Stabilisation of sewage sludge and vinasse bio-wastes by vermicomposting with rabbit manure using Eisenia fetida

Stabilisation of sewage sludge and vinasse bio-wastes by vermicomposting with rabbit manure using Eisenia fetida

Bioresource Technology 137 (2013) 88–97 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com...

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Bioresource Technology 137 (2013) 88–97

Contents lists available at SciVerse ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Stabilisation of sewage sludge and vinasse bio-wastes by vermicomposting with rabbit manure using Eisenia fetida María José Molina a,⇑, María Desamparados Soriano b, Florencio Ingelmo a,c, Josep Llinares d a

Centro de Investigaciones Sobre Desertificación-CIDE (CSIC-UV-GV), Carretera Moncada-Náquera Km 4.5, 46113 Moncada, Valencia, Spain ETSIAMN, Universidad Politécnica de Valencia, C/ Camí de Vera s/n, 46021 Valencia, Spain c Instituto Valenciano de Investigaciones Agrarias-IVIA, Carretera Moncada-Náquera Km 4.5, 46113 Moncada, Valencia, Spain d Institut d’Investigació Per a la Gestió Integral de les Zones Costaneres (IGIC-UPV), Paranimf 1, 46730 Grau de Gandia, Valencia, Spain b

h i g h l i g h t s  Rabbit manure and sewage sludge or vinasse mixtures tested as feed for E. fetida.  Quality of mixtures affected final products and worm’s growth and reproduction.  Worm number and weight rose in rabbit manure and sludge mixtures at low doses.  Soluble salt amount and composition were most important in biological parameters.  Worms recycled biowaste and biological parameters indicators of environmental risk.

a r t i c l e

i n f o

Article history: Received 11 November 2012 Received in revised form 5 March 2013 Accepted 7 March 2013 Available online 16 March 2013 Keywords: Rabbit manure Sewage sludge Vinasse Eisenia fetida Biological parameters

a b s t r a c t Changes in the chemical characteristics and biological parameters of Eisenia fetida were assessed by VER using (CO + VE) rabbit manure (Vo) spiked with sewage sludge (SS) or vinasse (V). Seven mixtures were used: Vo, control; Vo + SS at 10%, 30%, and 50% (SS1, SS2, and SS3); Vo + V at 10%, 30%, and 50% (V1, V2, and V3). SS vermicomposts had higher humus, nutrient and total metal contents, but less soluble salts (EC) than V vermicomposts. The number and weight of worms were higher in Vo, followed by SS, at decreasing doses. V3 showed the smallest number and size. The EC of the initial mixtures explained reduced weight, whereas EC and avP2O5 accounted for lower numbers. Vermicomposting is an efficient biowaste recycling technology, but the total amount and composition of soluble salts in food influence the quality of end products and are of primary importance for biological parameters of worms. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Vast amounts of organic waste are produced worldwide and disposing of such waste is a major environmental concern. Organic waste comprises under-utilised resources that can be recycled to help improve soil fertility in either agriculture or the restoration plans of degraded lands. This is particularly the case in semiarid Mediterranean areas, where soils are prone to degradation because of climatic characteristics, forest fires, urban pressure, Abbreviations: VER, vermicomposting; (CO + VE), combined pre-composting plus vermicomposting; EC, electrical conductivity; OC, organic carbon; HS, alkaliextractable humic substances; HA, humic acids; FA, fulvic acids; TKN, total Kjeldahl nitrogen; avP2O5, available phosphorous; HR, humification rate; CEC, cation exchange capacity; Vo, rabbit manure vermicompost; SS, rabbit manure plus sewage sludge vermicomposts; V, rabbit manure plus vinasse vermicomposts. ⇑ Corresponding author. Tel.: +34 963 424033; fax: +34 963 424160. E-mail address: [email protected] (M.J. Molina). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.03.029

industrialisation and intensive agriculture. Diminished soil fertility and productivity due to excessive soil erosion, loss of nutrients and organic matter in Mediterranean soils has generated interest in improving overall soil quality by adding organic matter. Some organic waste can be applied with no risk implied. Nevertheless, many others cannot be applied to soil before stabilisation because some components (nitrogen, phosphorous, metals, alcohols, etc.) may be noxious, and may lead to emission of greenhouse gases, affect plant growth and soil biota, or can even contaminate the surrounding ecosystem. To avoid the potential risk of noxious components and to biologically transform organic matter, most non-toxic, agricultural residues, animal manures, urban and industrial organic wastes can be used for composting purposes (Canet et al., 2008), vermicomposting if earthworms are involved (Yadav and Garg, 2011; Singh and Suthar, 2012) and combined composting followed by vermicomposting (Ndegwa and Thompson, 2001; Fornes et al., 2012).

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Vermicomposting is a low-cost, eco-efficient organic waste treatment technology which enhances stabilisation and humification through a non-thermophilic process. During the vermicomposting process, earthworms maintain aerobic conditions in organic waste, convert a portion of organic material into worm biomass and respiration products, and expel the remaining partially stabilised product. The vermicomposts and end products combining composting and vermicomposting can also result in a high quality humic product that can be used not only as a soil organic amendment, but can be physically, nutritionally and biochemically improved as compared to compost (Ndegwa and Thompson, 2001). Eisenia fetida is considered the most eurythermal species of epigeic earthworms. It is the favoured species for laboratory experiments given its ability to adapt to diverse environmental conditions (pH, temperature, moisture etc., Singh and Suthar, 2012) and its potential to recycle organic waste materials into value added products is well-documented (Yadav and Garg, 2011). However, information on the effect of diet on earthworms during the vermicomposting process is still limited. Some examples of noxious components of feed for E. fetida activity and vermicomposting processes are elevated pH and the salinity of some animal manure (Mitchell, 1997). Other components like metal contents may, or may not, prove noxious for worms depending on their bioavailability, and can also depend on pH, salinity and organic matter transformations (Ingelmo et al., 2012). Whatever the use of organic waste, potential environmental hazards must be considered. The growth and reproductive success of earthworms might provide an indication of potential environmental hazards, and may also help evaluate possible synergies or antagonisms with and between potentially hazardous chemicals of mixtures and worm species at the same time. Comparative studies on the composting efficiency and impairment of these vital earthworm functions under different feed conditions, such as use of heterogeneous waste combinations of cow dung, poultry droppings and food industry sludge employing E. fetida, have concluded that the quality of initial feed mixtures determine the physicochemical characteristics of vermicomposts (Yadav and Garg, 2011). Rabbit manure, sewage sludge and distillery vinasse are among the biowastes whose disposal causes environmental concern, and which require stabilisation before being employed as organic amendments for agricultural or environmental proposals. Combinations of pre-composted/vermicomposted rabbit manure with sewage sludge or winery-distillery vinasse as feed for E. fetida have not yet been studied. Thus extrapolating results from previous studies may prove difficult as they use different materials for mixtures and experimental conditions. In view of the above, the objectives of this study are to: (i) test the feasibility of vermicomposting to stabilise anaerobic sewage sludge and distillery vinasse mixed with pre-composted/vermicomposted rabbit manure at different ratios by evaluating the manurial value of final vermicomposts; (ii) investigate the effects of different substrate compositions on worm growth and reproductive success. 2. Methods 2.1. Biowaste, E. fetida collection and pre-composting/ vermicomposting rabbit manure The most relevant chemical data of fresh organic waste in the study area, considered to be noxious components of the materials used, were found in the scientific literature or in personal communications. These relevant data were: high salinity of fresh rabbit manure (17 dS m 1 EC1:5, Canet et al., 2008); metal contents of sewage sludge (FACSA, personal communication; Ingelmo et al., 2012); a combination of high moisture content, high salinity and

