Electronic Journal of Biosciences http://ejbios.co.in Volume 01(1), 2013 pp.

01-05

BACTERIA SHOWING PHOSPHATE SOLUBILIZING EFFICIENCY IN RIVER SEDIMENT

Paul, Dipak and Sankar Narayan Sinha Environmental Microbiology Research Laboratory, Department of Botany, University of Kalyani, Kalyani-741235, West Bengal, India Abstract
The occurrence and distribution of phosphate solubilizing bacteria (PSB) capable of dissolving insoluble phosphates were investigated in this study in the sediments of river Ganga near the effluent discharge point of Kesoram Rayon Factory, Tribeni, West Bengal, India. A total of 20 bacterial isolates were screened. All isolates were tested for phosphate solubilizing activity on Pikovskayas agar. Five different isolates exhibiting maximum formation of halos (zone of solubilization) around the bacterial colonies were selected for quantitative estimations of phosphate solubilization. Quantitative estimations for phosphate solubilization were analyzed for up to 5 days at an interval of 24h. Maximum solubilization of 144.36 g P ml-1 was observed after 5 days of incubation in isolate RPSB6, while isolate RPSB8 showed minimum phosphate solubilization (121.76 g P ml-1). The increase in solubilization coincided with the drop in pH. Many of these species showed wide range of tolerance to temperature. All isolates were tentatively identified as five different genera such as, Bacillus, Pseudomonas, Flavobacterium, Enterobacter and Aeromonas by morphological, physiological and biochemical tests. The presence of phosphate solubilizing bacteria and their ability to solubilize phosphate were indicative of the important role played by bacteria in the biogeochemical cycle of phosphorus and the plant growth in riverine ecosystems. Keywords: Phosphate solubilizing bacteria, River Ganga, Bacillus, Pseudomonas, Flavobacterium, Enterobacter, Aeromonas.

INTRODUCTION Phosphorus is a limiting nutrient though it is not a major component in fresh water riverine ecosystem. Major forms of phosphorus are either bound to or strongly adsorbed to silt and clay. Usually phosphorus occurs as phosphates in both inorganic and organic compounds [1,2,3]. Moreover, the uptake of phosphorus is generally dominated by bacteria [4]. Bacteria and other microbes assimilate inorganic phosphate and mineralize organic phosphorus compounds. The activities of microorganisms are involved in the solubilization and mobilization of phosphates. The preferred and apparently universally used substrate is orthophosphate. Phosphorus is solubilized mainly enzymatically or by acid hydrolysis. Though a large number of PSB like Pseudomonas, Serratia, Bacillus, Flavobacterium and Corynebacterium have been reported from various ecosystems [5,6], highest PSB population was found in fertile soils of forests, organic farming and rhizosphere as well as mangrove and shorelines samples [7,8]. PSB from sea sediments were reported to be capable of accelerating the dissolution of apatite phosphate within the phosphorus cycle and interacting with the carbon cycle [9]. Though many works have been done with PSB in marine ecosystem, studies of PSB in freshwater bodies particularly riverine ecosystem is scanty or lacking. The present study aims at studying the occurrence and determination of phosphate solubilizing activity of PSB in the river sediment of Ganga. MATERIALS AND METHODS Sampling site and Collection of Sediment Samples Sediment samples were collected from the river Ganga near the effluent discharge point of Kesoram Rayon Factory Ltd, Tribeni, West Bengal (230'56"N and 8824'54"E) with the help of Ekman dredge (Fig 1). The samples were aseptically transmitted to sterilized glass bottle and transported to the laboratory for further analysis.

Electronic Journal of Biosciences

Volume 01(1), 2013 pp. 01-05

Fig 1. Map of the river Ganga showing sampling site Isolation of Phosphate Solubilizing Bacteria Solubilization of precipitated tricalcium phosphate [Ca3(PO4)2] in unbuffered solid agar medium plates has been used widely as the initial criterion for the isolation of phosphate solubilizing microorganisms. The sediment samples were serially diluted, spread plated on Pikovskayas agar(gl-1) [glucose 10g, Ca3(PO4)2 5g, (NH4)2SO4 0.5g, KCl 0.2g, MgSO4 0.1g, MnSO4 traces, FeSO4 traces, yeast extract 0.5g, agar agar 20g, pH 7.0] and incubated at 30020C for 1-2 weeks. A total of 20 bacterial isolates with phosphate-solubilization halo zone on plates was purified with repeated culturing and maintained in 20% glycerol at -800C. All the subsequent experiments were conducted after raising fresh culture. Potential isolates which are showing more halo zones of phosphate solubilization (<9mm) were assessed for further characterization. Characterization and Tentative Identification of Isolated Strains Preliminary biochemical characterization was carried out as per standard methodology [10]. Growth versus incubation period was checked for maximum of 120 h and temperature range for growth was checked on nutrient broth from 10, 25, 37, 45 and 55C by inoculating 1 ml of an exponential culture (108 CFU ml-1) in 100 ml of nutrient broth and estimating the cell population at 12 h intervals. The identity of the isolate was revealed on the basis of carbon source utilization. Estimation of Phosphate Solubilization After qualitative estimation of the phosphate solubilizing activity which was carried out on Pikovskayas agar, quantitative estimation of phosphate solubilization was carried out as per standard methodology [11], by inoculating 1 ml of bacterial suspension (3107 cells ml1) in 50 ml of National Botanical Research Institute Phosphate broth (NBRIP) in Erlenmeyer flasks (150ml) that contained the following ingredients (gl-1): glucose 10.0g, Ca3(PO4)2 10.0g, MgCl2.6H2O 5.0g, MgSO4.7H2O 0.25g, KCl 0.2g, (NH4)2SO4 0.1g and incubating the flasks for 10 days at 30C. At the end of the incubation period, the cell suspension was centrifuged at 10,000 rpm for 10 minutes, and the phosphate content in the supernatant was spectrophotometrically determined by the ascorbic acid method [12]. All the studies were repeated on three times to confirm the results. Sterile water inoculated media was treated as blank. After incubation for 5 days, pH of the medium was recorded with a pH meter equipped with glass electrode.

