Abstract

This study was conducted to evaluate the treatment performance of the anoxic–oxic–anoxic processes in illuminated biofilm reactor (AOA-IBR) in removing organics and nitrogen contained in septic tank effluent. The 27 L of the AOA-IBR was illuminated with red light-emitting diode (LED) lamps (peak wavelength of 635 nm, intensity of 100 μmol/(m2s)). Three types of biofilm media, namely ball ring®, plastic sheets and zeolite beads, were placed in the anoxic, oxic and anoxic zones, respectively, of the reactor to support the growth of microalgal–bacterial biofilm. The AOA-IBR was continuously fed with septic tank effluent and operated at hydraulic retention times (HRTs) of 24, 48 and 72 h. The experimental results found the increases in chemical oxygen demand (COD), total nitrogen (TN) and ammonia nitrogen (NH4-N) removal efficiencies with increasing HRTs in which the HRT of 72 h resulted in 78.6, 72.8 and 90.6% removals of COD, TN and NH4-N, respectively. The effluent quality of the AOA-IBR could meet the ISO 30500 effluent standards for Non-Sewered Sanitation Systems. The predominant microalgal biofilm species was observed to be Oscillatoria sp., while Proteobacteria was the predominant bacterial phylum found in the biofilm growing in the reactor. The above results suggested the applicability of the AOA-IBR in improving septic tank treatment performance which should result in better water pollution control.

HIGHLIGHTS

  • The anoxic–oxic–anoxic processes in illuminated biofilm reactor (AOA-IBR) as an on-site post-treatment system could effectively treat chemical oxygen demand (COD) and total nitrogen (TN) concentrations in septic tank effluent.

  • The Stover–Kincannon model could be applicable to determine the kinetic values for COD and TN removal.

  • The observed major microalgal and bacterial groups were corresponding to the results of COD and TN removal.

INTRODUCTION

Decentralized wastewater treatment systems or on-site systems such as septic tanks are widely used in several areas of developing countries to treat blackwater (Taweesan et al. 2015). In Thailand, about 84% of the on-site systems are septic tanks, but their effluent characteristics were found to contain high concentrations of chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) and still do not meet the national effluent standards (Taweesan et al. 2015). Although leaching or drainage fields are employed to further treat the effluent of the on-site systems in most developed countries, due to land limitation and other constraints, the effluent of these on-site systems in developing countries is usually discharged directly into nearby environment, resulting in contamination of surrounding soil, surface and ground water (Richards et al. 2017). Therefore, for urban areas of developing countries, there is a need to develop a compact system to further treat the on-site effluent to reduce organics, nutrients and fecal microorganisms to meet the effluent discharge standards. The illuminated biofilm reactor (IBR) or photobioreactor (PBR) is a system that involves microalgal–bacterial symbiotic reactions in which oxygen produced by microalgae during photosynthesis is utilized by bacteria to decompose the organic compounds (Posadas et al. 2013; Boelee et al. 2014). Moreover, in the PBR, ammonia nitrogen (NH4-N) compound can be removed by biomass uptake as well as nitrification/denitrification reactions. The microalgal–bacterial PBR system has been applied to remove organic and nutrient matters from blackwater, but it was not effective in removing nutrient (TN and TP) concentrations because of the fluctuation in climatic conditions which resulted in limited natural sunlight for microalgal growth, high nutrient loading rates as well as short hydraulic retention times (HRTs) of the treatment systems (Surinkul et al. 2017). Therefore, there is a need to modify the PBR to promote the efficient growth of microalgae and nitrification–denitrification reactions for better nutrient removal.

Due to its high biomass density and promising treatment performance, the biofilm wastewater treatment reactor has been widely used as a post-treatment unit (Chaiwong et al. 2018). Several biofilm materials such as polyethylene and polyethylene terephthalate were employed to provide the surface area for the growth of biofilm microorganisms (Barwal & Chaudhary 2014; Surinkul et al. 2017). Furthermore, due to its high surface area for bacterial growth and rich adsorptive capacity, porous media, namely zeolite, has been applied to enhance TN removal in biofilm treatment systems (Yang et al. 2017). Moreover, to overcome the uncontrolled lighting conditions of the natural sunlight in the IBR, red light-emitting diode (LED) light (having wavelengths of 600–700 nm) was selected as an artificial light source to increase the growth rate of microalgal cultivation to achieve maximum rates of photosynthesis (Chaiwong et al. 2018).

