Bioresource Technology 320 (2021) 124404

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

Vermicomposting of livestock manure as affected by carbon-rich additives

(straw, biochar and nanocarbon): A comprehensive evaluation of
earthworm performance, microbial activities, metabolic functions and
vermicompost quality
Yune Cao a, Yongqiang Tian a, b, *, Qing Wu a, Jianshe Li a, Hongyan Zhu a
a
College of Agriculture, Ningxia University, Helanshan Xilu No. 489, Yinchuan 750021, China
b
College of Horticulture, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• The addition of straw promoted earth­

worm growth and cocoon production.
• The addition of biochar and nanocarbon
improved microbial activities.
• Both biochar and nanocarbon reduced C
and N losses as compared to straw.
• Straw combined with nanocarbon
showed the highest vermicompost
quality.

A R T I C L E I N F O A B S T R A C T

Keywords: Vermicomposting is an eco-friendly method for treating organic wastes. This study investigated the effects of the
C-rich additives addition of straw (S), biochar (B), nanocarbon (N), S + B and S + N to cow dung (CD) on earthworm (Eisenia
Vermicomposting efficiency fetida) performance, microbial properties and vermicompost quality. In general, the earthworm growth rate and
Earthworm performance
cocoon production were enhanced by straw addition, but were inhibited by biochar or nanocarbon addition.
Microbial activities
Overall vermicompost quality
However, biochar and nanocarbon increased microbial communities associated with organic matter decompo­
sition, and improved metabolic functions, enzyme activities and vermicompost properties. Moreover, addition of
straw in combination with nanocarbon resulted in the highest vermicompost quality index (VQI), and signifi­
cantly increased the biomass of three different test crops (radish, lettuce and pakchoi). These results indicated
that biochar and nanocarbon mainly improved microbial activities, while straw primarily enhanced earthworm
performance during vermicomposting. In addition, straw combined with nanocarbon can be used to enhance the
agronomic performance of vermicompost.

* Corresponding author at: College of Agriculture, Ningxia University, Helanshan Xilu No. 489, Yinchuan 750021, China.
E-mail address: [email protected] (Y. Tian).

https://doi.org/10.1016/j.biortech.2020.124404
Received 3 October 2020; Received in revised form 4 November 2020; Accepted 7 November 2020
Available online 12 November 2020
0960-8524/© 2020 Elsevier Ltd. All rights reserved.
Y. Cao et al. Bioresource Technology 320 (2021) 124404

