Spent mushroom substrate and cattle manure amendments enhance the transformation of garden waste into vermicomposts using the earthworm Eisenia fetida

Journal of Environmental Management 248 (2019) 109263

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Research article

Spent mushroom substrate and cattle manure amendments enhance the transformation of garden waste into vermicomposts using the earthworm Eisenia fetida

T

Xiaoqiang Gonga,b,1, Suyan Lia,*, Michael A. Carsonb, Scott X. Changb, Qian Wub,c, Li Wanga,1, Zhengfeng Anb, Xiangyang Suna,** a

College of Forestry, Beijing Forestry University, Beijing 100083, PR China Department of Renewable Resources, University of Alberta, Edmonton, Canada AB T6G 2E3 Key Laboratory of Grassland Resources, Ministry of Education PR China, College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, Hohhot 010018, China b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Cattle manure Garden waste Solid waste management Spent mushroom substrate Vermicompost

Garden wastes (GW) having high lignin contents could hinder the growth of earthworms and microorganisms in vermicomposting. This study investigated the Eisenia fetida-based vermicomposting of GW mixed with cattle manure (CM) and/or spent mushroom substrate (SMS) at different ratios of GW alone (control), 3:1 GW:SMS, 1:1 GW:SMS, 3:1 GW:CM, 1:1 GW:CM and 2:1:1 GW:SMS:CM to promote earthworm growth and improve the final vermicompost quality. In general, treatments with the addition of SMS and/or CM increased the survival rate, biomass, cocoon and juvenile numbers of E. fetida compared to the control. The addition of SMS and/or CM also significantly increased the activities of dehydrogenase, cellulase, urease, and alkaline phosphatase compared to the control. Furthermore, the addition of SMS and/or CM facilitated the decomposition of organic matter, cellulose and lignin, increased nutrient (N, P and K) concentrations, and accelerated nitrification compared to the control. The addition of SMS and CM led to greater chemical changes of the substrate compared to control. Heavy metal concentrations were increased in the final vermicomposts comparatively to the initial materials, but none of them exceeded the permissible limits. The highest germination index of Chinese cabbage and tomato seeds were both observed in the treatment of 2:1:1 GW:SMS:CM which reached 146.9 and 148.1. Overall, the 2:1:1 GW:SMS:CM treatment had the highest growth and reproduction rates of E. fetida, higher percentage degradation of organic matter, cellulose and lignin, as well as the best quality of the final vermicompost.

1. Introduction Garden waste (GW) is comprised of residues such as fallen leaves, grass cuttings, and bush and tree trimmings produced in urban areas (Gong et al., 2018a). Urbanization in China has led to a large quantity of GW being generated. In the city of Beijing alone, more than 2.37 million tons of GW are produced annually (Shi et al., 2013). Garden waste is usually disposed in a landfill or incinerated, with different environmental impacts. Although GW combustion leads to energy recovery and reduces the amount of GW, the incineration also releases various air polluting gases (NOx, VOC, etc.), into the atmosphere (Kannan et al., 2005). Moreover, another major drawback of the

incineration plant are the technical, and operation costs. As such, increasingly research is being conducted to develop environment-friendly recycling technologies for dealing with GW. Vermicomposting is a sustainable technique to convert organic wastes into a nutrient-rich humus-like product through the joint action of earthworms and microorganisms under aerobic conditions (Gong et al., 2017; John, 2010; Pattnaik and Reddy, 2013). A wide variety of organic wastes, such as sewage sludge, animal manure, fruit and vegetable wastes have been recycled as substrates through vermicomposting. Many earthworm species are normally used for vermicomposting, including Eudrilus eugeniae Kinberg, Perionyx excavatus Perrier, Lampito mauritii Kinberg, Lumbricus rubellus (Azizi et al., 2015;

*

Corresponding author. Mailing address: College of Forestry, Beijing Forestry University, P.O. Box no. 111, Beijing 100083, PR China. Corresponding author. E-mail addresses: [email protected] (S. Li), [email protected] (X. Sun). 1 Contributed equally to this work with: Xiaoqiang Gong, Li Wang. **

https://doi.org/10.1016/j.jenvman.2019.109263 Received 15 January 2019; Received in revised form 2 July 2019; Accepted 11 July 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.

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Gajalakshmi et al., 2001). Unlike other agricultural organic waste, GW is characterized by high content of lignin and cellulose, which are not preferred by most earthworm species (Ganesh et al., 2009; Ganihar, 2003). The most common and efficient way to enhance growth and reproduction of earthworms during vermicomposting, as well as generate a better product, is by mixing organic waste materials with other suitable additives, including biochar, straw, fly ash, and sawdust (Gong et al., 2018b; He et al., 2016). This was also confirmed by Sethuraman and Kavitha (2013) who indicated that 50% concentration of garden waste mixed with bedding material was ideal for the vermicomposting. In vermicomposting, cattle manure (CM) is usually considered an ideal supplementary material because it supplies a large amount of labile organic matter and non-assimilated carbohydrates, thus promoting growth and reproduction of earthworms (Bhat et al., 2016: Logakanthi et al., 2000). Moreover, CM is rich in microorganisms, protozoa, and nematodes, which accelerate the decomposition of organic waste (John Paul et al., 2011). Cattle manure also stimulates microbial growth and enzyme activities and increases the content of nutrients (N, P, K and micronutrients) in the vermicomposted material (Yadav et al., 2015). In order to improve the quality of vermicompost as well as the growth and fecundity of earthworms, another potential vermicompost additive is spent mushroom substrate (SMS). The SMS is a by-product generated from mushroom production, which contains a large amount of fungal mycelium and extra-cellular lignocellulosic enzymes along with various organic substances (carbohydrates, proteins, and fats), as well as a considerable quantity of inorganic nutrients such as ammonium nitrate, superphosphate, and potassium salts, (Fang et al., 2017; Lou et al., 2017). In contrast to this study focusing on garden waste, Azizi et al. (2011), Azizi et al. (2013) and Nik et al. (2009) have reported that adding an appropriate sawdust based SMS to CM or sewage sludge can be decomposed through vermicomposting by employing Lumbricus rubellus. The results of these studies have implied that applying additional SMS to materials may increase the growth and multiplication of earthworm, and improve the quality of final product. Hence, vermiconversion of garden waste supplemented with SMSs is comprehensively explored in the present study. This study investigated the feasibility of vermicomposting GW with the addition of SMS and CM. This study reports the changes in the physicochemical and enzymatic properties of the materials, and on the growth and reproduction of E. fetida during vermicomposting. The object of this study was to determine the best addition ratio of SMS and CM to GW during vermicomposting.

