates were prepared and maintained at
4  C for strain preservation.
2.3. Inoculum and cultural conditions
A spore disk of B. thuringiensis was used to inoculate a 250-mL
Erlenmeyer ask containing 50 mL of sterilized liquid culture medium with a slight modication of the formulation of Dulmage
(1970). Per liter, the medium contained 15 g of molasses, 10 g of
tryptone, 2 g of yeast extract, 3 g of MgSO4$7H2O, 0.02 g of
FeSO4$7H2O, 0.02 g of ZnSO4$7H2O, and 1 g of CaCO3. The initial pH

of the medium was adjusted to 7.2. The ask was incubated in a

shaker incubator (New Brunswick, Excella E24) at 30  C and
110 rpm for 12 h. A 2-L Erlenmeyer ask containing 1 L of the same
medium was then inoculated with 5% (v/v) inoculum from the
above mentioned ask. The ask was incubated in the same way
until sporulation was completed (5 d).
2.4. Characterization of the liquid medium and supernatant of B.
thuringiensis fermentation
The biomass was separated from the broth by centrifugation at
3823g for 15 min (Sigma, 2-16P), and the supernatant was lter
sterilized by passing it through a 0.45-mm membrane lter (Millipore Corp.) to produce a cell-free supernatant. The chemical and
biochemical oxygen demand (COD, BOD), total solids, carbohydrates, and total Kjeldahl nitrogen of the liquid medium and supernatant were analyzed according to the Standard Methods for the
Examination of Water and Wastewater (APHA, 1989). The characteristics of the liquid medium and cell-free supernatant used in this
study are presented in Table 1.
2.5. Soil sampling and characterization
The soil sample was obtained from one site in the town of
Marinilla, Antioquia, Colombia (N61102.200 W75170 30.100, altitude
2170 m). No specic permits were required for the eld studies
described, and the location is not protected in any way. The eld
studies did not involve endangered or protected species. The
sampling site had been used for intensive agricultural practices for
several decades and thus had been exposed to chlorpyrifos treatment over a long period. Soil samples were obtained on 30 January
2011 from one cover, a eld planted with green unripened banana
(Musa paradisiaca). Five replicates were obtained from plots
(1 m  1 m squares) that were at least 10 m apart from one another.
After removing the top 1e2 cm of the soil following the standard
procedure (Gupta, 2000), the soil from a depth of 2e15 cm was
mixed with a shovel, and approximately 2 kg of soil was sampled,
placed in plastic bags, and transferred to the laboratory. Within
120 h after sampling, the soils were sieved through a 2-mm mesh to
remove root fragments and large particles and homogenize the
particle size. Aliquots were used for microbiological and chemical
analyses. The physicoechemical parameters of the soil were
determined at the Soils Laboratory, Geoscience Department, National University of Colombia and are summarized in Table 2.
Briey, Soil texture was determined by the Bouyoucos hydrometer method using USDA textural triangle (Bouyoucos, 1962;
Gee and Bauder, 1986), pH was determined in 1:1 (wt/vol) diluted
water suspensions according to the American Society for Testing
Material (ASTM D4972, 2001), electrical conductivity was determined with a CO150 conductivity meter according to the

Table 1
Chemical characteristics of the liquid medium and the B. thuringiensis supernatant
(mean st. dev.).
Parameter

Sterile liquid medium

Supernatant from
fermentation

COD [mg L
1 O2]

BOD [mg L
1 O2]
Total solids [mg L
1]
Carbohydrate [g L
1]

26,636 666
23,580 3018
28,080 199
Sucrose 5.3 0.004
Glucose 1.1 0.017
Fructose 1.2 0.022
2,219 153

17,625 441
14,700 1882
22,480 186
Sucrose 2.8 0.055
Glucose 0.64 0.042
Fructose 0.71 0.027
1,573 109

Total Kjeldahl nitrogen

[mg L
1]

A.E. Aceves-Diez et al. / Journal of Environmental Management 157 (2015) 213e219

Table 2
Physico-chemical characteristics of the soil (mean st. dev.).

