Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting

Waste Management xxx (2017) xxx–xxx

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Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting Edmundo Arturo Pérez-Godínez a, Jorge Lagunes-Zarate a, Juan Corona-Hernández b, Martha Barajas-Aceves b,⇑ a Área de Biología, Departamento de Preparatoria Agrícola Universidad Autónoma Chapingo, Universidad Autónoma Chapingo, km. 38.5 Carretera Mexico-Texcoco, Chapingo, Edo de México, C.P. 56230 México, D.F., Mexico b Department of Biotechnology and Bioengineering, CINVESTAV-IPN, Av. IPN 2508, San Pedro Zacatenco, C.P. 07360 México, D.F., Mexico

a r t i c l e

i n f o

Article history: Received 30 October 2016 Revised 21 March 2017 Accepted 21 March 2017 Available online xxxx Keywords: Bokashi Population dynamic Organic C loss N loss Zoo animal dung Vermicomposting

a b s t r a c t Disposal of animal manure without treatment can be harmful to the environment. In this study, samples of four zoo animal dungs and one horse dung were pre-composted in two ways: (a) traditional composting and (b) bokashi pre-composting for 1 month, followed by vermicomposting for 3 months. The permanence (PEf) and reproductive potential (RP) of Eisenia foetida as well as the quality of vermicompost were evaluated. The PEf values and RP index of E. foetida were higher for samples pre-composted using the traditional composting method (98.7–88% and 31.85–16.27%, respectively) followed by vermicomposting (92.7–72.7% and 22.96–13.51%, respectively), when compared with those for bokashi pre-composted samples followed by vermicomposting, except for the horse dung sample (100% for both the parameters). The values of electrical conductivity (EC), cation exchange capacity (CEC), organic C, total N, available P, C/N ratio, and pH showed that both treatments achieved the norms of vermicompost (<4 mS cm
1, 40 cmol kg
1, 20–50%, 1–4%, 20, 5.5–8.5, respectively). However, the maturity indices of vermicompost, namely, organic matter loss, N loss, and CEC/organic carbon (OC) ratio indicated that bokashi precomposting followed by vermicomposting produced the highest values (98.7–70.7%, 97.67–96.65%, and 2.7–1.97%, respectively), when compared with the other method adapted in this study. Nevertheless, further studies with plants for plant growth evaluation are needed to assess the benefits and limitations of these two pre-composting methods prior to vermicomposting. Ó 2017 Published by Elsevier Ltd.

1. Introduction The daily amount of dung generated by zoo animals such as elephants and rhinoceros can reach large quantities (Dhimal et al., 2013). In Mexico, the management of increasing quantities of zoo animal dung is becoming a serious problem. While the dung from herbivores and carnivores is composted at the zoo in San Juan de Aragon, that from elephants, rhinoceros, hippopotamus, cats, and primates, reaching a quantity of 26.71 tons year
1 (data from San Juan de Aragon), is disposed using the general waste management system of the Board of Xochiaca, Mexico. From an ecological point of view, conventional methods of waste disposal such as land filling, open dumping, or open burning

⇑ Corresponding author. E-mail address: [email protected] (M. Barajas-Aceves).

are unsustainable owing to loading and production of certain toxic substances and gases from the wastes, which may have potential adverse effects on the environment, health, and biodiversity (Seongwon et al., 2012). Several researchers have reported that disposal of animal manure without any treatment can cause serious environmental problems such as contamination with excessive inorganic salts and pathogens, emission of hydrogen sulfide, ammonia, and other toxic gases, loss of N, and nitrate pollution of drinking water (Dalton and Hardy, 2003; Follett and Hatfield, 2001; Hubbard et al., 2004; Hutchison et al., 2005). In addition, these environmentally unhealthy waste disposal methods may also lead to under-utilization of the nutrients present in the waste and consequently, economic loss (Shröder et al., 2004). The conversion of waste into beneficial materials is an important component of resource recovery and recycling principles. Scientific utilization of organic solid wastes can provide nutrients for plant growth as well as improve soil physical properties (Scotti1 et al., 2015).

http://dx.doi.org/10.1016/j.wasman.2017.03.036 0956-053X/Ó 2017 Published by Elsevier Ltd.

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

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Fresh organic dung materials from zoo animals cannot be applied to soil until they have been sufficiently biostabilized. It has been reported that application of immature organic materials to soil may affect plant growth owing to N starvation and production of toxic metabolites (Bernal et al., 2009; Moral et al., 2009). Moreover, immature organic wastes are a source of pathogens, produce putrid odor, and have a negative impact on the health and development of plants (El Jalil et al., 2001). These wastes have not been fully exploited owing to the unavailability of viable technology for their economic recycling. Vermicomposting is a biological waste management technology by which organic fraction of wastes is decomposed by microorganisms and earthworms under controlled environmental conditions to a level that can be handled, stored, and applied to agricultural fields without adverse impacts on the environment (Aira et al., 2002). This method may be an eco-friendly and economically viable technology for converting zoo animal dung into vermicompost. Bokashi is a Japanese term denoting ‘‘fermented organic matter.” It is an anaerobic fermentation process that produces a material that can be used as a ‘‘slow release” fertilizer in soil. During this process, complex structures are broken down by the microorganisms. However, owing to the lack of oxygen, the organic material is not completely broken down to CO2, water, and heat (anaerobic process). When compared with traditional composting, bokashi composting exhibits considerably lower energy losses and CO2 emission. The application of bokashi composts to soils increases the amount of microorganisms, improves the soil physical characteristics, and enhances the supply of nutrients to plants. When compared with traditional composting, bokashi composting can compost all types of food wastes such as meats, oily food, dairy, cheese, and bread (Suthamathy and Seran, 2013), does not produce putrid odor, does not have insect or rodent issues, does not cause loss of nutrients (Ahmed et al., 2014), produces minimal greenhouse effect, is faster, and produces end-products rich in beneficial microorganisms (Boechat et al., 2013). Moreover, while traditional composting requires a C/N ratio of approximately 30:1 for an optimal and rapid process, bokashi works best with higher C/N ratios (Merfield, 2013; Boechat et al., 2013). The microorganisms that participate in the fermentation process mostly use simple compounds as their food source, such as sugars, starch, and proteins, and generally do not use complex compounds such as cellulose or lignin. In other words, only materials with a relatively high C/N ratio (e.g., 10:1) such as food wastes, which also contain high levels of water, are suitable for fermentation. Therefore, the ideal materials for composting (with a C/N ratio of 25–30:1) may be difficult to ferment, and those with high carbon content may not be fermented at all. Similarly, materials suitable for fermentation may not be suitable for composting (Merfield, 2013). Thus, the aims of the present study include (1) determination of the permanence (PEf) values and reproductive potential (RP) of Eisenia foetida in five pre-composted zoo animal dung samples (using the traditional composting method and bokashi precomposting) before and after vermicomposting and (2) evaluation of the quality of the vermicompost obtained following the two methods of pre-composting.

