Vermicomposting of harvested waste biomass of potato crop employing Eisenia fetida: changes in nutrient profile and assessment of the maturity of the end products

The vermicomposting potential of waste biomass of potato crops that are generated at the time of harvesting was studied employing Eisenia fetida. The experiment was carried out in pots, and two treatments were applied during the study. In the first treatment, only potato plant biomass (PPB) was taken as the raw materials; whereas in the second treatment, a mixture of PPB with cow dung was engaged in the proportion of 5:1. The vermicomposted materials showed a reduction in C/N ratio, humification index, enhancement in nutrients profiles, ash contents, nitrogen-fixing, phosphate, and potassium solubilizing bacterial population. The macronutrient enhancement in the vermicompost samples was recorded 3.8–4.4-fold for total N, 5–5.6-fold in available P, 1.6-fold in total K, 5.2–6.2-fold in total Ca, and 1.6-fold in total Mg contents. The reduction in C/N was found in the range of 92.5–94.4% in the vermicompost samples. The scanning electron microscope (SEM) images showed higher disintegration in the vermicompost products when compared with initial raw material and compost samples. The addition of cow dung significantly enhanced the quality and quantity of vermicompost final products besides positively affecting the earthworm population and biomass by the end of 60 days of experimental trials.


Introduction
Potato (Solanum tuberosum) is an important crop under the Solanaceae family and is cultivated in several countries around the globe. It is the world's fourth most important food crop cultivated approximately in 19 million hectares of farmland and annual production of the crop has been recorded as more than 378 million tons globally (Devaux et al. 2020). India is the second-largest producer of potatoes (Scott et al. 2019;Rana and Anwer 2018) where approximately 1500 hectare per three lakh km 2 of land is used for its cultivation. The tuber or underground part of the crop is used for direct consumption or other industrial uses. The above-ground shoot/stem portions along with leaves of the crop that contribute towards the growth of underground tubers stop growing at maturity and die. These above-ground shoot/stem portions or biomass are not consumed by herbivores as it becomes dry or nonpalatable at the time of harvesting. According to an estimate, approximately 887.3 metric tons of dry waste biomass is generated in India per year after harvesting potato tubers (Bisht and Thakur 2019). These huge volumes of waste biomass are either thrown away to the dumping ground, sometimes brunt, or even left in the field for decomposition. Such unscientific methods of waste management contribute significantly towards environmental degradation besides causing loss of nutrients. The burning of any biomass releases both carbon dioxide and monoxide into the environment and causes health hazards and pollution (Cheng et al. 2014). It has been reported that the waste dumping sites are one of the largest sources of global anthropogenic methane emissions (Powell et al. 2016) and unmanaged dumping/landfilling of post-harvest biomass is responsible for the emission of 2.8 metric tons CH 4 /year or approximately 60 metric tons CO 2 -eq per year (Cardoen et al. 2015).
As the above-ground biomass of potato crop has no other use at the time of harvesting (Cardoen et al. 2015), therefore, vermicomposting could be the better option for management of these wastes. Vermicomposting is considered as a superior method among all other composting techniques as it is costeffective, eco-friendly, and very effective for sanitization of solid waste more particularly against the waste of biological origin (Lim et al. 2016;Sahariah et al. 2019). It is a biooxidative process in which earthworms and microbes along with other degradable communities interact and accelerate the decomposition process of organic waste (Patwa et al. 2020). The vermicompost, the end product of vermicomposting process, has been reported for several beneficial effects in the soil such as improvement in physical, chemical, and biological conditions and can be used effectively to achieve sustainable agricultural growth . Besides, vermicompost also enhances health-related secondary metabolites in plants (Das et al. 2018). The application of vermicompost along with other organic amendments significantly improves the radical scavenging and antibacterial activity of the plant's leaf (Das et al. 2016). The environmental impact such as greenhouse gas emission like methane is significantly lower and negligible during vermicomposting as compare to traditional composting (Nigussie et al. 2016). The efficacy of vermicomposting for the management of different types of bio-waste including post-harvest biomass of agricultural fields has already been established by several workers around the globe. For example, the efficacy of vermicomposting has been tested in wheat crop residues (Suthar 2009), rice husk (Lim et al. 2012), citronella bagasse (Boruah et al.2019), paper industry sludge , and many others. However, the success of vermicomposting depends on several factors such as nature, origin, palatability, nutrients profile of substrate materials, type of earthworms, and other environmental parameters that mainly include temperature and moisture. It has been reported that quality and the quantity of vermicompost output entirely depends on the types of waste/ substrate materials and the earthworm species used for the process (Deka et al. 2011a, b). Nevertheless, the reports on the vermicomposting potential of waste biomass of potato crop fields that are generated at the time of harvesting are found to be very scanty. Therefore, an understanding of their use in waste conversion technologies is necessary to provide new insights on sustainable utilization and effective management of waste.
The Eisenia fetida is an epigeic earthworm and vermicomposting potential of the species is well documented in the literature (Boruah et al. 2019). The small size, short life span, high reproductive rate, and wide adaptability of Eisenia fetida makes them highly suitable for use in vermicomposting systems (Saba et al. 2019). The vermicomposting abilities of Eisenia fetida have been studied in wastes of different biological origin such as fruit and vegetable processing waste (Sharma and Garg 2017), cow dung and waste paper mixture (Unuofin and Mnkeni 2014), a mixture of wheat straw, horse and sheep manure (Biabani et al. 2018), and many others. However, studies about the vermicomposting potential of Eisenia fetida on harvested waste biomass of potato crop are still limited.
Considering the above-mentioned facts, the present investigation was taken to study the vermicomposting potential of Eisenia fetida for the conversion of waste biomass of potato crops into a value-added product. The efficacy of the overall vermicomposting process was judged based on the changes in physicochemical, nutrients, and microbial profiles, growth of earthworm, stability parameters such as ash contents, C/N ratio, humification index, and scanning electron microscope (SEM) imaging of the end products.

