Journal of Bioscience and Bioengineering VOL. 117 No. 6, 763e768, 2014 www.elsevier.com/locate/jbiosc
Bio-augmentation as a tool for improving the modified sequencing batch biofilm reactor Kai Yang,1 Bin Ji,1 Hongyu Wang,1, * Huaiyu Zhang,2 and Qian Zhang3 School of Civil Engineering, Wuhan University, Wuhan 430072, China,1 Central and Southern China Municipal Engineering Design and Research Institute, Wuhan 430010, China,2 and School of Civil Engineering and Architecture, Wuhan University of Technology, Wuhan 430070, China3 Received 18 July 2013; accepted 6 November 2013 Available online 15 December 2013
Biological treatment of domestic sewage was accomplished in a pilot-scale modified sequencing batch biofilm reactor (SBBR) bio-augmented with consortium of 5 strains of indigenous bacteria (genus Pseudomonas and Bacillus). The reactor consisted of fibrous filler in the upper and ceramsite filter media in the bottom. It demonstrated to have a short hydraulic residence time (HRT) for 10 h and good quality effluent to cope with low C/N ratio domestic wastewater. The biofilm attached fibrous fillers mainly contributed to contaminants removal. Bio-augmentation dramatically enhanced the removal efficiency of chemical oxygen demand (COD), total phosphorus (TP), and especially total nitrogen (TN), which increased respectively from 80.3%, 58.1% and 41.3% to 83.7%, 67.8% and 58.7%. Polymerase chain reaction (PCR)denaturing gradient gel electrophoresis (DGGE) technique indicated the 5 strains’ survival in the reactor and that Bacillus cereus strain ZQN2 was the most dominant bacteria. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Modified sequencing batch biofilm reactor; Bio-augmentation; Domestic sewage; Nitrogen removal; Denaturing gradient gel electrophoresis]
In a traditional sequencing batch biofilm reactor (SBBR) wastewater is treated by bacteria growing on media packed in the reactor body and the packing serves as filter media to minimize the concentration of suspended solids in the effluent (1). SBBR is a highly effective reactor and widely tested to cope with different high salinity wastewater (2), high carbon wastewater (3), toxic wastewater (4), and especially to treat domestic wastewater with satisfactory results of chemical oxygen demand (COD) removal efficiency of 60%e80% (5,6). New mechanisms were proposed such as simultaneous nitrification, phosphorus uptake and denitrification (7,8) during aerobic stage. However, problems exist with the quite high effluent suspended solids (SS) of around 45 mg L1 and long hydraulic residence time (HRT) of approximate 15 h (5,6). In comparison, traditional SBBR usually needs a quite long stage of settling over 1 h. To solve the problems, a modified SBBR is proposed with a filter layer added to the bottom of the reactor (Fig. 1) to cut HRT and to improve the quality of effluent. This reactor is based on biological aerated filter prototype (9) and principles of cake filtration (10). The reactor consists of an upper biofilm reactor component and a lower filter bed component. In addition, bio-augmentation method is investigated to enhance contaminants removal. Bio-augmentation is a process of application of indigenous or allochthonous wild type or genetically modified organisms to polluted sites or bioreactors to accelerate the removal of undesired contaminants. When an efficient
* Corresponding author. Tel.: þ86 27 61218623; fax: þ86 27 68775328. E-mail address: [email protected] (H. Wang).
