Biodegradation of organochlorine pesticides by bacteria

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International Biodeterioration & Biodegradation 59 (2007) 239–244
www.elsevier.com/locate/ibiod
Biodegradation of organochlorine pesticides by bacteria grown in
microniches of the porous structure of green bean coffee
B.E. Barragán-Huertaa,b, C. Costa-Pérezc, J. Peralta-Cruza, J. Barrera-Cortésb,
F. Esparza-Garcı́ab, R. Rodrı́guez-Vázquezb,
a
Department of Environmental Systems Engineering, National School of Biological Sciences, National Polytechnic Institute,
Carpio y Plan de Ayala 11340 DF, México
b
Department of Biotechnology and Bioengineering, CINVESTAV, Av. Instituto Politécnico Nacional 2508 AP 14-740, Col. Zacatenco CP 07360, México
c
Department of Chemical Engineering, University of Salamanca, Plaza de la Merced s/n, 37008 Salamanca, Spain
Received 3 March 2005; received in revised form 13 October 2005; accepted 1 November 2006
Available online 5 January 2007
Abstract
In this paper, the authors propose a model for DDT biodegradation by bacteria grown in microniches created in the porous structure
of green bean coffee. Five bacteria isolated from coffee beans, identified as Pseudomonas aeruginosa, P. putida, Stenotrophomonas
maltophilia, Flavimonas oryzihabitans, and Morganella morganii. P. aeruginosa and F. oryzihabitans, were selected for pesticide
1
degradation. Bacteria were selected according to their ability to grow on mineral media amended with: (a) glucose (10 g l ), (b) peptone
1
1
1
(2 g l ), and (c) ground coffee beans (2 g l ). These three media were supplemented with 50 mg l of 1,1,1-trichloro-2,2-bis (4chlorophenyl) ethane (DDT) and endosulfan. GC/MS analysis demonstrated that the greatest DDT removal was obtained in the medium
supplemented with coffee beans, where 1,1-dichloro-2,20 -bis (4-chlorophenyl)ethylene (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethane
(DDMU) and 2,20 -bis (p-chlorophenyl)ethanol (DDOH) were detected. DDMU is a product of the reductive dechlorination of DDE,
which in this system could be carried out under the anaerobic conditions in microniches present in the porous structure of the coffee
bean. This was supported by scanning electron microscopy. Green bean coffee could be used as a nutrient source and as a support for
bacterial growth in pesticide degradation.
r 2006 Published by Elsevier Ltd.
Keywords: Coffee; DDT; Flavimonas; Pseudomonas; Microniche
1. Introduction
Although some persistent organochlorine pesticides have
been banned from agricultural and public health use during
the past few decades, high concentrations of DDT and its
metabolites have been found in soil, water, and sediment
samples (Shen et al., 2005; Miersma et al., 2003; Yañez
et al., 2002; Bould, 1994). Furthermore, other insecticides,
such as endosulfan and lindane, are currently in use
throughout the world (EPA, 2002) and their presence in
air, water, and soil is a problem of great concern. Reducing
their levels in the environment has therefore become an
important goal.
Corresponding author. Tel.: +52 55 50613316; fax: +52 55 50613313.
E-mail address: rrodrig@cinvestav.mx (R. Rodrı́guez-Vázquez).
0964-8305/$ - see front matter r 2006 Published by Elsevier Ltd.
doi:10.1016/j.ibiod.2006.11.001
The biochemical and molecular modes of pesticide
degradation by microorganisms have been well documented (Singh et al., 1999; Kumar et al., 1996). Organochlorine
pesticides possess halogen electron withdrawing groups
that generate electron deficiency in the molecule, which
thus resists aerobic degradation (Rieger et al., 2002).
However, these compounds can be attacked more readily
under reductive conditions, which could be enhanced by
the addition of auxiliary electron donors. For example, a
number of studies have reported a rapid rate of DDT to
DDD reduction in soil under reducing conditions when a
readily available energy source, such as alfalfa, barley
straw, or glucose, is present (Aislabie et al., 1997).