1. Introduction 2017) focused only on earthworm performance or microorganisms in

vermicompost, but not both. Furthermore, little information is available
The world population is growing at 1.7% annually and is expected to on how CRMs influence microorganisms in earthworm gut. Thus, the
increase from 7.7 billion in 2019 to 9.7 billion in 2050 (Leridon, 2020). novelty of this work lies in studying how CRMs simultaneously influence
The rapid population growth has led to an increasing demand for live­ earthworms, earthworm gut microorganisms and vermicompost micro­
stock products around the world. It is estimated that the livestock sector organisms. In addition, nanocarbon, a novel C-rich material that has
occupies approximately one-third of the global terrestrial surface been increasingly used to improve soil and water quality (Yarima et al.,
excluding the Antarctica (Dangal et al., 2017). Accordingly, a huge 2020), has not yet been reported in studies regarding vermicomposting.
amount of livestock manure is produced annually in different regions of Therefore, investigating the efficiency of nanocarbon in enhancing
the world. Global livestock manure production is estimated at about vermicompost quality was also the novelty of this study.
6,252 million tons per year (approximately equal to 113 million tons of During the decomposition process of organic matter, nutrient
N per year; FAO, 2018). Generally, livestock manure can be used to transformation is dominated by microbial metabolism function (Metcalf
improve soil physiochemical properties and increase crop yields, et al., 2016). Yet the microbial metabolic functions in vermicomposting
because it contains significant contents of organic matter and nutrients system are still poorly understood. In the present study, it was hypoth­
(Schlegel et al., 2017). However, since livestock manure normally con­ esized that CRMs might regulate the activities and functions of micro­
tains pathogens, heavy metals and antibiotics, direct land application organisms in both earthworms and vermicompost during
can easily result in various environmental risks such as disease vermicomposting of livestock manure, and therefore affect the quality of
spreading, soil pollution and groundwater contamination (Zubair et al., the final vermicompost products. To test this hypothesis, a vermi­
2020). More seriously, the current trends in livestock production composting experiment was conducted to evaluate the influence of
intensification promote the occurrence of the phenomenon associated CRMs (i.e. straw, biochar, nanocarbon, straw combined with biochar,
with the direct discharge of manure into the environment (Pedizzi et al., and straw combined with nanocarbon) on the vermicomposting of
2018). Consequently, there are increasing concerns regarding the livestock manure (i.e. cow dung). The objectives of this study were to (1)
disposal of livestock manure in an environmentally-friendly manner. examine whether CRMs can promote the activities and functions of
Vermicomposting, an eco-friendly process involving joint action of microorganisms in either earthworm body or vermicompost and further
earthworms and mesophilic microorganisms for treating and stabilizing enhance vermicompost quality and (2) to reveal the potential mecha­
organic wastes (e.g. livestock manure), has gained worldwide attention nisms underlying CRM-induced improvement of the activities and
due to its excellent ability to alleviate the environmental burdens functions of microorganisms.
associated to waste management (Devi and Khwairakpam, 2020; Huang
et al., 2020). This technique offers four main advantages in terms of 2. Materials and methods
organic waste disposal: (i) accelerating the biodegradation of chemical
toxicants by improving microbial and enzymatic activities (Bhat et al., 2.1. Vermicompost materials and experimental design
2018); (ii) reducing the toxicity of heavy metals by decreasing their total
concentration and bioavailability (Wang et al., 2017); (iii) suppressing The experimental earthworms (Eisenia fetida) were collected directly
microbial pathogens via enhancing microbial diversity and functions from stock cultures maintained by the authors at the Vermicomposting
(Huang et al., 2020); and (iv) converting organic wastes into excellent Research Center of Ningxia University, Yinchuan, Ningxia, China. Cow
bio-fertilizers with abundant humic-like compounds and diverse pro­ dung was obtained from a cattle farm of Yinchuan, Ningxia. Corn straw
biotics that are helpful for agricultural use (Sanchez-Hernandez and was collected from an agricultural field situated in Helan country,
Domínguez, 2019). In spite of these benefits, the efficiency of vermi­ Ningxia. Biochar, which was produced from coconut husks, was pur­
composting is strongly determined by the characteristics of organic chased from a commercial pyrolysis plant located in Shangqiu, Henan.
wastes (Bhat et al., 2018), which means that unsuitable waste properties Nanocarbon, which was made from processed coconut husks, was pur­
can restrict earthworm performance and associated microbial activities. chased from a commercial charcoal factory in Shanghai, China. The
Therefore, improvement of characteristics of the initial raw materials is coconut husk biochar was used in this study because it is a common
of crucial importance to ensure vermicomposting efficiency and thereby amendment that can efficiently improve compost/vermicompost quality
vermicompost quality. (Velasco-Velasco et al., 2011; Malińska et al., 2017). The reason for the
In recent years, several techniques have been developed to enhance use of the coconut husk nanocarbon is that nanocomposite has been
the efficiency of vermicomposting of organic wastes. One efficient increasingly used to improve soil and water quality (Yarima et al.,
technique involves the addition of C-rich materials (CRMs) to enhance 2020).
earthworm performance and to regulate vermicomposting process. For Six different experimental media were prepared using cow dung,
instance, Malińska et al. (2017) observed that the addition of biochar corn straw, biochar and nanocarbon. The treatments considered were (i)
efficiently promoted the growth and reproduction of earthworms by cow dung (CD), (ii) CD plus 30% C from straw (CD + S), (iii) CD plus
alleviating toxicity of heavy metals to earthworms during vermi­ 30% C from biochar (CD + B), (iv) CD plus 30% C from nanocarbon (CD
composting of sewage sludge. Velasco-Velasco et al. (2011) demon­ + N), (v) CD plus 15% C from straw plus 15% C from biochar (CD + S +
strated that the addition of extra straw enhanced vermicompost fertility B), and (vi) CD plus 15% C from straw plus 15% C from nanocarbon (CD
through suppressing ammonia volatilization during sheep manure ver­ + S + N). The biochar and nanocarbon were added in dry weight basis.
micomposting. Despite these advances, there remain a number of The addition rate of straw was suitable for vermicomposting trials, based
unanswered questions in terms of the potential mechanisms underlying on the comprehensive consideration of the results of Sharma and Garg
CRM-induced improvement of vermicomposting efficiency. Particularly, (2018) and Contreras-Ramos et al. (2005). With respect to biochar and
it is still unclear how CRMs might affect the activities and functions of nanocarbon, their application rates were considered based on the equal
microorganisms in both earthworms and vermicompost during vermi­ amount of total C input. Vermicomposting was carried out indoors using
composting. Thus, it is crucial to comprehensively evaluate the role vermicomposting vessels (diameter 47.5 cm and depth 26 cm). There
played by microorganisms in the vermicomposting process towards were three replicates for each treatment and each replicate contained
disposal of organic wastes amended with CRMs. four vermicomposting vessels. In total, seventy-two reactors (6 treat­
The process of vermicomposting is controlled not only by the per­ ments × 3 replicates × 4 vermicomposting vessels) were used. The
formance of earthworms but also by the activities of microorganisms in culture media added to the vessels were CD, CD + S, CD + B, CD + N, CD
both earthworm gut and vermicompost. However, prior CRM-related + S + B and CD + S + N. A total of 5.4 kg of different raw material mixes
vermicomposting studies (Velasco-Velasco et al., 2011; Malińska et al., was added to the individual vermicompost vessels. Four hundred

