Assessment of Arsenic Tolerant Bacteria from Arsenic Contaminated Groundwater in Nawalparasi District of Nepal

Abstract

worldwide public health issue, including Nepal. To assess arsenic tolerant bacteria from arsenic contaminated drinking water, this research study was carried out from June 2007 to January 2008. Eighty-six water samples were collected from one municipality and six VDCs’ wards in Nawalparasi district of Nepal having arsenic concentration above WHO guidelines (10 ppb).

Physico-chemical and microbiological parameters of water quality were determined to be poor. The pH of 11.63% water samples was found below WHO guideline. Sixty two percent (53/86) samples in Nawalparasi district showed arsenic level above Nepal drinking water quality standard (NDWQS) (50 ppb). Filtered water samples showed that 30.16% (19/63) was above NDWQS guideline. Comparison between arsenic concentration of influent and effluent water showed significant difference (p<0.05). Within the last 10 years, 66.27% (57) tubewells were constructed whereas 52.63% (30/57) tubewells constructed within 6-10 years. Moderately positive correlation (r=0.23, p=0.83) was found between arsenic concentration and age of tubewells. At 36-95 feet depth range, 50 (58.14%) tubewells were above NDWQS and 70 (81.39%) tubewells above the WHO guideline. Results showed weekly negative correlation (r=-0.12, p=0.24) indicating arsenic concentration of groundwater decreases as the depth of tubewell increases.

Skin manifestations were observed in 12 (34%) females and 18 (33%) males, skin manifestations and gender difference was found statistically insignificant (p>0.05). Arsenicosis cases were highest (33.3%, 10/30) in 32-41 age groups. Most of the patients (30%) suffered from diffused arsenical melanosis and 20% patients from both diffused arsenical melanosis and keratosis. Mild skin manifestation were observed in most, 66.7% (20/30) patients followed by 7 (23.3%) moderate and 3 (10%) severe. Percentage of arsenicosis cases was higher among respondents using tubewell that were built >10 years (41%) than that were built ≤10 years (32%).

The microbiological analysis of groundwater revealed the presence of total coliforms in 98.84% samples (85) of influent water from tubewell and 71.43% (45/63) effluent water from filter. Among the isolates, E. coli (37%) was found to be predominant in influent water which was followed by P. aeruginosa (16%), Citrobacter spp. (16%) and K. pneumoniae (10.5%). Similarly, E. coli (39.21%) was found predominant in effluent water which was followed by P. aeruginosa (19.61%), K. oxytoca (13.72%) and Citrobacter spp. (13.72%). Among thirteen arsenic tolerant bacteria isolated from plate count agar incorporated with As (V) >50 ppm, the most predominant isolates were unidentified NP1 (Bluish green) (19.51%) followed by Pseudomonas spp. (16.38%), Bacillus cereus (11.15%). The mean colony count of samples (<300 cfu/ml) was highest in 10 ppm (216 cfu/ml) and lowest in 200 ppm (70 cfu/ml). The highest MIC value of As (III) was obtained for E. coli (mean=15 ppm) followed by NP1 (Bluish green) (mean=10.71ppm). Similarly the highest MIC value of As (V) was obtained for Pseudomonas spp. (mean=560 ppm) followed by E. coli (mean=360ppm).

Plasmids that were extracted from 8 arsenic tolerant E. coli and one arsenic sensitive E. coli and run in agarose gel which showed 8 types of plasmid profiles of size ranging from 3.1 to 16.21 kb. The most frequent band was 14.17 kb (in four isolates) followed by 6.16 kb (two isolates) and 8.5 kb (two isolates). Apparently a size difference was observed in overall plasmid profiling. Large size bands were obtained from arsenic tolerant bacteria than arsenic sensitive bacteria (4.36Kb).