In this study, it was hypothesized that effective TN removal in the anoxic–oxic–anoxic processes in illuminated biofilm reactor (AOA-IBR) could be achieved by enhancing nitrification/denitrification reactions, biomass uptake and increased adsorption by zeolite. The AOA-IBR was divided into three zones, i.e. anoxic, oxic and anoxic in series. The first zone received combined influent and recycled effluent from the 3rd zone. The nitrification reactions were designed to occur in the aerobic zone (2nd zone), while the denitrification reactions were supported to occur in the first and third zones. The experiments aimed to evaluate the performance of the AOA-IBR system in removing both COD and TN from a septic tank effluent and to determine the related kinetic constants applicable for design and operation of the AOA-IBR system.

METHODS

Wastewater characteristics

The septic tank effluent used as influent wastewater in this study was collected three times a week from septic tanks of some households located near the AIT campus, Pathumthani, Thailand, and preserved in the refrigeration room (under temperature of 5 °C) before feeding to the AOA-IBR. The septic tank effluent characteristics (Table 1) had average total suspended solids (TSS), COD, TN, NH4-N and TP concentrations of about 1,065, 693, 169, 132 and 24 mg/L, respectively. The N:P ratio of the wastewater was observed to be 8:1 which was in the recommended range of 8–45 for microalgal–bacterial systems (Cuellar-Bermudez et al. 2017). The average NOX-N (nitrate (NO3-N) and nitrite (NO2-N)) concentration was less than 1.0 mg/L.

Table 1

Characteristics of the influent wastewater

ParametersUnitsValue
pH – 7.63 ± 0.17 
DO mg/L 0.20 ± 0.4 
Temperature °C 28 ± 1.2 
TSS mg/L 1,065 ± 862 
COD mg/L 693 ± 130 
TNa mg/L 169 ± 23 
NH4-N mg/L 132 ± 19 
TP mg/L 24 ± 13 
ParametersUnitsValue
pH – 7.63 ± 0.17 
DO mg/L 0.20 ± 0.4 
Temperature °C 28 ± 1.2 
TSS mg/L 1,065 ± 862 
COD mg/L 693 ± 130 
TNa mg/L 169 ± 23 
NH4-N mg/L 132 ± 19 
TP mg/L 24 ± 13 

Number of samples (n) = 33.

aNOX-N less than 1.0 mg/L.

Experimental setup

The AOA-IBR used in the experiment was made of acrylic material with a dimension of 0.15 × 0.40 × 0.60 m (width:depth:length) and a working volume of 27 L (Figure 1). It included an ANX1 (anoxic1) of 3 L, oxic or AOX (aerobic) of 18 L and ANX2 (anoxic2) of 6 L, in series (Metcalf & Eddy 2014). Due to their commercial availability, affordability and technical characteristics, three types of biofilm media including ball ring®, plastic strip and zeolite beads, as illustrated in Table 2, were selected. Two types of the biofilm media, namely ball ring® and plastic strip, were placed in the ANX1 and AOX zones, respectively, while the ANX2 zone was filled with zeolite beads for biofilm growth and simultaneous adsorption. The AOX zone was operated with four submerged red LED lamps (Bogdan® J&Y Trading, Thailand) having a peak wavelength of 635 nm and light intensity of 100 μmol/(m2s), suitable for the growth of microalgae (Chaiwong et al. 2018). Aeration with a flow rate of 7.6 L/h was also provided in the AOX zone to enhance mixing condition and maintaining dissolved oxygen (DO) concentrations higher than 2 mg/L, suitable for nitrification reactions (Metcalf & Eddy 2014).

Table 2

Characteristics of biofilm media used in the experiments

PropertiesConfigurations
ANX1 zoneAOX zoneANX2 Zone
Types Ball ring®a Plastic strip Zeolite 
Biofilm media Polyethylene Polyethylene terephthalate Zeolite 
Size (mm) 35 × 35 (Length × Height) 120 (Ø) 1.80 (Ø) 
Specific area (m2/m3200 210 43 
Porosity (%) 93 92 47 
Photo    
PropertiesConfigurations
ANX1 zoneAOX zoneANX2 Zone
Types Ball ring®a Plastic strip Zeolite 
Biofilm media Polyethylene Polyethylene terephthalate Zeolite 
Size (mm) 35 × 35 (Length × Height) 120 (Ø) 1.80 (Ø) 
Specific area (m2/m3200 210 43 
Porosity (%) 93 92 47 
Photo    

Ø, diameter (mm).

aBio Media and Water System Co., Ltd.

Figure 1

Schematic diagrams of the AOA-IBR experimental setup.

Figure 1

Schematic diagrams of the AOA-IBR experimental setup.

