International Journal of Process Wastes Treatment Vol. I, No. I, January-June 2009, pp.

71-81

. I I PWT
PubUahed by luten.liona! ScIence PraI

.J

Growth and Fecundity of Eisenia Fetida Earthworm During Vermicomposting of Food Industry Sludge
Anoop Yadav*l, Ackmez Mudhoo**& Vinod K. Garg*2

ABSTRACT This paper reports the growth and fecundity of an epigeic earthworm Eisenia fetida during the transformation of food industry sludge (FIS) spiked with biogas plant slurry (BPS) into vermicompost. The results showed that Eisenia fetida was unable to service in 100% FIS. Addition of some other organic waste to FIS was necessary during vermicomposting. Addition ofFIS with BPS in smaller quantities (20% to 30% on dry weight basis) had no adverse effect on the growth and fecundity of Eiseniafetida because biomass gains by earthworms in 20% FIS was higher than control (100% BPS). The maximum growth rate and cocoon production by Eiseniafetida was 16.21:1:0.64 mg worm-I day-' and 0.21 cocoons worm-I day-I respectively in a 80% BPS + 20% FIS feed mixture. Whereas, minimum growth rate and cocoon production by Eisenia fetid a was 8.14:1:0.32 mg worm-I day-Iand 0.07 cocoons worm-I day-' respectively in feed mixture no. 7 (40% BPS + 60% FIS), a higher percentage of FIS in the feed mixture significantly affected the biomass gain and cocoon production by Eisenia fetida. Finally it is concluded that vermicomposting can be an alternate waste treatment route for final management of food industry sludge. Keywords: Vermicomposting; Eiseniafetida; Biomass growth; Cocoon production; Biogas plant slurry.

1. INTRODUCTION Food processing and packaging industries have emerged as one of the most polluting industries in recent years (Kirby et aI., 2003; Murto et aI., 2004). The authors have observed that most of the food industries dispose their wastewater treatment sludges (Fatta et aI., 2004) by improper and indiscriminate disposal methods (Dieu, 2006; Jardin, 2006; Arvanitoyannis and Ladas, 2008) which not only cause pollution but also lead to the loss of nutrients present in them (Elvira et al., 1985, Wang et al., 2005). Vermicomposting has been reported as a viable, cost-effective, and rapid technique for the management of the organic wastes (Kaushik and Garg, 2003; Maboeta and van Rensburg, 2003; Banu et al., 2005; Contreras-Ramos et al., 2005; Sun et al., 2006; Suthar,2007a). The product, vermicompost is a well stabilised, aestheticallypleasing, fmely divided peat-like material with excellent structure,porosity,aeration, drainage and enhanced moisture holding capacity with the capability of enhancing plant growth (Hashemimajd et aI., 2004; GutierrezMiceli et al., 2007; Zaller, 2007). Padmavathiamma et aI., (2008)concluded in their study that vermicompostamended acid-agriculture-soil significantly improved the yield, biometric character and quality of banana, cassava and cow-pea and vermicompost application stimulated root growth, facilitating nutrient absorption and thereby favouring higher yield. Several reports are available of the use of earthworms in the management of sewage sludge and it has been established that earthworms hasten the composting process (Loh et al., 2005; Nogales et aI., 2005; Nair et aI., 2006; Campitelli and Ceppi, 2008; Martin-Gil et al., 2008) with production of a better quality of
Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India, E-mails:.yadavanoop@rediffmail.com.&2vinodkgarg@yahoo.com Department of Science (Chemistry Division), Universal College, Department B, SSR Road, Mauritius, E-mail: ackmezchem@yahoo.co.uk
Com-I-E\ Vijay Jha\Journals-2009\004-lnlernational Journal of Process Wastes Treatment, I(I), (January-June 2009)\5. S. Mukherjee

