Isolation of Nitrate and Phosphate Removing Bacteria from Various Environmental Sites

Problem statement: Nitrate and phosphate are two major pollutants due to anthropogenic activity like excessive use of fertilizers in agric ulture. Their contamination has emerged as a global problem and its potential threat is marked on the e nvironmental sustainance as well as on the public health. Approach: The objective of the current study is to isolate ef ficient nitrate and phosphate removing microbes from various environmental sites that have been selected on the basis of the nature of polutants received by them and their water quali ty assessment. These well characterized isolates could in future be used for the remediation of wast e water. 30 different sites were screened using culture based method. The nitrate and phosphate rem oving abilities of the microbes were checked in enriched medium (Himedia M439) after 16 h of incuba tion at 37°C. Results: 7 efficient isolates were obtained from rhizosphere of Water lily, Marine bea ches, Paddy field and Raw sewage canal. The highest nitrate removal (88.3%) was shown by isolat e (WBUNB009) from raw sewage canal and the highest phosphate removal (82.9%) was shown by isol ate (WBUNB004) from rhizosphere of Water lily. Morphologically all the isolates were gram positive bacilli as reconfirmed by environmental scanning electron microscopy. Biochemically as well as physi ologically they differ from each other. Conclusion/Recommendation: This study leads to the isolation of efficient nit rate and phosphate removers from environmental origin. The phosphate removing e ffici ncy is much higher than the type strain under identical condition. These native microbes might be responsible for main taini g the phosphate and nitrate levels at the 30 sites investigated inspite of the rec ived pollution load. These isolates could be th e potential bioremedial agents for other sites with high nitrat e nd phosphate contamination level.


INTRODUCTION
Nitrate and Phosphate are recognized as the major nutrients which are required by living organisms for their physiological processes. They are most commonly added as fertilizer to enhance the quality of soil. However they have emerged as most abundant pollutants in the world due to their excess usage. The traditional agricultural practices like dry farming with marginal irrigation, flood plain farming and random application of fertilizers are considered as diffused sources of nitrate and phosphate in soil and aquifers. Besides this, the irregular rainfall during different seasons and the stream flow pattern causes seepage of these contaminants from soil to surface and ground water (Whitmore et al., 1992;Jorgensen, 1999;Giupponi et al., 1999;Agrawal, 1999;Krishnaswamy et al., 2009).
The cultivation patterns like terrace farming results in nitrate leaching into aquifers (Nakasone and Yamamoto, 2004;Kinoshita et al., 2003).
Increased levels of nitrate up to 400 ppm have been detected in groundwater (Filintas et al., 2008). Possible sources of nitrate pollution include manure, agricultural fertilizer, industrial effluent, domestic wastewater, septic systems, human waste lagoons, animal feedlots and native soil organic matter, as well as geologic sources (Jin et al., 2004) Other point sources of nitrate are municipal sewage canals, septic tanks, sewage dumping grounds (Wakida and Lerner, 2005;2006). The mining tailings, industrial effluent from nuclear reactors, radioactive waste processing units mainly those dealing with compounds like plutonium or thorium nitrate (Singleton et al., 2005). Nitrate contamination is a global problem and stands as second most dangerous pollutant after the pesticides. High concentration of nitrate in drinking water is a threat especially to infants, causing methemoglobinemia, also called "blue baby syndrome. The carcinogenic effect of nitrate is also reported. Concentration higher than 10ppm in drinking water may also cause stomach cancer in infants (Jin et al., 2004). EPA has demarcated the maximum contaminant level to be 10 ppm for NO 3 -N and 45 ppm for NO 3 concentration. A similar guideline of 50 ppm as NO 3 has been set by the WHO and the European Community (EC). Several conventional technologies adopted for nitrate removal are ion exchange resins, electro dialysis, reverse osmosis and distillation which substantially increase the cost of operation. Therefore the cost-effective alternative lies in the biological denitrification process (Pinar et al., 1997;Eckford and Fedorak, 2002).
The addition of phosphorus as phosphate fertilizers in soil in excessive amount causes serious environmental problems in the form of eutrophication which uses up large amounts of oxygen. The main sources of phosphate in aquatic environment is through household sewage water containing detergents and cleaning preparations, agricultural effluents containing fertilizers as well as industrial effluent from fertilizer, detergent and soap industries (Pradyot, 1997). Phosphate is generally present as polyphosphate and orthophosphate. The concentration of phosphate in water bodies vary from 0.005-10 ppm depending on the source of phosphate near the water body. On one hand digestive problems occur from extremely high levels of phosphate, on the other, phosphate levels greater than 1.0 may interfere with coagulation in water treatment plants. The EPA has fixed standard phosphate levels as 0.015 ppm for water supply, 0.025 ppm for aquatic life, 0.05 ppm for lakes and 0.02 ppm for mountain lakes (Kotoski, 1997). Microbial strategies are currently being used for the removal of excess phosphate load in waste water since it is an attractive alternative to chemical processing (Krishnaswamy et al., 2009).
The objective of the present study is to isolate efficient nitrate and phosphate reducing microbes from different environmental water bodies. Further characterization of these isolates would lead to the development of an array of novel organisms which can reduce nitrate and phosphate load in water bodies of various environmental sites leading to bioremediation.

