Evaluate the Shelf Life of Irradiated Carrier Based Pseudomonas Biofertilizer Stored At Different Temperatures at Different Intervals

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1 Available online at Thirumal et al Int. J. Pure App. Biosci. 5 (4): (2017) ISSN: DOI: ISSN: Int. J. Pure App. Biosci. 5 (4): (2017) Research Article Evaluate the Shelf Life of Irradiated Carrier Based Pseudomonas Biofertilizer Stored At Different Temperatures at Different Intervals G. Thirumal 1*, R. Subhash Reddy 2, S. Triveni 3 and M.H.V. Bhave 4 1,2,3 Department of Agricultural Microbiology and Bioenergy 4 Department of Statistics & Mathematics College of Agriculture, Prof Jaya Shankar Telangana State Agricultural University, Rajendranagar, Hyderabad , Telangana, India *Corresponding Author thirumalgummula18@gmail.com Received: Revised: Accepted: ABSTRACT In this study focus on shelf life of different carrier based bioinoculants sterilized by irradiation, for this Pseudomonas cultures collected from different resource labs, then cultures were studied morphologically and biochemically for purity confirmation. The selected Pseudomonas (RGP-1) isolate was multiplied in large quantities in appropriate culture broth by incubating at 28 ± 2 C in an incubator shaker till they attained log phase with a cell load of l l0 9 cfu ml -1. For biofertilizer production Vermicompost, Vermiculate, Lignite, and Sodium Alginate carriers were selected and carriers sterilized by gamma irradiation at a dose rate of 5.0 kgy for 1h then carriers were used for bioinoculant preparation and stored at different temperatures i.e. 4ºC and 28± 2 C. In first month log 10 values at 4ºC 9.65 (Vermicompost), 9.76 (Sodium Alginate), 9.64 ( Lignite), 9.67 (Vermiculate) and 28± 2 C 9.85 (Vermicompost), 9.92 (Sodium Alginate), 9.80 ( Lignite), 9.90 (Vermiculate) showed. Even though bioinoculant populations were higher at first month gradually deceasing upto 8 th month, at the end ( 8 th month) of the experiment log 10 values were at 4ºC 8.40 (Vermicompost), 9.05 (Sodium Alginate), 8.00 ( Lignite), 8.70 (Vermiculate) showed which is highest value then minimum cfu g -1 (7.6 log 10 ) viable count of powdered form of carrier based biofertilizer and 28± 2 C was different viable count obtained 7.80 (Lignite) showed upto 6 th month, 7.60 (Vermicompost), 8.16 (Sodium Alginate), 8.00 (Vermiculate) showed upto 7 th month only. Key words: Pseudomonas, Irradiated sterilization, Different temperatures, Sodium alginate. INTRODUCTION Organic farming has emerged as an important priority area globally in view of the growing demand for safe and healthy food and long term sustainability and concerns on environmental pollution associated with indiscriminate use of agrochemicals. Cite this article: Thirumal, G., Reddy, R.S., Triveni, S. and Bhave, M.H.V., Evaluate the shelf life of irradiated carrier based pseudomonas biofertilizer stored at different temperatures at different intervals, Int. J. Pure App. Biosci. 5(4): (2017). doi: Copyright August, 2017; IJPAB 2158

