Chemistry and Ecology ISSN: 0275-7540 (Print) 1029-0370 (Online) Journal homepage: http://www.tandfonline.com/loi/gche20 Performance evaluation of isoproturon-degrading indigenous bacterial isolates in soil microcosm Krishna Giri, J. P. N. Rai, Shailesh Pandey, Gaurav Mishra, Rajesh Kumar & Deep Chandra Suyal To cite this article: Krishna Giri, J. P. N. Rai, Shailesh Pandey, Gaurav Mishra, Rajesh Kumar & Deep Chandra Suyal (2017): Performance evaluation of isoproturon-degrading indigenous bacterial isolates in soil microcosm, Chemistry and Ecology, DOI: 10.1080/02757540.2017.1393535 To link to this article: http://dx.doi.org/10.1080/02757540.2017.1393535 View supplementary material Published online: 28 Oct 2017. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gche20 Download by: [University of Florida] Date: 28 October 2017, At: 04:18 CHEMISTRY AND ECOLOGY, 2017 https://doi.org/10.1080/02757540.2017.1393535 RESEARCH ARTICLE Performance evaluation of isoproturon-degrading indigenous bacterial isolates in soil microcosm Krishna Giria, J. P. N. Raib, Shailesh Pandeya*, Gaurav Mishraa, Rajesh Kumara and Deep Chandra Suyalc Rain Forest Research Institute, Jorhat, India; bDepartment of Environmental Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar, India; cDepartment of Microbiology, G. B. Pant University of Agriculture and Technology, Pantnagar, India Downloaded by [University of Florida] at 04:18 28 October 2017 a ABSTRACT ARTICLE HISTORY Isoproturon (IPU)-degrading soil bacteria were isolated from herbicide-applied wheat fields. These isolates were identified using cultural, morphological, biochemical and 16S rRNA sequencing methods. 16S rRNA sequences of both the bacterial isolates were compared with NCBI GenBank data base and identified as Bacillus pumilus and Pseudoxanthomonas sp. A soil microcosm study was carried out for 40 days in six different treatments. Experimental results revealed maximum 95.98% IPU degradation in treatment 6 where bacterial consortia were augmented in natural soil, followed by 91.53% in treatment 5 enriched with organic manure as an additional carbon source. However, only 14.03% IPU was degraded in treatment 1 (control) after 40 days. In treatments (2–4), 75.59%, 70.92% and 77.32% IPU degradation was recorded, respectively. IPU degradation in all the treatments varied significantly over the control. 4-Isopropylaniline was detected as IPU degradation by-product in the medium. The study confirmed that B. pumilus and Pseudoxanthomonas sp. performed effectively in soil microcosms and could be employed profitably for field-scale bioremediation experiments. Received 10 June 2017 Final Version Received 13 October 2017 KEYWORDS Biodegradation; isoproturon; soil microcosms; B. pumilus; Pseudoxanthomonas sp 1. Introduction Excessive pesticide application in agriculture and public health sector has resulted in the contamination of terrestrial and aquatic ecosystems. Pesticides undergo spillage during production, transportation and application in the crop fields and cause serious environmental pollution. Due to recalcitrant nature, pesticides persist in the environment for longer periods and cause deleterious effects in flora and fauna. Pesticide build-up in the environment is also hazardous for human health and ecosystem functioning. Phenylurea herbicide isoproturon is used for pre- or post-emergence broad-leaved weed control in agriculture . Widenfalk et al.  and Vallotton et al.  reported that IPU and its metabolites are harmful to the aquatic environment; however, Hoshiya et al.  found it carcinogenic for human health. According to the official survey conducted by Directorate of Plant CONTACT Krishna Giri email@example.com Rain Forest Research Institute, Jorhat, Assam 785 001, India Supplemental data for this article can be accessed at https://doi.org/10.1080/02757540.2017.1393535 *Present address: Forest Research Institute, Dehradun 248001, India © 2017 Informa UK Limited, trading as Taylor & Francis Group Downloaded by [University of Florida] at 04:18 28 October 2017 2 K. GIRI ET AL. Protection, Quarantine and Storage, Govt. of India from 2005–2006 to 2009–2010, IPU is among the most consumed pesticide in the country with 7163 metric tonnes consumption per annum . Although IPU has been banned in several countries or restricted to use 1.2 kg ha−1 year−1 since 2003, but in several countries it is still extensively being used. U.S. Nagar district of Uttarakhand is known as Tarai agro-ecosystem and one of the major food production zones in the state. Due to large-scale agricultural operations in this region, pesticide consumption is also very high. Pesticide spray in various crops leads to air, water and soil