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

individuals of earthworms with an initial average weight of 0.189 g were determined according to the method detailed in Schinner and von Mersi
introduced into each vermicomposting vessel. During the experimen­ (1990), while cellulase, urease, catalase and phosphatase were analyzed
tation period, the moisture was maintained at 60–66% by sprinkling the by methods described in Qu et al. (2020).
culture media with distilled water when required. Moreover, the
ambient temperature ranged from 20 to 30 ◦ C within the tolerance limit
2.4. Overall vermicompost quality assessment and bioassay
of Eisenia fetida (0–40 ◦ C).
To evaluate the overall quality of the final vermicompost, a vermi­
2.2. Sampling procedure, earthworm parameters and vermicompost compost quality index (VQI) was calculated by integrating key in­
properties dicators using the method described in Moharana and Biswas (2016).
Briefly, parameters associated with vermicompost quality (i.e. earth­
Based on the life cycle of Eisenia fetida, the duration of the experi­ worm performance, nutrients, enzyme activities and microbial proper­
ment was 30 days. On average, earthworm individuals reached maturity ties) were used to establish a minimum dataset (MDS) of the key quality
at day 30 and body weight at maturity ranged between 0.5 and 0.7 g. indicators based on principal component analysis (PCA). To calculate
Samples collected on days 10, 20 and 30 after initiating the vermi­ the VQI, the key quality indicators were ranked in ascending (for those
composting experiment were recognized as early, medium and late having positive effects on vermicompost quality) or descending (for
vermicomposting periods, respectively. At each sampling time, vermi­ those having negative effects on vermicompost quality) order. The VQI
composts were turned and mixed, and a sample of 200 g was taken from ∑
was calculated according to the equation VQI = ni=1 Si Wi , where Wi is
each vessel. Earthworms were separated from feedstocks carefully with
the PC weighting factor, and Si presents the indicator score for variable i.
a sterilized tweezer. Earthworm biomass was measured on a fresh
It was assumed that higher VQI meant better vermicompost quality.
weight basis, while earthworm population was counted manually ac­
Moreover, three test crops of radish, lettuce and pakchoi were selected
cording to the dump and hand sort method (Devi and Khwairakpam,
for bioassay (plant growth test) (Qu et al., 2020).
2020). Meanwhile, the cocoons produced from each worm were calcu­
lated. The growth, survival, and fecundity of earthworms were calcu­
lated by methods described in Sharma and Garg (2018). 2.5. Statistical analysis
The vermicompost output was calculated as described in Boruah
et al. (2019). To evaluate the changes in vermicompost properties, Data were subjected to analysis of variance (ANOVA) carried out by
various parameters were quantified including pH, electrical conductiv­ SPSS 19.0 (SPSS Inc., Chicago, USA). Whenever the ANOVA showed
ity (EC), total organic carbon (TOC) and nutrient elements. The pH and significant differences at P ≤ 0.05, multiple comparisons were done
EC were determined in a 1:10 (w/v) water-soluble extract (Sharma and using Tukey’s honestly significance difference (HSD) post hoc test. The
Garg, 2018). TOC was determined by the dichromate oxidation and non-metric multidimensional scaling (NMDS) was applied to visualize
titration method, while total N (TN) was measured using the Micro- microbial community, function and activity data. In addition, Pearson
Kjeldahl method (Sparks et al., 1996). The losses of TOC and TN of correlations were run among chemical properties and microbial activ­
the vermicompost were calculated using the equation Q loss rate = (Q1 ities or metabolic functions in vermicompost.
× M1 – Q2 × M2)/(Q1 × M1) (Qu et al., 2020), where Q1 and Q2 are the
initial and final TOC (or TN) concentrations, respectively, and M1 and 3. Results and discussion
M2 represent the initial and final dry weights, respectively. Available N
(ammonium N plus nitrate N) was analyzed using a continuous flowing 3.1. Earthworm growth performance and reproduction
analyzer (TRAACS2000, USA; Sparks et al., 1996). Available P was
measured using the molybdenum-blue colorimetry, while available K The earthworm performance reflects the dynamic of the vermi­
was determined by the flame photometry (Sparks et al., 1996). The composting process (Boruah et al., 2019). The results of earthworm
concentrations of nutrient elements P, K, Ca, Mg, S, B, Cu, Fe, Mn, Zn growth rate, biomass gain and cocoon production are presented in
and Mo were determined using the Inductivity Coupled Plasma Atomic Table 1. Clearly, the initial biomass of earthworms ranged from 188.3 to
Emission Spectrometry (ICP-AES) (Tian et al., 2012). 190.0 mg/worm on average, and did not show significant differences (P
> 0.05) among treatments. However, earthworm biomass increased
2.3. Microbial communities, activities and functions gradually over the course of vermicomposting due to earthworm

Microbial communities in both earthworms and vermicompost were Table 1

determined using the high-throughput sequencing as described in Wei Earthworm growth performance and fecundity during vermicomposting.
et al. (2018). Briefly, DNA were extracted from samples at the early, Treatments Initial Maximum Growth Net Cocoons/
medium and late vermicomposting periods, using the Power Soil DNA biomass/ biomass/ rate/ biomass worm
Kit (MoBio Laboratories Inc., CA, USA) according to the manufacturer’s worm worm (mg) worm/ gain/
instructions. After determining the concentration and quality of (mg) day (mg) worm
(mg)
extracted DNA, the total genomic DNA samples were used to amplify the
16S rRNA gene using two primers 338F (5′ -ACTCCTACGGGAGGCAG­ CD 188.3 ± 427.3 ± 11.95 ± 239.0 ± 0.29 ±
0.3a 20.3c 1.00c 20.0c 0.12c
CAG-3′ ) and 806R (5′ -GGACTACHVGGGTWTCTAAT-3′ ). Then, PCR
CD + S 188.6 ± 588.8 ± 20.01 ± 400.2 ± 0.52 ±
reactions were conducted and the PCR products were purified and 0.3a 17.8b 0.88b 17.7b 0.02b
sequenced on an Illumina MiSeq platform. The quality filtered se­ CD + B 188.4 ± 304.3 ± 5.79 ± 115.9 ± 0.00 ±
quences were binned into operational taxonomic units (OTUs) at the 0.3a 29.9d 1.49d 29.8d 0.00d
97% sequence similarity level. Rarefaction was performed using the CD + N 188.5 ± 220.9 ± 1.62 ± 32.4 ± 0.00 ±
0.5a 7.4e 0.34e 6.9e 0.00d
QIIME to discern levels of OTU richness, Shannon diversity and the CD + S + B 190.0 ± 680.8 ± 24.53 ± 490.7 ± 0.74 ±
Chao-1 of total species richness. To predict metabolic functions of mi­ 0.9a 18.9a 0.94a 18.9a 0.02a
crobial communities, PICRUSt was used to generate a functional profile CD + S + N 189.8 ± 287.0 ± 4.86 ± 97.2 ± 0.00 ±
from 16S rRNA data (for more details, see Wei et al., 2018). 1.2a 17.6de 0.87de 17.5de 0.00d
In addition to microbial communities and functions, vermicompost CD: cow dung; S: straw; B: biochar; N: nanocarbon. Values are means ± standard
enzymes were also assessed at the early, medium and late vermi­ errors. Values followed by the same lowercase letter are not significantly
composting periods to reflect microbial activities. Invertase was different (P > 0.05).