Table 1 Initial physicochemical characterization of the garden waste (GW) spent mushroom substrate (SMS) and cattle manure (CM) used in the vermicomposting process. Values are means ± standard error (n = 3). Parameters

GW

SMS

CM

pH EC (dS m−1) TOC (%) Cellulose (%) Lignin (%) TN (g kg−1) TP (g kg−1) TK(g kg−1) NO3−-N (mg kg−1) Cu (mg kg−1) Zn (mg kg−1) Cr (mg kg−1) Pb (mg kg−1) Cd (mg kg−1) Ni (mg kg−1) Moisture content (%)

7.2 ± 0.1 1.1 ± 0.1 46.8 ± 0.5 38.9 ± 1.9 26.1 ± 0.2 10.8 ± 0.1 2.8 ± 0.3 4.0 ± 0.2 130.7 ± 4.8 16.1 ± 0.5 62.4 ± 3.4 0.3 ± 0.1 11.5 ± 0.9 0.02 ± 0.00 1.5 ± 0.1 32.7 ± 0.9

6.6 ± 0.1 2.3 ± 0.1 38.1 ± 1.3 27.7 ± 0.8 15.4 ± 0.7 15.6 ± 1.0 7.4 ± 0.1 6.1 ± 0.5 83.3 ± 2.6 13.4 ± 0.7 14.2 ± 2.3 0.4 ± 0.0 7.7 ± 0.9 0.41 ± 0.04 5.5 ± 0.6 54.9 ± 1.8

8.8 ± 0.1 1.7 ± 0.1 31.2 ± 1.0 17.7 ± 0.6 8.8 ± 0.5 22.4 ± 0.7 8.7 ± 0.2 9.1 ± 0.1 211.6 ± 6.9 30.4 ± 1.6 179.5 ± 20.9 3.9 ± 0.2 31.7 ± 1.7 0.69 ± 0.04 38.4 ± 1.5 36.4 ± 0.5

EC = electrical conductivity; TOC = total organic carbon; TN = total nitrogen; TP = total phosphorus; TK = total potassium.

and chemical properties of the GW, SMS and CM are presented in Table 1. 2.2. Treatments and experimental design This work was conducted at the Forest Science Company Limited Nursery of Beijing Forestry University, Beijing, China. Garden waste and SMS were chopped and sieved (5 mm opening size) before use in the experiments to improve the homogeneity and the digestibility of the material by the earthworms. For the vermicomposting trial, a total of six combinations of GW, SMS, and CM mixtures were prepared, with GW as the main component (≥50%) in all of the mixtures: 1) GW, garden waste (control); 2) GW + SMS3:1, 3:1 ratio (dry weight based same below) of garden waste:spent mushroom substrate; 3) GW + SMS1:1, 1:1 ratio of garden waste:spent mushroom substrate; 4) GW + CM3:1, 3:1 ratio of garden waste:cattle manure; 5) GW + CM1:1, 1:1 ratio of garden waste:cattle manure; 6) GW + SMS + CM2:1:1, 2:1:1 ratio of garden waste:spent mushroom substrate:cattle manure.

2. Materials and methods 2.1. Materials for vermicomposting

To set up the experiment, 4 kg (dry weight basis) of each mixture was filled in a plastic container (52 × 68 × 39 cm in length × width × height), which had four holes (10 mm diameter) in the bottom for drainage. The moisture content of the mixture in each container was measured using a digital analyzer MS-70 (A&D Co., Ltd., Tokyo, Japan) and maintained at 65–70% by sprinkling distilled water as required during the entire period of pre-composting and vermicomposting. Mixtures were pre-decomposed for 21 days to make the substrate palatable to earthworms (Frederickson et al., 1997). After precomposting, 40 non-clitellated earthworms (Eisenia fetida) with fresh weights ranging from 155 to 162 mg were introduced into each container. The top and bottom of containers were covered with 1 mm nylon mesh to contain the earthworms, and secured with a rubber band. All containers were incubated in a greenhouse under identical ambient conditions (temperature 23.17–28.41 °C). Each treatment was replicated three times, for a total of 18 experimental units in a fully randomized design. The biomass of adults, number, cocoon number, and juvenile number of earthworms were monitored every week, for ten weeks following the methods of Gong et al. (2018b). In this study, all adult earthworms were separated manually from the composted materials in

Garden wastes, consisting of fallen leaves (approximately 30%), grass clippings (approximately 20%), and branch cuttings of woody plants (approximately 50%), were obtained from a municipal GW treatment plant in Chaoyang District, Beijing, China. Air-dried CM was collected locally from a cattle farm in Tongzhou District, Beijing, China. The SMS was obtained from Beijing Jingpuyuan Biological Engineering Co., Ltd. (China), and is the residual material after the cultivation of Pleurotus ostreatus, with the SMS containing sawdust (about 30–40%, by weight), straw (about 10–30%), and wheat bran (about 10–20%). In this study, the earthworm Eisenia fetida was selected for vermicomposting because it tolerates a wide range of pH, temperature, and moisture content. However, low quality organic waste may cause earthworms to lose weight and increase mortality during vermicomposting, producing a lower quality vermicompost (Ganesh et al., 2009). Healthy non-clitellated E. fetida earthworms of uniform size were obtained from a commercial earthworm breeding farm in Shunyi District, Beijing, China. Because non-clitellated earthworm is more sensitive to the environment variation and has relatively longer life cycle, this study chose to use non-clitellated earthworm. The physical 2

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vermicompost samples was determined by the triphenyl tetrazolium chloride method by colorimetry using a spectrophotometer set at 485 nm wavelength as described by Tiquia (2005). The seed germination index (GI) of Chinese cabbage (Brassica parachinensis) and tomato (Lycopersicon esculentum L.) were determined following the method described in Gong et al. (2017) and were used to examine the final vermicomposts for phytotoxicity. The morphological characteristics of raw materials and end products of GW were studied with a scanning electron microscopy. The airdried samples were sputter coated (Quorum Technologies SC7620, Berkshire, U.K.) with a gold thickness of 30 nm to obtain SEM images on a Hitachi S–3400N (Tokyo, Japan) scanning electron microscopy, at an accelerating voltage of 5 kV at various levels of magnification.

each container. Adult earthworms were then counted, washed with distilled water to remove any adhering waste materials, weighed, and then returned to the containers. During this process, juveniles and cocoons contained in the composted material were located and counted directly within the material to reduce disturbance. Other data on earthworm biology, i.e., maximum individual biomass gain, maximum individual growth rate, and mortality rate were also measured. Vermicompost samples were collected weekly. Fresh samples (30 g) were taken from each vermicomposting container before materials were uniformly mixed during earthworm removal. Half of the samples were oven dried at 65 °C and finely milled to determine pH, electrical conductivity (EC), the concentrations of total carbon (TOC), cellulose, lignin, total nitrogen (TN), total phosphorus (TP), total potassium (TK), NO3−-N, Cu, Zn, Cr, Pb, Cd, and Ni. The other half of the sample was kept at 4 °C and used to determine germination index and enzyme activities including cellulase, urease, phosphatase, and dehydrogenase within 3 days of sampling to avoid the decrease in enzymatic activities (Verchot and Borelli, 2005). During the end of experiment, garden waste in different treatments were extracted and air dried for scanning electron microscope (SEM) analysis.

2.4. Statistical analysis The reported results are the mean of three replicates with standard error (SE). Enzyme activities were analyzed by repeated measures analysis of variance (ANOVAR) in which the type of material was set as a between-subject factor, and time was set as a within subject factor. When sphericity assumptions (Mauchly's test) could not be met, the Huynh–Feldt correction was employed to adjust the P values whenever values of Ɛ were close to 1. Mean values of the enzyme activities were compared by Tukey's HSD post-hoc test to determine which treatments differed. One-way ANOVA with Tukey's HSD post-hoc test were employed to test the effect of material type on earthworm growth and fecundity, and on the chemical properties of the vermicomposts, on seed germination at individual sampling times. The probability level used for statistical significance was P < 0.05 for all tests. Prior to conducting the ANOVA, the normality of distribution and homogeneity of variance were checked with Shapiro-Wilk and Levene's test, respectively. SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.

