Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is a component of S-triazine. Its characteristics make it a pollutant of ecosystems and a probable human carcinogen. The present study evaluated volcanic pumice stone as a suitable media for biological growth and biofilm development in a fixed-bed sequencing batch reactor (FBSBR) for atrazine removal from aquatic environments. The FBSBR was fed with synthetic wastewater containing sucrose and atrazine at four hydraulic retention times to assess biodegradation of atrazine by a microbial consortium for removal from aquatic environments. The maximum efficiency for atrazine and soluble chemical oxygen demand removal were 97.9% and 98.9%, respectively. The results of this research showed that the Stover–Kincannon model was a very good fit (R2 > 99%) for loading atrazine onto the FBSBR. Increasing the initial concentration of atrazine increased the removal efficiency. There was no significant inhibition of the mixed aerobic microbial consortia by the atrazine. Atrazine degradation depended on its initial concentration in the wastewater and the amount of atrazine in the influent. Although this system shows good potential for atrazine removal from aqueous environments, that remaining in the effluent does not yet meet international standards. Further research is required to make this system effective for removal of atrazine from the environment.

ABBREVIATIONS

     
  • ATZ

    atrazine

  •  
  • BOD5

    biochemical oxygen demand

  •  
  • BHI

    brain-heart infusion broth

  •  
  • COD

    chemical oxygen demand

  •  
  • DO

    dissolved oxygen

  •  
  • EMB

    eosin methylene blue

  •  
  • FBSBR

    fixed-bed sequence batch reactor

  •  
  • HPLC

    high-performance liquid chromatograph

  •  
  • HRT

    hydraulic retention time

  •  
  • IARC

    International Agency for Research on Cancer

  •  
  • MSM

    mineral salts medium

  •  
  • MLSS

    mixed liquor suspended solids

  •  
  • OLRs

    organic loading rates

  •  
  • SEM

    scanning electron microscopy

  •  
  • SRT

    solids retention time

  •  
  • SCOD

    soluble chemical oxygen demand

  •  
  • TSS

    total suspended solids

  •  
  • VSS

    volatile suspended solids

  •  
  • VPS

    volcanic pumice stone

  •  
  • VAL

    volumetric atrazine load

  •  
  • VAR

    volumetric atrazine removal

  •  
  • VOL

    volumetric organic loads

  •  
  • VOR

    volumetric organic removal

INTRODUCTION

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine, ATZ) is probably the most commonly-used herbicide in agriculture (Ghosh & Philip 2004; Baghapour et al. 2013; Nasseri et al. 2014). It is a member of the S-triazine group of herbicides and is a probable human carcinogen (the International Agency for Research on Cancer (IARC) has concluded that atrazine is a group 2B carcinogen) (Baghapour et al. 2013; Dehghani et al. 2013). Although many European countries have banned its use, ATZ is still used in China, the USA, and Iran, and it has been detected at higher than recommended levels (0.1 ppb) throughout aquatic environments (Nasseri et al. 2009, 2014; Baghapour et al. 2013).

Physicochemical methods such as coagulation/flocculation, electrolysis, adsorption, membrane filtration, ion-exchange, irradiation, and advanced oxidation have been tested for removal of ATZ from water and wastewater. None, however, has proven effective because most of these methods transfer contaminants to another phase rather than mineralizing them into less toxic and harmful products. High cost and energy consumption and generation of hazardous sludge, which requires safe disposal, are the major drawbacks of these treatment methods. Biological processes are of interest because they are cost effective, reliable, and environmentally beneficial (Ghosh & Philip 2006; Abigail & Das 2012; Baghapour et al. 2013; Nasseri et al. 2014).

Biofilm reactors are of interest for the treatment of wastewater containing biorecalcitrant, inhibitory, and toxic compounds. The immobilization of microorganisms on the surface of biofilm carriers is increasingly used as a biological treatment strategy. Their decreased sensitivity to toxic loads, increased catalytic stability, longer microbial residence time, greater tolerance to oligotrophic conditions, and lower biomass washout risk are the advantages of using attached cells over their suspended counterparts (Wei et al. 2008; Metcalf et al. 2010; Baghapour & Jabbari 2011). The authors have carried out a series of research projects to evaluate and develop novel applications for attached-growth biofilm to enhance removal of ATZ from aquatic environments. The present study builds on earlier research and successful testing by the authors.

Baghapour et al. (2013), by using a submerged biological aerated filter at different organic loadings and Nasseri et al. (2014). by using an anaerobic submerged biological filter in four concentration levels of atrazine, investigated the effects of hydraulic retention time (HRT) on the efficient treatment of wastewater bearing ATZ. These studies have shown that when HRT reached 24 h, ATZ removal increased significantly. A summary of research performed on microbial degradation of ATZ is presented in Table 1.

