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Arsenic-resistant Bacteria Isolated from Agricultural Soils of Bangladesh and Characterization of Arsenate-reducing Strains

S.P. Bachate*
Department of Food Science and Microbiology, University of Milan, Milan, Italy

L. Cavalca*
Department of Food Science and Microbiology, University of Milan, Milan, Italy

V. Andreoni
Department of Food Science and Microbiology, University of Milan, Milan, Italy

Abstract/ Executive Summary:

Aims: To analyse the arsenic-resistant bacterial communities of two agricultural soils of Bangladesh, to isolate arsenic-resistant bacteria, to study their potential role in the arsenic transformation and to investigate the genetic determinants for arsenic resistance among the isolates.Methods and Results: Enrichment cultures were performed in a minimal medium in the presence of As(III) and As(V) to isolate resistant bacteria. Twenty-one arsenic-resistant bacteria belonging to different genera of Gram-positive and Gram-negative bacteria were isolated. The isolates, with the exception of Oceanimon as doudoroffii Dhal Rw, reduced 2 mmol l)1As(V) completely to As(III) in aerobic conditions. Putative gene fragments for arsenite efflux pumps were amplified in isolates from Dhal soil and a putative arsenate reductase gene fragment was amplified from Bacillus sp. from Rice soil. Conclusions: Phylogenetically diverse arsenic-resistant bacteria present in agricultural soils of Bangladesh are capable of reducing arsenate to arsenite under aerobic conditions apparently for detoxification purposes. Significance and Impact of the Study: This study provides results on identification, levels of arsenic resistance and reduction of arsenate by the bacterial isolates which could play an important role in arsenic cycling in the two arsenic-contaminated soils in Bangladesh.

Keywords: Agricultural soil of Bangladesh, Arsenate reduction, Arsenic resistance, Arsgenes, 16SrRNA gene

Location: Satkhira District, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Bacteria

Objectives:

The aim of the present work was (i) to isolate and identify the culturable arsenic-resistant bacterial populations present in arsenic-contaminated paddy rice and lentil field soils of Bangladesh; (ii) to evaluate the arsenic transformations performed by the arsenic-resistant isolates and (iii) to check for the presence of genetic determinants for arsenic resistance among isolates to understand the resistance mechanism involved.

Methodology:

