ARTICLE IN PRESS 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: email@example.com (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, ARTICLE IN PRESS 240 B.E. Barragán-Huerta et al. / International Biodeterioration & Biodegradation 59 (2007) 239–244 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). ARTICLE IN PRESS B.E. Barragán-Huerta et al. / International Biodeterioration & Biodegradation 59 (2007) 239–244 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. ARTICLE IN PRESS B.E. Barragán-Huerta et al. / International Biodeterioration & Biodegradation 59 (2007) 239–244 242 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. ARTICLE IN PRESS B.E. Barragán-Huerta et al. / International Biodeterioration & Biodegradation 59 (2007) 239–244 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. 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