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1 Available online at Life Science Archives (LSA) ISSN: Volume 2; Issue - 6; Year 2016; Page: DOI: /lsa Research Article PHOSPHATE SOLUBILIZATION AND ALKALINE PHOSPHATASE ACTIVITY OF Pseudomonas fluorescence ISOLATED FROM GROUNDNUT FIELD SOIL S. Anand* 1, A. C. Gomathi 2, R. Anantha Lakshmi 3 and K. Kaviyarasu 3 Department of Biochemistry, Sacred Heart College (Autonomous), Tirupattur , Vellore District, Tamil Nadu, India. Abstract The Alkaline phosphatase is a hydrolase enzyme obtained from Plant Growth Promoting Rhizobacteria (PGPR) which was isolated from soil. The PGPR solubilized the phosphate and produce phosphatase enzyme in soil and supports the plant growth. The Alkaline phosphatase enzyme producing bacteria was isolated by serial dilution method and identified based on the morphological characteristics and biochemical reactions. The isolates were tentatively identified as Pseudomonas fluorescens. Out of twenty two isolates, fourteen showed the phosphate solubilization by producing the halo zone by which it can be determined that they are have been shown to develop the solubilization of insoluble The solubilization effectiveness and solubilization capability for the fourteen colonies isolates were assessed after seven days of incubation beyond which no further increase in phosphate solubilization was seen and zone of inhibition (mm). The maximum phosphate solubilizing Pseudomonas fluorescens was centrifuged and the crude enzyme of alkaline phosphatase (ALP) was obtained. The ALP activity was optimized at different parameters (ph, Temperature, Incubation time, Carbon sources, and Nitrogen sources) and it was observed that the maximum ALP activity was recorded at ph 8, 37 C, 48 hrs, Dextrose as carbon source and Ammonium nitrate as nitrogen source. Article History Received : Revised : Accepted : Introduction Alkaline phosphatase (EC ) is a hydrolase enzyme and a ubiquitous enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins and alkaloids. Since soil sample rhizosphere represents a complex of living groups it is considered that soil alkaline phosphatase, that are responsible from extracellular and intracellular enzyme activities. It is produced by soil microorganisms and soil fauna and the activity of soil alkaline phosphatase hydrolysis of esters and anhydrous * Corresponding author: S. Anand E.mail: srianandsrinivasan@gmail.com Key words: Alkaline phosphatase, Pseudomonas fluorescens, Rhizobacteria, Halo zone, Pikovskaya s Medium. organic substance fertilisation in soil, the type of fertilisation and nutrient organization, soil microbiological activity, organic substance, soil ph, soil moisture and varieties of higher plant species (Tamas et al., 2002). Therefore, the activity of enzymes is generally connected to the concentration of soil Phosphorus (Bettina Eichler et al., 2004). Rhizobacteria that can be present in the soil or in nodules formed on the roots of legumes. In root nodules, they form an interdependent association with the legume, locating nutrients from the plant and producing nitrogen in a process known has a nitrogen fixation. The Extracellular Plant Growth Promoting Rhizobacteria (epgprs) may occur

2 S. Anand /Life Science Archives (LSA), Volume 2, Issue 6, Page 820 to 826, in the rhizoplane or in the spaces between the cells of root cortex while ipgprs finds usually inside the specialized nodular structures of root cells (Kukreja Girish et al., 2010). field soil (Arachis hypogaea L.) by using rhizobacteria (Pseudomonas fluorescens) and the alkaline phosphatase activity was optimized at different parameters (ph, Temperature, Incubation time, Carbon sources, and Nitrogen sources). Plant growth promoting rhizobacteria promote plant growth directly by any often due to their capability for nutrient supply (Nitrogen, 2. Materials and Methods Phosphorus, Potassium and Essential minerals). The Collection of the Groundnut Soil Sample The bacterial species such as Arthrobacter, The Groundnut (Arachis hypogaea L.) Agrobacterium, Azotobacter, Azospirillum field soil sample was