215

2.7. Chemical extraction and HPLC analysis of chlorpyrifos

degradation

Characteristic
Texture
pH
Electrical conductivity [dS m
1]
Ca2 [cmolc kg
1]
Mg2 [cmolc kg
1]
K [cmolc kg
1]
Na [cmolc kg
1]
Phosphate [mg kg
1]

1
NH
4 -N [mg kg ]
Total nitrogen [%]
Cl
[mg kg
1]

1
]
NO

2 eN [mg kg

1
NO

]
3 eN [mg kg

1
Sulfate [mg kg ]
Organic matter [% volume]

Sandy loam (74% sand, 16% silt, 10% clay)

5.5 0.010
0.18 0.004
5.85 0.041
1.80 0.001
0.58 0.004
0.01 0.001
49 0.010
43 0.816
0.555 0.012
34.05 0.285
0.85 0.041
18 0.816
8 0.001
15.5 0.489

manufacturer's instructions (Hach Company, Loveland, CO, USA).

The chloride ion concentration was determined by colorimetric
method using mercury thiocyanate (Pansu and Gautheyrou, 2006).
After extraction with 1 N ammonium acetate at pH 7.0, the total
Ca, Mg, Na and K concentrations were determined by atomic absorption spectrometer, according to the Colombian Technical
Norm (NTC) 5349 (ICONTEC, 2008). After extraction from the soil
using Bray No. 2 solution, phosphorus was measured colourimetrically based on the reaction with ammonium molybdate according to indications of NTC 5350 (ICONTEC, 2005). Furthermore,
ammonium (NH
4 ), nitrite and nitrate were quantied colourimetrically after KCl extraction from the soil, according to the
protocol of Strickland and Parsons (1972). Sulfate was determined
by turbidimetric method using BaCl2 and gelatin, after digesting
the soil sample with di-acid mixture (perchloric acid and nitric
acid 1: 2 v/v) (Kalra, 1971). Total organic matter was determined
by wet oxidation with potassium dichromate and dosage titration
e Walkley Black method, as described by Pansu and Gautheyrou,
2006. Total N was determined by the Kjeldhal method (Pansu
and Gautheyrou, 2006).
2.6. Biodegradation and biostimulation experiments
The dried and sieved soil used for fortication showed no residual pesticide contamination. Three replicate samples (200 g) of
soils were treated with chlorpyrifos (140 mL; 100 mg L
1) and
supplemented with the supernatant of Bacillus thuringiensis
fermentation (BtS). The soils were well mixed using a metal
spatula to yield a nal concentration of 70 mg active ingredient/kg
dry soil. The pesticide concentration, 70 mg kg
1, was selected to
represent the highest dose of chlorpyrifos achieved in agricultural
applications and is equivalent to 7.0 kg/ha of active ingredient
incorporated at a soil depth of 1 cm. The control was created using
the same fortication protocol but with the addition of water
instead of BtS to the chlorpyrifos-contaminated soil. Furthermore,
sterile soils with and without BtS supplementation were used to
study the effect of the organic matter present in this organic waste
on the degradation of chlorpyrifos and to determine the physical
and chemical losses of pesticide that may have occurred during
the experiment. Contaminated soils were transferred to sterile
amber glass containers (200-mL capacity), which were kept
covered but unsealed and incubated at 20  C. The treated soils
were sampled periodically for 80 d and analyzed by highperformance liquid chromatography (HPLC) for chlorpyrifos and
TCP concentrations, the number of microorganisms, and total soil
microbial activity.

