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oxic (A-MAO) process to treat municipal wastewater
Accepted Manuscript Biological nutrient removal and molecular biological characteristics in an anaerobic- multistage anaerobic/oxic (A-MAO) process to treat municipal wastewater Xiao Huang, Wenyi Dong, Hongjie Wang, Shilong Jiang PII: DOI: Reference:
S0960-8524(17)30829-5 http://dx.doi.org/10.1016/j.biortech.2017.05.161 BITE 18187
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
22 March 2017 19 May 2017 25 May 2017
Please cite this article as: Huang, X., Dong, W., Wang, H., Jiang, S., Biological nutrient removal and molecular biological characteristics in an anaerobic- multistage anaerobic/oxic (A-MAO) process to treat municipal wastewater, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.161
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Biological nutrient removal and molecular biological characteristics in an anaerobicmultistage anaerobic/oxic (A-MAO) process to treat municipal wastewater
Xiao Huang a,
Wenyi Dong a,b,
Hongjie Wang a,b *, Shilong Jiang
a.
School of Civil and Environmental Engineering, Harbin Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China b. Shenzhen Key Laboratory of Water Resource Utilization and Environmental Pollution Control, Shenzhen, 518055, China * Corresponding author: Hongjie Wang School of Civil and Environmental Engineering, Harbin Institute of Technology Shenzhen Graduate School Shenzhen 518055, China Tel. / Fax: +86 0755 26032718 E-mail: [email protected] (H. Wang).
Abstract This study aimed to present an anaerobic- multistage anaerobic/oxic (A-MAO) process to treat municipal wastewater. The average COD, NH4+-N, TN, and TP removal efficiency were 91.81%, 96.26%, 83.73% and 94.49%, respectively. Temperature plunge and C/N decrease have a certain impact on the modified process. Characteristics of microbial community, function microorganism, and correlation of microbial community with environmental variables in five compartments were carried out by Illumina Miseq high-throughput sequencing. The differences of microbial community were observed and Blastocatella, Flavobacterium and Pseudomonas were the dominant genus. Nitrosomonas and Nitrospira occupied a dominant position in AOB and NOB, respectively. Rhodospirillaceae and Rhodocyclaceae owned a considerable proportion in phosphorus removal bacteria. DO and COD played significant roles on affecting the microbial components. The A-MAO process in this study demonstrated a high potential for nutrient removal from municipal wastewater. Keyword: A-MAO process, Biological nutrient removal, Molecular biological characteristics, High-throughput sequencing，Microbial community 1. Introduction Eutrophication, as one of the most urgent issue to limit the economic development and pose a threat to the safety of drinking water resources, has been widely concerned in recent years. The nutrients (nitrogen and phosphorus) in effluent of waste water treatment plants (WWPTs) have been confirmed a main reason that cases receiving surface water eutrophication (Kowalkowski et al., 2012). A large number of WWPTs have been built up for several decades to meet the population and economic development in china. Currently, the standard for municipal sewage discharge is getting more and more stringent. The discharge has increased from standards first class B to class A. Nitrogen and phosphorus are the important control parameters and their control has become the urgent problem for sewage treatment. Biological nutrient removal (BNR) processes are effective for nitrogen and phosphorus removing and own economic advantages compared with other ways. Some biological treatment processes, such as anoxic/oxic (AO), anaerobic/anoxic/oxic (A 2O), multistage AO and 5-stage Bardenpho process (phoredox process), et al., have been developed for nitrogen and phosphorus removal. For these traditional biological nitrogen and phosphorus removal processes, carbon sources play a restrictive role due to the competitive effect on carbon sources was occurred between denitrifying bacteria and phosphorus removal bacteria (Wang et al., 2006; Wang et al., 2015; Barker and Dold, 1996). In China, especially in the south, the ratio of chemical oxygen demand (COD) to total nitrogen (TN) in influent is lower. Therefore, it is difficult to achieve stable removal of nitrogen and phosphorus and meet discharge standards. For low carbon to nitrogen ratio (C/N) wastewater, one of the most effective ways was adding additional carbon sources. However, this method increases the operating cost. Besides, some advanced wastewater treatment methods followed BNR processes, such as post-denitrification biofilter (Li et al., 2016; Shi et al., 2015), chemical phosphorus removal (Kim