Materials and methods
Collection of potato plant biomass (PPB), cow dung (CD), and Eisenia fetida The potato plant biomass (PPB) and fresh cow dung (CD) were collected locally from nearby harvested crop field and livestock farm of Gauhati University, Guwahati, Assam, India. For use in the experiment, the PPB was shade dried and cut into small pieces of 5 cm size. The individuals of Eisenia fetida were procured from Krishi Vigyan Kendra, Guwahati, Assam, India. The stock culture of Eisenia fetida was maintained in the laboratory (Boruah et al. 2019).

Experimental design
The experiment was carried out in the pots of 2 L capacity (diameter 17 cm and depth 18.5 cm) in laboratory conditions. A bed (3 cm thick) for earthworm was prepared at the bottom of each pot by putting a small layer of sand and CD over the small bricks and stones. The vermicomposting experiment was conducted employing two treatments, and also three replicas were maintained for each treatment. In one treatment, PPB alone was applied as the substrate material and in the other PPB and CD mixture was engaged in a ratio of 5:1 (Deka et al. 2011a) to ensure the minimum use of CD with PPB. Previous authors have suggested the use of CD with biowaste because it serves as the initial nutrients for earthworms and helps in microbial proliferation and enzyme activities during vermicomposting . In both the treatments, an equal amount of raw materials (100 g on a dry weight basis) was taken and kept for 10 days of predecomposition to avoid the adverse effects of heat released during the early phase of decomposition and also to create a conducive environment for earthworms activity  in the substrate mixture. Then, 30 individuals of Eisenia fetida were introduced to the substrate mixture from the stock culture. A similar control setup without earthworms was maintained for comparison of the results. A periodic sprinkling of water was carried out to maintain the moisture levels at 60-65% in the experimental pots. The average temperature was 30 ± 2°C during the entire 60 days period of experimental trials. The vermicompost outputs as obtained from each treatment were calculated out on a dry weight basis whereas the earthworm population was counted manually and biomass was measured on a fresh weight basis.