consortium is added to the initial indigenous microbial community of a contaminated medium, versatile enzymes are likely to be produced to enhance its biopurification abilities (11). Previous study was mostly related to one single bacteria bio-augmentation to remove specific pollutants, including transgenic bacteria (12). For example, Pseudomonas sp. strain HF-1 (13) was used to degrade nicotine while Pseudomonas stutzeri strain TR2 (14) was used to reduce nitrous oxide. In this article consortium of 5 strains of Gammaproteobacteria were adopted to treat domestic wastewater including 3 strains of Pseudomonas genus and 2 strains of Bacillus genus. It was found that Pseudomonas and Bacillus genera were promisingly effective for wastewater treatment. For instance, Pseudomonas sp. KW1 and Bacillus sp. YW4 were found to be the most efficient in terms of nitrate reduction in synthetic nitrate-rich wastewater (15). Pseudomonas aeruginosa was found to be able to produce nitrite reductase, coded by genes nirQ, to catalyze a key reaction of the denitrification pathway, namely the reduction of nitrite to nitric oxide (16,17). Bacillus cereus was the most effective microbe for simultaneous bio-oxidation of ammonia and manganese in biological aerated filter system (18), and able to occur simultaneous aerobic nitrification and denitrification (19), with a remarkable removal rate of nearly 100% of nitrate nitrogen in 24 h (20). However, most tests are still in the lab scale, and we need to put it to application. For this purpose, after having developed modified SBBR in a pilot-scale to treat domestic wastewater and determined proper process parameters based on the previous study, we added the highly effective 5 strains of bacteria to strengthen the domestic wastewater treatment.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.11.006
764
YANG ET AL.
J. BIOSCI. BIOENG., TABLE 1. Characteristics of the raw water.
Average Standard deviation
Chemical oxygen demand (mg L1)
Total nitrogen (mg L1)
Total phosphorus (mg L1)
Turbidity (mg L1)
pH value
109.87 26.22
20.64 2.35
2.41 0.76
160.23 23.51
7.12 0.67
from the bioreactor and dried for 2 h at 105 C. The total weight of the dried fibers was measured and the weight of the attached biomass was obtained by subtracting the original weight of the fibers. The attached biomass concentration was finally expressed as mg L1 by considering the total number of fibers and the total volume of the reactor.
FIG. 1. Sketch of modified SBBR. 1, long handle filter nozzle; 2, ceramsite filter media; 3, fibrous filler; 4, sampling point; 5, overflow pipe; 6, intake pipe; 7, pipeline pump; 8, backwashing water valve; 9, effluent regulating valve; 10, effluent electronic valve; 11, gas flowmeter; 12, air compressor; 13, intake electromagnetic valve; 14, control cabinet; 15, influent electromagnetic valve.
MATERIALS AND METHODS Wastewater characteristics The raw wastewater used in the study was obtained from the outlet of grit chamber of a municipal wastewater treatment plant, Wuhan, China. The influent characteristics of the bioreactor are presented in Table 1. Reactor operation As shown in Fig. 1, the reactor is a 4.0 m high organic glass column, with 23.5 cm inner diameter, below which was a long handle filter nozzle in the middle of retainer plate with dozens of holes with a diameter of 0.9 mm, whose function mainly was aeration and backwashing. The upper layer was filled with a depth of 0.3 m fibrous filler (1512 kg m3 density, 0.4 mm diameter, 35e40 mm length, 90% porosity) as a biofilm reactor and the lower layer was filled with a depth of 1.0 m ceramsite filter media (1e2 mm diameter, 2260 kg m3 density, non-uniform coefficient K80 ¼ d80/d10 < 2.0) as a filter bed. The highest water level (H1) and the lowest water level (H2) were controlled by floating ball valve according to processing capacity. Through the operation of a control cabinet, raw water flowed by the pipeline pump into the reactor, mixing and standing for 30 min. The aeration time was 70 min. Finally after settling for 10 min, the effluent water was discharged from the reactor. The parameters were controlled as follows: dissolved oxygen (DO) concentration of upper reactor