MATERIALS AND METHODS

Instant organic fertilizer (IOF) preparation: The Instant organic fertilizer was prepared by collecting the vegetable and fruit waste collected from the canteen at the university campus. These were sun dried for a week and ground to get a homogenous powder. The newly prepared fertilizer was analysed for its NPK and moisture content.

Experimental set up: Four pots of 5 kg capacity were prepared for each experimental plant; the pot contained a 10 g IOF, 1 g chemical fertilizer (urea), 250 g compost and a control (with out fertilization), respectively. The experimental plant selected was spinach. In each pot 6 seeds were sown and left for germination. The fertilization was done for every 7 days.

Examination of microbial diversity: The increase utilization of chemical fertilizer has diminished the normal microbial flora in soil that plays an essential role in recycling of biomaterials. Hence the soil treated with IOF, compost and chemical fertilizer were analysed for the diversity of bacteria, fungi and actinomycetes population in soil, using dilution plate technique with suitable media like nutrient agar, potato dextrose agar and actinomycetes isolation agar, respectively.

Determination of plant growth promoting activity: Growth promoting activities of soils treated with different fertilizers were tested. The plumule lengths of spinach seedlings were measured initially and were sown in the soils and soon after the development of plumule; it was taken out and soaked in water. After 24 h the plumule length was measured.

Effect of IOF, chemical fertilizer, compost on spinach growth: The plant fertilized with Instant organic fertilizer, compost and chemical fertilizer were analysed for its growth for a period of 45 days. The parameters examined are height, fresh and dry weights, pH, leaf size, diameter, protein concentration, chlorophyll concentration, total phenols and free radical scavenging activity. The values were recorded in triplicates. At harvest time (after 45 days) the leaves were collected and subjected to analysis.

Estimation of protein and chlorophyll: The protein content was estimated by the method of Lowry et al. (1951) and Samman et al. (2008). The chlorophyll content of the sample was also determined by the method of Arnon (Tan et al., 2009).

Determination of total phenols: The total phenols were estimated by the method of McDonald et al. cited by Wu et al. (2005). The diluted plant extract was taken in different concentrations and was made up to 1 mL with distilled water. To the samples 2.5 mL of 20% sodium carbonate solution and 0.5 mL of Folin’s solution was added and kept for incubation for 40 min. The absorbance was recorded at 725 nm. Tannic acid in different concentrations was used as reference standard. Total phenol values are expressed in terms of tannic acid equivalents.

Determination of hydrogen peroxide (H₂O₂) scavenging activity of plant extract: Hydrogen peroxide scavenging activity of plant extract was determined using a modification of the method of Ruch et al. cited by Tejada et al. (2009). Solution of H₂O₂ was prepared in phosphate-buffered saline (PBS, pH 7.4). H₂O₂ concentration was determined spectrophotometrically from absorbance at 230 nm. Different concentrations of plant extract corresponding to extract stock solution were added in 4 mL distilled water and are then added with 0.6 mL hydrogen peroxide PBS solution. Absorbance of H₂O₂ at 230 nm was determined 10 minutes later against a blank solution containing plant extract in PBS without H₂O₂.

DPPH radical scavenging activity: DPPH radical scavenging activity was carried out by the method of Molyneux (Asal, 2010). To 1.0 mL of 100.0 μM DPPH solution in methanol, equal volume of the test sample in methanol of different concentration was added and incubated in dark for 30 min. The change in colouration was observed in terms of absorbance using a spectrophotometer at 515 nm. One milliliter of methanol instead of test sample was added to the control tube. Different concentration of ascorbic acid was used as reference.

Statistical analysis: Results calculated from triplicate data were expressed as Means±SD. The data were compared by Least Significant Difference (LSD) test using statistical analysis.

