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Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR
Accepted Manuscript Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR Qian Chen, Jinren Ni, Tao Ma, Tang Liu, Maosheng Zheng PII: DOI: Reference:
S0960-8524(15)00191-1 http://dx.doi.org/10.1016/j.biortech.2015.02.022 BITE 14594
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 November 2014 1 February 2015 7 February 2015
Please cite this article as: Chen, Q., Ni, J., Ma, T., Liu, T., Zheng, M., Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.02.022
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Revised Manuscript submitted for Bioresource Technology
1/31/2015
Manuscript Number: BITE-D-14-06531
Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR
Qian Chen, Jinren Ni∗, Tao Ma, Tang Liu, Maosheng Zheng
Department of Environmental Engineering, Peking University; Key Laboratory of Water and Sediment Sciences, Ministry of Education, Beijing 100871, China
∗
Corresponding author: Jinren Ni
Yiheyuan Road, Peking University, Beijing 100871, China; Tel.: +86-10-62751185; Fax: +86-10-62756526; E-mail address: [email protected]
Abstract PCN bacteria capable of heterotrophic-aerobic nitrogen removal was successfully applied for bioaugmented treatment of municipal wastewater in a pilot-scale SBR. At an appropriate COD/N ratio of 8, the bioaugmentation system exhibited stable and excellent carbon and nutrients removal, the averaged effluent concentrations of COD, NH4+-N, TN and TP were 20.6, 0.69, 14.1 and 0.40 mg/L, respectively, which could meet the first class requirement of the National Municipal Wastewater Discharge Standards of China (COD<50 mg/L, TN<15 mg/L, TP<0.5 mg/L). Clone library and real-time PCR analysis revealed that the introduced bacteria greatly improved the structure of original microbial community and facilitated their aerobic nutrients removal capacities. The proposed emerging technology was shown to be an alternative technology to establish new wastewater treatment systems and upgrade or retrofit conventional systems from secondary-level to tertiary-level.
Keywords: nutrients removal; heterotrophic-aerobic nitrogen removal bacteria; bioaugmentation; municipal wastewater treatment; 16S rRNA gene libarary
1. Introduction Nitrogen and phosphorus have become the key factors leading to the eutrophication of receiving waters. Therefore, increasingly stringent environmental regulations are carried out to decrease their discharges, which are creating an urgent need for technological solutions to enhance nutrients removal, especially the nitrogen removal, in the existing secondary wastewater treatment plants (WWTPs) (Jin et al., 2012). Conventional nitrogen removal consists of two steps: nitrification by autotrophs under aerobic conditions and denitrification by heterotrophs under anaerobic conditions. With this technology to retrofit or upgrade the WWTPs, supplemental tertiary treatment facilities and extra land are always necessary owing to the low rate of nitrification and the different requirement for nitrifiers and denitrifiers. However, most old WWTPs in China use conventional activated sludge systems. They are requested to satisfy the first class (level A) requirement of the National Municipal Wastewater Discharge Standards (COD<50 mg/L, TN<15 mg/L, TP<0.5 mg/L). As a result, they will face enormous challenge for modifying their treatment systems because of the limitation of land and capital (Brepols et al., 2008). Hence, the demand for more cost-efficient and less land-occupied technology is especially acute. Recently, bacteria capable of heterotrophic nitrification and aerobic denitrification, such as Thiosphaera pantotropha, Alcaligenes faecalis, and Bacillus sp., etc., has been investigated as potential microorganisms in biological nitrogen removal systems (Guo et al., 2013; Joo et al., 2005; Zhang et al., 2012). These microorganisms, due to their high growth rate and ability to remove ammonium and nitrate aerobically, have a great number of advantages as applied for the removal of nitrogen: i) procedural simplicity, where nitrification and denitrification can take place simultaneously; ii) less acclimation
problems; iii) lesser buffer quantity needed because alkalinity generated during denitrification can partly compensate for the alkalinity consumption in nitrification (Gupta, 1997). However, most of these studies have focused on the characteristics of single strain and the nitrogen removal efficiency in lab-scale reactors by single strain (Joo et al., 2006; Shoda & Ishikawa, 2014; Yao et al., 2013). To date, the effectiveness of adding these kind of bacteria into actual activated sludge systems to enhance nitrogen removal and the possibility of using these kind of bacteria to directly upgrade the WWTPs has never been evaluated. Previously, the heterotrophic–aerobic nitrogen removal abilities of Agrobacterium tumefaciens LAD9, Comonas testosteroni GAD3 and Achromobacter xylosoxidans GAD4 were described (Chen & Ni, 2011). The present study aimed to determine whether augmentation with this consortium could successfully enhance nitrogen removal in a pilot-scale SBR for municipal wastewater treatment and retrofit the original system without any supplementary structures. Particular attention was paid to investigate the interactions between inoculated microorganisms and indigenous microbial communities and to reveal the possible biochemical mechanisms for nutrients removal on the basis of ecological data. This study is of particular importance to establishing new wastewater treatment systems and upgrading or retrofitting conventional systems from secondary-level to tertiary-level. 2 Materials and methods 2.1. Bacteria and growth condition Three
heterotrophic
nitrification-aerobic
denitrification
bacteria,
named
Agrobacterium tumefaciens LAD9, Comonas testosteroni GAD3 and Achromobacter xylosoxidans GAD4, were used in the experiment. They were isolated from the sludge for the treatment of landfill leachate (Chen & Ni, 2011). All the strains were fermented
with the synthetic mineral medium and freeze-dried into powder. The mixture of them (1:1:1), named PCN bacteria, were used for the inoculation. 2.2. Pilot-scale SBR Two parallel pilot-scale SBR systems were constructed in the Yanshi WWTP (Henan province, China). The SBR was proportionally scaled as the reaction tank in the WWTP, with a size of 830 (length) ×400 (width) ×500 (height) mm and a working volume of 165 L (Figure 1). 2.3. Wastewater and seeding sludge The influent wastewater was directly introduced to the SBR reactor from the regulating tank in the WWTP. The composition of the influent was given in Table 1. Both reactors were initially inoculated with activated sludge taken from the aeration tank of the WWTP. The seeding sludge had a mixed liquor suspended solids (MLSS) concentration of 4.9 g/L, a mixed liquor volatile suspended solids (MLVSS) concentration of 2.58 g/L and a sludge volume index (SVI) of 46.9 mL/g. 2.4. Operation strategy The SBRs were operated with a cycle of 6 h. The cycle consisted of 5 phases: 1 h of influent feeding, 3 h of aeration, 1 h of settling, 10 min of drawing, and 50 min of idle period, which was consistent with the actual operation of the WWTP. The volumetric exchange ratio was 50% and dissolved oxygen (DO) was maintained at 2-3 mg/L. After one week stabilization, the two reactors were operated with different protocols: (1) R1, control SBR, with no extra bacteria addition; (2) R2, bioaugmented SBR, seeded with 3 g PCN bacteria made in 2.1. The operation was divided into two phases, first phase (0-20 d): both reactors were fed with raw wastewater; second phase (21-53 d): sodium succinate was used as the extra carbon source and directly added to the reactor at the beginning of every aeration period. The pH and DO values were
monitored at a regular time interval during the operation. For long operation of reactor R2, 50 g of sodium succinate was directly added to the reactor at the beginning of every aeration period. The solid retention time (SRT) of reactor R2 was kept at 12-15 d and the mixed liquor suspended solids (MLSS) in the main reaction zone were maintained at about 4000 mg/L throughout the experiment. 2.5. Analytical methods COD, MLSS (mixed liquid suspended solids), ammonium (NH4+-N), total nitrogen (TN) and total phosphorus (TP) were analyzed following the procedures in the standard methods.