for aeration was 2e3 mg L1, filter speed was 5 m h1, HRT was 10 h (2 h each cycle), and sludge retention time (SRT) was 50 d. The tests were operated outdoor under ambient climatic conditions with the temperature at 16e25 C. The reactor was inoculated with about 30 L activated sludge of the plant with a concentration of 3000 mg L1. Then the whole reactor was submerged into feed water and aerated for 1 h to achieve complete mixing. After conditioned for 10 h, wastewater in the reactor was discharged entirely. Fresh influent was continuously injected into the reactor, accompanied by the discharge line opened to maintain a steady flow. Aeration was applied consistently from the bottom of the reactor. This process continued for about 15 days until the attached biofilm could be found on fibrous filler by microscopical examination. Then the reactor was operated as mentioned above. After about 7 days, the removal of COD and TN were stabilized. Backwashing procedure was designed as follows: air scour for 2 min, combined air scour and water backwash for 2 min, water backwash for 5 min. The backwash air and water velocities were 16 and 5 L m2 s1, respectively. The average operation before a backwash was 3 days and the maximum duration was 7 days. After backwashing, excess sludge was discharged from the bioreactor in order to achieve the desired SRT. Control experiment without biomass To estimate the contribution of activated sludge and biofilm on organic pollutants and nutrients removal in the bioreactor, the control experiment was conducted. The fibrous fillers with biofilm were replaced with new fibrous fillers. After a stable operation of 24 h, samples of three cycles were analyzed. Analysis methods COD, total nitrogen (TN), total phosphorus (TP) and biomass concentrations of the samples were determined according to the standard methods (21). Water pH was measured by an 828 Orion pH meter. Dissolved oxygen (DO) and water temperature were measured with a 52 YSI DO meter. Turbidity was analyzed with a 2100P Hach photoelectric turbidity meter. The suspended biomass concentration was determined as mixed liquid suspended solids (MLSS). To measure the attached biomass on the fibrous fillers, about 1% of fibrous fillers were collected
Consortium of bacterium The test adopted five mixed strains of bacteria. Two strains of bacteria separated from lake sediments were named Pseudomonas sp. enrichment culture clone W1 and W2; while the other three were separated named P. aeruginosa strain ZQP4, B. cereus strain ZQN2 and ZQN6. They all belong to Gammaproteobacteria. Strains W1 and W2 were autotrophic and separated with the medium (0.3 g L1 KNO3, 0.2 g L1 MgSO4, 0.2 g L1 CaCl2, 0.3 g L1 K2HPO4, 0.5 g L1 KH2PO4, 1.0 g L1 NaCl, 2.0 g L1 NaHCO3, 0.8 g L1 FeSO4$7H2O, at pH 6.8). While strain ZQP4, ZQN2 and ZQN6 were heterotrophic and separated with the medium (5.0 g L1 sodium citrate, 1.0 g L1 K2HPO4, 1.0 g L1 KH2PO4, 0.2 g L1 MgSO4$7H2O, and 2.0 g L1 KNO3, at pH 7.2). After cultured and enriched, they were mixed in the same proportion. After sludge seeding and start up in the modified SBBR, at the end of 30th day of operation, the mixed bacteria were added to the reactor with the proportion of 1e150. PCR-DGGE Taking the biological biofilm as samples (approximately 0.2 mL) (22), we treated it by centrifugal and oscillation three times with dd H2O (doubledistilled water), TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl and 0.01 g mL1 PVP, at pH 10.0) and phosphate-buffered saline (PBS; 130 mM NaCl, 7 mM NaHPO4, 3 mM NaH2PO4, at pH 7.4) respectively. Then it was stored it in a 1:1 mixture of PBS and 96% ethanol at 20 C (23). The total DNA of mixed bacteria was extracted by bacteria gDNA Isolation Mini Kit (Watson Biotechnologies, Inc., Shanghai, China) as described in the manufacturer’s instructions. Amplification of V3 region of 16S rRNA genes (rDNA) was tested in a touchdown PCR (TD-PCR) assay. The PCR mixture (total volume of 20 mL) contained the following components: 0.5 mL of each primer (100 mM, BSF338-GC 50 -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG-30 and BSR518 50 - ATT ACC GCG GCT GCT GG -30 ), 1.7 mL of dNTPs (2.5 mM for each), 0.2 mL BSA (20 mg mL1), 