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low pH (Vinicola del Oeste S.A., personal communication). The experimental design included available knowledge on the different results that composting, vermicomposting or combined treatments can offer to overcome the aforementioned noxious components. Fresh rabbit manure, procured from a local farmer, was selected as the bulking and initial substrate for worms after an appropriate precomposting/vermicomposting process. It was precomposted for 21 days, heaped on outdoor unpaved ground, exposed to a temperature between 29 and 32 °C, and was periodically aerated and moistened when necessary. The heat generated in the initial decomposition stages made the temperature rise up to >60 °C. After the 21-day period, the precomposted rabbit manure served as bedding and feed for E. fetida for 8 weeks. Worms adapted well to the substrate, as visually observed by the number of individuals and cocoons. This precomposted/vermicomposted rabbit manure was collected in large-sized plastic pot containers to be taken to the laboratory in order to be mixed with sewage sludge or vinasse after previously separating worms. The vinasse used was lees cake vinasse (90% moisture, pH 5, 6 dS m 1 EC) obtained from a winerydistillery industry in San Antonio de Requena (Valencia, E Spain). The used sewage sludge was an anaerobically digested dewatered sludge cake (80% moisture, pH 8, 2.2 dS m 1 EC, 825 g kg 1 Zn, 276 g kg 1 Cu) obtained from a municipal wastewater treatment plant (FACSA, Castellón, E Spain). 2.2. Mixture preparation, vermicomposting and worm development Seven mixtures were prepared: the precomposted/vermicomposted rabbit manure alone was used as the control (Vo); Vo + sewage sludge at 10%, 30%, and 50% (wet weight basis) (SS1, SS2, and SS3, respectively); Vo + vinasse at 10%, 30%, and 50% (wet weight basis) (V1, V2, and V3, respectively). Each treatment was replicated three times. A homogenisation process of the mixing materials and the mixtures was carefully carried out. Next, 1500 g (wet weight) of each mixture were placed into 3 L plastic circular containers (20 cm long; 15 cm diameter) with a fine nylon mesh at the top and pierced lids for aeration. Forty-five clitellated and non-clitellated earthworms, which emerged from the beds they had adapted to for 8 weeks, were added to the mixtures. The moisture content was maintained at 85% throughout the vermicomposting period by periodically sprinkling adequate amounts of tap water, and containers were kept in a dark room at 25 °C. Neither rabbit manure nor biowaste was added during this vermicomposting period. At the end of 8 weeks, the activity of the worms in the mixtures was clearly observed after a black colour with a uniformly disintegrated structure had developed. The earthworms produced in each container during the experiment were separated from the substrate material. The organic substrate in the container was emptied, dried at 60 °C and analysed in the laboratory. 2.3. The biological parameters of worms The number and biomass of the worms were determined when vermicomposting finished. Worms were separated by hand, manually counted and weighed on a live weight basis. Biomass was determined by washing them in distilled water and drying them on paper towels. They were weighed in a water-filled weighing boat on a Sartorius analytical balance model CPA224S. This was done to prevent the worms from desiccating, which affects weight. 2.4. Chemical analysis The following chemical characteristics were determined in the initial mixtures and in the final vermicomposts: moisture content,

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organic carbon (OC), pH, electrical conductivity (EC), total nitrogen (TKN), C:N ratio, available phosphorous (avP2O5), and total contents of Zn(II), Cu(II), Ni(II) and Cd(II). The final vermicomposts were also analysed for humus fractionation in humic substances (HS), Fulvic acids (FA) and Humic acids (HA), the HA:FA ratio, the humification rate (HR) and cation exchange capacity (CEC). Organic carbon (OC) was determined by oxidation with potassium dichromate (Nelson and Sommers, 1996). pH and EC were measured with a glass electrode by respectively using the 1:2.5 and 1:5 sample:water ratios. EC values were referred to as a standard temperature of 25 °C. Total nitrogen (TKN) and available phosphorous (avP2O5) were measured by the micro-Kjeldahl method (Jackson, 1973) and the methodology by Olsen et al. (2002), respectively. Humus fractionation in HS, FA and FA was determined following the method proposed by Zaccheo et al. (2002). HR was calculated according to the following expression: HR = (100 ⁄ (HS/OC). Total micronutrients and heavy metals were extracted after the digestion of samples with HNO3:HClO4, (Polkowska-Motrenko et al., 2000), determined in a Perkin Elmer ICP/-5500. CEC was established according to Harada and Inoko (1980). 2.5. Statistical analysis The SPSSÒ statistical package (Windows Version 19.0, IBM, Chicago, USA) was employed. The probability levels used for statistical significance were P < 0.05. All the results reported in the text are the mean of three replicates. A two-way analysis of variance (ANOVA) was used to analyse the significant differences among mixtures. A Tukey’s HDS-test was performed as a post hoc to identify the homogeneous type of mixtures for the various parameters. A discriminant analysis (DA) was conducted to classify the initial and final groups of mixtures according to the most relevant changing chemical parameters. To analyse the underlying variation in the chemical parameters of the final vermicomposts, data were subjected to a principal component analysis (PCA), which was done using the correlation matrix (rotation Varimax) of the determined chemical parameters. A stepwise linear regression analysis was used to ascertain the relationships between worms’ biological parameters and the mixtures’ chemical composition. 3. Results and discussion 3.1. Chemical characteristics of precomposted/vermicomposted rabbit manure and of its mixtures with biosolids Table 1a provides the results for the chemical properties characterising the seven groups of initial mixtures. Type of mixture, dose of added biosolids and their interaction were statistically significant for almost all the properties. This finding suggests that different chemical feed scenarios for worm activity resulted after mixing the treated rabbit manure (Voi) as a bulking agent by adding the type of biosolids and dose. 3.1.1. Precomposted/vermicomposted rabbit manure It proved difficult to compare the chemical characteristics of the control Voi with the other organic wastes used in the literature because data about employing fresh rabbit manure after a combined precomposting/vermicomposting process as a substrate and bulking agent for SS and V mixtures are lacking. The comparisons made with other data (Nogales et al., 2005) reveal that: TKN and avP2O5, as obtained in Voi, were between those achieved with the initial manure used as a control and the 16-week vermicompost; the OC contents came close to the initial manure; the C:N ratio, EC, and zinc and copper came close to the final vermicompost. These results are supported to an extent by the results recently

obtained after comparing chemical changes by vermicomposting and combined composting + vermicomposting techniques (Fornes et al., 2012). These authors worked on composting/vermicomposting tomato crop residues and almond shell mixtures. They reported a C:N ratio of around 13, and they observed chemical changes which were attributable to both composting and worm activity. When compared with vermicomposted cow manure (Singh and Suthar, 2012), Voi was characterised by lower OC and TKN contents, and by a 10-fold higher concentration of avP2O5. According to the C:N values as the maturity index established for composts (<20, preferable <10, Bernal et al., 2009), Voi can be considered a mature product. By considering that soils are a natural habitat for terrestrial worms under optimal moisture and temperature conditions, the chemical characteristics of the study area soils were taken as a reference point to evaluate the quality of Voi as feed for worms and to make further comparisons with other mixtures. Voi macronutrient contents (OC, TKN and avP2O5) approached the organic-mineral interlayer of natural fertile calcareous soils in the study area in Valencia (Molina, personal communication). The C:N ratio of around 17 was between that considered in soils when organic matter was transformed by humification processes (around 10), whereas the C:N ratio of 25 was considered optimal for Eisenia vermicomposting. (Ndegwa and Thompson, 2000). However, Pramanik and Chung (2011) reported that lower C:N ratios facilitated E. fetida feeding on mixtures with fly ash and vinasse at increasing doses which, in turn, raised the organic matter decomposition rate because E. fetida worms preferred organic substrates that are rich in TKN content. Regarding pH and EC, the Voi values approached those of calcareous non-saline non-sodic soils. Moreover, total Zn(II) and Cu(II) contents were respectively around two and three times higher than the 100 mg kg 1 zinc and the 20 mg kg 1 copper estimated from the observed contents in natural soils in the study area (Molina, personal communication) if extrapolated to OC contents as high as those in Voi. The chemical data obtained for Voi indicate that precomposted/vermicomposted rabbit manure is good quality growing media for plants and worms. 3.1.2. Chemical characteristics of mixtures with biosolids The OC contents in Voi (468 g kg 1) were significantly higher than those for S1i and V1i, which were also higher than the OC contents in S3i, V2i and V3i (401–378 g kg 1). An alkaline pH, ranging from 8.4 to 8, characterised the Voi, SS1i, SS2i, SS3i and V1i mixtures, and was significantly higher than the pH in V2i and V3i (6.7 to 6). pH and EC values correlated negatively (R = 0.98). As a result, the different mixtures also differed in terms of the type of cations, which increased EC (mainly Ca2+ and Na+) and pH (mainly Ca2+). When compared with Voi, the EC in the V2i and V3i mixtures was significantly higher and increased with dose (3.8–4.7 dS m 1). In the SSi mixtures, EC did not vary in accordance with dose, while the mean values were similar to those in Voi. The TKN in the SS3i and SS2i mixtures (38 to 28 g kg 1) was significantly higher than in the remaining mixtures. Higher avP2O5 contents were seen for SS3i, followed by those found for SS2i, V2i and V3i, with mean values of between 5 and 6 g kg 1. The available phosphorous contents in Voi, S1i, and V1i were significantly lower than for the remaining mixtures. The C:N ratios of around 17 characterised Voi, S1i and V2i, and were significantly higher than those for V2i and V3i. The lowest C:N ratio was observed for SS2i and SS3i. The Zn(II) contents of SS3i, SS2i and SS1i (in this order) fell in the 407 to 266 mg kg 1 range, and were significantly higher than Zn(II) in Voi (248 mg kg 1). The lowest content was observed for Vi mixtures. Similar trends were noted for Cu(II), with values ranging from 188 to 118 mg kg 1, from mixtures SS3i to SS1i, to the lowest content in Vi mixtures (around 27 mg kg 1), Voi had intermediate Cu(II) contents and the SSi mixtures showed the highest Ni(II)

