Probiotic-assisted bioconversion of agro-waste into organic liquid NPK fertilizer: Fermentation dynamics and efficacy evaluation on paddy under pot and field conditions

Introduction

Large quantities of agro waste are generated like poultry litter, groundnut cake, greengram flour, rock phosphate, pressmud, wood ash, etc. If not properly disposed, it causes environmental pollution, greenhouse-gas emission and soil degradation. While these residues are abandoned, they still have favourable compositions in macronutrients (N, P, K) and organic materials for sustainable valorization. Low-cost agro-by products can be converted into organic liquid NPK fertilizer which can reduce pollution, promote circular bio-economy and provide an alternative to conventional fertilizers. Inorganic fertilizers are used to improve soil fertility but intensive inorganic fertilizer usage in agriculture causes so many health problems and unrecoverable environmental pollution. [1] . On the other hand, use of organic fertilizers results in increased soil organic matter, reduced erosion, better water infiltration and aeration, higher soil biological activity as the materials decompose in soil, and increased yields after the year of application (residual effects) [2] . During the fermentation process of organic sources, microorganisms can also decompose organic matter and produce many nutrients such as nitrogen, phosphorus, and potassium that crops can absorb [3] . Integrated consumption of organic manures and chemical fertilizers has the potential to provide greater sustainability in crop production, maintain improve soil fertility, and enhance the efficiency of growth, yield and nutrients uptake of rice cultivars [4] . Synergistic combination of waste biomass and plant-growth-stimulating microorganisms creates bioorganic fertilizers that reduce dependence on primary raw materials and synthetic fertilizers. They may alleviate issues such as the dispersal of antibiotic resistance and the persistence of recalcitrant residues in current compost and AD-based fertilizers, reducing soil pollution and enhancing crop yield, and soil health [5] The study uses probiotics to convert agro waste into a liquid fertilizer with higher NPK. Lactobacillus species in addition to Saccharomyces species, which is isolated from milk and bread, plus confirmed with 16S rRNA sequencing, mixed with Azospirillum, phosphate solubilizing bacteria, and potassium mobilizing bacteria obtained from Tamil Nadu Agricultural University. This microbial consortium not only helps dissolve nutrients but also helps microbes produce metabolites in aerobic fermentation during a 30 days period at 30 °C and pH 7.0 in sterilized molasses medium along with coconut milk, curd, seaweed gel and gibberellic acid. The fermentation produced a liquid full of nutrients-it holds NPK in even amounts as well as good metabolites like IAA, gibberellic acid, cellulase as well as phosphatase. The best mixture, which had groundnut cake, rock phosphate in addition to potassium humate, reached 15% NPK. It also contained many bioactive metabolites.

Materials and Methods

Collection and Pre-treatment of Agro-waste Materials

Agro waste materials and agro residues rich in NPK such as poultry litter, greengram flour, groundnut cake, rock phosphate, prom, bone meal, Potassium humate, wood ash, pressmud were collected and sun dried to reduce moisture content. Then the wastes and residues were pulverized to pass through a 2 mm sieve (Fig. 1). 27 formulations using these agro wastes were made and fermentation proceeds. 12 g of each substrate was weighed and added to 600 mL of a sterilized fermentation molasses medium within a 1 L flask. The medium was composed of molasses (30 g), peptone (1g), yeast extract (1g) per L of water and sterilized at 121 °C for 20 minutes to eliminate contaminants prior to inoculation (Fig. 2).

Isolation and Maintenance of Microbial Strains

Probiotic strains used in the study Lactobacillus and Saccharomyces were isolated from milk and bread respectively (Fig. 3) (Fig. 4) and subjected to biochemical characterization and 16s rna and ITS sequencing. The cultures were maintained separately in selective media. de Man Rogosa and Sharpe (MRS) broth for Lactobacillus and Yeast Peptone broth for Saccharomyces. Azospirillum, Phosphobacteria Bacillus megaterium and potash bacteria were also obtained from TNAU, Coimbatore and maintained in selective media. NFB (Nitrogen free bromothymol malate broth) for Azospirillum, Pikovaskya broth for phosphobacteria and GYC (Glucose Yeast Extract Calcium carbonate) media for potash bacteria were made and cultures were inoculated and maintained under controlled conditions.

Fermentation Protocol

To the fermentation media in 1 Litre flasks coconut milk 20 mL, coconut water 10 mL, curd 5 mL, seaweed gel 5 mL and gibberellic acid 0.01% were added. 1 ml of all the five microbial strains were inoculated. The temperature and pH was maintained 30°C and 7.0 throughout the fermentation process. Aerobic fermentation was maintained by covering the flasks with cotton plug and avoiding external contamination [6] . Fermentation carried out over one month period. During that time, the flasks were shaked twice a week for well distribution of organic sources and microbes.

Nutrient and Biochemical Characterization of Liquid Fertilizer

The NPK content of the fermentation liquid was tested after 30 days after filtration of the liquid from the solid components.  Nitrogen was estimated by kjeldahl method involving digestion, distillation, neutralization and titration with HCl  Phosphorus (P) was determined by spectrophotometry by absorbance of the molybdenum blue solution at a specific wavelength, typically in the range of 600-900 nm.  Potassium (K) was Determined by flame photometry method.

