Assessment of Shelf Life and Quality of Biofertilizers using Tricalcium Phosphate as an Anticaking Agent and Aluminium Silicate as the Inert Carrier

Plants for their survival and normal functioning require different minerals from soil. This can be achieved by normal physiological process such as osmosis. There are certain microorganisms which are present in soil and help in accelerating nutrient availability to plants. These soil microorganisms when used as an inoculam and when provided to unfertile soil increase the fertility of the soil. Thus they are named as biofertilizers. Biofertilizers are generally carrier based microbial preparations which contain beneficial microorganisms in a viable state for certain period of time and which when applied to seed or soil application enhances plant growth through nutrient uptake and also elevates growth hormone production (Brahmaprakash et al., 2012). There are different definitions available in literature for biofertilizers. According to the definition proposed by Vessey (Vessey 2003), biofertilizers are substances which contain living microorganisms which, when applied to seed, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply or availability of primary nutrients to the host plant. Seed inoculation of pigeonpea (Cajanus cajan) + mungbean (Phaseolus radiatus) with Rhizobium and phosphorous solubilizing bacteria (PSB) recorded significantly higher nutrient uptake (N and P₂O₅) (Singh et al., 2013). Thus Rhizobium based biofertilizer along with Phosphate solubilizing bacteria (PSB) are important for plant growth.

Biofertilizers help in making the soil environment rich in all kinds of micro- and macro-nutrients via number of processes such as nitrogen fixation, mineralization as phosphate and potassium solubilization, release of plant growth regulating substances, production of antibiotics and biodegradation of organic matter in the soil (Sinha et al., 2014; Sivakumar et al., 2013) which provide efficient nutrient uptake and help in improving plant tolerance towards drought and moisture stress (Abdelraouf et al., 2013). Sometimes combine treatment of urea and Rhizobium resulted in maximum plant growth as seen in cluster bean (Gul et al., 2019). In India Rhizobium was the first microbial inoculant, which was introduced as biofertilizer in the beginning of the seventies with the introduction of soybean into the country. Azospirillum and Azotobacter were added to the list in mid- nineties. Phosphate solubilising biofertilizer (PSB) was introduced in late nineties (Yadav et al., 2014). It is studied in Rhizobium based liquid biofertilizer that on addition of different polymeric additives; polyvinyl pyrrolidone, gum arabic and glycerol it support growth and promote survival of liquid inoculants (Rhizobium sp. strain MB1503) during the storage. (Sherawat et al., 2017).

Biofertilzer once made for commercial production purposes should pass the entire quality mandate. Quality of biofertilizer is one of the most important factors which has to be maintained, before it reaches to the farmers, thus proper quality control is required. It has to be properly assessed in laboratory itself where it is initially prepared that shelf life of biofertilizer should be for long time. According to forum for nuclear cooperation in Asia (FCNA) quality is meaning the number of selected microorganism in the active form per gram or milliliter biofertilizer. (Biofertilizer Manual by FNCA Biofertilizer, 2006).

It is necessary that biofertilizer produced should be of high standard quality and should pass all the quality mandates. It becomes utmost important to evaluate the produced inoculum from commercial units comparable with some reference values so it can be ensured that protocols are strictly followed as recommended by recognized laboratories. This is most crucial as several handling errors may occur during product generation at industrial level thus resulting in poor product quality. This may lead to quite dissatisfaction to both producers and users. Thus, specific protocols for checking the quality of microbial biofertilizers should be there for proper monitoring of commercial biofertilizers. Biofertilizers should have prolonged shelf life. The Biofertilizer should not form clumps when mixed with the carrier. It should be free flowing so that it could mix well with the soil. To overcome this problem anticaking agents are routinely added to biofertilizers during their initial production. In recent years concern has been developed to find alternative to the mineral and chemical fertilizers for increased yield of crops. Since chemical fertilizers imposes health and some environmental consequences. There is need to replace these chemical fertilizers with other available alternatives. It is the crop nutrient uptake and crop yields that are the principal factors that determine optimal fertilization practices (Ju et al., 2011). Microbial inoculant is used as biofertilizers in recent past in agriculture sector which represents an attractive environmentally friendly alternative (Suyal et al., 2016). This new approach to agriculture farming is often referred to as sustainable agriculture.

Formulation of liquid biofertilizer inoculants
 
The strains used for liquid biofertilizer formulation were Azospirillum, Pseudomonas and Rhizobium. Malic acid, dipotasiumhydrogen phosphate (K₂HPO₄), Ferrous sulphate (FeSO₄) Calcium chloride (CaCl₂), Manganese sulphate (MnSO₄), Sodium molybdate (Na₂MoO₄), Sodium chloride (NaCl) and Magnesium sulphate (MgSO₄) were used to culture Azospirillum. Glycerol, dipotasiumhydrogen phosphate (K₂HPO₄), Sodium chloride (NaCl) and Magnesium sulphate (MgSO₄) were used to culture Rhizobium. Glycerol, peptone, dipotasiumhydrogen phosphate (K₂HPO₄) and Magnesium sulphate (MgSO₄) were used to culture Pseudomonas respectively. The sterilized broths were inoculated with 2% pre-inoculam of the respective strains; pH maintained 6.8 for Azospirillum and Rhizobium, 7 for Pseudomonas and incubated at 28°C on a reciprocatory shaker for 40 hours for Azospirillum and Rhizobium and 20 hours for Pseudomonas. After incubation period microscopic observation is done to check for bacterial growth, contaminations and pH of the culture is also checked. Now Colony forming unit (CFU) of the overnight cultured broth is estimated using plate count method. Further fresh sterilized prepared media were inoculated with 1.0 ml respective overnight grown mother culture and incubated in BOD incubator at 28°C for 40 hours and 20 hours respectively. Mixing of the given cultivated respective strain is done in inert carrier aluminium silicate and tricalcium phosphate is used as anticaking agent in different proportion viz 5%, 10%, 15% and 20% of the inert carrier. Shelf life of the formulation is checked using serial dilution and plate count method. This work is carried out in Biotechnology Laboratory in School of Applied and Life Sciences, Uttranchal University Dehradun under Institutional funded project. The project was started in November 2019.
 
