The application of vermifiltration could reduce the load of chemical and biological pollutants present in wastewater, reducing the pressure over water requirements and allowing the reclamation of the treated water. In the present study, vermifiltration has shown a great potential for chemical pollutants and pathogen removal in wastewater through the synergistic interactions of earthworms and microorganisms. The results of a pilot-scale study showed a higher percentage removal of biochemical oxygen demand (88%), chemical oxygen demand (78%), total suspended solids (83%) and log removal of fecal coliforms (2.61), fecal streptococci (2.50), Salmonella (2.20) and Escherichia coli (2.48) to the levels considered acceptable for reuse in irrigation purposes. Specifically, earthworms in the vermifilter were able to transform insoluble organic material to soluble form followed by selective digestion of the material to finer size, and further degradation by the microorganisms in the reactor. In vitro antimicrobial assay tests also showed that the present microflora had strong inhibitory efficiency against Staphylococcus aureus, E. coli, Pseudomonas aeruginosa and Klebsiella aerogenes. The observed inhibitory effect was found to be responsible for the phenomenon mentioned above, with release of antimicrobial substances by earthworms and associated microflora that showed antimicrobial potency against pathogenic bacteria. The kinetics evaluation showed the predominance of a first order removal model during vermifiltration.

INTRODUCTION

In recent years, the magnitude of the problem of sewage treatment and management has increased due to augmenting industrialization and urbanization. The main problem of pollution is excessive sewage generation and its discharge in the nearby water bodies (Belmont et al. 2004). Available water resources in developing regions are becoming increasingly scarce because of increasing population, changing precipitation patterns and degradation of existing sources of water. In order to address these issues, wastewater is now being looked on as a resource of water and energy, rather than a waste (Jadhav et al. 2014). The reuse of treated wastewater is an alternative resource of water, since freshwater scarcity and lack of access to safe water is a huge problem today (García-Fernández et al. 2015). Water recycling is reusing treated wastewater for beneficial purposes such as agricultural irrigation, and offers resources and financial savings. Wastewater must be treated before discharge or for reuse as it may contain industrial and agricultural chemical pollutants and also a wide range of pathogens, i.e. bacteria, viruses and fungi. Wastewater treatment can be tailored to meet the water quality requirements for a planned reuse, which includes the treatment of wastewater in an environmentally sound and economically effective manner (Belmont et al. 2004). Consequently it is necessary to seek a low cost, decentralized and sustainable technology that addresses the treatment and management of wastewater. One such approach is vermifiltration, which is a biotechnological process that enables the treatment of wastewater into nutrient rich, pathogen free effluent through the combined action of earthworms and microorganisms.

The treatment of wastewater utilizing earthworms was first suggested over 20 years ago due to its ability to biodegrade a variety of organic materials. The late Prof. Jose Toha at the University of Chile first described the utilization of earthworms for wastewater treatment, which has been termed vermifiltration, in 1992. It is one of the most promising technologies, which has gained widespread popularity over the last decade, for wastewater treatment. Vermifiltration is a comparatively new technology for wastewater treatment, which adopts a modern concept of ecological design and extends the existing chain of microbial metabolism by introducing earthworms (Zhao et al. 2010). Vermifilters (VFs) are an engineered wastewater treatment system that encompasses different treatment modules including biological, chemical and physical processes, and this technology has been successfully used to mitigate environmental pollution by removing a wide variety of pollutants from wastewater. One of the major barriers to water treatment and reuse is apprehension regarding the health hazard of wastewater exposure to the public. Out of all the contaminants in wastewater, pathogens are of major concern because of their ability to cause diseases in humans. Human pathogens are typically present in domestic sewage and need to be removed during wastewater treatment (Arias et al. 2003). Pathogens, being natural hazards to the well-being of humans, cause infectious diseases. One of the important goals of vermifiltration is the inactivation of pathogens during treatment to minimize the public health risks associated with reclaimed water (Li et al. 2013a, 2013b).