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

growth, and was differently influenced by various additives. Specif­ production) when applied in conjunction with straw (compare CD + S +
ically, maximum biomass was significantly higher in CD + S and CD + S B versus CD + S), during cow dung vermicomposting (Table 1). The
+ B but lower in CD + B, CD + N and SC + S + N, as compared to CD. negative impact of biochar alone might be attributed to the recalcitrant
Moreover, the highest maximum worm biomass was observed in CD + S character of biochar, which is less palatable to earthworms than cow
+ B (588.8 mg/worm on average), while the lowest maximum biomass dung (Liesch et al., 2010). This could be partly supported by the results
was recorded in CD + N (220.9 mg/worm on average) after vermi­ reported by Ashwood et al. (2017), who found that the food palatability
composting. Similar trends were observed in either growth rate or net is a key factor in determining the survival of earthworms. With respect
biomass gain. Together, these results indicate that addition of corn straw to the positive influence of biochar along with straw, it could be
alone or in combination with biochar enhanced earthworm performance explained by the promoting effects of biochar on straw decomposition
during vermicomposting of cow dung. Similar results were reported (Zhou et al., 2019), which can make straw more palatable to earthworms
earlier by Sharma and Garg (2018), who found that rice straw mixed (Sizmur et al., 2017). Similarly, Malińska et al. (2017) reported that
with cow dung could maintain earthworm growth and fecundity, biochar amendment significantly increased the number of cocoons
thereby facilitating the vermicomposting process. Presumably, straw during vermicomposting of sewage sludge mixed with woodchips. Un­
addition can lead to the enhancement of earthworm performance expectedly, nanocarbon showed extremely adverse effects on earth­
through two main mechanisms, i.e., (i) enhancing food consumption by worm performance (Table 1). This was attributed to toxic effects of
earthworms through accelerating organic matter decomposition which nanomaterials on earthworm survival and antioxidant enzymes (Zhao
can make feedstock more palatable to earthworms (Wu et al., 2018) and et al., 2017). It was noted that either biochar or nanocarbon alone
(ii) creating favorable conditions for earthworm gut microbiota by negatively affected earthworm performance by reducing earthworm
regulating the C/N ratio in feedstocks (Suthar and Ram, 2008). growth and cocoon production (Table 1). One possible explanation for
Interestingly, biochar, when applied alone, exhibited adverse effects this phenomenon is that biochar and nanocarbon store C in a recalcitrant
on earthworm performance (compare CD + B versus CD), but it pro­ form that is generally not palatable to earthworms (Liesch et al., 2010;
moted earthworm growth and reproduction (as reflected by cocoon Ashwood et al., 2017). Another possible explanation is the fact that the

Fig. 1. Selected chemical properties in the initial feedstock and final vermicompost as affected by various carbon-rich additives. CD: cow dung; S: straw; B: biochar;
N: nanocarbon. EC: electrical conductivity; TOC: total organic carbon; TN: total nitrogen; C/N: the ratio of carbon to nitrogen; TP: total phosphorus; TK: total
potassium; AN: available nitrogen; AP: available phosphorus; AK: available potassium. Bars represent standard errors. The same lowercase letter over each bar
represents no significant difference (P > 0.05) between treatments.