Microalgal–bacterial biofilm cultivation

The AOX zone of the AOA-IBR system was inoculated with 14 L of a microalgal consortium collected from an algal pond water in AIT campus, and 10 L of activated sludge samples (MLSS 0.9 g/L) taken from the AIT wastewater treatment plant (Chaiwong et al. 2018). The microalgal–bacterial cultivation was conducted under red LED lamps (14.4 Watt) and aeration, similar to those described in the ‘Experimental setup’ section. The cultivation was carried out for 30 days to achieve the biofilm biomass concentration of about 0.6 g/L (2.8 g/m2media), sufficient for the growth of the microalgal–bacterial biofilm on the plastic strip media before start-up of the AOA-IBR experiments (Chaiwong et al. 2018).

Operating conditions

The AOA-IBR, continuously fed with the septic tank effluent (24 h/day), was operated under various HRTs of 24, 48 and 72 h for 72 days with 100% internal recirculation from ANX2 to ANX1 (Yan et al. 2016), as shown in Table 3. The organic loading rate (OLR) and the nitrogen loading rate (NLR) were varied from 0.69 to 0.25 gCOD/(L d) and 0.19 to 0.05 gN/(L d), respectively. Additionally, the AOX zone of the AOA-IBR was continuously illuminated (24 h/day) by the red LED light in order to result in better photosynthetic oxygen production for bacterial utilization in wastewater treatment efficiency and higher microalgal biomass productivity.

Table 3

Operational conditions of the AOA-IBR experiment

HRT (h)Operation period (day)OLRs (gCOD/(L d))NLRs (gN/(L d))
24 12 0.69 0.19 
48 22 0.34 0.08 
72 38 0.25 0.05 
HRT (h)Operation period (day)OLRs (gCOD/(L d))NLRs (gN/(L d))
24 12 0.69 0.19 
48 22 0.34 0.08 
72 38 0.25 0.05 

Analytical methods

Wastewater analysis

Influent and effluent samples of the AOA-IBR were collected three times a week to measure DO, pH and temperature using a multiparameter portable meter (HATCH-HQ40d, USA). Analysis of TSS, volatile suspended solids (VSS), COD, total Kjeldahl nitrogen (TKN), NH4-N and TP concentrations were according to Standard Methods for the Examination of Water and Wastewater (APHA 2017) (see Supplementary Material, Table S1). Meanwhile, the collected samples were filtered with GF/C filters with a pore size of 0.45 μm before the analysis of NO3-N and NO2-N concentrations by a portable analyzer (HACH DR-2700, Germany) using the reagents of NitraVer®6 Nitrate and NitriVer2 Nitrite Reagent Powder Pillows, respectively, according to the manufacturer procedures (©Hach Company/Hach Lange GmbH, USA). Accordingly, the TN concentration in the wastewater was equal to combined TKN, NO3-N and NO2-N concentrations. In addition, in order to determine TN and NH4-N removal efficiencies of the ANX1+AOX and ANX2, the effluent from each zone of the AOA-IBR (see sampling points in Supplementary Material, Figure S1) was also sampled for the wastewater analysis according to the above-mentioned methods. Light intensity of the red LED lamps was measured by a quantum meter (Model MQ-200, USA). The laboratory analysis was undertaken at the Environmental Engineering and Management laboratory, AIT, Pathumthani, Thailand.

Microalgal–bacterial species analysis

At the end of the AOA-IBR operation at the HRT of 72 h, the suspended and attached-growth biomass samples were collected from the AOX and ANX2 zones of the AOA-IBR for analysis of the microalgal–bacterial species. The biofilm biomass was randomly scraped from biofilm media at 5, 25 and 40 cm depths of the AOX and ANX2 zones. The collected biomass samples from the AOX were then diluted in 1 L of distilled water, fixed with Lugol acid and stored at 4 °C before analysis of microalgal species at the Thailand Institute of Scientific and Technology Research (TISTR) laboratory, Pathumthani, Thailand, by using the microscopic method (microalgal strain identification and microalgal cell count under a microscope) according to APHA (2017) (see Supplementary Material, Table S1). The bacterial species of the suspended and biofilm biomass samples were analyzed by next-generation sequencing (NGS) platform (Illumina's Hiseq) (Ibekwe et al. 2017). Briefly, the samples were firstly centrifuged prior to total DNA extraction by using a CTAB/SDS method (Demeke et al. 2010). Polymerase chain reactions (PCRs) (targeting bacterial genes of 16S rRNA) were amplified by using specific primer, e.g. 16S V4: 515F-806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R (Ibekwe et al. 2017), with the Phusion® high-fidelity PCR master mix (New England Biolabs, UK). Sequencing analysis was performed on Illumina platform (Hiseq) with similarity more than 97% and was assigned to the same operational taxonomic units (OTUs) via Uparse software (Uparse v7.0.1001 http://drive5.com/uparse/) (see Supplementary Material, Figures S2 and S3).