Table 1

Results of previous studies on ATZ removal

  Performance
 
Initial concentration   
Operating condition/microorganism type Atrazine removal (%) HRT of ATZ (mg/L) Reference 
Aerobic/natural consortia 99 1 d Wide range Baghapour et al. (2013)  
Aerobic/pure culture/Nocardia 60 6 d 30 Giardina et al. (1982)  
Aerobic/Agrobacterium radiobacter, J14a 94 3 d 50 Struthers et al. (1998)  
Aerobic/ Pure culture/Pseudomonas 99 3 wk Wide range Yanze-Kontchou & Gschwind (1994)  
Biostimulation with nutrients/Pseudomonas 80 10 d 30 Masaphy & Mandelbaum (1997)  
Facultative anaerobic bacterium 47 1 wk 75 Jessee et al. (1983)  
Aerobic/Pseudomonas sp. ADP 75 4 d 0.01 Shapir et al. (1998)  
Anaerobic/natural consortia 51 1 d Wide range Nasseri et al. (2014)  
  Performance
 
Initial concentration   
Operating condition/microorganism type Atrazine removal (%) HRT of ATZ (mg/L) Reference 
Aerobic/natural consortia 99 1 d Wide range Baghapour et al. (2013)  
Aerobic/pure culture/Nocardia 60 6 d 30 Giardina et al. (1982)  
Aerobic/Agrobacterium radiobacter, J14a 94 3 d 50 Struthers et al. (1998)  
Aerobic/ Pure culture/Pseudomonas 99 3 wk Wide range Yanze-Kontchou & Gschwind (1994)  
Biostimulation with nutrients/Pseudomonas 80 10 d 30 Masaphy & Mandelbaum (1997)  
Facultative anaerobic bacterium 47 1 wk 75 Jessee et al. (1983)  
Aerobic/Pseudomonas sp. ADP 75 4 d 0.01 Shapir et al. (1998)  
Anaerobic/natural consortia 51 1 d Wide range Nasseri et al. (2014)  

Yang et al. (2009) studied a simple consortium of Klebsiella sp. A1 and Comamonas sp. A2 isolated from sewage from a pesticide mill in China. The bacteria were able to use ATZ alone as a source of carbon and nitrogen. The consortium showed high ATZ-mineralizing efficiency and about 83.3% of the initial ATZ had degraded after 24 h. Contrary to earlier reports on microorganisms, the consortium was insensitive to common nitrogenous fertilizers. The ATZ was completely mineralized despite the presence of urea, (NH4)2CO3, and (NH4)2HPO4 in the medium.

Wang & Xie (2012) studied ATZ removal from contaminated soil and water using Arthrobacter sp. and showed that this strain of bacteria was able to remove ATZ at a wide range of pH values (4 to 11) and temperatures (25 to 35°C). The addition of an external source of carbon and nitrogen increased bacterial growth and ATZ degradation rates.

Atrazine has shown carcinogenic and mutagenic effects, and removal of ATZ from the environment is a major problem (Ghosh & Philip 2006; Nasseri et al. 2009). Previous research has attempted to control the transport and fate of ATZ in the soil and aquatic environments. The results of these studies suggest that biological methods are more economical and cost-effective (Nasseri et al. 2009; Baghapour et al. 2013). The present study investigated the removal of different concentrations of ATZ from aqueous environments at different HRTs using a consortium of microorganisms in a fixed-bed sequencing batch reactor (FBSBR) reactor with volcanic pumice stone (VPS) as packing media.

MATERIALS AND METHODS

Chemicals and reagents

All chemicals were of analytical grade and were purchased from Merck (Germany). Atrazine standard was supplied by Sigma Aldrich (USA). A stock solution of 30 mg/L analytical grade ATZ was prepared by dissolving 3 mg of solid standard ATZ (99.9% purity) in 100 mL methanol. Working solutions were prepared by diluting an appropriate volume of the stock solution in methanol.

The standard solution was stored in a freezer at −20°C. Dichloromethane was used as a solvent with analytical grade reagent (99.5% purity). Stock solutions were prepared by dissolving the required amounts of chemicals in distilled water. All stock solutions except ATZ were autoclaved at 120°C for 20 min and kept at 4°C. All stock solutions were kept separately and were not mixed with other stocks to prevent precipitation. Atrazine solution was prepared (concentration: 1.0 to 10.0 mg/L) by dissolving a known quantity of ATZ in distilled water and shaking it intermittently for at least 5 d. Cartridge ATZ solution was covered with aluminum foil and kept at 4°C in the dark to prevent photolytic degradation (Baghapour et al. 2013).

Preparation of media

VPS was measured to determine the average approximate size. Physicochemical characterization was performed to verify the chemical resistance of the media by placing it in glass beakers containing either tap water, acidic (pH = 4.9), or basic (pH = 9.2) solutions for 30 d. The media were then removed from the solution, rinsed repeatedly with distilled water, dried in an oven at 60°C for 24 h, cooled in a desiccator, and then reweighed. A weight loss of 2.5% and 2.2% was recorded for samples placed in the acidic and basic solutions, respectively (Natarajan et al. 2015).

Biological filter set-up

The experiments were performed at pilot scale. Figure 1 is a simplified flow-diagram of the pilot plant. The model consisted of a Plexiglas column with a 100 mm internal diameter as the FBSBR reactor. The effective height of the reactor was 55 cm and the free board was 5 cm.
Figure 1

FBSBR reactor. 1-Heater, 2-Mixer, 3-Feed Tank, 4-Peristaltic Pump, 5-Control Unit, 6-Feed Control Valve, 7-Decanter (Sampling) Valve, 8-Discharge Sludge Port, 9-Packing Media, 10-FBSBR Reactor, 11-Air Compressor, 12-Effluent Tank.