Pot Culture Experiment and Field trails

Pot culture experiments was carried out in the Pot culture yard of Faculty of Agriculture, Annamalai University. Surface sterilized paddy seeds were sown in 10 kg pots filled with clam loam soil. The experimental set up is Completely Randomized Design (CRD). 27 factorial combinations along with 50% RDF taken as 27 treatments. With these a control and inorganic fertilizer treatment also carried out. Totally 29 treatments were carried out.

Treatment details given in Table 1. The liquid organic fertilizer was foliar sprayed and applied in soil at three growth stages 15, 30 and 45 days after transplanting. Growth parameters like plant height, number of tillers per plant and chlorophyll content was measured at 60 days after transplanting (DAT). Field trail was conducted in Kottapakathuveli region of Villupuram District in 30 cents area. Randomized Block Design experimental setup was carried out for 29 treatments and 3 replications with plot size 5 x 2 and spacing 20 cm x 10 cm. The organic liquid fertilizer was foliar sprayed and applied in soil at critical growth stages. Mean value of Plant growth parameters like plant height, number of tillers per plant, grain yield, 1000 grain weight was recorded at 120 days after transplanting.

Results and Discussion

Characterization of Lactobacillus and Saccharomyces strains 16S rRNA and ITS sequencing for Lactobacillus and Saccharomyces were compared to those of known sequences using BLAST and the GenBank database.

Sequence phylogenetic analysis revealed that Lactobacillus LF1 was closely related to 12 known strains of Limosilactobacillus fermentum (Fig. 5) and Saccharomyces SCB-1 was closely related to and 10 known strains of Saccharomyces boulardii (Fig. 6).

Nutrient

Composition of Fermented Liquid Formulations

The NPK content of all the 27 formulation was tested by standard techniques and among them the formulation containing groundnut cake + rock phosphate + potassium humate recorded the highest NPK of 15% (N-4.5%, P-5%, K-5.5%) (Table . 2). Microbial fermentation of nitrogen rich groundnut cake was previously studied to support higher nitrogen release [7] . Rock phosphate fermentation in the presence of phosphobacteria was also studied for phosphate solubilization [8] . Potassium humate provided slow release of K complemented by potassium mobilizing microbes. These findings are similar with [9] , who reported that the insoluble K and structural inaccessible forms of K complexes are mobilized and solubilized due to the excretion of a wide range of organic acids by microbes.

Phytohormone and Enzyme Production

Indole 3 acetic acid and Gibberellic acid in the formulation of groundnut cake + rock phosphate + potassium humate which contains 15% NPK were tested using High Performance Liquid Chromatography (HPLC) while Cellulase and Phosphatase activity were tested by Bradford Protein estimation method. The results obtained shows that IAA, 28.6 µg/mL, GA 3, 19.3 µg/mL, Cellulase 4.8 IU/mL and Phosphatase 3.6 IU/mL were recorded in that specific formulation. The presence of probiotics contributes to IAA and gibberellin production [10] .

Plant Growth Performance in Pot Culture and Field Trails

Plant Growth parameters of Paddy (Oryza Sativa) ADT-37 at pot culture and Field Trails were carried out at 60 and 120 days after transplanting respectively (Fig. 7) (Fig. 8). Results showed that Treatment T 21 (the formulation containing groundnut cake + rock phosphate + potassium humate + 50% RDF) recorded the highest plant height 89.4 cm, 14.4 tillers per plant and Chlorophyll content (SPAD value 47.9) in pot culture studies (Table 3) and also T 21 recorded maximum values of plant height, number of productive tillers per plant and 1000 grain weight in field studies compared with control and inorganic fertilizer treatment (Table 4).

Soil Health and Microbial Response

Soil from 29 pots were analysed after harvesting of paddy for available NPK, organic carbon and microbial population. Results favoured T 21 treatment (50% organic + 50% RDF) that produces 0.48% available NPK, 0.68% organic carbon and 2.17 x 10 6 CFU/g of soil. These results were significantly higher compared to inorganic treatment and control (Table 5). Thus, the diverse microbial population promote better soil health and organic matter content.

Conclusion

The current research offers scientific knowledge on the feasibility in exploiting probiotic-assisted fermentation as a green technique for upscaling NPK liquification from agrowaste. The combination of groundnut cake, rock phosphate and potash humate was found to be the best among 27 combinations tested for obtaining high plant nutrients (15% NPK) and also enriched with microbial metabolites (IAA, gibberellic acid, cellulase, phosphatase). Field and pot experiments on the paddy (var. ADT 37) was reported that T 21 significantly improved plant growth, tillering, and grain yield at least similar to full chemical fertilizer application. Furthermore, its application benefited soil health after harvest by improving soil organic carbon and microbial biomass, confirming the potential long-term agronomic advantages. The treatment T 21 (50% organic + 50% recommended chemical fertilizers) recorded the maximum yield which proves the suitability of these formulations in Integrated Nutrient Management (INM) systems. Considering the increasing soil deterioration and dependence on fertilizers, these biologically enhanced liquid fertilizers may offer a step toward more sustainable food systems.

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