Production of nutrient-enriched vermicompost from aquatic macrophytes supplemented with egg shell, bone meal, banana peel, and tea waste: Assessment of nutrient changes, phytotoxicity, and earthworm biodynamics

Vermicompost is an organic fertilizer rich in nutrients, benecial microbes, and plant growth hormones that not only enhances the growth of crops but also contributes to the improvement of the physicochemical and biological properties of the soil. However, its lower nutrient content makes it less preferable among farmers and limits its applicability. The present study, evidently the rst of its kind, was investigated to enrich the nutrient content of vermicompost by supplementing the macrophyte biomass with cow manure and organic nutrient supplements (egg shell, bone meal, banana peel, and tea waste). Results showed an enhanced TKN (2.87%), TP (0.86%), TK (3.74%) and other nutrients in vermicompost amended with cow manure and nutrient supplements. Highest biomass gain (710-782 mg), growth rate (11.83-13.04 mg), and reproduction rate (3.34-3.75 cocoons per worm) was also observed in T2 and T3, indicating that amending bulking agent and nutrient supplements not only enhance the nutrient content of the nal product but also improve overall earthworm activity. The stability and maturity of vermicompost, as indicated by C/N (< 20) and GI (> 80), indicates that vermicompost obtained is suitable for agricultural applications. It is concluded that amendment of cow manure and organic nutrient supplements results in producing mature and nutrient enriched vermicompost suitable for sustainable agricultural production.


Introduction
Vermicomposting is considered an economically viable, socially acceptable, and environment friendly technique that uses earthworms to transform any type of organic waste into highly valuable organic fertilizer (Singh et Gusain and Suthar (2020a) observed that the amendment of cow manure is suitable for the transformation of these weeds into vermicompost using the earthworm Eisenia fetida. Further, the addition of cow manure, at least in small amounts, is essential for the growth and fecundity of earthworms , which aids in the recovery of nutrients from them.
Organic manures, such as farmyard manure, compost, and vermicompost, are utilized as organic fertilizers to improve soil fertility and crop yield. However, the low nutrient content, bulkiness, less availability in the market, lack of awareness among farmers of their bene cial effects, and handling challenges of these organic manures discourage farmers from using them more frequently (Sindhu et al. 2020; Yatoo et al. 2021). The quality and recycling speed of vermicompost can be improved with the incorporation of different organic substrates such as banana peel, egg shell, bone meal, and certain microbial inoculants as well as by maintaining optimum temperature, pH, and moisture (Yatoo et al. 2020). Various researchers have investigated the possibility of augmenting vermicomposts with additional nutrient rich organic and inorganic materials as a remedy to the poor nutrient content of organic fertilizers. To enrich, researchers have added organic materials such as green manure plants (Balachandar et al. 2020;Karrmegam et al. 2021), biofertilizer microbes (Karmegam and Rajasekar 2012), and inorganic materials like rock phosphate (Unuo n and Mnkeni 2014), and y ash (Bhattacharya et al. 2012; Mupambwa et al. 2015). However, mixing organic with inorganic substrates has been observed to cause a general decrease in microbial activity, which could jeopardize the e cacy of the vermidegradation process (Mupambwa and Mnkeni 2018).
Nutrient recovery from organic wastes such as kitchen trash, agricultural waste, and municipal waste is critical for waste management and environmental protection (Yatoo et al. 2020). However, when these organic wastes are inappropriately disposed of, signi cant amounts of nutrients stored in them are lost or diminished (Soobhany 2019). These essential nutrients might be recovered via vermitechnology and used as nutrient-rich fertilizers in agricultural elds to improve soil fertility.
Eggshells, a vital source of calcium, are frequently thrown out as waste from households, hotels, and other establishments (Gaonkar and Chakraborty 2016). When this calcium-rich substrate is applied to the soil, it not only nourishes the soil with calcium, but also increases its pH (Gaonkar and Chakraborty 2016). In the recent years, with the improvement of revenue and dietary acceptance, increasing egg consumption is noticed as eggs are recognized as high quality protein source. For the year 2018, global egg production was 78 million metric tonnes (MMT), resulting in roughly 8.58 MMT of eggshells, which are typically deposited in land lls as waste, leading numerous environmental problems and therefore should be carefully managed (Waheed et al. 2019). Another important biowaste is banana peel, which, if not adequately managed, can pollute the environment (Budhalakoti 2019). Potassium (9.39 percent of DW) is the most abundant element in banana peel, followed by magnesium, calcium, sodium, and other minerals (Aboul-Enein et al. 2016). Traditionally, banana peel has been utilized as a fertilizer by simply decomposing it to improve soil nutrients. Kalemelawa et al. (2012) found increased levels of K and N in compost amended with banana peel, indicating that banana peel has a strong potential as a source of K and N. Tea waste is another important biowaste that is commonly thrown in open areas after tea processing, where it contributes to toxic gases, soil, water pollution, and unpleasant surroundings (Khayum et al. 2018;Cooper et al. 2011). Recently, Bhuvaneswari et al. (2021) have reported that tea waste can be managed by vermicomposting and have shown an improvement in nitrogen content. Another signi cant organic resource and a key source of phosphorus, i.e. bone meal, is also discarded as waste. Because of the scarcity of the P reserves based on rock phosphate, it is critical to seek out organic sources of phosphorus. Mäkelä et al. (2020) reported that organic P sources like bone meal is a potential alternative to arti cial P fertilizers because they can induce functional mycorrhizal symbiosis with roots, which improves P absorption and utilization e ciency in crops. Baker et al. (1989) also con rmed via a greenhouse experiment that bonemeal is a better source of P than regularly used rock phosphate. Thus, vermicomposting of organic waste at the point of generation should be given top priority in handling the waste, which will help in reducing transportation costs, disease transmission risks, GHG emissions, water pollution, and land space for dumping, besides producing organic fertilizer that can be utilized as a soil supplement (Yatoo et al. 2020).
Hence, the main aim of the current study was to produce enriched vermicompost from free-oating macrophytes by amending it with easily available kitchen waste such as tea waste (source of nitrogen), banana peel (source of potassium), and bone meal (source of phosphorus). All the major nutrients have been targeted. The study might provide a strategy to utilize kitchen waste to produce useful enriched vermicompost in order to achieve sustainable agriculture while at the same time avoiding organic wasterelated environmental issues in an eco-friendly way.