Figure 1

FBSBR reactor. 1-Heater, 2-Mixer, 3-Feed Tank, 4-Peristaltic Pump, 5-Control Unit, 6-Feed Control Valve, 7-Decanter (Sampling) Valve, 8-Discharge Sludge Port, 9-Packing Media, 10-FBSBR Reactor, 11-Air Compressor, 12-Effluent Tank.

The column was filled with an immobilized biofilm VPS support of equal height and diameter. The VPS was used as a biofilm support material because of its high porosity (up to 70%) and low cost compared to other synthetic packing media. The physical specifications of the model were: external diameter, 160 mm, internal diameter, 100 mm; height, 60 cm; total volume, 4.7 L; and effective volume, 3.9 L. Tables 2 and 3 list the physical and chemical properties of the media. To prevent interference from light (photocatalytic) and algae growth, the column was covered with aluminum foil. A control pilot was also used to increase the accuracy of the project and eliminate the effects of interfering factors.

Table 2

Physical properties of media

D10 25 mm 
D30 33 mm 
D60 39 mm 
Cu = D60/D10 1.5 
Cc = (D30)2/(D60 × D101.1 
Porosity 60–90% 
Density 0.6–1.2 
Specific surface area 28 m2/gr 
D10 25 mm 
D30 33 mm 
D60 39 mm 
Cu = D60/D10 1.5 
Cc = (D30)2/(D60 × D101.1 
Porosity 60–90% 
Density 0.6–1.2 
Specific surface area 28 m2/gr 
Table 3

Chemical properties of media

Chemical composition Value (%) 
Silica (SiO265.75 
Aluminium oxide (Al2O312.43 
Ferric oxide (Fe2O32.88 
Lime (CaO) 1.70 
Manganese oxide (MnO) 1.11 
Sodium oxide (Na2O) 3.59 
Potassium oxide (K2O) 4.47 
Titanium dioxide (TiO21.54 
Others 6.53 
Chemical composition Value (%) 
Silica (SiO265.75 
Aluminium oxide (Al2O312.43 
Ferric oxide (Fe2O32.88 
Lime (CaO) 1.70 
Manganese oxide (MnO) 1.11 
Sodium oxide (Na2O) 3.59 
Potassium oxide (K2O) 4.47 
Titanium dioxide (TiO21.54 
Others 6.53 

Synthetic wastewater

The synthetic wastewater used to feed the bioreactor was a mixture of sucrose and tap water with a soluble chemical oxygen demand (SCOD) of 1,000 ± 13.4 mg O2/L. Fluctuations in pH were controlled using 0.5 mol/L sodium bicarbonate. Table 4 shows the composition of wastewater used to feed the pilot reactor during the test period. Synthetic wastewater was injected into the FBSBR reactor by a peristaltic pump. Previous research has shown that the maximum removal efficiency of ATZ biodegradation occurs at 32°C (Abigail & Lakshmi 2012; Baghapour et al. 2013). Accordingly, the temperature was controlled in the reservoir at 32 ± 0.7 °C using an electric heater.

Table 4

Chemical composition of synthetic wastewater (Baghapour et al. 2013; Nasseri et al. 2014)

Component Concentration (mg/L) 
Nutrients 
 NaHCO3 20 
 MgSO4.7H2
 KH2PO4 
 CaCl2.2H2
 FeSO4.7H20.2 
 ZnCl2 0.1 
 CoCl2 0.1 
 NiCl2 0.1 
 CuSO4.5H20.001 
 H3BO3 0.2 
 MnSO4 0.5 
 (NH4)2HP2O4 50 
 C12H22O11 Variable (600–900) 
Atrazine Variable (1, 5 and 10) 
Component Concentration (mg/L) 
Nutrients 
 NaHCO3 20 
 MgSO4.7H2
 KH2PO4 
 CaCl2.2H2
 FeSO4.7H20.2 
 ZnCl2 0.1 
 CoCl2 0.1 
 NiCl2 0.1 
 CuSO4.5H20.001 
 H3BO3 0.2 
 MnSO4 0.5 
 (NH4)2HP2O4 50 
 C12H22O11 Variable (600–900) 
Atrazine Variable (1, 5 and 10) 

Startup and system operation

The column was filled with synthetic wastewater that had SCOD equal to 10,000 mg O2/L. Seeding was done with aerobic bacteria collected from the activated sludge system of a domestic wastewater treatment plant in Yazd. The air compressor was then turned on and the reactors began work under batch conditions. Under aerobic conditions, oxygen was added to the mixed bacteria to stimulate growth and production of enzymes that could oxidize or degrade the target pollutant.

The sludge was fed with wastewater for 1 month to acclimatize the system to the changing environment and was then used for further experimentation. During this period, very low concentrations of ATZ were added to further acclimatize the microorganisms to the operational conditions. The bacterial adaptation stage lasted about 32 d. During this time, the wastewater inside the reactors was changed five times and the pH, dissolved oxygen (DO), and temperature were recorded as being 7.3 ± 0.6, 4.1 mg/L, and 32 ± 0.7°C, respectively. The decrease in the SCOD was also measured daily. The results of the measurements will be presented in the corresponding sections.