et al., 2015), and membrane technology (Yang et al., 2010) were applied to decrease the effluent concentration of nitrogen and phosphorus further. Nevertheless, these methods not only need additional carbon sources, increase the construction cost and operating cost, but also result in wasting occupied area. For some existing WWTPs, there is no enough area to increase the treatment structures. Thus, modifying the conventional treatment process (e. g. A2O or 5-stage Bardenpho process) and excavating their treatment efficiency fully are necessary. In the previous studies, there were many modifying ways to remedy the disadvantage of conventional activated sludge system. Zeng et al. (2011) set up a pre-anoxic zone before A2O process and a better biological nutrients removal was obtained than the conventional A 2O process. Zhao et al. (2016) put forward a pre-denitrification process of anaerobic/anoxic/aerobic nitrification, which are operated as a two sludge system and exhibited an excellent phosphorus and nitrogen removal performance. In this study, an A-MAO process was put forward to remedy the defects of 5-stage Bardenpho process. Two important adjustments were conducted to improve the process performance, i.e., a varying internal recycle and distribution of influent. (i) The influent was divided into two parts, The carbon source in influent was sufficiently used for nitrogen and phosphorus removal, which reduces treatment cost. (ii) Adding influent into the 2nd anoxic zone brought about a problem that NO3--N will be out of limit in effluent. For reducing NO 3--N concentration, an adjustment of internal recycle was carried out to improve denitrification. For revealing the relationships between composition of microbial communities and pollutants removal high throughput sequencing method was conducted in this study. There were a lot of researches on microbial community characteristics, which have been studied. However, most biochemical characteristics, microbial community, and functional microorganisms in wastewater treatment have focused on different wastewater treatment plants (Hu et al. 2012), environmental conditions (Zou et al. 2014), and operating conditions (Duan et al. 2013; Liu et al. 2016) rather than different units (or compartments) in one complex wastewater treatment process. Many microbial communities involve in the removal of nitrogen, phosphorus and organic matter and the generation of many heterotrophic bacteria are short to many minutes or hours. How the microbial communities change when pollutants are removed in the different units? Whether a necessary connection of environmental factors and microbial communities in different units is existent or not? Therefore, it is necessary to discuss the composition of the microbial community, and then explain the pollutant degradation mechanism from the angle of microbial community. In this study, the performance of an anaerobic- multistage anaerobic/oxic (A-MAO) process was monitored and demonstrated its ability to remove TN and TP efficiently with an effluent superior to Chinese integrated wastewater discharge standard first-A standard, even under low C/N. In addition, microbial composition, diversity and abundance in different compartments were analyzed by high throughput sequence technology. It was found that there were some differences of microbial diversity and abundance among five compartments and the change of microbial community had significant association with environmental factors. Thus, this study is aiming to present an A-MAO process and reveal the functional species that are about TN and TP removal during the operation process.
2. Materials and Methods
2.1 Experimental system and operational conditions A lab-scaled A-MAO system and 5-stage Bardenpho process (shown in Fig. 1and Fig. 1S), was consisted of three parts: an influent tank, an A-MAO reactor (with a working volume 192 L) and a settler. The A-MAO reactor(shown in Fig. 1), which was made of colorless Plexiglas, was separated into five compartments in sequence to create anaerobic (13.6 L)/first anoxic (30.6 L)/first oxic (54 L)/ second anoxic (37.4 L)/second oxic zones (56.4 L). The influent was pumped into anaerobic and the second anoxic zone with 60% and 40% distribution ratio by peristaltic pumps. To keep sludge suspension, mechanical stirrers were installed at the top of the anaerobic and two anoxic zones. Besides, sand bubble diffusers which play roles in supplying air and mixing, were installed at the bottom in two oxic zones. Sewage mixed liquor returned from second oxic zone to first anoxic zone to improve the nutrients removal and the sludge was from secondary settler to anaerobic. The compartments of 5-stage Bardenpho process was same with A-MAO process, the influent was only pumped into anaerobic and mixed liquor returned from first oxic zone to first anoxic zone. Two systems were operated at natural temperature and treated actual sewage. The mean influent was maintained 240 L d-1, the total hydraulic retention time (HRT) was 19.2 h and each zone was 1.36, 3.06, 5.4, 3.74 and 5.64 h in sequence. The sludge was discharged from secondary settler every day and kept sludge retention time (SRT) 16 d. The sewage mixed liquor and sludge recycle ratio was at 100% and 85%, respectively. Mixed liquor suspended solids (MLSS) concentration was 3400 mg/L. The dissolved oxygen (DO) concentration was controlled at 5-6 mg/L in both oxic zones. 