0.35 mL Taq polymerase (5 U mL1), 2 mL of genomic DNA, 2 mL 10 PCR buffer and 12.75 mL H2O. TD-PCR program was executed under the following conditions: initial denaturation, 94 C for 5 min; cycle at 94 C for 30 s, 62 C for 1 min, 72 C for 1 min; followed by 19 cycles, when annealing temperature was decreased by 0.5 C in every other cycle; 94 C for 30 s, annealing 55 C for 1 min, 72 C for 2 min, 15 cycles; a final extension step at 72 C for 10 min; cooling and holding at 4 C. PCR products were verified in 1% (w/v) agarose gel. PCR products of 16S rDNA V3 region were mixed DNA fragments with the same base-pair length of about 260 bp, which were separated by denaturing gradient gel electrophoresis apparatus. PCR products (12 mL) blended with 4 mL 10 loading buffer were loaded onto an 8% (w/v) polyacrylamide gel containing a modified 35e68% gradient of denaturant (100% denaturant correspond to 5.6 M urea and 32% (v/v) deionized formamide), which was operated in 1 TAE buffer at 60 C and 100 V for 12 h. The polyacrylamide gels were treated with modified Brant’s silver staining method (24). The DGGE band was cut and the DNA was amplified with primer BSF338 and BSR518 by conventional PCR (94 C for 5 min; 94 C for 30 s, annealing 55 C for 1 min, 72 C for 2 min, 30 cycles; 72 C for 10 min; cooling and holding at 4 C). Verified amplification products were linked with pMD 18-T Vector and added into competent cells of Escherichia coli TOP10. Positive clones were picked and sequenced, and the sequence information was submitted to NCBI database and compared with other microorganisms of 16S rRNA sequence.
RESULTS AND DISCUSSION Variation of biomass and organic loading Fig. 2A shows the variations of biomass and organic loading rates in the bioreactor over the operational period. From the figure, it can be seen that the concentration of MLSS slightly ranged from 1004 mg L1 to 1620 mg L1 and that the biofilm remained stable at appropriate 2780 mg L1. The increase of activated sludge was mainly derived from the biofilm detachment. And the activated sludge decreased as a result of the discharge of sludge after backwashing. The backwashing resulted in a slight detachment of biofilm (
Bio-augmentation as a tool for improving the modified sequencing batch biofilm reactor Kai Yang,1 Bin Ji,1 Hongyu Wang,1, * Huaiyu Zhang,2 and Qian Zhang3 School of Civil Engineering, Wuhan University, Wuhan 430072, China,1 Central and Southern China Municipal Engineering Design and Research Institute, Wuhan 430010, China,2 and School of Civil Engineering and Architecture, Wuhan University of Technology, Wuhan 430070, China3 Received 18 July 2013; accepted 6 November 2013 Available online 15 December 2013
Biological treatment of domestic sewage was accomplished in a pilot-scale modified sequencing batch biofilm reactor (SBBR) bio-augmented with consortium of 5 strains of indigenous bacteria (genus Pseudomonas and Bacillus). The reactor consisted of fibrous filler in the upper and ceramsite filter media in the bottom. It demonstrated to have a short hydraulic residence time (HRT) for 10 h and good quality effluent to cope with low C/N ratio domestic wastewater. The biofilm attached fibrous fillers mainly contributed to contaminants removal. Bio-augmentation dramatically enhanced the removal efficiency of chemical oxygen demand (COD), total phosphorus (TP), and especially total nitrogen (TN), which increased respectively from 80.3%, 58.1% and 41.3% to 83.7%, 67.8% and 58.7%. Polymerase chain reaction (PCR)denaturing gradient gel electrophoresis (DGGE) technique indicated the 5 strains’ survival in the reactor and that Bacillus cereus strain ZQN2 was the most dominant bacteria. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Modified sequencing batch biofilm reactor; Bio-augmentation; Domestic sewage; Nitrogen removal; Denaturing gradient gel electrophoresis]