Kinetic model

The Stover–Kincannon model is commonly used for determining kinetic constants based on organic and nutrients removal rates against OLR in the AOA-IBR as shown in Equation (1). This kinetic model was originated for determining kinetic constants of a rotating biological contactor. Later, the model has been applied to determine kinetic constants of both the suspended and biofilm microorganism in other types of the bioreactor (Yang et al. 2015; Derakhshan et al. 2018).
formula
(1)
in which, So and Se are the influent and the effluent substrate concentrations (mg/L), Umax is the maximum utilization rate (g/(L d)), KB is the saturation value constant (g/(L d)), V is reactor volume (L), Q is the flow rate (L/d). The experimental data obtained from this study were used to determine the KB and Umax values of the AOA-IBR system for comparison with other biofilm systems and application of the results.

Statistical analysis

The statistical comparisons among data obtained from the experiments of the AOA-IBR were analyzed by using a variance analysis (ANOVA) in SPSS software (V.16.0).

RESULTS AND DISCUSSION

At the steady-state conditions (relatively constant effluent COD and TN concentrations for 4 weeks), the average pH values in the ANX1, AOX and ANX2 zones were observed to be 7.7 ± 0.18, 8.1 ± 0.08 and 8.1 ± 0.26, respectively, while the DO concentrations were 0.2 ± 0.03, 3.7 ± 1.17 and 0.4 ± 0.12 mg/L, respectively (n = 33) (see Supplementary Material, Table S2). The AOX zone with the DO concentrations more than 2 mg/L was considered suitable for growth of aerobic and nitrifying bacteria (Metcalf & Eddy 2014), while the DO concentrations of less than 1.0 mg/L in the ANX1 and ANX2 could support denitrifying bacteria to remove NO3-N and NO2-N in the wastewater via denitrification reactions (Shen et al. 2009). Accordingly, the average pH value and DO concentration of the effluent samples were found to be 8.0 ± 0.18 and 0.94 ± 0.32 mg/L, respectively, (see Supplementary Material, Table S2). Probably due to continuous lighting and aeration, there were no statistical differences among pH value and DO concentrations in the AOA-IBR under various HRTs. The average temperature of 28 ± 0.91 °C was recorded during the experimental period.

Treatment performance of the AOA-IBR system

As shown in Table 4, the TSS, COD, TN and NH4-N removal efficiencies were used to evaluate the treatment performance of the AOA-IBR system operating at HRTs of 24, 48 and 72 h.

Table 4

Treatment performance of the AOA-IBR system

HRT (h)Parameters
TSS (n = 33)
COD (n = 33)
TN (n = 33)
NH4-N (n = 33)
Removal (%)Effluent (mg/L)Removal (%)Effluent (mg/L)Removal (%)Effluent (mg/L)Removal (%)Effluent (mg/L)
24 98.3 ± 0.7 12.6 ± 5.7 74 ± 1.9 176.3 ± 18.1 60 ± 14.6 75.6 ± 24.6 75 ± 3.9 34.5 ± 3.7 
48 94.4 ± 5.1 27 ± 15.1 76 ± 9.2 164.3 ± 46.1 67 ± 4.3 54.2 ± 11.5 79 ± 3.6 26.8 ± 5.1 
72 94.4 ± 3.4 14.8 ± 4.1 79 ± 6.9 145.5 ± 47.3 73 ± 2.3 42.2 ± 15.7 91 ± 3.4 11.1 ± 4.4 
HRT (h)Parameters
TSS (n = 33)
COD (n = 33)
TN (n = 33)
NH4-N (n = 33)
Removal (%)Effluent (mg/L)Removal (%)Effluent (mg/L)Removal (%)Effluent (mg/L)Removal (%)Effluent (mg/L)
24 98.3 ± 0.7 12.6 ± 5.7 74 ± 1.9 176.3 ± 18.1 60 ± 14.6 75.6 ± 24.6 75 ± 3.9 34.5 ± 3.7 
48 94.4 ± 5.1 27 ± 15.1 76 ± 9.2 164.3 ± 46.1 67 ± 4.3 54.2 ± 11.5 79 ± 3.6 26.8 ± 5.1 
72 94.4 ± 3.4 14.8 ± 4.1 79 ± 6.9 145.5 ± 47.3 73 ± 2.3 42.2 ± 15.7 91 ± 3.4 11.1 ± 4.4 

Because there was no significant difference (P < 0.05), the average TSS removal efficiency of three experimental conditions of the AOA-IBR was found to be 95.7 ± 2.3%, resulting in the average effluent TSS concentration of 18.1 ± 7.8 mg/L which could meet the type B effluent standard (≤30 mg/L) of the ISO 30500: Non-Sewered Sanitation Systems (ISO 2018) and the domestic effluent standard of Thailand (≤50 mg/L) (PCD Thailand 2017). Because the last zone (ANX2) of the AOA-IBR was filled with the zeolite beads, the TSS was retained in the reactor by the filtration of the zeolite beads.