2 Though the use of chemical inputs in cultures accelerate the decomposition of agriculture is inevitable to meet the growing organics residues and agricultural by products demand for food in world, there are through various processes and gives healthy opportunities in selected crops and niche areas harvest of crops 2. where organic production can be encouraged to tape the domestic export market. Biofertilizers MATERIAL AND METHODS are being essential component of The present study was carried out in the organic farming are the preparations Department of Agricultural Microbiology & containing live or latent cells of efficient Bioenergy, College of Agriculture, strains of nitrogen fixing, phosphate Rajendranagar, PJTSAU, and Hyderabad. Pure solubilizing or cellulolytic micro-organisms cultures of Plant Growth Promoting used for application to seed, soil or Pseudomonas isolates collected from different composting areas with the objective of laboratories. Screening and characterization of increasing number of such microorganisms isolates were done with multiple beneficial and accelerate those microbial processes properties then the efficient PGPR isolate was which augment the availability of nutrients selected for preparation of carrier based that can be easily assimilated by plants. biofertilizers. Selection of carrier material is very important COLLECTION OF Pseudomonas while preparing biofertilizers. Although, there ISOLATES FROM DIFFERENT are no clear cut criteria for the selection of SOURCES carrier materials but some general Promising bacterial isolates are collected from characteristics should be present in the different laboratories and these isolates were material which is going to be used as a carrier tested for their purity and preservation in Dept. for biofertilizer such as it should be cost of Agricultural Microbiology & Bioenergy, effective, contain non-toxic compounds and College of Agriculture, PJTSAU high organic content, easy to process, more Rajendranagar, Hyderabad. than 50% water holding capacity, high IDENTIFICATION OF BACTERIAL buffering capacity, sticky in nature and ISOLATES PURITY CHECKING. available in bulk quantity. A variety of Morphological and Biochemical materials can be used as carrier but the need of Characterization hour is to find out the most suitable carrier The isolated bacteria were studied for their which fulfils all the above stated properties. 1 morphological like gram reaction, Carrier based biofertilizers proved to pigmentation, cultural characteristics and be the best over the agrochemicals and are biochemical characteristics like Indole showing the tremendous effect on the global production, methyl red, voges-praskaure s test, agricultural productivity, from the last two citrate utilization test, oxidase, catalase and decades. Rectifying the disadvantages of the sugar fermentation tests. carrier based bio fertilizer liquids biofertilizer Screening for plant growth promoting are developed which would be the alternative properties for the cost effective sustainable agriculture. Screening will be carried out for different Biofertilizer production in India during the plant growth promoting properties such as period , is MT, in Andhra mineral solublization like Phosphorus Pradesh is MT. Solubilisation 3, Zinc Solubilization 4, The knowledge of applied microbial Potassium releasing 5, Plant growth promoting inoculums is long history which passes from substances such as IAA production 6, generation to generation of farmers. It started biocontrol activity such as HCN production 7 with culture of small scale compost production and Siderophore production 8 and antifungal that has evidently proved the ability of activity with soil born plant pathogens all ten biofertilizer. This is recognize when the isolates were checked for their purity and then Copyright August, 2017; IJPAB 2159

3 studied for the colony morphology and Directions pigmentation. The cell shape and gram Suspend grams of dehydrated medium reaction were also recorded as per the standard in 1000 ml distilled water containing 15 ml of procedures given by 9 glycerol. Heat to boiling to dissolve the Gram s staining. medium completely. Mix well. Sterilize by A loopful of inoculum from young culture was autoclaving at 15 lbs pressure (121 C) for 15 taken, mixed with water, and placed in the minutes. Aseptically pour into sterile Petri center of the slide. The suspension was spread plates. out on slide using the tip of inoculation needle Collection of Carrier Materials to make a thin suspension. The smear was Sodium alginate collected from Department of dried in air and fixed through mild heating by Agricultural Microbiology and Bioenergy passing the slide 3 to 4 times over the flame. College of Agriculture Rajendranagar, The smear was then flooded with Crystal Hyderabad. Vermiculate collected from violet solution for 1 min and washed gently Navaratna Crop Science Pvt Ltd, Cherlapally, Hyderabad. Lignite collected from with flow of tap water. Then the slide was Department of Agricultural Microbiology and flooded with Iodine solution. After incubation Bioenergy College of Agriculture at room temperature for 1 min, Iodine solution Rajendranagar, Hyderabad. Vermicompost was drained out followed by washing with collected from NIRD Rajendranagar, 95% decolorizer. After that, it was washed Hyderabad. with water within 15 to 30 sec and blot Physico-chemical properties of carriers carefully. The smear was incubated with For the preparation of bioformulation, the Saffranin solution for 1 min. The slide was collected different carriers such as lignite, washed gently in flow of tap water and dried vermicompost, sodium alginate and in air. The slide was examined under vermiculite were tested for their moisture microscope at 100X power with oil immersion content and ph at initial and end of the and data was recorded. experiment. Cultural Characterization Irradiation Sterilization Morphological characteristics of the colony of Lignite, vermicompost, vermiculate, and each isolate were examined on specialized sodium alginate was sterilized by gamma medium. Cultural characterization of isolates irradiation at a dose rate of 5.0 kgy for 1h. observed by different characteristics of (Gamma irradiation was carried out at the colonies such as shape, size, elevation, surface, Irradiation unit PJTSAU, Hyderabad). margin, colour, odour, pigmentation etc were Preparation of Biofertilizers recorded. Suspend 12.8 grams in 1000 ml distilled water. Growing of culture Sterilize by autoclaving at 15 lbs pressure King's medium B Base (121 C) for 15 minutes. Mix well and dispense Kings Medium B Base is recommended for into sterile test tubes. The selected isolate was multiplied in large quantities in culture broth non-selective isolation, cultivation and by incubating at 28±2 C in an incubator pigment production of Pseudomonas species shaker till they attained log phase with a cell Composition load of l l0 9 cfu ml -1 and were used for Ingredients Gms / inoculant preparation. The individual carrier Litre materials were powdered and the ph was Proteose peptone brought to neutral by adding CaCO 3 then Dipotassium hydrogen phosphate sterilized by gamma irradiation at a dose rate Magnesium sulphate. Heptahydrate of 5.0 kgy for 1h after then mixed with the Agar log phase culture (l l0 9 cfu ml -1 ) of the selected Final ph (at 25 C) 7.2±0.2 plant growth promoting bacterial isolate viz., Copyright August, 2017; IJPAB 2160