2.3. Sample analysis The electrical conductivity (EC) and pH were determined in an aqueous solution (sample:dionized water, 1:10 wt:volume) using a conductivity meter (DDS-11A, Shanghai Leici-Chuangyi Instrument Co. Ltd., Shanghai, China) and a pH meter (Starter 3C, Ohaus Instrument Co. Ltd., Shanghai, China), respectively. This suspension was shaken on a mechanical shaker at 230 rpm for 30 min and allowed to stand for an hour prior to pH and EC measurement (Unuofin and Mnkeni, 2014). The wet oxidation method of Yeomans and Bremner (1988) was used for determination of total organic carbon (TOC). Cellulose was determined by using the HNO3–ethanol method as shown in Liu (2004). Lignin content was analyzed following the 72% (v/v) H2SO4 procedure described by Liu (2004). Total nitrogen (TN) was analyzed following a modified semi-micro Kjeldahl method, employing an automatic Kjeldahl analyzer (KDY-9830; Beijing Tongrunyuan Mechatronics Technology Co., Ltd., Beijing, China). For the determination of total phosphorus (TP) and total potassium (TK), 0.1 g of dry sample was digested with 98% (v/v) sulfuric acid and 30% (v/v) hydrogen peroxide (Bian et al., 2016). Total phosphorus (TP) was measured by the anti-Mo-Sb spectrophotometry method using a UV spectrophotometer (UV-120-02; Shimadzu Scientific Instruments, Kyoto, Japan). Total potassium (TK) was determined using a flame photometer (425, Spring Instrument Equipment Co., Ltd., Shanghai, China). Extractable NO3−-N concentrations was measured by mixing samples with 2 M KCl in a 1:5 sample:solution ratio (w:v) and shaking for 30 min. The extracts were filtered (0.45-μm Millipore membrane filter) and filtrates analyzed for extractable NO3−-N concentrations using a continuous flow auto-analyzer (Auto Analyzer 3, Germany) (Xiao et al., 2010). Metal concentrations were determined according to the method proposed by Ko et al. (2008) by digesting 1 g of the dried and ground samples using HNO3 and HClO4 mixed at an 5:1 ratio (v:v). The Cu, Zn, Cr, Pb, Cd and Ni concentration in the resulting solution was determined by inductively coupled plasma mass spectrometry (Prodigy, Leeman Laboratories Inc., USA). Cellulase activity was analyzed according to the method of GómezBrandón et al. (2008) and was quantified from the glucose generated by incubating the samples (5 g fresh weight) with carboxymethyl cellulose sodium salt (0.7%) for 24 h at 50 °C. Urease activity was assayed by determining the amount of ammonia released after incubation with urea in a citrate buffer at 37 °C for 3 h, following the method of Pramanik et al. (2007). Phosphatase activity (alkaline) was measured using the p-nitrophenyl phosphate method described by Pramanik et al. (2007), using colourimetric determination of the hydrolysis of p-nitrophenol at 37 °C for 30 min. Dehydrogenase activity of the

3. Results and discussion 3.1. Earthworm growth, reproduction, and survival During the ten weeks of vermicomposting, the earthworm biomass in all treatments increased over time and reached peak biomass in week 6–8 (Fig. 1a). Following peak biomass, adult earthworm biomass decreased in all treatments until the end of the incubation. These changes in earthworm biomass may have reflected the greater availability of food at the start of the incubation and the reduction of food over time (Chaudhuri and Bhattacharjee, 2002). The substrate type added significantly altered E. fetida biomass during the ten weeks of vermicomposting, with maximum individual biomass (P < 0.05), maximum individual biomass gain (P < 0.05), and maximum individual growth rate significantly (P < 0.05) higher in all SMS and CM addition treatments than in the control (Table 2). Among all substates tested, the highest earthworm biomass and growth rate were found in the GW + SMS + CM2:1:1 treatment. Final individual earthworm biomass was significantly influenced by treatments (P = 0.002). Final individual earthworm biomass was significantly (P < 0.05) higher for GW + SMS1:1, GW + CM3:1, GW + CM1:1 and GW + SMS + CM2:1:1 than for the control, but no difference was found between the control and GW + SMS3:1 treatments (Table 2). The formation of cocoons was first observed in the 2nd week in GW + CM1:1 and GW + SMS + CM2:1:1, in the 3rd week in GW, GW + SMS3:1, and GW + CM3:1, and in the 4th week in GW + SMS1:1. The number of cocoons increased consistently till week 6–7, with a gradual decrease thereafter until the end of the experiment (Fig. 1b). Maximum cocoon number per container was significantly higher in the GW + SMS3:1, and GW + SMS + CM2:1:1, treatment but significantly lower in the GW + SMS1:1 treatment, when compared with the GW control (one-way ANOVA, F5,12 = 34.13, P < 0.001; Tukey HSD post3

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hoc test, P < 0.05) (Table 2). Juveniles were first observed in the 4th week in GW + CM1:1 and GW + SMS + CM2:1:1, in the 5th week in GW, GW + SMS3:1 and GW + CM3:1, and in the 6th week in GW + SMS1:1, followed by a sharp increase peaking at week 10 in all treatments (Fig. 1c). Maximum juvenile number per container was significantly higher (one-way ANOVA, F5,12 = 35.38, P = 0.002; Tukey HSD post-hoc test, P < 0.05) in GW + CM1:1 and GW + SMS + CM2:1:1 than in GW, but was significantly lower in GW + SMS1:1 (Table 2) than in GW. The GW + SMS + CM2:1:1 treatment had the highest cocoon and juvenile numbers when compared with other treatments. Total earthworm mortality (%) after ten weeks of vermicomposting was significantly (one-way ANOVA, F5,12 = 27.78, P < 0.001; Tukey HSD post-hoc test, P < 0.05) higher in GW than in all other treatments (Table 2) with a minimum increase in mortality of 2.36 times that of the highest mortality rate treatment (GW + SMS1:1). The lowest earthworm mortality rate was found in GW + SMS + CM2:1:1 and was 8.44 times lower than the control (GW). The motility of earthworm was observed in all treatments, even in the optional ratio of GW:CM. Similar result was also obtained in other studies (Vig et al., 2011). The addition of CM can improve the living condition of earthwarms, however the exhaust of food will eventually cause the decrease of earthworm number. Taken together, these results indicate that the addition of SMS and CM to GW significantly increased E. fetida growth, reproduction, and survival for all treatments, excluding GW + SMS1:1 which had a lower number of cocoons and hatchings. These findings agree with those of Haynes and Zhou (2016) who demonstrated that the addition of an appropriate ratio of CM (25%) to municipal garden waste can increase the survival, growth, and reproduction of E. fetida. A possible explanation is that the addition of cattle dung may have provided substrate and a better environment to the E. fetida than GW alone (Bhat et al., 2016). Azizi et al. (2013) also suggested that SMS provided large amounts of edible fungi as an additional nutritious food source for earthworm during vermicomposting of sewage sludge and could increase the survival, growth, and reproduction of earthworms. While the addition of SMS to GW at a high rate (1:1) significantly decreased E. fetida reproduction, Jun et al. (2012) found that increasing soil salinity significantly inhibited the fecundity of E. fetida, and no cocoon was produced in soil with EC of 5.25 or 7.35 ds m−1. Thus, these reproduction and fecundity decreases for E. fetidain by this treatment were likely due to the high EC which is possibly the result of SMS addition in high concentration (Chong and Danny, 1994). The high EC value could be due to the use of gypsum together with sawdust, straw and wheat bran in SMS (González-Marcos et al., 2015). 3.2. Altered enzyme activities Dehydrogenase activity (DHA) has been widely used as overall microbial activity indictor in compost systems, because it is an intracellular enzyme involved in oxidative phosphorylation. Dehydrogenase activity increased gradually till week 3 in all treatments, and then tended to gradually decrease (Fig. 2a). There was a significant effect of treatment (repeated measures ANOVA, F5,12 = 46.04, P < 0.001) and time (repeated measures ANOVA, F10,120 = 303.86, P < 0.001), but no interaction effect of time and treatment (repeated measures ANOVA, F50,120 = 1.30, P = 0.124) on dehydrogenase activity (Table 3). The dehydrogenase activity was the highest in GW + SMS + CM2:1:1, and all addition treatments were significantly (repeated measures ANOVA, Tukey HSD post-hoc, P < 0.05; Table 4) higher than in the control (GW). These results are consistent with Meng et al. (2017), who revealed that the addition of SMS increased dehydrogenase activity during the composting of sewage sludge. Similarly, Gusain et al. (2017) had reported an increase in dehydrogenase activity by incorporation CM into aquatic weed Pistia during composting. Means in a column followed by different letters denote significance