Nitrite
(NO2--N)
N-(1-naphthyl)-ethylene spectrophothometry,
and
diamine
respectively.
nitrate
(NO3--N)
were
spectrophothometry Dissolved
oxygen
(DO)
determined and
using
ultraviolet
concentration
was
determined by a DO meter (YSI-550A, YSI, USA). Activated sludge suspension was serially diluted with sterile distilled water and spread onto beef-extract peptone agar plates in duplicate. The number of bacterial colonies was counted after the plates were incubated at 30 oC for 3 days. Limited by specific PCR primers for PCN bacteria, bromothymol blue (BTB) plating method were used to demonstrate the possible abundance of PCN bacteria, which was initially proposed by Takeya et al. (2003) for the purpose of isolating aerobic denitrifiers. The plates contained nitrate and the pH indicator BTB. If there were aerobic denitrifiers, the pH value in the medium would increase due to the depletion of nitrate by denitrification and the colonies would turn blue. The pH of the medium was initially controlled within 7.0 to 7.3. The plates inoculated with the activated sludge suspension were aerobically incubated at 30 oC for 3 days, and then aerobic bacteria for nitrate metabolism were monitored by examining the blue colonies or halos formed owing to the increasing pH of the medium. The number of positive strains were counted and
regarded as the possible abundance of PCN bacteria. 2.6. Microbial ecology in the reactors 2.6.1. DNA extraction Genomic DNA was extracted by the 3S DNA isolation kit (Shenergy Biocolor, Shanghai) under the manufacture’s instruction. The size and concentration were estimated by agarose gel electrophoresis (1%) and then stored at -20 oC in a freezer for further application. 2.6.2. Construction of 16S rRNA gene clone libraries and sequence analysis Two clone libraries were constructed from reactors R1 and R2. PCR amplification was performed with 27F and 1492 R primer pairs and performed under the following conditions: 94 oC for 5 min, followed by 30 cycles 94 oC for 60 s, 55 oC for 60 s and 72 o
C for 60 s, and a final extension step at 72 oC for 10 min. The PCR products were
separated by agarose (0.8%) gel electrophoresis and purified with Qiaquick PCR Gel Extraction Kit (QIAGEN, Stanford, CA, USA). The purified DNA fragments were cloned using the pGEM-T Easy Vector System (Takara, Dalian, China) with TOP10 competent E. Coli cells, and plated on LB (Luria-Bertani) plates supplied with ampicillin. Colonies were randomly picked, cultured overnight in LB broth supplemented with ampicillin, and then sequenced in Invitrogen Inc. (USA). Obtained sequences were compared with available sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Phylogenetic reconstructions were performed in MEGA 6.0 (Tamura et al., 2013) using the neighbor-joining (NJ) algorithm, with bootstrap values calculated from 1000 replicate runs. 2.6.3. Quantification of amoA genes by real-time PCR amoA genes of ammonia-oxidizing bacteria (AOB) were amplified from the
environmental
DNA
extracted.
Primer
(5’-GGGGTTTCTACTGGTGGT-3’)
pairs
and
amoA-1F amoA-2R
(5’-CCCCTCKGSAAAGCCTTCTTC-3’) were used. Real-time PCR was conducted using an ABI 7300 Sequence Detector (Applied Biosystems). The 20 µL reaction volume contained 10 µL SYBR® Green PCR Master Mix (Takara, Dalian, China), 0.5 µL of each primer and 2 µL of 5-fold diluted extracted DNA as a template. Amplifications were carried out as follows: 50 oC for 2 min, 95 oC for 10 min, followed by 45 cycles of 15 s at 95 oC, 45 s at 50 oC, 1.5 min at 72 oC. Melting curve analysis was performed at the end of PCR runs to check the specificity of the products. PCR products amplified from extracted DNA with the primers for real-time PCR assays were gel-purified and ligated into the pMD 18-T Vector (Takara, Dalian, China), with an access number of L08752.1. The resulting ligation products were transformed into E.coli cells. After reamplification with the vector, the positive clones were selected to extract plasmid DNA (Axygen, USA) and used as amoA gene standards. Tenfold serial dilutions of a known copy number of the plasmid DNA were subjected to real-time PCR to generated an external standard curve. 3. Results and discussion 3.1. Effectiveness of bioaugmentation in the pilot-scale SBRs Two parallel SBR reactors were used to investigate the performance and efficacy of bioaugmentation during continuous operation. 3.1.1. First stage (with raw wastewater) After the inoculation of PCN bacteria, R1 (control reactor) and R2 (bioaugmented reactor) had similar behavior for 22 days with regards to COD, NH4+-N, and TN removals (Figure 2).In R1, averaged effluent concentrations of COD, NH4+-N, and TN were 18.5, 0.48, 34.6 mg/L, respectively; while in R2, they were 24.8, 0.42, 33.2 mg/L,
respectively. It was suggested that PCN bacteria were not effective at this condition. From previous studies, the competition between the introduced bacteria and the indigenous species, and the insufficiency of substrate have been considered as most possible reasons for the failure of the bioaugmentation (Bouchez et al., 2000). As a result, batch experiments were carried out to investigate activities of the inoculated bacteria in the received ecosystem. First, activated sludge taken from R1 and R2 were separately diluted and plated on the BTB medium to detect the possible abundance of PCN bacteria in the reactors. The result showed that the number of BTB positive bacteria in R2 was 10-times higher (3.3×105 CFU/mL) than that in R1 (6.7×104 CFU/mL), suggesting PCN bacteria was probably active in the reactor. Meanwhile, batch experiment with the bioaugmented sludge and the target wastewater was also conducted in shake flasks with or without the supply of sodium succinate, a favorable carbon source to PCN strains (Chen & Ni, 2012). Without external carbon source, the TN removal efficiency by the sludge was only 42%. However, with 5.0 g/L of sodium succinate feeding, it was enhanced to 80% (data not shown). It could be concluded that the insufficient substrate availability (low COD/N ratio in the influent) was the main reason for the failure of bioaugmentation. 3.1.2. Second phase (with succinate as the external carbon source) According to the above inference, sodium succinate, an extra carbon source, was directly added into R1 and R2 tanks at the beginning of every aeration period. It was observed that no matter with or without the sodium succinate, R1 and R2 reached similar and high removal efficiencies for COD and NH4 +-N (Fig. 2(A) and 2(B)). However, it was significantly different for the TN removal (Fig. 2(C)). TN removal efficiencies in R2 reached 66.0%, 67.1%, 69.0% and 80.5% at external carbon dosages of 30, 40, 45 and 50 g, respectively, which were higher than those in R1 (57.9%, 60.0%,