In a traditional sequencing batch biofilm reactor (SBBR) wastewater is treated by bacteria growing on media packed in the reactor body and the packing serves as filter media to minimize the concentration of suspended solids in the effluent (1). SBBR is a highly effective reactor and widely tested to cope with different high salinity wastewater (2), high carbon wastewater (3), toxic wastewater (4), and especially to treat domestic wastewater with satisfactory results of chemical oxygen demand (COD) removal efficiency of 60%e80% (5,6). New mechanisms were proposed such as simultaneous nitrification, phosphorus uptake and denitrification (7,8) during aerobic stage. However, problems exist with the quite high effluent suspended solids (SS) of around 45 mg L1 and long hydraulic residence time (HRT) of approximate 15 h (5,6). In comparison, traditional SBBR usually needs a quite long stage of settling over 1 h. To solve the problems, a modified SBBR is proposed with a filter layer added to the bottom of the reactor (Fig. 1) to cut HRT and to improve the quality of effluent. This reactor is based on biological aerated filter prototype (9) and principles of cake filtration (10). The reactor consists of an upper biofilm reactor component and a lower filter bed component. In addition, bio-augmentation method is investigated to enhance contaminants removal. Bio-augmentation is a process of application of indigenous or allochthonous wild type or genetically modified organisms to polluted sites or bioreactors to accelerate the removal of undesired contaminants. When an efficient
* Corresponding author. Tel.: þ86 27 61218623; fax: þ86 27 68775328. E-mail address: [email protected] (H. Wang).
consortium is added to the initial indigenous microbial community of a contaminated medium, versatile enzymes are likely to be produced to enhance its biopurification abilities (11). Previous study was mostly related to one single bacteria bio-augmentation to remove specific pollutants, including transgenic bacteria (12). For example, Pseudomonas sp. strain HF-1 (13) was used to degrade nicotine while Pseudomonas stutzeri strain TR2 (14) was used to reduce nitrous oxide. In this article consortium of 5 strains of Gammaproteobacteria were adopted to treat domestic wastewater including 3 strains of Pseudomonas genus and 2 strains of Bacillus genus. It was found that Pseudomonas and Bacillus genera were promisingly effective for wastewater treatment. For instance, Pseudomonas sp. KW1 and Bacillus sp. YW4 were found to be the most efficient in terms of nitrate reduction in synthetic nitrate-rich wastewater (15). Pseudomonas aeruginosa was found to be able to produce nitrite reductase, coded by genes nirQ, to catalyze a key reaction of the denitrification pathway, namely the reduction of nitrite to nitric oxide (16,17). Bacillus cereus was the most effective microbe for simultaneous bio-oxidation of ammonia and manganese in biological aerated filter system (18), and able to occur simultaneous aerobic nitrification and denitrification (19), with a remarkable removal rate of nearly 100% of nitrate nitrogen in 24 h (20). However, most tests are still in the lab scale, and we need to put it to application. For this purpose, after having developed modified SBBR in a pilot-scale to treat domestic wastewater and determined proper process parameters based on the previous study, we added the highly effective 5 strains of bacteria to strengthen the domestic wastewater treatment.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.11.006
764
YANG ET AL.
J. BIOSCI. BIOENG., TABLE 1. Characteristics of the raw water.
Average Standard deviation
Chemical oxygen demand (mg L1)
Total nitrogen (mg L1)
Total phosphorus (mg L1)
Turbidity (mg L1)
pH value
109.87 26.22
20.64 2.35
2.41 0.76
160.23 23.51
7.12 0.67
from the bioreactor and dried for 2 h at 105 C. The total weight of the dried fibers was measured and the weight of the attached biomass was obtained by subtracting the original weight of the fibers. The attached biomass concentration was finally expressed as mg L1 by considering the total number of fibers and the total volume of the reactor.
FIG. 1. Sketch of modified SBBR. 1, long handle filter nozzle; 2, ceramsite filter media; 3, fibrous filler; 4, sampling point; 5, overflow pipe; 6, intake pipe; 7, pipeline pump; 8, backwashing water valve; 9, effluent regulating valve; 10, effluent electronic valve; 11, gas flowmeter; 12, air compressor; 13, intake electromagnetic valve; 14, control cabinet; 15, influent electromagnetic valve.