2.2 Seed sludge and wastewater characteristics The seed sludge used in this study was collected from the secondary sedimentation tank at the Luofang Municipal wastewater Treatment Plant (first phase project) (Shenzhen, China). Where Adsorption (aerobic aeration)-Biodegradation (anaerobic/anoxic/oxic, A2O) process (AB process) was employed to treat 100,000 m3/d wastewater. The wastewater source was collected from the drain well of Harbin Institute of Technology Shenzhen Graduate School (Shenzhen, China) to an intermediate tank, and then pumped into an influent tank. The main characteristics of influent wastewater were summarized in Table 1. 2.3 Pollutant mass balance The pollutants balance is shown in Fig. 2S and calculated according to Eqs. (1) to Eqs. (5). Q1·SINF C,N,P +Qr·Sset C,N,P =(Q1+Qr)·S(ANA-AN1)+SANA C,N,P (1) (Q1+Qr)·S(ANA-AN1) + q·SO2 C,N,P =(Q1+Qr+q)·S(AN1-O1) C,N,P +SAN1 C,N,P (2) (Q1+Qr+q)·S(AN1-O1) C,N,P =(Q1+Qr+q)·S(O1-AN2) C,N,P +SO1 C,N,P (3) (Q1+Qr+q)·S(O1-AN2) C,N,P + Q2·Sinf C,N,P =(Q1+Qr+ Q2+q)·S(AN2-O2) C,N,P +SAN2 C,N,P (4)
(Q1+Qr+ Q2+q)·S(AN2-O2) C,N,P =(Q1+Qr+ Q2+ q)·SO2 C,N,P +SO2 C,N,P (5) Where, Q1, Q2, Qr, and q present the flow of two step-feed, return sludge, and mixed liquid return; SINF C,N,P and Sset C,N,P are the concentration of pollutants of influent and effluent; S(ANA-AN1) S(AN1-O1) C,N,P, S(O1-AN2) C,N,P, S(AN2-O2) C,N,P, SANA C,N,P, are pollutant concentrations of anaerobic to first anoxic, first anoxic to first oxic, first oxic to second anoxic, and second anoxic to second oxic, respectively. SO2 C,N,P is pollutant concentration after second oxic zone; SAN1 C,N,P, SO1 C,N,P, SAN2 C,N,P, and SO2 C,N,P denote the removal amount of pollutants changed in anaerobic, first anoxic , first oxic, second anoxic, second oxic zones, respectively; C, N, P present the concentration of COD, NH4+-N, TN, and TP, respectively. C,N,P,
2.4Microbial community analysis The total activity sludge DNA from five different compartments (Sample named as ANA, AN1, O1, AN2, and O2 and represented samples are taken from anaerobic, first anoxic, first oxic, second anoxic, and second-oxic compartments, respectively) are extracted using the Power Soil DNA kit (Mobio, CA, USA), and following the method described in Simister et al. (2015). Nanodrop Spectrophotometer (2000c, Thermo, USA) was used to checked DNA density and 5 µL DNA sample was taken to electrophoresis detection by 1% agarose gel electrophoresis and Polymerase chain reaction (PCR) amplification was conducted by PCR instrument (9700, GeneAmp® ABI, USA) using the DNA extracts as a template, and the V4 region of 16S rDNA was amplified with universal primers 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACCAGGGTATCTAAT-3’). All PCR reactions were carried out in 20 μL reactions with 4 μL 5×FastPfu Buffer, 2.5mM dNTPs, 0.4μL FastPfu Polymerase, 0.2μL BSA, 0.8 μL of forward and reverse primers, and about 10 ng template DNA .Thermal cycling consisted of initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 45 s. Finally 72 °C for 5 min. PCR products were monitored with 2% agarose gel electrophoresis. After PCR products were purified and fluorescence quantitative detection, sequencing was determined by using two-terminal sequencing (Paired-End) method and constructing a small fragment library on an Illumina MiSeq plantform by Majorbio company (Shanghai, China). Paired-end reads from the original DNA fragments were merged with the software of Trimmomatic and FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/) (Magoč et al., 2011) and sequencing reads were assigned to each sample according to the unique barcodes. The richness and diversity of microbial communities which include ACE index, Chao 1 index, Simpson index, and Shannon index, were calculated and analyzed using Mothur version v.1.30.1 (http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity) (Schloss et al., 2011). In order to study the species diversity of information in different samples, the sequences were assigned to operational taxonomic units (OTUs) by Usearch software (vsesion 7.1 http://drive5.com/uparse/) and all the sequence column similarity within the scope of the threshold (> 97%) sequence were grouped together and regarded as one OTU. The rarefaction curves, principle component analysis and Venn diagram were generated basic on OTUs. Graphical representation of the relative abundance of microbial diversity at phylum and genus levels was counted basic on the taxonomic data. The sequences of main functional microorganisms were analyzed by BioEdit software. A phylogenetic tree was constructed using Mega 4.0 software.