Fig. 1. Changes in (a) mean individual earthworm mass, (b) number of cocoons, and (c). number of juveniles during the vermicomposting of different type of mixture materials. GW, garden waste; SMS, spent mushroom substrate; CM, cattle manure. Each symbol indicates the means + standard error (n = 3).

4

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Table 2 Growth, reproduction, and survival of the earthworm Eisenia fetida. Values are means ± standard error (n = 3). Means in a row followed by different letters are significantly different at P < 0.05 according to Tukey's HSD test. GW, garden waste; SMS, spent mushroom substrate; CM, cattle manure. Parameter

GW

Initial individual biomass [1] (mg worm−1) Maximum individual biomass [2] (mg worm−1) Maximum individual biomass gain [2]-[1] (mg worm

−1

Maximum individual growth rate (mg worm−1 day−1) Final individual biomass (mg worm−1) Mortality rate after 10 weeks (%) Maximum number of cocoons (per container) Maximum number of hatchings (per container)

)

161.2 ± 1.4a 552.6 ± 7.3d 390.2 ± 5.8d 8.0 ± 0.1d 434.3 ± 10.3c 63.3 ± 3.6a 318 ± 8c 1057 ± 42b

GW + SMS3:1

156.0 ± 3.9a 687.0 ± 9.3b 531.0 ± 9.5b 12.6 ± 0.2b 473.9 ± 21.2bc 22.5 ± 4.3bc 418 ± 33ab 1339 ± 60ab

GW + SMS1:1

160.8 ± 0.7a 631.2 ± 5.8c 470.3 ± 5.7c 11.2 ± 0.1c 502.0 ± 11.9ab 26.8 ± 3.6b 153 ± 13d 454 ± 60c

GW + CM3:1

159.5 ± 1.0a 677.7 ± 11.1bc 518.2 ± 11.7bc 12.3 ± 0.3bc 518.8 ± 15.1ab 18.3 ± 3.6bc 385 ± 10bc 1362 ± 117ab

GW + CM1:1

156.8 ± 2.9a 722.4 ± 12.8b 565.7 ± 14.5b 13.5 ± 0.4b 529.2 ± 12.5ab 14.2 ± 4.2bc 391 ± 26bc 1413 ± 26a

GW + SMS + CM2:1:1

155.2 ± 2.4a 782.0 ± 14.8a 626.8 ± 15.0a 14.9 ± 0.4a 559.2 ± 6.8a 7.5 ± 2.9c 499 ± 18a c ± 63a

One-way ANOVA F

P-value

0.81

0.565

22.80

＜0.001

24.10

＜0.001

36.20

＜0.001

7.42

0.002

27.78

＜0.001

34.13

＜0.001

35.38

＜0.001

treatments having consistently higher (repeated measures ANOVA, Tukey HSD post-hoc, P < 0.05; Table 4) activity levels than in GW. No significant interaction effect of treatment and time on alkaline phosphotase activities was observed (repeated measures ANOVA, F50,120 = 0.51, P = 0.995). Throughout vermicomposting, enzyme activities (cellulase, urease, and alkaline phosphatase) in the GW + SMS + CM2:1:1 treatment were significantly higher than that in other treatments (repeated measures ANOVA, Tukey HSD post-hoc,

in repeated measures ANOVA followed by Tukey's HSD test, P < 0.05. Cellulase, urease, and alkaline phosphotase activities rapidly increased in all six treatments with vermicomposting time, peaking in weeks 1–2, 3–4, and 2–3, respectively (Fig. 2b–d). Material type had significant effects on cellulase (repeated measures ANOVA, F5,12 = 50.46, P < 0.001; Table 3), urease (repeated measures ANOVA, F5,12 = 127.01, P < 0.001), and alkaline phosphotase activities (repeated measures ANOVA, F5,12 = 24.93, P < 0.001) with all

Fig. 2. Change in the (a) dehydrogenase activity (DHA), (b) cellulase activity, (c) urease activity, and (d) alkaline phosphatase activity in different treatments during vermicomposting. GW, garden waste; SMS, spent mushroom substrate; CM, cattle manure. Each symbol indicates the means + standard error (n = 3). 5

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et al., 2017) for the growth and development of E. fetida. In addition, the pH values of all final vermicomposts were within the satisfactory range (7.0–8.5) for agricultural use (Masó and Blasi, 2008). The electrical conductivity (EC) value gradually increased in all three treatments from 0 to 7–8 weeks and then sharply decreased until 10 weeks (Fig. 3b). The increase in EC could be due to the mineralization of organic matter leading to the release of phosphate, ammonium, K+, and other soluble mineral salts in available forms (Kaviraj and Sharma, 2003). The subsequent decrease in electrical conductivity values could be as a result of volatilization of ammonia and formation of insoluble compounds which reduced the number and mobility of ions (Huang et al., 2004). All amended vermicomposts had significantly (one-way ANOVA, F5,12 = 144.77, P < 0.001; Tukey HSD post-hoc, P < 0.05) higher EC values than the GW alone at the end of vermicomposting (Table 5). The final (week 10) EC of all vermicomposts (except in GW + SMS1:1) did not exceed 4 dS m−1, a level which is considered an upper threshold for plants with medium salinity sensitivity (Lasaridi et al., 2006).

Table 3 Repeated measures ANOVA for changes in dehydrogenase activity, cellulase activity, urease activity, and alkaline phosphatase activity in different treatments during the ten weeks of vermicomposting. Source of variation −1

d.f.