pollution as well as harmful impacts on beneficial micro flora and non-target insects. Besides, surface and ground water contamination is another environmental risk associated with pesticide use . Biodegradation is the cost-effective and eco-friendly approach for pesticide removal from the environment over physical and chemical methods. Soil microbes harbour a wide array of genes and enzymes which can detoxify recalcitrant xenobiotics in environmentally innocuous end products. Soil bacteria and fungi utilise these pesticides as a source of energy and capable of mineralisation in CO2 and water which are non-toxic to the environment. Biochemical and enzymatic reactions transform the original structure and toxicological properties of complex organic compounds and convert them in to non-toxic forms. Therefore, microbial biodegradation of pesticides is the potential area of bioremediation to eliminate them from various segments of the environment. Considering these harmful effects of IPU, elimination of its build-up in the environment is imperative . Microbial degradation plays an important role in pesticide dissipation from the soil, which could prove to be a reliable cost-effective remediation technique for IPU abatement [7–8]. Several studies have already reported IPU degradation by bacterial isolates vis-a-vis repeated exposure to this herbicide over a longer period of time [9–10]. Bacterial degradation of IPU in the contaminated soils is influenced by various physico-chemical and environmental factors . Variety of soil bacteria and fungi has been isolated from different regions in the world which can utilise IPU as a carbon source . However, only Sphingomonas sp., Methylopila sp. and Sphingobium sp. strains have shown the complete IPU mineralisation potential [12–14]. Laboratory microcosms are a good compromise between field experiments, which are often hampered by a high environmental variability and a high cost, and standard laboratory tests of which the results are rarely representative of the real world, because the experimental conditions and the organism tested are not site-specific. Finally, as laboratory microcosms are relatively small, it is possible to produce many replicates and to vary experimental conditions once at a time in order to more firmly establish the casual relationship between a toxicant and its effects on microbial communities under different abiotic conditions . Considering the herbicide biodegradation potential of Bacillus pumillus and Pseudoxanthomonas sp. in the laboratory experiments, the present investigation was undertaken to evaluate their IPU degradation performance in soil microcosms experiment. 2. Materials and methods 2.1. Chemicals and reagents Analytical grade [(99.5% purity) 3-(4-isopropylphenyl)-1-methylurea (IPU)] and its major metabolites [N-(4-isopropylphenyl)-N′ -methylurea] (MDIPU), [N-(4-isopropylphenyl) urea] CHEMISTRY AND ECOLOGY 3 (DDIPU) and 4-isopropylaniline (4IA) were procured from Sigma Aldrich, USA. All the stock solutions were prepared in methanol. Acetonitrile, dichloromethane and all other chemicals used during this investigation were of analytical grade and purchased from Himedia, Laboratories India. The composition of mineral salt medium (MS) used for the enrichment, isolation and IPU degradation contained (gL−1) KH2PO4, 0.5; K2HPO4, 1.5; NH4NO3 1.0; MgSO4, 7H2O 0.2; NaCl 1.0; and FeSO4 0.025. Nutrient agar medium contained (gL−1): Peptone 10; Yeast extract 5.0 and NaCl 5.0 [12,16]. Downloaded by [University of Florida] at 04:18 28 October 2017 2.2. Isolation and identification of IPU-degrading bacteria Surface soil samples (0–30 cm) depths were collected from IPU-applied wheat fields, Norman E. Borlaug Crop Research Centre, Pantnagar, India and brought to the laboratory for isolation of bacteria. IPU-degrading bacterial strains were isolated using enrichment culture method, where IPU concentration in growth medium was gradually increased from 50 to 400 mg L−1 and screened under laboratory conditions for their IPU tolerance capacities. Maximum growth of these two bacterial isolates was observed in 200 mg L−1 initial IPU concentration. Based on the laboratory experiments, two bacterial isolates K1 and K2 were chosen for their performance evaluation in the soil microcosm. These bacterial isolates were identified based on cultural, morphological, biochemical and 16S rRNA partial sequencing. 