Collection of feedstocks and earthworms for vermicomposting
The earthworm species Eisenia fetida was obtained from the vermicomposting unit of the Soil Science Department, Sher-e-Kashmir University of Agricultural Science and Technology, Wadoora Sopore, Jammu and Kashmir, India. Eisenia fetida was selected because of its high speed of bioconversion, wide range of temperature tolerance, high reproduction rate, etc. The earthworm was mass cultured in a plastic bin using partially decomposed cow manure as a culturing medium and then used for vermicomposting studies.
Macrophytes (Floating i.e., Azolla, Lemna, and Salvinia), which are abundantly present in the lakes of Kashmir, were collected form the famous Dal lake, Jammu and Kashmir. After collection, they were transferred to the lab, where they were washed with tap water to remove mud and other undesirable items, and nally transferred to the vermiculture unit. Macrophytes were then shade dried for one week, as they contain signi cant amount of water, to make them suitable for vermicomposting. One-week old cow manure, obtained from a local farmyard, was used as a bulking agent as it decreases the toxicity of feeding substrate, makes it more feasible for earthworms and enhances the decomposition rate of organic waste (Yuvaraj et al. 2019). For nutrient enrichment, different organic substrates, i. e. banana peel, bone meal, egg shell, and tea waste, were obtained from the university cafeteria (Kashmir University) and boys' hostel (Zadibal, Srinagar). The physico-chemical characteristics of different feeding substrates like cow manure, tea waste, egg shell and banana peel, used in vermicomposting, are presented in table 1.

