Characterization of wastewater
The physico-chemical parameters of the untreated domestic effluent were evaluated following the standard protocols, as mentioned by APHA⁷ and was compared to the standard permissible limits given by CPCB/WHO, as given in Table 1. pH is the measure of hydrogen ion concentration in water and is thus the assessment of the water being acidic or alkaline in nature. In the present study, effluents were found to be slightly alkaline as the pH was 7.68 for Institutional and 7.80 for the city sample, respectively; the standard being 6.5-7.5. Temperature for both the samples was almost near to the standard value (35-37°C). Colour for STP1 sample was light grey and for STP2 sample was grey, visibly indicating poor water quality. Similarly, pungent smell of samples indicated release of harmful gases, leading to bad odour. Electrical conductivity (EC) is the ability of the water to carry electric current because of the presence of ions, thereby reducing its possibility for agricultural use.

EC values were 2.21±0.02mScm^-1 and 2.3±0.1 mScm^-1, for institutional and city samples, respectively; much beyond the standard limit. TDS values were 902±1.08mgL^-1 and 1127±2.12 mgL^-1 respectively, whereas TSS values were 65.086±0.05 mgL^-1 and 74.048±0.16mgL^-1 for STP1 and STP2 samples, respectively; but was within the expected standard limits. Hence, the Total Solids (TS) were found to be 1202±0.5 mgL^-1 and 1248±0.12 mgL^-1 for site I and site II samples, respectively; which was almost double to that prescribed in the standard limits. Total solids (suspended and dissolved) values indicate the nature of salinity of the wastewater. Total alkalinity indicates the level of carbonates and bicarbonates in water and its value was 31.84±0.05 mgL^-1 and 33.62±0.05 mgL^-1, for site I and site II samples, respectively, and was within the expected range. Turbidity is the measure of water clarity; that indicates the degree of light scattering by the suspended solids. Its values were 65.36±0.002NTU and 71.12±0.009NTU respectively, for the two samples, which was much higher than the standard level. Hardness (calcium and total) of water is due to the presence of calcium and magnesium in it. Its values were again much higher than the standard values, indicating a high content of calcium, magnesium, carbonate, bicarbonate and chloride ions. Dissolved oxygen (DO) values indicate the degree of pollution in the water bodies. The water quality is enhanced by the presence of DO, whereas, its values were found to be 3±0.08mgL^-1 and 2±0.02mgL^-1, for site I and site II samples, respectively. Biochemical oxygen demand (BOD) is the amount of oxygen needed by aerobic biological organisms to break down organic material present in a water sample at a certain temperature over a particular time period. Its values in the present samples were 56±1.5mgL^-1 and 90±1.8mgL^-1, respectively, higher than the expected standard values. Chemical oxygen demand (COD) is the amount of oxygen depleted on account of decomposition by microbes to a level detrimental to the aquatic life. Its values of both the samples were 308±1.4mgL^-1 and 340±1.9mgL^-1, respectively, which was higher than the expected standard limit of 250mgL^-1. Total nitrogen was found to be above the expected range, however, sulphate and phosphate were much below the standard permissible limit.

Identification and characterization of the bacterial isolates
35 bacterial isolates were isolated and purified from the standard isolation procedure. Then, they were identified on the basis of physiology, morphology and biochemical assays. The isolates were analysed for their morphological parameters, covering colony colour, shape, size, margin elevation and edge and opacity. The strains were also tested for their gram nature by following the standard gram staining protocol^15,16. The cell count was done by calculating the CFU (colony forming unit) per millilitre (mL) by counting the colonies grown on nutrient agar plates. The parameters studied are all summarised in Table 2. After the characteristic physiological and morphological analyses, the bacterial isolates were then characterized by biochemical tests, namely; amylase, cellulase, H₂S, urease, citrate, methyl-red, voges-proskauer, gelatin, indole, carbohydrate catabolism, triple sugar iron, catalase and casein tests (Table 3).

Following the biochemical chara-cterization, the bacteria giving maximum positive results for the biochemical assays and showed better growth in the wastewater sample were chosen to be efficacious and used for consortia development. The selected bacteria were identified by following the Bergey’s manual¹⁷. The recognized bacteria were, namely, Bacillus species (B15), Achromobacter species (B24) and Comamonas species (B27).

The synergistic/antagonistic pattern of the prepared consortium
The consortium was developed for efficient and faster biodegradation of complex organic matter in the wastewater. The four selected bacteria were used to formulate four possible consortia with three bacterial strains in each consortium. To evaluate the compatibility of these four bacteria together, the growth of microbial cells in the consortia were checked in the nutrient broth by measuring optical density, OD₆₀₀ nm (Table 4) as well as on nutrient agar plates (Fig. 2). As seen in Fig. 2(b), in Consortium 2, all the three bacterial strains showed no antagonistic phenomenon and grew well together in synergism, when incubated at 35°C for 48 hours¹⁶ as compared to consortia plates in Fig. 2(a), (c) and (d) in which all the constituting bacteria grew in an antagonistic manner.