Soil samples contaminated with chlorpyrifos were removed on

days 0, 20, 40, and 80 for degradation analysis. Chlorpyrifos and TCP
were extracted from 10 g of soil with 30 mL of solvent (ethyl acetate) in a separate ask. The resulting suspensions were incubated
in a shaker incubator (New Brunswick, Excella E24) at room temperature and 240 rpm for 4 h, after which the supernatant was
removed. The solvent was evaporated to dryness in a vacuum
evaporator at 30  C. Next, 7 mL of a mobile phase consisting of a
mixture of water, acetonitrile, and glacial acetic acid (50:50:1, v/v)
was used to reconstitute the samples for HPLC analysis, and 20 mL
(injection loop was 20 mL) of each sample was injected into an HPLC
system (Agilent 1100 series) equipped with a diode array detector.
Analysis was conducted on a Supelco Spherisorb ODS-2 column
(250  4.6 mm; Waters). The separation of chlorpyrifos and TCP
was performed using an HPLC gradient system elution. The mobile
phase was acidied water (1% glacial acetic acid), and the acetonitrile gradient ow rate was programmed to 1 mL/min. The
gradient began at 50% acetonitrile and then increased to 90%
acetonitrile at 3 min. Next, the system returned to 50% acetonitrile
at 8.5 min and was held for 2 min for re-equilibration. The eluents
were monitored by UV detection at a wavelength of 299 nm for
chlorpyrifos and at 290 nm for TCP. The chromatographic analysis
was performed at ambient temperature. Each sample replicate was
injected three times to determine the average peak area. The percentage degradation of the compounds was calculated as the difference between the peak areas of the experimental and control
samples, where the peak area of the control was used as the 100%
point of reference.
2.8. Number of microorganisms in soil
Identical sets of soils (treatment and control) were set up in
triplicate to determine the total number of bacteria, fungi, and
actinomycetes. The samples were collected from these soils on days
0, 20, 40, and 80. One gram of soil was removed and mixed with
9 mL of 0.9% saline (NaCl). The suspensions were serially diluted to
10
7 and plated on plate count agar (PCA; g/L: casein peptone, 5.0;
yeast extract, 2.5; dextrose, 1.0; agar, 14.0; pH 7.1 0.2) for bacteria;
Sabouraud-glucose agar (g/L: casein peptone, 10.0; glucose, 20.0;
agar, 17.0; pH 5.6 0.2) for fungi, and starch-casein agar (g/L: soluble starch, 10.0; casein, 0.3; KNO3, 2.0; NaCl, 2.0; K2HPO4, 2.0;
MgSO4$7H2O, 0.05; CaCO3, 0.02; FeSO4$7H2O, 0.01; agar, 15.0; pH
7.0 0.2) for actinomycetes. The number of cultivable bacteria,
fungi, and actinomycetes was determined by measuring the number of colony forming units per gram of soil (CFU g
1) after incubation at 35  C for 2 d, 30  C for 5 d, and at 30  C for 8 d,
respectively.
2.9. Total microbial activity in soil
The total microbial activity in the soils (treatment and control)
was measured by monitoring uorescein diacetate hydrolysis (FDA)
as described by Schnrer and Rosswall (1982), with modications.
Briey, 2 g of soil collected from glass containers on days 0, 20, 40,
and 80 was incubated in a 250-mL Erlenmeyer ask containing
49.75 mL of sterile 60 mM sodium phosphate buffer, pH 7.6. The
reaction was started by adding 0.25 mL of a FDA solution
(2 mg mL
1) and incubated in a shaker incubator (New Brunswick,
Excella E24) at room temperature and 200 rpm. A490 was
measured using a UV/Vis spectrophotometer (Thermo Scientic,
Spectronic Genesys 2) after the removal of the soil by centrifugation
and ltration. The samples were analyzed in triplicate. The

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A.E. Aceves-Diez et al. / Journal of Environmental Management 157 (2015) 213e219

concentration of the released uorescein was calculated using a

calibration curve with standard quantities of FDA (0.5e5 mg mL
1),
and the results were expressed as mg FDA g
1 h
1. Samples exhibiting absorption values above 0.8 were diluted before the nal
absorbance determinations.
2.10. Statistical analyses
Chromatographic analyses of chlorpyrifos degradation, microorganism counts (bacteria, fungi, and actinomycetes), and total
microbial activity in the soils were performed in triplicate. All data
are presented as means st. dev. The statistical signicance of the
data were evaluated by one-way analysis of variance (ANOVA). A pvalue of less than 0.05 was considered statistically signicant unless otherwise indicated. If an overall signicance was detected,
Tukey's multiple range tests were performed.
3. Results and discussion
3.1. Supernatant of B. thuringiensis fermentation as biostimulating
agent
In general, most of the culture media used in the industrial
fermentation of biotechnologically relevant microorganisms, such
as B. thuringiensis, contain a high level of nutrients because the aim
is to ensure high biomass production. However, as shown in Table 1,
after the fermentation process is completed, the supernatant is
converted into wastewater with a high organic content
(BOD 14,700 1882 mg L
1 and COD 17,625 441 mg L
1) and
therefore cannot be discarded without pretreatment. Furthermore,
this wastewater exhibits high biodegradability (BOD/COD > 0.6),
indicating a high content of easily biodegradable hydrocarbon
compounds such as glucose, polysaccharides, and cellulose.
Because the organisms rst use the compounds mentioned above
for growth and energy, an alternative could be to use the supernatant as a biostimulating agent to promote the growth of native
microbial populations in the bioremediation of soils contaminated
by pesticides, such as chlorpyrifos.
3.2. Effect of B. thuringiensis supernatant on the degradation of
chlorpyrifos and TCP
The present research work evaluated the potential of BtS for the
bioremediation of chlorpyrifos in contaminated soil using microcosm experiments. The soil collected from the glass containers,
having been dried and homogeneously mixed, was sampled for
extraction and analyzed further to determine the chlorpyrifos and
TCP concentrations. In the recovery assay, the average percentage
recoveries of chlorpyrifos and TCP were 92.7% and 92.4%,
respectively.
The degradation of chlorpyrifos in the supplemented and nonsupplemented non-sterile soils is illustrated in Fig. 1. The data
indicate that chlorpyrifos degraded rapidly in the soils within the
rst 10 d and continued to degrade throughout the 80d experiment. The percentage degradation of chlorpyrifos in the
soil supplemented with BtS was 73.74% at the rst harvest period.
After 40 d, the amount of chlorpyrifos remaining in the supplemented soil was only 16.53 mg kg
1, corresponding to a 76.39% loss
of chlorpyrifos. At the end of the experiment, the chlorpyrifos in the
soil was almost completely degraded, with a degradation percentage of 83.1%. Compared to the control soil, the concentration of
the insecticide was 39.38% lower in soil supplemented with BtS.
According to these results, the degradation of chlorpyrifos was
signicantly enhanced (p < 0.05) in BtS-supplemented soil relative
to that in the control soil (Fig. 1a). Meanwhile, after 80 d of