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Table 1 (a) Chemical characteristics of initial mixtures. (b) Chemical characteristics of final vermicomposts. (c) Chemical changes after vermicomposting (difference final minus initial). (d) Biological parameters of E. fetida at the end of the process. (Mean ± SD, N = 3). (a) Parameter

Voi

SS1i

SS2i

SS3i

V1i

V2i

Moisture OC pH1:2.5 EC1:5 TKN avP2O5 C:N Cu Zn Ni Cd ANOVA

70 ± 4b 468 ± 23a 8.4 ± 0.2a 1.70 ± 0.20c 27 ± 3c 4.13 ± 0.15c 17 ± 2a 59 ± 2b 248 ± 30b 6.7 ± 8.9b 0.7 ± 0.3b Biosolid

71 ± 3b 447 ± 21ab 8.3 ± 0.3a 1.80 ± 0.20c 28 ± 2c 4.47 ± 0.15c 16 ± 1ab 118 ± 14b 266 ± 35b 17.3 ± 5.0a 0.8 ± 0.1b F = 254.5 P < .001

73 ± 5b 422 ± 27ab 8.2 ± 0.1a 1.87 ± 0.15c 30 ± 2ab 5.23 ± 0.15b 14 ± 1c 139 ± 22b 332 ± 34ab 20.9 ± 5.3a 0.9 ± 0.0a Dose

75 ± 3b 401 ± 15b 8.0 ± 0.1ab 2.00 ± 0.10c 33 ± 2a 6.03 ± 0.25a 12 ± 1c 188 ± 26a 407 ± 53a 25.9 ± 5.9a 1.1 ± 0.2a F = 3.169 P < .001

72 ± 2b 446 ± 15ab 7.9 ± 0.2ab 2.13 ± 0.15c 26 ± 2c 4.27 ± 0.15c 17 ± 2a 28 ± 2c 129 ± 14c 4.4 ± 0.1b 0.4 ± 0.1c Biosolid ⁄ Dose

83 ± 6a 378 ± 27b 6.7 ± 0.3c 3.83 ± 0.21b 25 ± 2c 5.67 ± 0.31ab 15 ± 2b 26 ± 5c 98 ± 13c 4.7 ± 0.3b 0.3 ± 0.1c F = 3.393 P < .001

(b) Parameter

Vof

SS1f

SS2f

SS3f

V1f

V2f

Moisture OC pH 1:2.5 EC1:5 TKN avP2O5 C:N Cu Zn Ni Cd (c) Parameter Moisture OC pH1:2.5 EC1:5 TKN avP2O5 C:N Cu Zn Ni Cd (d) Parameter End life weight Number of worms

42 ± 2c 373 ± 10a 7.8 ± 0.1b 1.95 ± 0.08d 25 ± 1a 4.30 ± 0.30c 15 ± 1b 39 ± 4d 144 ± 12a 0,8 ± 0.1c 0.1 ± 0.0c Vof–i

42 ± 1bc 336 ± 7b 7.7 ± 0.2b 2.66 ± 0.40c 23 ± 1ab 6.03 ± 0.46b 14 ± 0c 94 ± 9c 145 ± 15a 18.0 ± 3.0a 0.2 ± 0.0c SS1f–i

28 ± 1.5c 94 ± 31ab 0.6 ± 0.2 a 0.30 ± 0.01ab 2.1 ± 3.2ab 1.70 ± 3.8ab 2±3 20 ± 5.2a 103 ± 32c 6 ± 2.0ab 1.0 ± 0.1a Vof

SS2f–i

28 ± 1b 111 ± 28a 0.5 ± 0.1a 0.90 ± 0.50b 4.1 ± 2.9ab 1.60 ± 0.32b 2±1 24 ± 5.0a 121 ± 40c 0.7 ± 0.4b 0.6 ± 0.1a SS1f

a

707 ± 28 424 ± 23a

44 ± 3b 331 ± 7b 7.7 ± 0.1b 2.84 ± 0.18c 25 ± 2a 7.23 ± 0.74a 13 ± 1d 136 ± 13b 140 ± 14a 17.7 ± 3.5a 0.5 ± 0.1b

SS3f–i

29 ± 2b 91 ± 28ab 0.5 ± 0.1a 1.00 ± 0.30b 4.9 ± 3.6ab 2.00 ± 0.60c 1±1 2.1 ± 3.6c 191 ± 37ab 3.2 ± 4.0b 0.4 ± 0.1ab SS2f

b

611 ± 26 386 ± 23ab

45 ± 2b 312 ± 9c 7.6 ± 0.1a 2.83 ± 0.18c 25 ± 1a 7.43 ± 0.40a 12 ± 0d 159 ± 13a 162 ± 16a 17.3 ± 3.1a 0.8 ± 0.1a

V1f–i

30 ± 1b 88 ± 20ab 0.4 ± 0.1a 0.80 ± 0.20b 8.7 ± 1.2a 1.40 ± 0.70b 1±1 28 ± 4.0b 245 ± 41a 8.5 ± 3.0a 0.2 ± 0.2ab SS3f

bc

550 ± 21 328 ± 20ab

43 ± 1 b 366 ± 9a 7.0 ± 0.1c 2.9 ± 0.10c 25 ± 2 4.90 ± 0.50c 14 ± 1c 32 ± 2d 140 ± 15a 11.0 ± 2.6b 0.4 ± 0.1b

551 ± 25 250 ± 15b

508 ± 22 315 ± 31bc

48 ± 3 a 307 ± 11c 8.2 ± 0.1a 4.16 ± 0.15a 18 ± 1 4.40 ± 0.26c 17 ± 1a 21 ± 2d 130 ± 5b 4.0 ± 1.0c 0.8 ± 0.1a V3f–i

33 ± 2b 32 ± 33b 1.1 ± 0.2b 0.40 ± 0.2a 2.7 ± 1.6ab 1.4 ± 2.50a 1±1 3.1 ± 7.0c 24 ± 5d 0.3 ± 0.7b 0.2 ± 0.1b V2f

c

81 ± 6a 401 ± 27b 6.0 ± 0.2d 4.73 ± 0.15a 26 ± 3c 5.25 ± 0.13b 16 ± 6b 28 ± 2c 52 ± 7c 6.3 ± 1.7b 0.2 ± 0.1d

