Cow dung and tea waste amendments enhance the biotransformation of textile mill sludge into vermicomposts using Eisenia fetida


 Vermicomposting of textile mill sludge (TMS) with cow dung (CD) and tea waste (TW) as amendments was done in seven different combinations using Eisenia fetida for 90 days. Results revealed that pH decreased from 7.68–8.63 to 7.09–7.59. TOC content and C/N ratio reductions were in range of 15.71–20.08% and 39.33–50.05% respectively (P < 0.05). The macronutrients in the form of TN, TP and TK increased 0.38–0.64 folds, 1.07–2.27 folds and 0.56–1.98 folds respectively after the end of bioconversion process (P < 0.05), among increases in ash content and EC. The biomass and cocoon production of E.fetida increased significantly (P < 0.05), while increasing mortality rate of earthworms was observed in treatments containing 50% or more TMS content. The bacterial population beneficial for degradation of organic matter increased significantly over initial substrates (P < 0.05). Increased humification index in the end product indicated better maturity of vermicompost as observed in treatments containing higher proportions of amendments. The addition of amendments favored earthworm activity which significantly decreased the heavy metal concentration (Fe, Cu, Pb, Zn) in the end product. The study concluded that sustainable utilization of TMS could be achieved for the cleaner and enriched vermicompost production by addition of amendments CD and TW in proportions of 50% and above.