MATERIALS AND METHODS Wastewater characteristics The raw wastewater used in the study was obtained from the outlet of grit chamber of a municipal wastewater treatment plant, Wuhan, China. The influent characteristics of the bioreactor are presented in Table 1. Reactor operation As shown in Fig. 1, the reactor is a 4.0 m high organic glass column, with 23.5 cm inner diameter, below which was a long handle filter nozzle in the middle of retainer plate with dozens of holes with a diameter of 0.9 mm, whose function mainly was aeration and backwashing. The upper layer was filled with a depth of 0.3 m fibrous filler (1512 kg m3 density, 0.4 mm diameter, 35e40 mm length, 90% porosity) as a biofilm reactor and the lower layer was filled with a depth of 1.0 m ceramsite filter media (1e2 mm diameter, 2260 kg m3 density, non-uniform coefficient K80 ¼ d80/d10 < 2.0) as a filter bed. The highest water level (H1) and the lowest water level (H2) were controlled by floating ball valve according to processing capacity. Through the operation of a control cabinet, raw water flowed by the pipeline pump into the reactor, mixing and standing for 30 min. The aeration time was 70 min. Finally after settling for 10 min, the effluent water was discharged from the reactor. The parameters were controlled as follows: dissolved oxygen (DO) concentration of upper reactor for aeration was 2e3 mg L1, filter speed was 5 m h1, HRT was 10 h (2 h each cycle), and sludge retention time (SRT) was 50 d. The tests were operated outdoor under ambient climatic conditions with the temperature at 16e25 C. The reactor was inoculated with about 30 L activated sludge of the plant with a concentration of 3000 mg L1. Then the whole reactor was submerged into feed water and aerated for 1 h to achieve complete mixing. After conditioned for 10 h, wastewater in the reactor was discharged entirely. Fresh influent was continuously injected into the reactor, accompanied by the discharge line opened to maintain a steady flow. Aeration was applied consistently from the bottom of the reactor. This process continued for about 15 days until the attached biofilm could be found on fibrous filler by microscopical examination. Then the reactor was operated as mentioned above. After about 7 days, the removal of COD and TN were stabilized. Backwashing procedure was designed as follows: air scour for 2 min, combined air scour and water backwash for 2 min, water backwash for 5 min. The backwash air and water velocities were 16 and 5 L m2 s1, respectively. The average operation before a backwash was 3 days and the maximum duration was 7 days. After backwashing, excess sludge was discharged from the bioreactor in order to achieve the desired SRT. Control experiment without biomass To estimate the contribution of activated sludge and biofilm on organic pollutants and nutrients removal in the bioreactor, the control experiment was conducted. The fibrous fillers with biofilm were replaced with new fibrous fillers. After a stable operation of 24 h, samples of three cycles were analyzed. Analysis methods COD, total nitrogen (TN), total phosphorus (TP) and biomass concentrations of the samples were determined according to the standard methods (21). Water pH was measured by an 828 Orion pH meter. Dissolved oxygen (DO) and water temperature were measured with a 52 YSI DO meter. Turbidity was analyzed with a 2100P Hach photoelectric turbidity meter. The suspended biomass concentration was determined as mixed liquid suspended solids (MLSS). To measure the attached biomass on the fibrous fillers, about 1% of fibrous fillers were collected