2. Materials and methods

arid climate with low degree of humidity, an annual average precipitation of 651.8 mm, and an average annual temperature of 17 °C. The dung samples were collected from elephants (EL), rhinos (RH), lions (LI), and hippos (HI) in the zoo. In addition, a dung sample from horses (HO) was also collected from the zoo and used as the control. All the samples were stored in plastic bags (measuring 1.03 m  0.525 m with a 40-kg capacity) at room temperature prior to analysis. The amount of dung produced by each zoo animal was noted to be as follows: EL, 42 kg day
1; RH, 24.66 kg day
1; HI, 6.7 kg day
1; and LI, 4.7 kg day
1. 2.2. Earthworm E. foetida was selected for the vermicomposting procedure because of its high voracity, high reproductive capacity, easy handling, ability to adapt to adverse conditions, i.e., wider tolerance for temperature (Gajalakshmi and Abbasi, 2004; Gupta and Garg, 2008), and ability to live in organic waste with high moisture content. The worms were obtained from the company SEyVAO (Texcoco, Mexico State), and were on a diet of cow dung and agricultural waste mixture. 2.3. Pre-composting It is well known that pre-composting is essential to avoid mortality of worms during vermicomposting owing to the presence of toxic compounds such as ammonia or salts and pathogens in the manure (Gunadi et al., 2002). In the present study, the animal dung samples were subjected to two types of organic pre-composting before vermicomposting, namely, the traditional composting method and bokashi pre-composting. The study was conducted under a completely randomized experiment design with 10 treatments: five treatments of traditional pre-composting and bokashi pre-composting, respectively. Each treatment was performed in triplicate, totaling to 30 experiments. The samples subjected to traditional composting method were indicated as HO, EL, RH, HI, and LI, while those subjected to bokashi pre-composting were denoted as BHO, BEL, BRH, BHI, and BLI (bokashi pre-composting, followed by vermicomposting of HO, EL, RH, HI, and LI, respectively). For the traditional composting method, two bags (80 kg) of each animal waste sample were placed in square-shaped spaces of 1  1  0.10 m (L  W  H). The manure was composted as indicated by López-Jiménez et al. (2003) by adding water and turning it every day to lower the temperature (<30 °C, which took 1 month) and stabilize the pH. For bokashi pre-composting, two bags of each animal waste were mixed with two sacks of straw, 15 kg of cisco (charcoal obtained from bone heated in the absence of air), 5 kg of wheat bran, 200 g of yeast, and 400 g of brown sugar (80 g dung kg
1 mixture ratio) (Restrepo, 2007). The bokashi pre-composting treatment was also performed in spaces of 1  1  0.10 m (L  W  H). The piles were covered with plastic to prevent loss of moisture. The maximum and minimum temperatures for the traditional composting method were 45 °C–33 °C and 23 °C–25 °C, respectively, whereas those for bokashi pre-composting were 65 °C–55 °C and 30 °C–27 °C, respectively. In all the treatments, the samples were mixed every day and the moisture was adjusted if necessary, and kept in shade.

2.1. Samples 2.4. Treatments The animal dung samples were collected from the zoo in San Juan de Aragon, Mexico, which is located in the northeastern part of Mexico City on one side of the Forest of San Juan de Aragón (99° 050 0000 longitude west and 19° 360 and 19° 3280 0000 latitude north) at 2240 m above sea level. The area experiences a semi-

After pre-composting, the samples were subjected to vermicomposting. A completely randomized experimental design was employed with 10 treatments. Each treatment was performed in triplicate, totaling to 30 experiments. The HO was used as the

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

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control. Once the temperature of the composites was <30 °C, black plastic rectangular boxes (51  36  30 cm L  W  H) were filled to the top with the composites (3 kg). All the samples were moistened (60% in grip test), placed in black plastic boxes (51  36  30 cm (L  W  H), with holes in the bottom to prevent flooding with leachate), inoculated with 50 clitelum worms (E. foetida, each with an initial average weight of 0.36 g) according to SEyVAO criteria, and re-moistened to 80%. The SEyVAO registered that the daily consumption of E. foetida is equivalent to its own weight. Thus, it was estimated that 50 worms weighing 25 g in total would consume 3 kg of pre-composting substrate for approximately 120 days. The traditional compost samples subjected to vermicomposting were indicated as VHO, VEL, VRH, VLI, and VHI, and the bokashi pre-compost samples subjected to vermicomposting were denoted as VBHO, VBEL, VBRH, VBLI, and VBHI. Following incubation for 3 days, the PEf, biomass, and RP of E. foetida were determined using 30 random worms. The RP was calculated as follows (Schuldt, 2006): RP ¼

capsule average  worms average=capsule  % juveniles  % sub
adults 50 inoculated worms

The worms average/capsule was determined using 12 capsules per treatment. The capsules were placed in 8  8 cm boxes containing 8 g of pre-compost and moistened with 10 ml of tap water. The boxes were covered with black plastic to avoid light and incubated for 3 days (Schuldt, 2006). The number of juveniles, biomass gain, clitelium development, and cocoon production exhibited by E. foetida in each vermicomposting treatment were monitored weekly for 90 days by considering all the four ‘‘ages” of the worm (capsules, juveniles, sub-adults, and adults) (Schuldt, 2006). For differentiating juveniles, subadults, and adults, the color and length of the worm were taken into account (Garg et al., 2005). The biomass gain was determined by weighing 30 randomly selected earthworms per treatment. The number of capsules, sub-adults, and juveniles were ascertained at three points diagonally in each experimental unit, with a sample area of 26.3% with respect to the total. 2.5. Chemical analysis The pH and electrical conductivity (EC) were determined by using a double distilled water suspension of each sample (at a ratio of 1:10 (w/v)). The total organic matter (OM) was measured by dichromate digestion (Walkley and Black, 1934), total N was evaluated using the Micro-Kjeldahl method (Bremner and Mulvaney, 1982), and hydrolyzable and orthophosphate P was determined using the colorimetric method with molybdenum in sulfuric acid (Murphy and Riley, 1962). The cation exchange capacity (CEC) was measured using barium chloride-triethanolamine (PrimoYúfera and Carrasco-Dorrien, 1990), and the moisture content, EC, pH, and bulk density were evaluated using methods described elsewhere (NMXF-109-SCFI, 2008). All the measurements were performed after 1 month of pre-composting and 3 months of vermicomposting using 250 g of samples from each treatment. Bulk density evaluation was conducted after 3 months only. The rates of OM or N mineralization during composting of zoo animal dung were measured by determining the loss of OM or N after 1 and 3 months of vermicomposting. The losses of organic carbon (OC) (OM loss) and total nitrogen (TN loss) were calculated as follows (Paredes et al., 2000):