2.5 Analytical methods Samples were collected from the influent tank and secondary settler once three days. Besides, samples in anaerobic, first-anoxic, first-oxic, second-anoxic and second-oxic zones were collected once a week. COD, NH4+-N, NO3--N, TN, TP, MLSS and MLVSS were analyzed according to Standard Methods (APHA, 2005). DO, pH, temperature were monitored online by using WTW pH/Oxi 340i meter with DO and pH probes (WTW, Germany). 2.6 Statistical analysis Three replicates were conducted for each sample and average values were calculated and showed in charts. Origin 8.6 software was used for drawing figures 3. Results and discussion
3.1 Overall performance of organics and nutrients removal In this study, the lab-scale reactor was operated for a period of 220 days. The start-up process took about 50 days to reach steady (Fig. 2). When the removal efficiencies reached a satisfactory hill, it was identified reaching steady-state period. On the other hand, the overall system, after 50 days cultured, performed steadily with reference to the pollution removal. It is interesting that a sudden cooling which was appeared one week (94th to 100th day) and C/N changed from 8-10 to 5-6 (166th -172th day) were observed during the operation period. Under unsteady-state and steady-state periods, the concentration of COD, TN, NH4+-N and TP in influent and effluent as well as removal efficiencies are shown in Fig. 2 COD, NH4+-N and TN removal efficiencies were lower compared to steady-state, but removal efficiencies were also highly satisfactory during the unsteady-state. TP removal efficiency, however, during the unsteady-state was range from 50% to 80% and not satisfactory. In the steady-state the effluent concentration of COD, TN, NH 4+-N and TP from the A-MAO system all met the integrated wastewater discharge standard (class A discharge standards, GB18918-2002, China) which were lower than 50.0, 15.0, 5.0 and 0.5 mg L-1, respectively. The results from the lab-scale study suggest that the A-MAO system can be used efficiently for organics and nutrient removal for municipal wastewater. The COD removal of the lab-scale A-MAO system during the whole operation period (220 days) is presented in Fig. 2 A. The influent COD concentration was in the range of 315.6 - 660.7 mg L-1 with an average value of 513.3 mg/L in the first 160 d. Afterwards, The COD concentration decreased to 323.1 - 448.3 mg L-1 (average value was 377.2 mg L-1) from 160th to 220th days and the C/N of two periods were 8-10 and 5-6, respectively. During the start-up process, the removal efficiency rose from 80.1% to 97.5% gradually and effluent concentration were steady with the influent concentration increasing. Besides, the COD removal efficiency in this A-MAO system kept as high as 80% and effluent was lower than discharge standard despite of the variation of COD concentration in common situation. The effluent, however, rapidly worsened when sudden cooling (94th to 100th day) and C/N droped from 8-10 to 5-6 (166th -217th day),
and then quickly returned to a stable state. The influent COD load fluctuated from 0.4 to 0.8 kg COD (m3·d)-1, the removal efficiency remained around 90%. According to the 220 days research, the system reflected a good effect for COD removal. Fig. 2 B and Fig. 2 C show the NH4+-N and TN concentration in influent and effluent as well as the removal efficiencies during the 220 days experiment period. The excellent NH4+-N removal was achieved in the whole treatment process, with the average effluent concentration and removal efficiency (without sudden cooling and C/N drop) of NH4+-N was 2.75 mg L-1 and 94.6%, respectively. No matter start-up and steady-state periods, the effluent NH4+-N remained a favorable concentration and a weak effect for C/N. In contrast, the effluent and removal efficiency of TN were unsatisfactory during start-up period. The effluent concentration varies 20 to 50 mg L-1, higher than the discharge standard of 15 mg L-1 obviously. After 50 d start-up period, the average effluent concentration 9.66 mg L-1 and removal efficiency 84.3% were obtained. However, with the C/N decrease from 8-10 to 5-6, the effluent TN showed a large fluctuations in the first 20 days, and the removal efficiency range from 74.57% to 94.27%, and remained the level of discharge standard (15 mg L-1). Results indicated C/N decrease did not inhibit the TN removal, enough carbon sources were offered for denitrification. TP removal efficiency was shown in Fig. 2 D，as we can see, TP removal efficiency maintained rising trend at initial operation period and reached stability until 40th day. The effluent concentration was below 0.5 mg L-1 at steady-state period and exceeded 90% removal efficiency was achieved. The comparative study of Bardenpho process and A-MAO process was conducted to demonstrate the advantages of modified process. From Table S1, there were no changes for COD and NH4+-N removing among three processes, the mean removal efficiency was higher than 93% and 97%, respectively. The removal efficiencies of TN and TP were significantly increase by A-MAO process compared with 5-stage Bardenpho process. Hence, the A-MAO, which was modified depending on 5-stage Bardenpho process, was effective for denitrification. 