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International Journal of Process Wastes Treatment (IJPWT)

compost as compared to traditional composting methods. Butt (1993) showed that paper mill sludge had no deleterious effects on the earthworms, although worm growth rate was poor. Elvira et al., (1998) reported the growth and reproduction of Eisenia andrei in mixed paper-pulp mill sludge and cattle waste. But there are only a few studies on the influence of industrial sludges on the growth and fecundity of earthworms (Gunadi et al., 2002, 2003; Garg et al., 2005). Earthworms feed on organic matter and utilize a small amount of that for their body synthesis and excrete a large part of the consumed materials in a partially digested form as worm casts (Loh et al., 2005). Eiseniafetida is one of the most promising earthworm species used for vermicomposting in northern India (Garg et a/., 2006). There are abundant reports on the response of earthworms to different type of organic substrates, but there is a lack of information about the effect of feed composition on earthworm growth and fecundity of earthworms. The potential of utilization of an earthworm species for vermicomposting and vermiculture depends on its survival (Aira et al., 2006; Majumdar et al., 2006), growth (Reinecke and Viljoen, 1990; Dominguez et al., 2000; Gunadi et al., 2002; Pizl and Novcikovci,2004) and fecundity (Garcia and Fragoso, 2002; Gunadi et al., 2002) in different feeds. Keeping this in view, the present study was designed and conducted to assess the growth and fecundity of Eisenia fetida in food industrial sludges mixed with biogas plant slurry during the vermicomposting experiments. 2. MATERIAL AND METHODS

2.1 Materials Food industry sludge was procured from the wastewater treatment plant of a food industry located at Bahadurgarh, India. It was dried in shade before use in vermicomposting experiments. The main characteristics ofFIS are given in Table I. Anaerobically digested biogas plant slurry (BPS) was procured from post methanation storage tank of an on-farm biogas plant located at the village Agroha, Hisar, India. The raw material used in the biogas plant was cow dung. The main characteristics of BPS are also given in Table 1. All physico-chemical analyses were carried out as reported earlier in Garg et a/., (2006). Healthy
Table 1 Initial Physico-Chemical Characteristics of BPS and FIS Parameter pH Electrical Conductivity (ds m'l) Ash content ( g/kg) Total Organic Carbon (g/kg) TKN (glkg) Total Phosphorus (g/kg) Total Potassium (glkg) C:N ratio Total Sodium (glkg) Total Calcium (glkg) Fe (mg/kg) Cu (mglkg) Cr (mg/kg) Zn (mglkg) Cd (mg/kg) Ni (mg/kg) Pb (mg/kg) BPS 7.8:1:0.60 1.4:1:0.1 186.6:1:12.5 471:1:11.0 6.2:1:0.2 5.6:1:0.3 4.21:1:0.2 75.9:1:3.2 1.95:1:0.2 1.4:1:0.09 1077:1:15 22.7:1:1.4 127.5:1:4.6 31:1:5.0 6.3:1:0.7 8.7:1:2.2 28.9:1:9.5
FIS 6.4:1:0.40 1.6:1:0.2 280:1:8.5 417:1:6.7 14.7:1:0.3 12.34:1:0.3 2.34:1:0.1 28.7:1:1.5 10.31:1:0.5 3.85:1:0.3 1447:1:13 174.9:1:11.0 255.5:1:2.9 318.8:1:46.0 9.1:1:0.3 47.7:1:2.8 62.6:1:3.0

Growth and Fecundity of Eisenia Fetida Earthworm DuringVermicomposting of Food Industry Sludge