Introduction
Industrial waste management has been a rising concern due to the rapid industrialization and demand driving factors like per capita expenditure and increasing urbanization. The textile industry plays a major role in expanding Indian economy and is the 2nd largest exporter of textiles and clothing in the entire world. The textile exports are expected to reach US$ 82 billion in 2021 from US$ 39 billion in 2019 ( https://www.ibef.org/industry/indian-textiles-and-apparel-industry-analysis-presentation ). The fast fashion, falling prices and disposable clothing have led to a rise in consumerism which require large quantities of oil, water and non-renewable resources for production of synthetic bres and toxic chemicals that are applied onto those bres for dyeing, another water intensive operation. It is estimated that, globally, around 80-100 billion unused garments are sent to land lls every year ( https://textilevaluechain.in/2020/04/10/textile-waste-management/ ).
There are 2324 textile industries in total in India as per Central Pollution Control Board (2007) in which the annual arti cial dye production is about 700,000 tonnes (Robinson et al., 2001). The dyes in the textile industry have been classi ed into ve different groups on the basis of chemical nature -triphenyl methane, azo, polymeric, heterocyclic and reactive dyes (Banat et al., 1996). 830 million m 3 / year of fresh water is utilized by Indian textile industries for different wet operations like bleaching, desizing, dyeing, etc. (Jegatheesan et al., 2016), which expels out colorful e uent at the end. In order to remove color and traces of cotton and dyes, chemicals like ferric chloride, alum, polyelectrolyte and lime are added in E uent Treatment Plants (ETPs), where accumulation of sludge occurs in primary and secondary clari ers (Jahagirdar et al., 2013). The consumption of 50 m 3 /hr of water results in 1-10 ton/day of sludge generation on wet basis (Balan and Monteiro, 2001).This textile mill sludge (TMS) is very troublesome for environment and is composed of complex dyes, perishable organics, surfactants, heavy metals, auxiliaries and polycyclic aromatic hydrocarbons persistent in nature (Liang et al., 2017).
Approximately, 4.65 MT of TMS was produced in China in 2016 as per China Environment Statistical Yearbook (Man et al., 2018). Destructive methods such as open burning, land lling practices and incineration is illegally practiced by industries in management of TMS which results in hazardous air pollution. The stringent waste disposal regulations/ restrictions imposed by the controlling authorities has resulted in textile sludge disposal in open dumps, agricultural elds, along the railway tracks and roadsides and in unorganized land lls, thereby causing public health hazards through soil and ground water contamination. Cost effective technologies are required for the safe disposal of textile industry sludge and usage of nutrients present in it, thus, promoting circular economy and sustainability in the total environment. The textile mill sludge has high organic and nutrient contents and could well be utilized as fertilizer/ soil conditioner in soils with low yielding capacities (Rosa et al., 2007a).
Being a popular beverage, the global consumption of tea is about 3.5 million tonnes per year (Mahaly et al., 2018) and it has been estimated that the black tea and green tea production is expected to reach 3.5MT and 4.14MT in 2027 respectively at an annual growth rate of 7.5% and 2.5% respectively (http://www.fao.org/3/BU642en/bu642en.pdf). Tea waste (TW) is disposed of in larger quantities in the environment, just like TMS. Researchers have used TW in bioremediation processes as novel adsorbent, but only few studies have used it in vermicomposting process (Badhwar et al., 2020;Hussain et al., 2018;Kaur et al., 2014;Mahaly et al., 2018). Cow dung has also been used by most of the researchers as bulking agent and amendment in the process of vermicomposting (Badhwar et al., 2020;Negi and Suthar, 2018;Sharma and Garg, 2020;Suthar, 2010). Badhwar et al. (2020) used major proportions of cow dung (60%) in all the treatment units for the bioconversion of paper mill sludge and tea waste using Eisenia fetida. The amendments, thus, play a crucial role in bioconversion of biosolids such as TMS. The addition of amendments/ bulking substrates is helpful for survival of earthworms as they may not be able to survive the harsh conditions of different raw wastes.
Amendments decrease the harmful effects of substrates that could alter the vermicomposting process in their absence. This study was intended to investigate the effectiveness of both TW and CD as amendments in vermicomposting of TMS.
Vermicomposting is an eco-friendly sustainable method of biotransformation of different organic and nonhazardous agro-industrial wastes. The waste fed to earthworms gets fragmented and aerated after passage through gut of earthworms, and is excreted out in the form of earthworm castings. These castings are rich in macro and micro-nutrients required for plants' growth and conditioning of soil (Lavelle et al., 2006). These castings are formed as a result of biochemical actions like enzymatic digestion, enrichment of nitrogen and transport of organic and inorganic materials, taking place in the earthworm gut in association with microbial action (Edwards and Lofty, 1972). The mutual relationship of earthworms with ingested micro ora results in high enzymic activity and effective degradation of organic matter available in substrates. The selection of species of earthworm is crucial before initiating vermiconversion process. Eisenia fetida is an epigeic earthworm that has good tolerance to different non-hazardous industrial wastes and has been used by various researchers in vermicomposting of wastes such as bakery industry sludge, tannery sludge, pulp and paper sludge, rice straw, etc. (Sharma and Garg, 2018;Ravindran et al., 2008;Yadav and Garg, 2019;Yuvaraj et al., 2020). Vermiculture technology is odourless, cost-effective, sustainable and much preferable than traditional composting in terms of availability of nutrients (Suthar, 2009a).
The biotransformation of different industrial wastes using a variety of earthworms has been reported by researchers in recent past (Bhat et al. 2015;Sharma and Garg, 2020;Negi and Suthar, 2018;Yuvaraj et al. 2018). Vermistabilization of paper mill sludge was done using earthworm species Perionyx excavatus which showed 48.51% reduction of TOC with maximum TP and TK contents of 83.33% and 73.33% respectively in PMS: CD = 1:1, while 100% CD (control mixture) showed maximum TN content of 88.23% (Yuvaraj et al., 2018). Singh et al. (2010a) vermicomposted bio-sludge from beverage industry in a short period of 110 days using Eisenia fetida and concluded that it has to be mixed with cattle dung in 50:50 ratio considering the toxicity of sludge. Vermicomposting of textile mill sludge spiked with horse dung and cow dung using Eisenia fetida resulted in increase in nitrogen and phosphorus contents and decrease of C/N ratio from 66. 1-148.3 to 20.4-26.9 in the nal substrate after six-month pilot-scale study done by Garg et al. (2009). Ravindran et al. (2008) reported increase in earthworm Eisenia fetida biomass from 12.5 to 50 g and maximum decrease of C/N ratio to 15.5 after biotransformation of animal esh (ANFL) generated from tannery industry into nutrient-enriched vermicompost using cow dung and agricultural residues in 3:1:1 mixing ratios in feed mixtures. Therefore, considering leaching and production of primary and secondary pollutants generated from conventional disposal methods of open dumping, land lling and incineration and human-ill effects associated with them, zero-waste technology of vermicomposting is most preferable and feasible method of industrial waste management and regulating recovery of resource from waste. Therefore, the main objectives of this study were (1) to investigate the variation in physico-chemical parameters after addition of cow dung and tea waste as amendments for vermicomposting of textile mill sludge using earthworm Eisenia fetida, (2) evaluate biological growth patterns of earthworms and bacterial population bene cial for the enhanced mineralization rate of substrate, (3) access the e cacy of amendments and earthworms in terms of heavy metal removal and maturity of vermicompost determined by humi cation index.

