Feasibility of sequential anaerobic-aerobic integrated settler-based biofilm reactor for onsite treatment of domestic wastewater

The present study investigates the applicability of sequential anaerobic aerobic integrated settler-based biofilm reactor (SAABR) for the onsite treatment of domestic wastewater. The main aim of the study is to overcome the inherent flaws in an anaerobic system as well as enhancing the effluent quality by means of aerobic post-treatment. The sequential system consisted of an anaerobic settler is followed by two biofilters, anaerobic as well aerobic in series. The first biofilter is anaerobic and the second one is aerobic. The system was run on two hydraulic loading conditions (steady and non-steady flow) with a 24-hour hydraulic retention time (HRT) for the anaerobic system and a 2-hour HRT of aerobic filters. It was found that the performance of the system at steady flow stood at 93.9 ± 2.6, 93.3 ± 1.8, 91.2 ± 4.2, 75.8 ± 3.8 and 98.7 ± 1.1% in terms of total suspended solids (TSS), biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN) and faecal coliform (FC), respectively while at non-steady flow, it decreased slightly. The Field Emission Scanning Electron Microscope (FESEM) showed the presence of anaerobic bacteria in the system. The system is able to overcome the major flaws of the anaerobic systems and able to deliver high effluent quality. The study demonstrated that the sequential system can be a sustainable alternative for the onsite treatment of domestic wastewater, particularly in rural areas of the developing countries like India.


Introduction
Onsite wastewater treatment systems are widely used for treating domestic wastewater in the areas where any centralized treatment facility is not available [1,2].Due to increasing population, industrialization and lack of water management system, water quality is going to become a major problem in future [3].In developing countries like India where 68.84% of population resides in villages [4] no proper wastewater treatment facility is available at onsite level [5,6].People still rely mostly on conventional septic tank, which has become outdated.Most of the domestic wastewater is directly disposed into open water bodies [7], which further deteriorates the quality of water resources [8].
At the onsite level, anaerobic treatment is one of the most cost-effective and viable treatment options available [9] for the treatment of domestic wastewater because of low investment in construction as well as operation and maintenance [10].It is also relatively easy to operate.The sludge formation is low [11] and can also be used to generate energy in the form of biogas [12].Anaerobic treatment is not limited to onsite level.It is also used at centralized, peri urban and industrial level [13,14].
Anaerobic filters are very effective in removing solids and organic matter by anaerobic bio mechanism wherein the organic materials are converted into biogas and other stable end products [15].Biogas is basically, mostly of methane and carbon dioxide.After digestion, the end products are stabilized and separated into liquids and solids [16].Organically bounded nitrogen converts into ammonium and sulphate, which reduce the formation of hydrogen sulphide [17].Anaerobic treatment is generally less efficient in removal of nutrients [10,11,18], and microbiological contaminants [12,13].It requires post treatment to maintain the local standard disposal norms or reuse norms [14,15,19].Post treatment can be made more effective with the help of aerobic treatment [11], which removes the remaining pollutants (organic matter and nutrients) present in the anaerobic effluent [17].When aerobic treatment unit is connected with an anaerobic filter, it works as a bio-mechanized two-step sequential (anaerobic and aerobic) treatment system [17,20].
Sequential anaerobic-aerobic wastewater treatment technology is a revolutionary method that successfully treats wastewater by integrating two different treatment stages into a single system.This technique synergizes the benefits of anaerobic and aerobic processes in order to accomplish complete pollution removal and resource recovery.Sequence of anaerobic and aerobic systems requires less energy, less operation and maintenance cost [9,21].
An important attribute of this technology is its ability to handle varying organic loads and fluctuating influent conditions, making it appropriate for both constant and variable wastewater flow scenarios [22].Compared to traditional approaches, the sequential arrangement optimises microbial activities at each stage, resulting in improved treatment efficiency and lower sludge production.The technology's application is versatile, including decentralised wastewater treatment systems, onsite treatment systems, industrial effluent treatment, and municipal sewage treatment [11].Notably, including a settler or separator in the system allows efficient solids-liquid separation, which improves reactor stability and prevents biofilm washout [23].
Much research has demonstrated the effectiveness of sequential anaerobic-aerobic treatment in terms of organic matter removal, nutrient removal, and overall pollutant degradation.But at the onsite level, it is very limited.Where challenges such as reactor design, nutrient removal, microbial removal, and long-term system stability remain, ongoing research highlights the potential of this technology as a viable and effective response to solve these problems in modern wastewater challenges.Considering its effectiveness and energy-efficient approach to the treatment of domestic wastewater, sequential anaerobic-aerobic wastewater treatment technology can be adopted at the onsite level.This technology has the potential to tackle water pollution issues while also providing valuable resource recovery by combining the benefits of anaerobic and aerobic processes in a single system [23,24].
Over the past few decades, increasing concerns over water scarcity, pollution, and environmental degradation have accelerated the research for innovative and sustainable wastewater treatment technologies [25,26].Among these approaches, the sequence of anaerobic and aerobic processes with a compact and efficient system has attracted substantial attention due to its potential for pollutant removal and resource recovery [27].A possible solution to the challenges associated with wastewater management is the viability of using a sequential anaerobic-aerobic integrated settler-based biofilm reactor for the on-site treatment of domestic wastewater [18,28].
Based on literature, we designed a sequential anaerobic-aerobic integrated settler-based biofilm reactor (SAABR) for treating domestic wastewater at onsite level.In this research, the entire study was done with the labscale setup of the SAABR.The SAABR is connected in series with an anaerobic rector (ISBRintegrated settler based anaerobic biofilm reactor) and aerobic filter.Both the filters are attached growth type filters working on the principle of upward flow mechanism, having HRT of 24 h in case of ISBR and 2 h HRT in case of aerobic filters.It was run on actual sewage (generated from a hostel) under steady (PFF1 -peak flow factor1) and nonsteady flow (PFF2 -peak flow factor2) conditions [29,30].The results obtained were quite impressive and fulfil the disposal standard of NGT-2019(Nation Green Tribunal) India.Hence, the objectives of this research were to evaluate the long-term performance, stability of the sequential system, and Insite examination of accumulated sludge using FESEM analysis.This research could be beneficial in rural and impoverished areas where centralised facilities are unavailable.It can make a significant contribution to reducing water pollution.By implementing the onsite sanitation systems provides cost-effective and long-term wastewater treatment solutions while protecting community health, preserving the environment, and supporting skill development in local communities.