RESULTS

The NPK content in the newly produced Instant organic fertilizer were found to be found to be 1.18, 5 and 3.06 mg g^-1, respectively. The moisture content was found to be 3.3%. The pH of the fertilizer was 6.0-6.2. The ash percentage was found to be 11%. These results evident that Instant organic fertilizer produced has required amount of essential nutrients to facilitate plant growth. The spinach seeds soaked in different fertilized soils showed marked difference in plumule length. Table 1 indicates the soil treated with different fertilizer have different growth promoting activity with a good increase in seeds treated with Instant organic fertilizer. Data represented in Table 2 showed that, growth parameters of leaf vegetable spinach were greatly influenced by fertilization treatments. A gradual increase in all the parameters studied (germination period, leaf number, leaf area, plant height, fresh weight, dry weight), progressed with the plant age. Significant differences among the fertilized and control plants were observed. And less difference were found between the plants fertilized with chemical, instant organic fertilizer and compost. Instant organic fertilizer has positive effect on the content of photosynthetic pigments and protein. The sustainable release of nutrients provided the plant to have increased protein and chlorophyll concentration. The content was not much significantly higher in Instant organic fertilizer when compared with chemical treated plant (Table 3). The statistical analysis showed a positive and highly significant, R² = 0.062 (H₂O₂) and R² = 0.079 (DPPH) for instant organic fertilizer treated spinach and showed significant differences in phenolic content and high scavenging effect when compared to other fertilized plant (Table 4). These experimental data proved that the newly formulated Instant organic fertilizer is best in all aspects of plant growth (Fig. 1a-d). The inorganic fertilizer also gave the similar results; however, the use of inorganic fertilizer may cause problems in human health and environment.

Table 1:	Growth promoting activity of different fertilizer treated soils

Table 2:	Effect of different fertilizers on spinach growth for a period of 45 days

Table 3:	Effect of different fertilizers on protein and chlorophyll content in spinach
Protein and chlorophyll concentration is expressed as mg mL-1 and μg mL-1, respectively

Table 4:	Total phenolics and antioxidant activity of spinach
All data were average (±SD) of three replicates

Fig. 1(a-d):	Fertilizer effects on spinach growth. Cultivation was carried out for 45 days; (a) Normally grown spinach, (b) Instant organic fertilizer treated spinach, (c) Compost grown spinach and (d) Chemical fertilizer treated spinach

DISCUSSION

The present study, recorded higher microbial population (bacteria, fungi, actinomycetes) in instant organic fertilizer fertilized soil than with other fertilized soils was clearly depicted in Fig. 3. This may be due to existing temperature and pH in instant organic fertilizer might invite large number of microbes which obviously facilitates the easier degradation of wastes therefore enriching soil properties. Any microbial species release phytohormones and may stimulate plant growth. The microbial diversity in agricultural soil is quite discriminating now days by the continuous application of chemical fertilizers. These data confirms that newly formulated Instant organic fertilizer showed higher growth effects like chemical and organic compost. Chicken manure and farm yard manure, had a synergestic effect on both fresh weight and dry weights of tomato shoots and agricultural compost gave the lowest growth of tomato growth, yield, chemical constituents and mineral nutritional contents and might be due to high pH, losses of leaching and volatilization under soil poor condition. A promotion effect may be due to instant, compost organic and inorganic might be due to the fact that N is a constituent of chlorophyll molecule.

Fig. 2:	Relationship between phenol content and antioxidant activity of spinach

The role of natural dietary antioxidants in disease prevention has been the focus of much investigation. The significant differences are due to the over-fertilization of compost and chemical fertilizer might reduce the health benefits from Spinach. This is an agreement with Mitchell (2007), experimented with tomatoes. Figure 2 shows the relationship between the phenolic content and antioxidant activity in spinach, the accelerated antioxidant activity was associated with high phenolics value.

Fig. 3:	Enumeration of microbial diversity in soils

Toor et al. ( 2006) investigated three inorganic and 2 organic fertilizer methods to determine if nitrogen availability affects the production of polyphenols in plants. Dauda et al. (2005) showed higher level of some individual phytochemicals in organically grown lettuce but not significantly different. No supports was found for higher levels of antioxidants and polyphenols in the conventionally grown vegetables.

CONCLUSION

The instant organic fertilizer used in the study gave the highest results in the physical properties of spinach and also in the protein, chlorophyll concentration and antioxidant activity, equivalent to compost grown spinach. It was therefore to be expected to use Instant organic fertilizer by the farmers since composting operation should have appropriate background and techniques for producing high-quality composts without creating odour and other environmental problems. The main disadvantage of composting is variable costs; which include labour, fuel, electricity and maintenance charges. Hence the study clearly showcases an innovative idea as well as a new approach for the utilization of agricultural waste in the formulation of economical instant organic fertilizer.

ACKNOWLEDGMENTS

This study was supported by School of Chemical and Biotechnology, SASTRA University, India. We are grateful to the Vice-chancellor, Prof. R. Sethuraman, for the providence to carry out the research.