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Evaluation of different packaging materials for microbial inoculants.

Biofertilizer are products containing live or latent cells of efficient strains of microorganisms used for application to seed, soil or composting with an objective of increasing number of such microorganisms and accelerate those microbial process which augment the availability of nutrients that can be easily assimilated by plants. Effect of biofertilizer application in different crops have different functional traits such as plant growth and productivity, nutrient profile, plant defense and protection with special emphasis to its function to trigger various growth and defense related genes in signaling network of cellular pathways to cause cellular response and there by crop improvement (Bharadwaj et al, 2014).

Different kinds of biofertilizers are recommended for agriculture such as nitrogen fixers, phosphorus solubilizers, phosphorus mobilizers, plant growth promoters and biocontrol agents. Variety of materials used as carriers has been shown to improve the survival and biological effectiveness of inoculants by protecting bacteria from biotic and abiotic stresses (Van Veen et al., 1997).

Packaging of biofertilizer has been defined as microbial inoculants are covered by any materials in order to enhance its protection, handling, delivery and presentation of biofertilizer, from producer to user or farmer. Commonly metals (aluminium, aluminium foil and laminates films) and plastics (polythene, polypropylene, polyolefin and polyesters) are used as packing material for biofertilizer. Packaging plays a vital role in protecting and maintain viable cell as it is transported from the lab to land. The inoculants require protection from climatic conditions such as temperature, humidity, precipitation and solar radiation, which directly or indirectly influence the survival of microbial inoculants during storage.

Packaging materials prevents any wastage such as leakage or deterioration which may occur during storage, transportation and distribution. It also facilitate the labeling, which helps to the farmer to identify the biofertilizer and instruct them how to use it, how much to apply, to which crop it is intended etc. Good Packaging materials should possess some of the properties such as stable towards gamma irradiation, autoclavable, high gas exchange capacity and not allow high rates of moisture loss for packing of biofertilizer. These attributed materials must be used to ensure that inoculants reaches the farmer field in viable state. Hence, quality of packing materials is an important role in biofertilizer production technology. But virtually no information is available on the effect of packaging materials on microbial inoculants, by keeping all these points in view, the present research work was conducted with the following objectives. 1. To study the suitability of different packaging materials. 2. To study the survival of microbial inoculants.

MATERIALS AND METHODS

Bradyrhizobium japonicum, Bacillus megaterium and Pseudomonas fluorescens were grown in Yeast Extract Mannitol Broth (YEMB), Pikovskaya's broth and King's B broth respectively. They were mixed uniformly and separately with sterilized lignite at the rate of 1:3 (170 ml of broth culture in 500 g of lignite) under aseptic condition and packed in aluminium and polythene covers, sealed with the help of electronic sealer. Similarly talc based biofertilizer were prepared at the rate of 1:5 (100 ml of broth culture in 500 g of talc) and packed in aluminium and polythene covers.

Details of Experiment

In this experiment, three microorganisms, two carrier materials and two packaging materials were used. There were four treatments and three replications.

Survivability of microbial inoculants in different treatment

Survival study was done to estimate the microbial population in different treatments at 0, 15, 30, 60, 90, 120, 150 and 180 days of storage by standard plate count technique.

RESULTS AND DISCUSSION

Physico--chemical properties of carrier materials

Physical and chemical properties of carrier materials such as colour, particle size, water holding capacity (WHC), bulk density (BD), pH and electrical conductivity (EC) were analyzed. Colour of lignite was black with 106 [micro]m particle size and it recorded 62.26 per cent of water holding capacity, 0.64 g/cc bulk density, 4.06 pH and 0.50 dS/m EC. Talc was white in colour with particle size of 106 pm. It recorded 72.25 per cent of water holding capacity, 0.82 g/cc bulk density, 9 pH and 0.62 dS/ m of EC. Gade et al. (2014) reported similar outcomes regarding physical and chemical properties of lignite and talc.

Survival of Bradyrhizobium japonicum, Bacillus megaterium and Pseudomonas fluorescens in different carrier materials

There were differences in population of B. japonicum in lignite and talc based formulations. Survival of B. japonicum was better in lignite compared to talc based formulations. At the end of storage period [log.sub.10] 7.58 cfu/g and [log.sub.10] 6.97 cfu/g were observed in lignite and talc based formulations of B. japonicum respectively (Table 1). The results revealed that maximum population was observed in lignite throughout the storage period. This clearly supports the findings of Kalaivani (1998) that lignite supported higher survival of P. fluorescens and B. japonicum than peat.