Materials and methods
The sequential anaerobic-aerobic laboratory-scale unit was divided into two separate filters.The first was the ISBR, which works on the principle of anaerobic treatment.The second was an aerobic filter, which acts as a post treatment facility and needs extended aeration to function.

Configuration of the anaerobic (ISBR) filter
The ISBR is a multi-stage anaerobic treatment system having a working volume of 57 Litres.It has been manufactured using transparent acrylic sheets.This ISBR system consists of three rectangular chambers

Configuration of aerobic filter
The aerobic filter has been fabricated with the help of transparent acrylic sheets.It has a working volume of 4.7 Litres.The framework of the filter is cylindrical having unit dimensions of 450 mm × 150 mm (L × D).The manufacturing details of Mutag BioChip TM are 30 mm diameter in size, 1.1 mm thickness, model no.PP22, MoC-Virgin PE material and surface area-to 5,500 m 2 /m 3 as shown in figure 1.

Selection of filter media
The filter media were carefully chosen for their exceptional qualities, such as a high surface area that promotes active biofilm activity and assures long-term durability.This method took into account their compatibility with wastewater properties as well as their adherence to demanding regulatory norms.The success of these media in inducing biofilm activity in similar applications verified their efficacy.By combining these media, the wastewater treatment system not only outperforms in pollutant removal but also improves biological treatment processes via active biofilm growth.This meticulous choice ensures a long-term and effective wastewater treatment solution that meets operational requirements while also protecting the environment [23].