The highest COD removal efficiency of 79 ± 6% was achieved in the AOA-IBR operating under the HRT and OLR of 72 h and 0.25 gCOD/(L d), respectively, corresponding to the average effluent COD concentration of 145 ± 47 mg/L which also comply with type B effluent standard (≤150 mg/L) of the ISO 30500. However, the lower COD removal efficiencies of 76 ± 14% and 74 ± 2% (P < 0.05) were found in the AOA-IBR operating under the HRT and OLR of 48 and 24 h and 0.34 and 0.69 gCOD/(L d), respectively. Due to the relatively stable influent COD concentration of 693 ± 130 mg/L during these three experimental stages, increasing HRT (or decreasing OLR) resulted in increased COD removal efficiencies in the AOA-IBR (R2 = 0.98). The COD concentrations in the influent wastewater were mainly degraded by aerobic heterotrophs in the AOX zone, while some of the COD concentrations were unutilized in denitrification processes by denitrifiers in the ANX1 and ANX2 zones (Posadas et al. 2013; Boelee et al. 2014) corresponding to the results of the bacterial community (Figure 5).

Similarly, the highest TN removal efficiency of 73 ± 12% was observed in the AOA-IBR operating under the HRT and NLR of 72 h and 52 gN/(L d), respectively (Table 4), whereas the lower TN removal efficiencies of 67 ± 5% and 60 ± 17% (P < 0.05) were found with the HRT of 48 and 24 h, respectively, similar to the previous study of Karapinar-Kapdan & Aslan (2008). TN removal of the AOA-IBR was also found to meet the ISO 30500 (≥70% more than TN removal efficiency). The NH4-N removal efficiencies were found to be 91 ± 4, 79 ± 4 and 75 ± 5% under the HRT of 72, 48 and 24 h, respectively (Table 4), indicating effective nitrification reactions and microalgal uptake of NH4-N in the AOX zone. Moreover, the average NO3-N concentration in the AOX zone was found to increase from less 1 than to 29 mg/L, indicating the occurrence of nitrification processes, corresponding to the results of the bacterial community as revealed in the ‘Bacterial community in the AOX and the ANX2 zones’ section. Because the pH value of about 8 was not high enough, the NH4-N removal via NH3 volatilization in the AOA-IBR was considerably negligible (Valero & Mara 2007). Additionally, the TN and NH4-N removal efficiencies of 49 and 81%, respectively, were found in ANX1 and AOX zones of the AOA-IBR at the HRT of 72 h, while 24 and 10% were removed in the ANX2 zone (Figure 2). Because of the suitable condition (DO < 2 mg/L) in the ANX1 zone, some TN could be removed via denitrification processes corresponding to the data of bacterial community as described in the ‘Bacterial community in the AOX and the ANX2 zones’ section. The bacterial community in the AOX and the ANX2 zones, while some TN was assimilated into the microalgal–bacterial biofilm in the AOX zones (Boelee et al. 2014). Additionally, some TN could be removed by denitrification in the inner layers of the biofilm, and further investigation in this respect is recommended. Some previous studies have reported that the red LED light is effective for the growth of microalgae in the microalgal based treatment systems (Zhang et al. 2017; Chaiwong et al. 2018). Moreover, because about 24 and 10% of TN and NH4-N were found to be removed in the ANX2 zone containing the zeolite media (Figure 2), it could be hypothesized the denitrification reaction (Yang et al. 2017).

Figure 2

(a) TN and (b) NH4-N removals in the AOA-IBR system.

Figure 2

(a) TN and (b) NH4-N removals in the AOA-IBR system.

Kinetics model of the AOA-IBR system and its application

The Stover–Kincannon model was applied to determine the maximum utilization rate (Umax) and saturation value constant (KB) for COD and TN removal in the AOA-IBR by considering the linear plot of VQ/(SoSe) against V/(QSo), as shown in Figure 3(a) and 3(b), respectively. From Figure 3(a), the kinetic values of the Umax and KB for COD removal in the AOA-IBR were calculated to be 5.00 and 3.56 gCOD/(L d), respectively (R2 = 0.94), which were relatively higher than other biofilm reactors, namely the biofilm photobioreactor (BPBR) (Surinkul et al. 2017; Chaiwong et al. 2018), the hybrid microalgal membrane photobioreactors (HMPBR) and the microalgal membrane photobioreactors (MPBR) (Derakhshan et al. 2018; Table 5). The high kinetic values for COD removal in the AOA-IBR could be due to the combination of the aerobic (AOX) and anoxic (ANX1 and ANX2) zones which enhanced organic biodegradation and denitrification reactions (Zhou et al. 2017), respectively. In addition, the Umax and KB values for TN removal in the AOA-IBR were calculated to be 0.59 and 0.76 gN/(L d), respectively, with R2 of 0.92 (Figure 3(b)), higher than the Umax and KB values of the BPBRs treating synthetic wastewater (Karapinar-Kapdan & Aslan 2008) and septic tank effluent (Chaiwong et al. 2018), the bio-diatomite biofilm reactor (BBR) treating polluted raw water (Yang et al. 2015), as revealed in Table 5. The high Umax and KB values indicated the high efficiencies of the AOA-IBR system in recovery COD and TN concentration from wastewater.