Physicochemicals, biological, and nutrient analysis
All the samples of vermicompost, compost, and raw materials were dried and analyzed in triplicate. Although the raw materials (i.e., PPB and PPB + CD mixture) were sample analyzed separately, the results were averaged and presented together. The pH and electrical conductivity (EC) were measured in 1:5 (w/v) water suspension using digital pH (Biochem PM79) and conductivity meter (Systronics 304) respectively. For estimation of ash contents, Nelson and Sommers (1982) method was used. Walkey and Black titration method was employed for the estimation of total organic carbon (TOC) contents of the samples (Jackson 1967). Total Kjeldhal nitrogen (TKN) was determined by the micro Kjeldhal method (Jackson 1967). The C/N values were obtained from the total organic carbon and nitrogen content of the samples. The available phosphorus (AP) was determined spectrophotometrically (Shimadzu UV 1601) following the stannous chloride method (APHA 1998). The total potassium (TK) and sodium (T Na) were determined in a flame photometer employing the acid digestion method (APHA 1998). The humification index was determined by employing the method as outlined by Boruah et al. (2019). The Ca, Mg, Zn, Fe, Cu, Mn, Cd, Ni, Co, and Cr contents were analyzed by atomic absorption spectrophotometer (Shimadzu AA 7000) after acid digestion of the samples. The total population of nitrogen-fixing, phosphorus, and potassium solubilizing bacteria was estimated by the pour plate and serial dilution techniques (Dubey and Maheshwari 2005;Boruah et al. 2019), and the results have been expressed as the number of colony-forming unit per gram (CFU g −1 ). For enumeration of nitrogen fixing bacterial population, Jensen agar media was used. On the other hand, Pikovskya agar and Aleksandrow agar media were used respectively for estimation of phosphorus and potassium solubilizing bacterial population.

Scanning electron microscopy (SEM)
For SEM analysis, the samples of vermicompost, compost, and raw materials were oven-dried at 70 ± 2°C and then powdered. The SEM analysis was performed by putting the samples uniformly over the metallic sample holder aided with a double-sided adhesive carbon tape and then coated with gold using a sputter coater (Bhat et al. 2015). The surface morphology of the samples was recorded at different magnifications and micrographs were obtained (Gemini, Sigma-300 series).

Statistical analysis
SPSS (version 18) was used for statistical analysis of data. Vermicompost production of the two treatments was compared by an independent sample t test (two-tailed, P < 0.05). Differences in physicochemical properties were compared by pair t test. The results of earthworm productivity, microbial population, and humification index were compared by ANOVA, LSD test (P < 0.01).