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

higher pH in biochar (pH 10.20) and nanocarbon (pH 10.33) likely compared to the initial feedstock (Fig. 1d), primarily because of the dry
contributed to continued death of earthworms, because many species of matter degradation (Jain et al., 2018). Despite that, significant negative
earthworms show intolerance to alkaline residues with preference for effects of all additives were observed on TN concentration in both the
residues typically at neutral pH (Liesch et al., 2010). Nowadays, many initial feedstock and final vermicompost (Fig. 1d). This comes as no
studies regarding additive strategies in vermicomposting focused on the surprise, as cow dung (19.3 g kg− 1) had a higher concentration of TN
mitigation of several issues like odor and greenhouse gas (GHG) emis­ than C-rich additives (2.6–13.7 g kg− 1) used in this study. The additive-
sions, but ignored the responses of earthworms. Thus, it is critical for induced reduction in TN could have profound influences on microbial
future research to address the effects of additives on earthworm activities, because N is essential for heterotrophic microorganisms.
performance. Additionally, it could also affect earworm performance because N reg­
ulates microbe-mediated organic matter decomposition which can make
3.2. Vermicompost properties and losses of TOC and TN feedstock more palatable to earthworms (Wu et al., 2018). Because of
the decreased TOC and increased TN over the vermicomposting course
The EC, an index used to reflect salt concentration in a sample (Yadav (Fig. 1c, d), the C/N ratio for all treatments was lower in the final ver­
and Garg, 2019), decreased during vermicomposting process in all micompost than in the initial feedstock (Fig. 1e). The reduction in C/N
treatments (Fig. 1a). This decrease in EC could be explained by the ratio throughout vermicomposting was found to be lower for CD + S
volatilization of ammonia and the precipitation of mineral salts (Huang (10.1%), CD + B (24.7%) and CD + N (31.4%) but higher for CD + S + B
et al., 2004). The EC in the final vermicompost was decreased for the CD (47.8%) and CD + S + N (38.2%), as compared to CD (33.2%), indi­
+ B and CD + N treatments compared to CD (Fig. 1a). Addition of cating the interaction effects of straw with biochar or nanocarbon. The
biochar or nanocarbon alone decreased the EC significantly in the final higher decrease in C/N ratio could be attributed to the suitability of
vermicompost (compared CD + B and CD + N versus CD), due to the feedstock materials, which stimulates microbial and earthworm’s ac­
high salt-absorbing capacity of C-rich materials (Amini et al., 2016). tivity (Boruah et al., 2019). Notably, the C/N ratio in the final vermi­
Moreover, the decreased in EC could be attributed to the enhancing compost was under 20 for all treatments, which meets the desired
effects of C-rich materials on microbial activities (Velasco-Velasco et al., quality of a vermicompost (Devi and Khwairakpam, 2020). Similar to
2011; Zhou et al., 2019), which could promote microbes to immobilize TN, the total P (TP) and K (TK) concentrations in the final vermicompost
soluble salts into microbial biomass components. Notably, the EC of the was increased for all treatments compared to the concentration in the
final vermicompost was in the range of 2.17–2.99 mS cm− 1 among initial feedstock (Fig. 1f, g), because of the degradation of dry matter
treatments (Fig. 1a). This indicates that all vermicomposts can be safely during vermicomposting (Jain et al., 2018; Wu et al., 2018). It was noted
applied to the agricultural soils, because the threshold value of EC for a that both TP and TK in the final vermicompost were significantly lower
material is 3.0 mS cm− 1 for its safe application (Soumaré et al., 2002). in treatments associated with biochar or nanocarbon than in CD and CD
Similar to EC, the pH decreased in all treatments over the course of + S (Fig. 1f, g). This phenomenon could be explained by the fact that
vermicomposting (Fig. 1b). Interestingly, the pH of the final vermi­ biochar and nanocarbon were less biodegradable compared to cow dung
compost was not significantly influenced (P > 0.05) by each additive and straw (Liesch et al., 2010). As a result, addition of biochar or
when applied alone, but it was significantly increased by straw in nanocarbon resulted in lower levels of dry matter degradation and
combination with biochar and decreased by straw along with nano­ thereby decreased TP and TK concentrations as compared to their
carbon. The pH value in a vermicompost depends on the acidic content respective controls. In the final vermicompost, available N was
of feedstock and reactions occurred during the vermicomposting pro­ increased addition of biochar alone (compare CD + B versus CD;
cess. Generally, increase in pH is primarily due to the release of Fig. 1h), while available P was increased by addition of straw alone
ammonia, which is generally conducted by the metabolism of the (compare CD + S versus CD; Fig. 1i). With respect to available K, it was
ammonia oxidizing microorganisms (Qu et al., 2020), while decrease in increased in the final vermicompost by all C-rich additives as compared
pH is because of the production of organic/inorganic acids by micro­ to untreated CD (Fig. 1j). Collectively, these results indicate that addi­
organisms during the process (Wong et al., 2001). Thus, addition of tion of C-rich additives could enhance the availability of a specific
straw along with biochar might promote microbial ammonification, nutrient in vermicompost and thereby improve the agronomic perfor­
while addition of straw in combination with nanocarbon probably mance of vermicompost when it is used as a fertilizer or an amendment.
enhanced microbe-mediated organic matter decomposition to generate Due to the decomposition of organic matter during vermicomposting
humic/fulvic acids (Swarnam et al., 2016). Notably, the biochar- and (Devi and Khwairakpam, 2020), obvious TOC and TN losses were
nanocarbon-induced increase in pH might have adverse impacts on observed in all treatments (Fig. 1k). Specifically, the TOC loss ranged
earthworm performance because the growth and reproduction of from 55.4% in CD + N to 76.8% in CD + S, while the TN loss ranged from
earthworms could be inhibited by the alkalization of culture media 36.0% in CD + B to 80.4% in CD + S. Of note, both TOC and TN losses
(Liesch et al., 2010). were significantly lower in treatments associated with biochar or
Since C is the source of energy for both earthworms or microorgan­ nanocarbon than in CD + S. This is partly because biochar and nano­
isms during various metabolic activities like respiration (Sharma and carbon can bound CO2 and NH3 to their surface through physisorption
Garg, 2017; Devi and Khwairakpam, 2020), the concentration of TOC (Andreoli et al., 2015; Qu et al., 2020). Furthermore, adding recalcitrant
showed a decreasing trend during the vermicomposting process C-rich materials such as biochar and nanocarbon can help to immobilize
(Fig. 1c). For the final vermicompost, the TOC concentration was some of the N and reduce dry matter loss (Qu et al., 2020). Notably, the
increased by addition of nanocarbon alone and decreased by addition of addition of biochar showed improvement in the degradation of straw,
straw alone or in conjunction with biochar or nanocarbon. The but with manure it presented the opposite result (Fig. 1k). This could be
nanocarbon-induced increase in TOC was attributed to the much higher due to that straw had higher C/N ratio than manure and therefore might
TOC of nanocarbon (918.4 g kg− 1) as compared to cow dung (231.2 g be more easily degraded and lost in the form of CO2.
kg− 1). Since organic C can enhance microbial growth and enzyme ac­
tivities, increased TOC imply that nanocarbon addition might enhance 3.3. Microbial communities, metabolic functions and enzyme activities
microbial activities in vermicompost. However, because nanocarbon
stores C in a recalcitrant form, its enhancing effects on TOC had negative The 16S rRNA sequencing was performed to characterize the bac­
effects on earthworm growth and reproduction (Table 1). With regards terial community composition at three different vermicomposting pe­
to straw, it could easily lead to TOC loss due to its promoting effects on riods (Fig. 2). Overall, Proteobacteria, Actinobacteria, Firmicutes,
organic matter decomposition, which was conducted by microorganisms Chloroflexi, Bacteroidetes and Gemmatimonadet were the dominant phyla
(Wu et al., 2018). The TN concentration increased in final vermicompost that accounted for 88%-98% of the total sequences. The addition of

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Fig. 2. Phylum (a) and genus (b) levels composition of the bacterial community as affected by various carbon-rich additives. CD: cow dung; S: straw; B: biochar; N:
nanocarbon. Genera of all samples with greater than 0.5% abundance are listed.