OM loss ð%Þ ¼ 100
100

½X 1 ð100
X 2 Þ ½X 2 ð100
X 1 Þ

N loss ð%Þ ¼ 100
100

ðX 1 N 2 Þ ðX 2 N 1 Þ

where X1 and X2 are the initial and final ash content, respectively, and N1 and N2 are the initial and final TN concentration, respectively. The ratio of CEC/OC was calculated to describe the degree of humification (Roig et al., 1988). 2.6. Statistical analysis All the results presented are the mean of triplicate measurements. The data were analyzed using repeated measures analysis of variance (ANOVA) because the sample collection was nondestructive. The results obtained were compared using Duncan’s new multiple range test. The values with P < 0.05 were considered as statistically significant. Statistical analysis was done with the help of SPSS version 24.0 software programs. Correlations were calculated between different variables. To obtain additional information on the relationships, behavior, and source of contamination, factor analysis (FA) was performed using Varimax normalized rotation (Kleinbaum et al., 1988). 3. Results and discussion 3.1. Characterization of animal dung The amount of dung produced by the zoo animals examined in the present study was as follows (in kg day
1): EL, 42; HI, 6.73; RH, 24.7; and LI, 4.6, reaching a total of 29 tons year
1. These wastes are disposed using the general waste management system of the Board of Xochiaca. Table 1 shows the chemical analysis of the raw dung from the zoo animals and horse. It can be noted that the lion dung had the highest EC, total P, and TN, and the lowest pH; the hippo dung had the lowest total P and EC; and the elephant dung had the highest pH and OC and lowest TN content, when compared with all the other dung samples (Table 1). All the treatments with traditional pre-composting did not reach the thermophilic temperature, because pathogen removal was not ensured. However, some studies have provided evidence of suppression of pathogens (Monroy et al., 2008). Monroy et al. (2009) demonstrated that the decrease in coliforms caused by the presence of earthworms in the low-dose reactors was entirely owing to the gut transit effect. 3.2. Permanence of E. foetida The PEf of E. foetida after 3 days of incubation (determined using 50 worms) showed that vermicomposting after pre-composting of animal dung using the traditional composting method had higher % PEf values, when compared with that after pre-composting of animal dung using the bokashi method, except for horse dung (Table 2). Bhat et al. (2015) showed that Eisenia fetida significantly decreased their growth and reproduction at high bagasse concentration. The HO, VHO, and VBHO samples presented the highest number of worms, whereas VLI and VBLI samples had the lowest and zero PEf, respectively. The absence of worms or corpses of E. foetida in VBLI may be because of their death after a few days from the start of the experiment. The results of P and PEf for treatments of LI with traditional or bokashi methods (Table 2) and the high content of N (46%) in LI (Table 1) suggest that E. foetida could not adapt to these conditions of the fed mixtures. In a previous study, Chan and Griffiths (1988) showed that E. foetida could not survive in untreated pig manure, and attributed this mortality to the presence of ammonia. Nevertheless, when the ammonia content was

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

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Table 1 Characterization of zoo animal dung.

Lion Hipo Horse Elephant Rhino

Moisture %

pH

EC ms

C tot %

Total N %

P mg g
1

C/N Ratio

60.00 ± 2.54 86.62 ± 1.84 66.02 ± 3.56 78.21 ± 1.30 80.75 ± 1.62

6.35 ± 0.04 7.64 ± 0.12 7.70 ± 0.03 7.80 ± 0.02 7.41 ± 0.06

4.98 ± 0.84 1.16 ± 0.14 2.31 ± 0.13 2.35 ± 0.28 4.45 ± 0.11

30.68 ± 0.87 50.21 ± 0.21 48.50 ± 0.13 51.79 ± 0.70 47.13 ± 0.55

45.91 ± 0.59 20.62 ± 0.86 21.66 ± 0.85 11.40 ± 1.18 27.03 ± 3.05

2.95 ± 0.57 2.25 ± 0.09 2.43 ± 0.47 2.51 ± 0.20 3.04 ± 0.43

0.67 2.44 2.24 4.54 1.74

% (w/w), values after ± are the standard error of the mean of three replicates.

Table 2 Permanence of E. foetida and reproductive potential index after three days inoculation with the two pre-composting treatments: traditional composting or bokashi. % Permanence

Reproductive potential index

VHO VBHO VEL VBEL VRH VBRH VHI VBHI VLI VBLI

100 100 97.3 92.7 98.7 72.7 88.0 85.3 72.7 0

35.71 26.85 31.85 22.96 25.44 20.46 16.27 13.51 – –

V (Vermicompost), B (Bokashi), HO (Horse), EL (Elephant), RH (Rhino), HI (Hippo), LI (Lion). – not determined.

controlled with calcium sulfate and urine salts were eliminated, the substrate was found to be suitable as a feed for the worms. All the pre-composted zoo animal dung samples subjected to vermicomposting presented the highest RP index (Table 2), except VHI and VBHI, which had the lowest RP index. These results suggest that the RP index depends on the quality and nature of the raw animal waste that provides ingestible substrates to E. foetida (Neuhauser et al., 1980). 3.3. Biomass and behavior of E. foetida

200

150

VHO VEL VRH

100

Eisenia foetida biomass (g)

Treatment

250

VHI

50

0 0

2

3

4

5

6

7

8

250

200

VBHO

150

VBEL VBRH

100

VBHI

50

0 0

The maximum E. foetida weight was noted in VHO, whereas the minimum E. foetida weight was observed in VLI, VBHO, and VBRH (Fig. 1). There were no significant differences in E. foetida weight between VEL and VRH. With regard to bokashi and vermicomposting treatments, VBHO and VBEL presented the highest E. foetida weight, whereas VBRH exhibited the lowest E. foetida weight (Fig. 1). It can be observed that bokashi pre-composting resulted in a higher decrease in the weight of the worms throughout the incubation period, when compared with the traditional composting method (Fig. 1). Some previous studies have demonstrated that the biological activities of the worms slow down with the progression of vermicomposting (1 month), owing to the depletion of readily available N, which is converted to NO3 (Atiyeh et al., 2000). Thus, the combination of vermicomposting and bokashi pre-composting reflects the rapid degradation of organic materials during the fermentation process developed at the stage of bokashi pre-composting. The rapid degradation could result in losses of N owing to volatilization (Hitman et al., 2013), which in turn could affect the growth of E. foetida. In contrast, the number of E. foetida juveniles increased through the treatment period in all the samples, except in VLI, following both traditional composting and bokashi pre-composting (Fig. 2). This trend reflected the expansion of the E. foetida population. The highest number of juveniles was observed in VBHO and VBHI, whereas the lowest number of juveniles was noted in VBRH, VRH, and VBEL. However, the weight values were not significant in VLI

1

1

2

3

4

5

6

7

8

Time (weeks) Fig. 1. Biomass of E. foetida following pre-composting of zoo animal dung. (a) Traditional composting method and (b) bokashi pre-composting (B), followed by vermicomposting (V). HO (Horse), EL (Elephant), RH (Rhino), HI (Hippo), LI (Lion).

and null in VBLI (Fig. 2). Vermicomposting after pre-composting using the traditional composting method resulted in a lag phase of 3 weeks in all the samples, except in VHI. A similar lag phase was also observed during vermicomposting after bokashi precompositing in all the samples, except in VBRH, in which the lag phase was 4 weeks (Fig. 2). These findings are in accordance with those reported by Gómez-Tovar (1999), who indicated that the capsules hatched after 21 weeks. When compared with the samples subjected to the traditional composting method, the number of juveniles increased in VBHO and VBHI and decreased in VBRH and VBEL (Fig. 2), suggesting that the chemical composition of zoo animal dung changed during the treatment period. For instance, the pH of the zoo animal dung samples and amended substrates in bokashi, high C/N ratio, and production of toxic ammonium, nitrate oxide, and CO2 were noted to affect the biomass and number of E. foetida (Flegel and Schreder, 2000). The highest weight and number of juveniles of E. foetida noted in HO pre-composted with traditional or bokashi method was used as the reference. In a previous study, Garg et al. (2005) and Edwards

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

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350 300 250

b cb

200

VHO VEL

cd

VRH

Number of Eisenia foetida juvenile

150

VHI

d

VLI

100 50

f 0 0

1

2

3

4

5

6

7

8

350

a

300 250

a

200

VBHO

cd

VBEL

150

VBRH VBHI

100 50

e

0 0

1

2

3

4

5

6

7

8

Time (weeks) Fig. 2. Behavior of E. foetida juveniles following pre-composting of zoo animal dung. (a) Traditional composting method and (b) bokashi pre-composting (B), followed by vermicomposting (V). HO (Horse), EL (Elephant), RH (Rhino), HI (Hippo), LI (Lion).