In both formulations, viable cells of B. megaterium increased upto 60 days and later declined. Lignite based inoculants recorded maximum population ([log.sub.10] 7.19 cfu/g) than talc based inoculants ([log.sub.10] 6.93 cfu/g) at the end of storage period. (Table 1). The results are in agreement with the finding of Sangeetha and Stella (2012), that survival rate of B. subtilis increased initially upto 60 days later declined towards the survival period.

Pseudomonas fluorescens recorded its survival from [log.sub.10] 7.36 to [log.sub.10] 6.87 cfu/g in lignite formulation. Talc based inoculants recorded its survival from [log.sub.10] 7.1 to [log.sub.10] 7.07 cfu/g. The data is statistically on par between the carrier materials during storage period except 60 days of storage (Table 1). A similar finding was reported by Gade et al. (2014) talc as a carrier, which supports the maximum number of viable cells of P. fluorescens than lignite. Similarly Chandar et al. (2013) reported that higher cfu in talc based formulation of P fluorescens as compared to lignite or vermiculite based formulation.

Survival of Bradyrhizobium japonicum, Bacillus megaterium and Pseudomonas fluorescens in different packaging materials

Maximum survival of B. japonicum was observed at 90 days ([log.sub.10] 8.32 cfu/g) which were placed in aluminium cover as compared to polythene bag ([log.sub.10] 7.65 cfu/g). However, at the end of six months of storage maximum viable cells of B. japonicum recorded in polythene bag ([log.sub.10] 7.34 cfu/g) than aluminium cover ([log.sub.10] 7.21 cfu/g) (Table 2). The results were on par between the packaging materials upto 60 days and later, showed a significant difference. It may be due to properties of packaging materials which include gas exchange property, thickness, density of material etc. Polythene bag had good gas exchange property and less thickness compared to aluminium cover. Roughley (1968) confirmed that the Rhizobium needed a gas exchange through a packing materials. Strijdom and Deschodt (1976) reported a maximum survival of Azospirillum sp. (3.5 x [10.sup.8] cfu/g) in steam sterilized peat, which was packed in low density polythene bag (0.31 - 0.32 mm).

There were significant differences in population of B. megaterium with respect to packaging materials after 30 days of storage. B. megaterium declined during storage period from [log.sub.10] 7.31 to [log.sub.10] 6.72 cfu/g which packed in aluminium cover. In case of polythene bag, population gradually increased upto 60 days then slowly declined, during storage from [log.sub.10] 7.31 to [log.sub.10] 7.42 cfu/g of B. megaterium was observed (Table 2). The results are in agreement with the finding of Prihast (2013) who reported that highest population of Bacillus sp. and Paenibacillus in plastic bag (polythene bag) with 50 to 60 per cent of water than aluminium foil. There were differences in population of P. fluorescens with respect to packaging materials. Survival of P fluorescens was better in aluminium cover compared to polythene bag. At the end of storage period [log.sub.10] 7.09 and [log.sub.10] 6.86 cfu/g were observed in aluminium and polythene bag respectively (Table 2). According to results obtained, aluminium cover was a suitable packing materials for P. fluorescens than polythene bag. The result were on par with work done by Callaghan et al. (2006) who reported that greater survival of P. fluorescens in aluminium foil than gas transferable bag.

It is evident from this investigation, lignite or talc can be a carrier material for the survival of microbial inoculants. With regard to packaging materials, polythene bag was the best packaging material for B. japonicum and B. megaterium whereas aluminium cover was the best for P. fluorescens. However, both packaging materials were not harmful for survival of microorganisms and maintain good viable cells as per the BIS standards upto 180 days of storage.

REFERENCES

(1.) Bharadwaj, D., Ansari, M. W., Sahoo, R. K. and Tuteja, N., Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microbial Cell Factories, 2014; 13: 66-72.

(2.) Callaghan, M. O., Swaminathan, J., Lottmann, J., Wright, D. A. and Jackson, T. A., Seed coating with biocontrol strain Pseudomonas fluorescens F113.New Zealand Plant Prot., 2006; 59: 80-85.

(3.) Chandar, K., Gangopadhyay, S. and Godara S.l., Studies on shelf life of Trichoderma spp. and Pseudomonas fluorescens in different formulating materials. Plant Dis. Res., 2013; 28: 53-57.

(4.) Gade, R. M., Chaithanya, B. H. and Khurade, K. C., A comparitive study of different carriers for shelf life of Pseudomonas fluorescens. Int. Quartery J. Life Sci.,2014; 9: 287-290.