various C-rich additives resulted in obvious differences in the bacterial KEGG pathway databases also exhibited significant differences among
community composition in both earthworm and vermicompost. Specif­ treatments, although the magnitude of difference was relatively low
ically, for the bacterial community in earthworm, addition of straw (Fig. 3a). The relative abundance of sequences related to metabolism
alone increased the relative abundances of Actinobacteria and Chloroflexi pathway explained more than 79% during the whole vermicomposting
in early and late periods and those of Proteobacteria and Bacteroidetes in process. Some metabolic pathways were overrepresented in earthworm
medium period, while addition of biochar alone increased the relative or vermicompost under C-rich additive-related treatments compared
abundance of Actinobacteria in early and late periods and that of Firmi­ with the untreated control (CD). Obviously, the relative abundance of
cutes in medium period. With respect to nanocarbon, it increased the sequences related to carbohydrate metabolism was significantly
relative abundances of Firmicutes and Chloroflexi in medium and late increased by addition of biochar or nanocarbon alone in earthworm in
periods, respectively. Moreover, combined application of straw with medium period, and by addition of nanocarbon alone or in combination
either biochar or nanocarbon increased the relative abundance of Pro­ with straw in vermicompost in early period, as compared to the un­
teobacteria in earthworm in all three vermicomposting periods (Fig. 2a). treated control. Similar results were reported in an earlier study (Zhou
Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes play a et al., 2019) that recalcitrant C-rich materials such as biochar enhanced
considerable role in organic matter mineralization and nutrient cycling the carbohydrate metabolism associated with the degradation of cellu­
(Metcalf et al., 2016; Awasthi, et al., 2018). In addition, some members lose and hemicellulose, and thereby resulted in the acceleration on
of Actinobacteria, Bacteroidetes and Firmicutes are also able to degrade amino acid production and humic substance synthesis during compost­
lignocellulose into short chain fatty acids and to break complex organic ing process. Furthermore, the level 3 KEGG ortholog function pre­
polymers down into smaller molecules (Azman et al., 2015; Awasthi, dictions showed that, in most situations, the abundance of sequences
et al., 2018). Notably, it has been demonstrated that Acidobacteria, assigned into biosynthesis of ansamycins (antibiotics) increased for CD
Bacteroidetes, Chloroflexi, Firmicutes and Proteobacteria are active phyla + S, CD + B and CD + S + B compared to CD (Fig. 3b). Simultaneously,
in earthworm gut contents (Wüst et al., 2011). Collectively, various C- the abundance of genes associated with biosynthesis of vancomycin
rich additives could stimulate the growth of different microbial taxa that group antibiotics was generally higher in CD + S and CD + S + B than in
are capable of accelerating lignocellulose degradation, organic matter CD. These results suggested that addition of straw and/or biochar could
mineralization and nutrient cycling in earthworm. An obvious differ­ enhance the potential of vermicompost to suppress the growth of
ence in the bacterial community composition was observed between phytopathogenic microorganisms, because antibiotic biosynthesis is an
earthworm and vermicompost (Fig. 2a). In particular, vermicompost important characteristic of plant beneficial bacteria (Tsuge et al., 1996).
generally had a higher relative abundance of Firmicutes as compared to Clearly, the NMDS plots based on microbial communities and metabolic
earthworm. The Firmicutes was also identified to be a dominant phylum functions exhibited distinct separations between treatments in both
in various kinds of organic waste compost (Awasthi et al., 2018). C-rich earthworm and vermicompost (Fig. 4a). Moreover, significant correla­
additives also significantly affected the bacterial community composi­ tions were observed between several chemical properties (e.g. TOC,
tion at the genus level (Fig. 2b). For instance, in medium period, Bacillus which is influenced by C-rich additives) and microbial communities or
in earthworm was enriched by addition of biochar or nanocarbon alone, metabolic functions (P < 0.05; Fig. 4b). Thus, C-rich additives could
while Unidentified_OM2_clade in vermicompost was strongly increased regulate microbial activities and thereby influence the vermicomposting
by addition of biochar alone. This indicates that biochar and nanocarbon process.
could help to enhance the vermicomposting process, because Bacillus Enzymes are major mediators of various degradative processes.
can rapidly colonize on lignocellulolytic substrates and has a strong Overall, effects of C-rich additives on vermicompost enzyme activities
tendency to degrade complex organic matter under aerobic condition changed over the course of vermicomposting. Specifically, urease, an
(Awasthi et al., 2018). enzyme involved in the catalytic decomposition of urea (Du et al., 2019),
Similar to microbial communities, the metabolic functions based on was enhanced by addition of various C-rich additives except nanocarbon

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

Fig. 3. Biochemical metabolic pathways (a) and the level 3 KEGG ortholog function predictions (b) of the bacterial community as affected by various carbon-rich
additives. CD: cow dung; S: straw; B: biochar; N: nanocarbon. The level 3 KEGG ortholog function predictions about the relative abundance of the 20 most metabolic
functions are listed.

Fig. 4. Non-metric multidimensional scaling (NMDS) plots visualizing data associated with microbial communities or metabolic functions (a) and relationships
between chemical properties and microbial communities or metabolic functions in vermicompost (b). In Fig. 4a, CD: cow dung; S: straw; B: biochar; N: nanocarbon.
In Fig. 4b, EC: electrical conductivity; TOC: total organic carbon; TN: total nitrogen; C/N: the ratio of carbon to nitrogen; TP: total phosphorus; TK: total potassium;
TCa: total calcium; TMg: total magnesium; TB: total boron; TCu: total copper; TFe: total iron; TMn: total manganese; TZn: total zinc; TMo: total molybdenum. *P <
0.05, **P < 0.01.

