The Gradual depletion of organic carbon and total soluble protein in the Beejamrit solution upon days of incubation
In any biological system, organic carbon and proteins are considered to be the principal contributors to the C/N ratio. Several lines of evidence imply that the stoichiometry of the C/N ratio in the system regulates the composition of the microbial community and in particular, governs the rate of its decomposition (Ashraf et al. 2020; Zhao et al. 2018). To study the microbial dynamics, we were initially interested in analyzing the status of the organic carbon and total soluble protein i.e., nutrient sources of the Beejamrit solutions collected after different days of incubation. It may be noteworthy here that the Beejamrit preparation has been widely recommended for use after overnight incubation. In this study, we observed that the concentration of the organic carbon (in percent value) of the Beejamrit solution gradually gets depleted after 1-day of incubation (P < 0.0001) (Fig. 1a). This result strongly indicates that the organic matters decompose into simpler forms of biomolecules in the system and subsequently, gets emitted mostly as CO2, CH4 (Grandy and Neff 2008; Berg 2000). It is also evident from this study that the depletion of organic carbon was found to be more rapid after 2-days of decomposition (P < 0.0001). Similarly, we observed that total soluble protein in the Beejamrit solution decreases progressively after 1-day of incubation (P < 0.0001) (Fig. 1b). This observation suggests the proteolytic degradation of the proteins into peptides and amino acids which are then released as NH3, N2O, and NO2 from the system. Interestingly, the depletion of soluble protein was found to be more significant after 2-days of incubation as similarly observed in organic carbon decomposition (P < 0.0001).
Decomposition leads to pH changes in the Beejamrit solution
We next sought to determine the pH of the Beejamrit solutions collected after different days of incubation. It was reported earlier that pH regulates the composition of the microbial population in the system and therefore, the decomposition rate (Jin et al. 2018). This study showed a gradual fall in pH upon more days of incubation (P < 0.0001) (Fig. 2). The initial alkaline pH of the Beejamrit solution (8.51 ± 0.03) indicates the presence of cow urine and limes in the system. We further noticed that the pH of the Beejamrit solution becomes neutral after 7-days of incubation. Importantly, the pH of the Beejamrit solution approaches 7.49, which is near to physiological pH, after 4-days of incubation. It was reported earlier that the neutral pH promotes the growth of most microbes, particularly the plant-growth promoting bacteria in vitro (Ahemad et al. 2011). Hence, it would be fascinating to study the dynamics of the microbial population in relation to pH, organic carbon, and protein content of the Beejamrit input upon days of incubation.
Microbial population dynamics in Beejamrit
To identify the microbial diversity and also to determine its dynamics, the Beejamrit solutions collected after different days of decomposition were serially diluted in appropriate concentrations and were subsequently plated over different selective nutrient agar media by following standard methods (Please see Supplementary Table 2 for details). It was observed that the overall bacterial population of the Beejamrit input does not increase considerably up to 2-days of initial decomposition (Fig. 3a). However, the bacterial population multiplies significantly after 3-days of incubation (P < 0.001) and approaches its highest number in terms of the colony-forming unit after 5-days of incubation (2.43 ± 0.03x108). After 5-days of incubation, the bacterial population is surprisingly showing a decline. On the other hand, the populations of the actinomycetes and fungi do not change significantly up to 2-days and 1-day of incubation respectively. However, both these populations were found to be gradually decreased thereafter upon its further incubation (P < 0.0001) (Fig. 3b-3c). Together, these findings indicate that Beejamrit is a poor nutrient medium for microbial multiplication over a longer period.
Beejamrit is a rich source of plant-beneficial bacteria
Plant-beneficial microbes, such as the free-living nitrogen fixers (FNFs), the phosphate solubilizing bacteria (PSBs), the potassium solubilizing bacteria (KSBs), and the IAA producers, are known to promote plant growth (Ahemad et al. 2011; Hayat et al. 2010). In this work, we, however, focused our study on free-living nitrogen fixers (FNFs) and phosphate solubilizing bacteria (PSBs) because of their role in producing plant growth regulators such as the indole acetic acid (IAA) (Please see Discussion for details).
The study reveals that the population of free-living nitrogen fixers (FNFs) gradually grows with days of decomposition. However, its population changes substantially after 3-days of incubation (P < 0.0001) and it reaches a maximum after 4-days of incubation (2.30 ± 0.12x107) (Fig. 4a). We next extend our study on phosphate solubilizing bacteria (PSBs) to elucidate their role in phosphorus mineralization into plant-available forms. Importantly, we report in this study that the level of available phosphorus content in Beejamrit increases gradually and reaches its peak in concentration after 4-days of incubation (151 ± 3.54 mg per ml). However, the available form of phosphorus gets depleted after 5-days of decomposition (Supplementary Fig. 1). Surprisingly, we also observed here that the population of PSBs increases significantly after 1-day of incubation (P < 0.0001), and it reaches a maximum after 5-days of incubation (3.63 ± 0.09x106) (Fig. 4b). This result suggests that the population of PSBs has a strong relationship with the level of available phosphorus content in Beejamrit. Together, these findings indicate that the Beejamrit solution would yield optimum performance when applied to the crop field after 4-days of its incubation.
Beejamrit is an excellent source of the indolic class of plant growth regulator
It is well known that indolic compounds help to promote cell elongation and cell division and hence, regulate plant growth (Uggla et al. 1996). Tryptophan, one of the twenty natural amino acids, is a known precursor for the synthesis of indole acetic acid (IAA) in vivo (Ahemad et al. 2011). In this study, we report the depletion of soluble protein in the Beejamrit solution (Fig. 1b). We assume that the protein decomposes into simpler amino acids and hence, supplies tryptophan continuously to the system. On the other hand, it was reported that the Beejamrit solution is an excellent organic source for seed treatment. In addition, foliar application of Beejamrit has been shown to boost plant growth, particularly in vegetables and fruit development (Chadha et al. 2012; Devakumar et al. 2014). Based on these data, we next sought to determine the IAA content of the Beejamrit solution. The study reveals that the IAA concentration in the Beejamrit solution rises gradually upon days of incubation (P < 0.0001) (Fig. 5). It is also important to note that the IAA content of Beejamrit solution reaches its highest concentration (20.33 ± 0.06 µg per ml) after 4-days of decomposition (Fig. 5). This concentration of the IAA is comparable to the recommended hormonal dose of IAA sprayed in crops and also, its effect on increasing the growth and yield of mungbean as reported earlier (Karamany et al. 2019). Taken together, this study establishes that the Beejamrit solution is an excellent source of indolic compounds including IAA.