Dechlorination of p,p0 -DDT to 1,1-dichloro-2,20 -bis
(4-chlorophenyl)ethylene (DDE), DDD, 1-chloro-2,2-bis
(4-chlorophenyl) ethane (DDMU), DDMS, and DDNU,
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by Enterobacter aerogenes, P. fluorescens, Escherichia coli,
and Kleibsiella pneumaniae under anaerobic conditions has
been reported (Lal and Saxena, 1982).
Among these compounds, DDE has traditionally been
considered as a dead-end DDT-metabolite, which is
metabolized in co-metabolism with biphenyl by P. acidovorans and Terrabacter sp. (Hay and Focht, 1998;
Aislabie et al., 1999). Thus, a complex set of environmental
conditions (redox potential, pH, co-substrate, pollutant
concentration, etc.), is required for DDT mineralization
(Bidlam and Manonmani, 2002; Aislabie et al., 1997;
Nadeau et al., 1994).
Recently, agro-industrial wastes have been used to
enhance toxic recalcitrant organopollutant biodegradation
(Chávez-Gómez et al., 2003; Molina-Barahona et al., 2005;
Pérez-Armendáriz et al., 2004; Aslan and Turkman, 2005).
The use of agricultural wastes in bioremediation processes
is highly advantageous, as these wastes can provide a
nutrient source as well as a support for microorganisms.
The reuse of these wastes could also solve problems related
to their disposal.
Due to a surplus of coffee on the world market, coffee
producers have committed themselves to removing the
lowest quality coffee, i.e., ‘‘defective’’ coffee or triage, from
the market to stabilize prices. In Mexico, 5% of total coffee
exports (about 12,000 tons annually) is withheld from the
market (Federal Official Gazette (FOG), 2001). In contrast
with other agricultural wastes, green bean coffee is rich in
nutrients such as carbohydrates, vitamins, minerals,
proteins, and alkaloids (Belitz and Grosch, 1999; Mazzafera, 1999; Rogers et al., 1999; Nunes and Coimbra, 2001;
Kilmartin and Hsu, 2003; Oosterveld et al., 2004; Campa
et al., 2004; Franca et al., 2005), which would be advantageous to bacterial growth in bioremediation processes.
For these reasons, the goal of this research was to study
the role of microniches in the porous surface of the green
coffee bean in DDT and endosulfan biodegradation by
bacteria associated with the beans. Changes in the surface
of the coffee beans during cultivation were investigated by
SEM analysis.
2. Materials and methods
2.1. Reagents and chemicals
Technical-grade DDT and endosulfan were supplied by Teckchem and
Velsimex Company (Mexico). The compositions of the following
pesticides were determined by GC–MS: p,p0 -DDT (62%), o,p0 -DDT
(30%), DDE (8%); endosulfan I (65%), and endosulfan II (35%).
Green bean coffee (Coffea arabica) from Veracruz was supplied by the
Mexican Coffee Council (Consejo Mexicano del Café). Beans were airdried, ground, and sieved (10–20 mesh). All chemicals used were reagentgrade and solvents were HPLC-grade (Merck).
2.2. Culture medium
The composition of the mineral salts medium (MSM) used was (g l1):
Na2HPO4, 2.4; KH2PO4, 2.0; NH4NO3, 0.1; MgSO4 7H2O, 0.01; CaCl2,
0.01. The pH was adjusted to 6.5 (Radehaus and Schmidt, 1992). The
medium was sterilized by autoclaving at 121 1C for 20 min.
2.3. Isolation of organochlorine pesticide degrading bacteria from
coffee beans
The procedure to isolate bacteria consisted of the addition of 0.1 g of
ground coffee beans to a flask containing 50 ml of MSM medium, with
200 mg l1 of pentachloronitrobenzene (PCNB) as the organochlorine
pesticide model and as a fungal inhibitor. Cultures were incubated at 30 1C
and shaken at 100 rpm for 7 days. Two milliliters were then transferred to
a fresh MSM medium containing 200 mg l1 of PCNB and incubated
under the same conditions. Cultures from the fifth transfer were plated on
nutrient agar and incubated for 24 h at 30 1C. Colonies were isolated on
the basis of morphological, culture, and biochemical characteristics using
BBL Crystal GP System (Becton, Dickinson and Co., 2005).
