INTRODUCTION:

Compost is a mixture of organic matter that has decayed or has been digested by the organisms used to fertilize the soil and to provide nutrients. Composting is considered as a natural biological process, carried out under controlled aerobic conditions¹. At simplest level, the process of composting requires making a pile of wetted natural matter known as green waste followed by its breakdown into humus after an intermediate period². Under perfect conditions, composting proceeds through three noteworthy stages. An underlying, (i) mesophilic stage, in which compost bacteria consolidate carbon with oxygen to deliver carbon dioxide and energy, increasing the temperature of the composting mass up to 44 °C.

As the temperature rises, (ii) thermophilic stage begins, in which the decomposition is completed by different thermophilic microorganisms under high temperatures. After the thermophilic stage, the microorganisms that were pursued away by the thermophiles relocate over into the compost and digesting the more resistant organic materials. The final stage of the composting process is called the curing stage, A long curing period (e.g., a year after the thermophilic arrange) includes a safety net for pathogen devastation³.Fungi are responsible in the curing stage of composting process. Their main contribution to a compost pile is to breakdown cellulose and lignin, (Complex polymer which cannot be degraded by the organisms take part in the mesophilic and thermophilic stages)⁴.

Compost has a variety of benefits in rainfall penetration, which reduces water runoff and soil erosion⁵. It can also be used in horticulture in a wide range of contexts. Compost mixed with sand at a specific ratio can be used to create an enriched mix for landscape beds or raised bed gardens⁶. It plays a vital role in maintaining the soil ecology by attracting earthworms and providing them a healthier diet. The presence of earthworms, redworms, centipedes, sow bugs, and other soil critters shows that compost is a healthy living material⁷.

Compost stability refers to the rate of microbial activity in the compost and is well evaluated by different respirometric measurements and/or by studying the transformations in the chemical characteristics of organic matter of the compost⁸. Compost maturity generally refers to the degree of decomposition of phytotoxic organic substances produced during the active composting phase and to the absence of pathogens and viable weed seeds. Both these properties are critical for the quality and marketability of the final product⁹.

Compost teas are aqueous extracts prepared by mixing compost in water in a specific ratio¹⁰.Compost tea is composed of water extractable components such as mineral supplements, organic acids, active microorganisms primarily bacteria, fungi, protozoa and other microbial metabolites¹¹. Various reports have suggested the application of compost tea has significantly enhanced the plant productivity by improving plant nutrient status and by diminishing disease incidence¹². In addition, an increasing body of experimental evidences indicated that compost tea can suppress some plant diseases, such as botrytis on green beans, strawberries etc¹³.Aerated compost tea (ACT) is water-based oxygen rich extract containing high populations of beneficial aerobic bacteria, nematodes, fungi, and protozoa, which can be utilized to bioremediate toxins. It has been associated with shorter brewing time¹⁴.

Generally one gram of compost will contain 80-90% of bacteria and the remaining 10% includes actinomycetes, fungi molds and yeasts¹⁵. Compared with bacteria and mycorrhizal fungi, the use of yeasts as plant growth promoting agents has been under-exploited¹⁶. Recent reports showed that yeast is capable of producing ammonia in order to make the plants accessible for the easy uptake of atmospheric nitrogen¹⁷. In addition, the production of indole 3-acetic acid (IAA) (a principal hormone that regulates various developmental and physiological processes in plants), solubilisation of zinc and phosphorous (essential micronutrients for the plant growth) by yeast were reported¹⁸.

MATERIALS AND METHODS:

Collection of compost:

Compost samples were collected from nursery of VIT university, Vellore, Tamilnadu (12.9165°N, 79.1325° E). The collected compost sample was manually cleaned and passed through a stainless steel sieve (2 mm) for consistency and stored in polythene bags at room temperature.