To ensure microbial activity at this stage, surface cultivation of mixed liquor suspended solids (MLSS) in the bioreactor was frequently done in a mineral salts medium (MSM) containing ATZ. The MSM preparation method was performed as recommended by Rezaee et al. (2011).

Experiments

After microbial adaptation was complete, the reactor was operated in cycles of 24, 12, 6 and 3 h. The system was controlled using timer switches (Theben, Germany). Each cycle comprised 4 phases. In phase one, the reactor was continuously fed for 15 min. In phase two, the reactor was aerated for 1,365, 645, 285 and 105 min, depending on the cycle duration. In phase three, settling occurred for 45 min and, in phase four, the effluent was discharged for 15 min.

Wastewater with a concentration of SCOD = 1,000 mg O2/L was injected into the reactor by a peristaltic pump at different ATZ concentrations to approximate the high range of ATZ concentrations in the ecosystem. Three ATZ concentrations (1, 5 and 10 mg/L) were selected, and discharges corresponding to different HRTs and volumetric organic loads (VOLs) were used in the reactor. The operational scheme of the system for 12 phases (runs) is presented in Table 5.

Table 5

Operational scheme of runs at 32°C

Run Cycle time (h) Initial conc. of atrazine (mg/L) Initial conc. of SCOD (mg O2/L) Initial conc. of BOD5 (mg O2/L) DO (mg/L) pH 
24 994 ± 19 278 4.6 ± 0.157 7.01 
992 ± 12 257 4.6 ± 0.902 7.19 
10 997 ± 11 239 4.6 ± 0.176 7.14 
12 1,005 ± 12 311 4.7 ± 0.122 7.08 
1,007 ± 10 261 4.5 ± 0.173 7.04 
10 1,010 ± 15 222 4.7 ± 0.501 7.00 
1,001 ± 5 290 4.8 ± 0.144 7.09 
998 ± 8 249 4.7 ± 0.116 6.98 
10 1,004 ± 14 210 4.6 ± 0.105 7.11 
10 1,002 ± 13 300 4.5 ± 0.198 7.10 
11 998 ± 9 249 4.5 ± 0.139 7.07 
12 10 995 ± 8 228 4.5 ± 0.057 7.44 
Run Cycle time (h) Initial conc. of atrazine (mg/L) Initial conc. of SCOD (mg O2/L) Initial conc. of BOD5 (mg O2/L) DO (mg/L) pH 
24 994 ± 19 278 4.6 ± 0.157 7.01 
992 ± 12 257 4.6 ± 0.902 7.19 
10 997 ± 11 239 4.6 ± 0.176 7.14 
12 1,005 ± 12 311 4.7 ± 0.122 7.08 
1,007 ± 10 261 4.5 ± 0.173 7.04 
10 1,010 ± 15 222 4.7 ± 0.501 7.00 
1,001 ± 5 290 4.8 ± 0.144 7.09 
998 ± 8 249 4.7 ± 0.116 6.98 
10 1,004 ± 14 210 4.6 ± 0.105 7.11 
10 1,002 ± 13 300 4.5 ± 0.198 7.10 
11 998 ± 9 249 4.5 ± 0.139 7.07 
12 10 995 ± 8 228 4.5 ± 0.057 7.44 

Sampling was carried out atleast twice; all results were obtained from the bioreactors at steady state. The supernatant from one entire cycle was collected in a container and the mixed liquor was sampled at the end of aeration time. When the reactor reached a steady state for residual ATZ and SCOD, the efficiency of ATZ and SCOD removal was determined. The parameters measured were residual ATZ concentration, SCOD, biochemical oxygen demand (BOD5), pH, DO, MLSS, total suspended solids (TSS), volatile suspended solids (VSS), and temperature. The pH, DO, and temperature were measured every cycle at a specific HRT. BOD5 measurements were carried out for each run to obtain the BOD5/SCOD rates. These parameters were included in the list of measurements to ascertain the proper operation of the system and stability of the reactors. Unless otherwise specified, analysis of the parameters was carried out according to Standard Methods for the Examination of Water and Wastewater (APHA 1998).

Atrazine extraction and determination

Atrazine was extracted from wastewater using the liquid–liquid extraction method suggested by Ghosh & Philip (2004). Dichloromethane (SG 1.32; ATZ solubility 28 g/L at 25°C) was used as the extractant. Extraction efficiency using this method was 91 ± 0.94%. The ATZ was measured using a high-performance liquid chromatograph (HPLC) (model UV-2487; Waters, USA) using a UV/VIS detector at a wavelength of 220 nm and a HPLC pump (Dionex Summit P580).

Analysis was carried out using the method reported by Yang et al. (2009). The analytes were filtered through a 0.22 μm nylon syringe filter. The ATZ concentration was determined using a reversed phase C18 column (0.5 μm; 4.6 × 250 mm; Spherisorb W; Waters, USA). The injection volume was 20 μL. The column operated at room temperature; the mobile phase was an 80%–20% methanol gradient with water, the flow rate was 0.5 mL/min, and the peak retention time was 12 min. Before each run, the instruments were standardized using the anticipated ATZ concentration range. Six ATZ standards were prepared by serial dilution in advance and stored in amber bottles in a refrigerator at 4°C until use. To check the build-up of ATZ in the biofilm, the method suggested by Ghosh & Philip (2004) was utilized.