Bacterial isolates were plated separately on MSM medium containing
50 ppm DDT, 50 ppm endosulfan, or 200 ppm PCNB. Cultures that were
able to grow in all pesticides tested were used for further studies.
2.4. Preparation of bacterial inoculum for biodegradation studies
The strains selected were pre-grown on liquid MSM amended with
2 g l1 of peptone and incubated at 30 1C at 100 rpm for 48 h. The
inoculum was then centrifuged (5000 rpm, 20 min). To remove residual
nutrients, cells were washed twice by centrifugation (5000 rpm, 20 min)
using 15 ml of 0.85% NaCl. Washed cells were resuspended in 0.85%
NaCl (Okeke et al., 2002). A standard curve of Abs600 nm vs. dry biomass
l1 was obtained.
2.5. Effect of carbon source on bacterial growth and pesticide
biodegradation
Assays were performed in 125-ml conical flasks containing 50 ml of
MSM medium amended with glucose (10 g l1), peptone (2 g l1), and
ground coffee beans (2 g l1). Sterilized coffee grains (g-radiation) were
added after autoclaving the MSM medium. Media were spiked with
technical DDT or endosulfan (40 ml of acetone/ethanol) to give 50 mg l1
final concentration (Siddique et al., 2003).
Media were inoculated with 5 ml biomass (Abs600 ¼ 0.6; 0.3 g dry
biomass l1) and incubated in the dark on an orbital shaker (100 rpm) at
30 1C for 7 days. The addition of 17 g NaCl to the medium was used to
stop bacterial growth. Two experimental sets (for cell growth and
degradation tests) were prepared in duplicate.
A biomass (g l1) versus time curve was determined for a 5-ml aliquot
of culture broth as described above. A control for the determination of
pesticide recovery efficiency was carried out under the same culture
conditions, but pesticides were added after bacterial inactivation.
The experimental and control samples were extracted with dichloromethane. The extracts were dehydrated by passing them through
anhydrous Na2SO4 and concentrated using a rotary evaporator. The
residue was re-dissolved in 5 ml of methanol and 2 ml were injected into the
GC/MS. The GC-MS consisted of a Varian Saturn 3 with an electron
multiplier as detector and an SPI injector. A DB-5 fused silica capillary
column (30 m 0.25 mm) was used. The temperatures in the injector and
the transfer line were 250 and 280 1C, respectively, and the operating
conditions were: flow rate 1 ml min1, column temperature 140 1C for
2 min followed by ramping at 5 1C min1 to 240 1C and then maintained at
240 1C for 27 min. Ionization was carried out at 70 eV. Quantification of
the chromatographic peaks was performed using external standards.
Metabolites were identified by comparison with MS spectra library
records (NIST/EPA/NIH, 1998).
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2.6. Statistical analysis
Data were interpreted through analysis of variance (po0:05), multiple
range analysis (least significant difference), and correlation analysis. Each
experiment was performed in duplicate. Treatments were considered
significant when po0:05. Statistical Analysis System Software (SAS, v
6.08 (8), SAS Institute S.A de C.V, Mexico) was used.
3. Results and discussion
Green bean coffee is a material rich in nutrients that
promotes the growth of microorganisms (Avallone et al.,
2001). The magnitude and diversity of the microbial
populations associated with the natural processing of
coffee (C. arabica) have previously been assessed, and
members of the genera Aeromonas, Pseudomonas, Enterobacter, Serratia, Cellulomonas, Arthobacter, Microbacterium, Dermabacter, and Lactoballis (Silv et al., 2000) have
been identified. The fungi that have been isolated include
Cladosporium, Fusarium, Penicillium, and Aspergillus (Silv
et al., 2000).