Characterization of compost:

The collected compost was analyzed for physiochemical and biological parameters. The physical parameters such as temperature and colour were recorded at the time of collection.

pH and electrical conductivity (EC):

The pH and electrical conductivity (EC) of the compost were measured in a 1.5:2.5 (w/v) suspension of compost and distilled water.

Estimation of organic carbon:

The organic carbon in the compost was estimated by the procedure described by Walkley and Black, (1934). In brief, 20 ml of concentrated sulphuric acid and 10 ml of 1 N potassium dichromate were added to 0.1 g of compost and incubated for 30 min at room temperature. After incubation, 30 ml of distilled water was added and titrated against 0.5 N ferrous ammonium sulphate (FAS) until the colour change was noticed using ferroin as an indicator.

Total organic carbon (%) =

where, N: normality of FAS (0.5N), T: volume of FAS used in sample titration (ml), S: volume of FAS used in blank titration (ml), ODW: oven dry sample weight (0.1 g).

Estimation of total nitrogen:

The total nitrogen content of the compost was calculated using the following equation

Total nitrogen (%) = Organic carbon x 162

Estimation of total phosphate:

The phosphorous content of the compost was estimated by Olsen’s method. A pinch of Darco-G 80 charcoal and 50 ml of 0.5 N sodium bicarbonate was added to 5 g of compost and the contents were mixed and incubated for 30 min at room temperature. The sample was filtered and 5.0 ml of filtrate was added to 10 ml of 1.15 % (w/v) ammonium molybdate and mixed until the evolution of carbon dioxide (CO₂). To the above mixture, 20 ml of distilled water and 2.0 ml of stannous chloride were added and the volume was made up to 50 ml with distilled water. The sample was analysed spectrometrically at 660 nm and the concentration of phosphorous was quantified using standard curve ploted using dipotassium phosphate (K₂HPO₄) using the following formula.

Average phosphate (ppm) = Concentration of phosphate from std graph x volume of the extractant

Estimation of Potassium:

The amount of potassium in the compost was determined by Merwin and Peach method (1951). To 25 ml of 1 M neutral ammonium acetate, 5 g of compost was added and mixed. The sample was filtered and filtrate fed into the atomizer of flame photometer. The available amount of potassium in the compost was determined from the standard curve by given formula

Available potassium (ppm) = standard curve reading x total dilution

Rate of Respiration:

Respiration is directly related to the metabolic activity of a microbial population. It plays a considerable role in the stability of compost. it is viewed as a critical element for the estimation of compost maturity and also used for the monitoring of the composting process. Fresh compost sample (5 g) was added to a beaker and sealed in a 1 L vessel containing 50 ml of 3 M sodium hydroxide (NaOH) solution. The samples were incubated at room temperature (30±2°C) for 10 days. During the incubation, the evolved CO₂ was captured by NaOH solution and the solution was titrated against hydrochloric acid (HCl) using phenolphthalein as an indicator.

Milligrams (mg) C or CO₂ =

where, V_o : The titre value of blank,V₁ : The titre value of test.

Level of Humification:

One gram of compost sample was added to Erlenmyer flask containing 10 ml of distilled water. The samples were mixed and filtered using a Whatmann filter paper and filtrate was analyzed spectroscopic ally at 472 nm and 664 nm. The level of humification (Q) was determined using the following formula,

Q (%) =

Estimation of phytotoxicity of the compost and seed germination response:

Two sets of experiments were performed to examine the presence of phytotoxic compounds in the compost. Germination test was carried out in the petriplates using an aqeous extract of compost prepared by mixing 10 g of compost in 100 ml of distilled water. The fresh aquoues extracts were added to the petridishes containing 10 seeds (raddish) placed on Whatmann filter paper. The seeds were allowed to germinate for 7 days at room temperature. Similar experiment was performed using tap water as control. After incubation, the results were recorded. The rate of germination and germination index were calculated using the following formulae,

Rate of germination (mg of CO₂/ g of substrate) =

Germination index (%) =

where G: rate of germination, L : shoot length, G_C : rate of germination in control, L_C : shoot length in control

Isolation of yeast from compost:

Yeast were isolated from the compost by serial dilution on YPD media using spread plate technique. Plates were incubated at 28°C for 72 h and observed for the appearance of yeast colonies. Well separated colonies were selected, subcultured and used for further studies.