Experimental setup and vermicomposting
To investigate the effect of cow manure and nutrient supplements on the nutrient and earthworm dynamics, three treatments were prepared: T1, T2 and T3. Each experiment was carried out in triplicates in plastic bins having dimensions of 56.5 x 39.5 x 16.5 cm, under dark room conditions. All waste mixtures were added adequate amount of water and left for three weeks to pre-decompose. Pre-decomposition is recommended since it makes the substrate more palatable for earthworms and leads to the release of heat and toxic gases (Sharma and Garg 2019), which could be toxic to earthworms. During pre-decomposition, waste mixtures were remixed and turned several times to disseminate more heat and toxic gases and ensure uniform withering of the substrate. After pre-decomposition, 20 non-clitellated earthworms with an average individual weight ranging from 154-163 mgs were collected from the stock culture and introduced into each vermibed. To maintain moisture and aeration, prevent earthworms from escaping, and protect them from predators like ants and rats, each vermibin was covered with perforated plyboard (Fig. 1). Perforated plyboards also provided a dark environment inside the vermibins, as earthworms prefer a dark environment because they are photosensitive. Throughout the experimentation period (60 days), appropriate moisture (60-70%) was maintained by sprinkling an adequate quantity of tap water.

Physico-chemical analysis of initial substrates and nal vermicompost
The initial substrates and nal vermicomposts from different vermibeds were analyzed for pH, EC, total organic carbon (TOC), total Kjeldahl nitrogen (TKN), total phosphorous (TP), total potassium (TK), sodium (Na), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) using standard methods. For pH and EC, a substrate to water ratio of 1:10 (w/v) was used. Five grams of sample were mixed with 50 ml of distilled water and kept on shaker for 45 minutes for proper mixing. The mixture was then ltered through Whatman lter paper, and the pH and EC were determined using a digital pH and conductivity meter. Total Kjeldahl nitrogen (TKN) was measured by the standard method of Kjeldahl digestion and distillation using Kjeldahl apparatus (Kelplus) (Tandon 2005). Total organic carbon (TOC) was estimated by loss on ignition method as described by Tandon (2005). Total potassium (K) and sodium (Na) were estimated using a ame photometer, whereas total phosphorus (P) was estimated using the vanado-molybdate method (Tandon 2005). The concentrations of Cu, Zn, Fe, and Mn were analyzed using the Atomic Absorption Spectrophotometric method. The C/N ratio was calculated by dividing the TOC by the total nitrogen content. All reagents and chemicals used during analytical work were of AR grade. The physico-chemical characteristics of initial substrates and nal vermicomposts are presented in tables 1 and 2.

Growth and reproduction of earthworms
For individual biomass gain of the earthworms, the clitellated worms were carefully taken out of the different vermibeds, washed with tap water, blotted with lter paper, and weighed with a digital electronic balance. The earthworm biomass was recorded from the start of the experiment up to 60 days. After measuring the biomass, earthworms were promptly reintroduced into the respective bins. For the reproduction of earthworms, cocoons and juveniles produced were counted from each vermibed at the end of the experiment. On the basis of the data obtained on the biomass, cocoons, and juveniles produced, other parameters like maximum biomass achieved, net biomass gain, growth rate (mg worm -1 day -1 ), reproduction (cocoons worm -1 ), and fecundity rate (juveniles' worm -1 ) were also calculated for different vermibeds. During the experimentation period, earthworm mortality was also calculated.

Phytotoxicity test using seed bioassay
Seed bioassay test was used to screen the phytotoxicity of vermicomposted materials using Fenugreek (Mithi) seeds. Vermicompost was collected from different vermibins and mixed with soil in a 1:1 ratio and taken in rectangular plastic bins. Garden soil was taken as an experimental control. In each setup, 21 fenugreek seeds were sown, irrigated with tap water, and maintained in the dark for seven days. All setups were kept in triplicates. After one week, the germination index (GI) was calculated using the relative seed germination (RSG) and relative root growth (RRG) values following the method of Zucconi et al. (1981).