60.3% and 66.8%), indicating the bioaugmented reactor was more effective for TN removal. The improved nitrogen removal in the bioaugmented reactor could be attributed to the contribution of PCN bacteria. In addition, the removal percentage of TN was correlated well with the carbon supply and there was a remarkably improvement at a carbon dosage of 50 g (COD/N ratio approximately equal to 8.0). At this condition, the concentration of effluent TN in R2 was averaged at 11.2 mg/L, which could meet the first class (level A) requirement of the National Municipal Wastewater Discharge Standards of China (TN<15 mg/L). When the carbon dosage was lower than 50 g, the variations of TN removal efficiency were insignificant. Nevertheless, much better effluent quality would be achieved when the carbon dosage was further increased (data not shown). It was suggested that the activity of PCN bacteria in the received activated sludge system could be significantly prompted when the COD/N ratio reached to 8.0. However, the optimal COD/N ratio (approximate 20.0) for heterotrophic-aerobic nitrogen removal by PCN bacteria was much higher than that value (Chen & Ni, 2012). This difference revealed that the introduced PCN bacteria could coordinate with other indigenous species to achieve high efficiency of nitrogen removal, which resulted in the reduction of carbon requirement. Moreover, comparison was made between full-scale SBR, the bioaugmented SBR, and representative improved SBR systems for nitrogen removal (Table II). It was shown that TN removals in all the improved systems were much better than that in the conventional SBR, while the bioaugmentation was most convenient and simple method for the improvement. Besides, at similar COD/N ratios, TN removal efficiency in the bioaugmented SBR was comparable to it in the systems accompanied with complex control strategy. These preliminary findings indicated that satisfactory effluent quality could be achieved through bioaugmentation.
Furthermore, the performance of carbon and nitrogen transformation during a typical SBR-cycle in R2 was investigated at the carbon dosage of 50 g (Fig. 3). It was demonstrated that at the beginning of the aeration period (1 h), COD concentration was rapidly increased to 234 mg/L because of the addition of succinate sodium into the tank. After that, COD, NH4 +-N and NO3--N concentrations were synchronously decreased during the initial 30 min aeration, indicating aerobic total nitrogen removal had took place in the reactor. When the COD was exhausted (1.5h), the removed ammonium was almost converted to nitrate and the accumulation amount of NO3--N maintained at 11.1 mg/L after 99% removal of NH4+-N, suggesting conventional nitrifiers played a major role at this period. Regarding the nitrogen balance, nitrogen loss at the aerobic period reached to 79.1%, which was comparable to the conventional simultaneous nitrification and denitrification studies (Chiu et al., 2007). Aerobic denitrification and assimilation probably contributed to this nitrogen loss. 3.2. Long term operation of the bioaugmented SBR Long-term operation was conducted in R2 to evaluate the stability and the frequency of bacteria adding. Because NH4+-N and TN concentrations of raw wastewater were relatively stable, 50 g of sodium succinate was added into the aeration tank every cycle to change influent COD and COD/N ratios. As depicted in Fig. 4(B) and 4(C), the removal percentages of COD and NH4 +-N was maintained at 85.8% and 98.8%, respectively. Fig. 4(A) revealed that the system performed excellently on TN removal, with an averaged effluent TN concentration of 14.1 mg/L, which meet well with the national standard. The exceptions occurred on days of 28th, 37th and 51th owing to the big shocks of nitrogen load, but the system recovered quickly by itself without supplementary addition of PCN bacteria. More importantly, remarkable performance of TP removal was observed in this system (Fig.
4(D)). TP removal efficiency reached 85.0% and the averaged effluent concentration was 0.40 mg/L, which could meet the first class (level A) requirement of the National Municipal Wastewater Discharge Standards of China (TP < 0.5 mg/L). According to the PO43--P removal profile during a whole SBR cycle (data not shown), the decrease of PO43--P concentration was synchronous with the degradation of carbon and nitrogen at the aerobic phase. It could be speculated that the excellent uptake of phosphorus at the aerobic condition was mainly caused by the high removal of nitrogen, where nitrogen and phosphorus were co-metabolized by the PCN bacteria. These findings indicated that this bioaugmentation system was high-stability and had excellent performance on nutrients removal. Furthermore, successful application of bioaugmentation technology for wastewater treatment depends on the adaptation of microbial strains with the indigenous microorganisms, which means the introduced microbial strains should survive and keep activity in the received systems and do not influence the function of indigenous degrading microorganisms. As a result, daily repeated adding (Boon et al., 2002) or embedding strategy (Bouchez et al., 2009) was often adopted to avoid the influence of native microorganism and to evade the dilution effect of the withdrawals. However, it greatly enhanced the operation cost in actual applications. In this study, PCN bacteria were successfully introduced to the activated sludge system through one time addition and exhibited excellent accommodation and compatibility with the received ecosystem, which was of great importance for actual application. In conclusion, simultaneous COD and nutrients removal was achieved aerobically through bioaugmentation with PCN strains. As a result, it has many advantages over the conventional systems: i) nitrification and denitrification could take place simultaneously in the aeration tank and then it was unnecessary to recirculate the nitrate rich liquid to
the anoxic tank for anaerobic denitrification or construct additional advanced treatment systems for nutrients removal; ii) it was highly flexible to be merged into existing biological treatment systems to enhance nutrients removal efficiency without the requirement for extra land occupancy; iii) the resistance capability to fluctuations would be greatly enhanced because the maximum growth rates of the heterotrophic nitrifiers are five to ten times than those of the autotrophic nitrifiers. In view of the above consideration, the proposed bioaugmentation strategy should be very promising owing to its incomparable advantages such as reducing energy consumption and waiving additional structures over conventional systems designed for upgrading or retrofitting WWTPs. 3.3. Microorganism community 3.3.1. Biomass Culture-depend technique was used to compare the biomass between reactors R1 and R2. The results demonstrated that the number of total bacteria in reactor R1 (1.71×10 7 CFU/mL) was similar to that in R2 (1.46×107). However, the number of BTB positive bacteria in reactor R2 (2.53×105 CFU/mL) was significantly higher than that in R1 (4.99×104 CFU/mL), indicating the addition of PCN bacteria changed the composition of bacterial community. 3.3.2. Phylogenetic Analysis The two 16S rDNA clone libraries from reactors R1 and R2 were constructed to evaluate the composition of bacterial community and investigate the community shift resulted by the bioaugmentation. Estimation of species coverage and diversity were carried out for the two 16S rDNA clone libraries. Good coverage was found in both libraries (70.8% for R1 and 70.6% for R2 ). Sequences analysis showed that 36 distinct OTUs and 34 distinct OTUs