A number of earlier studies have demonstrated the capability and applicability of VFs for organics, suspended solids (SS), nutrients and heavy metals removal. Different lab- to pilot-scale studies have shown the stabilization of wastewater and sludge from wastewater treatment plants (Sinha et al. 2008; Li et al. 2009; Xing et al. 2010; Wang et al. 2011; Kumar et al. 2014). Previous studies by Arora and co-workers focus on the treatment efficacy and microbial community dynamics of VFs for wastewater treatment, and use of VFs for removal of pathogens, bacteria, fungi and actinomycetes populations. These studies demonstrated the benefits of using VFs for disinfection of domestic wastewater polluted with pathogens, and explained the possible mechanism behind the removal (Arora et al. 2014a, 2014b, 2014c). Given the importance of vermifiltration, to our knowledge there has been no research done on the disinfection potential and kinetics evaluation of VFs for wastewater treatment. This contribution will experimentally undertake this aspect at a pilot scale.

This study is the first to unveil the dynamics of complex kinetics evaluation of microbial communities in a VF undergoing different stages of treatment as well as in biofilms associated with treatment steps in vermifiltration. The aim of the present study is to assess the performance and pathogen removal efficiency of a pilot scale VF, followed by antimicrobial activity assay of the isolated microflora from the VF. The assessment includes the continuous monitoring of physico-chemical and microbiological parameters, along with the kinetics evaluation of microbial communities.

MATERIALS AND METHODS

Reactor set-up

A pilot scale reactor made up of plastic material, was set up in the Environmental Engineering Laboratory at the Department of Civil Engineering, Indian Institute of Technology Roorkee, India. The schematic diagram of the reactor is shown in Figure 1. The reactor was divided into two chambers; one being a VF with earthworms and another being a geofilter (GF) without earthworms (as a control reactor). It consisted of filter bed material (river-bed material), a wastewater storage tank, a wastewater distribution system and an effluent collection system. Both the chambers were 80 cm long and 40 cm wide with a depth of 80 cm, and had 70 cm of packed bedding of different filter materials. At the top, an empty space of 10 cm was kept for aeration purposes and a porous fiber cloth material was placed for uniform distribution of wastewater to the VF. The filter bed consisted of four layers. The description of the filter bed layers is illustrated in Table 1. The first layer consisted of mature vermigratings with a 20 cm depth. The second layer consisted of sand (1–2 mm) filled to the depth of 20 cm. These 2 layers comprised the active layer, where earthworm sp. Eisenia fetida was inoculated at 15,000 worms/m2 stocking density. The third and fourth layers consisted of gravels of size 4–6 mm and 12–14 mm, respectively, to a depth of 15 cm each. These two layers were the supporting layers. The pump and constant head tank were installed to collect and transfer the influent (domestic sewage) to the reactor. Municipal wastewater was collected from a nearby sewage pumping station and stored in the wastewater tank. The distribution system collected the wastewater from the tank and distributed it uniformly over the VF at a hydraulic loading rate of 1.5 m3/m2/d continuously. Based on the flow rate and reactor configurations, the hydraulic retention time of the reactor was found to be 3.5 h. The effluent (treated water) was collected from the bottom in the effluent collection tank.
Table 1

Description of filter bed layers

Layers (from the top)Filter materialParticle sizePorosityDepth
Layer 1 Active layer Vermicasts + Mature vermigratings 600–800 μm 60% 20 cm 
Layer 2 Second layer Coarse sand 1–2 mm 35% 20 cm 
Layer 3 Third layer Small Gravels 4–6 mm 40% 15 cm 
Layer 4 Supporting layer Large Gravels 12–14 mm 43% 15 cm 
Empty space     10 cm 
Total depth     80 cm 
Layers (from the top)Filter materialParticle sizePorosityDepth
Layer 1 Active layer Vermicasts + Mature vermigratings 600–800 μm 60% 20 cm 
Layer 2 Second layer Coarse sand 1–2 mm 35% 20 cm 
Layer 3 Third layer Small Gravels 4–6 mm 40% 15 cm 
Layer 4 Supporting layer Large Gravels 12–14 mm 43% 15 cm 
Empty space     10 cm 
Total depth     80 cm 
Figure 1

Schematic diagram of a pilot-scale VF.

Figure 1

Schematic diagram of a pilot-scale VF.

