Environ Sci Pollut Res DOI 10.1007/s11356-014-3456-9
Potentiality of Eisenia fetida to degrade disposable paper cups— an ecofriendly solution to solid waste pollution Karthika Arumugam & Seethadevi Ganesan & Vasanthy Muthunarayanan & Swabna Vivek & Susila Sugumar & Vivekanadhan Munusamy
Received: 28 April 2014 / Accepted: 14 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of the present study was to subject the postconsumer waste, namely paper cups for vermicomposting along with cow dung in three different ratios for a period of 90– 140 days employing Eisenia fetida. The post-consumer wastes are a menace in many developing countries including India. This waste was provided as feed for earthworms and was converted to vermicompost. Vermicompost prepared with paper cup waste was analyzed for their physicochemical properties. Based on the physicochemical properties, it was evident that the best manure is obtained from type A (paper cup/cow dung in the ratio 1:1) than type B (paper cup/cow dung in the ratio 1.5:0.5) and type C (paper cup/cow dung in the ratio 0.5:1.5). The results showed that earthworms accelerated the rate of mineralization and converted the wastes into compost with needed elements which could support the growth of crop plants. The predominant bacterial strains in the vermicompost were characterized biochemically as well as by 16S ribosomal RNA (rRNA) gene sequencing. The bacterial strains like Bacillus anthracis (KM289159), Bacillus endophyticus (KM289167), Bacillus funiculus (KM289165), Virigibacillius chiquenigi (KM289163), Bacillus thuringiensis (KM289164), Bacillus cereus (KM289160), Bacillus toyonensis (KM289161), Acinetobacter baumanni (KM289162), and Lactobacillus pantheries (KM289166) were isolated and identified from the final compost. The total protein content of E. fetida involved in vermicomposting was extracted, and the banding pattern was analyzed. During final stages of vermicomposting, it was Responsible editor: Philippe Garrigues K. Arumugam : S. Ganesan : V. Muthunarayanan (*) : S. Vivek : S. Sugumar : V. Munusamy Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India e-mail: drvasan[email protected]
V. Muthunarayanan e-mail: [email protected]
observed that the earthworm did not act on the plastic material coated inside the paper cups and stagnated it around the rim of the tub. Further, the degradation of paper cup waste was confirmed by Fourier transform infrared spectroscopy analysis. Hence, vermicomposting was found to be an effective technology for the conversion of the paper cup waste material into a nutrient-rich manure, a value-added product. Keywords Vermicomposting . Paper cups . Eisenia fetida . 16S rRNA . SDS-PAGE . FT-IR
Introduction Solid waste management in Indian cities has emerged as one of the major concerns over the past few years. The rise in urban population and economic growth together with the absence of an effective management mechanism has been stated to be responsible for the current unfortunate status of solid waste management in India (Federation of Indian Chambers of commerce and Industry Federation of Indian Chambers of commerce and Industry FICCI, 2009). Urban India generates 1,88,500 t of municipal solid waste per day at per capita rate of 500 g/person/day reported by National Solid Waste Association of India (www.nswai.com) Mumbai, India. The growth of industries and ever increasing human population have led to an increased accumulation of waste material, of which paper waste is one of the major components. The household generates around 2.6 million tones of recyclable waste per annum, out of which 1.3 million tones are contributed by paper alone. The paper waste includes newspaper, magazines, books etc., of this post-consumer waste that refers to the waste which cannot be reused or recycled anymore (Karthika et al. 2014). The common and highly
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generated post-consumer waste includes packaging material, old magazine, waste clothes, used batteries, etc. Therefore, the post-consumer waste needs much more attention in developing countries like India. Most importantly, disposable paper cups are found in many places like office canteens, fast food restaurants, railway stations, and festival areas (Plate 1). Unfortunately, paper cups have a significant negative effect on the environment which might nullify any of the benefits of using them. During the manufacturing process, paper cups are laminated with a plastic resin called polyethylene (PE) for the purpose of keeping the beverages warm and to make the cups leak proof. But, the plastic material prevents the cups from being recycled (Kennedy 2012). Every paper cup coated with plastic resin ends up in landfill and not get decomposed. Overall 6.5 million trees are cut down to make 16 billion paper cups which result in 253 million pounds of waste (Whirely, 2006). Every tree used for making paper cups is also removed permanently from the ecosystem, and no longer could they absorb carbon dioxide, produce oxygen, or filter groundwater. Lack of awareness might be one of the prime reasons for the unorganized disposal of such products which end up in pollution. Awareness camps are also needed to make people understand that there exits an alternative for the post-consumer waste management in an ecofriendly manner. To overcome this, there is a need for an efficient technology which has the potential to minimize the burden on the landfill, as well as to reduce the environmental hazards due to improper solid waste disposal. Composting may be one of the options which involves the accelerated degradation of the post-consumer waste by microorganisms under controlled condition (Renuka and Garg 2008; Kaviraj and Sharma 2003). And also, the joint action of earthworm and microorganism will be an efficient and fastidious alternative for the biooxidation of the paper cup waste material. Therefore, discarded paper cup wastes can be a good feed for earthworm employed for the
vermicomposting process. From environmental and economic point of view, vermicomposting technology appears to be an efficient and sustainable method to dispose such wastes (Chris et al. 2006; Ndegwa and Thompson 2001). Hence, the aim of the present study was to subject paper cup wastes along with cow dung and Eisenia fetida popularly called manure worm for vermicomposting. Further, the end product was subjected to estimation of various physicochemical parameters like total organic carbon (TOC), total Kjeldahl nitrogen (TKN), total organic matter (TOM), C/N ratio, total phosphorus (TP), pH, electrical conductivity (EC). The bacteria involved in the vermicomposting process were also identified by amplifying the 16S ribosomal RNA (rRNA) gene sequence. The changes in the protein profile of the engaged earthworms were also studied. Further, FT-IR analysis was performed with vermicomposted samples to characterize the nature of the end products.