2.3. Biodegradation of isoproturon in soil microcosm Soil microcosm study was carried out using bacterial isolates viz. K1 and K2. Six microcosm treatments with different combinations were prepared using 500 mL capacity Erlenmeyer flasks. Treatment 1 (control) contained 200 g soil and 200 mg kg−1 IPU. The quantity of soil and IPU were kept constant in all the treatments. Treatment 2 was inoculated with K1 at the rate of 5% (v/w) concentration. Treatment 3 was inoculated with K2 bacterial isolate at the rate of 5% (v/w). Treatment 4 was inoculated with a consortium of K1 + K2. Treatment 5 contained consortium of K1 + K2 and sterilised organic manure @ 100 g kg−1 of soil. The soil used in treatments 1–5 was sterilised twice. However, treatment 6 contained natural (unsterilised) soil, IPU and consortium of both the bacteria. Adequate amount of water was added to maintain 40% moisture content of the soil, because 40% moisture content has been reported as optimum for bacterial growth. All the components were thoroughly mixed; flasks were covered with perforated paraffin film and kept in ambient temperature for 40 days. Autoclaved distilled water was added at regular interval to compensate water losses. The experiment was carried out in triplicates and 5 g soil samples from each treatment were withdrawn aseptically at 5, 10, 15, 20, 25, 30, 35 and 40 days interval for the determination of IPU degradation. 2.4. IPU extraction and analysis Residual IPU was extracted and processed using previously described methodology . The IPU extracts were analysed using Dionex HPLC equipped with auto sampler and Acclaim 120, C18 5 µm 4.6 × 250 mm column. Acetonitrile/water (75:25 v/v) at a flow rate of 1 mL min−1 was used as mobile phase. The solutes were detected using UV 4 K. GIRI ET AL. detector at 243 nm . The HPLC analysis was carried out in the College of Fisheries, G.B. Pant University of Agriculture and Technology, Pantnagar, India. Minimum detection limits of IPU, [N-(4-isopropylphenyl)-N′ -methylurea] (MDIPU), [N-(4-isopropylphenyl) urea] DDIPU and 4IA were 0.75, 0.69, 0.86 and 0.89 µg L−1 respectively. Maximum recovery efficiency of the extraction method was 95.61%. 2.5. Statistical analysis The standard error of means and analysis of variance was calculated using MS Excel Worksheet, 2007 and experimental data were expressed at 95% confidence level. Downloaded by [University of Florida] at 04:18 28 October 2017 3. Results 3.1. Isolation and identification of bacterial isolates IPU-degrading bacterial strains K1 and K2 were identified using cultural, morphological (Figure 1), biochemical and 16S rRNA sequencing method (Table 1). The results of biochemical tests (carbohydrate utilisation) is given in the supplementary data with the original source of citation [6,16]. K1 strain showed 99% sequence similarity with B. pumilus and identified as B. pumilus GenBank accession No. KF279694. However, K2 showed 95% sequence similarity with Pseudoxanthomonas japonensis and designated as Pseudoxanthomonas sp. GenBank accession No. KF279695. 3.2. Biodegradation of isoproturon in soil microcosm After 40 days incubation, 14.03% IPU was degraded in treatment 1 (control). Treatment 2, inoculated with B. pumilus showed 75.59% degradation. IPU degradation rate was also measured at every 5 days interval and found 3.77 mg/day in treatment 2. Treatment 3 inoculated with Pseudoxanthomonas sp. showed 70.92% degradation at the rate of Figure 1. Gram staining images of (a) Bacillus pumilus and (b) Pseudoxanthomonas sp. CHEMISTRY AND ECOLOGY 5 Table 1. Cultural, morphological and molecular characteristics of IPU-degrading bacterial isolates. K1 K2 Moderate Irregular Filamentous Raised Rough White Moderate Circular Entire Convex Smooth Off white Gram positive (+) Long rods Joint Sequence similarity 99% 95% Gram negative (−) Small rods Single GenBank accession number KF279694 KF279695 Identification Bacillus pumilus Pseudoxanthomonas sp. 3.54 mg/day. The similar trend was also observed in the laboratory experiments where B. pumilus degraded more IPU than Pseudoxanthomonas sp. [16,6]. Treatment 4, which contained sterilised soil amended with IPU and consortium of B. pumilus + Pseudoxanthomonas sp. degraded 77.32% IPU (3.86 mg/day) at the end of the experiment. 91.53% IPU degradation was recorded in treatment 5, containing consortium of B. pumilus + Pseudoxanthomonas sp. and organic manure as an additional carbon source. However, maximum 95.98% IPU degradation was recorded in treatment 6, containing natural soil and consortium of both the bacterial isolates. IPU degradation in this treatment was achieved at the rate of 4.57 mg/day after 40 days incubation (Figures 2 and 3). Statistical analysis of the data revealed that IPU degradation in all the treatments varied (P < 0.05) significantly in comparison to control. Initially IPU degradation was very slow; as incubationpreceded degradation increased significantly. IPU degradation rate in control treatment was 0.70 mg/day after 40 days (Figure 3) which was negligible in comparison to other five treatments. The findings of this experiment revealed that IPU degradation was dependent in different growth phases of bacterial isolates. Initially IPU degradation in all the treatments was slow. Once bacterial isolates adapted in the microcosm IPU degradation enhanced significantly (Figure 2). 