collected from Agricultural Bacillus, Burkholderia, Caulobacter, land in Andiyur village, Uthangarai (Taluk), Chromobacterium, Erwinia, Flavobacterium, Kirshnagiri (District), Tamil Nadu, India. Micrococcous, Pseudomonas and Serratia are belongs to PGPR (Ahemad and Kibret, 2014; Isolation of Bacteria Sonam Sharma et al., 2011). The PGPR belongs The bacteria present in soil sample were to the Allorhizobium, Bradyrhizobium and isolated by Serial dilution technique (Spread Rhizobium which can symbiotically fix plate method). Volume of 0.5 g of soil sample atmospheric nitrogen in plants (Rajankar et al., was dissolved in 5 ml of sterilized distilled 2007; Arvind Kumar, 2016). The Pseudomonas water, mixed well and considered the diluted soil sp. is a plant growth promoting biofertilizer. sample as 5-1 to 5-8 (Each tube containing 4.5 ml Induced systemic resistance, biological control of water and 0.5 ml of sample). Then, last four of pathogens, it genus of Gram negative, tubes (5-5 to 5-8 ) and Nutrient agar plate were Aerobic Gamma proteobacteria, belonging to the used for taken for spread plate technique. A family Pseudomonadaceae and comprising 191 volume of 0.1 ml of sample was poured into validly describe species. The inoculation of plate and spread by using L-Rod evenly over the different strains had Pseudomonas fluorescens agar surface and then incubated at 37 C for 24 negative, neutral or positive effects on hrs. After incubation, the bacterial colonies were rhizosphere hydrolase activity (Dilfuza selected and streaked on Nutrient agar slants to Egamberdieva et al., 2011). It has multiple get the isolated pure colonies. Further flagella, and can be found in the soil and in purification was carried out to identify the water, and it s require aerobe, but certain strains colonies in the plate. are capable of using nitrate instead of oxygen as a last electron acceptor during cellular Screening of phosphatase Enzyme respiration. Optimal temperatures for growth of The PVK media was poured into a plate. Pseudomonas fluorescens are C. It is Each bacterial culture was streaked in the centre also a non saccharolytic bacterial species. The of a PVK plate and incubated at 37 C for 24 specific name Pseudomonas fluorescens refers to hours. Phosphate solubilisation was assessed up the microbe's secretion of a soluble fluorescent to 24 hours by measuring the zone of clearing pigment called pyoverdin, which is a type of the area of solubilisation of bacteria surrounding siderophore. The Pseudomonas genus have been the developed bacterial colony. applied to cereal seeds or directed to soils as a way of preventing the growth or founding of Plate Assay crop pathogens (Hanudin, 2016). The 100 ml of PVK broth culture was Microorganisms with phosphate solubilizing prepared and centrifuged in 3000 rpm for 10 likely increase the availability of soluble minutes. The supernatant was considered as phosphate and improve the plant growth by crude enzyme of phosphatase. The crude enzyme improving biological nitrogen fixation, number was poured into PVK plate and incubated at 37 of nodules, dry weight of nodules, yield C for 24 hrs and the presence of halo zone has components, grain yield, nutrient availability and been reported that the bacteria are capable uptake in crop (Sonam Sharma et al., 2011). The around the colonies upto 24 hrs by measuring objective of this present study was to isolated the zone of solubilisation. alkaline phosphatase enzyme from Groundnut

3 S. Anand /Life Science Archives (LSA), Volume 2, Issue 6, Page 820 to 826, Chemical Assay Alkaline phosphatase activity was measured spectrophotometrically by monitoring the release of p-nitrophenol from p-nitrophenyl phosphate (pnpp) at 400 nm. A typical reaction mixture contained 100 µl of enzyme diluted in 2 ml of 200 mm Tris buffer (ph 8.5), 100 µl of 5 mm CaCl 2, 100 µl of 500 µmol pnpp and the final volume was made upto 3 ml using distilled water. Blank was prepared by mixing 2 ml of 200 mm Tris buffer (ph 8.5) and 1 ml of distilled water. The reaction was performed at 37 C for 30 min and stopped by addition of 500 