Results and discussion
Vermicompost production Figure 1 represents the results of vermicompost production from the PPB and PPB + CD mixtures. The vermicompost production was recorded at 47.33% from PPB and 54.11% from PPB + CD mixture indicating a significant enhancement in the outputs after the addition of CD. This result agrees with the previous findings of Mohapatra et al. (2019) who have reported about 39-54% vermicompost production during vermicomposting of paper mill sludge. Several factors are found to be associated with the vermicompost production which includes types, initial population, reproductive and metabolic activities of the earthworms, physicochemical, nutrient composition and amount of resource materials, and finally time duration of experimental periods (Sharma and Garg 2018). Besides, the addition of CD with PPB enhances the palatability of substrate (Negi and Suthar 2018) and gives a better environment for the fecundity of the earthworms (Xie et al. 2016) which ultimately helps in the higher production of vermicompost. Changes in physicochemical profiles (pH, EC, ash, TOC, and Na) The results of physicochemical analysis including pH, EC, ash, TOC, and Na are presented in Table 1. There was a marginal decrease in the pH values towards the neutral range in both compost and vermicompost samples when compared with the initial raw materials. The pH values in the compost samples were found in the range of 6.73-7.11 whereas in the vermicompost samples, it was within 6.63-6.93. The variation in pH values among the different samples was not significant. Earthworms can neutralize the pH level in the end products (i.e., vermicompost). The decrease in the pH level in the vermicompost samples has also been reported by earlier workers (Suthar et al. 2017). The decrease in pH values is associated with the mineralization of nitrogen and phosphorus and production of CO 2 and NH 3 during the vermicomposting process due to the humification process (Cao et al. 2016). Further, the shifting of pH during vermicomposting is a dynamic process and dependent on the initial substrate materials used in the process which can be attributed to the production of different intermediate species of organic acids (Yuvaraj et al. 2019;Karmegam et al. 2019). The pH is an important parameter for determining the quality of compost/ vermicompost, and a pH level near the neutral range indicates the stabilization of the vermicomposting end products (Esmaeili et al. 2020;Pérez-Godínez et al. 2017).
The EC contents have increased significantly in both vermicompost and compost samples as against the initial values of raw materials (Table 1). The increase in EC level was 1.3 fold in compost and 1.9 fold in vermicompost samples. The bioconversion of organically bound nutrients into available forms, the release of different soluble salts, ammonium, and other inorganic ions/compounds have enhanced the EC values in the vermicomposted materials (Lukashe et al. 2019;Karwal and Kaushik 2020). Further, higher contents of EC in the vermicompost samples than the compost counterpart may be attributed to the differences in the rate of mineralization and accumulation of ions (Negi and Suthar 2018). Nevertheless, the EC levels were found within the permissible limits of organic product application and not beyond the safe limits of phytotoxicity (Li et al. 2012).
The loss in TOC contents in the vermicompost samples was found within 71.2-75.6% whereas it was 42.5-46.5% in the case of compost/control. The TOC loss was found greater in the samples of PPB + CD treatment. The C mineralization, humification, and decrease of mass in the substrate materials during composting and vermicomposting cause a reduction in TOC in the final products (Negi and Suthar 2018). Besides, earthworms bring chemical modifications in the substrate materials and provide a favorable environment for the proliferation of microbes (Aira et al. 2007). This joint activity of earthworms and microbes brings higher consumption of organic carbon as their energy source (Devi and Khwairakpam Environ Sci Pollut Res 2020) and causes higher falls in TOC in the vermicomposting systems. Moreover, it has been suggested that earthworms efficiently consume the pre-decomposed waste materials and bring higher decomposition of organic matter which ultimately causes more reduction in TOC in the vermicompost samples (Esmaeili et al. 2020). The present finding shows the conformity with earlier findings (Khaket et al. 2012) who have reported up to 63.73% reduction in TOC values in the vermicompost samples. Besides, a reduction in TOC in the range of 78.5-86% has been reported by Boruah et al. (2019) which can be attributed to the substrate condition that influences the microbial and worm's activities during the vermicomposting process. The compost samples have recorded about 1.3-fold increase in the ash value whereas in the case of vermicompost samples it was found to be 1.6-fold. The addition of CD with raw materials also results in a significant enhancement in ash level in the end products (Table 1). The present finding has shown similarity with the previous works of Devi and Khwairakpam (2020) who have observed the increasing ash contents in the vermicomposted samples of Lantana camara. The degradation of cellulose, hemicelluloses, lignin; release of CO 2 ; and mineralization of the waste materials during vermicomposting enhance the ash values in the final products (Chatterjee et al. 2016).
The Na contents have decreased significantly in the final products and are found in the range of 90.85-110.85 mg/kg and 50.5-60.52 mg/kg in the compost and vermicompost samples respectively. The decrease in Na content in the vermicompost samples has been also reported by the previous workers (Mohapatra et al. 2019) which could be attributed to the absorption of Na by the earthworms during the vermicomposting process. Nevertheless, the possibility of Na volatilization due to the activity of microbes and earthworms during vermicomposting also cannot be ignored.