incubation, in the sterile soils mixed with 70 mg kg
1 of chlorpyrifos and with and without BtS supplementation, the pesticide
was degraded very slowly with time (approximately 12%) as shown
in Fig. 1c. A similar result was reported by Racke et al. (1996), who
found that the degradation of chlorpyrifos was of 13.5% in a sandy
loam sterile soil. Thus, the organic matter present in the BtS did not
play a signicant role in the degradation of chlorpyrifos, indicating
that the biodegradation occurred as a result of the stimulation of
the native microorganisms in the supplemented soils. The results
obtained in the present study are in agreement with those of a
study conducted by Gupta and Baummer (1996), who showed that
the biodegradation of atrazine in soil supplemented with poultry
litter occurs through microorganisms. Similarly, Singh et al. (2003)
and Tortella et al. (2012) suggest that the degradation of chlorpyrifos in soil is carried out by soil microorganisms. However, as
shown in Fig. 1a, a decrease in the efciency of chlorpyrifos
degradation occurred by day 20. Similar results were reported by
Vischetti et al. (2008) and Tortella et al. (2012), who observed that
the degradation rate of chlorpyrifos decreased at high concentrations of this. The above-mentioned nding can be attributed to the
high accumulation of TCP in chlorpyrifos-contaminated soil
(Fig. 1b), which can inhibit chlorpyrifos-degrading microorganisms
due to the previously reported antimicrobial properties of TCP
(Racke et al., 1990; Singh et al., 2003).
The formation of TCP was detected in supplemented and nonsupplemented non-sterile soils, as shown in Fig. 1. In general, a
greater accumulation of this metabolite was observed during the
rst 20 d of incubation, which was when the maximum chlorpyrifos degradation was observed. At this time, the accumulation
percentage of TCP was signicantly higher (p < 0.05) in the control
soil (13%) than in the supplemented soil. After 20 d, the degradation
of TCP was achieved in both the supplemented and nonsupplemented soil but in greater proportion in the soil supplemented with BtS, as shown in Fig. 1b. After 80 d, the TCP in the
supplemented soil was partially degraded (30.34%). Compared to
the control soil, the concentration of this metabolite in the soil
decreased 13% more in the BtS-supplemented soil.
Interestingly, an increased degradation of chlorpyrifos in the
initial 20 d of the experiment in soil supplemented with BtS should
result in a high concentration of TCP compared to the control soil.
However, contrary to our expectations, a higher degradation of TCP
in the supplemented soil than in the control soil was presented, as
shown in Fig. 1b. The greatest decrease in the concentration of TCP
from 20 to 40 d along with a decrease on the efciency of chlorpyrifos degradation over the same period in soil supplemented
with BtS suggests that the toxic effect of TCP at very high concentration induced a metabolic stress in chlorpyrifos-degrading microorganisms and led them to redirect their metabolism towards
degradation of TCP rather than chlorpyrifos. The sterile soils with
and without BtS supplementation accumulated and degraded TCP
very slowly over time (Fig. 1d). The research ndings thus show
that BtS can efciently promote chlorpyrifos and TCP degradation
in soil through the biostimulation of native microbial communities.
3.3. Effects of B. thuringiensis supernatant on soil microbial
populations
The effects of BtS on soil bacterial, fungal, and actinomycete
populations are shown in Table 3. In general, all microbial populations were signicantly (p < 0.05) improved by BtS supplementation throughout the experiment. The bacterial population
was enhanced by 136.80%, 144.42%, and 140.38% compared to the
control soil after 20, 40, and 80 d, respectively. The trend for the
fungal population in BtS-supplemented soils was similar to that for
the bacterial population, but the enhancement was greater: 86.61%,

