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Available online at http://www.urpjournals.com International Journal of Research in Environmental Science and Technology Universal Research Publications.

Anbalagan and S. Manivannan* Department of Zoology, Annamalai University, Annamalainagar, 608 002, Tamilnadu, India. *Corresponding Author: sarosubamani@rediffmail.

1 Available online at International Journal of Research in Environmental Science and Technology Universal Research Publications. All rights reserved ISSN Original Article Effect of organic additives on the microbial population and humic acid production during recycling of fly ash through vermitechnology M. Anbalagan and S. Manivannan* Department of Zoology, Annamalai University, Annamalainagar, , Tamilnadu, India. *Corresponding Author: Received 28 August 2012; accepted 09 October 2012 Abstract Fly ash is generated in huge amounts and cause serious hazards to the environment. In this experiment, different proportions of fly ash (FA)with organic additives i.e. cow dung (CD), press mud (PM) and crop residue (CR) were used as food for epigeic earthworm Eisenia fetida to standardize the recycling technique of these wastes and to study their effect on microbial population and its activity and humic acid content after vermicomposting. The results suggested that the total microbial population of vermicompost was significantly higher than initial substrate and worm unworked natural compost. The microbial activities of vermicompost obtained from all the treatments (T 1 -T 8 ) were significantly increased after vermicomposting. Humic acid content in the vermicompost was also higher than initial substrate and worm unworked natural compost. The maximum humic acid content was registered in T 5 treatment followed by T 8 treatment. Results revealed that E. fetida had considerable effects on T 5 treatment during vermicomposting. Periodical analysis of above mentioned microbial properties and humic acid content of final vermicompost indicated that equal proportion of FA, CD, PM and CR are probably the optimum composition to obtain best quality vermicompost Universal Research Publications. All rights reserved Key words: Vermicomposting, E. fetida, microbial activity, humic acid, fly ash. INTRODUCTION Vermicomposting is a kind of composting process, carbon dioxide producing decomposition process under controlled condition [1] (Hoitink and Kuter, 1986), involving composting earthworms [2] (Reinecke et al., 1992). During vermicomposting, earthworms fragment the organic waste, stimulate microbial activity and increase rates of mineralization, rapidly converting the wastes into humuslike substances having diverse microbial population [3] (Elvira et al., 1998). According to Lazcano et al [4] (2008) after transit of organic substrates through earthworm guts, rate of CO 2 evolution, bacterial plate count and soluble organic carbon content of wastes were increased. The ability of epigeic earthworms to consume and breakdown a wide range of organic residues such as sewage sludge, animal wastes, crop residues and industrial refuse is well known [5] (Hartenstein and Bisesi, 1989). The exotic epigeic species, like Eudrilus eugeniae [6] (Ashok, 1994), Eisenia fetida [7] (Manivannan et al, 2004) and Perionyx excavates [8] (Suthar, 2007) are usually being used for vermicomposting. Yasir et al [9] (2009) showed that changes in bacterial community play a major role during vermicomposting. In addition to bacteria, fungi especially cellulolytic fungi also play an important role during vermicomposting. Population of cellulolytic fungi was found to be increased during vermicomposting of different organic wastes [10] (Pramanik et al., 2007). As the end product i.e. vermicompost is pathogen free, odourless and rich in plant nutrients as compared to conventional compost. Agricultural utilization of vermicompost will help in recycling the plant nutrients to soil and also avoid soil degradation. Agricultural utilization of vermicompost will also add to the economy by reducing the load on inorganic fertilizer and increasing the plant yield. Moreover using vermicompost as organic amendment will help in maintaining the sustainability of ecosystem. Fly ash, the inert residue obtained from complete combustion of coal, causes environmental hazards and creates problems occupying large storage areas [11] (Nass et al., 1993). Therefore, it is important to overcome these problems not only by safe disposal but also through conversion of these materials to value-added products [12] (Chattopadhyay and Bhattacharya, 2000). Bhattacharya and Chattopadhyay [13] (2004) tried different proportions of cow manure and fly ash to reveal the best composition for 96