Start-up and operation of the system
The laboratory scale sequential system was installed in open environment near the hostel STP of Manipal University Jaipur, Rajasthan, India, which worked on actual sewage.In order to make the designed system effective and quick, inoculation was needed.If we had started the system without inoculation, the time required by favourable bacteria to grow would have increased considerably.Keeping all these things in mind, the inoculation was done in the ISBR system.The sludge for inoculation was taken from the biogas digester plant located at Manipal University Jaipur, Rajasthan, India.The TSS and VSS concentration of sludge was 14341 and 7130 mg L −1 , respectively and favourable temperature of Jaipur were the main reasons behind achieving the stability within 50 days..The sludge was added to the tank as 35% of the total volume [4].The entire system was meticulously wrapped in a protective black polythene sheet to ensure total coverage.Following that, the system was started by putting a mixture of diluted sewage (50% sewage and 50% water) collected from Manipal University Jaipur's 1 MLD Sewage Treatment Plant.A peristaltic pump was used to introduce sewage into the system.

Operational time and analysis
Throughout the duration of the six-month investigation, biweekly samples were collected from both the system's inlet and outlet.The Environmental Engineering Lab of Manipal University Jaipur analysed these samples.The system operated for 162 days under PFF1 conditions and 18 days under PFF2 conditions.Samples were additionally analysed at Jagdamba Laboratory, an institution accredited by the National Accreditation Board for Testing and Calibration Laboratories (NABL), to assure the validity of the results.On these samples, comprehensive analyses were performed on parameters such as pH, temperature, TSS, BOD, COD, orthophosphate, and NH 4 -N.

Analytical procedure and instruments
The study included a comprehensive analysis of multiple parameters using the standard methods specified in the water and wastewater examination procedures (APHA et al, 2005) [19].The Hach-USA HQ 40d multiparameter instrument was utilized to measure pH and temperature.The COD analysis involved digestion in the COD Digester (DRB-200) and subsequent UV-vis Spectrophotometry (Model No. DR6000 Hach-USA) without RFID.This method was also used to calculate OP and NH 4 + -N.Using the Persulfate Digestion Test 'N Tube method: Hach Method 10072, a range of 10-150 mg L −1 N −1 of total nitrogen was determined.
Regular measurements of microbial concentration were required for microbial analysis, with a focus on signalling organisms as the primary area of interest.On a weekly basis, the Multiple Tube Fermentation Technique [19] was utilised to count the total coliforms present in the sample.Coliforms found in faeces were analysed with the help of M-FC Broth Base (HIMEDIA-M1111).Each month, samples were analysed for the presence of pathogenic bacteria such as E. coli, Salmonella, and Shigella.Through the use of the membrane filtration method, both total coliforms and faecal coliforms were successfully extracted.Salmonella was isolated with high chrome-enhanced salmonella agar (HIMEDIA-M 1466), while shigella was recovered with Shigella broth base (HIMEDIA-M 1326).E. coli was isolated using an EMB agar base (HIMEDIA-M 301).This stringent testing protocol assured a comprehensive evaluation of the microbial content and the presence of pathogens in the samples.

Raw wastewater characteristics
The actual sewage generated from the hostel was fed into the system whose characteristics are shown in table 1.The organic loading rate during the study ranged from 0.22 to 0.71 (Kg.COD / m 3 .day) and the BOD to COD ratio was 0.61, which indicated the stability of biological treatment.The microbial quantity was quite consistent in raw sewage whose average concentrations in terms of total coliform, faecal coliform and e-coli were observed to be 5.6 × 10 8 ± 1.6 × 10 8 (MPN / 100 ml), 9.2 × 10 7 ± 2.1 × 10 7 and 9.0 × 10 6 ± 1.0 × 10 6 (CFU/100 ml) respectively during the study.The concentrations of pathogenic bacteria such as Salmonella and Shigella were found to be 6.8 × 10 6 ± 1.8 × 10 6 and 8.3 × 10 5 ± 1.3 × 10 4 (CFU / 100 ml) respectively.