were obtained from the bioaugmented sludge and the control sludge clone libraries, respectively. It was important that a significant number of the OTUs comprised of a single clone, indicating high levels of bacterial diversity. Shannon-Weaver indices were observed to be 4.23 in R1 and 4.25 in R2. This high similarity suggested that bioaugmentation did not result in the transformation of community diversity. Furthermore, the 16S rDNA sequences of two clone libraries were combined to analyze the relationship of bacterial communities between the two reactors, which were presented in a figure of taxonomic classification (Fig. 5). The results showed that the bacterial communities were distinctively different from each other. The bacterial community in the control reactor was dominated by Bacterodietes (25%), Acidobacteria (18.8%), Planctomycetes (16.7%) and Proteobacteria (10.4%). However, the abundance of phylum Acidobacteria was decreased significantly after bioaugmentation (3.92%). In contrast, the abundance of phyla Chlorobi (15.7%) and Firmicutes (11.8%) were increased significantly after bioaugmentation. Among the different classes of Proteobacteria, β-proteobacteria exhibited the highest diversity in both reactors. It was remarkable that phyla Planctomycetes, Chloroflexi, and Actinobacteria were disappeared, while phyla Verrucomicrobiales and Gemmatimonadetes were newly represent after bioaugmentation. Results in literatures showed that members of the phyla Planctomycetes, Chloroflexi, and Actinobacteria could induce the occurrence of anaerobic nitrogen removal (Chiellini et al., 2013; Pizzetti et al., 2011). In this aerobic nitrogen removal systems, they were all degenerated. Members of the phylum Gemmatimonadetes
have
been
identified
as
Gram-negative,
aerobic,
polyphosphate-accumulating microorganisms found in vermicompost (Vaz-Moreira et al., 2008), and members of the phylum Verrucomicrobia have been widely reported in wastewater treatment systems for nitrogen removal (Yu & Zhang, 2012).
These preliminary findings suggested that the bioaugmentation accelerated the variation of inoculated microorganism community and facilitated the improvement of the community function. For instance, the concentration of AOB amoA gene in the bioaugmented system was 8.40×108/g sludge, while it was 2.85×10 8/g sludge in the control system. It was also demonstrated by previous studies (Boon et al., 2003) that bioaugmentation could promote bacterial community shift and make the community distinct from the original system, because of changes in interior (such as invasion of inoculated strains) and exterior environment (such as change of pH condition). Moreover, there was no sequence identical (>97% similarity) with the inoculated PCN strains (Fig. 6), indicating the introduced PCN bacteria did not become dominant in the reactor. However, high aerobic nitrogen removal efficiency was obtained through bioaugmentation in this study. Similar results were present in the bioaugmentation studies on refractory organic matter degradation, which revealed that the inoculated strains were undetectable in the end and the removal of target pollutants was still in effect (Bai et al., 2011; Munakata-Marr et al., 1997). It could be speculated that the introduced PCN bacteria could synergize with other indigenous microorganisms to achieve good treatment results. 4. Conclusions PCN bacteria capable of heterotrophic-aerobic nitrogen removal was used for the bioaugmented treatment of municipal wastewater in a pilot-scale SBR. At an appropriate COD/N ratio of 8.0, the produced effluent could satisfy the first class requirement of the National Municipal Wastewater Discharge Standards of China without any extra system modification. The bioaugmentation improved the microbial community structure and facilitated their capabilities of aerobic nutrients removal. The proposed bioaugmentation strategy could be considered as an effective
and economical alternative technology for the treatment of municipal wastewater and the upgrade or retrofitting of the WWTPs.
Acknowledgements Financial support from National Natural Science Foundation of China (Grant No. 51208007), Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20120001120101) and Guangzhou Scientific and Technologic Project for Water Service (Grant No. GZSK-FW-1201) are fully appreciated.
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Figure Captions Fig. 1. SBR reactor configuration. Fig. 2. Comparison of treatment efficiencies for (A) COD, (B) NH4+-N, and (C) TN in R1 and R2. Symbols: ●, influent; ▲ , effluent in R1; ○, effluent in R2; -, extra carbon dosage. Fig. 3. COD and nitrogen removal profiles in a typical SBR cycle in R2 at an extra carbon dosage of 50 g. Symbols: ○, pH; □, COD; ▲ , NH4+-N; ■ , NO3--N; ●, NO2--N. Fig. 4. Long-term performance of the bioaugmented system with external carbon addition. (A) TN removal; (B) COD removal; (C) NH4+-N removal; (D) TP removal. Symbols: ■ , influent; ○, effluent; ▲ , dosage of sodium succinate. Fig. 5. Phylum or class-level (for Proteobacteria) phylogenetic affiliations for sequences retrieved from reactors R1 and R2. Fig. 6. Neighbor-joining trees showing the phylogenetic affiliation between PCN strains and selected 16S rDNA OTU sequences (only OTUs comprising ≥2 clones are shown) from reactors R1 and R2. The scale bar indicates the number of nucleotide substitutions per site. Three 16S rDNA sequences, named LAD9, GAD3 and GAD4, were derived from PCN strains.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Table 1 Compostion of the municipal wastewater. Component
Level
Average value
Total COD (mg/l)
101-254
140
NH4+-N (mg/l)
30.4-66.6
50.2
Total N (mg/l)
30.6-72.4
51.6
Total P (mg/l)
1.10-5.60
2.69
pH
7.50-8.07
7.81
Table 2 Comparison of nitrogen removal efficiencies between full-scale SBR, the bioaugmented SBR, and representative improved SBR systems.
Process
Influent
Municipal Full-scale SBR wastewater Lab-scale SBBR Lab-sclae
Synthetic wastewater Domestic
A2N-SBR wastewater Lab-scale Synthetic intermittently wastewater aerated SBR Lab-scale Artificial MBR Pilot-scale SBR
wastewater municipal
Inoculum
Activated sludge Activated sludge Acitivated sludge Activated sludge Single bacteria Activated sludge+PCN
Average Average Influent N COD/N TN removal ratio (%) 34
8.8
58%
30
10
87.8%
37
7.9
83%
125
8
71.2%
120
8.3
53%
51.6
7-8
80.5%
Reference
(Fernandes et al., 2013) (Jin et al., 2012) (Wang et al., 2009) (Mansouri et al., 2014) (Yao et al., 2013) This study
Graphical abstract
Highlights
Bioaugmented treatment of municipal wastewater in a pilot-scale SBR. Heterotrophic-aerobic nitrogen removal bacteria (PCN) used for bioaugmentation. Addition of PCN bacteria improved the original bacterial community structure. Bioaugmentation enhanced the aerobic nutrients removal ability of original system.