Collection of earthworms, CD, TMS and TW
The epigeic earthworms were randomly collected from the Water Treatment Plant of college campus. The cow dung (urine-free) was brought from a local shed. The textile mill sludge was collected from Khurana Textile Mills, Ludhiana. The tea waste was collected from all kitchens of college campus and nearby tea stalls. Both TMS and TW were air-dried for 5 days, grinded and sieved through 2mm mesh for further use.

Experimental design
In all, seven different treatment combinations of CD, TMS and TW were set-up on dry weight basis and 5 kg of the nal mixture was made in each treatment. Plastic containers of size 50.8 cm x 34.3 cm x 15.25 cm were used for the study and triplicates of each treatment were prepared for drawing statistical inferences. The following treatments were prepared: Treatment I (T1) -100% CD Treatment II (T2) -75% CD + 25% TMS (3:1) Treatment III (T3) -50% CD + 25% TMS + 25% TW (2:1:1) Treatment IV (T4) -25% CD + 25% TMS + 50% TW (1:1:2) Treatment V (T5) -25% CD + 50% TMS + 25% TW (1:2:1) Treatment VI (T6) -75% TMS + 25% TW (3:1) Treatment VII (T7) -100% TMS The treatment combinations were so prepared to analyze the suitability of TMS in the process of bioconversion, considering the varying C/N ratio of TMS. All the treatments were pre-composted for 15 days by maintaining 70 ± 5% moisture content and turning the mix at regular intervals for proper aeration and degradation mechanism. After 15 days, 30 earthworms E.fetida (average biomass = 287.4 ± 0.57 mg/ worm) were added in each treatment and all the treatments were kept in a dark room at room temperature and 75-85% moisture content. The samples were collected at the 45th day and 90th day of study and were dried for 1 day at 60°C in hot-air oven, before being kept in airtight plastic containers for further testing and analysis of physico-chemical parameters (pH, EC, Ash content, TOC, TN, TP, TK and C/N ratio). The biological properties such as cocoon population, mortality rate and biomass changes of earthworms were also analysed at the end of vermicomposting as suggested by Negi and Suthar (2013).

Physico-chemical analysis
The measurement of pH and EC was done using procedure given by Tandon (2017). For pH analysis, digital pH meter was used. Distilled water aqueous extract of vermicompost (1:10 w/v) was used for pH, while it was ltered using Whatman lter paper No. 42 for EC analysis using digital conductivity meter. Ash content and TOC were determined using Nelson and Sommers method (1982). Macro Kjedahl method by Humphries (1956) was used for TN determination. Tandon (2017) was used to analyze TP (diacid digestion method -9:4 mixture of HNO 3 :HClO 4 ) and TK content (acid digestion method, followed by ame photometry), while C/N ratio was simply obtained by dividing TOC content with TN content of respective samples. The humi cation index was determined according to Zbytniewski and Buszewski (2005). 1 g sample was shaked with 50 ml of 0.5M NaOH for 2 hours, left overnight and centrifuged at 3000 revolutions/minute for 25 minutes using REMI R-8C Laboratory centrifuge. Finally, Agilent Cary 60 UV -VIS spectrophotometer was used to check the absorbance of supernatants at wavelengths 472 nm (A472) and 664 nm (A664). The humi cation index is evaluated by the ratio of A472/A664. Heavy metals (Fe, Cu, Pb, Zn) were determined by employing the method as outlined by Pedersen and van Gestel (2001). 1g sample was digested with HNO 3 and HClO 4 mixture and diluted with deionised water. After ltering with Whatman no.42 lter paper, the supernatant was estimated using Atomic Absorption Spectrophotometer (Agilent Technologies Spectra 240FS AA).