4IA was accumulated as IPU degradation by-product in the medium. However, no other reported metabolites were detected in the medium (Table 2). Treatment 1 Treatment 4 Treatment 2 Treatment 5 Treatment 3 Treatment 6 120 100 % Degradation Downloaded by [University of Florida] at 04:18 28 October 2017 Cultural characteristics Size Shape Edge of colony Elevation of colony Surface of the colony Colour Morphological characteristics Gram reaction Shape of cells Arrangement of cells 16S rRNA sequence analysis 1 2 80 60 40 20 0 -20 5 10 15 20 25 Time (days) 30 35 40 Figure 2. Biodegradation of isoproturon in soil microcosm: (error bar shows ± SE, n = 3). Downloaded by [University of Florida] at 04:18 28 October 2017 6 K. GIRI ET AL. Figure 3. Rate of isoproturon degradation in different treatments. Table 2 Concentration of 4 IA accumulated in the medium. Time (days) Treatment-1 Treatment-2 Treatment-3 Treatment-4 Treatment-5 Treatment-6 5 10 15 20 25 30 35 40 ND ND ND 0.02 ± 0.01 0.06 ± 0.1 1.03 ± 0.03 1.5 ± 0.2 2.04 ± 0.02 ND ND 0.82 ± 1.0 1.06 ± 0.3 1.72 ± 0.5 1.98 ± 0.4 2.01 ± 1.3 2.5 ± 1.04 ND 0.7 ± 0.3 0.98 ± 0.2 1.02 ± 0.7 1.32 ± 0.5 1.53 ± 1.0 187 ± 0.6 2.21 ± 0.91 ND 0.92 ± 1.3 1.08 ± 0.5 1.23 ± 0.3 1.75 ± 1.2 2.08 ± 1.3 2.43 ± 1.0 2.95 ± 1.28 ND 1.34 ± 0.6 1.87 ± 1.2 2.69 ± 1.20 3.16 ± 1.3 4.60 ± 1.15 4.88 ± 1.5 5.06 ± 2.2 ND 1.46 ± 1.3 1.98 ± 1.20 2.91 ± 1.8 3.56 ± 1.3 4.78 ± 1. 0 5.06 ± 1.4 6.30 ± 1.01 Note: Values are given in mg L−1; ND: not detected; ±SE, n = 3. 4. Discussion Soil micro flora plays an important role in detoxification and mineralisation of complex organic pollutants and serves as best candidates for pesticide bioremediation. Microcosm experiments are small-scale ecosystems, and reproducible for testing the biodegradation potential of microbes before large-scale bioremediation studies, further environmental conditions can be controlled in laboratory-based microcosm studies in order to acclimatise microorganisms under suboptimal environmental conditions . In present investigation IPU degradation in treatment 1 (control) was negligible than other five treatments which is attributed to chemical hydrolysis/reactions of the compound [17–18]. IPU degradation by B. pumilus in treatment 2 was more than treatment 3 inoculated with Pseudoxanthomonas sp. These observations depict that B. pumilus harbour more efficient IPU-catalysing enzymes than Pseudoxanthomonas sp. In treatment 4 more IPU degradation is accounted to cooperative metabolic activities by bacterial consortium. Enhanced pesticide degradation by microbial consortium has been previously reported as an effective method of harnessing catabolic potential of natural micro flora . Organic amendment in the treatment 5 enhanced IPU degradation potential of bacterial consortium. Mostly these organic Downloaded by [University of Florida] at 04:18 28 October 2017 CHEMISTRY AND ECOLOGY 7 amendments contain peptides, amino acids, carbohydrates and lipids, which stimulate microbial growth under pesticide stress and toxic environments, simultaneous enhancement of pesticide uptake and degradation . Exogenous addition of electron donors could enhance the herbicide degradation and mineralisation in environmentally benign forms . Apart from essential growth stimulators, organic amendments serve as a rich source of nitrogen, carbon and other primary nutrients for microbial activities in pesticide-polluted environments . El Fantroussi and Agathos  also reported that addition of energy sources or electron donors may benefit both indigenous and exogenous degraders. The IPU degradation potential of bacterial consortia increased significantly when it was augmented in natural soil where other microbes were present. Maximum IPU degradation was observed in treatment 6 in the presence of natural indigenous soil microbes. This might be due to synergistic metabolic relationship between natural micro flora and inoculated consortia which is a good indicator for application of these bacterial strains in field-scale bioremediation. This is further opined that bacterial consortium obtained more conducive environmental conditions in the natural soil for IPU degradation than sterilised soil treatments. However, some studies reported that pesticide degradation was declined when enriched bacteria were augmented in natural soil . This could be because