Preparation of sludge for FESEM
The morphology of the sludge was investigated using a Field Emission Scanning Electron Microscope (FESEM).
The sample was initially fixated in a phosphate buffer solution containing 2.5% (w/v) glutaraldehyde for one hour at 4 °C.Afterward, it was sterilised through a series of acetone-water solutions (ranging from 10% to 100%).Each mixture was given 10 min to equilibrate prior to critical point drying.Aurum was applied to the samples prior to FESEM analysis [31,32].Using FESEM (NOVA NANO SEM-450), the analysis was conducted.

Physico-chemical characteristics
At the beginning of the study, the removal efficiency of the sequential system was 36% to 60% in terms of COD, but over time its removal efficiency increased, and later the removal efficiency of the system was stabilized.Several samples were also found in which the COD value suddenly increased or decreased, yet the removal efficiency did not change much.The fluctuations in COD value at the inlet were due to hostel holidays, party, and other hostel activities that affected the sewage concentration observed during the experiment.
The system operated nonstop, with daytime temperatures fluctuating between 22 °C and 43 °C, while nighttime temperatures ranged from 18 °C to 22 °C.Samples were collected between 9 a.m. and 11 a.m., during which the temperature varied from 26 °C to 37 °C, a range optimal for wastewater treatment [33].After collecting the sample, we analysed all the necessary parameters within 24 h.The physicochemical characteristics of the SAABR are detailed in table 2.
Table 2 presents the outcomes obtained from the sequential system, distinctly showcasing the outcomes from both the anaerobic and aerobic reactors.Notably, the concentration after the removal of pollutants by this anaerobic reactor was 32.85 mg /l, 29 .83mg l −1 , and 73.68 mg l −1 , especially in terms of TSS, BOD, and COD, which are much lower than the inlet.Remarkably, these values fulfil disposal standards.The anaerobic part of this system makes it effective against shock loading.Under the shock loading condition (PFF2), the performance of the system was not much affected.On the other hand, for TN and TP, the performance of the system was not good on this parameter, which shows the limitations of the anaerobic system [34].
Integration of aerobic system promoted to increase the efficacy of anaerobic wastewater treatment.The subsequent interaction between the anaerobic and aerobic technologies synergistically works to further enhance the quality of treated wastewater from the anaerobic reactor; thus, the average removal efficiency of ammonia and total nitrogen was 70.77 ± 3.65% and 75.79 ± 3.84% respectively, which could not have been achieved by using only anaerobic treatment [11].We could increase the removal efficiency of ammonical nitrogen by 33.94% and that of total nitrogen by 47.06% by maintaining 2 mg/l minimum oxygen at 2 h HRT through aerobic treatment with power consumption of only 8.4 units/month.
During the study, the pH of the effluent in the aerobic chamber (7.80 ± 0.40) was found to be higher than that of the effluent of the anaerobic chamber (7.69 ± 0.35).Decrease in the pH value in anaerobic chamber and subsequently increased in the pH value in the effluent is mainly due to oxidation process in aerobic reactor.The value of pH is decreased due to the processes of hydrolysis, acidogenesis and acetogenesis [35].Later these acids are converted into methane, carbon dioxide, and ammonia [2].The methane leaves the reactor while the carbon dioxide forms bicarbonate inside the anaerobic system, which increases the pH and alkalinity of the effluent as compared to the effluent present in the inlet.In the aerobic chamber the effluent's pH increases due to the presence of a certain group of nitrifying bacteria, which convert ammonia into nitrate via nitrite [36].The nitrifying bacteria require approximately a neutral pH of (7-8) to grow [37].These autotrophs use carbon dioxide as a carbon source, which plays an important role in the removal of nutrients.This indicates that complete nitrification can be expected in the reactor at pH 7 to 8 [38].