Soil characteristics and bacterial counts in soils Soil samples (0–20 cm) were collected from a paddy rice field (Rice soil) and lentil field (Dhal soil) from the Satkhira District (Bangladesh) and the samples were chemically analysed by Martinet al.(2007). The various forms of arsenic content of the soils (total, water-soluble and oxalate-extractable) were determined by following the sequential extraction method of Wenzelet al.(2001).For microbiological analysis, eight samples of each soil were randomly collected and then mixed together to form two composite samples. Each composite sample was put into plastic bags, transported to the laboratory where they stored moist in the dark at 4°C in polyethylene bags for 2 weeks until the analyses. An initial soil extraction was performed by adding45 ml of sodium pyrophosphate solution (2 g l^-¹) to 5g of wet soil. Serial tenfold dilutions of soil samples were prepared in 1:20 saline solution (Jensen 1962) for each soil sample (Pochon and Tardieux 1962). Heterotrophic bacteria were determined by plating aliquots (1 ml) of the various dilutions into 1:10 diluted Tryptic Soy Agar (TSA⁄10) medium in double sets of pour plates. Cycloheximide (0Æ1gl)1) was added to the media to inhibit fungal growth. To analyse the arsenic tolerance level of the bacterial community in the soil (Kunitoet al.1999), the number of arsenic-tolerant bacteria was determined by plating 1 ml of the various dilutions of soil on TSA⁄10medium supplemented with 0Æ77 mmol l)1either of sodium arsenate, As(V), or sodium arsenite, As(III) mixed in the medium prior to pouring the plates. The arsenic tolerance of the heterotrophs was expressed as a percentage of growth on TSA⁄10 without arsenic addition. All incubations were carried out at 30°C for 10 days. The data were subjected to analysis of variance (ANOVA), and treatment means were compared by Duncan’s multiple range test. All analyses were performed at the P£0.05 level. Enrichment cultures and isolation of arsenic-resistant strains To evaluate the arsenic resistance, soil bacteria were grown in the presence of high concentration of arsenic to determine the response to arsenic stress. Soil samples(10 g) were added to flasks containing Tris mineral medium (TMM) at low phosphate content (Mergeay et al.1985), supplemented with 6 g l^-1of sodium gluconate (TMMG) and with 20 mmol l^-¹of As (V) or 5 mmol l^-¹. of As(III). Flasks were incubated under shaking(150 rev min)1)at30°C for 5 days and 10 ml of each of these enrichment cultures were transferred into a fresh medium and then incubated under the same conditions.After three subsequent transfers, aliquots of cell suspensions (15 ml) were centrifuged (10°C for 20 min at13 000g) and cell pellets were then subjected to DNA extraction as specified below. To isolate arsenic-resistant bacteria, appropriate dilutions of enrichment cultures were plated on TSA⁄10 containing 20 mmol l)1of As(V)or 5 mmol l^-¹of As (III) and the plates were incubated at30°C for 5 days. After incubation, colonies with different morphologies were selected from the plates. Single colonies were streaked to purity on the same medium. Strains were maintained in glycerol stocks at -70°C. Prior to use, the strains were grown to mid-exponential phase in TMMG at 30°C with shaking. DNA extraction methods DNA from enrichment cultures was extracted by a conventional, not organic solvent method (G-NOME BIO101, QBiogene, Illkirch Cedex, France). Cell suspension solution (1Æ85 ml) was added to the pellet (50 mg of fresh weight) obtained from freshly grown liquid culture and50ll of RNase was added to the sample. Then the sample was incubated at 55°C for 2 h in the presence of 100llcell lysis solution and 25ll of Proteinase mix. Samples were precipitated in the presence of 8 ml 100% ethanol and the DNA pellet was resuspended in 200llTE(10 mmol l^-1Tris HCl pH 8Æ0 and 1 mmol l^-1 Na2 EDTA pH 8.0). DNA from pure strains was extracted from TMMG overnight-grown liquid cultures by Proteinase K digestion (1 mg ml^-1) according to Cavalcaet al. (2004). The DNA-containing supernatant was withdrawn and put in sterile microtubes. Cloning Clone libraries of hypervariable V3 16S rRNA regions of the DNA from As(V) (20 mmol l^-1) and As (III) (5 mmol l^-¹) enrichment cultures of both soils were prepared in order to describe species composition. V3 fragments were amplified as described above and PCR products were purified by QIA quick PCR purification columns (Qiagen, Germany) and ligated into pCRÒII-TOPOTA cloning vector (Invitrogen). After chemical transformation into library efficiency DH5aÔcompetent cells(Invitrogen), 50 white colonies from each experiment were screened for the presence of the correct size insert colony PCR using vector-specific primers (M13 forward and reverse). Sequence analysis was performed on representative clones showing different HhaI (GE Health-care, Sweden) restriction fragment length polymorphisms.Analogously, PCR products of eubacterial 16S rRNA and arsenic resistance gene fragments amplified from isolated strains were cloned into pCRÒII-TOPO TA cloning vector. All the selected clones were directly sequenced by using M13 forward and reverse primers. Sequence actions were performed by using the TaqDye-DeoxyTerminator Cycle Sequencing kit (Applied Biosystems, USA) and analysed by automatic DNA sequencer 310Aapparatus (Applied Biosystems).

Source of Information: Journal of Applied Microbiology 107 (2009) 145–156 ISSN 1364-5072

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Achievement/Findings:

The results obtained in this study give evidence of the presence of phylogenetically diverse arsenic-resistant bacteria in agricultural soils and that the resistance levels of these bacteria are regardless of the arsenic contamination. Although the direct role of these bacteria in arsenic mobilization was not studied, the ability of the isolates to reduce As (V) suggests that these bacteria can potentially mediate arsenic transformations in these soils and their role in the arsenic cycling in these soils may become relevant with changing environmental conditions.

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