V3f

49 ± 4 a 345 ± 9b 7.7 ± 0.0b 3.46 ± 0.06b 23 ± 1ab 4.20 ± 0.46c 15 ± 0b 29 ± 2d 122 ± 9b 5.0 ± 1.0c 0.5 ± 0.1b V2f–i

29 ± 1b 79 ± 22b 0.8 ± 0.3a 0.80 ± 0.20b 0.3 ± 2.2c 0.63 ± 0.5ab 3±2 4.9 ± 4.2c 11.8 ± 3d 6.5 ± 2.7d 0.1 ± 0.1b V1f

bc

V3i

32 ± 2a 93 ± 29ab 2.1 ± 0.2c 0.60 ± 0.30a 8.4 ± 2.8a 0.80 ± 0.50a 2.1 ± 2.1 7.5 ± 8.5c 78 ± 11d 2.3 ± 0.4ab 0.5 ± 0.1c V3f

c

458 ± 22 271 ± 26bc

351 ± 40d 197 ± 25c

Moisture: %; OC: g kg 1; EC: dS m 1; TKN: g kg 1; avP2O5: g kg 1; Cu, Zn, Ni and Cd: mg kg 1; Life weight: mg/worm. Vo, precomposted/vermicomposted rabbit manure; SS1, SS2 and SS3, Vo plus sewage sludge at 10%, 30% and 50%, respectively. V1, V2 and V3, Vo plus vinasse at 10%, 30% and 50%, respectively. i, initial mixtures; f, final vermicomposts. A minus sign preceding data in Table 1c indicates a decrease of the parameter in the final vermicompost. ⁄ Different letters in a row are significant at P < 0.05 (Tukey’s HDS-test).

contents, which increased with the sludge dose from 17 to 26 mg kg 1. The Ni(II) in Voi showed high variability with mean values of around 7 mg kg 1, which were close to those in the Vi mixtures. Finally, Cd(II) content was between 1.1 and 0.8 mg kg-1 for the SSi mixtures, followed by Voi. The lowest cadmium concentrations were recorded for the Vi mixtures (0.2–0.4 mg kg 1). By mixing Voi with sewage sludge at increasing doses, the OC and C:N ratios in the mixtures clearly lowered, and EC, TKN, avP2O5, and metal content increased proportionally to the dose added, whereas moisture and pH remained similar to Voi. Mixing Voi with vinasse reduced the OC contents and pH, but increased EC and avP2O5 in the mixtures proportionally to the dose added. Conversely, TKN remained similar to Voi, while the C:N ratio and the moisture content increased inversely and proportionally to the dose added, and the metal contents decreased proportionally to the added dose of vinasse.

3.2. Potential of E. fetida in organic waste stabilisation and biosolids recycling The final vermicomposts were odour-free, darker, finer and more homogeneous than the initial mixtures. Table 1b offers the main properties analysed after the 8-week worm activity on both the control treatment (Vof) and the biosolid mixtures (SSf and Vf). 3.2.1. Chemical characteristics of final vermicomposts The mean values and statistical results reveal significant differences among the final vermicomposts. The moisture contents in V2f and V3f were around 6% higher than in the rest. The OC contents in Vof, V1f and SS1f (from 373 to 345 g kg 1) differed from those of SS1f, SS2f and V2f (345 to 331 g kg-1), and were significantly lower in SS3f and V3f. pH varied from alkaline in V3f (8.2) to neutral in V1f. When compared with Vof, the EC of all the

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biosolid mixtures was higher, and it was significantly higher in the V3f and V2f (4.2 and 3.5 dS m 1) than in the SSf mixtures. TKN in V3f, V2f and SS1f (from 18 to 23 g kg 1) was significantly lower than in Vof and the remaining SSf mixtures (25 g kg 1). The highest avP2O5 contents were observed for SS3f, SS2f and SS1f (from 7.4 to 6 g kg 1, in this order), which differed from the rest (4.9 to 4.2 g kg 1). Four groups of vermicomposts were distinguished in a decreasing gradient of the C:N values: V3f (17); V2f and Vof (15); SS1f and V1f (14); SS2f and SS3f (<14). SS3f, SS2f, SS1f, V1f and Vof had similar Zn(II) contents (162– 140 mg kg 1), which were significantly higher than those in V3f and V2f (130–122 mg kg 1). The lowest content was found for the Vf mixtures. In SS3f and SS2f, Cu(II) came close to Zn(II) contents (159 and 136 mg kg 1, respectively), whereas in SS1f, Cu(II) content (94 mg kg 1) was not as close to the Zn(II) concentration (145 mg kg 1). The SSf mixtures presented similar Ni(II) contents (around 17 mg kg 1) and differed from 11 mg kg 1 in V1f, while these two last groups differed from that formed by V2f, V3f and Vof (<5 mg kg 1). Finally, Cd(II) contents were higher for SS3f and V3f (0.8 mg kg 1) and differed from the SS2f, V1f and V2f (0.5 mg kg 1) group, whereas both groups differed from SS1f and Vof (<0.2 mg kg 1). The obtained results suggest that the chemical characteristics of the initial mixtures varied after vermicomposting, and that the magnitude and sign of changes may depend on the chemical characteristics of the initial mixtures interacting with the worms and microorganisms implied in the process. 3.2.2. Changes in the chemical characteristics of mixtures after vermicomposting The differences between the properties of the initial mixture and the final vermicomposts (Table 1c) reveal that earthworms have a statistically significant effect on most of the chemical parameters characterising the initial mixtures. The magnitude of the change (absolute values in Table 1c) varied with dose. The direction of change (sign of the values in Table 1c) depended on the most relevant parameters that differentiated between the V and SS mixtures as compared to the control (Vo). As already mentioned (Section 3.1.2.), these parameters were increased salinity (phosphate anions), pH and metal contents decreased with dose in the V mixtures, while salinity, pH and metal contents increased with dose in the SS mixtures. On the whole, EC in the SSf mixtures was lower than in Vf mixtures, but was higher than in Vo. Lorencova et al. (2012) reported that phosphate salts have a general inhibitory effect on the growth of Gram-positive bacteria, but Gram-negative bacteria show resistance according to their chain length and pH (i.e., short chain such as monophosphates, or long chain such as polyphosphates under a low or high pH). The Vo, V, and SS mixtures differed in salinity, pH and avP2O5 concentration, whose chemical structure could also differ. Concerning pH and metals, from V2 to V3, the main changes were observed for pH and Zn(II), which increased in the final vermicomposts, whereas lesser variation was noted for Cu(II) and Ni(II) (covaried with Cu) except for V3. The drop in the Cu(II) concentration in the V3f mixture, despite Cuf–i having high variability, suggested that this metal (along with Ni, which exhibited much lower variation in V3f–i) may accumulate in worm tissue. These results are interpreted as the result of increased salinity and phosphate concentration in the mixtures, probably with dissolved organic and inorganic matter originating from vinasse (Pont and Adholey, 2007) under reducing pH and metal contents. This scenario would diminish the metabolic function of the microorganisms/worm system to finally result in the chelation of Zn and Cu metal ions in more stable forms in V1f and V2f (Lorenkova et al., 2012), except Cu and Ni in V3f. The process in the V mixtures is interpreted in the light of the logical ion interchange processes between the salts in solution and those in the solid phase of the