Analysis

The sampling frequency was twice a week. The influent and effluent samples were collected in sterile plastic bottles and analyzed within 6 h for microbiological parameters and 24 h for other chemical parameters. The pH, temperature and dissolved oxygen (DO) of samples were measured daily in situ using a Hach multi-parameter kit (Hach, USA). Biochemical oxygen demand (BOD) was measured by the azide modification method and chemical oxygen demand (COD) was measured using the potassium dichromate method (American Public Health Association [APHA] 2005). Total coliforms (TC), fecal coliforms (FC), and fecal streptococci (FS) were evaluated using Lauryl Tryptose broth, A1 broth, and Azide Dextrose broth, respectively (APHA 2005). The population of Escherichia coli was enumerated by standard plate count on MacConkey agar media after incubation for 24–48 h at 37 °C (Bhatia et al. 2012). The population of Salmonella was enumerated on the plates of Modified Semisolid Rappaport–Vassiliadis media after incubation for 17–24 h at 42 °C (Bhatia et al. 2012). The total bacteria count was determined using Nutrient agar media after incubation for 24–48 h at 37 °C. Total fungi were determined using Potato Dextrose agar, for 48–72 h at 28 °C. The actinomycetes population was determined on starch casein agar media for 72 h at 28 °C (Kadam et al. 2009). All the specific media were obtained from Vikas Scientific Co. supplied from Hi-Media Laboratory Pvt. Ltd., India. All the analysis followed the Standard Methods for examination of water and wastewater (APHA 2005).

Antibacterial activity assay

The spread plate technique was used to isolate microorganisms from the active layer of the VF. Well-isolated colonies were selected and sub-cultured to a new petriplate. Cells from the new colony were then picked up with an inoculating needle and transferred to an agar slant for maintenance of pure culture. Pure bacterial cultures were identified morphologically by microscopy (shape, gram staining and motility) and biochemically by biochemical identification tests (Sugar utilization, Indole production, Citrate utilization, Methyl Red-Voges Proskauer test, Triple sugar iron utilization, Oxidase production, Catalase production, Coagulase test) according to Bergey's manual of determinative bacteriology (Holt et al. 1994). Pure cultures of fungal isolates were identified using both macroscopic (cultural) and microscopic (morphological) features (Barnett & Hunter 1998). For actinomycetes, cultural characteristics of cells were recorded and morphological observations were made with a microscope (Shirling & Gottlieb 1966; Kadam et al. 2008).

The isolated microorganisms were tested for antibacterial activity against the known bacterial culture by the agar well diffusion method as described by Parekh & Chanda (2007) and Sethi et al. (2013). Twenty-four-hour fresh cultures of Gram-Positive Staphylococcus aureus (ATCC 29213), Gram-negative E. coli (ATCC 25922), Klebsiella pneumoniae (NCIM2719) and Pseudomonas aeruginosa (ATCC 27853) were swabbed by sterilized cotton swab and lawns were prepared over the agar surface on the petriplate. All of these strains are pathogenic in nature, procured from the Vikas scientific co., Roorkee, India. The molten Mueller-Hinton agar (Hi-media) was inoculated with 100 μL of the inoculum (108 CFU/mL) and poured into the petriplate. Two wells were made in the inoculated plates using a sterile cork borer (0.85 cm). About 80 μL of cell-free supernatant was added in the first well and antibiotic streptomycin (50 mcg) was added in the second well as positive control for the experiment. Plates were then incubated at 37 °C for 24 h. After 24 h, the zones of inhibition were measured in millimetres (mm). Species showing diameters between 12 and 16 mm were considered to be moderately active and with more than 16 mm were considered to be highly active. The experiment was done in triplicate and the mean values are presented. The average diameters of the inhibition zone surrounding the discs were measured visually, and did not include the diameter of the paper disc.

Kinetics evaluation

Four different kinetics models have been used to fit the experimental data obtained during the vermifiltration: (1) a log-linear according to the Chick’ law (Equation (1)). This model reduces disinfection to a bimolecular chemical reaction in which microorganisms are treated as molecular species. There is an extensive application of this equation in other disinfecting agents as well, i.e. chlorine, ozone, hydrogen peroxide and chloramines. (2) A double log-linear kinetics (Equation (2)), with a first stage of very fast (k1 > k2) inactivation and a second phase of attenuated inactivation (k2). (3) A log-linear region followed by a ‘tail’ (Equation (3)). The ‘tail’ shape represents the bacterial population remaining at the end of the experiment due to the presence of a population of cells resistant to the treatment. (4) An initial delay or very smooth decay at the beginning (‘shoulder’), attributed to loss of cells' viability after the accumulation of oxidative damage during the process, followed by a log-linear decrease (Equation (4), Garcia Fernandez et al. 2015).
formula
1
formula
2
formula
3
formula
4
where N/N0 is the microorganism concentration reductions, ki is the disinfection kinetic rate and t is the time of treatment, Nres is the residual population density, and SL = Shoulder length (min−1).