Bene cial bacterial population analysis
Standard pour plate and serial dilution method were used to evaluate the total number of colony forming units (CFU) of nitrogen xing, phosphorus and potassium solubilizing bacteria. The procedure was followed as per Dubey and Maheshwari (2005). For preparation of stock solution, 1 g sample was taken in 10 ml deionised water and serial dilutions up to 10 − 6 were made. 1 ml of aliquot was poured onto plates containing agar media, i.e., Jensen's medium (N 2 -xing), Pikovskaya's agar (P-solubilizing) and Aleksandrow agar (K-solubilizing) respectively.
The plates were then incubated at 30°C for 48 hours. The bacterial population was obtained by multiplication of number of cells per plate with the dilution factor. The results were expressed as CFU/g.

Decomposition rate
The rate of decomposition of substrates is equal to the ratio of reduction in the weight of substrate after the end of study (i.e., after 90 days) to the initial weight of substrate just before the addition of earthworms (i.e., on 0th day) (Goswami and Kalita, 2000).
where, W i = Initial weight of substrates before addition of earthworms and W f = Final weight of substrates after vermicomposting 2.5. Statistical analysis SPSS software version 22.0 was used to evaluate the statistical signi cance between treatment units and their relation to physcio-chemical parameters. Two-way analysis of variance (ANOVA) was done to nd the mean value difference at signi cant level (P 0.05), followed by Tukey's honestly signi cant difference (HSD) multiple comparison test. All the data has been expressed as mean ± standard error of mean (SEM).

Physico-chemical characteristics of raw CD, TMS and TW
The physico-chemical parameters of CD, TMS and TW were presented in Table 1. The TMS and CD were slightly alkaline while TW was acidic in nature. Maximum Ash content was to be seen in TW (87.62 ± 0.3 %), while maximum C/N ratio was seen in TMS (111.48 ± 1.25) and clearly indicates the imbalance of organic matter and nitrogen content. Therefore, CD and TW were added to TMS for ful llment of nutrient content and degradation of organic matter in the process of vermicomposting. Moreover, the compost maturity and biological growth patterns of earthworms were also assessed after addition of amendments. The main characteristics of 'fresh' solid textile mill sludge observed by Kaushik and Garg (2003) were: total solids (192 g/kg); pH (8.4); TOC (138 g/kg); TKN (0.66 g/kg) and C/ N ratio (230). The difference in characteristics in comparison with our study could have been due to textile mill sludge age of both studies.

Decomposition rate (%)
The decomposition rate of all the treatments after 90 days of vermicomposting with E. fetida, was found to have statistically signi cant difference (P 0.05) as shown in Fig. 1. The decreasing order of rate of decomposition was: T1 (61.7%) > T2 (55.8%) > T3 (52.9%) > T4 (48.4%) > T5 (35.7%) > T6 (21.0%) > T7 (7.3%) and clearly depicted unsuitability of higher TMS content treatments (T5, T6 & T7) to earthworms in the degradation process. This could well have been due to toxicity and varying C/N ratio of TMS that earthworms were not able to consume and die. Contrarily, treatments T1, T2, T3 & T4 showed good rate of degradation which can be attributed to higher proportions of amendments in the form of CD and TW. The rate of decomposition is also said to depend on the species of earthworms inoculated in vermicomposting and on the physico-chemicals characteristics of substrates (Prakash and Karmegam, 2010).