Mixing of Bio-fertilizer with its carrier (Aluminum silicate) and anticaking agent (tri calcium phosphate).
 
Each and every bio-fertilizer sample has to be mixed with an appropriate carrier in which the microbial activity remains stable for some period. After production of the liquid broth culture of each biofertilizer (Azospirillum, Pseudomonas and Rhizobium), the bioinoculant is mixed with its carrier i.e. aluminium silicate aseptically and packed. The ratio for mixing is fixed so that we can have particular moisture content which is required for microbial growth. Sometimes anticaking agent is also used to prevent clump formation. Generally clumps get formed due to high moisture content. In the present study tri calcium phosphate (TCP) is tested in different proportion (i.e. 5%, 10%, 15% and 20% of inert carrier Aluminium silicate) for Azospirillum, Pseudomonas and Rhizobium to be used as anticaking agent.
 
For estimation of shelf life of particular bio-fertilizer
 
For every bio-fertilizer shelf life is to be determined which gives an idea of using that particular sample before its activity gets demolished. The self-life of common carrier based biofertilizer is around six months. (Brar et al., 2012)  In the present studies powder formulation shelf life of three independent biofertilizer formulation containing viz Azospirillum, Pseudomonas and Rhizobium is taken at different intervals like 15 days, 30 days and 90 days respectively.  For powdered samples shelf life is taken by serially diluted that media and plate count method.
 
Method for shelf life estimation
 
In powder formulation of bacterial culture shelf life estimation is done using 10 gm of sample aseptically and put it into 90 ml normal saline. After mixing serial dilution is done and plate the diluted media on Nutrient agar plates. After incubation when results comes we estimate the decrease in microbial activity on the basis of previous data. When we get the least number of colonies after certain interval of time. Thus we can calculate its shelf-life (i.e. it can be best used before that date). So by using this technique one can easily find out the shelf life of a particular biofertilizer sample and hence we can get the exact information regarding its use.

The shelf life of Rhizobium based bioinoculant with tricalcium phosphate (TCP) as an anticaking agent
 
After mixing anticaking agent (TCP) to respective bioinoculant it is seen that TCP is reducing clump formation in generally all the percentage (viz 5%, 10%, 15% and 20%) used to the inert carrier. On day one i.e. the very first day of mixing TCP to bioinoculant highest no of colonies were seen in 15% (2 x 108 cfu/gm) followed by 10% and 20% (2 x 108 cfu/gm each) of TCP. After 90 days on the basis of CFU result highest no of colonies were seen in 10% TCP (9 x 108 cfu/gm) followed by 5% TCP (4 x 107 cfu/gm) (Table 1).


 
The shelf life of Pseudomonas based bioinoculant with tricalcium phosphate (TCP) as an anticaking agent
 
After mixing anticaking agent (TCP) to respective bioinoculant it is seen that TCP is reducing clump formation in generally all the percentage (viz 5%, 10%, 15% and 20%) used to the inert carrier. At very first day of mixing TCP to bioinoculant highest no of colonies were observed in 10%, 15% and 20% (1 x 109cfu/gm each) followed by 5% (9 x 108 cfu/gm). After 90 days based on the CFU result highest no of colonies were seen in 10% TCP (2 x 108 cfu/gm) followed by 5% TCP (1 x 107 cfu/gm) (Table 2).



From table data it is found that when percentage of TCP is increased CFU count of the biofertilizer powder also first increases (in 10% TCP) and then decreases afterwards this may be due to 10% TCP concentration is favoring the growth of the Pseudomonas as compared to other percentage of TCP.
 
The shelf life of Azospirillum based bioinoculant with tricalcium phosphate TCP as an anticaking agent
 
After mixing anticaking agent (TCP) to respective bioinoculant it is seen that TCP is reducing clump formation in generally all the percentage (viz 5%, 10%, 15% and 20%) used to the inert carrier. On the very first day of mixing TCP to bioinoculant highest no of colonies was observed in 15% (9 x 107cfu/gm) followed by 20% (2 x 108 cfu/gm). After 90 days based on CFU result highest no of colonies were seen in 10% TCP (6 x 107 cfu/gm) followed by 5% and 15% TCP (3 x 107 cfu/gm each) (Table 3).

The present studies showed that liquid biofertilizer inoculants when mixed with anticaking agent tricalcium phosphate and aluminium silicate as an inert carrier the clump formation has been reduced in all the proportions used. Nevertheless, shelf life of bioinoculant is also important as it states stability of the biofertilizers. It was seen in our studies that 10% TCP as an anticaking agent reduces clump formation in the tested bioinoculants and also it has shown good growth as estimated from CFU even after 90 days in all the bioinoculant (Azospirillum, Pseudomonas and Rhizobium.) tested. Thus 10 % tricalcium phosphate can be used as an anticaking agent in the stated biofertilizers for maximum efficacy.