Phosphorus removal efficiency of the sequential system was found to be 82.33 ± 2.89, which was only 32.5% in the anaerobic chamber due to the presence of phosphate accumulating organism (PAO), which are unable to reproduce in the anaerobic environment.They also have an unusual behaviour for intake of volatile fatty acids under normal conditions, which enables them to store intracellular carbon compounds.These compounds expand upon entering the aerobic chamber and thus complete the supply of phosphate [39].
During the steady-state flow period (PFF1), which spanned from day 0 to day 160, we encountered both the initial unstable phase and the period after 50 days when the reactor was virtually stabilized.During this phase, sewage was fed into the reactor at a constant rate and without turbulence.However, our analysis showed natural daily variations in organic loading rates.As shown in figure 2, the unregulated organic content of real sewage caused perceptible fluctuations in the BOD and TSS inlet concentration graphs.Despite these fluctuations, the efficacy of the reactor improved significantly after stabilization.Following the stabilization phase, the reactor demonstrated remarkable efficacy in removing pollutants during PFF1, reaching a 90% removal rate for both BOD and TSS.
In case of PFF2 shock loading, the average removal efficiency of COD, TSS and BOD seemed to decrease slightly from PFF1, which can be clearly seen in figure 2. The inlet concentration of BOD fluctuated from 310 to 385 mg L −1 and TSS from 305 to 410 mg L −1 between 160 to 180 days, but at this condition the reactor maintained the effluent quality below the disposal standard of MoEFCC 2017.This is really appreciable that the system maintains its stability even under hydraulic and organic loading conditions, which can be attributed to its design, flow pattern and good hydraulic efficiency.
Figures 2(a) and (b) show the pollutants removal efficiency of the system in terms of BOD and TSS with their inlet and outlet concentrations over time.The system remained stable even after large fluctuations in the concentrations of raw sewage and the average removal efficiency of the system was found to be 93.33 ± 1.84% and 93.87 ± 2.63% in terms of BOD and TSS respectively.After 50 days, it was found that the system had almost maintained its stability.The average concentrations of BOD and TSS at the outlet were found to be 21.73 ± 4.98 and 24.41 ± 10.83 mg / l respectively, which fulfilled the disposal criteria of Indian standards at onsite level.The system was run on PFF1 for 160 days, after which the system was shifted to PFF2 for 20 days, resulting in slight decrease in the performance of the system.
In the temperature study related to COD removal efficiency, it was observed that the biodegradation of organic matter remained relatively unaffected by temperature variation, as evident from figure 3(a).However, the high temperature helped the reactor stabilization process, and the reactor became stable quickly.Because the temperature during the first 50 days was higher than the temperature of the remaining days.As the reactor stabilized, the atmospheric temperature decreased.However, the stability of the reactor was advantageous; Even with fluctuations between 27 and 32 °C, the removal efficiency remained consistently high at around 90%.This stability had minimal effect on the shock loading condition.Figure 3(b) shows the relationship of COD removal efficiency with the organic loading rate (excluding initial 15 days) of the system.The linear trend line shows the in which the overall COD removal efficiency of the system appears to be increasing with an increase in organic  loading rate in maximum cases.The highest removal efficiency was found to be 95% at the organic loading rate of 0.48 kg.COD / m 3 and lowest 67% at the organic loading rate of 0.34 kg.COD / m 3 [2].However, some of the data is below the trend line which shows samples taken during the initial phase when the reactor was trying to stabilize.