Table 5

Comparison of kinetic constants of Stover–Kincannon model for COD and TN removals

SystemType of wastewaterSubstrateKinetic constantUnitValuesR2References
AOA-IBR Septic tank effluent COD Umax gCOD/(L d) 5.00 0.94 This study 
KB gCOD/(L d) 3.56 
TN Umax gN/(L d) 0.59 0.92 
KB gN/(L d) 0.76 
BPBR Blackwater COD Umax gCOD/(L d) 0.17 0.93 Surinkul et al. (2017)  
KB gCOD/(L d) 0.19 
BPBR Septic tank effluent COD Umax gCOD/(L d) 0.41 0.95 Chaiwong et al. (2018)  
KB gCOD/(L d) 0.58 
TN Umax gN/(L d) 0.03 0.85 
KB gN/(L d) 0.04 
HMPBR Secondary-treated effluent COD Umax gCOD/(L d) 3.31 0.99 Derakhshan et al. (2018)  
KB gCOD/(L d) 3.39 
MPBR Secondary-treated effluent COD Umax gCOD/(L d) 1.70 0.99 Derakhshan et al. (2018)  
KB gCOD/(L d) 1.99 
BBR Polluted raw water TN Umax gN/(L d) 0.33 0.94 Yang et al. (2015)  
KB gN/(L d) 0.41 
BPBR Synthetic wastewater TN Umax gN/(L d) 0.01 0.93 Karapinar-Kapdan & Aslan (2008)  
KB gN/(L d) 0.01 
SystemType of wastewaterSubstrateKinetic constantUnitValuesR2References
AOA-IBR Septic tank effluent COD Umax gCOD/(L d) 5.00 0.94 This study 
KB gCOD/(L d) 3.56 
TN Umax gN/(L d) 0.59 0.92 
KB gN/(L d) 0.76 
BPBR Blackwater COD Umax gCOD/(L d) 0.17 0.93 Surinkul et al. (2017)  
KB gCOD/(L d) 0.19 
BPBR Septic tank effluent COD Umax gCOD/(L d) 0.41 0.95 Chaiwong et al. (2018)  
KB gCOD/(L d) 0.58 
TN Umax gN/(L d) 0.03 0.85 
KB gN/(L d) 0.04 
HMPBR Secondary-treated effluent COD Umax gCOD/(L d) 3.31 0.99 Derakhshan et al. (2018)  
KB gCOD/(L d) 3.39 
MPBR Secondary-treated effluent COD Umax gCOD/(L d) 1.70 0.99 Derakhshan et al. (2018)  
KB gCOD/(L d) 1.99 
BBR Polluted raw water TN Umax gN/(L d) 0.33 0.94 Yang et al. (2015)  
KB gN/(L d) 0.41 
BPBR Synthetic wastewater TN Umax gN/(L d) 0.01 0.93 Karapinar-Kapdan & Aslan (2008)  
KB gN/(L d) 0.01 
Figure 3

Stover–Kincannon model of COD (a) and TN (b) removal in the AOA-IBR system.

Figure 3

Stover–Kincannon model of COD (a) and TN (b) removal in the AOA-IBR system.

Hence, the obtained kinetic values for COD and TN removal in the AOA-IBR could be substituted into Equation (1) to become Equations (2) and (3), respectively, which could be used to estimate the effluent COD and TN concentrations of the AOA-IBR as well as to design size of the AOA-IBR for treatment of septic tank effluent.

COD removal:
formula
(2)
TN removal:
formula
(3)

Microalgal community in the AOX zone of the AOA-IBR system

Due to the presence of both suspended and biofilm biomass in the AOX zone, its samples were collected for analysis of the major microalgal species. The predominant biofilm microalgal species was Oscillatoria sp. (83.3% or 1.27 × 104 filaments/mL), while Scenedesmus sp. (85.2% or 1.94 × 104 colony/mL) was predominant in suspended microalgal species, as shown in Figure 4. Other microalgal species densities are detailed in Supplementary Material, Table S3. These two microalgal species were also found to be predominant in other microalgal systems used for wastewater treatment, especially for TN removal via the microalgal biomass assimilation as well as producing oxygen (Boelee et al. 2014; Rasheedy et al. 2017).

