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The study entitled “Development of encapsulated formulation of PGPR mix-I and its evaluation” was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to develop calcium alginate based encapsulated formulation of PGPR mix-I and its evaluation for slow release and biodegradation.
The component cultures of PGPR mix-I were procured from the Department of Agricultural Microbiology, College of Agriculture, Vellayani for standardization of protocol for preparation of calcium alginate based encapsulated bead formulation of PGPR mix-I. Encapsulated bead formulation of PGPR mix-I was prepared by standard procedures.
An experiment was carried out to standardize the protocol for preparation of calcium alginate based encapsulated formulation of PGPR mix–I in completely randomized design with different treatments such as 10% Standard starch, 15% Standard starch, 10% Wheat flour, 15% Wheat flour, 10% Talc, 15% Talc and control treatment as 2% Sodium alginate alone in three replications.
Consistent viable count was recorded in encapsulated formulation amended with 10% Standard starch. It exhibited maximum viable count of each of the component cultures of PGPR mix-I as a result of three month population study. A significant decline of total viable population in control treatment was observed in each month compared to encapsulated formulation amended with 10% Standard starch.
Based on the population study, encapsulated formulation of PGPR mix-I 10% Standard starch amended was adjudged as the best combination of filler material and hence the shelf life studies of the same had to be continued at monthly intervals at room temperature and refrigerated conditions for six months by serial dilution and plate count method. Significant viable count was recorded in encapsulated beads stored at room temperature condition throughout the shelf life study.
The moisture content of beads were also monitored during standardization and shelf life study. During standardization study, moisture content of PGPR mix-I encapsulated beads of
each treatment was monitored for a period of three months at monthly intervals at room temperature and it showed a significant variation among treatments in each month. A reduction in moisture content of beads was observed from first month to the end of sixth month in all treatments. Beads amended with 10% Standard starch showed a moisture content of 13.37%, 12.07%, 11.72% and 11.45% after 24 hours of drying, first, second and third month respectively.
During shelf life study, 10% Standard starch combination at refrigerated condition showed moisture content in the range of 12.83% to 11.45% while at room temperature the same has recorded values in the range of 12.07% to 10.70%.
Evaluation of rate of release of immobilized bacteria from encapsulated beads was determined as per the procedure described by Bashan (1986) and the number of released bacteria was determined by the plate count method in respective selective medium. The higher cfu of component cultures of PGPR mix-I was observed after gentle shaking at 32⁰C for 24hours (T1) in75ml of sterile saline solution.
Evaluation of biodegradation of encapsulated beads was studied at weekly intervals in sterile and non-sterile soil with PGPR mix-I inoculated and non-inoculated beads with three replications each (Bashan, 1986). Both the sets were observed weekly for their rate of biodegradation. As per biodegradation scale values like 0, ˃0-0.5, ˃0.5-1, ˃1-2, ˃2-2.5 or 3 was assigned according to the degree of visible degradation which indicates no visible degradation,onset of degradation, slight visible degradation on bead edges, one-half to three-fourth of the beads degraded, 90% of beads become mushy, full degradation (beads are disintegrated into small pieces or not found in the nylon bag) repectively (Bashan, 1986). The PGPR mix-I inoculated beads with bacteria in non-sterile soil showed highest scale of biodegradation throughout the biodegradation study (mean value 1.34) and beads without bacteria in sterile soil showed the lowest scale (mean value 0.52). Kruskal-Wallis rank sum test was done and there was a significant difference between treatments and so multiple comparison was done using Dunn test. During all the four weeks of biodegradation study, treatment T1 (beads with PGPR mix-I in non sterile soil) recorded the highest biodegradation and T4 (beads without PGPR mix-I in sterile soil) recorded the least biodegradation. Treatments T2 (beads with PGPR mix-I in sterile soil) and T3 (beads without PGPR mix-I in non sterile) were on par with both the treatments T1 and T4 in all the four weeks.
Treatment wise evaluation of biodegradation of beads was done with Kruskal-Wallis rank sum test and gives a chi-squared value of 46.205 with df = 15 and p-value = 4.932e-05. There was a significant difference between treatments and so multiple comparison was done using Dunn test. Treatment T4 (beads with bacteria in non sterile soil during fourth week) showed significantly different from treatment T13 (beads without bacteria in sterile soil during first week). In the present investigation, calcium alginate based encapsulated beads of PGPR mix-I amended with 10% Standard starch exhibited maximum viable count of component cultures of PGPR mix-I throughout the three months period of standardization study. In terms of evaluation of shelf life and moisture retention during storage, beads stored under room temperature condition was found to be better. The rate of release of component cultures of PGPR mix-I from the encapsulated formulation was more during the first 24-48 hours. Biodegradation studies of encapsulated beads of PGPR mix-I revealed that the beads inoculated with PGPR mix-I in non sterile soil showed highest biodegradation throughout the period of investigation.