µl of 4 M sodium hydroxide. One unit of phosphatase is the amount which hydrolyses 1 µ mol of substrate per min. The standard curve obtained by absorbance of p-nitrophenol (0-500 µ mol) at 400 nm was used for quantification of enzyme activity. Optimization of Enzyme The activity of alkaline phosphatase enzyme was optimized under various parameters viz., ph, Temperature, Incubation time, Carbon source and Nitrogen source. ph The culture was prepared in various ph ranges (ph 4, ph 5, ph 6, ph 7, ph 8 and ph 9), and incubated at 37 C for 24 hrs. After incubation, this was centrifuged at 10,000 rpm for 10 mins. A volume of 100 µl of supernatant was taken and 2 ml of Tris buffer, 100 µl of PNP, 100 µl of calcium chloride and 700 µl distilled water were added and incubated at 37 C for 30 min and stopped by addition of 500 µl of 4 M sodium hydroxide. The optical density was read at 400 nm using UV-VIS spectrophotometer. Temperature The culture was prepared and incubated at various temperature ranges (27 C, 32 C, 37 C, 42 C, 47 C and 52 C) and incubated at appropriate temperature for 24 hrs. After incubation, this was centrifuged at 10,000 rpm for 10 mins. A volume of 100 µl of supernatant was taken and 2 ml of Tris buffer, 100 µl of PNP, 100 µl of calcium chloride and 700 µl distilled water were added and incubated for 30 min and stopped by addition of 500 µl of 4 M Sodium hydroxide. The optical density was read at 400 nm using UV-VIS spectrophotometer. Incubation Time The culture was prepared in various Incubation time ranges in hours (0 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs and 72 hrs) and incubated at 37 C for appropriate hours. After incubation, this was centrifuged at 10,000 rpm for 10 minutes. A volume of 100 µl of supernatant was taken and 2 ml of Tris buffer, 100 µl of PNP, 100 µl of calcium chloride and 700 µl distilled water were added and incubated at 37 C for 30min and stopped by addition of 500 µl of 4 M Sodium hydroxide. The optical density was read at 400 nm using UV-VIS spectrophotometer. Carbon Source In PVK medium, 0.2 g of Dextrose, Maltose, Starch, Lactose and Sucrose instead of Ammonium nitrate and Peptone was added. Then, the media was sterilized and the culture was added. The culture was incubated at 37 C for 24 hours. After incubation, the culture was centrifuged at 10,000 rpm for 10 minutes. A volume of 100 µl of supernatant was taken and 2 ml of Tris buffer, 100 µl of PNP, 100 µl of calcium chloride and 700 µl distilled water were added and incubated for 24 hrs and stopped by addition of 500 µl of 4 M Sodium hydroxide. The optical density was read at 400 nm using UV-VIS spectrophotometer. Nitrogen Source In the PVK medium, 0.01 g of Casein, Peptone, Ammonium Chloride, Ammonium nitrate and Urea instead of Ammonium sulphate and yeast extract are added. Then, the media was sterilized; culture was added and incubated at 37 C for 24 hrs. After incubation, the culture was centrifuged at 10,000 rpm for 10 mins. A volume of 100 µl of supernatant was taken and 2 ml of Trish buffer, 100 µl of PNP, 100 µl of calcium chloride and 700 µl distilled water were added and incubated for 24 hrs and stopped by addition of 500 µl of 4 M Sodium hydroxide. The optical density was read at 400 nm using UV-VIS spectrophotometer. 3. Results and Discussion The collected sample has the possibility of occurrence of phosphate solubilizing and alkaline phosphatase producing bacteria from agriculture land. The isolated bacteria were identified as species Pseudomonas sp. and