Characterization of yeast for PGP traits:

Isolated yeast strains were characterized for various plant growth promoting traits (PGP traits) such as enzymatic activities (cellulase, tannase and chitinase), phosphate and zinc solubilization, ammonia production, HCN production and IAA production.

Enzymatic analysis:

Cellulase activity:

Cellulase activity of yeast isolates were determinate by inoculated 24 h old cultures on Carboxy methyl cellulose (CMC) agar. The plates were incubated at 28±2°C for 7 days. At the end of incubation, the plates were flooded with Gram’s iodine and the appearance of yellow coloured zone around the yeast colonies indicates the production of cellulase

Tannase activity:

Yeast isolates were streaked on media containing 1.5 % of malt extract, 0.5 % of tannic acid and 2.3 % of agar agar. The plates were incubated at 30˚C for 7 days. After incubation, 1 % of Ferric chloride was assimilated onto the above medium and the activity was visualized based on the formation of clear or transparent zone around the yeast colony.

Chitinase activity:

Chitinase activity of yeast isolates were determinate by inoculated 24 h old cultures on YPD agar amended with 1.2 % of colloidal chitin. The plates were incubated at 28±2 °C for 7 days. The formation of hallow zone around the yeast colonies indicated the production of chitinase enzyme.

Phosphate solubilization:

Qualitative estimation of solubilisation of tricalcium phosphate in broth was carried out by innocuating the yeast isolates in National botanical research institute phosphate (NBRIP) broth amended with 0.5 % of tricalcium phosphate (pH-7.0) and the broth was incubated for 7 days on rotatory shaker at 28±2 °C. After incubation, culture broth was centrifuged at 10,000 rpm for 30 min. To the 5 ml of supernatant, 2 drops of p-nitrophenol and 8 ml of ammonium molybdate-citric acid reagent were added. The contents were incuabated for 20 min and the volume was made up to 25 ml using deionised water. The available phosphorous was determined colorimetrically at 882 nm against the control.

Zinc solubilization:

The yeast isolates were grown in NBRIP media supplemented with 0.5 % of insoluble zinc compounds such as zinc oxide and zinc carbonate (pH-7.0) to study the zinc solubilization activity of yeast. The broth was incubated at 28±2°Con shaker incbator for 7 days. After incubation, culture broth was centrifuged at 10,000 rpm for 30 min The available zinc in the sample was determined using atomic adsorption spectroscopy (AAS).

Ammonia Production:

Yeast isolates were examined for the ammonia production by inoculating 24 h old cultures in glycerol agar medium (GM) containing 0.01% of (w/v) bromocresol purple. The pH of the media was adjusted to 5.0 and the plates were incubated at 28±2°C for 7 days. After incubation, ammonia production was visualization of bluish colour zone around the yeast colony.

HCN Production:

HCN production by isolated yeast were qualitatively analysed by following the method of Bakker and Schipper. The yeast islolates were grown on YPD medium amended with 4.4 % of glycine. A sterile Whatman filter paper soaked in 2.5 % of picric acid (prepared in 12.5 % (w/v) of sodium carbonate) was placed on the upper lid of the petri plate. The dishes were sealed with parafilm and incubated at 28 °C for 72 h. The change in colour on the filter paper was observed.

Indole acetic acid (IAA) production:

IAA production by yeast isolates was qualitatively estimated by inoculation 24 h old cultures on YPD broth supplemented with 0.1 % (W/V) of L-tryptophan. The pH was adjusted to 6.0 and the cultutres were incubated for 7 days at 28±2 °C on shaker incubator. After incubation, samples were centrifuged at 3000 rpm for 10 min. To 0.5 ml of the supernatant, 2.0 ml of salkowski reagent was added and incubated for 20 min. The samples were observed for development of pink colour.