73

non-clitellated hatchlings weighing 100-150 mg (live weight) were randomly picked up for the subsequent experiments from stock cultures maintained by authors in the laboratory taking cow dung(Moisture (%), 69.4; Ash content (gkg-I), 195; pH (1: 10 ratio), 8.2; TOC (g kg-I), 467; TKN (g kg-I), 7.7; C: N ratio, 60.6; TK (g kg-I), 4.8 and TAP (g kg-I),3.3) as culture medium. The worms were adapted to laboratory conditions (Temperature 25:f:3C, moisture 70:f:l0%) before inoculating into the reactors. 2.2 Experimental Setup Seven vermireactors were established having different ratios of FIS and BPS. 250 g of each feed mixture were filled in circular plastic containers (diameter 16em, depth 10 cm) on dry weight basis. The composition ofFIS and BPS in different vermireactors is given in Table2. Each vermireactor was establishedin triplicates. All feed mixtures were pre-composted for 3 weeks in order to remove volatile toxic substances and semicompost the feed so it becomes palatable to worms. After 3 weeks, 10 non-clitellated hatchlings of Eisenia fetida were inoculated in each vermireactor. All the vcrmireactors were kept in dark at a controlled temperature of25:f:3C.The moisture content was maintained at 70:f: 10% by periodic sprinkling of distilled water throughout the study period and by covering the vermireactors with jute covers to limit evaporative losses of moisture. During the study period no extra feed was added at any stage. Biomass gain, clitellum development, hatchlings and cocoon production by the earthworms in each feed mixture were measured and recorded on weekly interval for 13 weeks. The feed in the container was turned out, and earthworms and cocoons were separated from the feed by hand sorting, after which they were counted and weighed after washing with water and drying them by paper towels. Then all earthworms and the feed were returned to their respective container. The worms were weighed with full gut.
Table 2 The Composition of Food Industry Sludge (FIS) and Biogas Plant Slurry (BPS) in Different Vermireactors VermireactorNo. 1 2 3 4 5 6 7 Description 100% BPS 90% BPS + 10% FIS 80% BPS + 20% FIS 70% BPS + 30% FIS 60% BPS + 40% FIS 50% BPS + 50% FIS 40% BPS + 60% FIS BPS(g) 250 225 200 175 150 125 100 FIS (g) 0 25 50 75 100 125 150

Statistical analysis: One-way ANOVAwas used to analyze the significant differences among different vermireactors for studied parameters. Tukey's t-test was also performed to identify the homogeneous type of vermireactors for the various parameters. The probability levels used for statistical significance were p < 0.05 for the tests. 3. RESULTS AND DISCUSSIONS

Experiments were conducted in 100% FIS but Eiseniafetida could not tolerate the 100% FIS. It has been reported that fresh sludge can be toxic for earthworms by hindering their growth and reproduction potential (Hartenstein, 1981;Dominguez et ai., 2000; Srivastava el ai., 2005; Gal et ai., 2008; Suthar, In Press). This inhibitory effect could be due to odour and the presence of toxic compounds and heavy metals originating from the process of sludge stabilization. Research findings on the toxicity of heavy metals on earthworms' growth and reproduction rates may support and explain the toxicity to earthworms observed in the present

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International Journal of Process Wastes Treatment (lJPWT)

study.Neuhauser et al., (1985) found that Cd, Cu, Ni, Pb, and Zn caused stress on earthworm populations and ledto significantspecies weight losses with increasing concentrations of the latter metal salts. Spurgeon et aI., (2005) found out and concluded that Cu and Cd significantly influenced and affected earthworm reproduction. Helling et al., (2000) found that earthworm growth and cocoon production were significantly reduced at copper oxychloride exposure concentrations of 8.92 mg/kg and higher, this corresponding to copper levels of about 8.5 mg/kg and higher. Lock and Jansscn (2002a) found that the 21 day ECsofor the cocoon production of E. fetida was 362 mg Ni/kg dry wt. and 892 mg/kg dry weight for Cr (Lock and Janssen, 2002b). Since the Cu, Ni and Cr levels in the frcsh sludge studied presently averaged to 174.9 mg! kg, 47.7 mg/kg and 255.5 mg/kg, respectively, and the levels of Cd, Zn and Pb were also relatively high, the presence of these metals in the fresh sludge may have possiblycausedtoxicity to the Eiseniafetida populations and suppress their growth and reproduction. So, addition of some other organic waste was essential for the survival of the earthworms in the FIS. The addition of other organic waste to the vermicomposting reactors would have increased the organic matter contents (Suthar, In Press) and consequently showed bcttcr growth performances (Suthar, In Press) with respect to the 100% FIS vermicomposting scenario. The vcrmicomposting process converted a portion of the organic waste into worm biomass and respiration products, and expelled the remaining partially stabilized product i.e. vermicompost (Benitez et al., 1999). There was little mortality observed during the initial stages ofvermicomposting ofFIS and BPS mixtures. A similar observation was made by Gajalaksmi et al., (2002) in their study of the high-rate composting-vermicomposting of water hyacinth by Eichhornia crassipes whereby it was found that there was no earthworm mortality during the first four months in spite of the high animal densities in the reactors. Figure 1 represents the survival of Eisenia fetida in different feed mixtures during experimental period. 100% survival was observed in feed mixture for vermireactor no. 2 and 3. However, in the other feed mixtures there was 5-10% mortality. This mortality may be due to a non-acceptability of feed by a portion of the worm population. Fayolle et aI., (1997) have also reported high mortality at the beginning of experimcnt with young worms during vermicomposting of paper waste.
100