MATERIALS AND METHODS
Sampling: Water samples were collected from different environmental sites to screen for nitrate and phosphate removing organisms. The sites were selected on the basis of the pollutants received by them. Main focus was on sites, which according to pollutants received were expected to have high load of nitrate and phosphate but showed low concentrations of these. Presumably these are the sites which host the nitrate and phosphate removers. The list of sites has been given below Table 1.
Environmental parameters: Different physical, chemical and biological parameters were assessed for the water samples from each site to understand the different concentration of pollutants in them. The physical and chemical parameters were assessed as per the protocol laid down by Central Pollution Control board of India. The biological parameter of the sites were assessed by studying the normal population of the sites. 10 microbes were selected which are commonly present in waste water (viz Escherichia coli, Enterobacter aerogenes, Shigella flexneri, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis, Salmonella sp. and Staphylococcus aureus.) and the water from the sites were serially diluted and spread on media specific for the microbes. The different media used were HiChrome E.coli coliform Selective agar base (HiMedia M-1294 for Escherichia coli, Enterobacter aerogenes, Shigella flexneri, Klebsiella pneumonia); HiChrome UTI agar base (HiMedia M-1353 for Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis); Salmonella differential agar base (HiMedia M-1078 for Salmonella sp) and HiChrome Aureus agar base (HiMedia M-1468 for Staphylococcus epidermidis and Listeria monocytogenes).

Cultivation medium and growth conditions:
Since one of the main objectives of our study is to isolate nitrate reducing microbes therefore the screening for microbes were done in high nitrate containing medium. The water samples of all the above mentioned sampling sites were serially diluted and plated on media containing 2000ppm of nitrate followed by overnight incubation at 37°C to isolate microbes which can survive in high nitrate concentration. Further selection was made on the basis of morphology. The cultures were re-streaked three or more times to obtain pure colonies. The isolates were also grown in Nitrate Broth (Himedia M439-500G) and maintained at 37°C in 150rpm shaking condition. The final selection of the pure isolates was on the basis of their nitrate removing efficiency from the liquid culture.

Morphological characterization:
The initial morphology of the isolates were determined by using light microscope (1000X magnification on a Zeiss Axiostar Plus microscope) following simple staining using 5% Crystal Violet. The Gram nature of the isolate was determined by differential staining as per standard procedure. Dimensions of the isolates were determined using Environmental scanning electron microscopy (FEI QUANTA 200 MARK 2 at 15 kV) as per the protocol.

Biochemical characterization:
The ability of the isolates to produce enzymes like DNase, oxidase, lipase, catalase and amylase was determined. The tests for the first five enzymes were done according to the protocol of Nandy et al. (2007) the amylase test was done on 1% starch agar plate and incubated at 37°C for overnight followed by flooding of the plate with iodine solution. The substrate utilization profile of the isolates were checked as per manufacturer's protocol using substrate utilization kits (HiMedia KB009).

Antibiotic assay:
The response of the isolates towards 18 different antibiotics (HiMedia) were checked according to the procedure reported by Nandy et al. (2007) Nitrate removal: The isolates were inoculated (2% inoculum) in Nitrate broth and incubated for 16 h at 37°C in a shaking incubator 150 rpm. The cell free supernatant was taken for estimation of nitrate removal after harvesting the culture at 8609×g for 10min. 200 µL of Salicyalic acid (5% Salicylic acid in H 2 SO 4 ) and 40 µL of cell free supernatant was added and vortexed. The tubes were incubated in dark for 10 min. The reaction was stopped by addition of 2 mL of 4N NaOH. Optical density of this solution was measured after 20 min at 420 nm. The O.D was then compared to the standard curve prepared with known concentrations of NaNO 3 (100-1000 ppm) to determine the concentration of Nitrate remaining in the medium (Cataldo et al., 1975).
Phosphate removal: Phosphate can be detected by spectrophotometric method by conversion of the phosphates to Molybdophosphoric acid Complex (MOP) by Ammonium molybdate, followed by reduction of the MOP Complex by Sn 2+ of SnCl 2 to give a blue coloured complex. For our study, at first a standard curve of phosphate was prepared by using standard solutions of phosphate from 0.05-0.5 ppm. 10 mL of cell free supernatant of bacterial sample (2% inoculum grown in Nitrate broth for 16hrs at 37°C) was diluted in 60 mL of distilled water in a 250 mL conical flask. 2 mL of Ammonium molybdate reagent was added followed by 4 drops of Stannous chloride. The solution was shaken well and volume was made upto 100 mL. The blue colour which developed indicated presence of phosphate which could be measured spectrophotometrically at 660 nm. The unknown concentration of phosphate was determined by comparing with the standard curve (Krishnaswamy et al., 2009).