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RESULTS AND DISCUSSION A total of 20 bacterial isolates were isolated. All isolates were screened for phosphate solubilizing activity on Pikovskayas agar. Five different isolates exhibiting maximum formation of halos (zone of solubilization) 10-13 mm zone of phosphate solubilization after 5 days incubation around the bacterial colonies were selected for quantitative estimations of phosphate solubilization (Table 2). Morphological and biochemical characterization performed were presented in Table 1. Based on these characters, the isolates were assigned to different genera like Bacillus, Pseudomonas, Flavobacterium, Enterobacter and Aeromonas though molecular analysis based on 16S rRNA gene sequencing are necessary for final characterization. The details of the pH and amounts of soluble phosphorus in the medium due to 120 h of incubation were presented in Table 2. The solubilization of Ca3(PO4)2 in the liquid medium by different strains was accompanied by a significant drop in pH (4.17 to 4.83) from an initial pH of 7.00.2 after 120 h were recorded. The soluble-P concentration in the medium ranged between 113.72 to 144.36 g P ml-1 with variations among different isolates. In the blank treatment without addition of any bacterial isolate, no soluble-P was detected or any drop in pH was observed. The maximum P-solubilization was recorded by Pseudomonas RPSB6, followed by Aeromonas RPSB9, Bacillus RPSB5, Enterobacter RPSB8 and Flavobacterium RPSB7. Maximum drop in pH was associated with higher levels of P-solubilization, Pseudomonas RPSB6 where pH was decreased to 4.17 from initial pH 7.0. Comparatively higher amounts of soluble-P (144.36 g P ml-1) were detected in the medium. The maximum P-solubilization was detected after 120 h of incubation. The decrease in pH clearly indicates the production of organic acid and phosphatase, which is considered to be responsible for phosphate solubilization activity (Table.2). Generally phosphates precipitate due to abundance of cation in the sediment of aquatic bodies making phosphorus mostly unavailable to aquatic flora. Phosphate solubilizing bacteria as a potential supplier of soluble form of phosphorus have a great advantage for aquatic plants. Usually anoxic conditions of the sediments beneath the aerobic zone would tend to favour desolation of non soluble phosphate through sulphide production. Phosphate solubilizing bacteria were recorded from all the samples near rayon factory effluent discharge area, among which five isolates of different genera showed positive phosphate solubilizing activity. Production of halo zones on solid agar and release of phosphate in the medium would be ascribed to the release of organic acids by the microbes [6]. The ability of phosphate solubilizing bacteria to quantitative dissolve phosphate and the pH of the liquid medium with various phosphorus sources after 5 days incubation were measured using spectrophotometer. It showed that all of the tested isolates were able to dissolve the inorganic phosphate of Ca3(PO4)2 within the media of Pikovskaya. The highest concentrations of dissolved phosphate were found in Pseudomonas RPSB6 with the amount of 144.36 g P ml-1. The lowest concentration was found in Flavobacterium RPSB7 with an amount of 113.72 g P ml-1. CONCLUSIONS From this study it is concluded that the isolated 5 bacterial genera were found to be efficient strains for inducing phosphate solubilizing activity. PSB are widely distributed in different niches and sediment was found to be the most efficient carrier. Therefore, these isolates might serve continuously to fertilize a niche by solubilizing insoluble phosphorus compounds especially in environments where a low concentration of phosphorus causes various limitations. The role of PSB to phosphorus transfer can be analyzed by means of these bacteria in the sediments. It is expected that such studies will potentially increase our awareness of the roles of microorganisms in aquatic ecosystems and the process of entrophication. ACKNOWLEDGEMENT The authors thank to University of Kalyani for providing necessary facilities for doing this research. Authors acknowledge the financial support received under the grant from DST PURSE, New Delhi, India for this study.

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Table 1. Biochemical characteristics of phosphate solubilizing bacteria isolated from river sediment Parameters Bacillus sp Pseudomonas Flavobacterium Enterobacter Aeromonas RPSB5 sp sp sp sp RPSB6 RPSB7 RPSB8 RPSB9 Cell shape Rod Rod Rod Rod Rod Gram reaction + Motility + + + + + Growth at 5% + + + NaCl Catalase + + + + + Oxidase + + + + IMViC test Indole + production Methyl red + Voges+ + + Proskauer Citrate + + + + Urease + H2S + + production + + + NO3reduction Gelatine + + + + liquefaction Starch + + hydrolysis HughF O/F O/F F Leiffson (O/F) reaction Utilization of carbon source Glucose + + + + + Fructose + + + Sucrose + + + + Raffinose + Cellobiose + Xylose + Mannitol + + Sorbitol + + Dulcitol + + + indicates presence or positive; - indicates absence or negative; O = Oxidation; F= Fermentation

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Table 2. Phosphate solubilization with respect to zone of solubilization, in vitro activity and pH decline Isolates Phosphate solubilization (g P ml-1) pH of the Incubation Phosphate Calcium medium period (h) solubilization phosphate zone (mm) Ca3(PO4)2 solubilization RPSB5 11 123.18 4.47 120 RPSR6 13 144.36 4.17 120 RPSB7 10 113.72 4.83 120 RPSB8 11 121.76 4.50 120 RPSB9 12 129.92 4.44 120

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