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Feed m:.\ture:\o Figure 1: Survival Percentage of Eisellia Felicia in Different Feed Mixtures

Figure 2 shows the growth of Eiseniafetida in studied feed mixtures over the observation period. It was found that the increase in biomass was Icsser in those fced mixtures which had higher percentage ofFIS. Initial increase in biomass in all the vermircactors (Tripathi and Bhardwaj, 2004; Rosa et al., 2007) was followed by a stabilization period and, then, worm biomass started decreasing at the later stages. Our results are supported by Sangwan et al., (2008) as the latter have equally reported an initial increase in

Growth and Fecundity of Eisenia Fetida Earthworm DuringVermicomposting of Food Industry Sludge

75

biomass and decrease at later stages during vermicomposting of sugar mill sludge. The overall impression was that FIS and BPS mixtures containing 80% BPS + 20% FIS were better feed than 100% BPS in terms of earthworm biomass gain over the experimental period. Highest mean worm biomass 781:f: 74 mg worm1 was recorded in feed mixture no. 3 (20%BPS + 80%FIS) and lowest mean worm biomass was 565:f:4.0 mg worm-1in the feed mixture no. 7 (40%BPS + 60%FIS). The maximum biomass was attained in 6th,7th and 8thweek in different feed mixtures (Fig. 2). Maximum net biomass gain was observed in feed mixture no. 3 (681:f:40 mg/worm) and minimum was observed in feed mixture no.7 (456:f:3.7mg/worm) (Figure 3).
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Figure 3: Initial, Maximum and Net Biomass of Eisel/ill Fetitlll in Different Feed Mixtures

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International Journal of Process Wastes Treatment (IJPWT)

Thegrowth rate,expressedin termsof mg weightgainedworm-Iday-Ihas been consideredas a good index to compare the growth of earthworms in different environments (Gupta and Garg 2008a). The growth rates achieved during the vermicomposting ofFIS have been depicted in Figure 4. The highest growth rate was observed in feed mixture no. 3 (20%BPS +80%FIS; 16.21 :!:0.64 mg worm-I day-I) and minimum growth rate (8.14:!: 0.32 mg worm-I day-I) was in feed mixture no. 7 (40%BPS +60%FIS). A comparison of the data showed that the growth rate in the feed mixture no 1 and 4 was not significantly different from each other. Likewise, in feed mixture no. 6 and 7, the growth rates were not significantly different from each other. The growth rate in feed mixture no 2, 3 and 5 were significantly different from each other (p < 0.05). Although the highest worm biomass gained per unit of the feed waste was recorded in feed mixture no. 3 (2.72 :!:0.17 mg g-I feed), it was not significantly different from other feed mixtures (Figure 5). 18 16
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Growth and Fecundity of Eisenia Fetida Earthworm During Vermicomposting of Food Industry Sludge