Changes in macronutrient composition (TKN, AP, TK, Ca, and Mg)
The results of TKN, AP, TK, Ca, and Mg contents are also presented in Table 1. The result showed a 2-fold increase in TKN values in the compost sample. In vermicompost samples, TKN enhancement was found in the range of 3.8-4.4fold by the end of the experimental trials. Moreover, there was a significant enhancement in TKN contents in the vermicompost samples obtained from the PPB + CD mixtures over the samples obtained from PPB only. The nitrogen enhancement in the final products was mainly governed by some gut-associated phenomena of earthworms such as the release of hormones and enzymes during the vermicomposting process (Gómez-Brandón and Domínguez 2014). Besides, earthworm's body fluid, mucus, excretory products, and decaying tissues of the dead earthworms also determined the nitrogen level in the vermicompost products (Bhat et al. 2015). These results agreed on the previous findings where it was reported that enhancement in TKN contents may vary depending on the substrate/raw materials and or other organic amendments used for vermicomposting (Sudkolai and Nourbakhsh 2017;Karmegam et al. 2019). An increase in AP was recorded 3.9-4.4-fold in compost and 5.0-5.6-fold in vermicompost samples than the initial substrate materials. The addition of CD with the PPB has significantly enhanced the AP level in both compost and vermicompost samples. It has been suggested that joint action of earthworms and microbes was responsible for the enhancement of AP in the vermicompost products, and these activities became accelerated in presence of cow dung (Deka et al. 2011a, b). Besides, the higher level of AP in the vermicompost samples could also be attributed to the activities of phosphate solubilizing bacteria (Mupambwa et al. 2016) and the release of fecal phosphatase enzyme by earthworm's gut microbes (Singh and Kalamdhad 2016). The concentration of TK had increased up to 1.6-fold in the vermicompost samples but marginally decreased in the compost than the initial value found in the raw materials. The physical breakdown of substrate materials and release of endogenic and/or exogenic enzymes due to the activities of earthworms and microbes causes a rise in total K in the vermicompost samples . Further, the decrease in TK values in the compost/control sample may be attributed to the poor mineralization of the substrates besides some microbes might utilize the already available potassium for their metabolism. The increase in total Ca was recorded 2.5-fold in compost whereas it was 5.2-6.2-fold in vermicompost samples as against the initial values found in raw materials. Similarly, there was a single fold increase in total Mg in compost samples as against 1.5-fold in the vermicompost materials. The variations in Ca and Mg contents observed in the raw material, compost, and vermicompost samples were statistically significant. These findings can be corroborated with the previous works of Malińska et al. (2016) who have reported that the waste mineralization process during vermicomposting releases the Ca and Mg from bind to free form and enhanced their level in the end products.

Changes in C/N values
The C/N values have declined sharply in both the compost and vermicompost products when compared with the raw material samples (Fig. 2). The decline in C/N values was found in the range of 72.5-73.8% in the compost and 92.5-94.4% in the case of vermicompost samples. The enhanced level of nitrogen and organic matter decomposition during the vermicomposting helps in a higher reduction of C/N values in the end products (Singh and Kumar 2017). The present results are corroborated with the previous findings of

Humification Index (HIX)
The results of HIX are presented in Fig. 3. The average value of humification index in the initial substrate materials was 6.24 ± 0.78, and it was 2.44 ± 0.21 and 2.66 ± 0.3 respectively in the compost samples of PPB and PPB + CD. On the other hand, the HIX values were found to be 1.94 ± 0.4 and 1.61 ± 0.2 in the vermicompost samples of PPB and PPB + CD mixture respectively. Humification index is an indicative parameter for compost/vermicompost stabilization. The joint action of microbes and earthworms are probably responsible for the decomposition and stabilization of waste materials, and this process becomes more accelerated in the presence of CD resulting in a greater reduction of HIX in the vermicompost samples of PPB + CD treatment. The HIX below 5 is an indication of a high level of organic matter humification in waste material (Ravindran et al. 2013).