(5.) Kalaivani, R., Studies on plant growth promoting rhizobacteria. M.Sc. (Agri.) Thesis, Tamil Nadu Agricultural University, Coimbatore, 1998; 152.

(6.) Prihast, T., Testing the quality of biofertilizer Illetrisoy. Amer. J. Microbiol. Res., 2013; 2: 63-67.

(7.) Roughley, R. J., Some factors influencing the growth and survival of root nodule bacteria in peat culture. J. Appl. Bacteriol., 1968; 31: 259-265.

(8.) Sangeetha, D. and Stella, D., Survival of plant growth promoting bacterial inoculants in different carrier materials. Int. J. Pharm. Biol. Arch, 2012; 3: 170-178.

(9.) Strijdom, B. W. and Deschodt, C. C., Carriers of rhizobia and the effect of prior treatment on the survival of rhizobia. In: Symbiotic Nitrogen Fixation in Plants. Nutman, P.S., (eds). International Bilogical Programme 7. Cambridge University press, Cambridge, 1976; 151-168.

(10.) Van Veen, J. A., Van Overbeek, L. S. and Van Elsas, J. D., Fate and activity of microorganisms introduced into soil. Microbiol. Molecular Biol. Rev., 1997; 61: 121-135.

P.S. Arpitha and G.P Brahmaprakash

Department of Agricultural Microbiology, UAS, GKVK, Bengaluru--560 065, India.

(Received: 15 January 2016; accepted: 28 February 2016)

* To whom all correspondence should be addressed.
Table 1. Survival of Bradyrhizobium japonicum, Bacillus megaterium
and Pseudomonasfluorescens in different carrier materials

              Bradyrhizobium japonicum   Bacillus megaterium

Duration      Lignite   Talc       CD    Lignite   Talc       CD
of storage                                                    @ 5 %
(Days)
              [log.sub.10] cfu/g         [log.sub.10] cfu/g

0              7.40     7.20       NS    7.36     7.26        NS
15             8.81     7.15      0.16   7.28     7.28        NS
30             8.42     7.31      0.18   8.01     8.02        NS
60             8.82     7.69      0.07   8.60     8.08        0.11
90             8.12     7.85      0.16   8.35     7.75        0.17
120            7.96     7.69      0.14   8.23     7.58        0.16
150            7.64     7.06      0.15   7.26     7.02        NS
180            7.58     6.97      0.35   7.19     7.19        0.25

              Pseudomonas fluorescens

Duration      Lignite   Talc       CD
of storage                        @ 5 %
(Days)
              [log.sub.10] cfu/g

0              7.36     7.10      0.19
15             7.23     7.15       NS
30             7.25     7.28       NS
60             7.87     8.16      0.18
90             7.65     7.74       NS
120            7.50     7.56       NS
150            7.04     7.14       NS
180            6.87     7.07       NS

Where: NS - Non Significant
Note: Values are mean of three replications

Table 2. Survival of Bradyrhizobium japonicum, Bacillus megaterium
and Pseudomonas fluorescens in different packaging materials

                 Bradyrhizobium japonicum

Duration      Aluminium   Polythene   CD @ 5
of storage      cover        bag        %
(Days)
                    [log.sub.10] cfu/g

0               7.30        7.30        NS
15              7.95        8.00        NS
30              7.83        7.89        NS
60              8.25        8.26        NS
90              8.32        7.65       0.16
120             8.16        7.49       0.14
150             7.26        7.43       0.15
180             7.21        7.34        NS

                    Bacillus megaterium

Duration      Aluminium   Polythene   CD @ 5
of storage      cover        bag        %
(Days)
                    [log.sub.10] cfu/g

0               7.31        7.31        NS
15              7.28        7.28        NS
30              7.28        8.75       0.14
60              7.76        8.92       0.11
90              7.51        8.60       0.17
120             7.44        8.37       0.16
150             6.78        7.86       0.38
180             6.70        7.42       0.25

                 Pseudomonas fluorescens

Duration      Aluminium   Polythene    CD @ 5
of storage      cover        bag         %
(Days)
                    [log.sub.10] cfu/g

0               7.23         7.23        NS
15              7.10         7.28        NS
30              7.15         7.38       0.21
60              8.72         7.31       0.18
90              8.25         7.15       0.19
120             7.83         7.23       0.27
150             7.19         6.99       0.14
180             7.09         6.86       0.20

Where: NS - Non Significant
Note: Values are mean of three replications
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Author:Arpitha, P.S.; Brahmaprakash, G.P.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2016
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