A glass house experiment was conducted to develop phospho-vermicompost using different combination of organic residues with rock phosphate and earthworms at the Soil Science Division, BINA, Mymensingh during 2019-2020. The experiment was conducted in a Completely Randomized Design with eight treatments and three replications. The treatments were as follows:
T1: 50% Cowdung (CD) + 50% Mustard straw (MST) T2:50% CD + 50% Water hyacinth (WH)
T3:50% CD + 50% Rice straw (RST) T4: 50% CD + 25% WH + 25% RST
T5:50% CD + 50% MST + 4% Rock phosphate (RP)
T6:50% CD + 50% WH + 4% RP T7: 50% CD + 50% RST + 4% RP
T8: 50% CD + 25% WH + 25% RST + 4% RP
Rock phosphate (@ 4%) was mixed well with the mixtures of organic residues as treatment plan into the plastic pots (50 L) and all the pots were pre-incubated until three weeks. After softening and partial decomposed of residues then 150 earth worms (Eisenia foetida or Red wiggler earthworms) were released into all the plastic pots. All the pots were covered with gunny bag to make dark condition. Small amount of water was sprayed if necessary to avoid the dryness of the residues. After 90 days of incubation, the residues were completely decomposed and no bad odor was observed. So phospho vermicomposts were ready for collection. Then the samples were collected from each pot for chemical analysis and estimation of earthworms, total bacteria, phosphate solubilizing bacteria and phosphatase activity. The prepared samples were analyzed for pH, organic carbon, total N, P, K and S and available P. Plant residues, cowdung and prepared phospho-vermicompost were analysed with the standard methods which were as follows: pH of prepared phospho-vermicompost: pH was measured by a glass electrode pH meter using phospho-vermicompost : water suspension of 1:2.5 as described by Jackson (1967).
Organic carbon from organic residues and prepared phospho-vermicompost: Organic carbon was determined by wet oxidation method as described by Black (1965) from residues, and phospho- vermicompost. The underlying principle was used to oxidize the organic matter with an excess of 1N K2Cr2O7 in presence of conc. H2SO4 and conc. H3PO4 and to titrate the excess K2Cr2O7 solution with 1N FeSO4. The results were expressed in percentage (Page et al., 1989). Total N from residues and prepared phospho- vermicompost: Total N content was determined following micro-Kjeldahl method as described by Jackson (1967) from residues and phospho- vermicompost. Each sample was digested with H2O2, conc. H2SO4 and catalyst mixture (K2SO4:CuSO4.5H2O: Se in the ratio of 100:10:1). After completion of digestion, making the volume to 100ml. Distillation was performed with adding of 40% NaOH into the digest. The distillate was received in 2% boric acid (H3BO3) solution and 4 drops of mixed indicator of bromocresol green and methyl red solution. Finally, the distillate was titrated with standard H2SO4 (0.01N) until the color changed from green to pink. Then amount of N was calculated. Total P, K and S from organic residues and prepared phospho-vermicompost: Total P, K and S were determined following micro-Kjeldahl method as described by Jackson (1967). About 0.5 g of samples was transferred into dry clean 100 ml kjeldahl flasks. 10 ml of di-acid mixture (HNO3:HClO4 = 2:1) wereadded into the flask. After leaving for a while the flasks were heated at temperature slowly raised to 200°C. The contents of the flaks were boiled until they became sufficiently clear and colorless. After cooling the digests were transferred into 100ml volumetric flaks and the volumes were made up to the mark with distilled water. Phosphorus was determined by developing blue color by SnCl2 reduction of phosphomolybdate complex and measuring the intensity of color by calorimetrically at 660 µm wave length and the readings were calibrated to the standard P curve. Potassium was determined from the aliquot by flame photometer (Black, 1965) and calibrated with a standard curve. Sulphure was determined from aliquot by calorimetrically at 440 µm wave length (Williams and Steinbergs, 1959) and the readings were calibrated to the standard curve.