Materials and methods Field sites and sampling The paper cup wastes were collected from the disposal site of Bharathidasan University campus, Tiruchirappalli, Tamil Nadu, India. The earthworm E. fetida hatchling and ciliated adults were randomly picked for experiment from several stock cultures containing 500–2,000 earthworms maintained in the laboratory with cow dung as a culturing medium. Preparation of experimental media Three 5-l circular plastic tubs (diameter 40 cm, depth 9 cm) were filled with the feed mixture as given below and were subjected for vermicomposting process. Type A (1:1 ratio): 1,000 g of shredded paper cup wastes+1,000 g of cow dung slurry Type B (1.5:0.5ratio): 1,500 g of shredded paper cup wastes+500 g of cow dung slurry Type C (0.5:1.5): 500 g of shredded paper cup wastes+ 1,500 g of cow dung slurry
Plate 1 Paper cup disposal site
The mixtures were mixed manually to facilitate predecomposition up to 20 days. At the end of 21 days, 20 earthworms with an average weight of 0.15 to 0.2 g each were introduced into each feed mixture. The moisture content was maintained at 60–80 % throughout the study period by sprinkling adequate quantity of water. The whole assay was performed under room temperature in triplicates.
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Physicochemical characterization of the vermicompost Homogenized samples (free from earthworms, hatchling, and cocoons) from feed mixture tubs (types A, B, and C) were drawn after 1, 4, 8, 12, 16 and 19 weeks. pH, EC, TOC, TOM, C/N, TKN, and TP of the samples were analyzed (Tandon 2009; Senesi 1989). Microbial study Total heterotrophic bacterial (THB) population of the vermicompost samples was quantified by serially diluting 1 g of freshly weighed sample up to 10-3 to 10 -7 dilution and 100 μl of each dilution was spread on nutrient agar plate (Yasir et al. 2009). From the bacterial colonies, the genomic DNA was obtained using the total genomic DNA extraction mini kit (Real Biotech Corporation, Chennai). The amplification of the 16S rRNA gene was performed using universal primers (27 F 5′-AGAGTTTGATCMTGGC TCAG-3′ and 1492R 5′-TAC GGYTACCTTGTTAC GACTT-3′). The total reaction mixture of the PCR consisted of 50 μl with the following ingredients: master mix 25 μl, template 10 μl, forward primer 1 μl, reverse primer 1 μl, and sterile millQ 13 μl to final volume. The PCR reaction conditions were initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 1.30 min, extension at 72 °C for 2.30 min followed by final extension for 7 min (Vivas et al. 2009).
obtained pellet, 1 ml of ice cold 0.1 % TCA was added and centrifuged at 12,000 rpm for 15 min. The supernatant was discarded, and the pellet was added to 1 ml of ice cold acetone followed by centrifugation at 12,000 rpm for 15 min. The pellet was resuspended in minimum volume of PBS and was stored at −20 °C. The concentration of protein was quantified using Bradford method (Bradford 1976). The protein thus extracted was resolved using 12 % SDS-PAGE. The gel was stained with coomassie staining (G-250).
Statistical analysis The significant differences in the physicochemical parameters among types A, B, and C were analyzed by one-way ANOVA using SPSS software version 16.0, and the differences were significant at p< 0.05. Data were expressed as mean±SEM. FTIR spectroscopy About 5 mg of dried vermicompost sample was mixed with 400 mg of potassium bromide (KBr) and was compressed under vacuum for 10 min. The FTIR spectra were recorded on potassium bromide pellets between 4,000 and 400 cm−1 at a rate of 16 nm/s using Perkin Elmer 1,600 FTIR spectrometer (Zainab et al. 2009).
DNA sequence analysis Results and discussion DNA was sequenced in both directions, and consensus sequence was generated. The amplified 16S rRNA sequence was compared with the nucleotide sequence present in the GenBank using the standard BLASTN site at NCBI server (http://ww.ncbi.nlm.nih.gov/ BLAST) (Stephen et al. 1990) From the aligned sequence, a neighbor-joining phylogram was constructed using MEGA software. Protein estimation In this study, the protein content of E. fetida was extracted using phosphate buffer saline (PBS). The whole earthworm was homogenized with 2 ml of PBS followed by centrifugation at 12,000 rpm for 15 min. The supernatants were collected and again centrifuged at 12,000 rpm for 15 min. The final supernatants were collected and stored at −20 °C. To 100 μl of supernatant, 12.5 μl of 100 % trichloroacetic acid (TCA) was added and centrifuged at 12,000 rpm for 15 min. To the
Characterization of vermicompost During the process of vermicomposting, earthworms mineralize the waste material and convert a part of it as the worm biomass and the rest is egested as nutrientrich vermicompost. The appropriate feed composition for the earthworm could be optimized based on the manural value of the vermicompost. To establish this, physicochemical analysis of the vermicompost samples on the 1st, 4th, 8th, 12th, 16th, and 19th week were p er f o r m e d , a nd th e r es u l t s w er e p r e s en t e d i n (Fig. 1a, b). The vermicompost obtained after 19 weeks of earthworm activity was much finer, odor free, and brown in color. As compared to the 1st week, TOC of the final vermicompost was remarkably reduced. The TOC values in types A, B, and C were 39, 40, and 59 %, respectively (Fig. 1a). Lavelle et al. (1989) have reported that during vermicomposting process, TOC reduction to the range of
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Fig. 1 a, b Changes in the physicochemical parameters of types A, B, and C. Mean values obtained from the 19th week samples are significantly different from each other with the level of significance at *p