77

The earthworms attained sexual maturity after 3rdweek in feed mixture number 1 and 2; after 4thweek in feed mixtures 3, 4,5,6 and 7. Sexual maturity and cocoon production patterns in studied feed mixtures have been illustrated in Figure 6. Neuhauser et ai., (1980) suggested that food availability and population density determined the time to reach sexual maturity for earthworms. The time needed for clitellum developmenthas a direct relationship with nutrient availability. The cocoon production started in 5thweek in feed mixture 1 and 3; in 6thweek in feed mixtures 2 and 4; and in 7thweek in remaining feed mixtures (Figure 6). Maximum numbers of cocoons were registered in feed mixture 3 (l07.2:i:1.63; 80%BPS + 20%FIS) that was significantly different from other feed mixtures; and minimum number of cocoons were registeredin feed mixtureno. 7 (21.2:1:1.31;40%BPS+ 60%FIS).Earthworms showed significantdifferences among feed mixtures for total cocoon production rate.
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Figure 6: Clitellum Development, Cocoon Production and Maximum Biomass Attained in Different Feed Mixtures

The number of cocoons produced per worm were in range of2. 1:1:0.36 (in feed mixture no. 7)-10.7:1: 1.37 (in feed mixture no. 3). The reproduction rate in feed mixture 1 and 2 was not significantly different (p < 0.05). The similar non-significant differences were observed in feed mixture 6 and 7. The variation in cocoon production rate among different feed mixtures could be attributed to the quality of feed. The cocoon production rate also fluctuated with time. This is in accordance with the findings of Gupta and Garg (2008b) who reported that initially cocoon production rate was higher and with the progress of time the cocoon production declined. This may be attributed to the non-availability ofthe feed material at the later stages of vermicomposting process. Edwards et ai., (1998) concluded that variations in rates of cocoon production in different feed mixtures could be related to the quality of the feed material. Suthar (2007b) reported that physico-chemical nature of feeding material is of primary importance for rearing of earthworms on organic waste resources. So, the difference in cocoon production, during the present study could be due to variation in the quality of substrate in each feed mixture. Also, microbes, which play an important role in earthworm diet, are influenced directly by the availability as well as quality of the organic matter supplied by the substrate. The increasing proportion of sludge tended to retard the growth performance of earthworms.

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International Journal of Process Wastes Treatment (IJPWT)

Maximum hatchlings were produced in feed mixture 2 (90%BPS + 10% FIS; 30.2:1:0.62) and minimum in feed mixture 6 (50%BPS + 50%FIS; 7.4:1:0.43) (Fig 7). Number of hatchlings recorded in feed mixture no. 2 were statistically different (p < 0.05) than in other studied feed mixtures. Number of hatchlings counted in feed mixture no 3 and 4 were not statistically different from each other. In similar manner, number of hatchlings in feed mixture no. 6 and 7 did not show significant difference. The numbers of hatchlings were significantly different in feed mixture no. I, 2 and 5 from other feed mixtures. The total hatchling biomass in different vermireactors was in range of 2783-798 mg. The maximum biomass was observed in feed mixture 2 (2783 mg), which was not significantly different from feed mixture I (2738 mg). The minimum hatchling biomass was recorded in feed mixture 6 (798 mg). 120
VI
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Figure 7: Number of Cocoons and Hatchlings During Vermicomposting in Different Feed Mixtures

4.

CONCLUSIONS

The success of a vermicomposting unit depends on the growth and fecundity ofthe earthworms in the used waste material.Our data demonstratethat feed compositioncan greatly influence the growth and reproduction of earthworm EiseniaJetida during vermicomposting. Earthworms were unable to survive in 100%FIS, so additionof someother organicwaste was necessaryto FIS before itsuse as a raw material in vermicomposting. The feed mixture 3 containing 20%FIS and 80%BPS appeared as a better option than 100% BPS in terms of earthworm biomass gain and reproduction. The cocoon production by the earthworm was also better in this feed mixture. The data support that EiseniaJetida can be used as a vermicomposting worm for FIS if it is mixed up to 20% with BPS.

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Growth and Fecundity of Eisenia Fetida Earthworm During Vermicomposting of Food Industry Sludge

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