Changes in pH and Electrical Conductivity (EC)
In this study, pH of substrates decreased after the end of 90 days vermicomposting period. The pH content signi cantly varied (P 0.05) between 7.68 ± 0.05 to 8.63 ± 0.05 at the 0th day (Table 2) and decreased to 7.54 ± 0.04 to 7.96 ± 0.02 (Table 3) and 7.09 ± 0.03 to 7.59 ± 0.05 (Table 4) after 45 days and 90 days of vermicomposting respectively. The production of humic and fulvic acids from microbial activity during organic matter degradation may have resulted in this decrease (Badhwar et al., 2020;Swarnam et al., 2016). The production of CO 2 as a result of metabolic activities may also have resulted in decrease of pH (Suthar, 2009b (Aira et al., 2006). It was also observed from decreasing rate of ash contents from treatments T2 to T7 that earthworms were not able to stabilize the increasing proportions of TMS to a good effect unlike T1. Khwairakpam and Bhargava (2009a) stated that there is a direct relationship between the ash content and rate of mineralization of the substrates. The results are comparable to Gupta et al. (2007) who vermicomposted water hyacinth and reported 16.5-56.5% increase in ash content.
The biotransformation of TMS using vermicomposting showed a signi cant decrease in the reduction of TOC in TN content in this process was seen from 0.18-1.06% on 0th day (Table 2) to 0.25-1.65% after 90th day (Table 4). Maximum TN content was seen in T1 and decreased with decreasing CD content and increasing TMS contents in treatments. This can be due to the higher TN of CD (Table 1) and mineralization of organics of TMS and TW along with the e cacy of earthworms in consuming CD as depicted by the maximum increase of 55.23% in their biomass (Table 5). Negi and Suthar (2020) have also reported the signi cance of adding CD with organic substrates and other suitable amendments in the bioconversion process and increment of TN content. The increase in TN in vermicomposts is also due to inclusion of mucus, body uid of earthworms, their excretory products and dead earthworm tissues (Bhat et al., 2015). The results of this study are similar to Karwal and Kaushik (2020) who reported maximum increase in TN content in 3:1 combination (Buffalo Dung + Pressmud: Fly ash) after vermicomposting for 90 days.
An increase in TP content of 1.07-2.27 folds was signi cantly seen after the end of study period (P 0.05) and treatment T2 (CD: TMS = 3:1) reported a maximum increase of 1.19 ± 0.03% (Table 4). The metabolism and growth of plants gets augumented with increase in TP content (Singh and Kalamdhad, 2016;Gong et al., 2019).
The enzymic (phosphatase) activity occurring in the earthworm gut with micro ora partially and the simultaneous release of phosphorus in castings of earthworm contribute in enhancement of TP content (Badhwar et al., 2020). Suthar (2006) used Perionyx excavatus for vermicomposting and reported a maximum increase in TP content of 72.8% in combination (guar gum industrial waste + cow dung + saw dust in 3: 1:1) after 150 days. The symbiotic relationship of earthworm's gut micro ora and secreted uids and mucus helps in release of available phosphorus and promotes effective decomposition of ingested substrates (Suthar, 2007).
After 90 days of study period, TK content increased signi cantly from 0.09-0.89% to 0.14-1.98% and treatment T3 recorded the maximum increase of 1.98 folds, while T7 showed minimum increase of 0.56 folds, as compared to initial values (P 0.05); (Table 2, 4). The production of organic acids from earthworm activity results in dissolution of K + ions in the mix, which could have increased the TK content in treatment units (Zhi-wei et al.,

2019
). An increase of 26.3% was also seen in 150 days study of Surendra (2007) who made vermicompost using vegetable waste and leaf litter with Perionyx sansibaricus.
The study done by Suthar (2010) in vermicomposting sugar mill industry sludge with three different amendments (biogas plant slurry, cow dung and wheat straw) also reported increase in TN (215.2%), TP (230.4%) and TK (253.3%). The increase in major nutrients in vermicompost is clearly attributed to the mutual activities performed by earthworms and microorganisms in converting complex organics into soluble forms (Badhwar at al., 2020). Figure 2 indicates the increasing nutrient content of treatment T3 (CD: TMS: TW = 2:1:1) and proved to a suitable combination for use in bioconversion using vermicomposting.

Carbon-Nitrogen balance (C/N ratio)
C/N ratio is a crucial indicator in the assessment of quality and maturity of compost and determines the humi cation rate of organic matter in the process of vermicomposting (Kaushik and Garg, 2003;Sharma and Garg, 2018). C/N ratio showed a signi cant decrease from initial level in all the treatment units (P 0.05) as seen in Tables 2, 3 and 4. Minimum decrease (39.33%) in C/N ratio was seen in T7 due to complete exposure of TMS to earthworms. This clearly indicates the role of amendments and mixing proportions of substrates in balancing the C/N ratio of substrates and higher rate of decomposition. The decrease is generally due to respiration of earthworms and microbes, leading to mineralization of liable organics (Hanc and Chadimova, 2014). C/N ratio in between 25-30 indicates intense stabilization of organic matter; below 20 represents superior quality of compost, while below 15 can be considered for application in agricultural elds (Edwards and Bohlen, 1996;Zhang and Sun, 2014