A.E. Aceves-Diez et al. / Journal of Environmental Management 157 (2015) 213e219

217

Fig. 1. Chlorpyrifos degradation in non-sterile soil with and without (control) B. thuringiensis supernatant (BtS) supplementation (a); accumulation of TCP in chlorpyrifoscontaminated non-sterile soil (b) after 80 d of incubation. Chlorpyrifos degradation in sterile soil with and without (abiotic control) B. thuringiensis supernatant (BtS) supplementation (c); accumulation of TCP in chlorpyrifos-contaminated sterile soil (d) after 80 d of incubation. Different letters refer to signicant differences between mean values
(n 3), as identied by Tukey's test (p < 0.05).

addition of nutrients or organic wastes to soils contaminated with

hazardous substances, such as herbicides, petroleum products, and
chloroanilines, exert a positive effect on the number and diversity
of native soil microbial populations, which contribute to pollutant
degradation (Evans et al., 2004; Tongarun et al., 2008; Kanissery
and Sims, 2011). Furthermore, the stimulatory effects of organic
waste have been evaluated and demonstrated through the quantication of the activities of critical soil enzymes, which are ultimately directly related to an increase in the native soil microbial
population, as observed in the present study (Tejada et al., 2011;
Kadian et al., 2012).

763.42%, and 102.78% compared to the control after 20, 40, and
80 d, respectively. The population of soil actinomycetes was
signicantly improved by 34.28%, 38.5%, and 74.82% compared to
that in the control soil after 20, 40, and 80 d, respectively.
The results revealed that BtS exerted a biostimulatory effect on
soil bacterial, fungal, and actinomycete populations throughout the
experiment. No previous report has assessed the stimulatory effect
of organic waste or nutrient addition on the growth of native microbial populations during pesticide biodegradation in soil using
conventional methods, such as a total plate count of native soil
microorganisms. However, some studies have shown that the

Table 3
Effect of B. thuringiensis supernatant on bacterial, fungal, and actinomycete populations in chlorpyrifos-contaminated soil (mean st. dev.).
Soil microorganisms

Treatment

Days after treatment (d)

Bacteria (106 UFC/g dw)

Control
BtS
Control
BtS
Control
BtS

1.39
1.53
11.4
11.3
6.80
6.42

Fungi (10 UFC/g dw)

Actinomycetes (105 UFC/g dw)

20

0.07
0.12
1.84
1.80
0.48
0.35

a
a
a
a
a
a

35.3
83.7
93.8
175
82.5
111

40

1.89 a
5.19 b
5.43 a
11.80 b
8.25 a
14.00 b

16.4
40.2
3.42
29.5
4.20
5.82

80

2.77
4.95
0.12
5.42
1.23
0.73

a
b
a
b
a
b

12.4
29.8
0.600
1.22
0.232
0.405

0.89
4.48
0.09
0.16
0.09
0.05

a
b
a
b
a
b

BtS: soil supplemented with B. thuringiensis supernatant. Control: non-supplemented soil. All values are the means st. dev. of triplicate samples. Means followed by the same
letter within a column are not signicantly different according to Tukey's test (p < 0.05).