Statistical analysis
The results were represented as mean ± standard deviation (SD). The mean and standard deviation for each parameter was calculated using the data of all the three replicates. Statistical software (SPSS, Version 22) was used to analyze the collected data. Analysis of variance (ANOVA) was also performed to determine whether there was a signi cant difference between different treatments for various physicochemical, germination index, earthworm growth, mortality, and fecundity parameters. Tukey's HSD multiple comparison test was used at the P < 0.05 signi cance level where there was a signi cant difference.

Physicochemical characteristics
The nal vermicomposts produced from different biowaste mixtures were stable and rich in nutrients, owing to the collaborative activity of earthworms and microorganisms. Table 2 displays the physicochemical characteristics of nal vermicomposts.

pH
The addition of cow manure maintains a pH that is conducive to earthworms and microbial activity. The pH of the nal vermicomposts after successful vermicomposting of 60 days was found in the neutral range (7.65-7.98). The neutral pH range was also found in earlier studies (Singh and Kumar 2017; Biruntha et al. 2020; Devi and Khwairakpam 2020). The maximum pH of 7.98 was found in T3, which was higher than T2 (7.81) and T1 (7.65) ( Table 2). The increase of pH in T3 was about 4.31% as compared to T1, which might be attributed to the addition of eggshells and cow dung. Eggshells have an alkaline pH and are often recommended for use in low pH soils (acidic soils). Gong
Regardless of the initial levels, T3 had the highest percentage of TKN, which could be attributed to the addition of tea waste and banana peel, both of which contain considerable amounts of nitrogen (Table  1). Other researchers have also claimed that the ultimate nitrogen concentration is often determined by the initial nitrogen content of the raw materials used for vermicomposting ( Fig. 2B), which was about 17.81% higher than T1, possibly due to the high phosphorus content of bone meal. Bone meal contains a signi cant amount of phosphorus and is considered a good source of phosphorus. Cow manure amendment was also responsible for higher TP content in T3 and T2 compared to T1, as cow manure, in addition to being a source of nutrients, aids in the proliferation of microorganisms involved in

Total Potassium
After 60 days of vermicomposting, total potassium was found in the range of 3.13-3.74% (  T1, T2, and T3, respectively, with a signi cant variation among different treatments (ANOVA; F = 5.8568, P < 0.05). The ash content increases during vermicomposting because the volatile fraction of organic substrate is released, leaving behind mineral residues that re ect the ash content (Singh and Kalamdhad 2016). The ash content was higher (13.09%) in T3 compared to T1, indicating nutrient enhancement and higher decomposition, which could be due to the addition of cow manure and other nutrient supplements that aid in the quick mineralization of organic wastes by microorganisms and earthworms. Better feasibility of feeding substrate leads to enhanced organic waste consumption by earthworms, which decreases TOC and increases ash content in the nal vermicompost (Devi and Khwairakpam 2021).

C/N ratio
The C/N ratio is an important indicator that signi es the vermicompost's maturity and stability. The C/N ratio in all the treatments was < 20, which is the desired value in vermicompost for soil applications (CPHEEO 2016). The difference in C/N ratio was highly signi cant (ANOVA; F= 44.6269, P = 0.0002; Tukey's HSD test, P < 0.05) across all treatments owing to the rate of microbial activity and earthworm assimilation, which is dependent on the appropriateness of food and a suitable C/N ratio in the feeding substrate. T3 had the lowest C/N ratio of 12.94, followed by T2 (15.50) and T1 (17.13) ( Table 2, Fig. 2D), indicating that the addition of bulking agent and nutrient supplements is important factor for the nal C/N ratio. Since the C/N ratio is calculated by dividing the carbon content with the nitrogen content of the same substrate, supplementing feeding substrates with high nitrogen content also reduces the C/N ratio.
The results were in consideration with Ananthavalli et al. (2019), who reported a C/N ratio of below 20 in sea-weed vermicompost when amended with cow manure. Biruntha et al. (2020) also suggested the inclusion of an appropriate bulking agent, especially cow manure, to reduce the C/N ratio, since a greater C/N ratio is not conducive to effective earthworm and microbial activity.