RESULTS AND DISCUSSION

Reactor performance

The average pH of the influent was 8.0 ± 0.1. The pH of VF effluent increased initially during the treatment, then decreased slightly, reaching out to be in the neutral range signifying the natural inherent ability of earthworms to act as a buffering agent and neutralize pH. The pH of GF effluent also improved, but it was not consistent on all days as observed in Figure 2(a). There was a significant variation in pH between VF and GF (t= 6.2, P < 0.001). Earthworms present in the VF have a buffering capacity to neutralize the pH of water slowly (Sinha et al. 2008). The mean DO concentration in the influent was 0.43 ± 0.4 mg/L. In VF effluent, the DO was observed to be 5.6 ± 1.5 mg/L while in GF effluent, initially DO appeared to be as high as 7.06 mg/L, but with time DO tended to decline and reached 1.0 mg/L as shown in Figure 2(b). DO varied for VF and GF effluent significantly (t = 3.3, P > 0.001). This suggests that earthworms are responsible for creating aerobic conditions inside the VF by their burrowing action, while the geological system in the GF tends to be anaerobic after few days. The increase in DO is also attributed to the dropping overflow of the VF layers, as water flowed through it by gravity (Wang et al. 2011).
Figure 2

Variation in (a) pH, (b) DO, (c) BOD and (d) COD during vermifiltration.

Figure 2

Variation in (a) pH, (b) DO, (c) BOD and (d) COD during vermifiltration.

The profile of BOD and COD for influent and effluent during the operation period is shown in Figure 2(c) and 2(d). The organic matter (OM) measured as average BOD in the influent was 311 ± 30 mg/L. The results show that earthworms can remove BOD loads by over 88% in VF, while BOD removal in GF (where only a geological and microbial system works) is around 74%. The percentage BOD removal (Figure 2(c)) was significantly higher in the VF as compared to the GF (t = 3.9 P < 0001). Higher BOD removal in the VF is attributed to the enzymatic degradation of the organic content of wastewater by the earthworms and aerobic microorganisms (Sinha et al. 2008). The mean COD removal in the VF is 78.2% (Figure 2(d)), indicating further OM degradation by earthworms, while in the GF it is 68%. The percentage of COD removal was significantly higher in the VF as compared to the GF (t = 2.7, P < 0.01). This is attributed to the enzymes present in the gut of the earthworms, which helps in degradation of chemicals that cannot be degraded by microbes alone. The symbiotic interactions of earthworms with microorganisms help in OM degradation, which leads to higher treatment efficiency. In this ecosystem, microorganisms survive and reproduce on the colloidal and dissolved OM in wastewater, and biofilm is formed on the surface of filter particles (Yang et al. 2013). The total suspended solids removal for the VF was observed as 73% while for the GF it was observed as 69%. Various physical, chemical and biological reactions take place in the vermifiltration process including the adsorption of molecules and ions, oxidation–reduction of OM, the behavior of earthworms and their synergetic effects with microorganisms (Arora et al. 2014a). Earthworms mainly feed on SS and microorganisms in wastewater and are capable of transforming insoluble organic materials to a soluble form, selectively digesting the particles to finer size, with further OM degradation.