90 80 70 60

VHO VEL VRH VHI VLI

50

Number of Eisenia foetida sub-adults

and Arancon (2004) showed that HO is one of the seven types of animal dungs that are more favorable for E. foetida growth. The E. foetida sub-adults appeared at 4 weeks in all the samples, except in VHO, in which they were noted at 3 weeks (Fig. 3). The number of E. foetida sub-adults in samples pre-composted using the traditional composting method exhibited the following trend: VHO > VRH > VEL > VHI. The number of E. foetida sub-adults in samples subjected to bokashi pre-composting at 6 weeks presented the following trend: VBRH > VBHI > VBHO > VBEL (Fig. 3). A higher number of capsules was observed at 1 week in VHO, VEL, and VRH, and at 2 weeks in VHI; however, in VLI, the number of capsules was almost zero (Fig. 4). The highest number of capsules was observed at 2 weeks in all the samples subjected to bokashi pre-composting, followed by a steep fall in the number of capsules at 5 weeks, reaching almost zero at 7 weeks. This steep decrease in the number of capsules coincided with the highest number of E. foetida juveniles and sub-adults, which might have been owing to the hatching of the cocoons (Kaushik and Garg, 2003). The bokashi pre-composting of zoo animal dung, except EL, was found to be an effective method to achieve a higher number of worms during vermicomposting.

40 30 20 10 0 0

1

2

3

4

5

6

7

8

90 80 70 60 VBHO VBEL VBRH VBHI

50 40 30

3.4. Statistical analysis of E. foetida biomass

20 10

The normality condition and homogeneity of variance were tested before repeated measures ANOVA analysis. Assumption of normality were tested using Shapiro-Wilk test (p > 0.05) (Shapiro and Wilk, 1965; Razali and Wah, 2011) and visual inspection of their histograms, normal Q-Q plots and box showed that the exam scores were approximately normally distributed for both treatments in the four animals dung. The homogeneity of variance in

0

0

1

2

3

4

5

6

7

8

Time (weeks) Fig. 3. Number of E. foetida suba-dults following pre-composting of zoo animal dung. (a) Traditional composting method and (b) bokashi pre-composting (B), followed by vermicomposting (V). HO (Horse), EL (Elephant), RH (Rhino), HI (Hippo), LI (Lion).

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

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60 50 40

VHO VEL VRH VHI VLI

Number of Eisenia foetida capsules

30 20 10 0

1

2

3

4

5

6

7

60 50 40 VBHO VBEL VBRH VBHI

30 20 10 0

1

2

3

4

5

6

7

Time (weeks) Fig. 4. Number of E. foetida capsules following pre-composting of zoo animal dung. (a) Traditional composting method and (b) bokashi pre-composting (B), followed by vermicomposting (V). HO (Horse), EL (Elephant), RH (Rhino), HI (Hippo), LI (Lion).

all the treatments was tested using Levene’s test and values of p (<0.05) showed that all variances of the samples are homogeneus. Assumption of sphericity did not meet in all the samples analyze. The Greenhouse-Geiser was used by the software to correct the p used in the statistical analysis. The results of repeated measures ANOVA (Table 3) shows that there was no significant effect of pre-composting treatment on E. foetida biomass in HO and RH animal dung. Although, the effect of time had a significant effect on all the treatments of the animal dung, time did not show interaction with the treatment. These results suggested that HO and RH dung animal significantly affected the E. foetida biomass. More measurements are needed to clarify the behavior of E. foetida growth in these two-animal dung. 3.5. pH, ash content, EC, and CEC The values did not exhibit a definite trend throughout the incubation period (Tables 3 and 4). The pH of the pre-composted samples, except RH, was lower than that of raw wastes (Tables 4 and 1). Both VRH and VBRH showed alkaline pH ranging between

7.84 and 9.40 after 3 months of vermicomposting (Tables 1 and 5). Similar values were also noted in VHI and VBHI samples, whereas VHO and VBHO showed an alkaline and a slightly acidic pH after 3 months and 1 month of vermicomposting, respectively (Tables 4 and 5). The increment in the pH values during vermicomposting has also been reported in some previous studies (Tripathi and Bhardwaj, 2004; Loh et al., 2005; Soobhany et al., 2015a). Among the studies on vermicomposting of zoo animal dung, Dhimal et al. (2013) reported that the pH value of elephant and rhino dung following vermicomposting and rhino dung subjected to bokashi pre-composting slightly decreased from alkaline towards neutral. The increase in pH in almost all the samples (except VLI and VBLI) during vermicomposting (Table 5) suggests the degradation and mineralization of organic nitrogenous compounds. The conversion of short-chain volatile fatty acids and organic nitrogenous compounds (such as proteins and amines) to ammonia during vermicomposting (Beck-Friis et al., 2003; Li et al., 2001; Tognetti et al., 2005) results in alkalization of the waste (Lim et al., 2012; Shak et al., 2014; Castillo et al., 2005). In the present study, the detection of both alkaline pH (VHO, VBEL, VRH, and VBRH samples) and neutral pH (VEL, VHI, VBHO, and VBHI samples) after 3 months of vermicomposting indicated generation of stable vermicomposts, because pH is known to increase during the latter stages of vermicomposting. Thus, neutral and partially alkaline pH values are usually indicators of stable vermicomposts (Majlessi et al., 2012). However, the possibility of increased pH could also be owing to the quality of water used to adjust the moisture of the vermicomposting treatments. The change towards acidic pH (VLI and VBLI, Tables 4 and 5) could be attributed to mineralization of N and P as well as formation of intermediate species of organic acids following the bioconversion of organic materials (Ndegwa et al., 2000; Garg et al., 2006a). The acidic pH conditions result in decreased bacterial activity and are not suitable for the worms, prompting them to escape to find better conditions or die (Rostami, 2011). Thus, different animal manure and treatments produce varied pH shifts (Loh et al., 2005; Ndegwa et al., 2000). However, the pH values in all the treatments after 3 months of vermicomposting were in a suitable range for the growth of the worms (5.5–8.5, NMX-FF109-SCFI-2008), except in VRH and VBEL, and were acceptable for optimum microbial activity (6.5–8.0), except in VLI, VBLI, and VBEL (Yadav and Garg, 2011b). While materials with a pH of 7 (pH 5.5–8.5) are preferred for the growth of worms (NMX-FF109-SCFI-2008), those from the tropics may have a pH of <7 and those from arid zones may have a pH of >7. Although worms can survive in a pH range of 5–9, a pH of 7.5–8.0 is considered to be the optimum (Pourzamani and Ghavi, 2017). Thus, these findings indicate that further studies are required to determine the best pH for E. foetida growth on zoo animal dung pre-composted with traditional or bokashi treatments. The ash content in all the pre-composted samples subjected to vermicomposting increased, when compared with those in raw animal waste (Tables 1, 4, and 5). This increase in the ash content