Isolation of predominant microbial strain

The biochemical methods used for microbial isolation were from Bergey's manual (Breed et al. 1957). To identify the dominant bacterial species, the biofilm samples were collected by separating the biomass from the biological carrier and homogenized in the physiological serum by a shaker for 5 min. Then, the 10−1 to 10−5 dilution solution was prepared from the obtained suspension; for initial separation, it was cultured on blood agar and eosin methylene blue (EMB) media using a spread plate and incubated in the incubator at 32°C for 1 to 2 d (Keinänen et al. 2004). After the growth of microorganisms, the colonies in the plates were counted and separated based on color, transparency, size, and appearance. To prepare the pure culture, every single colony was separately cultured in brain-heart infusion broth, EMB, and blood agar media and placed in the incubator at 32°C for 48 h. Then, Gram staining was performed on the bacterial isolates. Catalase, oxidase, citrate, urease, glucose, mannitol, and maltose sugar tests as well as pseudo F agar and psuedo P agar culture media were used and the microbiological reference sources was applied to identify and determine the identity of the predominant microbial strain (Marecik et al. 2007; Adawiyah 2008).

Scanning electron microscopy

Analysis of the biomass attached to the media was carried out by scanning electron microscopy (SEM). Samples were taken at the end of testing and prepared by fixing with 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 and 4°C over night. They were then dehydrated with ethanol from 60% to 100% in 20% increments for 10 min at each concentration. The samples were then dried at critical point (equilibrium of the gas and liquid phases of CO2), mounted, coated with gold, and examined by SEM (Dutta et al. 2014; Naz et al. 2014).

Modeling

Biological and mathematical models were used to determine the relationship between variables and evaluate the experimental results. The models were also used to monitor and predict performance and optimize the plant built at pilot/bench scale. It was confirmed that the criterion for biological growth system design is the VOL. The rate of substrate removal was obtained from the hyperbolic relations of the Stover–Kincannon function as (Equation (1)): 
formula
1
where (rSCOD) is the volumetric SCOD removal, (rmax) is the maximum rate of volumetric SCOD removal, (BSCOD) is the SCOD load per unit volume of the reactor, and (k) is the constant of half velocity. All parameters are in kgSCOD/m3d. The values for (BSCOD) and (rSCOD) were obtained as: 
formula
2
 
formula
3
where (Ci) is the SCOD concentration in the influent (kgSCOD/m3) and (Ce) is the SCOD concentration in the effluent (kgSCOD/m3) (Baghapour et al. 2013; Nasseri et al. 2014). Equations (2) and (3) and Tables 5 and 6 were used to compute the values of (BSCOD) and (rSCOD) under different conditions.

RESULTS AND DISCUSSION

During the system operation period, the HRT decreased from 24 to 12 to 6 h to 3 h. The respective organic loading rates (OLRs) in the reactor were set at 0.73, 1.46, 3.65, and 10.96 kgSCOD/m3.d. The most important parameters monitored were residual ATZ and SCOD, as described herein (Table 6). The SCOD of the inflow wastewater was 1,000 ± 13.4 mg O2/L. ATZ and SCOD removal are shown in Figures 2 and 3.
Table 6

Effluent concentration of ATZ and SCOD and removal efficiency from FBSBR at a steady state at 32°C

 Output conc. of atrazine Output conc. of SCOD BOD5MLSS Removal efficiency (%)
 
Run (mg/L) (mg O2/L) SCOD (mg/L) Atrazine SCOD 
0.073 ± 0.10 45 ± 2.2 0.52 4,635 92.7 95.4 
0.690 ± 0.07 18 ± 1.2 0.48 4,709 86.2 98.1 
0.070 ± 0.60 10 ± 1.3 0.49 4,401 99.3 98.9 
0.103 ± 0.89 58 ± 1.6 0.53 4,223 89.6 94.2 
0.830 ± 0.01 45 ± 2.9 0.49 4,957 83.4 95.5 
0.250 ± 0.08 37 ± 3.2 0.48 4,527 97.5 96.3 
0.352 ± 0.71 294 ± 1.8 0.49 4,400 64.8 70.6 
2.139 ± 0.43 279 ± 1.6 0.44 4,634 57.2 72.0 
2.380 ± 0.01 263 ± 1.2 0.45 4,316 76.2 73.8 
10 0.790 ± 0.08 428 ± 3.1 0.42 4,071 21.0 57.2 
11 4.190 ± 0.77 499 ± 1.1 0.4 4,816 16.2 49.9 
12 7.080 ± 0.02 484 ± 3.3 0.39 4,631 29.2 51.3 
 Output conc. of atrazine Output conc. of SCOD BOD5MLSS Removal efficiency (%)
 