Figure 4

Microalgal communities in the AOA-IBR system.

Figure 4

Microalgal communities in the AOA-IBR system.

Figure 5

Phylum of the bacterial group in the AOA-IBR system.

Figure 5

Phylum of the bacterial group in the AOA-IBR system.

Bacterial community in the AOX and the ANX2 zones

Both suspended and biofilm biomass samples in the AOX and the ANX2 zones were collected for examination of the predominant bacterial species and the results are shown in Figure 5. Additionally, the species diversity curves reveal about 40,000 of the sequence number which was retrieved from the NGS technique (see Supplementary Material, Figure S4).

In the AOX zone, the phylum of Proteobacteria (∼62%) was found to be the predominant suspended bacterial group, followed by Bacteroidetes (∼12%), Cloacimonetes (∼10%) and Firmicutes (∼8%), whereas the phylum of Cyanobacteria (∼52%) was observed to be the predominant biofilm bacterial group followed by the phylum of Proteobacteria (∼29%), Bacteroidetes (∼3%) and Nitrospirae (∼3%) (Figure 5). The cyanobacteria or blue-green algae were responsible for photosynthetic oxygen production for the heterotrophic bacteria to biodegrade and taking up TN in the wastewater (Rasheedy et al. 2017). Meanwhile, in the ANX2 zone, the phylum of Proteobacteria (∼50%) was found to be the predominant suspended bacterial group, followed by the phylum of Actinobacteria (∼14%), Choroflexi (∼11%), Chorobi (∼9%) and Bacteroidetes (∼8%), as illustrated in Figure 5; similarly, the predominant biofilm bacterial group in the ANX2 zone were observed to be the phylum of Proteobacteria (∼47%) followed by the phylum of Actinobacteria (∼15%), Bacteroidetes (∼10%) and Nitrospirae (∼9%). Hence, it could be hypothesized that the observed bacterial phylum of Proteobacteria, Bacteroidetes and Firmicutes, the typical heterotrophic bacteria (Ibekwe et al. 2017), were responsible for COD degradation (Ibekwe et al. 2017), while the phylum of Nitrospirae was responsible for nitrification processes in the biofilm AOA-IBR system (Wongkiew et al. 2017). Some of the Proteobacteria consisting of denitrifying bacteria (Wongkiew et al. 2017) were hypothesized to be responsible for denitrification processes for TN removal in the ANX2 zone.

The overall results of this study suggest the technical feasibility of applying the AOA-IBR system for treating septic tank effluent to meet the ISO 30500 effluent standards for Non-Sewered Sanitation Systems. However, more pilot and full-scale experiments should be further conducted to validate these experimental results and determining the system's cost-effectiveness.

CONCLUSIONS

Based on the experimental results obtained from this study, the following conclusion is made:

  • The AOA-IBR could be applied as an on-site post-treatment system to effectively treat COD and TN concentrations in septic tank effluent via combined microalgal and bacterial reactions such as aerobic biodegradation, biomass assimilation and nitrification/denitrification as well as adsorption by zeolite media.

  • The highest of COD and TN removal efficiencies were achieved in the AOA-IBR system operating at the HRT of 72 h, OLR of 0.25 gCOD/(L d) and NLR 0.05 gN/(L d).

  • The Stover–Kincannon model was applied to determine the kinetic values for COD and TN removal in the AOA-IBR for use in designing a suitable size of the AOA-IBR treating septic tank effluent.

  • The major microalgal groups in the AOA-IBR were found to be Oscillatoria sp. and Scenedesmus sp., while Proteobacteria and Nitrospirae were the predominant bacterial phylum responsible COD and TN removal, respectively.

ACKNOWLEDGEMENTS

This research was financially supported by the Bangchak Initiative and Innovation Center (BIIC), the Royal Thai Government (RTG) and the Asian Institute of Technology (AIT), Bangkok, Thailand.