Preparation and characterization of aerated compost tea:

Compost tea was prepared using aerated extraction method where compost and tap water were rmixed at a ratio of 1:10 (w/v) and manually agitated for 7 days while monitoring the pH. At the end of 7^th day, the suspension was filtered to remove unwanted solid particles. The pH and EC were analysed by the procedure described in the section 2.1. The prepared compost tea was characterized for water soluble nitrogen (WSN) by Bremar’s method and water soluble carbon (WSC) by Walkley and Black method, (1934).

Estimation of Water soluble nitrogen (WSN):

By following the methodology detailed in below, the water-soluble nitrogen in compost tea was estimated. Four grams of boric acid dissolved in hot water and the total volume was made up to 25 ml in volumetric flask. The complete sample was added to the Erlenmeyer flask containing 5 ml of compost tea and 8 ml of 40 % NaOH. The contents were titrated against concentrated H₂SO₄untill the bluish green turns to brown colour using bromocresol green and methyl red as indicators The percentage of water soluble nitrogen was calculated using the following equation.

% of WSN =

where, TV : test value, N: normality of acid.

Preparation of fortified compost tea and evaluation of their effect on the seed germination and seedling growth of different vegetable crops:

Compost tea was fortified with isolated yeast species in order enhance the efficiency of compost tea. The YPD broth containing7.5x10⁷ to 8.0x10⁹viable microbial cell counts of isolate 1 and 2 were added to the freshly prepred compost tea. The microbial enriched compost tea was maintained up to 7 days before application. The fortified compost tea and compost tea were sprayed on protray raised seedlings of radish (Raphanus sativus), tomato (Solanum lycosersicum) and chilli (Capsicum annuum). The rate of germination and plant growth parameters were recorded.

RESULTS AND DISCUSSION:

Characterization of compost:

The physical, chemical and biological properties of the collected compost are given in Table. 1. The EC value of the compost was recorded to be 2.50 mS/sec. The level of humification of the compost was revealed by the Q_4/6ratio, which was found to be 5.5% indicating the good level of humus content in the compost. The values of rate of respiration of the compost suggested that the compost was stable. The germination indices were recorded to be more than 1.0 which suggested the absence of phytotoxic compounds in the compost (Fig. 1 and Fig. 2)

Table 1: Physiochemical and biological properties of the compost.

Sl. No	Parameters	Compost values
1	pH	7.98
2	Electrical conductivity (mS/sec)	2.50
4	Temperature (°C)	31.0
5	Organic carbon (%)	3.80
6	Nitrogen (ppm)	615.6
7	Total phosphate (ppm)	2.4
8	Total potassium (ppm)	2000
9	Rate of respiration (mg of CO₂/g of substrate)	2.9
10	Level of humification (%)	5.5
11	Germination index (a) Petridish based expt (b) Pot based expt	1.1 1.3

Fig. 1: Seed germination response in Petri dishes

Fig. 2: Seed germination response

Isolation of yeast:

Two prominent yeast colonies namely, isolate 1 and isolate 2 were isolated from the compost and subcultured (Fig. 3) The isolates were maintained on YPD media for the preparation of compost tea.

Fig. 3: Isolation of yeast strains

Characterization of yeast for PGP traits:

All the yeast isolates were studied for plant growth promoting characteristics. The enzymatic activities viz., cellulase, tannase and chitinase were not observed in both the yeast isolates. Both the yeast isolates solubilize the insoluble tri-calcium phosphate and zinc sources (zinc oxide and zinc phosphate). The results revealed that the efficiency of solubilisation of insoluble phosphate and zinc compounds varied between the isolates (Fig. 4, 5 and 6). Ammonia excretion was observed in both the yeast isolates (Fig. 7), whereas HCN production was observed in isolate 2. Both the isolates were screened for IAA production and the isolate 1 was found to produce maximum IAA compared to isolate 2.