Pathogen removal

The pathogen removal performance is illustrated in Table 2. FC bacteria are indicators of fecal contamination and indicate the potential presence of pathogens associated with wastewater or sewage sludge. The amounts of FC found in treated effluent demonstrated a significant difference in the removal of pathogens. The concentration of FC decreased from 3.32 × 105 MPN/100 mL in the influent to 2.67 × 102 MPN/100 mL in the VF effluent. During the treatment process, however, the FC amount decreased slowly from the initial 3.32 × 105 to 3.1 × 105 within 24 h, then dramatically to 1.3 × 104 in the next 24 h, followed by a sharp decrease to 2.67 × 102 MPN/100 mL, below the WHO (1989) standards, up to the end of treatment. After 96 h, Salmonella were below detectable limits in the culture medium for all samples. Escherichia coli is the most representative bacterium in the group of FCs. During the study, 2.48 log removal of E. coli was observed in the VF. These results showed the possibility of an efficient pathogen removal process in vermifiltration. It was observed that the final treated effluent contained lower numbers of FC than the permissible limit (i.e. log 3 or 1,000 MPN/100 mL) specified by WHO (1989) for unrestricted irrigation reuse. Since most of the indicator organisms are removed from the influent, it seems that the treatment process may serve as an important technological process for disinfection. The possible reason for the pathogen removal may be due to the antimicrobial potency of the microorganisms against other pathogens, elaborated in the next section. This hypothesis is supported by Sinha et al. (2008) who demonstrated that the earthworm releases celeomic fluid from its body cavity, which possesses antibacterial substances which may inhibit the growth of pathogens.

Table 2

Pathogen removal performance of vermifiltration

Indicator organismsInfluentVF EffluentGF EffluentLog removal VFLog removal GF
TC 6.22 2.82 3.4 3.4 2.82 
FC 5.28 2.67 3.3 2.61 1.98 
FS 5.37 2.87 2.5 2.37 
E. coli 4.4 1.92 3.2 2.48 1.2 
Salmonella 3.87 1.67 2.2 0.87 
Indicator organismsInfluentVF EffluentGF EffluentLog removal VFLog removal GF
TC 6.22 2.82 3.4 3.4 2.82 
FC 5.28 2.67 3.3 2.61 1.98 
FS 5.37 2.87 2.5 2.37 
E. coli 4.4 1.92 3.2 2.48 1.2 
Salmonella 3.87 1.67 2.2 0.87 

The Indigenous population of total bacteria in the influent was 31.4 × 106 CFU/mL. The average bacterial number in the effluent increased within the initial 6 days, due to the availability of a large amount of OM or energy sources. Finally, the number reduced to 2.0 × 103 CFU/mL. The population of bacteria reduced by 3.35 log units in the VF. The fungal load in the effluent was reduced in the VF by 0.8 log units, and the actinomycetes population was reduced by 1.91 log units. In the VF, earthworm densities facilitated earthworm activity. Adequate oxygen and improved aerobic conditions due to the burrowing action of the earthworms helped the aerobic microorganisms to thrive in the microenvironment around the earthworm packing bed. Furthermore, the casts and mucus produced by the earthworms proved beneficial for the evolution of a diverse microbial community. There exists a diverse microbial community in the VF that favors the biodegradation of OM and pathogen removal during vermifiltration, however, with time the diversity of the microbial community decreases due to exhaustion of energy sources, or maybe due to inhibitory effects.

Antimicrobial activity assay

In the agar diffusion assay, the negative control (10 mmol/L sodium phosphate buffer, pH 7.0) did not show any inhibition, while the positive control ampicillin (50 μg) showed mean diameters of zones ranging from 38 to 40 mm. The mean zones of inhibition against bacteria ranged from 12 to 36 mm (Table 3). The results of the assay showed that most of the isolates were active against all four aerobic bacterial pathogens. In general, most of the bacterial strains showed higher activity against Gram-Negative bacteria than Gram-Positive bacteria, the reason for which was possible differences in the cell wall structure between Gram-Positive and Gram-Negative bacteria. The cell wall is a critical structure in all bacterial cells. Most bacteria could not live without them, because a cell wall protects bacteria from osmotic lysis. Often, the Gram-Positive cell wall appears as a broad, dense wall of 20–80 nm thickness and consisting of numerous interconnecting layers of peptidoglycan, so the Gram-Positive bacteria were less prone to be the target of antibacterial agents than the Gram-Negative bacteria. In addition, it was also found that bacterial strains isolated from the active layer of the VF exhibited significantly higher antibacterial activities than those in the bottom layers, further indicating the coding of inactive antibacterial genes for antimicrobial potency.