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

REFERENCES

REFERENCES
APHA
2017
Standard Methods for the Examination of Water and Wastewater
, 23rd edn.
United Book Press Inc.
,
USA
.
Boelee
N. C.
Temmink
H.
Janssen
M.
Buisman
C. J. N.
Wijffels
R. H.
2014
Balancing the organic load and light supply in symbiotic microalgal-bacterial biofilm reactors treating synthetic municipal wastewater
.
Ecological Engineering
64
,
213
221
.
Chaiwong
C.
Koottatep
T.
Surinkul
N.
Polprasert
C.
2018
Performance and kinetics of algal-bacterial photobioreactor (AB-PBR) treating septic tank effluent
.
Water Science and Technology
78
,
2355
2363
.
Cuellar-Bermudez
S. P.
Aleman-Nava
G. S.
Chandra
R.
Garcia-Perez
J. S.
Contreras-Angulo
J. R.
Markou
G.
Parra-Saldivar
R.
2017
Nutrients utilization and contaminants removal. A review of two approaches of algae and cyanobacteria in wastewater
.
Algal Research
24
,
438
449
.
Demeke
T.
Jenkins
G. R.
Demeke
T.
Jenkins
G. R.
2010
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits
.
Analytical and Bioanalytical Chemistry
396
,
1977
1990
.
Derakhshan
Z.
Mahvi
A. H.
Ehrampoush
M. H.
Ghaneian
M. T.
Yousefinejad
S.
Faramarzian
M.
Fallahzadeh
H.
2018
Evaluation of kenaf fibers as moving bed biofilm carriers in algal membrane photobioreactor
.
Ecotoxicology and Environmental Safety
152
,
1
7
.
Ibekwe
A. M.
Murinda
S. E.
Murry
M. A.
Schwartz
G.
Lundquist
T.
2017
Microbial community structures in high rate algae ponds for bioconversion of agricultural wastes from livestock industry for feed production
.
Science of The Total Environment
580
,
1185
1196
.
Karapinar-Kapdan
I.
Aslan
S.
2008
Application of the Stover–Kincannon kinetic model to nitrogen removal by Chlorella vulgaris in a continuously operated immobilized photobioreactor system
.
Journal of Chemical Technology & Biotechnology: International Research in Process, Environmental & Clean Technology
83
,
998
1005
.
Metcalf & Eddy
2014
Wastewater Engineering Treatment and Resource Recovery
, 5th edn.
McGraw-Hill
,
New York
.
ISBN:978-1-259-01079-8
.
PCD
2017
Standard Quality of Discharge for Wastewater in Thailand
.
Available from: http://www.pcd.go.th/info_serv/reg_std_water04. html#s3 (accessed 15 September 2017)
.
Posadas
E.
García-Encina
P. A.
Soltau
A.
Domínguez
A.
Díaz
I.
Muñoz
R.
2013
Carbon and nutrient removal from centrates and domestic wastewater using algal–bacterial biofilm bioreactors
.
Bioresource Technology
139
,
50
58
.
Rasheedy
A. G. M. S. H.
Farahat
M. A. D. A. Z.
Mohammed
T. A.
2017
The differential efficiency of Chlorella vulgaris and Oscillatoria sp. to treat the municipal wastewater
.
Journal of Biology, Agriculture and Healthcare
7
,
22
.
Richards
S.
Withers
P. J.
Paterson
E.
McRoberts
C. W.
Stutter
M.
2017
Potential tracers for tracking septic tank effluent discharges in watercourses
.
Environmental Pollution
228
,
245
255
.
Shen
J.
He
R.
Han
W.
Sun
X.
Li
J.
Wang
L.
2009
Biological denitrification of high-nitrate wastewater in a modified anoxic/oxic-membrane bioreactor (A/O-MBR)
.
Journal of Hazardous Materials
172
,
595
600
.
Surinkul
N.
Koottatep
T.
Chaiwong
C.
Singhopon
T.
2017
Modified cesspool system with upflow sludge tank and low-cost photobioreactor treating blackwater
.
Desalination and Water Treatment
91
,
329
335
.
Taweesan
A.
Koottatep
T.
Polprasert
C.
2015
Effective faecal sludge management measures for on-site sanitation systems
.
Journal of Water, Sanitation and Hygiene for Development
5
,
483
492
.
The ISO (the International Organization for Standardization)
2018
ISO/DIS30500: Non-Sewered Sanitation Systems. General Safety and Performance Requirements for Design and Testing. Team ANSI ISO, Switzerland
.
Valero
C.
Mara
D. D.
2007
Nitrogen removal via ammonia volatilization in maturation ponds
.
Water Science and Technology
55
,
87
92
.
Wongkiew
S.
Hu
Z.
Chandran
K.
Lee
J. W.
Khanal
S. K.
2017
Nitrogen transformations in aquaponic systems: a review
.
Aquacultural Engineering
76
,
9
19
.
Yan
X.
Han
Y.
Li
Q.
Sun
J.
Su
X.
2016
Impact of internal recycle ratio on nitrous oxide generation from anaerobic/anoxic/oxic biological nitrogen removal process
.
Biochemical Engineering Journal
106
,
11
18
.
doi:10.1016/j.bej.2015.11.005
.
Yang
G. F.
Feng
L. J.
Wang
S. F.
Yang
Q.
Xu
X. Y.
Zhu
L.
2015
Performance and enhanced mechanism of a novel bio-diatomite biofilm pretreatment process treating polluted raw water
.
Bioresource Technology
191
,
271
280
.

Supplementary data