mixtures (organic matter and worms) since E. fetida was exposed to food via intestines and through skin (Lukkari et al., 2005). The fact that the Zn(II) concentration in the Vf vermicomposts was higher than that in the initial mixtures could be explained if Zn(II) came from worms, which acquired it when adapting to the precomposted/vermicomposted Vo, but were interchanged by other cations from the soluble phase of the mixture. The covariation of Zn and Cd in the differences obtained at the low concentration of these two metals in the V2i and V3i mixtures might indicate, in accordance with Demuynck et al. (2007), E. fetida’s strategies to regulate body Zn concentration and to limit Cd toxicity. Thus, E. fetida’s metabolism may be negatively affected by increasing losses of Zn(II) as the main micronutrient for growth (Kizilkaya, 2004), and by the accumulation of Cu (and likely Ni and Cd) in its body at high dose of vinasse in the V3i mixtures. Regarding Zn, when the sign and the magnitude of the changes in Vf mixtures were compared to Vo, the sign changed and the absolute values were lower than in Vo, but the absolute value increased from V1f to V3f, thus supporting the idea of a reduced worm metabolism through lack of Zn(II). It is noteworthy that the more pH and Zn(II) increased, the more moisture, OC and TKN decreased. These results are interpreted in the light of a progressive reduction of the microorganism/worm functions with increased salinity and phosphate contents in the mixtures, along with low pH values. These functions affected the organic matter transformations, and induced worms to consume more water and nutrients to compensate for the increased osmotic potential caused by salinity (Lorenkova et al., 2012). Singh and Suthar (2012) indicated E. fetida’s intensified feeding activity under stress conditions. In addition to the chemical characteristics in the mixture reported in this work, other stressing conditions were the non-continuous feeding vermicomposting systems given the consumption of the fresh substrate (Nogales et al., 2005), or the laboratory-confined media where the avoidance mechanism of noxious components cannot be achieved by worms (Lukkari et al., 2005). As compared to Vo, from SS1 to SS3 the changes noted for Zn(II) were marked and diminished in the final mixtures. These changes were followed by a decrease in both OC and TKN concentrations, suggesting that microorganisms induce the degradation of organic matter in the mixtures, leading to mineralisation (increased EC) and humification (lower pH and increased carboxylic and phenolic groups), just as other works report (i.e., Fornes et al., 2012). When comparing Zn to Vo, the magnitude of changes was greater in SS2, and particularly in the SS3 mixtures, with a similar sign of change to that in Vo. Yet as compared to Vo, the drop in Cu(II) concentration was also similar in SS1f but was much higher in SS3f. This trend suggests that worms may have differentially accumulated additional amounts of Cu(II). The changes noted in Cu(II) and Zn(II) in SS3f are supported by those reported by Li et al. (2009), who observed increased bioavailability for Cu, but decreased bioavailability for Zn after the transit through the gut of worms, which would be the final forms of these metals at least in SS3f. Under alkaline pH, Owojori et al. (2009) reported a high concentration of Cu(II) and of soluble salts (like phosphates are in SS2i and SS3i), which had additive effects for the uptake and accumulation of available forms of Cu(II) in worm tissues and increased Cu(II) toxicity for worms. The high Cu(II)/Zn(II) concentration combined with high phosphate concentrations in SS2 and especially in SS3 might have caused synergistic effects which interfere with microbial/worms activity (Lukkari et al., 2005) for organic matter transformations, with the concomitant reduction of OC and TKN in vermicomposts as compared to the Vo and SS1 mixtures. The proximity of the changes between the Vo and SS1 mixtures supports a lesser antimicrobial effect of phosphate concentration on SS1f and SS2f than on SS3 (Lorenkova et al., 2012). Conversely, the type of soluble salts and pH in the V mixtures apparently caused much more

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Table 2 (a) The Discriminant Analysis (DA) results: coefficients of the Canonical Discriminating functions with the main chemical parameters that explain the differences between initial mixtures and final vermicomposts. (b) The Principal Component Analysis (PCA) results: factor loading of the chemical properties to evaluate the differences between final vermicomposts. Parameter

(a) DA Canonical Functions F1 (75% variance)

HS Cu HR avP2O5 HA FA CEC Ni TKN Zn HA:FA Cd OC pH Moisture EC C:N

(b) PCA Factors F2 (14% variance)

0.36

0.01

0.12 0.31

0.22 0.22

0.06

0.35

0.01 0.34

0.85 0.14

antimicrobial/worm effects than the combined effect of phosphate salts and metals of sewage sludges under an alkaline pH in the mixtures. The type of organic matter that is richer in TKN, and likely lipids and polycondensed aromatic structures (Reveillé et al., 2003), may have favoured the microorganisms/worm activity in SS mixtures which, to a point, coincides with the explanations provided by Pramanik and Chung (2011), who reported higher OC and TKN in cow manure vermicomposts for lime and microbes inoculation. Metal reduction and accumulation of worms have proven species-specific (Suthar et al., 2008). Therefore, the results obtained with other earthworm types may not be comparable with those of E. fetida vermicomposting, and may prove even less comparable under the specific quality of mixtures and the combined treatment processes used in this work. The increased metal content in Vf mixtures agrees with that observed by Yadav and Garg (2011). Metals accumulation in worm tissues depends on the amount and bioavailability of metals in food; that is, on the nature of the wastes in the mixture and on the chemical forms to which metals bond. The work of Kizilkaya (2004), on soils amended with sewage sludge at increasing doses, observed that the Zn(II) and Cu(II) contents in Lumbricus terrestris L tissues increased with the sludge dose added. According to Lukkari et al. (2005), Zn(II) and Cu(II) are trace elements which play an important physiological role in E. fetida (Zn in tissue growth; Cu in transporting substances to cells), and this worm adapts to the metal content in food by regulating the Cu:Zn ratio in tissues at a fairly constant level. The authors reported 30– 60 mg kg 1 Cu(II) and 100–200 mg kg 1 Zn(II) as the metal contents in worm tissues after soil treatment with Cu and Zn chlorides at Cu:Zn ratios from 79/138 to 267/407. Arnold et al. (2007) observed that Cu(II) content in worm tissues increased when the metal took the form of soluble salts (i.e., in V2 and V3 mixtures). EC, Zn(II) and Cu(II), and the other main parameters of the initial and final mixtures, like moisture, OC and TKN, were further employed for a DA analysis to gain a clear picture of the relations between the mixtures’ chemical composition, the way microorganism/worm systems act, and the resulting chemical composition in the final products. Two statistically significant (P = 0.001) canonical functions (Table 2a) explained a total variance of 89%. Function F1 associated EC (negative correlation with pH; data not shown), Cu(II), and Zn(II), EC and metals, all with negative correlations, with that of EC, and Cu(II) was stronger than that with Zn(II). F1 provided interesting information since it classified the groups of initial mixtures according to their most relevant

F1 (51% variance) 0.97 0.93 0.93 0.90 0.87 0.86 0.81 0.79 0.69 0.61 0.16 0.07 0.10 0.30 0.52 0.58 0.89

F2 (25% variance) 0.04 0.34 0.29 0.36 0.28 0.41 0.06 0.29 0.52 0.06 0.75 0.92 0.90 0.29 0.56 0.75 0.08

differences: increasing salinity, decreasing Zn(II) contents in the Vi mixtures at increasing vinasse doses, and lower salinity, yet increasing avP2O5 and Cu(II) and Zn(II) contents, especially Cu(II), in the SS mixtures with increasing sludge doses (Table 1a). This association of variables explained most of the total variance (75%). The concentrations of both soluble salts (and pH) and Cu(II) and Zn(II) were the main chemical parameters explaining the differences between groups of the initial and final mixtures according to F1. Function F2 integrated moisture content, OC and TKN, which all exhibited positive correlations. Moisture showed the highest coefficient, followed by OC and TKN. Although only 14% of total variance was explained, function F2 was as significant as F1 in explaining differences among groups. Interestingly, Zn(II) forms appeared to correlate more to TKN than to OC, which supports the enzymatic function of Zn(II) in the carboxypeptidase-protein metabolism of animals such as worms (Bolan et al., 2012). Moreover, F2 associated and ordered the variables in a gradient of the main needs for microorganisms and for worm activities and development: moisture, essential nutrients OC and TKN, followed by the main micronutrient, Zn(II), which E. fetida uses to adapt to metal contents in food by regulating the Cu:Zn ratio (Lukkari et al., 2005). Fig. 1a depicts the group scores according to functions F1 and F2. The V mixtures obtained negative scores and separated from the F1 axis in a gradient of increased salinity and decreasing metal contents. The SS mixtures, to the right of the F1 axis, obtained positive scores, indicating lower salinity (and higher pH) and higher metal contents. Moreover from SS1 to SS3, the score values increased in a gradient of salinity and metals content. The control treatment came close to the F1 axis, with Voi approaching the SS mixtures and Vof approaching the V mixtures. The differences in the F1 scores between each initial and final mixture reflected the magnitude and direction of changes in EC (and pH), and metal content after vermicomposting. Regarding F2, all the initial mixtures with vinasse (Vi) and sludge (SSi) (in this order) achieved higher scores than the control treatment (Voi), whereas the final vermicomposts obtained the lowest scores. The difference in the F2 scores between each initial and final mixture reflected mainly the changes in macronutrients, but also those in essential micronutrients such as Zn. As mentioned in Section 3.1.1., the chemical characteristics of Voi were generally appropriate for living organisms, except for metal content. According to the Cu:Zn ratio E. fetida uses to cope with metal content in food (30–60 mg kg 1 Cu(II)/100–200 mg kg 1

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Fig. 1a. Graphical representation (axis on a log scale) of initial mixtures (i) and final vermicomposts (f) according to the F1 and F2 functions of the DA (Table 2a). Small triangles in black indicate the centroid group (N = 3).