Table 3 Repeated measures ANOVA for Eisenia foetida biomass growth on zoo animal dung pre-composted in traditional and bokashi pre-composting method. Effect Time

Treatments

HO EL RH HI

Treatments x time

F (1, 6)

P

F (1, 6)

P

F (1, 6)

P

2.511 12.017 3.739 8.977

<0.188 <0.000 <0.125 <0.040

162.077 137.860 14.415 57.325

<0.00 <0.00 <0.019 <0.00

0.420 36.871 4.705 8.346

<0.552 <0.004 <0.096 <0.006

HO = horse, RH = rhino, EL = elephant, HI = hipho (p < 0.05 are significant, n = 3 in each treatment).

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

7

E.A. Pérez-Godínez et al. / Waste Management xxx (2017) xxx–xxx Table 4 Chemical analysis of dung zoo animal after one month pre-composting with two methods of pre-composting. Compost Traditional

Moisture %

pH

Ash %

EC mS cm
1

CEC cmol kg
1

OC %

Total N %

P mg g
1

C/N Ratio

HO RH LI EL HI

74.90 ± 2.31 67.31 ± 1.23 66.20 ± 1.10 78.50 ± 0.02 79.63 ± 0.65

6.63 ± 0.01 7.66 ± 0.01 5.96 ± 0.01 7.21 ± 0.77 6.55 ± 0.03

35.09 ± 1.00 61.15 ± 0.93 60.04 ± 0.4 49.01 ± 8.9 31.25 ± 3.85

10.06 ± 0.49 3.06 ± 0.01 2.11 ± 0.40 10.25 ± 2.76 3.81 ± 0.33

35.83 ± 6.13 40.21 ± 1.30 36.88 ± 1.65 15.63 ± 3.90 13.49 ± 0.24

37.65 ± 0.99 22.53 ± 1.93 23.18 ± 4.03 31.33 ± 8.25 45.65 ± 13.61

3.01 ± 0.21 1.67 ± 0.11 2.32 ± 0.23 2.63 ± 0.09 2.97 ± 0.35

3.16 ± 0.27 2.33 ± 0.06 2.51 ± 0.23 3.13 ± 0.44 2.63 ± 0.21

12.51 13.50 9.99 11.91 15.38

Bokashi BHO BRH BLI BEL BHI NMX-FF-109

65.11 ± 0.91 72.37 ± 1.76 58.67 ± 0.46 64.44 ± 3.37 76.44 ± 1.63 20–40

7.24 ± 0.01 6.99 ± 0.02 6.34 ± 0.02 7.87 ± 1.61 7.35 ± 0.07 5.5–8.5

42.11 ± 0.80 41.09 ± 1.87 60.83 ± 8.30 46.00 ± 8.25 34.38 ± 0.90 –

6.43 ± 0.25 6.67 ± 0.80 9.36 ± 0.14 9.95 ± 1.41 3.88 ± 0.09 4

35.42 ± 6.56 39.36 ± 0.53 28.13 ± 10.33 47.92 ± 8.33 18.33 ± 4.16 >40

33.58 ± 0.78 34.17 ± 2.07 22.72 ± 8.36 29.11 ± 8.90 38.06 ± 0.89 20–50

2.10 ± 0.23 2.01 ± 0.20 2.49 ± 0.13 2.18 ± 0.06 2.30 ± 0.16 1–4

2.83 ± 0.41 3.13 ± 0.06 2.44 ± 0.24 3.13 ± 0.15 2.46 ± 0.14 –

16.00 17.00 9.13 13.35 16.55 20

V = vermicompost, B = bokashi, HO = horse, RH = rhino, LI = lion, EL = elephant, HI = hippo, NMX-FF-109-SCFI-2008. Values after ± are the standard error of the mean of three replicates.

Table 5 Chemical analysis of dung zoo animal after three months vermicomposting. Compost Traditional

Moisture %

pH

Ash %

EC mS cm
1

CEC cmol kg
1

OC %

Total N %

P mg g
1

C/N Ratio

Bulk density g ml
1

VHO VRH VLI VEL VHI

79.90 ± 0.02 70.09 ± 3.57 70.51 ± 2.00 80.39 ± 0.53 74.23 ± 14.68

8.49 ± 0.035 8.89 ± 3.015 5.75 ± 0.036 7.03 ± 0.036 7.52 ± 0.036

33.50 ± 0.40 56.0 ± 4.50 59.12 ± 2.0 35.93 ± 0.31 33.10 ± 2.05

2.12 ± 0.01 1.38 ± 0.015 3.80 ± 0.005 2.34 ± 0.28 2.01 ± 0.03

52.03 ± 3.80 67.86 ± 2.19 79.11 ± 2.37 77.03 ± 1.08 72.14 ± 2.43

38.58 ± 0.23 26.84 ± 0.80 23.42 ± 1.16 37.17 ± 0.18 38.80 ± 1.19

2.59 ± 0.29 1.33 ± 0.05 2.11 ± 0.15 2.25 ± 0.33 1.93 ± 0.18

2.86 ± 0.13 2.06 ± 0.02 2.67 ± 0.33 1.88 ± 0.01 2.38 ± 0.42

14.90 20.18 11.10 16.52 20.11

0.40 ± 0.058 0.37 ± 0.031 0.33 ± 0.025 0.31 ± 0.081 0.24 ± 0.002

Bokashi VBHO VBRH VBLI VBEL VBHI NMX-FF-109

84.37 ± 13.42 75.00 ± 0.32 – 75.00 ± 0.32 78.97 ± 0.36 20–40

7.33 ± 0.030 7.84 ± 0.029 – 9.40 ± 0.096 7.65 ± 0.050 5.5–8.5

40.06 ± 0.73 49.21 ± 9.8 – 53.73 ± 1.36 36.74 ± 9.2 –

1.79 ± 0.04 2.70 ± 0.046 – 1.80 ± 0.04 3.11 ± 0.38 4

69.53 ± 4.38 70.74 ± 0.07 – 64.11 ± 0.72 71.41 ± 1.08 >40

34.77 ± 0.43 29.46 ± 11.46 – 38.80 ± 1.19 36.69 ± 5.34 20–50

1.82 ± 0.12 1.60 ± 0.06 – 1.93 ± 0.18 2.11 ± 0.15 1–4

1.89 ± 0.11 1.95 ± 0.05 – 2.38 ± 0.42 2.11 ± 0.002 –

19.10 18.41 – 20.18 20.05 20

0.39 ± 0.025 0.37 ± 0.031 – 0.31 ± 0.081 0.30 ± 0.015 0.40–0.90

V = vermicompost, B = bokashi, HO = horse, RH = rhino, LI = lion, EL = elephant, HI = hipho, – = not determined. NMX-FF-109-SCFI-2008. Values after ± are the standard error of the mean of three replicates.