S0960-8524(15)00191-1 http://dx.doi.org/10.1016/j.biortech.2015.02.022 BITE 14594
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 November 2014 1 February 2015 7 February 2015
Please cite this article as: Chen, Q., Ni, J., Ma, T., Liu, T., Zheng, M., Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.02.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised Manuscript submitted for Bioresource Technology
1/31/2015
Manuscript Number: BITE-D-14-06531
Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR
Qian Chen, Jinren Ni∗, Tao Ma, Tang Liu, Maosheng Zheng
Department of Environmental Engineering, Peking University; Key Laboratory of Water and Sediment Sciences, Ministry of Education, Beijing 100871, China
∗
Corresponding author: Jinren Ni
Yiheyuan Road, Peking University, Beijing 100871, China; Tel.: +86-10-62751185; Fax: +86-10-62756526; E-mail address: [email protected]
Abstract PCN bacteria capable of heterotrophic-aerobic nitrogen removal was successfully applied for bioaugmented treatment of municipal wastewater in a pilot-scale SBR. At an appropriate COD/N ratio of 8, the bioaugmentation system exhibited stable and excellent carbon and nutrients removal, the averaged effluent concentrations of COD, NH4+-N, TN and TP were 20.6, 0.69, 14.1 and 0.40 mg/L, respectively, which could meet the first class requirement of the National Municipal Wastewater Discharge Standards of China (COD<50 mg/L, TN<15 mg/L, TP<0.5 mg/L). Clone library and real-time PCR analysis revealed that the introduced bacteria greatly improved the structure of original microbial community and facilitated their aerobic nutrients removal capacities. The proposed emerging technology was shown to be an alternative technology to establish new wastewater treatment systems and upgrade or retrofit conventional systems from secondary-level to tertiary-level.
Keywords: nutrients removal; heterotrophic-aerobic nitrogen removal bacteria; bioaugmentation; municipal wastewater treatment; 16S rRNA gene libarary
1. Introduction Nitrogen and phosphorus have become the key factors leading to the eutrophication of receiving waters. Therefore, increasingly stringent environmental regulations are carried out to decrease their discharges, which are creating an urgent need for technological solutions to enhance nutrients removal, especially the nitrogen removal, in the existing secondary wastewater treatment plants (WWTPs) (Jin et al., 2012). Conventional nitrogen removal consists of two steps: nitrification by autotrophs under aerobic conditions and denitrification by heterotrophs under anaerobic conditions. With this technology to retrofit or upgrade the WWTPs, supplemental tertiary treatment facilities and extra land are always necessary owing to the low rate of nitrification and the different requirement for nitrifiers and denitrifiers. However, most old WWTPs in China use conventional activated sludge systems. They are requested to satisfy the first class (level A) requirement of the National Municipal Wastewater Discharge Standards (COD<50 mg/L, TN<15 mg/L, TP<0.5 mg/L). As a result, they will face enormous challenge for modifying their treatment systems because of the limitation of land and capital (Brepols et al., 2008). Hence, the demand for more cost-efficient and less land-occupied technology is especially acute. Recently, bacteria capable of heterotrophic nitrification and aerobic denitrification, such as Thiosphaera pantotropha, Alcaligenes faecalis, and Bacillus sp., etc., has been investigated as potential microorganisms in biological nitrogen removal systems (Guo et al., 2013; Joo et al., 2005; Zhang et al., 2012). These microorganisms, due to their high growth rate and ability to remove ammonium and nitrate aerobically, have a great number of advantages as applied for the removal of nitrogen: i) procedural simplicity, where nitrification and denitrification can take place simultaneously; ii) less acclimation
problems; iii) lesser buffer quantity needed because alkalinity generated during denitrification can partly compensate for the alkalinity consumption in nitrification (Gupta, 1997). However, most of these studies have focused on the characteristics of single strain and the nitrogen removal efficiency in lab-scale reactors by single strain (Joo et al., 2006; Shoda & Ishikawa, 2014; Yao et al., 2013). To date, the effectiveness of adding these kind of bacteria into actual activated sludge systems to enhance nitrogen removal and the possibility of using these kind of bacteria to directly upgrade the WWTPs has never been evaluated. Previously, the heterotrophic–aerobic nitrogen removal abilities of Agrobacterium tumefaciens LAD9, Comonas testosteroni GAD3 and Achromobacter xylosoxidans GAD4 were described (Chen & Ni, 2011). The present study aimed to determine whether augmentation with this consortium could successfully enhance nitrogen removal in a pilot-scale SBR for municipal wastewater treatment and retrofit the original system without any supplementary structures. Particular attention was paid to investigate the interactions between inoculated microorganisms and indigenous microbial communities and to reveal the possible biochemical mechanisms for nutrients removal on the basis of ecological data. This study is of particular importance to establishing new wastewater treatment systems and upgrading or retrofitting conventional systems from secondary-level to tertiary-level. 2 Materials and methods 2.1. Bacteria and growth condition Three
heterotrophic
nitrification-aerobic
denitrification
bacteria,
named
Agrobacterium tumefaciens LAD9, Comonas testosteroni GAD3 and Achromobacter xylosoxidans GAD4, were used in the experiment. They were isolated from the sludge for the treatment of landfill leachate (Chen & Ni, 2011). All the strains were fermented
with the synthetic mineral medium and freeze-dried into powder. The mixture of them (1:1:1), named PCN bacteria, were used for the inoculation. 2.2. Pilot-scale SBR Two parallel pilot-scale SBR systems were constructed in the Yanshi WWTP (Henan province, China). The SBR was proportionally scaled as the reaction tank in the WWTP, with a size of 830 (length) ×400 (width) ×500 (height) mm and a working volume of 165 L (Figure 1). 2.3. Wastewater and seeding sludge The influent wastewater was directly introduced to the SBR reactor from the regulating tank in the WWTP. The composition of the influent was given in Table 1. Both reactors were initially inoculated with activated sludge taken from the aeration tank of the WWTP. The seeding sludge had a mixed liquor suspended solids (MLSS) concentration of 4.9 g/L, a mixed liquor volatile suspended solids (MLVSS) concentration of 2.58 g/L and a sludge volume index (SVI) of 46.9 mL/g. 2.4. Operation strategy The SBRs were operated with a cycle of 6 h. The cycle consisted of 5 phases: 1 h of influent feeding, 3 h of aeration, 1 h of settling, 10 min of drawing, and 50 min of idle period, which was consistent with the actual operation of the WWTP. The volumetric exchange ratio was 50% and dissolved oxygen (DO) was maintained at 2-3 mg/L. After one week stabilization, the two reactors were operated with different protocols: (1) R1, control SBR, with no extra bacteria addition; (2) R2, bioaugmented SBR, seeded with 3 g PCN bacteria made in 2.1. The operation was divided into two phases, first phase (0-20 d): both reactors were fed with raw wastewater; second phase (21-53 d): sodium succinate was used as the extra carbon source and directly added to the reactor at the beginning of every aeration period. The pH and DO values were
monitored at a regular time interval during the operation. For long operation of reactor R2, 50 g of sodium succinate was directly added to the reactor at the beginning of every aeration period. The solid retention time (SRT) of reactor R2 was kept at 12-15 d and the mixed liquor suspended solids (MLSS) in the main reaction zone were maintained at about 4000 mg/L throughout the experiment. 2.5. Analytical methods COD, MLSS (mixed liquid suspended solids), ammonium (NH4+-N), total nitrogen (TN) and total phosphorus (TP) were analyzed following the procedures in the standard methods.