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

alone in medium and late periods. This result indicates that straw and the combined addition of straw and nanocarbon enhanced the degra­
biochar promoted the growth of urease-producing microorganisms dation of resistant cellulose compound at the early stage of vermi­
along with the vermicomposting process, because urease is a stable composting and thereby accelerated the vermicompost maturity.
endocellular enzyme whose activity depends on microbial biomass. Although effects of C-rich additives on vermicompost enzyme activities
Invertase, which facilitates the hydrolysis of sucrose and yields glucose, differed over the course of vermicomposting, the NMDS analysis showed
fructose and energy (Ge et al., 2020), was generally increased by addi­ distinct separations between treatments. Thus, C-rich additives could
tion of various C-rich additives. However, it was decreased by addition regulate enzyme activities and have a further impact on the vermi­
of biochar alone in medium period and by addition of straw, nanocarbon composting process.
and their combination in late period. This demonstrates that straw and
nanocarbon enhanced microbial activities primarily during the early
stage of vermicomposting, because a wide range of microorganisms can 3.4. Overall compost quality assessment and biological assay
produce invertase and utilize sucrose as the only carbon source. Phos­
phatase, an enzyme catalyzing the removal of phosphate groups from To comprehensive evaluate the overall quality of the mature ver­
organic compounds (Raut et al., 2008), was decreased by addition of C- micompost, we assessed the vermicompost quality index (VQI) by
rich additives in early period, but was increased by most C-rich additives integrating key vermicompost quality indicators, for which 28 vermi­
except nanocarbon alone in medium period. However, in late period, it compost quality parameters were used for principal component analysis
was only increased by addition of straw or biochar alone. This illustrates (PCA) analysis (Table 2). In this PCA, the first six PCs had eigenvalues >
that C-rich additives, particularly straw and biochar, first inhibited and 1 and explained 95.04% of the variance in the data. Within each PC
then enhanced microorganisms capable of producing extracellular reserved, the key indicators, which had highly weighted factor loadings
phosphatase during vermicomposting. Furthermore, it means higher (the absolute values were within 10% of the highest factor loading),
available P in the final vermicompost amended with straw and biochar, were retained in the minimum dataset (MDS). Key indicators included in
since phosphatase is able to mineralize organic phosphates into inor­ the MDS were Total B (TB) for PC1, and pH, phosphatase, OTU richness
ganic phosphates. Catalase, which catalyzes the decomposition of hy­ and Chao-1 index for PC2. Within PC3, PC4 and PC5, key indicators
droperoxides and organic peroxides (Jiang et al., 2010), was increased retained in the MDS were urease, catalase and total Fe (TFe), respec­
by addition of C-rich additives in late period. Thus, all C-rich additives tively. In the PC6, total K (TK) and Shannon diversity were found to be
could enhance the growth of catalase-producing microorganisms in the highly weighted and were retained for the MDS. The VQIs for various
final vermicompost to remove hydrogen peroxide. Cellulase, an enzyme treatments are represented in Fig. 5a. The relative order of performance
facilitating cellulose degradation (Raut et al., 2008), was enhanced by of these treatments in aggrading vermicompost quality was CD + S + N
addition of straw in conjunction with nanocarbon in early period and by (1.17) > CD + N (1.02) > CD + B (0.89) > CD + S + B (0.88) > CD
addition of biochar or nanocarbon alone in late period. This implies that (0.72) > CD + S (0.61). This indicates that recalcitrant C-rich materials,
such as biochar and nanocarbon, have the potential to enhance the

Table 2
Results of principal component (PC) analysis of vermicompost quality indicators.
PC1 PC2 PC3 PC4 PC5 PC6

Total eigenvalue 8.11 6.92 4.45 2.90 2.16 2.07

Percent (%) of variance 28.97 24.72 15.88 10.37 7.70 7.41
Cumulative (%) 28.97 53.68 69.56 79.93 87.64 95.04
Eigen vectors
Earthworm growth rate − 0.655 0.651 − 0.018 − 0.307 0.119 0.163
Cocoons/worm − 0.593 0.631 − 0.070 − 0.285 0.309 0.178
EC − 0.802 0.000 0.486 0.063 − 0.202 − 0.125
pH − 0.018 0.816 − 0.171 − 0.116 0.075 0.345
TOC 0.689 − 0.447 − 0.507 0.100 0.201 − 0.093
TN − 0.545 0.410 − 0.021 0.614 0.177 0.333
C/N 0.623 − 0.474 − 0.167 − 0.501 − 0.046 − 0.300
AN 0.545 0.407 − 0.518 0.475 − 0.171 − 0.111
AP − 0.292 0.563 − 0.182 − 0.422 − 0.368 − 0.322
AK 0.744 − 0.154 0.396 − 0.279 − 0.370 0.216
TP − 0.673 0.160 − 0.204 0.554 0.042 − 0.404
TK − 0.781 0.194 − 0.129 0.159 0.056 ¡0.522
TCa 0.429 0.548 0.568 0.121 0.378 − 0.168
TMg 0.390 0.520 0.611 0.166 0.365 − 0.121
TB 0.910 − 0.038 0.220 0.019 0.040 0.197
TCu 0.730 0.501 0.324 0.054 0.039 − 0.182
TFe 0.216 0.424 0.489 0.326 ¡0.572 0.216
TMn 0.240 0.533 0.502 − 0.127 0.498 − 0.359
TZn 0.577 0.606 0.395 − 0.027 0.193 − 0.204
TMo 0.587 0.101 − 0.162 − 0.374 0.225 − 0.097
Urease − 0.581 − 0.172 0.750 − 0.218 − 0.129 0.009
Invertase 0.235 0.474 0.554 0.264 − 0.475 0.323
Phosphatase 0.209 0.774 − 0.497 − 0.099 − 0.210 − 0.187
Catalase − 0.122 0.416 − 0.264 ¡0.715 0.066 0.429
Cellulase 0.751 0.022 − 0.444 0.485 − 0.026 0.035
OTU richness − 0.161 ¡0.778 0.244 0.080 0.438 0.305
Shannon diversity 0.04 0.521 − 0.503 0.211 0.355 0.538
Chao-1 index − 0.09 ¡0.837 0.391 0.157 0.262 0.163

Bold values indicate factor loadings correspond to the indicators included in the minimum dataset (MDS). EC: electrical conductivity; TOC: total organic carbon; TN:
total nitrogen; C/N: the ratio of carbon to nitrogen; AN: available nitrogen; AP: available phosphorus; AK: available potassium; TP: total phosphorus; TK: total po­
tassium; TCa: total calcium; TMg: total magnesium; TB: total boron; TCu: total copper; TFe: total iron; TMn: total manganese; TZn: total zinc; TMo: total molybdenum;
OTU: operational taxonomic units.