Run (mg/L) (mg O2/L) SCOD (mg/L) Atrazine SCOD 
0.073 ± 0.10 45 ± 2.2 0.52 4,635 92.7 95.4 
0.690 ± 0.07 18 ± 1.2 0.48 4,709 86.2 98.1 
0.070 ± 0.60 10 ± 1.3 0.49 4,401 99.3 98.9 
0.103 ± 0.89 58 ± 1.6 0.53 4,223 89.6 94.2 
0.830 ± 0.01 45 ± 2.9 0.49 4,957 83.4 95.5 
0.250 ± 0.08 37 ± 3.2 0.48 4,527 97.5 96.3 
0.352 ± 0.71 294 ± 1.8 0.49 4,400 64.8 70.6 
2.139 ± 0.43 279 ± 1.6 0.44 4,634 57.2 72.0 
2.380 ± 0.01 263 ± 1.2 0.45 4,316 76.2 73.8 
10 0.790 ± 0.08 428 ± 3.1 0.42 4,071 21.0 57.2 
11 4.190 ± 0.77 499 ± 1.1 0.4 4,816 16.2 49.9 
12 7.080 ± 0.02 484 ± 3.3 0.39 4,631 29.2 51.3 
Figure 2

ATZ removal in FBSBR reactors.

Figure 2

ATZ removal in FBSBR reactors.

Figure 3

SCOD removal in FBSBR reactors.

Figure 3

SCOD removal in FBSBR reactors.

The results presented in Figures 47 were obtained by substitution of the values of Table 7 into Equation (1). The FBSBR was designed using these diagrams. The values of volumetric atrazine removal and volumetric organic removal increased as volumetric atrazine load and VOL increased, but these relationships were not linear. The values for (k) and (rmax) were obtained using Curve Expert software, and are presented in Table 8.
Table 7

Volumetric load, removal of ATZ, and SCOD from FBSBR at 32°C

Run BATZ (kgatrazine/m3d) rATZ (kgatrazine/m3d) BSCOD (kgSCOD/m3d) rSCOD (kgSCOD/m3d) 
7.3 × 10−4 5.3 × 10−5 0.731 0.697 
3.6 × 10−3 3.0 × 10−3 0.731 0.717 
7.3 × 10−3 6.5 × 10−3 0.731 0.723 
1.4 × 10−3 1.5 × 10−4 1.462 1.377 
7.3 × 10−3 6.1 × 10−3 1.462 1.396 
1.4 × 10−2 1.3 × 10−2 1.462 1.407 
3.6 × 10−3 1.2 × 10−3 3.654 2.580 
1.8 × 10−2 1.6 × 10−2 3.654 2.631 
3.6 × 10−2 3.3 × 10−2 3.654 2.697 
10 1.1 × 10−2 8.6 × 10−3 10.965 6.276 
11 5.4 × 10−2 5.3 × 10−2 10.965 5.471 
12 1.1 × 10−1 1.0 × 10−1 10.965 5.628 
Run BATZ (kgatrazine/m3d) rATZ (kgatrazine/m3d) BSCOD (kgSCOD/m3d) rSCOD (kgSCOD/m3d) 
7.3 × 10−4 5.3 × 10−5 0.731 0.697 
3.6 × 10−3 3.0 × 10−3 0.731 0.717 
7.3 × 10−3 6.5 × 10−3 0.731 0.723 
1.4 × 10−3 1.5 × 10−4 1.462 1.377 
7.3 × 10−3 6.1 × 10−3 1.462 1.396 
1.4 × 10−2 1.3 × 10−2 1.462 1.407 
3.6 × 10−3 1.2 × 10−3 3.654 2.580 
1.8 × 10−2 1.6 × 10−2 3.654 2.631 
3.6 × 10−2 3.3 × 10−2 3.654 2.697 
10 1.1 × 10−2 8.6 × 10−3 10.965 6.276 
11 5.4 × 10−2 5.3 × 10−2 10.965 5.471 
12 1.1 × 10−1 1.0 × 10−1 10.965 5.628 
Table 8

Coefficients (k) and (rmax) for bioreactor at 32°C for Stover–Kincannon model

  Atrazine SCOD 
rmax, (kg/m3d) 4.963 2.052 
k, (kg/m3d) 3.224 6.464 
R2 0.997 0.965 
  Atrazine SCOD 
rmax, (kg/m3d) 4.963 2.052 
k, (kg/m3d) 3.224 6.464 
R2 0.997 0.965 
Figure 4

Atrazine loading of bioreactor at 0 to 0.11 kgATZ m3d and 32°C.

Figure 4

Atrazine loading of bioreactor at 0 to 0.11 kgATZ m3d and 32°C.

Figure 5

Atrazine loading of bioreactor at 0 to 5 kgATZ m3d and 32°C.

Figure 5

Atrazine loading of bioreactor at 0 to 5 kgATZ m3d and 32°C.

Figure 6

Organic loading of bioreactor at 0 to 11 kgSCOD m3d and 32°C.

Figure 6

Organic loading of bioreactor at 0 to 11 kgSCOD m3d and 32°C.

Figure 7

Organic loading of bioreactor at 0 to 25 kgSCOD m3d and 32°C.

Figure 7

Organic loading of bioreactor at 0 to 25 kgSCOD m3d and 32°C.

The equations for multivariable modeling were obtained using MATLAB software, and are presented in Table 9. Graphs were drawn using MATLAB software.