Scanning electron microscopic (SEM) evaluation
The SEM analysis provides an important insight on changes in the surface morphology of the raw materials during the vermicomposting process. The SEM analysis has been frequently used by researchers to determine the maturity of the composting/vermicomposting end products (Balachandar et al. 2021). The snapshots of SEM analysis showed the compact, robust and folk-like appearance of the initial substrate/ raw materials. On the other hand, there were significant changes in physical appearance in the vermicompost samples ensuring scattered, smaller, and disintegrated structure. A similar pattern of changes in physical appearance with less disintegration was also evident in the compost sample. Lim and Wu (2015) suggested that ingestion of waste materials through earthworm gut resulted in the fine texture/structure of the end products which may give scattered and smaller surface appearance in SEM micrograph. It has also been suggested that earthworms and their gut-associated microbes secret several enzymes to accelerate the biodegradation of substrate materials and bring changes in morphological structure indicating higher maturity of the vermicomposting end products (Srivastava et al. 2020;Balachandar et al. 2021).

Trace elements and heavy metals
The results of trace elements and heavy metals are presented in Table 2. The results showed enhancement in the concentrations of Zn, Fe, Mn, Ni, and Cr in both the compost and vermicompost samples. The increase was found within the range of 1.3-2.6-fold in the vermicompost and 1.05-1.3-fold in the compost samples. Similarly, there was a significant decrease in the Cu, Cd, and Co concentration in vermicompost samples than its initial level found in the raw material samples. The decrease was found in the range of 0.8-2.1-fold in the vermicompost samples whereas in the case of compost samples no significant changes have been observed. Earlier workers have established that the trace elements and heavy metal profile in the vermicomposting products can be diverse (Song et al. 2014), and accordingly, both increases, as well as a decrease in the concentrations of metals and the trace elements, are possible. It has been suggested that composting and vermicomposting enhanced the trace elemental concentrations due to loss of weight and volume in organic matter resulted from decomposition, the release of carbon dioxide, and mineralization processes which usually go faster in presence of earthworms (Malińska et al. 2016). On the other hand, a decrease in metals and trace elements concentration is due to the bioaccumulation of these elements in earthworm tissue (Panday et al. 2014). It has been suggested that earthworms release metallothionein isoform in the intestine and binds the excess metal ions by forming organo-metallic ligands which ultimately inhibits the release of metals during vermiconversion process (Maity et al. 2009;Goswami et al. 2013). Nonetheless, metal concentrations in the vermicompost samples were found within the range of international as well as Indian permissible levels for compost applications in agriculture and horticulture (Mohee and Soobhany 2014;Mandal et al. 2014).

Earthworm population and biomass
The results of earthworms' population and biomass are presented in Table 4. The results showed 1.3-and 1.7-fold increases in earthworm number and biomass in the PPB treatment. The addition of cow dung significantly enhances the earthworm numbers and biomass, and thus PPB + CD treatment recorded a 1.6-fold increase in earthworm numbers and 3.7-fold increase in biomass values over the initial level. The present findings are in agreement with the previous workers (Domínguez et al. 2019). The increase in worm population and biomass is linked with the survival, growth rate, and reproductive potential of the earthworms which again governed by the palatability and quality of the resource materials used in the vermicomposting process (Sharma and Garg 2019). It has been suggested that the substrate materials that contain a sufficient amount of metabolizable organic matter favor the earthworm's growth (Edwards 1988) which justifies the enhanced population as well as biomass in the CD added treatment. Besides, several studies have suggested that an increase in earthworm weight/biomass is associated with the microbial population as earthworms take microbes as their additional food (Bhat et al. 2015).

Conclusion
Vermicomposting of harvested waste biomass of potato crops is feasible by employing Eisenia fetida. The vermicomposting end products are enriched with plant's available macro and micronutrients and found superior to traditional compost. The concentration of toxic metals such as Cr was found within permissible limits in the vermicompost samples. The neutral pH and C/N ratio confirm the use of the vermicompost as a horticultural growing medium. The study brings into light the effective use of vermitechnology for the management of potato plant biomass and also boosts up the marketability of the end products.