Table 3

Antimicrobial activity of isolated bacteria against pathogens

IsolateSpecies nameAntibacterial activity against (zone of inhibition, mm)
S. aureusE. coliK. aerogenesP. aeruginosa
Aeromonas 22 34 12 29 
Citrobacter freundii 12 32 34 27 
Enterococcus faecium 30 32 34 25 
Enterococcus faecalis 14 31 28 28 
Micrococcus luteus 28 29 29 28 
Clostridium 27 30 20 39 
Pseudomonas aeruginosa 31 32 28 33 
Corynebacterium xerosis 29 33 30 23 
Bacillus cereus 12 37 21 23 
10 Bacillus thuringiensis 24 38 33 29 
IsolateSpecies nameAntibacterial activity against (zone of inhibition, mm)
S. aureusE. coliK. aerogenesP. aeruginosa
Aeromonas 22 34 12 29 
Citrobacter freundii 12 32 34 27 
Enterococcus faecium 30 32 34 25 
Enterococcus faecalis 14 31 28 28 
Micrococcus luteus 28 29 29 28 
Clostridium 27 30 20 39 
Pseudomonas aeruginosa 31 32 28 33 
Corynebacterium xerosis 29 33 30 23 
Bacillus cereus 12 37 21 23 
10 Bacillus thuringiensis 24 38 33 29 

Kinetics evaluation

All experimental data from the vermifiltration tests were fitted to the proposed Equations (14). Those results, which led to a minimum R2 coefficient, were accepted as the best statistical fitting. The results of these fittings are shown in Table 4, including R2 values. Kinetic parameters obtained for the different equations are comparable, as all are obtained under the same experimental and reactor conditions. As expected for disinfection during vermifiltration, first-order kinetics can be considered the most common behavior for all the results (Table 4). In the case of Salmonella and Actinomycetes populations, removal is represented by a biphasic model (model 2, double log-linear) and less frequently by linear behavior continued with a residual concentration (model 3, log-linear + tail) for total bacteria and total fungi. As all these equations are based on first-order kinetics (simple or modified) the kinetic constants can be directly compared to assess the best disinfection results. The ‘tail’ shape can be explained by the presence of the remaining population of bacteria, which were not removed due to the presence of the SS particles present in the wastewater aggregates around the bacteria cell, which shades it and prevents the attack on its cell wall surface.

Table 4

Microorganism inactivation rates (k) during vermifiltration

Microorganismsk1 (min−1)R12k2 (min−1)R22SL (min)Log (Nres)Model #
FC 0.033 ± 0.002 0.998 – – – – 
FS 0.013 ± 0.001 0.977 – – – – 
Salmonella 0.009 ± 0.001 0.983 0.003 ± 0.002 0.866 – – 
E. coli 0.024 ± 0.003 0.973 – – – – 
Total bacteria 0.005 ± 0.001 0.953 – – – 1.83 
Total fungi 0.051 ± 0.007 0.965 – – – 0.30 
Actinomycetes 0.069 ± 0.004 0.947 0.010 ± 0.001 0.970 – – 
Microorganismsk1 (min−1)R12k2 (min−1)R22SL (min)Log (Nres)Model #
FC 0.033 ± 0.002 0.998 – – – – 
FS 0.013 ± 0.001 0.977 – – – – 
Salmonella 0.009 ± 0.001 0.983 0.003 ± 0.002 0.866 – – 
E. coli 0.024 ± 0.003 0.973 – – – – 
Total bacteria 0.005 ± 0.001 0.953 – – – 1.83 
Total fungi 0.051 ± 0.007 0.965 – – – 0.30 
Actinomycetes 0.069 ± 0.004 0.947 0.010 ± 0.001 0.970 – – 

k = inactivation rate (linear regression of Log (concentration) versus time); R2 = regression coefficient; Model 1 = Log-lineal (k1); Model 2 = double log-lineal (k1, k2); Model 3 = Log-lineal (k1) + tail (Log (Nres)); Model 4 = shoulder (SL) + log-lineal (k1).

CONCLUSIONS

Vermifiltration is an ecofriendly waste management technology, which takes the privilege of both earthworms and the associated microorganisms to treat wastewater. The pilot-scale study demonstrated the reduction of pollutants with the in-built pathogen removal property. The concentrations of pollutants in the effluent reached the discharge standards of water for irrigation reuse. The pathogen removal is based on a first order kinetic model and is due to the inhibitory effects of microorganisms present in the VF. The vermifiltration technology is a cost-effective process, which is completely sustainable and profitable for sewage treatment with efficiency, convenience and potential for decentralized wastewater treatment.

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