4 Pseudomonas (RGP-1) in separate quantities Determination of viable bacterial population in of sterile carrier in shallow trays. The optimum the carrier based inoculants by serial dilution moisture content was adjusted to (30-40%) and plating technique. Influence of storage prior to preparation, followed by curing in temperature on the survival of the inoculants shallow trays for 24 hours in aseptic rooms as consortium in different carrier materials and then packed in high density opaque.the carrier based microbial inoculants polythene bag (12g) at the rate of 100g bag -1 prepared with different carrier material was and sealed. Individual inoculant was prepared kept in different temperature levels viz., Room by mixing with lignite (1:3v/w), vermicompost temperature (28±2ºC) and Refrigerator (4 C). (1:2v/w), vermiculite (1:2v/w) volumes of The surviving populations of PGPB at each culture broth with sterile carrier different temperatures were determined and materials. The populations of individual Plant population was enumerated by dilution plate Growth Promoting Rhizobacteria in the technique at different intervals i.e monthly inoculant carriers were assessed at monthly upto 8 months. intervals upto 8 months at different storage temperature at 4ºC 28 ± 2 º C. RESULTS AND DISCUSSION b. Preparation of Alginate based inoculant First month results revealed that log 10 values Pseudomonas (RGP-1) was grown in were 9.65, 9.85 (vermicompost), 9.76, 9.92 ( respective medium to get a population of sodium alginate ), 9.64, 9.80, (lignite), 9.67, lxl0 9 cfu ml -1 Sodium alginate beaded inoculant 9.90 (vermiculite) showed at 4 0 C, 28 ± 2 0 C was prepared as per the methods described storage temperature respectively. When by 10. Two gram of sodium alginate was added compared among carrier materials sodium to 100 ml of culture broth of PGPR and mixed alginate having highest population and lignite for 30 minutes in a magnetic stirrer. The having least population at both temperatures. mixture was added drop wise through a 10 ml Eight month results revealed that syringe into 100 ml sterile 0.1N CaCl 2 to log 10 values were 8.40, 6.80 (vermicompost), obtain uniform Alginate beads. One gram of 9.05, 7.35 (sodium alginate ), 8.00, 6.50, material contained 16 to 17 beads, each bead (lignite), 8.70, 7.00 (vermiculite) showed at approximately weighing 60mg. The beads 4 0 C, 28 ± 2 0 C storage temperature were washed twice in sterile distilled water respectively. and incubated for seven days in a From 1 st month to 8 th month psychrotherm (model environ shaker) Pseudomonas population gradually decreased incubator at 28±2 C to allow PGPR to at both temperatures (table no.1. and 2. ) As multiply inside the beads. The beads were per specification of biofertilizers, carrier again washed in sterile distilled water and air should be minimum cfu g -1 (log ) dried in Laminar air flow chamber under viable count of powdered form of carrier based aseptic condition. The Alginate beads were biofertilizer. So that the above results revealed then stored in polythene bags at room that at 4 0 C, lignite carrier based Pseudomonas temperature (28±2ºC) and refrigerator (4ºC) bioinoculants supported and maintained upto 8 months. optimum viable count log more than Treatments cfu g -1 (log ) viable count of T 1: S 1 C 1 O 1 (Irradiated Vermicompost with powdered form of carrier based biofertilizer Pseudomonas spp) upto 8 th month. but showed the viable count T 2: S 1 C 2 O 1 (Irradiated Sodium alginate with least among different carrier materilas, Pseudomonas spp) whereas sodium alginate, vermiculite and T3: S 1 C 3 O 1 (Irradiated Lignite with vermicompost based carrier bioinoculants Pseudomonas spp). supported and maintained optimum viable T4: S 1 C 4 O 1 (Irradiated Vermiculite with count log , 8.70, 8.40, respectively upto Pseudomonas spp), 8 th month fig no.1. Which is more than the Copyright August, 2017; IJPAB 2161