4 S. Anand /Life Science Archives (LSA), Volume 2, Issue 6, Page 820 to 826, Bacillus sp. The bacterial isolates were identified based on the morphological, cultural characteristics and colour. For initial growth of bacteria, PVK medium was used fixed with tricalcium phosphate as phosphate source, with analysis of agitation and aeration for 5 day allowed bacterial solubilization of P with in ph. This was followed by the dilution plating in order to isolate the single colonies. Out of fourteen isolates eleven showed halo zone around the colonies around the colony by which it can be concluded that they are solubilize phosphate and alkaline phosphatase enzyme producing capability when isolated bacteria inoculated on Pikovskaya s (PVK) agar medium (Kukreja Girish et al., 2010). Table - 1: Phosphate solubilization of Bacterial culture with halo zone (mm) of Groundnut field soil Soil Sample Ground nut soil sample Bacterial culture ( BS) Bacterial Culture Type Bacterial Growth (mm) Zone of Inhibition (mm) Solubilized Phosphate (mm) BS-2 Bacillus sp BS-3 Pseudomonas sp Bacillus sp BS-4 Bacillus sp BS-5 Pseudomonas sp Pseudomonas sp Bacillus sp BS-6 Pseudomonas sp BS-7 Bacillus sp The solubilization efficiency (E) and solubilization capacity of the one isolate was calculated after seven days of incubation outside which no further increase in phosphate solubilization was given in Table - 1. The given following data, the Bacterial sample formed a high level of halo zone around the colony. The range of solubilized phosphate is 7.0 mm (Amit Sagervanshi et al., 2012). The optimization of alkaline phosphatase enzyme activity was studied and the results were showed in Figure 1 to 5. Various parameters viz., ph, Temperature, Incubation time, Carbon source and Nitrogen source was studied in the present research. This increases the probability of a successful impact and so the rate increases. There is a certain temperature at which an enzyme catalytic activity, its greatest value or optimum temperature of the ALP enzyme (37 C) in the particular nm was seen (Figure - 1). There is a certain ph at which an enzymes catalytic activity, its greatest value or optimum ph range of the ALP enzyme (8.0) in the particular nm was seen in Figure - 2. It has been suggested that microorganisms which tend to decrease the ph of the medium during growth are efficient P- solubilization different to the decreasing ph for individual cultures upto 48 hrs and then obtaining reliability, the soluble phosphate concentrations continue to increase after 48 hrs. This clearly suggests that the ph drop is not the only factor for P-solubilization. An initial increase in phosphate concentration tailed by a slowly decrease in culture filtrate as observed by us has also been well documented by other manual workers. The high phosphatase activity of species, (0.82 U ml -1 ) and specific activity at 48 hrs would be responsible for its higher P-solubilizing possible in comparison to other cultures. The activities decreased slightly at 72 hrs interval, which might be due to the complete disappearance of species from the medium after 72 hrs of incubation. There is a certain carbon source which an enzymes catalytic activity and increased ALP activity its greatest value or optimum value of the carbon source (Dextrose 0.822) in the particular nm was seen (Figure - 4) in UV spectrophotometer. It also contain the nitrogen source and to increased the catalytic and enzyme activity, its greatest value or optimum value of the nitrogen source increased the catalytic and enzyme activity, its greatest value or optimum value of the nitrogen source (Ammonium nitrate and Peptone -

5 S. Anand /Life Science Archives (LSA), Volume 2, Issue 6, Page 820 to 826, ) in the particular nm was seen in UV spectra photometer (Figure - 5). ALP activities were recorded for Pseudomonas fluorescens, cultures than the alkaline conditions are more favourable for the solubilization of phosphates. ALP activity could be recorded by any of the cultures in broth enclosing higher concentration of available phosphate KH 2 PO 4 (5 g) as the only phosphorus source, which again proved the need of an insoluble phosphate source for the emission of phosphatases (Kumar et al., 2016) The above findings on phosphatase enzyme induction are in accordance with the P-solubilizing potential of the three cultures, proving (Test sample) as the best P-solubilizing possible of species (Anil Kapri and Lakshmi Tewari, 2010). Figure - 1: Optimization of alkaline phosphatase at different Temperature Figure - 2: Optimization of alkaline phosphatase at different ph ranges