Fig. 1b. Graphical representation of final vermicomposts in a common space with the variables composing the F1 and F2 factors of the PCA analysis (Table 2b). See Table 1 for codes.

Zn(II), Lukkari et al. (2005) and to the Cu:Zn ratio for Voi (60/248, means in Table 1a), worms need to regulate Cu:Zn when feeding on it. As function F2 indicates, the microorganisms/worm system to feed on Voi manages this by adapting their moisture, OC, TKN and Zn requirements (i.e., the F2 score changes from 1 to 10; see changes in Table 1c). This decrease parallels to the change in the F1 scores from Voi to Vof (from 1 to 1): Cu(II) lowered from 59 to 39 g kg 1, and Zn(II) from 248 to 144 g kg 1. If the effect of salinity and metals on the microbes living in the Voi mixture is attributed to F1, it follows that it is a response to the macronutrient variations (F2) and the chemical parameters in Vof.

In line with such reasoning, the relative changes in the F1 and F2 scores as compared to Vo were considered in order to compare the V and SS mixtures with the control Vo. According to F1, the chemical changes from V3i, V2i and V1i to their respective V3f, V2f and V1f were minimal, be it in a different direction as compared to Vo (see changes in Table 1c). Conversely, the changes in F2 were greater than for Vo. This result is interpreted to derive from the noxius components in vinasses, and from the amount of soluble salts and pH interfering with microbial processes (Lorencova et al., 2012) for organic carbon transformations, which lowered more markedly in the final product after worms fed on it. This interpretation is further supported by the humification parameters in the Vf vermicomposts, which were lower than in Vof (Section 3.2.3.; Table 3a). Conversely, the SS1i and SS2i mixtures changed more than V1i and V2i, with a similar direction of change to that of Vo; that is, towards negative F1 scores (a higher EC, lesser metal contents). For the SS mixtures, the change over the control Vo is interpreted as originating from the noxious components in sludge, phosphates and metals, which interfere with microbial processes (Lorencova et al., 2012), especially in the SS3i mixtures (7.43 g kg 1 and Cu:Zn ratio 188/ 407; Table 1a). The drop in the Zn(II) concentrations was more drastic from the SS1 mixtures to the SS3 mixtures. Clearly, different environment conditions for the mixtures induce different patterns in the microorganisms/worm system, resulting in a clear dependence of the type of biosolids and doses added to influence the final product’s characteristics. The reduction in moisture, OC and TKN is not surprising since any new food for worms was added during the process. The worm/microorganism system obtained energy from the substrate, which was progressively modified and exhausted due to not only loss of dry mass in terms of CO2, but also to moisture loss through evaporation. Nevertheless, some of these losses could be lesser if microorganisms contributed to the humification of organic matter, which would depend on substrate type (Ndegwa and Thompson, 2000). These results are in agreement with other works (Nogales et al., 2005; Aira and Domínguez, 2009), unlike the continuousfeeding processes where the incorporation of fresh organic matter resulted in increased OC and TKN contents (Fernández-Gómez et al., 2010). Different microbial populations and enzymatic activities are implied in these changes, as is the chemical composition of animal manures before their transit through earthworms (Aira and Domínguez, 2009; Singh and Suthar, 2012). Conversely, EC in mixtures with an alkaline pH tended to increase from Vof to SS3f. An increased EC is in agreement with that observed by Yadav and Garg (2011), and likely reflects both the type and maturity of the bulk agent used, the salinity and pH of the initial mixtures, as well as the mineralisation of organic matter. 3.2.3. Humification parameters and manurial value of the final vermicomposts Table 3a shows the parameters measured in the vermicomposts relating to the humification process. The C:N ratios were also included as an important maturity index to compare the different groups of vermicomposts. Standard chemical criteria for vermicompost maturity assessments were included to compare them with the data obtained. Although Voi was already a mature substrate rich in nutrients (Section 3.1.), worm activity induced changes, indicating further transformation and humification. This is revealed by the C:N ratio lowering from 17 to 15. This result agrees with those obtained by Singh and Suthar (2012), who reported a large microorganism population after vermicomposting. Pramanik and Chung (2011) indicated that the microorganism population in worms plays a key role in the decomposition of organic wastes. Aira and Domínguez (2009) stated the potential enzymatic activity of vermicomposts for further decomposition. After vermicomposting, the HA and FA

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Table 3 (a) Humus characteristics, humification indexes, C:N ratio, cation exchange capacity in the final vermicompost (Mean ± SD, n = 3), and reference values of vermicompost maturity. (b) The stepwise linear regression models results: linear dependence of the biological parameters of E. fetida on the chemical characteristics of the initial mixtures. (a) Parameter

Vof

SS1f

SS2f

SS3f

V1f

V2f

V3f

Reference values Bernal et al. (2009)

HS C:N HA FA HA:FA HR CEC

40.3 ± 1.2 15.0 ± 0.8 2.52 ± 0.1 1.51 ± 0.1 1.67 ± 0.1 14.0 ± 0.1 143 ± 0.9

42.1 ± 0.2 14.3 ± 0.3 2.63 ± 0.1 1.82 ± 0.1 1.44 ± 0.1 17.2 ± 0.4 155 ± 2.3

43.4 ± 0.6 13.2 ± 0.8 2.51 ± 0.1 2.50 ± 0.1 1.01 ± 0.1 19.6 ± 0.4 177 ± 16.9

44.5 ± 1.3 12.7 ± 0.4 2.57 ± 0.1 2.60 ± 0.1 0.99 ± 0.0 21.5 ± 0.1 146 ± 1.4

46.1 ± 0.8 14.5 ± 0.7 1.54 ± 0.1 1.66 ± 0.1 0.93 ± 0.1 11.3 ± 0.2 117 ± 0.3

48.1. ± 1.2 15.3 ± 0.4 1.65 ± 0.1 1.73 ± 0.1 0.96 ± 0.1 12.7 ± 0.4 121 ± 2.0

50.1 ± 0.4 17.5 ± 1.3 1.35 ± 0.1 1.39 ± 0.1 0.97 ± 0.1 11.6 ± 0.5 112 ± 1.3

660 620, preferable <10 61.25 P1.0 P7.0

(b) Dependent End-life weight Number individuals

Number individuals

Coefficient Constant EC1:5 Constant EC1:5 avP2O5 Constant EC1:5 Cu(II)

744 81.54 673 35.14 54.42 527 65.94 0.57

t

Sig.