Enrichment techniques were used to isolate pesticidedegrading bacteria with PCNB as an inducer; only five
bacteria were isolated, identified as: Pseudomonas aerugi-
a
nosa, Flavimonas oryzihabitans, P. putida, Stenotrophomonas maltophilia, and Morganella morganii. Only F.
oryzihabitants and P. aeruginosa were able to grow on
plates with DDT or endosulfan as the sole carbon source.
It has been reported that certain pesticides have
inhibitory effects on bacterial growth (Nawab et al.,
2003). Complete degradation of DDT at concentrations
up to 15 ppm in flasks, with shaking, has been achieved,
but inhibitory effects were observed at 50 ppm. A carbon
source other than the target chemical has been found to
influence the degradation rate of organic toxins. It has been
reported that the addition of sucrose, glucose, sodium
acetate, sodium succinate, and sodium citrate inhibits DDT
biodegradation (Bidlam and Manonmani, 2002) and also
that the presence of sodium acetate and sodium succinate
inhibits endosulfan degradation (Awasthi et al., 2000).
In this research, F. oryzihabitants and P. aeruginosa were
able to grow with different carbon sources when supplemented with 50 ppm of DDT or endosulfan (Figs. 1 and 2).
In all cases, biomass production was higher than 0.3 g l1
and reached a maximum between 24 and 48 h of incubation
at 30 1C and 100 rpm. Bacterial growth of both strains in
peptone supplemented with DDT and endosulfan, where
a
0.8
0.8
0.6
Biomass (g l -1)
Biomass (g l-1)
0.6
0.4
0.2
0.4
0.2
0
0
1
2
3
4
5
0
6
Time (d)
b
b
0.8
0.6
0
1
2
0
1
2
3
Time (d)
4
5
6
0.8
0.6
Biomass (g l -1)
Biomass (g l-1)
241
0.4
0.2
0.4
0.2
0
0
0
1
2
3
4
5
6
Time (d)
Fig. 1. Time course of F. oryzihabitants (a) and P. aeruginosa (b) cell
growth with different carbon sources: peptone 2 g l1 (~), glucose 10 g l1
(’), and ground coffee 2 g l1 (m), supplemented with 50 ppm endosulfan.
Values represent the mean of two different assays; error bars correspond
to the standard deviation.
3
Time (d)
4
5
6
Fig. 2. Time course of F. oryzihabitants (a) and P. aeruginosa (b) cell
growth with different carbon sources: peptone 2 g l1 (~), glucose 10 g l1
(’), and ground coffee 2 g l1 (m), supplemented with 50 ppm DDT.
Values represent the mean of two different assays; error bars correspond
to the standard deviation.
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242
the biomass production was between 0.34 and 0.44 g l1,
did not show significant differences (p40:05). In contrast,
biomass production by P. aeruginosa in medium with green
bean coffee was higher in medium supplemented with
endosulfan (0.53 g l1) than DDT (0.36 g l1). In addition,
F. oryzihabitans and P. aeruginosa biomass (0.26–0.6 g l1),
grown on glucose with 50 ppm of DDT or 50 ppm of
endosulfan, showed significant differences (po0:05). The
highest biomass production (0.6 g l1) was obtained with F.
Residual sustrate (%)
200
150
100
50
C2/AN1
C2/RB2
P/AN1
P/RB2
G10/AN1
G10/RB2
0
Carbon source/Bacteria
Fig. 3. Effect of carbon source on p,p0 -DDT (
) and DDE (
)
biodegradation by P. aeruginosa (AN1) and F. oryzihabitans (RB2). G10:
glucose 10 g l1; P: peptone 2 g l1; C2: ground coffee bean 2 g l1.
Temperature 30 1C, 100 rpm, 7 days incubation. Values represent the mean
of two different assays; error bars correspond to the standard deviation.
oryzihabitans in medium with glucose and endosulfan at
24 h.