Nitrite
(NO2--N)
N-(1-naphthyl)-ethylene spectrophothometry,
and
diamine
respectively.
nitrate
(NO3--N)
were
spectrophothometry Dissolved
oxygen
(DO)
determined and
using
ultraviolet
concentration
was
determined by a DO meter (YSI-550A, YSI, USA). Activated sludge suspension was serially diluted with sterile distilled water and spread onto beef-extract peptone agar plates in duplicate. The number of bacterial colonies was counted after the plates were incubated at 30 oC for 3 days. Limited by specific PCR primers for PCN bacteria, bromothymol blue (BTB) plating method were used to demonstrate the possible abundance of PCN bacteria, which was initially proposed by Takeya et al. (2003) for the purpose of isolating aerobic denitrifiers. The plates contained nitrate and the pH indicator BTB. If there were aerobic denitrifiers, the pH value in the medium would increase due to the depletion of nitrate by denitrification and the colonies would turn blue. The pH of the medium was initially controlled within 7.0 to 7.3. The plates inoculated with the activated sludge suspension were aerobically incubated at 30 oC for 3 days, and then aerobic bacteria for nitrate metabolism were monitored by examining the blue colonies or halos formed owing to the increasing pH of the medium. The number of positive strains were counted and
regarded as the possible abundance of PCN bacteria. 2.6. Microbial ecology in the reactors 2.6.1. DNA extraction Genomic DNA was extracted by the 3S DNA isolation kit (Shenergy Biocolor, Shanghai) under the manufacture’s instruction. The size and concentration were estimated by agarose gel electrophoresis (1%) and then stored at -20 oC in a freezer for further application. 2.6.2. Construction of 16S rRNA gene clone libraries and sequence analysis Two clone libraries were constructed from reactors R1 and R2. PCR amplification was performed with 27F and 1492 R primer pairs and performed under the following conditions: 94 oC for 5 min, followed by 30 cycles 94 oC for 60 s, 55 oC for 60 s and 72 o
C for 60 s, and a final extension step at 72 oC for 10 min. The PCR products were
separated by agarose (0.8%) gel electrophoresis and purified with Qiaquick PCR Gel Extraction Kit (QIAGEN, Stanford, CA, USA). The purified DNA fragments were cloned using the pGEM-T Easy Vector System (Takara, Dalian, China) with TOP10 competent E. Coli cells, and plated on LB (Luria-Bertani) plates supplied with ampicillin. Colonies were randomly picked, cultured overnight in LB broth supplemented with ampicillin, and then sequenced in Invitrogen Inc. (USA). Obtained sequences were compared with available sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Phylogenetic reconstructions were performed in MEGA 6.0 (Tamura et al., 2013) using the neighbor-joining (NJ) algorithm, with bootstrap values calculated from 1000 replicate runs. 2.6.3. Quantification of amoA genes by real-time PCR amoA genes of ammonia-oxidizing bacteria (AOB) were amplified from the
environmental
DNA
extracted.
Primer
(5’-GGGGTTTCTACTGGTGGT-3’)
pairs
and
amoA-1F amoA-2R
(5’-CCCCTCKGSAAAGCCTTCTTC-3’) were used. Real-time PCR was conducted using an ABI 7300 Sequence Detector (Applied Biosystems). The 20 µL reaction volume contained 10 µL SYBR® Green PCR Master Mix (Takara, Dalian, China), 0.5 µL of each primer and 2 µL of 5-fold diluted extracted DNA as a template. Amplifications were carried out as follows: 50 oC for 2 min, 95 oC for 10 min, followed by 45 cycles of 15 s at 95 oC, 45 s at 50 oC, 1.5 min at 72 oC. Melting curve analysis was performed at the end of PCR runs to check the specificity of the products. PCR products amplified from extracted DNA with the primers for real-time PCR assays were gel-purified and ligated into the pMD 18-T Vector (Takara, Dalian, China), with an access number of L08752.1. The resulting ligation products were transformed into E.coli cells. After reamplification with the vector, the positive clones were selected to extract plasmid DNA (Axygen, USA) and used as amoA gene standards. Tenfold serial dilutions of a known copy number of the plasmid DNA were subjected to real-time PCR to generated an external standard curve. 3. Results and discussion 3.1. Effectiveness of bioaugmentation in the pilot-scale SBRs Two parallel SBR reactors were used to investigate the performance and efficacy of bioaugmentation during continuous operation. 3.1.1. First stage (with raw wastewater) After the inoculation of PCN bacteria, R1 (control reactor) and R2 (bioaugmented reactor) had similar behavior for 22 days with regards to COD, NH4+-N, and TN removals (Figure 2).In R1, averaged effluent concentrations of COD, NH4+-N, and TN were 18.5, 0.48, 34.6 mg/L, respectively; while in R2, they were 24.8, 0.42, 33.2 mg/L,
respectively. It was suggested that PCN bacteria were not effective at this condition. From previous studies, the competition between the introduced bacteria and the indigenous species, and the insufficiency of substrate have been considered as most possible reasons for the failure of the bioaugmentation (Bouchez et al., 2000). As a result, batch experiments were carried out to investigate activities of the inoculated bacteria in the received ecosystem. First, activated sludge taken from R1 and R2 were separately diluted and plated on the BTB medium to detect the possible abundance of PCN bacteria in the reactors. The result showed that the number of BTB positive bacteria in R2 was 10-times higher (3.3×105 CFU/mL) than that in R1 (6.7×104 CFU/mL), suggesting PCN bacteria was probably active in the reactor. Meanwhile, batch experiment with the bioaugmented sludge and the target wastewater was also conducted in shake flasks with or without the supply of sodium succinate, a favorable carbon source to PCN strains (Chen & Ni, 2012). Without external carbon source, the TN removal efficiency by the sludge was only 42%. However, with 5.0 g/L of sodium succinate feeding, it was enhanced to 80% (data not shown). It could be concluded that the insufficient substrate availability (low COD/N ratio in the influent) was the main reason for the failure of bioaugmentation. 3.1.2. Second phase (with succinate as the external carbon source) According to the above inference, sodium succinate, an extra carbon source, was directly added into R1 and R2 tanks at the beginning of every aeration period. It was observed that no matter with or without the sodium succinate, R1 and R2 reached similar and high removal efficiencies for COD and NH4 +-N (Fig. 2(A) and 2(B)). However, it was significantly different for the TN removal (Fig. 2(C)). TN removal efficiencies in R2 reached 66.0%, 67.1%, 69.0% and 80.5% at external carbon dosages of 30, 40, 45 and 50 g, respectively, which were higher than those in R1 (57.9%, 60.0%,