32 11.32 67 9.00 14.50 42 11.14 0.20

23.360 7.202 9.974 3.904 3.704 12.533 5.920 2.783

.000 .000 .000 .001 .001 .000 .000 .012

HA = % C in humic acids; FA = % C in fulvic acids; HR = Humification rate = (100 ⁄ (HS/OC), g kg CEC = cation exchange capacity, cmol(+) kg 1.

contents for Vof were respectively 3-fold lesser and 2-fold higher than those reported by Fornes et al. (2012), and were respectively 2-fold lesser and 6-fold higher than those found for composted sewage sludge (Ingelmo et al., 2012). Since the raw materials and processes employed differed in the present research, it is not easy to compare the results, especially as regards FA contents, which were higher than the reference values obtained for FA content as a maturity index. In the present work, FA content probably includes not only organic substances of a low molecular weight, but also other organo-mineral compounds extracted as fulvic-like substances owing to the HS extraction method used. The low HA contents in Vof can be explained by the high stabilisation of humic substances resulting from the precomposting/vermicomposting treatment in Voi into humin forms. The humin fraction is considered the most stabilised organic matter and comprises mainly the by-products resulting from microorganism activity on organic matter (Zaccheo et al., 2002; Ingelmo et al., 2012). The CEC of Vof was 2.7 times higher than that reported by Fornes et al. (2012). The Vf mixtures had a lower CEC than Vof, suggesting an impoverishment of nutrients from V1f to V3f. The drop in CEC was followed by HA content progressively reducing and FA content increasing. These changes support the influence of the quality of the mixtures on the microorganisms/worm system explained in Section 3.2.2., and suggest an enhanced transformation process of the insoluble organic fractions in the mixture (bulk agent) to small or more soluble molecules in relation to the vinasse dose added. Conversely in the SSf vermicomposts, the higher CEC and the increased HA content indicate an enrichment in nutrients and microorganisms activity in transforming the mixture, resulting in higher HA contents vs. FA content. A greater sludge dose increased the CEC in SS2f, although microorganisms/worms activity did not seem to be negatively affected by the increase in the noxious components of sludge. In SS2f, HA lowered at the expense of FA. Both this reduction and that of the microorganisms/worms activity finally lowered CEC in SS3f. These results are in agreement with the chemical changes observed for SS mixtures and with the explanation provided for these changes (Section 3.2.2.). All the obtained vermicomposts can be considered mature according to the HS, C:N, and HR values. Nevertheless, the HA:FA ratios were much higher in Vof, SS1f and SS2f (in this order) which, according to this criterion, may be considered mature, although SS3f, and especially all the Vf vermicomposts, may not. The vermicomposts classification according

R2

Error

1

; HS = humic substances = (HA + FA), mg kg

0.73 0.52 0.73 0.52 0.66

1

; OC = % organic carbon;

to HA:FA clearly explains the magnitude and direction of the chemical changes from the initial mixtures to the final vermicomposts. The values of the obtained HA:FA ratios support the explanation of the greater, increasing stress for the microorganisms/worm system induced by the soluble salts concentration from V1 to V3, and that induced by the combination of salinity and metal content in the SS3 mixtures. The PCA analysis (Table 2b) grouped all the parameters measured in the final vermicomposts into two components to explain 76% of total variance (51% for F1 and 25% for F2). Fig. 1b provides the final vermicomposts classification, done in accordance with factors F1 and F2, along with the variables composing them. Note that the variables situated near each group in this figure are the most characteristic of this group among those composing the corresponding factor. The F1 PCA factor included the humification ratio, nutrient content (phosphorous, TKN, C:N ratio, CEC), and the total content of metals Cu(II), Ni(II) and Zn(II) in the final vermicomposts. The correlation among them was positive, except for the C:N ratio, which correlated negatively with the remaining parameters. Factor F1 clearly separated the SSf vermicomposts (to the right of the vertical axis in Fig. 1b) from Vof and Vf (to the left of the vertical axis). Moreover in the groups to the right, a gradient from SS3f to SS1f is clearly observed and indicates lower values of the variables composing F1. Cu(II) mainly correlated with HS and avP2O5, suggesting that available forms of this metal linked to extractable humic substances and phosphates in the vermicompost. This result agrees with that of Li et al. (2009), who reported increased Cu bioavailability in vermicomposted pig manure. Ni(II) associated with FA, thus confirming both its bioavailability and co-variation with Cu(II), as observed in the present work (Section 3.2.2.). Moreover, this association between Cu and Ni may indicate the still unexplored idea of Ni playing some micronutrient role for plants (as with Cu), as suggested by Bolan et al. (2012). Zn(II) correlated better with TKN, suggesting that Zn can bond to more stabilised organic forms containing nitrogen after passing through the worm’s gut, as other works report (Li et al., 2009), or even to the humin fraction when humic substances have stabilised in the maturation phase of the composting process (Ingelmo et al., 2012). Besides the differences in F1 scores, SS1f also separated from the remaining SSf through a higher HA:FA ratio (coefficient 0.751)

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and a lower EC (coefficient 0.745) than SS2f and SS3f, which clearly indicates the much better quality of the SS1f vermicompost. For the groups to the left, and according to factor F1, Vof displayed intermediate characteristics between SS1f and the Vf groups (with higher C:N ratios, and a higher EC). Moreover, factor F2 separated the Vof from the Vf vermicomposts given its higher OC and its lower Cd content. The data in Fig. 1b clearly helps classify the final vermicomposts according to their manurial value and their potentially noxious components, both of which depend on the initial mixtures’ chemical characteristics. The nutrient content in all the final vermicomposts can be considered adequate for their use as organic fertilizers, although the salinity level and low HA:FA ratio of the Vf vermicompost may be a matter of concern. The main concerns in the SS2f and SS3f vermicomposts could be Cu, Ni and Cd contents, or even more so, their bioavailability.

3.3. Biological parameters of E. fetida The biological parameters of E. fetida (Table 1d), respectively used as bioindicators of worm’s growth and reproductive success, varied among the different feed mixtures. The best results in terms of worm growth and reproduction were seen for Vof (706 ± 28 mg worm 1, 424 ± 26 worms), followed by the SS vermicomposts with decreasing doses of sewage sludge. The Vf mixtures proved less suitable for E. fetida than the SSf mixtures, especially at the highest vinasse dose added (V3f). The number of worms in the V3f mixtures was half that in Vof, suggesting that some parameters in V3f might be found at critical sublethal concentrations (LC50). Worms’ weight after the 8-week vermicomposting process fell in the same range as that obtained by Singh and Suthar (2012) for mixtures of cow manure and herbal pharmaceutical wastes, except for Vof, which was 1.2 times higher than the maximum weight reported in their work. The number of worms was between 1.2 and 2.2 times higher than the birth rates that the authors reported (between 2 and 6 earthworms/parental worm), with a larger number of worms counted in Vof. This difference in the number of worms is attributed to the different compositions of the assayed mixtures. The number of worms for SS1f, SS2f, and SS3f were respectively 9%, 23%, and 45% lower than for Vof. In vermibeds containing similar doses of vinasse (V1f, V2f, and V3f), the number of worms was respectively 25%, 34% and 54% lower than in Vof. Concomitantly to the reduced number of worms, earthworms’ weight was also lighter. From SS1f to SS3f, reduced control went from 13% to 22%, thus revealing that the quality of the SS3f mixtures affected the reproductive success more than worms’ growth. In V1f, V2f and V3f, worms’ weight respectively lowered by 28%, 35% and 50% as compared to Vof, thus affecting both reproduction and growth similarly. The observed trend of a reduction in the number and weight of worms with the dose of added vinasse is supported by the results reported in other works (Nogales et al., 2005), where worms’ biological parameters in manure alone were higher than in other substrates with different vinasse types. Among the initial mixtures’ parameters, EC (negatively correlated with pH) negatively influenced worms’ growth and explained 73% of variations (Table 3b). According to the data, the mean EC to reduce worms’ weight in V3f to half that in Vof was 4.7 dS m 1 (Table 1a), which suggests salt concentrations of around 3000 mg kg 1 being critical for worms’ growth under the experimental conditions of this work. This salt concentration was lower than the EC50 of 4985 mg kg 1 NaCl, which affected the growth of E. fetida observed by Owojori et al. (2009) after worms were exposed for 28 days. This difference might be due not only to the distinct composition of salts, but also to differences in the exposure period.