Table 9

Equations suggested for multivariable modeling

  Variable Suggested equation R2 
Effect of initial ATZ concentration and HRT on ATZ removal efficiency x = initial ATZ concentration f(x,y) = (−5.85) + (−4.37x) + (13.06y) + (0.49 × 2) + (−0.01xy) + (−37y20.95 
y = HRT 
Effect of initial ATZ concentration and HRT on SCOD removal efficiency x = initial ATZ concentration f(x,y) = (33.43) + (−0.72x) + (7.81y) + (0.04 × 2) + (0.03xy) + (−0.21y20.98 
y = HRT 
  Variable Suggested equation R2 
Effect of initial ATZ concentration and HRT on ATZ removal efficiency x = initial ATZ concentration f(x,y) = (−5.85) + (−4.37x) + (13.06y) + (0.49 × 2) + (−0.01xy) + (−37y20.95 
y = HRT 
Effect of initial ATZ concentration and HRT on SCOD removal efficiency x = initial ATZ concentration f(x,y) = (33.43) + (−0.72x) + (7.81y) + (0.04 × 2) + (0.03xy) + (−0.21y20.98 
y = HRT 

Sludge stabilization ratio

The sludge stabilization ratio (VSS/TSS) varied from 0.67 to 0.89 in the FBSBR reactor. Figure 8 shows the variation in VSS/TSS versus loading rates.
Figure 8

Characteristics of sludge in FBSBR reactor and sludge stabilization ratio.

Figure 8

Characteristics of sludge in FBSBR reactor and sludge stabilization ratio.

Biofilm morphology

Biofilm is a metabolically active matrix of cells and extracellular compounds. Figure 9 shows the SEM images of biofilm grown on the surface of packing media.
Figure 9

SEM images of surface of a virgin sample of VPS (left) and a VPS sample after biofilm formation (right).

Figure 9

SEM images of surface of a virgin sample of VPS (left) and a VPS sample after biofilm formation (right).

Dominant microbial strains

Table 10 shows the results of the isolated predominant microbial strains used for degradation of ATZ in an FBSBR reactor.

Table 10

Specifications of ATZ degrading bacteria

Name Appearance characteristics Blood agar EMB Catalase Oxidase Citrate Urease Indole Glucose Gelatinase Mobility VP MR H2Pseudo F Agar Pseudo P Agar 
Pseudomonas fluorescens Basil,
Gram-negative 
− − − − − − − − − − − 
Pseudomonas aeruginosa Basil,
Gram-negative 
− − − − − − 
Moraxella Coco bacilli, Gram-negative − − − − − − − − − − − − 
Acinetobacter Basil,
Gram-negative 
− − − − − − − − − − − 
Name Appearance characteristics Blood agar EMB Catalase Oxidase Citrate Urease Indole Glucose Gelatinase Mobility VP MR H2Pseudo F Agar Pseudo P Agar 
Pseudomonas fluorescens Basil,
Gram-negative 
− − − − − − − − − − − 
Pseudomonas aeruginosa Basil,
Gram-negative 
− − − − − − 
Moraxella Coco bacilli, Gram-negative − − − − − − − − − − − − 
Acinetobacter Basil,
Gram-negative 
− − − − − − − − − − − 

The potential for degradation by atrazine of a mixed aerobic consortium was evaluated at various ATZ concentrations and HRTs in an FBSBR reactor. The results are presented in Table 6. Figures 2 and 3 show that ATZ and SCOD removal efficiency increased as the HRT increased. These findings demonstrate that the solution containing ATZ was easily biodegraded and treated in the FBSBR reactor. ATZ removal efficiency was above 92% when high concentrations of ATZ influent were introduced to the FBSBR (runs 3 and 6). The majority of the input ATZ was consumed during these runs, as indicated by the low ATZ concentration in the effluent (<0.07 mg/L).

The treatment efficiencies achieved at the longer HRT (24 h) in the FBSBR fed with low, moderate, and high ATZ concentrations in the influent are summarized in Table 6. The decrease in ATZ and OLRs in the FBSBR indicate that, in comparison with other HRTs, ATZ and SCOD removal efficiency increased at longer HRTs. Figures 47 show, by the extent of ATZ loading for biological ATZ and the organic removal efficiency, that it was not highly effective. When the HRT was set to 24 h and the FBSBR operated until steady-state conditions were reached, the ATZ and SCOD removal efficiencies increased to 98.1% and 98.9%, respectively (Table 6). Figures 10 and 11 show that in the FBSBR, ATZ and SCOD removal efficiency depended on the HRT and initial ATZ concentration.
Figure 10

Effect of initial ATZ concentration and HRT on ATZ removal efficiency.

Figure 10

Effect of initial ATZ concentration and HRT on ATZ removal efficiency.

Figure 11

Effect of initial ATZ concentration and HRT on SCOD removal efficiency.

Figure 11

Effect of initial ATZ concentration and HRT on SCOD removal efficiency.

It can be concluded that a decrease in ATZ and organic loading positively affected FBSBR performance. This is likely the result of the increased exposure of the contaminants to the microbial consortium. This is consistent with the results obtained by Baghapour et al. (2013) and Nasseri et al. (2014).

Measurement of chemical oxygen demand (COD) is important for adherence to effluent discharge standards. COD represents the treatment potential of the reactor. In this study, the FBSBR showed an acceptable SCOD removal efficiency in all the experiments. The ATZ had no adverse effect on SCOD removal up to a concentration of 10 mg/L. SCOD decreased by 8% to 3% when the ATZ concentration increased to 5 mg/L, which is in agreement with the results reported by Ghosh et al. (2001). Because the FBSBR has not been used for removal of pesticides and is a new method for ATZ removal, there is no previous research for comparison; however, some similar studies are shown in Table 1.