Fig. 2. Representation of prepared consortia on nutrient agar plates; (a) Consortium 1, (b) Consortium 2, (c) Consortium 3 and (d) Consortium 4

Table 2:
Morphological characteristics and growth parameters of the isolated bacteria

SEM analysis for immobilization
The cross-section of the used plastic caps with and without immobilized bacterial consortium is shown in Fig. 3. The roughness of the surface of the caps was increased by forming small equal sized holes in them. As a result, the surface area was increased many-folds and thereby the immobilized consortium had increased reaction time on the effluent by enhanced growth. Differently shaped, constituting bacteria of the consortium was seen visibly in the SEM image, depicting the successful application of the plastic media for immobilization.

Fig. 3. SEM image of (a) plastic cap used for immobilization of consortium; (b) plastic cap with holes for enhanced immobilization and (c) immobilized consortium on the plastic caps.

Domestic effluent biodegradation analysis
In the present study, a closed and continuous laboratory scale bioreactor was developed, as illustrated in Fig. 1, firstly, for the growth of the developed consortium and then assessment of its biodegradation ability via the treatment of domestic effluent. The working experimental pH and temperature were noted as 7.4-7.5 and 37±5°C, respectively which were required for the optimum functioning of the bioreactor. Furthermore, optimization studies were done for flow rate and hydraulic retention time, as outlined in Table 5. Flow rate of 3, 5 and 8 mLmin^-1, respectively were applied and were assessed for optimization on the basis of reduction in BOD and COD values. 3mLmin^-1 of flow rate was less for the treatment process, however, could reduce much BOD and COD values, whereas, 8 mLmin^-1 of flow rate was higher and thus was not able to remediate the water efficiently. 5mLmin^-1 was found to have reduced the BOD and COD parameters sufficient to be applied in the bioreactor, hence was chosen. The hydraulic retention time optimization study was also done similarly on the basis of reduction in water quality parameters. Out of 12, 24, 48 and 72 hours of time provided for treatment of wastewater, 48 hours was selected as the optimum time needed for the appropriate treatment of water, accounting to the maximum reduction in BOD and COD values. 72 hours reduced the BOD and COD but were almost as much as reduced in 48 hours. Hence, 48hours retention time was chosen for time-consuming treatment.

Table 3:
Biochemical characterization of the isolated strains

(Amy-Amylase test, Cel-Cellulase test, H₂S-Hydrogen sulphide test, Ure-Urease test, Cit-Citrate test, MR-Methyl Red test, VP-Voges-Proskauer test, Gel-Gelatin test, Ind-Indole test, Carb-Carbohydrate catabolism test, TSI-Triple sugar iron test, Cat-Catalse test, Cas-Casein test.
Glu-Glucose, Lac-Lactose; + Low activity++; Moderate activity+++; Maximum activity;- No activity)

Table 4:
Bacterial consortia composition and their growth in effluent

Hence, with a regulated and optimized flow rate of 5mLmin^-1, about 1420mL of the working volume of wastewater in the primary tank was treated by the immobilized and exponentially grown consortium with a residence time of 4.76h. Thus, after about 6 hours of incubation time in the primary tank, the water was passed to the secondary tank, wherein the water was filtered through an inch thick layer of sawdust: rice husk (1:1) which provided the final filtration and cleaning of the wastewater before the collection. Also, after the treatment and filtration processes, some amount of settled sludge was collected in the sludge tank and finally the colourless, odourless and detoxified water was collected in the collection tank. Table 6 summarizes the effluent biodegradation parameters analysed before and after the treatment by the immobilized consortium 2 in the laboratory scale bioreactor. The effective treatment of the effluent by immobilized consortium 2 is justified by the synergistic action of the combined metabolic activities and interactions of the constituting strains in the consortium.

Table 5:
Effect of flow rate and hydraulic retention time in the bioreactor for effluent treatment

After 48h of treatment, physico-chemical parameters were determined and were found to have reduced drastically. pH was shifted from alkaline to close to neutral values for both STP1 and STP2 samples.  Reduction in electrical conductivity values in both the samples after primary treatment and filtration were associated with the use of ions by the consortium bacteria for their growth and further was adsorbed by the mixture of sawdust and rice husk during the filtration. The presence of total dissolved and suspended solids indicated a large amount of organic and inorganic matter present in the effluent, however, after the treatment, there was a huge reduction in these parameters which would be due to the use of organic matter by the bacteria for their growth and adsorption of the remaining on sawdust and rice husk mixture. Reduction in the turbidity values, biochemical oxygen demand (BOD) and chemical oxygen demand (COD), which are widely used as significant indicators of water quality, were clearly in justification with the fact that the bacteria consumed most of the organic matter and suspended particles for their growth and survival and thus lead to the reduction in these parameters. These vital measurements are important parameters for evaluating the suitability and practicality of the effluent for irrigation purpose. Reduction in the hardness values could possibly be due to filtration, helping in the removal of various ions like calcium, magnesium and others like sulphate, phosphate and nitrogen.

Table 6:
Physico-chemical characterization of the effluents after treatment in the bioreactor