F

P-value

5.12

46.04

＜0.001

10.120 50.120

303.86 1.30

＜0.001a 0.124a

5.12

50.46

＜0.001

10.120 50.120

298.25 1.85

＜0.001a 0.003a

5.12

127.01

＜0.001

10.120 50.120

577.14 2.89

＜0.001a ＜0.001a

5.12

24.93

＜0.001

10.120 50.120

114.76 0.51

＜0.001 0.995

−1

dw 24h ) Dehydrogenase activity (μg TPF g Between subjects Treatment Within subjects Time Time × Treatment Cellulase activity (mg glucose g−1 dw h−1) Between subjects Treatment Within subjects Time Time × Treatment Urease activity (mg NH4+ g−1 dw h−1) Between subjects Treatment Within subjects Time Time × Treatment Alkaline phosphatase activity (mg PNP g−1 dw h−1) Between subjects Treatment Within subjects Time Time × Treatment

3.3.2. Total organic carbon, lignin, and cellulose content A steady decrease in total organic carbon (TOC) content was recorded in all treatments with vermicomposting time (Fig. 3c). TOC loss is likely due to the mineralization of the organic matter through time. The percentage of reduction in TOC was significantly affected by treatments (one-way ANOVA, F5,12 = 66.70, P < 0.001; Table 5). The TOC content in different treatments decreased from an initial values 359.6–432.1 g kg−1 to final values188.1–339.3 g kg−1, corresponding to a TOC loss of 21.4% (GW), 36.8% (GW + SMS3:1), 27.2% (GW + SMS1:1), 32.0% (GW + CM3:1), 41.7% (GW + CM1:1) and 49.7% (GW + SMS + CM2:1:1). The percentage of reduction in TOC was significantly lower in the GW treatment than in the GW + SMS3:1, GW + CM1:1, and GW + SMS + CM2:1:1 treatments, but did not differ between the other treatments and the GW treatment (one-way ANOVA, Tukey HSD post-hoc, P < 0.05; Table 5). This result proves that the addition of SMS and/or CM can accelerate the loss of TOC during GW vermicomposting practices, and TOC reduction was greatest when combined addition of SMS and CM in treatment GW + SMS + CM2:1:1. SMS contains a large number of fungal strains and extra-cellular lignocellulosic enzymes such as lignin peroxidase, β-glucosidase, laccase, xylanase and cellulase and hemicellulose, potentially enhanced organic matter degradation (Phan and Sabaratnam, 2012). In addition, SMS is comprised of easily degradable carbon substrates which tend to benefit the microbial growth, resulting in a greater organic matter decomposition rate (Zhang and Sun, 2014). Suthar (2010) suggested that the microorganisms in CM produce many extracellular enzymes responsible for organic matter decomposition, explaining the higher rates of loss of TOC in treatments with CM. Furthermore, the addition of SMS and/or CM increased E. fetida numbers (Table 2, Section 3.1) increasing vermicompost turnover rates and subsequently enhancing microbial processes and lignocellulolytic enzymes activity, further accelerating the rate of decomposition. For all treatments, the lignin content appeared to slightly increase in the first week and then decrease gradually over time (Fig. 3d). The

a Huynh-Feldt adjusted values of P for within subject factors because of violation of spherecity (P value of Mauchly's test < 0.05).

P < 0.05). One possible explanation is that indigenous enzymes and microbes in the SMS and CM may have directly contributed to microbial and enzyme activities (Chiu et al., 2009; Liu et al., 2017). Another possibility is SMS and CM increased the supply of nutrients, especially available carbon and nitrogen, thus stimulating the rapid growth of the microorganism and microbial synthesis of enzymes (Álvarez-Martín et al., 2016; Lou et al., 2017). Furthermore, Aira et al. (2007) found that higher numbers of earthworms resulted in higher microbial biomass during vermicomposting, therefore the increasing growth and reproduction of earthworm by addition of SMS and/or CM (Fig. 1) may also increase microbial biomass and enzyme activities. 3.3. Physicochemical changes during vermicomposting process 3.3.1. pH and electrical conductivity (EC) values In general, treatments with CM added tended to have a higher pH, while treatments involving SMS had a lower pH compared to both the control and the GW + SMS + CM2:1:1 treatment (Fig. 3a). The increase in pH in the early stage can be attributed to the degradation of the organic compounds, which leads to the generation of ammonia, and the subsequent decrease in pH might be due to the production of intermediate organic acids (Hanc and Chadimova, 2014). The pH values in all treatments were within an acceptable range of 5–9 (Pérez-Godínez

Table 4 Effects of different treatments on enzyme activities throughout the vermicmposting period. (Values are means ± SE). Treatment

Dehydrogenase activity (μg TPF g−1 dw 24h−1)

Cellulase activity (mg glucose g−1 dw h−1)

GW GW GW GW GW GW

830.05 ± 73.79d 1056.24 ± 84.36c 1067.42 ± 99.27c 1184.42 ± 98.98bc 1312.63 ± 96.73ab 1396.54 ± 102.90a

3.59 5.84 5.45 4.82 4.41 6.39

+ + + + +

SMS3:1 SMS1:1 CM3:1 CM1:1 SMS + CM2:1:1

± ± ± ± ± ±

0.31e 0.43ab 0.39bc 0.39cd 0.36d 0.42a

Urease activity (mg NH4+ g−1 dw h−1) 10.58 14.73 13.16 17.32 16.12 18.33

GW, garden waste; SMS, spent mushroom substrate; CM, cattle manure. 6

± ± ± ± ± ±

1.22e 1.44c 1.35d 1.66ab 1.49b 1.76a

Alkaline phosphatase activity (mg PNP g−1 dw h−1) 45.90 53.87 50.71 56.61 54.99 60.29

± ± ± ± ± ±

1.94d 2.27bc 2.07c 2.31ab 1.99bc 2.24a

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Fig. 3. Evolution of (a) pH and (b) electrical conductivity (EC), (c) total organic carbon (TOC), (d) lignin, (e) cellulose, (f) total nitrogen (TN), (g) total phosphorus (TP), (h) total potassium (TK), and (i) NO3− in different treatments during 10 weeks of vermicomposting. GW, garden waste; SMS, spent mushroom substrate; CM, cattle manure. Values are means and standard error (n = 3).

P < 0.001; Tukey HSD post-hoc, P < 0.05) (Table 5). For both lignin and cellulose, the GW control had significantly (one-way ANOVA, Tukey HSD post-hoc, P < 0.05) lower percent lost than all addition treatments at the end of ten weeks, indicating more rapid degradation of lignin and cellulose with the addition of SMS and CM substrates. These results are consistent with the study of Zhang and Sun (2017) who composted GW in combination with CM and spent coffee grounds, showing that CM additions facilitates the degradation of cellulose and lignin in composting. As mentioned above, the increased earthworm growth and reproduction as well as the likely increase in numbers and/ or activity of microorganisms after SMS or/and CM addition, could have resulted in higher lignin degradation.

relative increase was probably caused by the removal of early degradable compounds such as free sugars, proteins, lipids, and starch, while the main cause of the later decreasing trend in the lignin content was the lignocellulolytic action of microbes (Gabhane et al., 2012). Lignin content in initial feed stocks was 17.2–27.6% and in vermicompost was 6.3–17.2%. The percent decrease in lignin content was in the range of 37.7–68.1% in different feed materials. The percent decrease in lignin content was significantly higher in the GW + SMS + CM2:1:1 than in the GW + CM1:1, GW + SMS1:1 and GW treatments, but did not significantly differ between the other treatments and the GW + SMS + CM2:1:1 treatment (one-way ANOVA, F5,12 = 18.42, P < 0.001; Tukey HSD post-hoc, P < 0.05) (Table 5). Similar to lignin breakdown, https://www.sciencedirect.com/science/article/pii/ S0960852416310963 Fig. 3e illustrates cellulose content in vermicomposts also decreased through time with an initial range of 26.2–36.8% cellulose and a final cellulose content of 7.1–20.1%. The percent decrease in cellulose content was in the range of 45.5–75.2% in different feed materials. The percent decrease in cellulose content was significantly higher in the GW + SMS + CM2:1:1 than in the GW + CM1:1, GW + SMS1:1 and GW treatments, but did not significantly differ between the other treatments and the GW + SMS + CM2:1:1 treatment (one-way ANOVA, F5,12 = 20.83,