of the natural bioprocesses or antagonistic growth relationship of microorganisms. Silva et al.  developed a combined bioaugmentation and biostimulation approach for the cleanup of soil contaminated with a high concentration of atrazine using Pseudomonas sp., and found that addition of citrate and succinate as biostimulant significantly increased herbicide degradation in contrary to Adenosine diphosphate (ADP) alone. Only 4IA was detected as a degradation by-product in the medium among the three reported metabolites of isoproturon. This is in agreement with the laboratory experiments of IPU degradation using these two bacterial isolates. Rest of the two metabolites may be produced below detection limits or utilised simultaneously by the bacterial consortium as reported by Weir et al. . 5. Conclusion Findings of the study revealed that B. pumilus and Pseudoxanthomonas sp. degraded significant amount of herbicide from all the treatments. The IPU degradation potential of bacterial consortia increased significantly when it was augmented in natural soil where indigenous microbes were present. A synergistic relationship between natural micro flora and inoculated consortia was established in this treatment. Such kind of relationship is a good indicator for application of these bacterial strains in field-scale bioremediation because in natural sites variety of other microbes interferes and alters the performance of laboratory-optimised microbes. Establishment of a positive synergy between laboratory-grown efficient culture and natural micro flora may be a boon for rapid clean-up of the pesticide-contaminated environmental site. Therefore, these soil bacterial isolates can be used further for fieldscale bioremediation of pesticide-contaminated soil and water environment. Acknowledgment The authors are thankful to Dr R. N. Ram, Professor and Head, Department of Fishery Biology, College of Fisheries for providing necessary facilities for HPLC analysis of the samples. This work was carried out at GBPUAT Pantnagar. 8 K. GIRI ET AL. Disclosure statement No potential conflict of interest was reported by the authors. Notes on contributors Downloaded by [University of Florida] at 04:18 28 October 2017 Dr. Krishna Giri is Scientist in the Shifting Cultivation Division at Rain Forest Research Institute, Jorhat, Assam. He obtained his Ph.D. in Environmental Sciences with specialization in Environmental Microbiology (Bioremediation) from G.B. Pant University of Agriculture and Technology, Pantnagar, India. His main area of interest is Environmental Microbiology and he is presently working on application of Plant Growth Promoting Rhizobacteria for soil fertility and crop productivity enhancement in degraded Shifting Cultivation areas in North East India. Dr. J .P. N. Rai is a Senior Professor and Head of Department, Environmental Sciences at G.B. Pant University of Agriculture and Technology, Pantnagar, India. He has more than 25 years of teaching and research experience in the field of Environmental Sciences and Bioremediation of industrial wastes. He has contributed more than 100 research papers in peer-reviewed journals of National and International repute and supervised 25 Ph.D. research scholars. Dr. Shailesh Pandey is a Scientist in the Forest Pathology Division at Forest Research Institute, Dehradun. He obtained his Ph.D. in Plant Pathology and Molecular Biology & Genetic Engineering from G.B. Pant University of Agriculture and Technology, Pantnagar, India. His main areas of interest focus on the population biology of plant pathogenic bacteria and fungi, molecular detection of microbes, biological control, and plant disease management. Dr. Gaurav Mishra is Scientist in the Silviculture and Forest Management Division at Rain Forest Research Institute, Jorhat, Assam. He obtained his Ph.D. in Soil Science from G.B. Pant University of Agriculture and Technology, Pantnagar, India. Presently, he is working on Carbon Sequestration and Soil Quality of Forest Soils in North East India. Shri Rajesh Kumar is a Scientist in the Forest Protection Division of Rain Forest Research Institute, Jorhat, Assam. He has expertise in the field of fungal taxonomy, especially higher fungi, microbiology and plant pathology. Dr. Deep Chandra Suyal is working as a Young Scientist (Scientific and Engineering Research Board, Department of Science and Technology, Govt. of India) in the Department of Microbiology at G.B. Pant University of Agriculture and Technology, Pantnagar, India. He did his Ph.D. from the same department on Microbial Genomics of Cold adapted Microbes. He has expertise in Molecular Microbial identification. 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