Available phosphorus from prepared phospho- vermicompost: Available phosphorus was extracted from the phospho-vermicompost samples by shaking with 0.5 M NaHCO3 solutions at pH 8.5 following the method of Olsen and Sommers (1982). The extracted phosphorus was determined by developing blue color by SnCl2 reduction of phosphomolydate complex and measuring the intensity of color calorimetrically at 660 ?m wave length and the readings were calibrated to the standard P curve. Counting of earthworms: Earthworms were counted at the end of the incubation i.e. at the mature of vermicompost from each pot. Visible earthworms were carefully sorted from the decomposted materials manually and counted. Estimation of total bacteria in prepared vermicompost: The dilution plate method was used for enumeration of total bacteria from prepared vermicompost (Subba Rao, 1993). The ingredients of the methods were soil extract –glucose agar medium in which 1.0 g glucose, 0.5g K2HPO4, 0.1 g KNO3, sterilized soil extract 100ml, distilled water 1000ml, pH adjusted 6.5 and agar 20 g and autoclaved. Soil extract was prepared by autoclaving 500 g of fresh soil with the addition of 1000 ml tap water at 1210C for 20 min at 15 PSI and filtered through Whatman No. 1 filter paper. Serial dilutions were prepared by weighing of 10 g vermicompost samples in 90 ml sterile water blank and shaked vigorously. This makes 10-1 dilution. After shaking, immediately transfered 1 ml of suspension from 10-1 dilution aseptically and discharge into 9 ml sterile water blank and shaked again. This makes a dilution of 10-2. Dilutions were made up to 10-7 followed by this procedure. Then 1 ml was taken from dilutions 10-4, 10-5, 10-6 and 10-7 and pour in sterilized Petri dishes with three replicates in each dilution. Each Petri dish contained about 15 ml of soil extract agar medium, melted previously and kept in a water bath at 450C. Quickly rotate the Petridish so as to mix the medium with the inoculum. Allow the agar to solidify. The plates were incubated inverted position for 5-7 days at 300C. The number of bacteria was calculated in per gram of dry vermicompost and expressed in colony forming unit (CFU g- 1vermicompost) and finally data were transferred in Log value as descrived by Cappuccino and Sherman (1999). Determination of phosphate solubilizing bacteria (PSB) in prepared phosphor-vermicompost: PSB populations were counted from the mature vermicompost at the end of the incubation. The samples were collected from each pot. The samples were processed immediately after collection, otherwise stored in plastic bags at 4 0C until processed. PSB were counted by serial dilution plate technique (Subba Rao, 1999) as like as above total bacterial count. One gram of vermicompost diluted in 9 mL sterile water blank which gave 10-1 dilution. Then 10-2 to 10-6 dilutions were spread on Pikovskaya’s solid medium containing tricalcium phosphate (TCP) (Pikovskaya, 1948). The colonies that appeared as discrete clear zones (halo zones) around them indicated the dissolution of TCP was assumed to be phosphate solubilizers and the PSB colonies were counted. PSB populations were expressed in the colony forming unit (CFUg-1 vermicompost) as described by Cappuccino and Sherman (1999) and finally data were transferred in value.
Assay of phosphatase activity in prepared phospho- vermicompost: The phosphatases are mainly of two types such as acid and alkaline phosphatases. Acid phosphatases exist in acidic condition while alkaline phosphatases exist in alkaline condition. All the vermicompost were exist in alkaline condition. Therefore, alkaline phosphatase activity (ALPA) were assayed from the vermicpmpost. Phosphatase activities were assayed according to the methods as described by Tabatabai and Bremner (1969). The activity of phosphatase enzyme was assayed by colorimetric method using p-nitrophenyl-phosphate (pNPP) as substrate. One gram of vermicompost was taken in a 30 mL test tube and 0.2 mL of tolune, 4 mL of modified universal buffer (MUB), 1 mL of p- nitrophenyl-phosphate solution were added and mixed the contents by shaking. All the test tubes were tightly closed by parafilm and incubated at 370C for one hour then turned the suspension yellow in colour. After incubation all the test tubes were brought out and removed the parafilm. 1 mL of 0.5M CaCl2 and 4 mL of 0.5 M NaOH were added into the test tube to stop the reaction. All the test tubes were swirled for few seconds and filtered the suspension through a Whatman No.1 filter paper. Absorbance of yellow coloured filtrate was taken at 420 ηm in spectrophotometer. Finally, ALPA was calculated by a reference to calibrate with the standard p-nitrophenol (pNP) and the results were expressed in terms of product of pNP formed g-1 vermicompost h-1. Statistical analysis: All data obtained were analyzed and processed using one-way ANOVA (Analysis of variance) with significance level of 0.05 through Statistix 10 software packages (Analytical Software, 2105 Miller Landing Rd,Tallahassee, FL 32312).