3.3.3. Total nitrogen, phosphorus, potassium content and NO3−-N The total nitrogen (TN) values exhibited an upward trend throughout the vermicomposting period (Fig. 3f). Initial TN content was 12.0–18.6 g kg−1, which increased to 17.3–32.3 gkg-1, an overall increase of 45.5–77.8% after 10 weeks. The final TN content was significantly (one way ANOVA, F5,12 = 109.60, P < 0.001: Tukey HSD post-hoc, P < 0.05) higher in all addition treatments than in the control (GW). The percent increase in TN content was significantly lower in GW control than in the GW + SMS + CM2:1:1, GW + CM1:1 7

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Table 5 Chemical characterization of waste materials at the beginning and at the end of the vermicomposting process using Eisenia foetida. Values are means ± SE (n = 3). Parameter

pH

EC (ds m−1)

TOC (g kg−1)

Lignin (%)

Cellulose (%)

TKN (g kg−1)

TP (g kg−1)

TK (g kg−1)

NO3−-N (mg kg−1)

GW

GW + SMS3:1

GW + SMS1:1

b

c

GW + CM3:1

GW + CM1:1

GW + SMS + CM2:1:1

One way ANOVA d.f.

F

P-value

Initial Final a Percent change (%) Initial Final (%) Initial Final (%) Initial Final (%) Initial Final (%) Initial Final (%) Initial Final (%) Initial Final (%) Initial

8.31 ± 0.00 7.86 ± 0.03b −5.4 ± 0.3a

8.26 ± 0.02 7.81 ± 0.03b −5.4 ± 0.6a

7.98 ± 0.04 7.60 ± 0.03c −4.7 ± 0.9a

8.34 ± 0.07 7.93 ± 0.01ab −5.0 ± 0.7a

8.62 ± 0.06 8.08 ± 0.04a −6.2 ± 0.9a

8.28 ± 0.06 7.99 ± 0.07ab −3.5 ± 0.6a

5.12 5.12 5.12

18.32 18.22 1.71

＜0.001 ＜0.001 0.206

1.69 ± 0.01d 2.51 ± 0.04e 48.2 ± 1.1a 432.1 ± 7.3a 339.3 ± 6.5a −21.4 ± 3.8d 27.6 ± 1.1a 17.2 ± 0.8a −37.7 ± 1.4c 36.8 ± 1.3a 20.1 ± 1.0a −45.5 ± 0.9c 12.0 ± 0.7e 17.3 ± 0.3e 45.5 ± 5.8b 3.3 ± 0.1c 4.2 ± 0.2d 29.2 ± 1.2b 4.7 ± 0.1c 6.8 ± 0.2d 45.1 ± 5.1b 226.7 ± 5.8c

2.47 ± 0.08b 3.32 ± 0.02bc 34.6 ± 4.1bc 411.1 ± 8.8ab 259.1 ± 6.1b −36.8 ± 2.9bc 23.8 ± 1.1ab 10.2 ± 0.4b −56.8 ± 2.1ab 33.8 ± 0.4ab 11.6 ± 0.9b −65.8 ± 2.2ab 12.4 ± 0.2de 21.3 ± 0.5d 71.8 ± 6.6a 4.6 ± 0.2b 7.3 ± 0.3bc 61.4 ± 8.4a 6.4 ± 0.3b 11.5 ± 0.4c 81.7 ± 7.0a 204.6 ± 4.1d

2.95 ± 0.04a 3.85 ± 0.04a 30.3 ± 2.8bc 389.1 ± 7.2bcd 283.1 ± 6.7b −27.2 ± 2.6cd 20.0 ± 0.5bc 10.4 ± 0.7b −48.3 ± 2.2bc 31.0 ± 1.7bc 12.8 ± 0.1b −58.3 ± 2.4b 13.9 ± 0.1cd 22.0 ± 0.5d 58.2 ± 3.9ab 5.9 ± 0.2a 8.5 ± 0.2ab 43.8 ± 4.7ab 8.0 ± 0.1a 13.8 ± 0.4b 72.5 ± 4.3ab 190.9 ± 3.9e

2.14 ± 0.04c 2.96 ± 0.03d 38.2 ± 1.6ab 395.7 ± 7.0bc 269.0 ± 6.6b −32.0 ± 1.3bcd 21.6 ± 1.1b 9.0 ± 1.2bc −58.7 ± 3.5ab 30.9 ± 1.0bc 10.2 ± 1.1bc −67.4 ± 2.5ab 15.6 ± 0.5bc 26.4 ± 0.4c 69.0 ± 5.9ab 4.4 ± 0.0b 7.2 ± 0.1c 63.6 ± 3.8a 6.8 ± 0.1b 12.8 ± 0.1bc 89.6 ± 1.1a 266.0 ± 4.3ab

2.52 ± 0.03b 3.39 ± 0.05b 34.7 ± 3.4bc 359.6 ± 4.0d 210.0 ± 11.7c −41.7 ± 2.7ab 17.2 ± 0.7c 7.9 ± 0.4bc −53.9 ± 2.9b 26.2 ± 0.7c 9.6 ± 0.6bc −63.1 ± 3.0b 18.6 ± 0.1a 32.3 ± 0.9a 73.8 ± 3.8a 5.9 ± 0.1a 9.2 ± 0.2a 56.7 ± 3.2a 8.8 ± 0.3a 16.5 ± 0.4a 87.1 ± 3.8a 277.1 ± 1.6a

2.55 ± 0.03b 3.17 ± 0.03c 24.4 ± 2.7c 374.6 ± 9.3cd 188.1 ± 10.0c −49.7 ± 2.9a 19.8 ± 0.4bc 6.3 ± 0.4c −68.1 ± 1.4a 28.5 ± 0.8c 7.1 ± 0.5c −75.2 ± 1.5a 16.3 ± 0.4b 29.0 ± 0.4b 77.8 ± 3.7a 5.7 ± 0.2a 9.7 ± 0.5a 68.3 ± 8.0a 8.4 ± 0.5a 16.2 ± 0.6a 92.9 ± 12.6a 253.0 ± 5.6b

5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12 5.12

87.80 144.77 8.18 11.93 43.34 66.70 17.40 27.61 18.42 11.89 33.38 20.83 39.54 109.60 5.57 43.43 57.46 7.06 36.83 83.09 6.92 60.98

＜0.001 ＜0.001 0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 0.007 ＜0.001 ＜0.001 0.003 ＜0.001 ＜0.001 0.003 ＜0.001

Final (%)

596.4 ± 20.0d 163.0 ± 2.3c

942.9 ± 33.0c 360.5 ± 7.4ab

900.7 ± 17.5c 371.4 ± 2.6a

1137.6 ± 27.9b 327.5 ± 3.6b

1356.6 ± 47.8a 389.5 ± 14.7a

1272.5 ± 52.5ab 402.7 ± 13.6a

5.12 5.12

61.29 97.61

＜0.001 ＜0.001

b

b

a

b

a Percent change (%) between in initial and final nutrient (element) concentration = ((Final concentration－initial concentration)/initial concentration) × 100. A positive value indicates an increase in concentration and a negative value indicates a decrease in concentration. b Means in a row followed by different letters denote significance in one way ANOVA followed by Tukey's HSD test, P < 0.05.