5 minimum cfu g -1 (log ) viable count comparing all carrier based bio inoculants of powdered form of carrier based shelf life sodium alginate Pseudomonas biofertilizer. carrier based biofertilizer showed highest At 28 ± 2 0 C storage temperature viable count upto 8 th moths at both storage lignite carrier based Pseudomonas inoculants temperatures viz., log Room supported and maintained optimum viable temperature (28±2ºC) and log count log upto 6 th month only, but Refrigerator (4 C ). sodium alginate, vermiculite and vemicompost But survival rate of Pseudomonas based carrier bioinoculants supported and cells was more upto 8 th month at 4 0 C maintained optimum viable count log , compared to 28 ± 2 0 C. This results revealed 8.00 and 7.60 respectively more than that 4 0 C storage temperature is best suitable cfu g -1 (log ) upto 7 th month and in for storage of carrier based inoculants because 8 th month showed viable count log ( of low level of moisture content in the carrier vermicompost ), 7.35 ( sodium alginate ), 6.50 inoculants stored at 28 ± 2 0 C temperature ( lignite ), 7.00 ( vermiculite ) fig no 2. When compared to 4 0 C. Table 1: Pseudomonas Population in different carrier based bioinoculants at 28±2ºC upto 8 th months VERMI SODIUM LIGNITE VERMICULITE MONTH COMPOST ALGINATE (28±2ºC) (28±2ºC) (28±2ºC) (28±2ºC) Table 2: VERMI SODIUM LIGNITE VERMICULITE MONTH COMPOST ALGINAT 4 ºC 4 ºC 4 ºC 4 ºC Copyright August, 2017; IJPAB 2162

6 pseudomonas population in log 10 values pseudomonas population in log 10 values Thirumal et al Int. J. Pure App. Biosci. 5 (4): (2017) ISSN: Pseudomonas population in different carrier based bioinoculants at 4ºC upto 8 months Months VERMICOMPOS T SODIUM ALGINATE LIGNITE Fig. 1: Pseudomonas population in different carrier based bioinoculants at 28±2 C upto 8 months Months VERMICOMPOS T SODIUM ALGINATE LIGNITE Fig. 2: CONCLUSION For the increasing the shelf life of Pseudomonas carrier based biofertilizers should be store at 4 C temperature then Room temperature (28±2ºC), and also for best carrier based biofertilizer production sodium alginate and vermiculite carriers were showed highest population with long time storage capacity at both temperatures viz., Room temperature (28±2ºC) and Refrigerator (4 C). ABBREVIATIONS RGP; Red Gram Pseudomonas, IAA; Indole Acetic Acid, PGPR; Plant Growth Promoting Rhizobacteria, HCN; Hydrogen Cyanide, PJTSAU; Professor Jayashanker Telangana State Agricultural University, NIRD; National Institute of Rural Development. carrier material and storage temperature on survivability of Rhizobium spp inoculants. Asian Journal of Plant Sciences. 10: (2011). 2. Abdul Halim, N.B., Effects of using enhanced biofertilizer containing N-fixer bacteria on patchouli growth. Thesis. Faculty of Chemical and Natural Resources Engineering University Malaysia Pahang. 145 (2009). 3. Pikovskaya, R.I., Mobilization of phosphorus in soil connection with the vital activity of some microbial species. Microbiologiya.17: (1948). 4. Saravanan, V.S., Subramoniam, S.R. and Raj, S.A. Assessing in Vitro solubilization potential of different zinc solubilizing bacterial (ZnSB) isolates. Brazilian Journal of Microbiology. 34: (2003). 5. Prajapati, K.B. and Modi, H.A., Isolation REFERENCES 1. Bazilah, A.B.I., Sariah, M., Abiddin, M.A.Z. and Yasmeen, S. Influence of and characterization of potassium Copyright August, 2017; IJPAB 2163

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