2 Mg, Na other micro-nutrients
Plants require micronutrients, like macronutrients, for their overall growth and yield. As a result, the presence of essential micronutrients in su cient quantity in vermicompost assures that it is suitable for agronomic use, allowing plants to grow and develop more effectively. One of the primary goals was also to enrich the micronutrient content of the nal vermicompost product as a result of the inclusion of various supplements.
The content of Mg among different treatments was observed in the range of 0.48-0.55%. A statistically signi cant variation was found among all the treatments (ANOVA; F = 6.5000, P < 0.05). Na was found to be 0. Organic waste mineralization, biomass volume reduction, which concentrates the metal levels, and the inclusion of a bulking agent are the variables that result in an enhancement in micro-nutrients in the nished product Rai et al. 2021). During vermicomposting, earthworms mineralize organic substrate, releasing organically bound metals in free forms (Song et al. 2014). Decomposition of organic waste also generates humic acids, which bind the metals even more tightly, reducing the risk of metal loss by leaching or bioaccumulation (Gusain and Suthar 2020a).
Taken together, the results of the present study incorporating cow manure and other organic nutrient supplements with aquatic macrophytes/wastes showed higher macro-and micro-nutrient contents, suggesting that the addition of organic nutrient supplements not only improves the nutritional status of vermicompost but also makes other parameters like C/N ratio, pH, EC, etc., desirable, which are required for a fertilizer to be used for agronomic purposes.  (Table 3). As shown in Table 3 for Eisenia fetida when organic wastes were spiked with cow manure and green manure plants, signifying that incorporating cow manure and other nutrient supplements not only enhances the quality of nal vermicompost but also supports earthworm biomass and fecundity. High protein and starch content in macrophytes (Gaur and Suthar 2017; Gusain and Suthar 2017), favorable C/N ratio by cow manure amendment, and high mineral content from bone meal, banana peel, and tea waste might have boosted the weight gain in Eisenia fetida.

GROWTH RATE
In all of the treatments, the growth rate exhibited an increasing trend, with the maximum growth rate in T2 (13.04 mg/worm/day), which was signi cantly (ANOVA, F = 23.039, P < 0.05) higher than T3 (11.83 mg/worm/day) and T1 (10.96 mg/worm/day). The maximum growth rate (13.04 mg/worm/day) in Eisenia fetida was higher than previous reports on vermicomposting of different organic wastes by

Reproduction rate
The ability of earthworms to reproduce successfully in a vermicomposting system indicates the viability of the feedstock for earthworm proliferation. After 60 days, the cocoon production rate of earthworms cultured in different setups ranged from 36.33 to 70.00. The highest number of cocoons were observed in T2 (70.00) containing macrophytes and cow manure, followed by T3 (48.0) and T1 (36.33) (Table 3. Fig.  3B). It is clear that the earthworm's growth and reproduction rates were signi cantly (ANOVA, P < 0.05) lower in T1 (macrophytes only), which might be attributed to the presence of secondary metabolites in macrophytes such as phenolics, alkaloids, and other compounds that make it less feasible for earthworms. When secondary metabolites are present in animal feed, it causes loss of appetite, lowers animal performance, reduces dry matter intake, and reduces nutrient digestibility in animals (Attia-Ismail 2005; 2015). Hendriksen (1990) also reported that earthworms prefer low polyphenol content plant food if accessible, implying that they have some detrimental impact on them. The results showed that while all waste mixtures promoted earthworm growth and reproduction, the addition of bulking agent like cow manure and/or other nutritional supplements signi cantly enhanced the growth and fecundity rate of Eisenia fetida, which might be owing to the feeding substrate's higher nutritional content and ideal C/N ratio. Biruntha  Similarly, the highest fecundity rate (cocoons worm −1 ) was observed in T2 (3.75 cocoons worm −1 ), followed by T3 (3.34 cocoons worm −1 ), and T1 (2.15 cocoons worm −1 ) (