Growth of E. fetida, cocoon production and mortality rate
The biological properties of Eisenia fetida showed signi cant improvement over its initial values in terms of biomass change and cocoon production (P 0.05) (  (Suthar, 2010). The cocoon production showed good progress with the increasing humic content of vermicompost. Maximum cocoons (33.50 ± 2.50) were seen in T2 followed by T3 (31.00 ± 1.50) and T1 (27.00 ± 1.00), while minimum was recorded in T7 (13.50 ± 0.50). The toxicity levels of different treatment units and varying proportions of amendments added to textile mill sludge may have affected rate of cocoon production. Maximum mortality rate of 44% was also seen in T7. Adverse effects in the survival and growth patterns of earthworms E.fetida was seen with increasing proportions of rice straw in study accomplished by Sharma and Garg (2018). Garg and Kaushik (2005) reported uctuations in production of cocoons with time and, i.e., it increased at the the start but declined after 8th week of vermicomposting. Factors such as porosity and water holding capacity of substrate also contribute to biomass change and earthworm population change.

Bene cial bacterial population
The total bene cial bacterial population of nitrogen xing, phosphorus and potassium solubilizing bacteria showed a signi cant increase in nal substrates (90th day) over initial substrates (0th day) as seen in Table 6 and is indicative of faster mineralization of organic matter (Sharma and Garg, 2018) (P 0.05). The results can be correlated with the ndings of Balachandar (2019) who reported a maximum bacterial population of 1.67 CFU x 10 8 g − 1 after vermicomposting pressmud with cow dung and nitrogenous green manures (Gliricidia sepium and Leucaena leucocephala) using Eudrilus eugeniae. The increase in bacterial population, however, decreased with the increasing TMS content in treatment units as seen in T5, T6 and T7. This clearly indicates the importance of both the amendments CD and TW in this bioconversion process as depicted by treatments T1, T2, T3 and T4 and the enzymic activities performed by earthworms in conjunction with microbes. The change in bacterial population is mostly dependent up on the nature of organic substrate and the symbiotic relationship between earthworms and microbes in the process of vermitechnology.

Humi cation index
The humi cation index of various treatment units for initial and nal substrates is presented graphically in Fig. 3. The changes in concentrations of heavy metals (Fe, Cu, Pb, Zn) was also evaluated and showed a signi cant decrease over initial substrates as seen in Table 7 (P 0.05). This reduction could have been due to the heavy metal accumulation within the gut of earthworms. Samal et al., 2019 stated that heavy metals have strong a nity to metal binding protein (metallothionein) available within the earthworms, which limits heavy metal effect in excreted casts. Suleiman et al., 2017 justi ed this decrease to the proli c defence mechanism of E.fetida who has higher metal accumulation capacity among other earthworm species that helps in effective bioremediation of industrial wastes. The addition of amendments CD and TMS also favored the decrease in heavy metals in this study as depicted by treatments T2, T3, T4 and T5. Some studies have also reported increase in heavy metal concentrations in the nal substrates (Yadav and Garg, 2011;Gong et al., 2019). Gong

Conclusion
The addition of amendments CD and TW (50% or more) in bioconversion of TMS using E.fetida accelerated degradation rate of substrates and enhanced growth patterns of earthworms. The increased bene cial bacterial population and higher humi cation index indicates high rate of organic matter degradation and better vermicompost maturity of the end product. The study observed that higher proportions of TMS (50% and above) hindered the performance of earthworms and increased their mortality rate. The addition of amendments favored the earthworm activity resulted in decrease of heavy metal concentrations in the nal vermicompost. The physicochemical characteristics indicated increased nutrient content and effective stabilization of organic matter of substrate. Therefore, vermicomposting of textile mill sludge can be done by addition of cow dung and tea waste as amending materials for eradication of disposal issues of industrial wastes, enriched vermicompost production and cleaner environment.
* [SEM= Standard error of mean and all the above mean values indicate that difference between treatments is statistically different] (P 0.05). Table 6 Beneficial nitrogen fixing, phosphate and potassium solubilizing bacterial population in different treatments on 0 th day and after 90 days of vermicomposting (Values are mean ± SEM of three replicates).