can be attributed to enhanced mineralization owing to microbial activity and earthworms (Gupta et al., 2007). The EC values of all the samples, except LI and RH, after 1 month of pre-composting were higher (2.11–10.06 mS cm
1) than those of the raw animal dung (1.16–4.98 mS cm
1) (Tables 1 and 4). Similarly, the EC values of all the samples after 1 month of bokashi pre-composting were higher (6.42–9.95 mS cm
1) than those recommended by NMX-F-109 (2008). Furthermore, the EC values of all the pre-composted samples after 3 months of vermicomposting varied between 1.4 and 3.8 mS cm
1 (Table 5) and were lower than those recommended by NMX-F-109 (2008) (<4 mS cm
1), suggesting generation of stable compost. The increase in the EC values after 1 month of pre-composting or 3 months of vermicomposting, when compared with those of raw animal dung, could have been caused by the release of different mineral ions such as phosphate, ammonium, and potassium ions through decomposition of organic substances or shredding of organic materials by E. foetida as reported earlier, respectively (Garg et al., 2006b; Sharma, 2003). In general, the EC values of all the samples after 3 months of vermicomposting were lower, when compared with those after 1 month of pre-composting, which may be owing to the decomposition of organic acids leading to higher pH (as mentioned earlier) and precipitation of soluble salts or leaching of salts (Lazcano et al., 2008; Lim and Wu, 2016). The CEC values increased in all the samples after 3 months of vermicomposting, when compared with those noted after 1 month of vermicomposting (Tables 4 and 5), indicating that the nutrient availability to plants was >40 cmol kg
1 in 3-month vermicompost, but not in 1-month vermicompost (NMX 2008; Tables 4 and 5). It

has been reported that the application of immature compost to soil, such as 1-month vermicompost, could cause serious damage to plant growth owing to N starvation and competition for oxygen, thus creating an anaerobic environment and insufficient biodegradation of OM, causing phytotoxicity (Brewer and Sullivan, 2003; Cooperband et al., 2003; Wu et al., 2000). The CEC values of bokashi pre-composted samples subjected to vermicomposting were higher, when compared with those of samples subjected to traditional composting and vermicomposting (Table 5). It has been demonstrated that the degree of decomposition of waste and CEC of compost are strongly correlated (Iqbal et al., 2010). The increase in CEC values during vermicomposting could be attributed to the accumulation of negatively charged compounds such as lignin-derived products and carboxyl and/or phenolic hydroxyl groups. Therefore, the CEC of the compost can be used to evaluate the degree of humification and/or maturity of composts (Roig et al., 1988). Nevertheless, the quality of OM added to the bokashi and animal manure should be taken into account (Schuldt, 2006).

3.6. P Content The amount of available P increased in all the samples, except in HO, LI, and BLI, after 1 month of pre-composting, when compared with that in raw animal dung (Tables 1 and 4). However, the content of P was lower in all the samples, except in VRH and VHI, at 3 months of vermicomposting, when compared with that in raw animal dung (Tables 1 and 5).

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

8

E.A. Pérez-Godínez et al. / Waste Management xxx (2017) xxx–xxx

The increase in the amount of P in the first month of precomposting may be owingIn contrast, the decrease in P content after 3 months of vermicomposting may be owing to the increase in the microbial activity during the digestion process and subsequent acceleration in OM mineralization (Kuczak et al., 2006) to the mobilization of P by OM and bacteria as well as the fecal phosphatase activity of the earthworms (Salas et al., 2003; Suthar, 2008; Tripathi and Bhardwaj, 2004). Moreover, pre-composting could also reduce the nutrient values of the vermicompost as a result of exhaustion of organic compounds (acting as the P source) owing to microbial activities (Castillo et al., 2010; Garg et al., 2008). 3.7. TN content and C/N ratio The C/N ratio showed that all the samples had a high TN content (Table 1), with the highest TN noted in LI, followed by RH. The TN contents in all the pre-composted and vermicomposted samples were the lowest, when compared with those in the raw manure (Tables 1, 4 and 5), which may be due to the volatilization of ammonia and readily available N compounds (Tremier et al., 2005). The increase or decrease in the TN content after 3 months of vermicomposting, when compared with that after 1 month of pre-composting, suggested the reduction in OC by mineralization of organic humic matter, leading to a relative increase in N (Francou et al., 2008). The reason for the variations in the TN content following vermicomposting of different wastes could be the quality of the substrates for E. foetida, along with their physical structure and chemical composition, which affect mineralization of organic nitrogenous compounds and the amount of N released from the compounds (Tognetti et al., 2007). Several studies have reported that vermicomposting causes a significant increase in the TN content after worm activity (Garg and Gupta, 2011; Yadav and Garg, 2011a; Soobhany et al., 2015b). However, it has been indicated that the N content in the vermicompost depends on the initial N present in the raw material and the degree of decomposition (Crawford, 1983). The C/N ratio of all the samples after 3 months of vermicomposting was higher, when compared with that of samples after 1 month of pre-composting (Tables 4 and 5), which may be due to the increase in the population of E. foetida (Fig. 1) (Ndegwa and Thompson, 2007). Nevertheless, the C/N ratio of 1-month pre-composted samples was higher than that of the raw manure (Tables 1 and 4). The C/N ratio of raw manure was between 3.5 and 17.0, while those of pre-composted samples subjected to vermicomposting ranged between 9.13 and 17 at 1 month (Table 4) and between 16.5 and 20.5 at 3 months (Table 5). These values (C/N ratio < 20) reflect the maturity and stabilization of OM (Table 5). Taken together, the variations in the chemicals, biomass, and number of E. foetida suggest that the stock density of the worms inoculated for all the treatments produces different effects on the degradation of the substrate (Mupambwa and Mnkeni, 2016). In the present study, a stock density of 15 g worm kg
1 was inoculated into the pre-composted material for all treatments. Gajalakshmi and Abbasi (2004) reported that the rate of decomposition during vermicomposting depends on the earthworm stock density, and that this effect tends to vary with the type of material subjected to vermicomposting. Similarly, Unuofin and Mnkeni (2014) demonstrated that E. foetida stock density had a highly significant effect on worm biomass, and that this effect tended to vary with time. Furthermore, they reported that an E. foetida stock density of 12.5 g worm kg
1 was optimum for the biodegradation of phosphate rock enriched cow dung waste paper mixtures, which maintained the lowest C/N ratio throughout the vermicomposting