2 recycling fly ash through vermicomposting. Fly ash are very poor in N and needed to be mixed with other, N rich, organic amendments in order to provide nutrients and an inoculam of microorganisms during vermicomposting. Therefore, in this experiment organic supplements mixed with fly ash in different proportions to study the changes in total microbial population and its activity and humic acid production after vermicomposting. The objectives of this experiment were to study suitability of organic supplements (cow dung, press mud and crop residue) for vermicomposting fly ash and to standardize right proportion of fly ash and organic supplements and efficiency of E. fetida for vermicomposting fly ash. MATERIALS AND METHODS Organic additives and earthworms: Fly ash (FA) was procured from the dumping site of thermal power station I, Neyveli Lignite Corporation (NLC), Tamil Nadu, India. Press mud (PM) was obtained from effluent treatment plant of E.I.D. Parry Sugar Mill located at Nellikkuppam, Tamil Nadu, India. Fresh cow dung (CD) and crop residue (CR) were collected from the agricultural farm, Faculty of Agriculture, Annamalai University, Tamil Nadu, India. The main physico-chemical characteristics of FA, CD, PM and CR are given in Table 1. The composting earthworm Eisenia fetida was cultured in the laboratory and were randomly picked for experimentation. Table 1. The main physico-chemical characteristics of FA, CD, PM and CR S.No Parameter FA CD PM CR 1 ph EC(dSm -1 ) TOC(gkg -1 ) TN(gkg -1 ) TP(gkg -1 ) TK(gkg -1 ) Ca(gkg -1 ) Mg(gkg -1 ) Zn(mgkg -1 ) Fe(mgkg -1 ) FA-Fly ash; CD-Cow dung; PM-Pressmud; CR-Crop residue Treatment design: Fly ash (FA) alone and in combination with cow dung (CD), press mud (PM) and crop residue (CR) were used as substrate for the studies. The CD and PM were dried in air at room temperature. FA was mixed with CD, PM and CR in different ratios in order to produce different treatments (dry weight proportion). The composition of FA, CD, PM and CR in different treatments are described in Table 2. One kg of substrate material was added to each circular plastic container (Vol. 10L, diameter 38cm, depth 14cm) for experimental trial. All the treatments were kept for 21 days prior to experimentation for thermal stabilization, initiation of microbial degradation and softening of substrate material (pre-composting). Twenty clitellated earthworms, Eisenia fetida was inoculated into each treatment, separately after 21 days of pre-composting. During the vermicomposting period, the moisture content of the substrate in each treatment was kept at 70 75% by periodic sprinkling of adequate quantity of water. The experimental treatments were kept in triplicate. Samples for periodical analysis were taken before inoculating earthworms and at the end of experimentation. Tablei2. The composition of fly ash and other organic waste S. No Treatments Composition a (dry weight basis) 1 T 1 FA alone 2 T 2 1 part FA:1part CD 3 T 3 1 part FA:1part PM 4 T 4 1 part FA:1part CR 5 T 5 1 part FA:1part CD:1part PM 6 T 6 1 part FA:1part CD :1part CR 7 T 7 1 part FA:1part PM:1part CR 8 T 8 1 part FA:1part CD:1part PM:1part CR FA-Fly ash; CD-Cow dung; PM-Pressmud; CR-Crop residue Microbial analysis Quantitative analysis of microbes: For the purpose of quantitative analysis of microbes, the samples were collected from gut of earthworms E.fetida reared in all the treatments (T 1 T 8 ), initial substrate, worm unworked natural compost (control) and vermicompost produced by E.fetida obtained from all the treatments (T 1 -T 8 ). The total microbial populations (bacteria, fungi and actinomycetes) were determined in the aforementioned samples by the following methods. Determination of total microbial populations: 1 gram of each substrate was diluted in one ml of sterile saline in a sterile test tube. The tubes containing substrate were shaken thoroughly in a Vortex mixture for 5 seconds. Three sets of Sabouraud Dextrose Agar (SDA) and Rose Bengal Agar (RBA) plates for fungal growth, three sets of Nutrient Agar (NA) and MacConkey Agar (MA) plates for bacterial growth and three sets of Actinomycetes Agar (AA) plates for actinomycetes growth were used for each substrate. The substrate inoculum in 0.01ml was spread on the surface of these media to estimate the number of bacterial, fungal and actinomycetes colonies. The fungal plates were incubated at 25 C to 37 C for 5-7 days, 37 C for hours of incubation for bacteria and 25 C to 35 C for days of incubation for actinomycetes. The different microbial colonies developing on the plates were estimated by counting. The number of colony forming unit (CFU) on the surface of the media was counted and expressed as CFU 10 6 g -1, according to the method described by Baron et al. [14] (1994). Determination of microbial activity (dehydrogenase activity): To determine the microbial activity (in terms of dehydrogenase activity), samples were collected from initial substrate, worm unworked natural compost (control) and vermicompost of all the treatments (T 1 T 8 ). Dehydrogenase activity was determined according to the method described by Stevenson [15] (1959). Extraction of humic acid (HA): The humic acid content was extracted by adopting the procedure as described by Schnitzer [16] (1978). 5 gram of fine sieved sample was dissolved in 100 ml of 0.5N NaOH. The liquid was shaken for one hour in a mechanical shaker and allowed to stand at room temperature for 24hrs. The dark brown liquid was 97