Statistical analysis:
The objective of the study being isolation of efficient nitrate and phosphate removers, the ideal situation would be a single isolate performing both the functions. The relation between nitrate and phosphate removal was investigated by using the Correlation Co-efficient as a measure of the association between the two variables. The correlation co-efficient measures the strength of the linear relationship between the variables.

Environmental parameters:
The physical parameters assessed were pH, Suspended solids, Turbidity level of the water body, Temperature, Odour and Colour (  (Table 4).

Cultivation medium and growth conditions:
The serial dilution of water samples from the above mentioned 30 sites on medium with 2000 ppm of nitrate gave 130 different colonies. Further selection was made on the basis of morphology since most of the nitrate reducers according to literature are bacilli therefore bacilli in long or short chain or isolated bacilli were selected.  19 such strains were then checked for their efficiency of nitrate removal from liquid culture. 7 bacilli were selected on the basis of nitrate removing efficiency for further characterization.
Morphological characterization: Simple staining of the microbes showed different morphology of microbes. The Environmental Scanning Electron Micrographs of the isolates (Fig. 1) show that WBUNB004, WBUNB005 and WBUNB006 (from rhizosphere of water lily) are bacilli in chain whereas WBUNB008 (from paddy field), SM2 (from marine beach) and WBUNB009 (from raw sewage canal) are short isolated bacilli and WBUNB007 (from marine beach) is long bacilli.Gram staining showed that all the isolates were gram positive in nature.

Biochemical characterization:
The biochemical characterization of the strains is represented in Table 5. All isolates except WBUNB007 produced oxidase and protease.      (Table 6).
The antibiotic susceptibility profile of the isolates were performed by using different antibiotic discs (Himedia). This study shows that the strains were resistant to antibiotics like Ceftazidime, Ampicillin, Methicillin, Rifampicin, Trimethoprim, Polymixin-B and Cefotaxime and sensitive to antibiotics like Chloramphenicol, Norfloxacin, Roxithromycin, Doxycycline hydrochloride, Gentamycin, Ciprofloxacin, Cefadroxil and Teicoplanin (Table 7).
Nitrate removal: The nitrate removal from the medium is the primary step for the reduction of nitrate though after removal the bacteria may use the nitrate by assimilatory or dissimilatory pathway. The result is represented in the form of percentage of nitrate remaining in the medium after incubation with the isolate for 16 h at 37°C (Fig. 2).

Phosphate removal:
The phosphate removal capacities of the isolates were checked in enriched medium in comparison with a type strain, Acinetobacter baumanii (MTCC 1425) known for phosphate removal obtained from MTCC. The result is represented in the form of percentage of phosphate remaining in the medium after incubation with the isolate for 16hrs in 37°C. The result indicates that all the isolates show better phosphate removal than Acinetobacter baumanii under the given set of conditions (Fig. 3).

Statistical analysis:
The nitrate removal by the isolates were found to be within 77-88%, the average removal being 85.3%. The phosphate removal by the isolates were found to be within 43.8-82.9%, the average being 63.71% while that of the type strain under similar conditions showed 31.9% removal. The correlation study of nitrate and phosphate showed a negative moderate correlation of (-) 0.5584 which implies that an efficient nitrate remover is not necessarily an efficient phosphate remover.

DISCUSSION
In this study we report the isolation of 7 strains with potential for nitrate removal. They could be used for bioremediation of nitrate contaminated sites leading to environmental protection. Phosphate removers isolated during the study were found to be more efficient than the type strain (Acinetobacter baumanii) under identical conditions. Here we report 7 gram positive bacterial isolates which are highly efficient in phosphate removal. Since the mechanism of phosphate removal in bacteria leads to the intracellular accumulation of polyphosphate granules, these could be used as potential candidates for sequestration of phosphate from environmental sites.

CONCLUSION
The study is a successful attempt to isolate efficient nitrate and phosphate removing bacteria from various environmental sites for remediation of waste water by reducing the nitrate and phosphate load. In addition optimization of the waste water treatment parameters by these isolates in future could not only lead to environmental protection but also sequestration of essential plant growth nutrients from the waste which in turn could be re used.