significantly higher in the GW + SMS + CM2:1:1, GW + CM1:1, GW + CM3:1 and GW + SMS3:1 treatments than in the GW treatment, but did not differ between the GW + SMS1:1 and GW (one way ANOVA, F5,12 = 6.92, P < 0.001; Tukey HSD post-hoc, P < 0.05). In both TP and TK the GW control had significantly (one way ANOVA, F5,12 = 57.46, P < 0.001, and F5,12 = 83.09, P < 0.001; Tukey HSD post-hoc, P < 0.05) lower values compared to all other treatments at the end of the vermicomposting period. Since phosphorus and potassium are not volatile, these increases can be attributed to mineralization of organic matter, which reduces the weight and volume of the vermicompost product (Zhou et al., 2018), effectively increasing the overall concentration. The addition of GW + SMS + CM2:1:1 produced the largest increases in TN, TK, and TP indicating that increased earthworm growth rates and potentially higher microbial activities by SMS and CM additions, subsequently accelerated waste degradation effectively increasing the overall nutrient concentrations. Moreover, higher nutrient concentration in amended treatments in general means a greater value as a fertilizer for soil and/or pot substrates. Nitrate (NO3−-N) content increased slightly in all treatments from week 0 to week 5 (Fig. 3i). Nitrifying bacteria were likely inhibited by an excessive amount of ammonia during the initial stages of vermicomposting (Cáceres et al., 2016) causing this slow accumulation of NO3−-N. Around week 5, the NO3−-N content increased rapidly in all treatments and attained its peak at week 10. The final NO3−-N content was significantly (one way ANOVA, F5,12 = 61.29, P < 0.001) higher in all addition treatments than in the control (GW). The vermicomposts showed 163.0–402.7% increment in NO3−-N contents compared to initial waste mixtures. The percent increase in NO3−-N content was significantly higher in all addition treatments than in the control (GW) (one way ANOVA, F5,12 = 97.61, P < 0.001: Tukey HSD post-hoc, P < 0.05).

and GW + SMS3:1 treatments, but did not significantly differ between the other treatments and the GW treatment. (one way ANOVA, F5,12 = 5.57, P < 0.001; Tukey HSD post-hoc, P < 0.05). This pattern has been seen elsewhere with nitrogen content increasing by 1.2–2.9 fold in rice straw and paper waste vermicomposting (Sharma and Garg, 2018). Deka et al. (2011) stated that the loss of organic carbon during decomposition is responsible for the increase in N content in the final product, and the conservation of N and subsequent decrease in C:N in litter decomposition is a commonly seen phenomena (Bosire et al., 2005). Earthworms can also increase the N content of the composting materials by adding nitrogenous excretory substances, enzymes, body fluids, mucous, and even decaying earthworm tissues (Suthar, 2010), while improving microclimatic conditions during composting, which increases the numbers of N-fixing bacteria in the vermicast (Suthar et al., 2012). As expected, an increasing trend was observed for both total phosphorus (TP) and potassium (TK) content in all treatments (Fig. 3g and h). These results were in agreement with the observations of Hait and Tare (2012), who reported that the TP and TK content increased by 30.1–51.9% and 39.9–69.8%, respectively, during vermicomposting of sewage sludge. Similar results were also obtained in the previous study by Cai et al. (2018). The final TP content in the vermicomposts ranged from 4.2 g kg−1 to 9.7 g kg−1, which represented a 29.2%–68.3% increase (compared to initial value). For different feeding materials, the percent increase in TP content was significantly higher in the GW + SMS + CM2:1:1, GW + CM1:1, GW + CM3:1 and GW + SMS3:1 treatments than in the GW treatment, but did not differ between the GW + SMS1:1 and GW (one way ANOVA, F5,12 = 7.06, P < 0.001; Tukey HSD post-hoc, P < 0.05). The final TK content in the final products ranged from 6.8 g kg−1 to 16.5 g kg−1, and the increase of TK during vermicomposting was 45.1–92.9% (compared to initial value). For different feeding materials, the percent increase in TK content was 8

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3.5. SEM analysis

Results indicate that SMS and/or CM can enhance the nitrification process in GW vermicomposting practices. The highest increment of NO3−-N content was obtained in the GW + SMS + CM2:1:1treatment. Application of SMS likely provided favorable micro-environment conditions and subsequently enhanced nitrifying bacteria growth and activity, thereby enhancing the conversion of NH4+-N to NO3−-N during the vermicomposting process (Meng et al., 2017). Cow manure has also been shown to promote the nitrification process; likely due to CM containing ample nitrifying bacteria and large amount of available nutrients (Zhang and Sun, 2017). In addition, as mentioned by Hait and Tare (2012), an increase in earthworm density (also seen in these treatments) has a positive effect on nitrification process of vermicomposting. Taken together, GW blended with SMS and/or CM increased the earthworm population (Fig. 1), which then favourably accelerated the nitrification process in the vermicomposting of GW.

Fig. 4 presents the SEM micrographs of the initial GW and its end products in different treatments. The raw GW had a relatively smooth surface without any pores and fragmentation before vermicomposting. After 10 weeks of vermicomposting, the external surfaces of all samples became rough, with the formation of cracks and holes with different sizes, likely due to the hydrolytic degradation process by the hydrolase secreted by the bacterial and fungal communities in both the substrate materials and earthworm gut (Soobhany et al., 2017). Treatment with SMS and CM addition remarkably increased the number of inhomogeneous pores and surface irregularities of GW samples compared to control, which indicating deeper biodegradation of the GW.

3.6. Germination index The germination index (GI) is commonly used to evaluate compost maturity and phytotoxicity. According to Zucconi et al. (1981), when a compost has a GI ≥ 80%, it is considered mature and non-phytotoxic. The GI values of cabbage and tomato seeds in vermicomposts of the GW + SMS3:1, GW + CM3:1, GW + CM1:1 and GW + SMS + CM2:1:1 treatments were higher (one way ANOVA, F5,12 = 36.52, P < 0.001; F5,12 = 17.73, P < 0.001; Tukey HSD post-hoc, P < 0.05) than in vermicompost of the GW treatment (Fig. 5). The highest GI values of cabbage and tomato seeds were both found in GW + SMS + CM2:1:1, which were 1.97 fold and 2.18 fold higher than in GW. All treatments with CM and/or SMS added reached a GI above 80%, indicating all of the final amended vermicomposts were mature and phytotoxic-free. In contrast, the GW control did not obtain a GI above 80% after ten weeks and can thus be considered immature and unfit for application. Taken together, these results showed that amending GW with SMS or CM (except in GW + SMS1:1) and especially with the combined addition of SMS and CM in treatment GW + SMS + CM2:1:1, the final vermicompost is mature after ten weeks and toxicity is greatly reduced compared to the un-amended control (GW). The reasonable explanation for this phenomenon was that the addition of SMS or/and CM resulted in the

3.4. Evaluation of metal concentrations in vermicomposts After the experiments were finished, vermicomposted materials showed a increase in metal contents. The increase ranged between 35.6 and 62.4% for Cu, 27.4–91.1% for Zn, 34.2–87.1% for Cr, 37.2–60.7% for Pb, 32.7–87.7% for Cd, and between 22.0 and 59.0% for Ni (Table 6). The concentration of metals following vermicomposing is well known as the mineralization and degradation of organic matter by earthworms and microorganisms leads to carbon loss as CO2, as well as compost mass and volume reduction; as a consequence the content of remaining metals is enhanced (Yadav and Garg, 2011). Despite the increase in metal concentrations in all vermicompost treatments, metal levels were well below the suggested United States compost limits (Brinton, 2000) and British compost standard (British Standards Institution, 2011) presented in Table 6. Thus, based on these guidelines, the vermicomposts produced from all six treatments in this study can be used although cumulative applications have to be carefully planned and made.