3 filtered through Whatman No.1 filter paper. The filtrate was collected in a glass jar, acidified with 6N HCl to ph1. After 3hrs the supernatant liquid (fulvic acids) was separated from the coagulate (humic acids) by siphoning off. Then the coagulate was dialysed extensively against distilled water till free of chloride and finally dried in hot air oven at 40 C. The humic acid contents are expressed in mg/5g substrates. Statistical analysis: All the reported data are the arithmetic means of three replicates. Two way analysis of variance (ANOVA) was done to determine any significant difference among the treatments at 0.05% level of significance. RESULTS AND DISCUSSION The total microbial population and its activity and humic acid content in these experiments are shown in Table 2 to 4. In the present study, the total microbial population of vermicompost was higher than initial substrate and worm unworked natural compost. Total microbial population ranged from 3.01±0.46 to 4.32±0.20 in the gut of E. fetida in all the treatments (T 1 T 8 ). Likewise, the total microbial population of vermicompost produced by E. fetida ranged from 3.13 ± 0.37 to 4.47 ± 0.16 in all the treatments (T 1 T 8 ). Among the different treatments, T 5 and T 8 treatments were found to have significantly (p<0.05) higher microbial population than other treatments (Table 3). Organic mixture was stabilized by mutual interaction between earthworms and microorganisms during vermicomposting [17] (Edwards and Fletcher, 1988). Data suggested that E. fetida was effective for vermicomposting fly ash with other organic waste mixture and they facilitate proliferation of microbial population, which in turn hastened the decomposition of feed materials. Hendrikson [18] (1990) and Fischer et al. [19] (1997) revealed that increase in fungal count during vermicomposting of different organic waste. They also reported that not all the microbes, present in the organic wastes, were killed during passage through the earthworm guts; in fact their population was increased in ejected materials. Aira et al. [20] (2006) reported that microbial population particularly fungal growth was activated in presence of earthworms, which in turn triggered cellulose decomposition during vermicomposting. Pramanik et al. [10] (2007) reported an increased population of bacteria and fungi in the cast of E. fetida and also they suggested that enhanced germination of microbial spores under the favourable conditions of earthworm guts. Table 3. Total microbial population in gut of earthworms, fly ash and organic waste mixture and vermicompost (Mean ± sd; n = 3) Total microbial population (CFU 10 6 g -1 ) Treatments Initial substrate Gut of worms Worm un worked natural compost Vermicompost T ± ± ± ± 0.31 T ± ± ± ± 0.11 T ± ± ± ± 0.47 T ± ± ± ± 0.37 T ± ± ± ± 0.16 T ± ± ± ± 0.40 T ± ± ± ± 0.21 T ± ± ± ± 0.25 Table 4. Microbial activity in initial, worm unworked natural compost and vermicompost produced by E. fetida (Mean ± sd; n = 3) Total microbial activity (μl 5g) Treatments Worm un worked natural Initial substrate compost Vermicompost T ± ± ± 0.61 T ± ± ± 0.27 T ± ± ± 0.15 T ± ± ± 0.31 T ± ± ± 0.29 T ± ± ± 0.27 T ± ± ± 0.19 T ± ± ± 0.42 In the present analysis, microbial activity was higher in the vermicompost, when compared to the initial substrate and worm unworked natural compost. The microbial activities of vermicompost obtained from all the treatments (T 1 -T 8 ) were increased significantly and especially T 5 (7.44 ± 0.29) and T 8 (7.35 ± 0.42) treatments were found to have significantly (p<0.05) higher microbial activity than other treatments (Table 4). Atiyeh et al [21] (2000) reported that during ingestion of organic substrates, earthworms not only fragment them, but also stimulate microbial activity and increase humic acids content by enhancing rates of mineralization. Earthworms produce an enormous amount of intestinal mucus composed of gluco-proteins and small glucosidic and proteic molecules [22] (Morris, 2005). The microbes entering the worm guts consume these nitrogenous compounds of mucus [23] (Zhang et al., 2000), 98