60.3% and 66.8%), indicating the bioaugmented reactor was more effective for TN removal. The improved nitrogen removal in the bioaugmented reactor could be attributed to the contribution of PCN bacteria. In addition, the removal percentage of TN was correlated well with the carbon supply and there was a remarkably improvement at a carbon dosage of 50 g (COD/N ratio approximately equal to 8.0). At this condition, the concentration of effluent TN in R2 was averaged at 11.2 mg/L, which could meet the first class (level A) requirement of the National Municipal Wastewater Discharge Standards of China (TN<15 mg/L). When the carbon dosage was lower than 50 g, the variations of TN removal efficiency were insignificant. Nevertheless, much better effluent quality would be achieved when the carbon dosage was further increased (data not shown). It was suggested that the activity of PCN bacteria in the received activated sludge system could be significantly prompted when the COD/N ratio reached to 8.0. However, the optimal COD/N ratio (approximate 20.0) for heterotrophic-aerobic nitrogen removal by PCN bacteria was much higher than that value (Chen & Ni, 2012). This difference revealed that the introduced PCN bacteria could coordinate with other indigenous species to achieve high efficiency of nitrogen removal, which resulted in the reduction of carbon requirement. Moreover, comparison was made between full-scale SBR, the bioaugmented SBR, and representative improved SBR systems for nitrogen removal (Table II). It was shown that TN removals in all the improved systems were much better than that in the conventional SBR, while the bioaugmentation was most convenient and simple method for the improvement. Besides, at similar COD/N ratios, TN removal efficiency in the bioaugmented SBR was comparable to it in the systems accompanied with complex control strategy. These preliminary findings indicated that satisfactory effluent quality could be achieved through bioaugmentation.
Furthermore, the performance of carbon and nitrogen transformation during a typical SBR-cycle in R2 was investigated at the carbon dosage of 50 g (Fig. 3). It was demonstrated that at the beginning of the aeration period (1 h), COD concentration was rapidly increased to 234 mg/L because of the addition of succinate sodium into the tank. After that, COD, NH4 +-N and NO3--N concentrations were synchronously decreased during the initial 30 min aeration, indicating aerobic total nitrogen removal had took place in the reactor. When the COD was exhausted (1.5h), the removed ammonium was almost converted to nitrate and the accumulation amount of NO3--N maintained at 11.1 mg/L after 99% removal of NH4+-N, suggesting conventional nitrifiers played a major role at this period. Regarding the nitrogen balance, nitrogen loss at the aerobic period reached to 79.1%, which was comparable to the conventional simultaneous nitrification and denitrification studies (Chiu et al., 2007). Aerobic denitrification and assimilation probably contributed to this nitrogen loss. 3.2. Long term operation of the bioaugmented SBR Long-term operation was conducted in R2 to evaluate the stability and the frequency of bacteria adding. Because NH4+-N and TN concentrations of raw wastewater were relatively stable, 50 g of sodium succinate was added into the aeration tank every cycle to change influent COD and COD/N ratios. As depicted in Fig. 4(B) and 4(C), the removal percentages of COD and NH4 +-N was maintained at 85.8% and 98.8%, respectively. Fig. 4(A) revealed that the system performed excellently on TN removal, with an averaged effluent TN concentration of 14.1 mg/L, which meet well with the national standard. The exceptions occurred on days of 28th, 37th and 51th owing to the big shocks of nitrogen load, but the system recovered quickly by itself without supplementary addition of PCN bacteria. More importantly, remarkable performance of TP removal was observed in this system (Fig.
4(D)). TP removal efficiency reached 85.0% and the averaged effluent concentration was 0.40 mg/L, which could meet the first class (level A) requirement of the National Municipal Wastewater Discharge Standards of China (TP < 0.5 mg/L). According to the PO43--P removal profile during a whole SBR cycle (data not shown), the decrease of PO43--P concentration was synchronous with the degradation of carbon and nitrogen at the aerobic phase. It could be speculated that the excellent uptake of phosphorus at the aerobic condition was mainly caused by the high removal of nitrogen, where nitrogen and phosphorus were co-metabolized by the PCN bacteria. These findings indicated that this bioaugmentation system was high-stability and had excellent performance on nutrients removal. Furthermore, successful application of bioaugmentation technology for wastewater treatment depends on the adaptation of microbial strains with the indigenous microorganisms, which means the introduced microbial strains should survive and keep activity in the received systems and do not influence the function of indigenous degrading microorganisms. As a result, daily repeated adding (Boon et al., 2002) or embedding strategy (Bouchez et al., 2009) was often adopted to avoid the influence of native microorganism and to evade the dilution effect of the withdrawals. However, it greatly enhanced the operation cost in actual applications. In this study, PCN bacteria were successfully introduced to the activated sludge system through one time addition and exhibited excellent accommodation and compatibility with the received ecosystem, which was of great importance for actual application. In conclusion, simultaneous COD and nutrients removal was achieved aerobically through bioaugmentation with PCN strains. As a result, it has many advantages over the conventional systems: i) nitrification and denitrification could take place simultaneously in the aeration tank and then it was unnecessary to recirculate the nitrate rich liquid to
the anoxic tank for anaerobic denitrification or construct additional advanced treatment systems for nutrients removal; ii) it was highly flexible to be merged into existing biological treatment systems to enhance nutrients removal efficiency without the requirement for extra land occupancy; iii) the resistance capability to fluctuations would be greatly enhanced because the maximum growth rates of the heterotrophic nitrifiers are five to ten times than those of the autotrophic nitrifiers. In view of the above consideration, the proposed bioaugmentation strategy should be very promising owing to its incomparable advantages such as reducing energy consumption and waiving additional structures over conventional systems designed for upgrading or retrofitting WWTPs. 3.3. Microorganism community 3.3.1. Biomass Culture-depend technique was used to compare the biomass between reactors R1 and R2. The results demonstrated that the number of total bacteria in reactor R1 (1.71×10 7 CFU/mL) was similar to that in R2 (1.46×107). However, the number of BTB positive bacteria in reactor R2 (2.53×105 CFU/mL) was significantly higher than that in R1 (4.99×104 CFU/mL), indicating the addition of PCN bacteria changed the composition of bacterial community. 3.3.2. Phylogenetic Analysis The two 16S rDNA clone libraries from reactors R1 and R2 were constructed to evaluate the composition of bacterial community and investigate the community shift resulted by the bioaugmentation. Estimation of species coverage and diversity were carried out for the two 16S rDNA clone libraries. Good coverage was found in both libraries (70.8% for R1 and 70.6% for R2 ). Sequences analysis showed that 36 distinct OTUs and 34 distinct OTUs