Baghapour et al. (2013) used a submerged biological aerated filter and attained 99% efficiency at a wide range of ATZ concentrations within 24 h. Ghosh & Philip (2004) used a sequential mode of operation and dextrose as the external carbon source for an initial ATZ concentration of 1 mg/L and attained 40% efficiency at 5 d. In a batch reactor with no external carbon and nitrogen, they attained 42% efficiency in 150 d. Comparison of the results of previous studies with those of the present study shows that the new system has a good ability to remove ATZ from aqueous environments.

There was no accumulation of ATZ in the biofilm and the loss of ATZ in the control reactor was negligible. This shows that ATZ removal from the system was caused by biodegradation. The high degradation rate of ATZ at comparatively high ATZ concentrations could be the effect of the concentration gradient. At a high concentration gradient, the pollutant had more time for exposure to penetrate the cell, which is essential for biodegradation.

BOD5 is a measure of oxidation occurring in response to microbial activity. BOD5/COD ratios are the common indicators of improved biodegradability, where a value of zero indicates non-biodegradability and an increase in the ratio reflects an improvement in biodegradability. In this study, the FBSBR increased BOD5/SCOD by more than 0.2 in all the experiments. Moreover, significant changes were observed in BOD5/SCOD with an increase in HRT.

Co-metabolic processes are used for bioremediation of the most persistent contaminants, such as ATZ. By utilizing primary carbon or nitrogen sources in co-metabolic processes, microbes produce enzymes or co-factors during microbial activities, which are responsible for degradation of the secondary substrates (toxic compounds: ATZ). The results obtained from the FBSBR showed that the co-metabolic process was effective in removing ATZ from the aqueous environment. Additional nitrogen sources (ammonium phosphate) also showed no adverse effect on ATZ degradation. Similar results have been reported by Yang et al. (2009). Overall, the results of modeling indicate that the Stover–Kincannon model showed a very good fit (R2 > 99%) for loading of ATZ in this system, which is in line with the findings of Baghapour et al. (2013).

It is evident that the biofilm played an important role in sludge stabilization. The lower VSS/TSS in the FBSBR can be attributed to greater solids retention. The effect of solids retention time (SRT) on sludge stabilization has been proven previously, and VSS/TSS is inversely related to SRT.

A variety of bacterial morphologies were observed in all samples. An increase in density was observed that was the result of both colonization and growth of dense cell clumps. Microorganisms colonized a significant portion of the surface, which could be attributed to the mixture of a bacterial layer and embedded particles. The dominant types of microorganism were isolated and identified as bacteria. The variety of bacteria and other microorganisms mean they played a more prominent role in biodegradation. According to the results of differentiative tests and their comparison with Bergey's manual of systematic and determinative bacteriology, the isolates were Pseudomonas fluorescens and Pseudomonas aeruginosa, which previous studies have reported as atrazine degrading bacteria. But Moraxella and Acinetobacter were identified to be effective in atrazine biodegradation, which earlier studies had not reported.

CONCLUSION

The present study investigated the ability of an FBSBR reactor to remove atrazine from an aqueous environment. The FBSBR was operated at four aerobic retention times to determine the optimum retention time for the highest ATZ and SCOD removal. The results demonstrate the feasibility of using VPS as a media and biofilm carrier for attached growth and suggest that the addition of a carrier improved ATZ and SCOD removal efficiency. SEM results showed an increase in the amount of biomass attached to the VPS.

The aerobic mixed biofilm culture was observed to be suitable for removal of ATZ from aqueous solutions. There was no significant inhibition of the mixed aerobic microbial consortia. Atrazine degradation depended on its initial concentration in the wastewater, the amount of ATZ in the influent, and the HRTs. The Stover–Kincannon model best described ATZ degradation in an aquatic environment using an FBSBR. Although this system shows good ability for removal of ATZ from aqueous environments, at high concentrations of ATZ it does not achieve international standards (0.1 ppb). Further research is required for adequate removal of atrazine from the environment. Possible strategies include using combination and hybrid treatment methods, and evaluating the effects of different sources of carbon and nitrogen in different concentrations that can be effective in removing atrazine from the aquatic environment.

ACKNOWLEDGEMENTS

This project was funded by the Faculty of Public Health, Shahid Sadoughi University of Medical Sciences (Grant 3613). The authors are grateful to the head of the Environmental Chemistry Laboratory for his help.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS' CONTRIBUTIONS

Seyed Mohammad Mazloomi, Zahra Derakhshan and Mohammad Faramarzian performed data collection, carried out statistical and technical analysis of data, participated in the design of the study and drafted manuscript. Mehdi Mokhtari and Mohammad Hassan Ehrampoush participated in the design of the study, the final revision of the manuscript and provided intellectual help for analyzing the data. Amir Hosein Mahvi participated in the design of the study, coordinated activities and revised the manuscript. All authors read and approved the final manuscript.

FINANCIAL DISCLOSURE

The authors declare that they have no financial disclosure.

FUNDING/SUPPORT

The authors declare that they have no funding/support.

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