4 Table 5. Humic acid content in initial substrate, worm unworked natural compost and vermicompost produced by E. fetida (Mean ± sd; n = 3) Humic acid content (mg / 5g) Treatments Initial substrate Worm un worked natural compost Vermicompost T ± ± ± 0.37 T ± ± ± 0.19 T ± ± ± 0.12 T ± ± ± 0.27 T ± ± ± 0.25 T ± ± ± 0.35 T ± ± ± 0.41 T ± ± ± 0.26 which mainly increase their activity, which in turn enables them to contribute enzymes to the digestive processes of the earthworms. These enzymes come with the ejected materials of earthworms. Hong et al [24] (2011) suggested that organic material passes through the earthworm gut, the resulting vermicast is rich in microbial activity, plant growth regulators and pest repellents. The epigeic earthworm, E. fetida, commonly known as a red wiggler, is a particularly efficient vermicomposting earthworm because it can consume its own body weight in food each day [25] (Tripathi and Bhardwaj, 2004). In E. fetida, a variety of intestinal microorganisms that produce enzymes, such as amylase, proteases, lipases, and cellulases, enhance the biodegradation of organic matter [20] (Aira et al., 2006). According to Barois and Lavelle [26] (1986) earthworm primes its symbiotic gut microflora with secreted mucus substances to increase their degradation of ingested organic matter and the release of assailable metabolites. Therefore, in the present study directly or indirectly earthworm enriches the substrate material with microbial activity. Table 5 shows the humic acid content of initial substrate, worm unworked natural compost and vermicompost produced by E. fetida. Humic acid content in the vermicompost was also higher than initial substrate and worm unworked natural compost. The maximum humic acid content (0.621 ± 0.25) was registered in T 5 treatment followed by T 8 treatment (0.611 ± 0.26). There was no significant (P<0.05) difference between T 5 and T 8 treatments. Earthworms, one of the major macro invertebrate groups and ecosystem engineers in temperate and tropical soils, have been shown to exert a very important role in the humification of organic matter [27] (Plaza et al., 2008). According to Atiyeh et al [21] (2000) earthworm fragment the organic substances, stimulate microbial activities greatly and increase rate of mineralization, rapidly converting the waste into humus-like substances. In general, the total microbial population, microbial activity and humic acid content of vermicompost were increased significantly (p<0.05) than initial substrate and worm unworked natural compost. During vermicomposting, earthworms fragment the organic waste substances, stimulate microbial activity and increase rates of mineralization, rapidly converting the wastes into humuslike substances having diverse microbial population [3].(Elvira et al., 1998). CONCLUSION In the present study, total microbial population (bacteria, fungi and actinomycetes) and its activity and humic acid content were found to have increased in the vermicompost of E. fetida obtained from all the treatments (T 1 -T 8 ) over initial substrate and worm unwormed natural compost. The significantly increased level of microbial population and its activity and humic acid content in the vermicompost could be due to the higher nutrient concentration in the substrate and cast, multiplication of microbes while passing through the gut of worms, optimal moisture and large surface area of casts ideally suited for better feeding, multiplication and activity of microbes and production of humic acid. Finally it was concluded that earthworms did not feed on raw fly ash and accepted it as a diet only when organic additives were spiked with it. Mixing of certain organic supplements as bulking agent in fly ash creates suitable microcosms for the earthworms. ACKNOWLEDGEMENT The authors are very thankful to the University Grants Commission (UGC, New Delhi) for the financial support in the form of a Major Research Project and also we thank Prof. Dr. M. Sabesan for encouragement. REFERENCES 1. H. A. J. Hoitink, G.A. Kuter, Effects of composts in growth media on soil borne pathogens, In: Y. Chen, Y. Avnimelech, (Eds.), The Role of Organic Matter in Modern Agriculture. Martinus Nijhoff Publishers, Dordrecht, Netherlands, 1986, pp A. Reinecke, S.A. Viljioen, R. J. Saayman, The suitability of Eudrilus eugeniae, Perionyx excavatus and Eisenia fetida (Oligochaete) for vermicomposting inbsouthern Africa in terms of their temperature requirements. Soil Biol. Biochem. 24 (1992) C. Elvira, L. Sampedro, E. Benitez, R. Nogales, Vermicomposting of sludges from paper-mill and dairy industries with Eisenia andrei: a pilot-scale study. Biores. Technol. 63(1998) C. Lazcano, M. Gomez-Brandon, J. 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Effect of Storage Duration in the Quality of Vermicompost

Research article erd Effect of Storage Duration in the Quality of Vermicompost NATTIRA KLEAWKLAHARN Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand CHULEEMAS BOONTHAI IWAI* Faculty of