Table 6 Concentrations of Cu, Zn, Cr, Pb, Cd, and Ni (mg kg−1) in the initial mixtures (week 0) and final vermicompost (week 10) obtained from different type materials. Values are means ± SE (n = 3). Means in a column followed by different letters are significantly different at P < 0.05 according to Tukey's HSD test for either the initial feed substrate or the final vermicompost. GW, garden waste; SMS, spent mushroom residues; CM, cattle manure. Treatment Initial mixtures GW GW + SMS3:1 GW + SMS1:1 GW + CM3:1 GW + CM1:1 GW + SMS + CM2:1:1 One-way ANOVA d.f. F P-value Vermicomposts GW GW + SMS3:1 GW + SMS1:1 GW + CM3:1 GW + CM1:1 GW + SMS + CM2:1:1 One-way ANOVA d.f. F P-value US limit rangex PAS 100 quality limity x y

Cu

18.1 17.7 17.2 22.6 26.9 22.3

± ± ± ± ± ±

0.6c 1.2c 1.0c 0.8b 1.8a 1.2b

5.12 34.12 ＜0.001 24.6 28.7 24.2 35.3 39.7 36.3

± ± ± ± ± ±

5.12 19.85 ＜0.001 1500 200

1.5c 3.5bc 1.4c 3.0ab 2.6a 2.6a

Zn

Cr

71.2 ± 2.7cd 59.9 ± 6.1d 45.2 ± 3.4d 107.3 ± 19.5b 141.3 ± 8.3a 92.8 ± 12.6bc

0.3 0.4 0.4 1.4 2.4 1.4

5.12 33.25 ＜0.001

5.12 104.15 ＜0.001

90.7 ± 6.4c 98.8 ± 9.1c 62.4 ± 6.6c 187.6 ± 44.0b 245.2 ± 10.4a 177.4 ± 7.8b

0.4 0.6 0.6 2.4 4.3 2.6

5.12 39.26 ＜0.001 2800 400

5.12 90.07 ＜0.001 1200 100

Brinton (2000). British Standards Institution (2011). 9

Pb

± ± ± ± ± ±

± ± ± ± ± ±

0.0c 0.0c 0.1c 0.1b 0.2a 0.3b

0.0c 0.0c 0.0c 0.4b 0.4a 0.5b

13.0 12.8 12.1 18.9 25.0 18.3

Cd

± ± ± ± ± ±

1.7c 0.4c 0.8c 0.7b 2.8a 1.5b

5.12 32.53 ＜0.001 17.8 21.2 17.7 29.9 43.7 29.4

± ± ± ± ± ±

5.12 35.55 ＜0.001 300 200

0.02 0.14 0.25 0.22 0.42 0.34

Ni

± ± ± ± ± ±

0.00e 0.01d 0.01c 0.01c 0.03a 0.01b

5.12 289.59 ＜0.001 2.1c 1.8c 1.4c 1.6b 5.7a 2.5b

0.02 0.24 0.39 0.39 0.71 0.63

± ± ± ± ± ±

5.12 780.23 ＜0.001 39 1.5

1.7 ± 0.1d 3.0 ± 0.3cd 4.2 ± 0.2c 12.7 ± 1.5b 24.7 ± 0.9a 14.8 ± 0.6b 5.12 414.53 ＜0.001

0.00e 0.01d 0.02c 0.01c 0.02a 0.02 b

2.1 ± 0.2d 4.4 ± 0.1d 5.6 ± 0.1d 19.0 ± 2.0c 37.7 ± 3.2a 23.5 ± 1.4b 5.12 215.37 ＜0.001 420 50

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Fig. 4. Scanning electron micrographs (SEM) of raw materials (a) and end products of garden waste in different treatments after ten weeks of vermicomposting; (b) GW, (c) GW + GW + SMS3:1, (d) GW + SMS1:1, (e) GW + CM3:1, (f) GW + CM1:1, (g) GW + SMS + CM2:1:1. GW, garden waste; SMS, spent mushroom residues; CM, cattle manure.

distinguished group, while the treatment of GW:SMS:CM 2:1:1 also separated with other treatments (Fig. 6). This result implied that the additives could significantly affect GW, and the treatment of GW:SMS:CM 2:1:1 confers the biggest change to GW. From the principal component analysis (PCA), the first three components chosen to examine the dataset explained 97.616% of the total variance, which represents most of the information on the variances. The first principal component represented 74.306%, the second principal components represented 14.301%, and the third principal components represented 9.009%. The first principal component (PC1) was strongly associated with most of the values, including all the mineral elements, earthworm biomass, and earthworm growth rate, while cellulase activity are the

accelerated decomposition of organic material, thus increasing the quantity of available nutrients in the final vermicompost, and this will presumably enhance seed germination and growth, as well as in reducing the content of toxic materials, such as extractable metals (Meng et al., 2018; Zhang and Sun, 2017). The similar study also reported that plant grew better under intermediate rates of GW and CM (e.g. 40–60% for CM) than GW (Sierra et al., 2013) . 3.7. Principal component analysis (PCA) The PCA indicated that all the 6 treatments could be roughly divided into four groups. GW with no additives stranded for one 10

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Fig. 5. The germination index (GI) of Chinese cabbage (A) and tomato (B) seeds in the final vermicompost as affected by spent mushroom substrate and cattle manure additions. Values are the means ± SE of three replications. For different parameters, bars with different letters are significantly different at P < 0.05 according to Tukey's HSD test. GW, garden waste; SMS, spent mushroom substrate; CM, cattle manure.

Conflict of interest The authors report no potential conflict of interest. Acknowledgements National Forestry Public Welfare Project of China “Development and Application of Technology of Converting Forestry Waste into Growing Media” supported this research (Grant No. 201504205). Xiaoqiang Gong would like acknowledge the scholarship from the China Scholarship Council. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jenvman.2019.109263. References

Fig. 6. Principal component analysis of 6 treatment based on 28 index.

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dominant variables in the second principal component (PC2) and alkaline phosphatase activity showed the highest weight in the third PC (Table S1). This tool reduced the number of dimensions from 28 variables to 3 principal components and retained most of the original information content of the dataset. 4. Conclusions This study indicates that vermicomposting of GW mixed with SMS and CM is a viable practice for the management of GW. The combined addition of SMS and CM (treatment GW + SMS + CM2:1:1) is more effective with GW vermicomposting than the addition of SMS or CM alone. Combined addition of SMS and CM increased the growth and fecundity of E. fetida, stimulated enzymatic activities in the vermicompost, increased degradation of lignin, and enhanced nitrification and nutrient (N, P and K) concentrations following ten weeks of vermicomposting. Additionally, vermicomposting of GW together with SMS and CM at rate 2:1:1 effectively promoted final vermicompost maturity and, as are reflected in the significantly higher GI values. Taken together, the results of this study recommend amending GW with SMS and CM together in a ratio of 2:1:1, respectively, in order to produce a better quality vermicompost from GW materials. These findings could provide key solutions to accelerate the vermicomposting process and improve the quality of final vemicompost product. 11

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