6 S. Anand /Life Science Archives (LSA), Volume 2, Issue 6, Page 820 to 826, Figure - 3: Optimization of alkaline phosphatase at different Incubation time Figure - 4: Optimization of alkaline phosphatase at different Carbon sources (mg) Figure-5: Optimize the alkaline phosphatase enzyme in Nitrogen sources (mg) 4. Conclusion For this study I conclude that, the phosphate solubilizing potential of Pseudomonas fluorescens, isolated from groundnut field soil were capable of utilizing phosphate (30 µg), producing the halo zone in PVK Medium. The Pseudomonas fluorescens further confirmed by their high ALP enzyme activities and also, these isolates brought about significant increase in the growth parameters of corn production under trials, suggesting their applicability for crop improvement. The ALP enzyme act on insoluble phosphates to form soluble phosphates thus provides phosphorous for plant growth and may provide high

7 S. Anand /Life Science Archives (LSA), Volume 2, Issue 6, Page 820 to 826, yield of oats, tea, banana, mustard, maize, rice, sorghum, barley, chickpea, soybean, groundnut, sugar beet, cabbage, corn, tomato and many number of human intestinal applications. The future plan of this study is then purification of alkaline phosphatase enzyme and its industrial application. 5. References 1) Ahemad. M and M. Kibret. (2014). Mechanisms and Applications of Plant Growth Promoting Rhizobacteria: Current perspective. Journal of King Saud University - Science, 26: ) Amit Sagervanshi, Priyanka Kumari, Anju Nagee and Ashwani Kumar. (2012). Media optimization for inorganic phosphate solubilizing bacteria isolated from Anand Agriculture Soil, Ashok and Rita Patel institute of integrated study and Research in Biotechnology and Allied Sciences, Jaipur. 3) Anil Kapri and Lakshmi Tewari. (2010). Department of Microbiology, G.B. Pant University of Agriculture & Technology, Pantnagar, Phosphate Solubilization potential and phosphatase activity of Rhizospheric Trichoderma spp. Brazilian Journal of Microbiology, 5 (6): ) Arvind Kumar. (2016). Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, India. Phosphate solubilizing bacteria in agriculture biotechnology: diversity, mechanism and their role in plant growth and crop yield, International Journal of Advancer Research, 8 (7): ) Bettina Eichler, Marria Caus, Ewald Schnug and Detloff Koppen. (2004). Soil acid and alkaline phosphatase activities in regulation to crop species and fungal treatment. Landbauforschung Völkenrode, 54: ) Dilfuza Egamberdieva, Giancarlo Renella, Stephan and Rafiq Islam Soil Enzymology Book, Enzyme activities in the rhizosphere of plants, ) Hanudin, Kurniawan Budiarto and Budi Marwoto. (2016). Pseudomonas fluorescens as plant growth promoting rhizobacteria and biological control agents for white rust disease in chrysanthemum, Indonesian Ornamental Crops Research Institute (IOCRI), West Java, Indonesia, ) Kukreja Girish, P, S. Bhute Shrikant, A. Mangate Sunil and N. Dhawale Manish. (2010). Exploring the Potential of Pseudomonas Species as phosphate solubilizer, plant growth promoter, biocontrol agent and pesticide degrader. Asian Journal of Experimental Biological Sciences, 3 (5): ) Kumar Anand, Baby Kumari and M. A. Mallick. (2016). Phosphate solubilizing microbes: an effective and alternative approach as biofertilizers, International Journal of Pharmacy and Pharmaceutical Science, 6 (5): ) Ponmurugan, P and C. Gopi. (2006). Distribution Pattern and Screening of Phosphate solubilizing Bacteria Isolated from Different food and forage Crops. 11) Rajankar, P. N., D. H. Tambekar and S. R. Wate. (. 2007). Study of phosphate solubilization efficiencies of fungi and bacteria isolated from saline belt of Pruna river basin. Research Journal of Agriculture and Biological Science, 3 (6): ) Sonam Sharma, Vijay Kumar and Ram Babu and Tripathi. (2011). Isolation of phosphate solubilizing microorganism (PSMs) from soil. Journal of Microbiology and Biotechnology Research, 1 (2): ) Tamas, H., J. Huttova, I. Mistrk and G. Kogan. (2002). Effect of carboxy methyl chitinglucan on the activity of some hydrolytic enzymes in maize plants. Chemical Paper Slovak Academy of Science Journal, 56 (5):