period. These results indicate that further studies are required to determine the optimum E. foetida stock density and type of precomposting method for each animal dung treatment. 3.8. Maturity index parameters The CEC/OC ratio of all the samples showed an increasing trend from 1 month of pre-composting to 3 months of vermicomposting (Table 6). The bokashi-pre-composted samples presented significantly higher CEC/OC ratios at 3 months of vermicomposting, when compared with those subjected to traditional precomposting and 3 months of vermicomposting (Table 6). Moreover, the CEC/OC ratios of all the samples, except VEL, at 1 month of pre-composting and those of VHO and VEL at 3 months of vermicomposting were <1.7 (Table 6). Several studies have proposed the use of the CEC/OC ratio to evaluate the degree of humification and maturity of composts (Cayuela et al., 2008; Raj and Antil, 2011; Roig et al., 1988). A CEC/OC ratio of 1.7 has been suggested to be the lowest limit for describing good humified manure (Roig et al., 1988). In the present study, samples subjected to bokashi pre-composting presented the highest CEC/OC ratios, indicating maximum humification (Table 6). Moreover, the CEC/OC ratios obtained suggested that all the samples, except VHO and VEL, at 3 months of vermicomposting were suitable for soil application. The %OM loss decreased in all the samples after 3 months of vermicomposting, when compared with that in samples after 1 month of vermicomposting, except in VEL, VBRH, and VBHI, which showed an increase in %OM loss (Table 6). Furthermore, the %OM loss in samples subjected to bokashi pre-composting and vermicomposting did not show a definite trend, and ranged between the values previously reported (50–72%) (Changa et al., 2003; Hao et al., 2004; Larney et al., 2006; Tiquia et al., 2000). However, VLI after 1 month and 3 months of vermicomposting (Table 6) presented a lower %OM loss (39.58%), whereas VEL at 3 months of vermicomposting and VBRH and VEL at 1 month and 3 months of vermicomposting showed higher %OM loss (81–89%), when compared with those reported in earlier studies. These results suggested that pre-composting resulted in increased microbial biomass causing higher rates of OM degradation, when compared with 3 months of vermicomposting, and that E. foetida may have increased the microbial biomass and accelerated the OM degradation rate (Ndegwa and Thompson, 2001). Readily available C was released throughout the vermicomposting period in all the samples subjected to bokashi pre-composting, when compared with those subjected to the traditional composting method (Table 7). Boechat et al. (2013) reported that an accelerated degradation occurred during bokashi pre-composting, supplying readily available nutrients to plants. Furthermore, Domínguez and Edwards (2004) demonstrated that the combined actions of earthworms and microorganisms caused OC loss from the substrate as CO2. Earthworms produce mucus and enzymes promoting microbial action, while the microorganisms provide extracellular enzymes within the worms’ guts (Domínguez and Edwards, 2004). Accordingly, in the present study, the sample VLI was resistant to degradation (Benito et al., 2003). Moreover, the %OM loss in all the samples was >42%, which has been suggested as an index for mature compost from agro-industrial waste combined with poultry waste (Raj and Antil, 2011). 3.9. %N loss The highest %N loss was noted in VBHO, followed by VBEL and VBHI at 1 month of pre-composting or 3 months of vermicomposting, when compared with that in VHO, VEL, and VHI, respectively (Table 6). Except VEL, all the samples presented higher %N loss at

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

9

E.A. Pérez-Godínez et al. / Waste Management xxx (2017) xxx–xxx Table 6 Chemical and soil microbial parameters of dung zoo animal after one month pre-composting and three months vermicomposting. Treatment

CEC/OC 1 month

Traditional Comp. a HO-bVHO RH-VRH LI-VLI EL-VEL HI-VHI

0.96 0.56 1.60 1.78 1.60

Comp.Bokashi BHO-VBHO BRH-VBRH BLI-VBLI BEL-VBEL BHI-VBHI Roig et al. (1988)

1.05 cd 1.41ba 1.20c 1.15c 0.48e 1.7

%MO loss a

d e b a b

b

% N loss

3 month

ratio

1 month

3 months

% decrease

1 month

3 months

% increase

1.35 2.68 3.40 1.34 1.86

e b a e ba

1.41 2.39 2.13 1.50 1.16

63.71 87.31 45.70 66.83 70.47

f a g e cd

61.06 78.58 39.58 81.67 68.64

f c g b e

4.16 10.0 13.39
22.21 2.60

93.50 94.48 94.52 93.81 94.55

d c c d c

94.13 98.44 96.36 94.11 96.20

d a c d c

0.67 4.02 1.91 0.32 1.72

2.00 cd 2.69 b – 2.39 c 1.97 cd 1.7

1.90 1.91 – 2.08 4.10

73.04 85.28 30.12 85.32 65.52 >42

c ab h ab e

70.66 89.65 – 82.80 71.58 >42

d a

3.26 -5.12 – 2.95
9.55

96.22 95.80 – 96.72 95.65

a b

96.57 97.48 – 97.67 96.65

c b

0.36 1.72 – 0.97 1.03

b d

a b

b c

Values in the same column followed by the same letter are not statistically significant at p  0.05, according to Duncan’s test. a One month pre-composting with common compost or bokashi pre-compost of animal dung. b 3 months vermicomposting with precomposting with common compost or pre-composting with bokashi.

Table 7 Correlation coefficient of number and weight of E. foetida and physic-chemical data. Treatment

OC

CEC

P

N

pH

EC

Common composta Num Biomass

0.895** 0.231


0.673**
0.432

0.110 0.111

0.307 0.148

0.496 0.633*


0.679**
0.377

Bokashib Num Biomass

0.036 0.178

0.235
0.340

0.019 0.073

0.454
0.661*


0.297 0.554

0.034
0.222

Num = number of E. foetida, weight = weight of E. foetida. a Number of samples = 14. b Number of samples = 12. * Significant at P < 0.05. ** At P < 0.001.

3 months of vermicomposting, when compared with those at 1 month of pre-composting. The increase in pH of all the samples at 3 months of vermicomposting, when compared with that at 1 month of pre-composting (Tables 4 and 5) suggests that this parameter is important for the loss of N as volatile ammonia at higher pH (Garg et al., 2006a). In contrast, the pH of VEL decreased after 3 months of vermicomposting, reflecting the decrease in %N loss (Table 6).

3.10. Relationship between the number and weight of E. foetida and chemical parameters The results of correlation coefficient analysis of the number and weight of E. foetida (Table 7) and those presented in Figs. 1 and 2 confirmed that the number of E. foetida was negatively affected by salt concentration (EC and CEC) and positively influenced by OC. Furthermore, the biomass of E. foetida was found to be affected by pH in all the samples that were not subjected to bokashi precomposting. In a previous study, Owojori et al. (2009) reported that E. foetida was the most sensitive species to salinity under controlled laboratory conditions and did not survive at an EC of 1.31 mS cm
1. In contrast, the biomass of E. foetida was observed to be negatively affected by the TN content in all the samples subjected to bokashi pre-composting. Thus, vermicomposting with high N content (low C/N ratio in vermicompost without pre-composting using the traditional composting method, Table 4) will result in N loss in the form of NH3 to the atmosphere. In addition, the high ammonia concentration is not suitable for the worms, prompting them to escape or affecting their growth (Rostami, 2011).

3.11. FA FA was conducted by evaluating the principal components and retaining only eigenvalues >1 (Kaiser’s criterion). This technique allows an important reduction in the number of variables, structures the association between different variables (Maiz et al., 2000), and creates new variables (factors), resulting in a considerably lower number of factors than the number of variables. In the present study, factor loadings <0.51164 were eliminated from the physicochemical data, which included the number and biomass of E. foetida (Table 8) smaller than 0.54109 and the index of maturity of vermicomposting zoo animal dung with E. foetida (Table 9). Factor 1 showed a positive association between P and N and E. foetida biomass and a negative association between P and N and

Table 8 Factor rotation matrix for physico-chemical data, number and biomass of E. fetida.