were obtained from the bioaugmented sludge and the control sludge clone libraries, respectively. It was important that a significant number of the OTUs comprised of a single clone, indicating high levels of bacterial diversity. Shannon-Weaver indices were observed to be 4.23 in R1 and 4.25 in R2. This high similarity suggested that bioaugmentation did not result in the transformation of community diversity. Furthermore, the 16S rDNA sequences of two clone libraries were combined to analyze the relationship of bacterial communities between the two reactors, which were presented in a figure of taxonomic classification (Fig. 5). The results showed that the bacterial communities were distinctively different from each other. The bacterial community in the control reactor was dominated by Bacterodietes (25%), Acidobacteria (18.8%), Planctomycetes (16.7%) and Proteobacteria (10.4%). However, the abundance of phylum Acidobacteria was decreased significantly after bioaugmentation (3.92%). In contrast, the abundance of phyla Chlorobi (15.7%) and Firmicutes (11.8%) were increased significantly after bioaugmentation. Among the different classes of Proteobacteria, β-proteobacteria exhibited the highest diversity in both reactors. It was remarkable that phyla Planctomycetes, Chloroflexi, and Actinobacteria were disappeared, while phyla Verrucomicrobiales and Gemmatimonadetes were newly represent after bioaugmentation. Results in literatures showed that members of the phyla Planctomycetes, Chloroflexi, and Actinobacteria could induce the occurrence of anaerobic nitrogen removal (Chiellini et al., 2013; Pizzetti et al., 2011). In this aerobic nitrogen removal systems, they were all degenerated. Members of the phylum Gemmatimonadetes
have
been
identified
as
Gram-negative,
aerobic,
polyphosphate-accumulating microorganisms found in vermicompost (Vaz-Moreira et al., 2008), and members of the phylum Verrucomicrobia have been widely reported in wastewater treatment systems for nitrogen removal (Yu & Zhang, 2012).
These preliminary findings suggested that the bioaugmentation accelerated the variation of inoculated microorganism community and facilitated the improvement of the community function. For instance, the concentration of AOB amoA gene in the bioaugmented system was 8.40×108/g sludge, while it was 2.85×10 8/g sludge in the control system. It was also demonstrated by previous studies (Boon et al., 2003) that bioaugmentation could promote bacterial community shift and make the community distinct from the original system, because of changes in interior (such as invasion of inoculated strains) and exterior environment (such as change of pH condition). Moreover, there was no sequence identical (>97% similarity) with the inoculated PCN strains (Fig. 6), indicating the introduced PCN bacteria did not become dominant in the reactor. However, high aerobic nitrogen removal efficiency was obtained through bioaugmentation in this study. Similar results were present in the bioaugmentation studies on refractory organic matter degradation, which revealed that the inoculated strains were undetectable in the end and the removal of target pollutants was still in effect (Bai et al., 2011; Munakata-Marr et al., 1997). It could be speculated that the introduced PCN bacteria could synergize with other indigenous microorganisms to achieve good treatment results. 4. Conclusions PCN bacteria capable of heterotrophic-aerobic nitrogen removal was used for the bioaugmented treatment of municipal wastewater in a pilot-scale SBR. At an appropriate COD/N ratio of 8.0, the produced effluent could satisfy the first class requirement of the National Municipal Wastewater Discharge Standards of China without any extra system modification. The bioaugmentation improved the microbial community structure and facilitated their capabilities of aerobic nutrients removal. The proposed bioaugmentation strategy could be considered as an effective
and economical alternative technology for the treatment of municipal wastewater and the upgrade or retrofitting of the WWTPs.
Acknowledgements Financial support from National Natural Science Foundation of China (Grant No. 51208007), Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20120001120101) and Guangzhou Scientific and Technologic Project for Water Service (Grant No. GZSK-FW-1201) are fully appreciated.
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Figure Captions Fig. 1. SBR reactor configuration. Fig. 2. Comparison of treatment efficiencies for (A) COD, (B) NH4+-N, and (C) TN in R1 and R2. Symbols: ●, influent; ▲ , effluent in R1; ○, effluent in R2; -, extra carbon dosage. Fig. 3. COD and nitrogen removal profiles in a typical SBR cycle in R2 at an extra carbon dosage of 50 g. Symbols: ○, pH; □, COD; ▲ , NH4+-N; ■ , NO3--N; ●, NO2--N. Fig. 4. Long-term performance of the bioaugmented system with external carbon addition. (A) TN removal; (B) COD removal; (C) NH4+-N removal; (D) TP removal. Symbols: ■ , influent; ○, effluent; ▲ , dosage of sodium succinate. Fig. 5. Phylum or class-level (for Proteobacteria) phylogenetic affiliations for sequences retrieved from reactors R1 and R2. Fig. 6. Neighbor-joining trees showing the phylogenetic affiliation between PCN strains and selected 16S rDNA OTU sequences (only OTUs comprising ≥2 clones are shown) from reactors R1 and R2. The scale bar indicates the number of nucleotide substitutions per site. Three 16S rDNA sequences, named LAD9, GAD3 and GAD4, were derived from PCN strains.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Table 1 Compostion of the municipal wastewater. Component
Level
Average value
Total COD (mg/l)
101-254
140
NH4+-N (mg/l)
30.4-66.6
50.2
Total N (mg/l)
30.6-72.4
51.6
Total P (mg/l)
1.10-5.60
2.69
pH
7.50-8.07
7.81
Table 2 Comparison of nitrogen removal efficiencies between full-scale SBR, the bioaugmented SBR, and representative improved SBR systems.
Process
Influent
Municipal Full-scale SBR wastewater Lab-scale SBBR Lab-sclae
Synthetic wastewater Domestic
A2N-SBR wastewater Lab-scale Synthetic intermittently wastewater aerated SBR Lab-scale Artificial MBR Pilot-scale SBR
wastewater municipal
Inoculum
Activated sludge Activated sludge Acitivated sludge Activated sludge Single bacteria Activated sludge+PCN
Average Average Influent N COD/N TN removal ratio (%) 34
8.8
58%
30
10
87.8%
37
7.9
83%
125
8
71.2%
120
8.3
53%
51.6
7-8
80.5%
Reference
(Fernandes et al., 2013) (Jin et al., 2012) (Wang et al., 2009) (Mansouri et al., 2014) (Yao et al., 2013) This study
Graphical abstract
Highlights
Bioaugmented treatment of municipal wastewater in a pilot-scale SBR. Heterotrophic-aerobic nitrogen removal bacteria (PCN) used for bioaugmentation. Addition of PCN bacteria improved the original bacterial community structure. Bioaugmentation enhanced the aerobic nutrients removal ability of original system.