Microbial characteristics
The microbial concentration in the sequential system is mentioned in table 3.During domestic wastewater treatment, removal of coliform group is important to know as it is generally categorised as pathogens.The removal efficiency of TC, FC and E. coli were observed as 96.97 ± 2.10, 98.69 ± 1.12 and 98.67 ± 2.19%, respectively.This substantially high removal efficiency can be attributed to the anaerobic-aerobic conditions created by the combination of the two mechanisms, namely physical (adding media property) [40] and organic (biological and chemical) [41].The disease-causing bacteria, such as salmonella and shigella, were also tested and the removal efficiency was 96.91 ± 1.84% and 94.04 ± 3.16% respectively.The removal efficiency in PFF 2 condition decreased slightly.

Morphology of sludge
In a FESEM study, the sample's surface is bombarded with electrons, producing high-resolution images that show the sample's topography in all of its exquisite complexity [42].FESEM can reveal the spatial distribution of anaerobic and aerobic microorganisms in the context of the sludge from the anaerobic-aerobic reactor.This information is essential for demonstrating the efficacy of the sequential treatment strategy since the existence of both microbial groups suggests that concurrent biological processes are occurring.The FESEM analysis of sludge was analysed to detect the biological morphology in the system as shown in figure 4. The FESEM images of anaerobic sludge in the sequential system are labelled as 4(a), 4(b) & 4(c).Figure 4(a) shows ring type and filamentous type bacteria.In figure 4(b), only filamentous cable bacteria are visible while in figure 4(c) a large swarm of small rod-shaped bacteria is visible, which has a huge contribution in anaerobic digestion and formation of biogas.The presence of filamentous cable bacteria is crucial in anaerobic wastewater treatment as they possess a unique ability to enhance electron transfer activities.This process facilitates the metabolic activities of microorganisms and expedites the breakdown of organic contaminants.These bacteria construct complex networks in anaerobic conditions, helping the decomposition of complicated organic compounds.
Conductive filaments facilitate the flow of electrons to terminal electron acceptors, such as sulfate and nitrate, which play a crucial role in anaerobic digestion processes.Filamentous cable bacteria play a crucial role in augmenting the efficacy of wastewater treatment procedures through their ability to catalyze redox reactions [43,44].Figure 4(d) is the FESEM image of sludge present in the aerobic filter, in which many micro-structures of granules, bacterial and algal cells are visible.FESEM examination of reactor sludge opens a window into the microscopic world of the biofilm, revealing the composition and structure of anaerobic and aerobic microbial populations.This data validates the biological treatment processes that occur within the reactor and aids in understanding the mechanisms that drive its efficiency [23].

Conclusions
The obtained results show the effectiveness of the sequential anaerobic-aerobic integrated settler-based biofilm reactor for the onsite treatment of domestic wastewater and consolidate their advantage over the conventional aerobic systems.The average removal efficiency of the sequential anaerobic-aerobic onsite treatment system in terms of TSS, BOD, COD, NH 4 + −N, TN, TP, TC, FC, and E. coli was 93.9 ± 2.6, 93.Furthermore, the FESEM analysis of the sludge formed within the reactor confirmed the presence of both anaerobic and aerobic microbial morphology.This observation lends strong support to the occurrence of concurrent biological treatment processes within the reactor, thereby strengthening its operational efficiency.The integrated approach of these studies validates the reactor's promise for efficient and successful onsite treatment of domestic wastewater.
. The unit dimensions of the first, second and third chambers are 288 mm × 280 mm × 450 mm (L × B × H), 141 mm × 280 mm × 450 mm (L × B × H) and 135 mm × 280 mm × 450 mm (L × B × H) respectively.The flow of the wastewater was in the upward direction throughout the ISBR.The chambers were interconnected with the help of vertical acrylic pipes as shown in figure 1.The second and third chambers of the ISBR were filled with aqwise carrier media and MBBR media respectively.The manufacturing details of aqwise carrier are 10 to13 mm diameter, 15 mm length, model no.ABC5, MoC-HDPE (Recycled high-density polyethylene) and surface area-650 m 2 /m 3 .The manufacturing details of MBBR media are 22 mm diameter, 25 mm length, model no.PP22, MoC-virgin polypropylene uv stabilized and surface area-250m 2 /m 3 [4, 16].

Figure 1 .
Figure 1.Schematic diagram of sequential system at laboratory scale.

Figure 2 .
Figure 2. Evaluation of the parameter concentrations at inlet and outlet with percentage removal (a) BOD and (b) TSS.

Figure 3 .
Figure 3. Evolution of the COD removal efficiency with variable (a) sewage temperature and (b) organic loading rate.

Table 1 .
Influent concentration of raw sewage.
a Average value ± standard deviation

Table 2 .
Average concentration and removal efficiency of the sequential anaerobic-aerobic treatment unit at steady-state condition.

Table 3 .
Results of microbial concentration and removal efficiency at steady-state condition.