Population buildup
At the end of the experiment, the highest population of E. fetida was found in T2 (45.33/vermibin), followed by T3 (31.33/vermibin) and T1 (30.67/vermibin). The results clearly suggest that all waste mixtures supported earthworm growth and fecundity, however, supplementing green waste with bulking agent and other nutrient supplements enhanced the overall population buildup of earthworms. The nal population build-up in vermi-setups is in uenced by hatchling success (Fig. 4), cocoon viability, and fecundity rate (Suthar et al. 2018). Earthworm mortality during vermicomposting also has a signi cant effect on the overall earthworm population buildup. A similar population build-up (35-50/vermibin) after 60 days of vermicomposting of forest litter waste with E. fetida was observed by Suthar and Griola (2014).

Mortality
The mortality of earthworms during the transformation of any waste into a stable end product is one of the most signi cant metrics that determines the appropriateness of the feeding substrate. Mortality usually occurs during the rst few weeks of vermicomposting of organic wastes because of lower microbial activity, low moisture retention, high C/N ratio of fresh feeding substrate, and non-availability of more pre-decomposed food preferred by earthworms. During the current study, signi cant mortality was detected with T2 having the lowest mortality rate of (6.67 %) which was statistically signi cant (ANOVA; F = 64.5000, P < 0.001; Tukey's HSD test, P < 0.05) than T1 and T3 (Table 3, Fig. 3D). In T3 (28.33%) overall mortality was observed, which could be attributed to the release of certain toxic metals during the decomposition of various feeding substrates. The high biomass residue might also be a potential factor for higher mortality in T3, since it is good to underfeed the worms but detrimental to overfeed the worms.
When more organic waste is introduced to the vermibin, earthworms are unable to convert it into a stable product, and anaerobic decomposition ensues, resulting in the emission of poisonous gases such as ammonia, methane, and nitric oxide, causing earthworm death (Negi and Suthar 2013 Zucconi et al. (1981). During the current study, the GI (%) of fenugreek seeds (Methi) in ready vermicompost collected from all treatments ranged from 95.61-110.08% (Fig. 5), indicating that the range was within the safe limit (> 80). The GI in T3 was signi cantly (ANOVA; F = 5.7386, P < 0.05) higher than T1 and T2 which might be attributed to the higher nutrient content of amended bulking agent and nutrient supplements. The results of GI showed that vermicomposting makes the aquatic weed biomass suitable for agricultural purposes. A recent study found that earthworms reduce the phytotoxicity of organic wastes during vermicomposting by depleting

Conclusion
The results of the present ndings signify that macrophyte biomass, in combination with cow manure and nutrient supplements can be utilized to produce enriched vermicompost with various environmental bene ts. The highest percentage of TKN, TP, TK and other micronutrients observed in treatment T3 suggested that amending macrophytes with suitable bulking agent and nutrient supplements aids in improving the overall quality of vermicompost. All treatments promoted earthworm growth and reproduction; however, the treatment containing only macrophytes had the lowest growth and reproduction rate, indicating that amendment of cow manure and other bulking agents not only supports earthworm activity but also reduces the toxicity of feeding substrates. Taken together, the study concludes that vermicomposting is a feasible approach for the management of macrophytes and that incorporation of cow manure and organic nutrient supplements is strongly recommended to accelerate the vermicomposting process and produce nutrient enriched vermicompost suitable for sustainable agricultural production.
Declarations Figure 1 Vermibins were covered with perforated plyboards to maintain moisture, aeration, and prevent earthworms from escaping out. The perforated plyboards also provided a dark environment for earthworms as they are photosensitive. Tukey's HSD test at P < 0.05).

Figure 3
Individual biomass gain (A), total cocoon production (B), cocoons per worm (C), and mortality (%) (D) of Eisenia fetida after vermicomposting of macrophytes amended with cow manure and nutrient supplements. Values are the mean of three replicates, and bars indicate standard error (SD). The different letters between treatments denote statistical signi cance (ANOVA; Tukey's HSD test at P < 0.05).