Fig. 4: Phosphate solubilisation by yeast isolates. Control (a), Isolate 1 (b) and Isolate 2 (c).

Fig. 5: Zinc oxide solubilisation by yeast isolates. Control (a), Isolate 1 (b) and isolate 2 (c).

Fig. 6: Zinc phosphate solubilisation by yeast isolates. Control (a), Isolate 1 (b) and isolate 2 (c).

Fig. 7: Ammonia excretion by yeast isolates. Control (a), Isolate 1 (b) and Isolate 2 (c).

Characterization of Compost Tea:

The physical and chemical properties of compost tea was recorded at the end of the fermentation period and are presented in Table. 2. It was obseved that compost tea was slightly alkaline. The variation in the pH during the fermentation period is presented in Fig. 8. The concentration of WSN and WSC were found to be in the acceptable limits. The GI of compost tea were above 1, suggesting the absence of phytotoxic compounds in the compost tea.

Table2: Characterization of the compost tea.

Parameters	Value
pH	7.64
Electrical conductivity (mS/cm)	1.5
Water soluble nitrogen (WSN)	0.56
Water soluble carbon (WSC)	4.2

Fig. 8: Variation in pH of the compost tea during fermentation

Effect of fortified compost tea on seed germination and seedling growth of different vegetable crops:

The stability of microbial enriched compost tea maintained up to 7 days of storage showed significantly higher number of viable cell counts as compared to compost tea. The application of fortified compost tea and compost tea significantly increased the rate of germination, plant growth parameters such as root length, shoot length and dry matter production in all the cases (Table. 3,4 and 5 and Fig. 9).An improvement was observed in radish, tomato and chilli growth parameterstreated with fortified compost tea as compared to the plants treated with compost tea and tapwater which signified the higher efficiency of fortified compost tea.

Table 3: Effect of fortified compost tea and compost tea on rate of germination of vegetable crops.

	Rate of germination (%)
	Control	Compost tea	Fortified compost tea
Radish	68	84	92
Tomato	62	89	97
Chilli	54	87	89

CONCLUSION:

The results of physical, chemical and biological properties of the composts showed that the compost is stable and matured. All the yeast isolated from the compost exhibited the IAA production, ammonia excretion, phosphate and zinc solubilisation activities. Among two yeast isolates, Isolate 2 was found to be capable of producing HCN. The major characteristics of compost teas was evaluated and the results suggested the absence of phytotoxic compounds in the compost tea. The compost tea fortified with yeast isolates showed significant increase in rate of germination and growth parameters viz., shoot length, root length and dry matter compared to control and compost tea. Hence, the obtained results state that, fortified compost tea can be used as a phytostimulant to increase the rate of germination and to enhance the growth of the vegetable crops by providing the necessary nutrients and growth factors for plant growth.

ACKNOWLEDGEMENT:

We would like to thank VIT University for providing laboratory facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 03.07.2017 Modified on 21.08.2017

Research J. Pharm. and Tech. 2017; 10(9): 3115-3122.

DOI: 10.5958/0974-360X.2017.00554.6

	Shoot length (cm)			Root length (cm)
	Control	Compost tea	Fortified compost tea	Control	Compost tea	Fortified compost tea
Radish	9.9	13.0	14.9	9.0	10.1	14.1
Tomato	11.2	14.9	15.6	5.2	7.5	11.0
Chilli	10.2	12.4	13.8	8.9	12.1	13.6

	Dry weight (mg/seedling)
	Control	Compost tea	Fortified compost tea
Radish	26.2	29.1	78.5
Tomato	70.4	83.5	88.9
Chilli	31.5	56.9	60.1