For all media tested, no significant endosulfan degradation by F. oryzihabitans was observed. In contrast,
endosulfan degradation by P. aeruginosa was shown at
51% with coffee bean medium and only 30% in glucose
and peptone media at 30 1C, 100 rpm, and 7 days of
incubation. Detoxification of endosulfan by aerobic
microorganisms often results in the formation of a toxic
endosulfan sulfate (Sutherland et al., 2000). Also, other
metabolites such as endosulfan diol or endosulfan ether
can be produced by microbial metabolism; however,
neither of these was identified by GC/MS in the culture
extracts. Removal of DDT was higher than that of
endosulfan, indicating that both bacteria preferentially
degrade aromatic compounds. A DDT concentration of
only 32–37% remained in the coffee bean culture medium
after 7 days of incubation (Fig. 3). Changes in the DDE
concentration indicate its production during DDT biodegradation, as well as its degradation, in the medium with
coffee bean addition. DDMU and 2,20 -bis (p-chlorophenyl)ethanol (DDOH) metabolites were also identified by GC/
MS in the culture broth.
Masse et al. (1989) and Quensen et al. (1998) reported
the conversion under anaerobic conditions of DDE to
DDMU through reductive dechlorination by bacteria and
marine sediments, respectively. In this research, experiments were run with shaking, where aerobic conditions are
expected. However, it is possible that inside the porous
structure of the coffee bean, anaerobic microniches could
be created, promoting the reductive dechlorination of DDE
Fig. 4. SEM photomicrographs at 1000 (a) green bean coffee inoculated with F. oryzihabitans in liquid media and 50 ppm DDT. Details at 5000 (b,
c). Temperature 30 1C, 100 rpm, 3 days incubation.
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to DDMU. Also, the DDOH peak in the chromatogram
indicates further degradation of DDMU (Singh et al.,
1999). In the medium with coffee beans, caffeine was
consumed by F. oryzyhabitans, according to the GC/MS
analysis.
Scanning electron microscopy of the coffee particles
showed a regular structure with a smooth external region,
rough zones, and open cavities (Fig. 4a). Micrographs at
early stages of culture (Figs. 4b and c) revealed colonization primarily in cavities and other regions of green bean
coffee sheltered from hydraulic shear forces. After 30 days
of growth, the coffee surface was completely colonized by
bacteria (Fig. 5b); at this time it was also possible to
observe coffee bean biodegradation (Fig. 5a). This
243
colonization pattern is probably the result of the adsorption properties, fluid dynamics, and system geometry, as in
granular activated carbon (GAC) systems (Massol-Deyá
et al., 1995). However, the coffee particles are a natural
support, which allow bacterial growth because of the
relatively high amount of carbon and other nutrients
(Belitz and Grosch, 1999). Microaerophilic biological
niches could be developed inside colonized coffee bean
cavities, due to the metabolic activity of microorganisms
(Beuninck and Rehm, 1988). This fact may explain the
DDD and DDE biodegradation in this system, since their
accumulation in the medium was not observed.
The combination of anaerobic and aerobic environments
enhances the mineralization of many electrophilic aromatic
contaminants such as organochlorine and azo compounds,
for which several strategies have been proposed (Field
et al., 1995). This is the first report to present evidence on
the creation of micro-environmental conditions by way of
structural microniches in natural waste, where it is possible
for reductive reactions to occur.
4. Conclusions
Bacteria isolated from defective green bean coffee with
traditional enrichment techniques using PCNB as an
organochlorine pesticide model were able to grow on and
to degrade DDT and endosulfan in liquid media. The
highest pesticide biodegradation was obtained when coffee
bean was added. The presence of certain metabolites such
as DDMU and DDOH suggests the formation of
anaerobic microniches in the porous structure of the coffee
bean. SEM photomicrographs at 3 days incubation showed
that colonization occurred mainly inside the coffee bean.
Thus, defective green bean coffee can be used as both a
nutrient source and a support for organochlorine pesticide
degrading bacteria in liquid media.
Acknowledgments
We would like to thank Ma. Esther Sánchez for taking
the microphotographs (ENCB-IPN), Dolores Dı́az Cervantes (CINVESTAV-IPN) for her technical help, and Ing.
Elvira Garcı́a (Consejo Mexicano del Café). This study was
supported by Consejo Nacional de Ciencia y Tecnologı́a
(Fondo Mixto TLAX-2003-C02-12416).
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