When relating the number of worms to the initial mixtures’ chemical parameters, the stepwise linear model (Table 3b) included the EC and avP2O5 as the main parameters that negatively affected worm reproduction. These two variables explain 73% of the variations found in the number of worms. Moreover, when avP2O5 was eliminated from the stepwise regression, the second selected variable to have a negative influence on the number of worms was Cu(II) concentration (R2 changed from 0.518 to 0.663). The obtained results suggest that not only the total concentration of soluble salts in food influenced worms‘ growth and reproduction, but also their cationic and anionic composition because both determined the metal uptake by worms, as observed in the present work. Anions such as phosphate or organic forms in the FA fraction of the assayed mixtures were observed to bond Cu(II) and other metals, such as Ni(II), and to negatively influence worm reproduction in accordance with their proportions. This agrees with the results reported by Arnold et al. (2007), who found greater metal uptake and earthworm mortality with increased amounts of labile Cu(II) (free ions together with weak inorganic Cu complexes, and small fractions of easily dissociable organic complexes). 4. Conclusions E. fetida vermicomposting recycles biowastes, but the quality of feed is of primary importance for worms’ growth and reproduction. The nutrient content in the final products implies that their use as organic fertilizers is adequate, although salt concentration and composition are main concerns for them to be utilised when based on worms’ biological parameters as indicators of environmental hazards. Rabbit manure proves a better natural food in terms of earthworm weight and reproduction. The elevated EC and avP2O5 in the mixtures are main disadvantages, and relate the poorest quality of the final products to the most marked reduction in the weight and number of worms. Acknowledgements This study has been financed partially by the Regional Valencian Government through Projects IIARCO2004-A-212 and IIARCO/2004/213, within which the study has been designed, which was finished within the framework of Projects CGL2009-14592C02-01 and CGL2009-07262/BOS from the Spanish Ministry of Education and Science, Spain. The authors would like to thank Helen Warburton for reviewing the English and the anonymous reviewers for their constructive comments. References Aira, M., Domínguez, J., 2009. Microbial and nutrient stabilization of two animal manures after the transit through the gut of the earthworm Eisenia fetida (Savigny, 1826). J. Hazard. Mater. 161, 1234–1238. Arnold, R.E., Hodson, M.E., Comber, S., 2007. Does speciation impact on Cu uptake by, and toxicity to, the earthworm Eisenia fetida? Eur. J. Soil Biol. 43, S230–S232. Bernal, M.P., Alburquerque, J.A., Moral, R., 2009. Composting of animal manures and chemical criteria for compost maturity assessment. A review. Bioresour. Technol. 100, 5444–5453. Bolan, N., Naidu, R., Brennan, R., Budianta, D., Sumner, M.E., 2012. Bioavailability of micronutrients. In: Huang, P.M., Li, Y., Sumner, M.E. (Eds.), Handbook of Soil Sciences. Resource Management and Environmental Impacts, 2nd ed. CRC Press, Boca Raton, pp. 61–80 (Chapter 11.5). Canet, R., Pomares, F., Cabot, B., Chaves, C., Ferrer, E., Ribó, M., Albiach, Ma R., 2008. Composting olive mill pomace and other residues from rural southeastern Spain. Waste Manage. (Oxford) 28, 2585–2592. Fernández-Gómez, M.J., Nogales, R., Insam, H., Romero, E., Goberna, M., 2010. Continuous-feeding vermicomposting as a recycling management method to revalue tomato-fruit wastes from greenhouse crops. Waste Manage. (Oxford) 30, 2461–2468. Demuynck, S., Grumiaux, F., Mottier, V., Schikorski, D., Lemière, S., Leprêtre, A., 2007. Cd/Zn exposure interactions on metallothionein response in Eisenia fetida (Annelida, Oligochaeta). Comp. Biochem. Physiol. C 145, 658–668.

M.J. Molina et al. / Bioresource Technology 137 (2013) 88–97 Fornes, F., Mendoza-Hernández, D., García-de-la-Fuente, R., Abad, M., Belda, R.M., 2012. Composting versus vermicomposting: a comparative study of organic matter evolution through straight and combined processes. Bioresour. Technol. 118, 296–305. Harada, Y., Inoko, A., 1980. The measurement of the cation-exchange capacity of composts for the estimation of the degree of maturity. Soil Sci. Plant Nutr. 26, 127–134. Ingelmo, F., Molina, M.J., Soriano, M.D., Gallardo, A., Lapeña, L., 2012. Influence of organic matter transformations on the bioavailability of heavy metals in a sludge based compost. J. Environ. Manage. 95, S104–S109. Jackson, M.L., 1973. Soil Chemical Analysis. Prentice Hall of India, New Delhi. Kizilkaya, R., 2004. Cu and Zn accumulation in earthworm Lumbricus terrestris L. in sewage sludge amended soil and fractions of Cu and Zn in casts and surrounding soil. Ecol. Eng. 22, 141–151. Li, L.Z., Wu, J., Tian, G., Xu, Z., 2009. Effect of the transit through the gut of earthworm (Eisenia fetida) on fractionation of Cu and Zn in pig manure. J. Hazard. Mater. 167, 634–640. Lorencova, E., Vltavska, P., Budinsky, P., Koutny, M., 2012. Antibacterial effect of phosphates and polyphosphates with different chain length. J. Environ. Sci. Health A 47, 2241–2245. Lukkari, T., Aatsinki, M., Väisänen, A., Haimi, J., 2005. Toxicity of copper and zinc assessed with three different earthworm tests. Appl. Soil Ecol. 30, 133–146. Mitchell, A., 1997. Production of Eisenia fetida and vermicompost from feed-lot cattle manure. Soil Biol. Biochem. 29, 763–766. Ndegwa, P.M., Thompson, S.A., 2000. Effect of C-to-N ratio on vermicomposting of biosolids. Bioresour. Technol. 75, 7–12. Ndegwa, P.M., Thompson, S.A., 2001. Integrating composting and vermicomposting in the treatment and bioconversion of biosolids. Bioresour. Technol. 76, 107–112. Nelson, D.W., Sommers, L.E., 1996. Total Carbon, Organic Carbon, and Organic Matter. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis, Part 2, 2nd ed, Agronomy 9. Am. Soc. of Agron. Inc. Madison, pp. 961–1010.

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Nogales, R., Cifuentes, C., Benítez, E., 2005. Vermicomposting of winery wastes: a laboratory study. J. Environ. Sci. Health B 40, 659–673. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 2002. Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. Circular US Department of, Agriculture, p. 939. Owojori, O.J., Reinecke, A.J., Rozanov, A.B., 2009. The combined stress effects of salinity and copper on the earthworm Eisenia fetida. Appl. Soil Ecol. 4, 277– 285. Polkowska-Motrenko, H., Danko, B., Dybczynski, R., Koster-Ammerlaan, A., Bode, P., 2000. Effect of acid digestion method on cobalt determination in plant materials. Anal. Chim. Acta 408, 89–95. Pont, D., Adholey, A., 2007. Biological approaches for treatment of distillery wastewater. A review. Bioresour. Technol. 98, 2321–2334. Pramanik, P., Chung, Y.R., 2011. Changes in fungal population of fly ash and vinasse mixture during composting by Eudrilus eugeniae and Eisenia fetida: documentation of cellulose isoenzymes in vermicompost. Waste Manage. (Oxford) 31, 1169–1175. Reveillé, V., Mansuy, L., Jardé, E., Garnier-Sillam, E., 2003. Characterisation of sewage sludge-derived organic matter: lipids and humic acids. Org. Geochem. 34, 615–627. Singh, P., Suthar, S., 2012. Vermicomposting of herbal pharmaceutical industry waste: earthworm growth, plant-available nutrient and microbial quality of end materials. Bioresour. Technol. 112, 179–185. Suthar, S., Singh, S., Dhawan, S., 2008. Earthworms as bioindicators of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: is metal bioaccumulation affected by their ecological category? Ecol. Eng. 32, 99–107. Yadav, A., Garg, V.K., 2011. Recycling of organic wastes by employing Eisenia fetida. Bioresour. Technol. 102, 2874–2880. Zaccheo, P., Ricca, G., Crippa, L., 2002. Organic matter characterization of compost from different feedstocks. Compost Sci. Utilizat. 10, 29–38.