Consortium of bacterium The test adopted five mixed strains of bacteria. Two strains of bacteria separated from lake sediments were named Pseudomonas sp. enrichment culture clone W1 and W2; while the other three were separated named P. aeruginosa strain ZQP4, B. cereus strain ZQN2 and ZQN6. They all belong to Gammaproteobacteria. Strains W1 and W2 were autotrophic and separated with the medium (0.3 g L1 KNO3, 0.2 g L1 MgSO4, 0.2 g L1 CaCl2, 0.3 g L1 K2HPO4, 0.5 g L1 KH2PO4, 1.0 g L1 NaCl, 2.0 g L1 NaHCO3, 0.8 g L1 FeSO4$7H2O, at pH 6.8). While strain ZQP4, ZQN2 and ZQN6 were heterotrophic and separated with the medium (5.0 g L1 sodium citrate, 1.0 g L1 K2HPO4, 1.0 g L1 KH2PO4, 0.2 g L1 MgSO4$7H2O, and 2.0 g L1 KNO3, at pH 7.2). After cultured and enriched, they were mixed in the same proportion. After sludge seeding and start up in the modified SBBR, at the end of 30th day of operation, the mixed bacteria were added to the reactor with the proportion of 1e150. PCR-DGGE Taking the biological biofilm as samples (approximately 0.2 mL) (22), we treated it by centrifugal and oscillation three times with dd H2O (doubledistilled water), TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl and 0.01 g mL1 PVP, at pH 10.0) and phosphate-buffered saline (PBS; 130 mM NaCl, 7 mM NaHPO4, 3 mM NaH2PO4, at pH 7.4) respectively. Then it was stored it in a 1:1 mixture of PBS and 96% ethanol at 20 C (23). The total DNA of mixed bacteria was extracted by bacteria gDNA Isolation Mini Kit (Watson Biotechnologies, Inc., Shanghai, China) as described in the manufacturer’s instructions. Amplification of V3 region of 16S rRNA genes (rDNA) was tested in a touchdown PCR (TD-PCR) assay. The PCR mixture (total volume of 20 mL) contained the following components: 0.5 mL of each primer (100 mM, BSF338-GC 50 -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG-30 and BSR518 50 - ATT ACC GCG GCT GCT GG -30 ), 1.7 mL of dNTPs (2.5 mM for each), 0.2 mL BSA (20 mg mL1), 0.35 mL Taq polymerase (5 U mL1), 2 mL of genomic DNA, 2 mL 10 PCR buffer and 12.75 mL H2O. TD-PCR program was executed under the following conditions: initial denaturation, 94 C for 5 min; cycle at 94 C for 30 s, 62 C for 1 min, 72 C for 1 min; followed by 19 cycles, when annealing temperature was decreased by 0.5 C in every other cycle; 94 C for 30 s, annealing 55 C for 1 min, 72 C for 2 min, 15 cycles; a final extension step at 72 C for 10 min; cooling and holding at 4 C. PCR products were verified in 1% (w/v) agarose gel. PCR products of 16S rDNA V3 region were mixed DNA fragments with the same base-pair length of about 260 bp, which were separated by denaturing gradient gel electrophoresis apparatus. PCR products (12 mL) blended with 4 mL 10 loading buffer were loaded onto an 8% (w/v) polyacrylamide gel containing a modified 35e68% gradient of denaturant (100% denaturant correspond to 5.6 M urea and 32% (v/v) deionized formamide), which was operated in 1 TAE buffer at 60 C and 100 V for 12 h. The polyacrylamide gels were treated with modified Brant’s silver staining method (24). The DGGE band was cut and the DNA was amplified with primer BSF338 and BSR518 by conventional PCR (94 C for 5 min; 94 C for 30 s, annealing 55 C for 1 min, 72 C for 2 min, 30 cycles; 72 C for 10 min; cooling and holding at 4 C). Verified amplification products were linked with pMD 18-T Vector and added into competent cells of Escherichia coli TOP10. Positive clones were picked and sequenced, and the sequence information was submitted to NCBI database and compared with other microorganisms of 16S rRNA sequence.
RESULTS AND DISCUSSION Variation of biomass and organic loading Fig. 2A shows the variations of biomass and organic loading rates in the bioreactor over the operational period. From the figure, it can be seen that the concentration of MLSS slightly ranged from 1004 mg L1 to 1620 mg L1 and that the biofilm remained stable at appropriate 2780 mg L1. The increase of activated sludge was mainly derived from the biofilm detachment. And the activated sludge decreased as a result of the discharge of sludge after backwashing. The backwashing resulted in a slight detachment of biofilm (