218

A.E. Aceves-Diez et al. / Journal of Environmental Management 157 (2015) 213e219

3.4. Effect of B. thuringiensis supernatant on soil enzymatic activity

One of the most widely used methods for assessing microbial
activity in soil supplemented with organic waste is FDA hydrolysis
nchez- Monedero
(Adam and Duncan, 2001; Gaspar et al., 2001; Sa
et al., 2008), which is a measure of lipase, protease, and esterase
activities (Federle et al., 1986; Federle, 1988). Fig. 2 shows the
evolution of FDA hydrolysis in supplemented and nonsupplemented non-sterile chlorpyrifos-contaminated soils during
the incubation period. Our data indicate that the application of
chlorpyrifos in supplemented and non-supplemented soil inhibited
the enzymatic activity. Similar observations were also made by
Tortella et al. (2010, 2012), who showed that hydrolytic activity was
affected by high concentrations (320e400 mg kg
1) of chlorpyrifos.
Furthermore, Dutta et al. (2010) observed a signicant decrease in
FDA hydrolysis when chlorpyrifos was added to soil at a high
dosage (50 mg kg
1). Likewise, the negative effects of chlorpyrifos
on the activities of other enzymes, such as dehydrogenase, urease,
phosphatase, and arylsulfatase, have been demonstrated (Pozo
et al., 1995; Pandey and Singh, 2006; Tejada et al., 2011; Kadian
et al., 2012). The inhibition of soil microbial activity observed in
our study may be attributed to a lack of active biomass among the
heterogeneous microbial population in the soil due to the antimicrobial properties of TCP, a major metabolic product of chlorpyrifos
(Racke et al., 1990), which peaked on day 20.
Nevertheless, our nding that the inhibitory effect of chlorpyrifos on enzyme activity could be counteracted by supplementation
with a nutrient-rich organic waste, such as BtS, is interesting. This
organic waste enhanced FDA hydrolysis by approximately 16% over
the entire experimental period when added to the chlorpyrifoscontaminated soil relative to the extent of hydrolysis observed for
the control, demonstrating that the supernatant had a high capacity to maintain and promote metabolic microbial activity due to
the large amount of nutrient present in the supernatant, despite the
addition of a high concentration of chlorpyrifos. Signicant differences (p < 0.05) between supplemented and non-supplemented
soil were observed. Previous studies have shown that supplementation with organic matter increases soil microbial activity (Moreno
et al., 1999; Masciandaro et al., 2000). Our results are in accord with
those of Tejada et al. (2011), who observed a stimulatory effect on
dehydrogenase, urease, phosphatase, and arylsulfatase activities
when various organic amendments were added to chlorpyrifoscontaminated soil. Similarly, Tortella et al. (2010) reported an increase in FDA hydrolysis when a biobed biomix was supplemented

with an inorganic fertilizer at low concentrations in chlorpyrifosdegradation studies. Moreover, our results are in agreement with
those reported by Kadian et al. (2012), who demonstrated that the
supplementation of chlorpyrifos-contaminated soil with different
organic amendments enhanced soil microbial activity as measured
by dehydrogenase activity.
The evaluation of FDA hydrolysis only provides a hint of the
effect of pesticides on soil microbial activity because pesticide
biodegradation is complex and involves diverse biological, chemical, and physical interactions. In addition, several studies have
shown that other enzymes can induce the degradation of pesticides
in soil, including hydrolases, carboxylesterases, and phosphatases
(Sutherland et al., 2004; Scott et al., 2008).
4. Conclusions
The use of organic wastes to improve degradation processes in
soils contaminated with pesticides has been studied recently. Our
results show that chlorpyrifos and its major degradation product,
TCP, were degraded more efciently when the contaminated soil
was supplemented with Bacillus thuringiensis supernatant and that
this degradation occurs by the stimulation of native microbial
communities. Conversely, it was observed that the application of
chlorpyrifos at a high concentration exerts a signicant inhibitory
effect on soil microbial activity, as measured by FDA hydrolysis. This
inhibition may be attributed to the antimicrobial effect exerted by
TCP on active microbial populations, whose concentration
remained practically unchanged from the day 40 to the end of the
experiment. The application of B. thuringiensis supernatant to the
chlorpyrifos-contaminated soil under laboratory conditions
improved the soil enzymatic activities compared to those of the
control soil over the 80-d experimental period. The same stimulatory effect was observed for the microbial populations evaluated.
Although the positive effect of organic waste on the degradation of
pesticides depends strongly on the chemical composition of the
waste, our research ndings indicate that B. thuringiensis supernatant is able to promote and sustain microbial populations and
consequently their enzymatic activities in removing persistent
pesticides from soil, which will lead to enhanced food safety.
However, further studies such as the interactions of the
B. thuringiensis supernatant with environment are still needed
before its application in actual eld-scale bioremediation. In addition, whereas current eld applications are increasingly making use
of the combination of several pesticide types, more researches with
pesticide mixtures are necessary in order to evaluate the biostimulating effect of B. thuringiensis supernatant.
Acknowledgments
This study was funded by the Committee for Research Development (CODI) at the University of Antioquia, Colombia (No.
201001). The authors also acknowledge the support provided by
Minkab Laboratories.
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