Treatment Number E. foetida Biomass E. foedida OC CEC P N pH EC C/N ratio

Factor 1

Factor 2

Factor 3


84*
9 53* 28
78* 89* 81*
8
3
59*


25 7 59*
2
37
21
43 93*
87* 51

4 80*
17 84*
29 5 35
4
14 35

Factor loadings greater than 0.51164 were flagged with a *OC = organic carbon, CEC = cationic exchange capacity, P = phosphorous, N = total nitrogen, EC = electrical conductivity.

Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036

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E.A. Pérez-Godínez et al. / Waste Management xxx (2017) xxx–xxx

Table 9 Factor rotation matrix for physic-chemical data and index of maturity of vermicompost zoo animal dung with E. foetida.

Treatment OC CEC P N pH EC C/N CEC/OC OM loss TN loss

Factor 1

Factor 2

Factor 3

82*
17 76*
82*
73* 6 4 61* 33 15 70*


13
2
44
36
48 92*
91* 57*
24 85* 33


14 98*
37 18 42 0
14 44
91*
4
60*

Factor greater than 0.54109 were flagged with a * OC = organic carbon, CEC = cationic exchange capacity, P = phosphorous, N = total nitrogen, EC = electrical conductivity, OM loss = organic matter loss, TN loss = total nitrogen loss.

CEC and C/N ratio (Table 8). These results confirmed that the biomass of E. foetida was affected by the C/N ratio, with the growth of E. foetida increasing with decreasing C/N ratio and vice versa (Gunadi et al., 2003; Ndegwa and Thompson, 2001). The increment in CEC in all the samples after 3 months of vermicomposting resulted from the increase in carboxyl and phenolic functional groups formed during humification (Lax et al., 1986), which are part of passive pool. In contrast, N and P are mineralizable nutrients that form the active pool, which are easily mineralized, leachable, or precipitated (Jouquet et al., 2011; Singh et al., 2010). Accordingly, a negative relationship exists between CEC and mineralizable nutrients such as N and P. Factor 2 presented a positive association between pH and biomass of E. foetida. However, EC was negatively associated with E. foetida biomass (Table 9). E. foetida increased the pH of the vermicompost as a result of higher mineralization and utilization of organic acids (Tripathi and Bhardwaj, 2004; Fares et al., 2005). The biomass of E. foetida increased with increasing pH and decreased with increasing EC. These results confirmed that the biomass of E. foetida was affected by the salt concentration (Fig. 1 and Tables 4 and 5), affirming that E. foetida is the most sensitive species to salinity. Factor 3 showed a positive association between OC and number of E. foetida. Fig. 1 showed the highest concentration of E. foetida in VEL, VHI, and VHO, which had higher %OC (37.17–38.80%), followed by VBHO, VBEL, and VBHI (%OC = 34.7–36.7%) (Table 5). These results suggested that samples that provided E. foetida with high amounts of easily metabolizable OM and nonassimilated carbohydrates favored its growth and reproduction (Edwards and Lofty, 2013; Gajalakshmi and Abbasi, 2004).

3.12. Factor rotation matrix for physicochemical data and maturity index Factor 1 presented a negative association between P and N and TN loss and a positive correlation between P and N and CEC and C/N ratio (Table 9). As mentioned earlier, N decreases during vermicomposting may be due to ammonification, ammonium volatilization, and denitrification (Bernal et al., 1996; Garg et al., 2006a). Both pre-composting and vermicomposting resulted in loss of C and N because of mineralization (Tables 4 and 5). The decomposition of animal dung was slow throughout vermicomposting, and the %N loss ranged between 0.32 and 4 (Table 6), indicating the slow mineralization of OM rich in lignin compounds and poor in N content, which promoted high humification (C/N ratios) (Sánchez-Monedero et al., 2002; Huang et al., 2006; Alburquerque et al., 2009) and increased CEC (Tomati et al., 2000). Accordingly, a

positive relationship resulted between the degree of humification and low N mineralization, and a negative association occurred between the degree of humification and N content and N loss in all the pre-composted samples before vermicomposting. Factor 2 showed a negative association between pH and EC and a positive correlation between pH and %OM loss and C/N ratio (Table 9). The increase in pH from 1 month (pH 6–7.3) to 3 months (pH 7.3–9.40) of vermicomposting (Tables 4 and 5) indicated that the negative charge resulted from the abundance of phenolic functional groups and OH alcohol groups (Stevenson, 1994). Thus, in the basic organic waste, the EC tends to decrease, resulting in a decrease in the basic cations (Sánchez-Hernández et al., 2007), producing a negative relationship between pH and EC. At neutral to basic pH, the OM is dominated by carbonate compounds (lignin and cellulose) as a consequence of increased humification (high C/N ratio). However, the progressive stabilization of the vermicompost from 1 to 3 months implicated that the %OM and %N loss gradually reduced owing to the strong decrease in the available C sources (Alburquerque et al., 2009) as well as increased mineralization (%OM loss). Factor 3 presented a negative association between OC and CEC/ OC and TN loss. This may be due to the increase in the degree of humification and mineralization with time during vermicomposting (Tables 6, 4, and 5) (Raj and Antil, 2011). 4. Conclusions The EC, CEC, OC, N, P, C/N, and pH levels of all the precomposted samples subjected to vermicomposting reached the norms of vermicompost. However, vermicomposting after bokashi pre-composting produced the highest values of %OM loss, %N loss, and CEC/OC ratio, when compared with those achieved by vermicomposting after pre-composting using the traditional composting method. These three parameters indicated higher mineralization and therefore higher microbial diversity and readily available nutrients for plants. Nevertheless, the RP of E. foetida in samples subjected to bokashi pre-composting followed by vermicomposting was lower, when compared with that in samples subjected to pre-composting using the traditional composting method followed by vermicomposting. Further studies are required to determine the optimal E. foetida stock density and the type of pre-composting for each animal dung treatment. Besides, the optimum pH for efficient growth of E. foetida needs to be ascertained. Thus, taken together, the two methods applied in the present study appear to be a good alternative to treat zoo animal dung waste and convert it into valuable nutritional material for plants. In the future, further experiments with plants should be conducted to determine the other advantages and disadvantages of using these two methods of pre-composting prior to vermicomposting. Acknowledgments The authors thank the directors of the installations of the zoo in San Juan de Aragón, México City, and Noemi Araceli Rivera Casado for her technical help. We would like to thank all anonymous reviewers for their constructive comments on the paper. The research did not receive any specific grant for funding agencies in the public, commercial or not for profit sectors. References Ahmed, F.F., Abada, M.A.M., Ali, A.H., Allam, H.M., 2014. Trials for replacing inorganic N partially in superior vineyard by using slow release N fertilizers, humic acid and EM. Stem Cell 5, 16–29. Aira, M., Monroy, F., Domínguez, J., Mato, S., 2002. How earthworm density affects microbial biomass and activity in pig manure. Eur. J. Soil Biol. 38, 7–10.

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Please cite this article in press as: Pérez-Godínez, E.A., et al. Growth and reproductive potential of Eisenia foetida (Sav) on various zoo animal dungs after two methods of pre-composting followed by vermicomposting. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.03.036