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

Fig. 5. Vermicompost quality index (VQI) and plant biomass (a), and the relationships between plant biomass and VQI. In Fig. 5a, CD: cow dung; S: straw; B: biochar;
N: nanocarbon. Bars represent standard errors. The same lowercase letter over each bar represents no significant difference (P > 0.05) between treatments. In Fig. 5b,
the Pearson correlation coefficient (r) is shown with the associated P value.

overall quality of vermicompost. The biochar-induced enhancement in vermicompost (Fig. 5a).

overall vermicompost quality could be explained by the fact that (i) Together, it seems that vermicomposting does not require addition of
biochar enhanced some microbial metabolic functions (e.g. carbohy­ recalcitrant C-rich materials if it is affecting earthworm. From this
drate metabolism) and thereby accelerated humic substance synthesis perspective, since the recalcitrant quality of the biochar and nanocarbon
during the vermicomposting process (Fig. 3; Zhou et al., 2019), and (ii) affects the microbial community of the process, it would be advanta­
the performance of earthworm was improved by biochar combined with geous to use these materials in composting instead of vermicomposting.
straw through increasing earthworm growth rate and cocoon production In particular, biochar and nanocarbon could stimulate the growth of
(Table 1). Interestingly, although addition of straw alone (CD + S) microorganisms (e.g. Bacillus) that has a strong tendency to degrade
resulted in the lowest VQI, addition of straw in combination with complex organic matter. However, the following question remain: Could
nanocarbon (CD + S + N) led to the highest VQI (Fig. 5a). This phe­ vermicompost quality be enhanced by recalcitrant C-rich materials
nomenon could be explained by the great potential of nanocarbon to without affecting earthworm performance? Future studies may be
improve substrate properties (e.g. improving strength, reducing enhanced by exploring the appropriate application time of C-rich ad­
cracking, and lowering the hydraulic conductivity; Alsharef et al., ditives during vermicomposting process. In addition, future studies need
2020). However, this remarkable vermicompost quality seems para­ to address the effects of additives on not only earthworm performance,
doxical given the bad performance of earthworm under the addition of but also microbial activities.
nanocarbon (Table 1). Presumably, nanocarbon might enhance vermi­
compost quality by improving microbial activities rather than earth­ 4. Conclusions
worm performance (Table 1; Figs. 2 and 3). This can be partly supported
by the results reported by Zhao et al. (2014), who found that addition of This study confirmed that recalcitrant C-rich additives (biochar and
nanocarbon significantly decreased the biomass and survival of earth­ nanocarbon) enhanced vermicompost quality primarily through
worm (Eisenia fetida) but increased the shoot dry mass of Festuca arun­ improving microbial activities rather than earthworm performance.
dinacea seedlings. With respect to labile C-rich additive (straw), it enhanced vermicompost
To further evaluate the vermicompost quality based on the agro­ quality by improving not only microbial activities but also earthworm
nomic performance of the mature vermicompost, three test crops of performance. The recalcitrant C-rich materials could be used to improve
lettuce, pakchoi and radish were selected for biological assay (Fig. 5a). the efficiency of composting instead of vermicomposting, if their addi­
Generally, the biomass of all three test crops was increased by addition tion has adverse effects on earthworm.
of biochar alone and straw in combination with biochar or nanocarbon
(compare CD + B, CD + S + B and CD + S + N versus CD). In addition,
the biomass of pakchoi was also increased by addition of nanocarbon Declaration of Competing Interest
alone (compare CD + N versus CD), while biomass of radish was also
increased by addition of straw or nanocarbon (compare CD + S and CD The authors declare that they have no known competing financial
+ N versus CD). Similar to the VQI, the biomass of lettuce and pakchoi interests or personal relationships that could have appeared to influence
generally exhibited the highest value under the combined treatment CD the work reported in this paper.
+ S + N. This result indicates that straw and nanocarbon had compound
effects on vermicompost quality and thereby the agronomic perfor­
Acknowledgments
mance of the mature vermicompost. Indeed, the VQI of the vermicom­
post was significantly and positively correlated with the biomass of the
The authors would like to acknowledge the financial support pro­
three test crops (r = 0.482, P < 0.001; Fig. 5b). Notably, although bio­
vided by the Key Research and Development Program of Ningxia
char or nanocarbon alone had adverse effects on earthworm perfor­
(2020BBF02010), the National Natural Science Foundation of China
mance (Table 1), these two C-rich additives generally had positive
(Project 31760569), and the Helan Mountain Scholars Program of
effects on both VQI and the agronomic performance of the mature
Ningxia University.

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Y. Cao et al. Bioresource Technology 320 (2021) 124404

Appendix A. Supplementary data Pedizzi, C., Noya, I., Sarli, J., Gonzalez-Garcia, S., Lema, J.M., Moreira, M.T.,
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Supplementary data to this article can be found online at https://doi. Qu, J., Zhang, L., Zhang, X., Gao, L., Tian, Y., 2020. Biochar combined with gypsum
org/10.1016/j.biortech.2020.124404. reduces both nitrogen and carbon losses during agricultural waste composting and
enhances overall compost quality by regulating microbial activities and functions.
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