3.2 Nutrients and organics removal efficiency in different compartments For full understanding of the organics and nutrients removal mechanism in the A-MAO system, pollutions concentration and utilization performance were tested in different treatment compartments. The evolution of COD pollutions in the A-MAO system is depicted in Fig. 3. It could be seen that great proportion (75.69%) of COD was removed in anaerobic zone. At the same time, 12.72% and 5.98% COD was utilized in the 1st anoxic and oxic compartments, respectively. 40% influent was fed into the 2nd anoxic compartment, and resulted in the increasing of COD concentration of effluent in the 2nd anoxic and oxic compartments. In fact, 29.20 g COD d-1 and 7.40 g COD d-1 were removed in the two compartments, respectively. The removal amounts of COD in 2nd anoxic and oxic compartments were higher than 1st anoxic and oxic compartments. NH4+-N in the influent was about 41.83-57.98 mg L-1, which was the mainly component of TN (about 95-98%) in the entire experiment period. NH4+-N concentration was decreased to 21.15 and 0.88 mg L-1 fleetly in anaerobic and anoxic compartments due to the dilution of sludge return and internal return. However, small part of ammonia in influent was removed (1.00 g d -1). The concentrations in 1st anoxic and oxic changed slightly. It is interesting that 1.35g d-1 and 1.65 g d-1 NH4+-N were removed in 1st and 2nd anoxic compartments, but only 1.61 g d-1 and 0.57 g d-1
NH4+-N were removed in 1st and 2nd oxic compartments. It may be because that the distribution of functional population was relatively similar in five units. When the oxygen was carried from 2nd oxic to 1st anoxic compartment, nitrifying flora utilized the dissolved oxygen (0.5 mg L-1) to convert NH4+-N to NO2--N or NO3--N in 1st anoxic unit. The change of TN was similar with NH4+-N that most of them were removed in anaerobic and anoxic compartments based on denitrification. TP in influent was 6.62 mg L-1 and rose to 19.29 mg L-1 in anaerobic compartment. Phosphorus-accumulating bacteria (PAO) absorbed small molecular organic to compound poly-β-hydroxybutyrate (PHB), then 5.75 g L-1 phosphorus was released and generated a phenomenon of concentration increasing in anaerobic compartment. Then a continuously decreasing trend was observed in the first anoxic (4.14 g L-1) and oxic compartments (0.99 g L-1). So some denitrifying phosphorus remove bacteria utilized NO3--N as the electron acceptor for denitrifying phosphorus removing. 3.3 Molecular biological characteristics in different compartments
3.3.1 Microbial community similarity analysis A total of 177728 effective sequence reads (34004, 34889, 34871, 40857, and 33107 for ANA, AN1, O1, AN2, and O2, respectively) were obtained from activated sludge samples in five activated sludge by the Illumina MiSeq plantform. Downstream analysis based on the assigning of gene sequences was conducted and 396, 417, 386, 414, and 385 OTUs were divided at a threshold scope of 97%. Rarefaction curves were used to standardize and compare the microbial community richness and sequencing quality (Amato, et al., 2013). As can be seen from Fig. S3 A, The cures of five samples nearly reached gentle which indicated that the sequencing numbers of five samples are reasonable and most microorganisms had been detected. The result could be proved by coverage index (the coverage indexes of all samples exceeded 99.76%) in Table 2. Venn diagram (Fig. S3 B), the cluster analysis (Fig. S3 C) of OTUs, principal component analysis (PCA) (Fig. S4) and non-metric multidimensional scaling (NMDS) (Fig.S5) could effectively evaluate the similarity and difference of microbial communities. From Venn diagram, 75.87% of total OTUs were shared among all samples. In two oxic zones nearly 90% microbial communities were similar and only about 5% was changed in both anoxic zones. A high microbial community similarity was detected in five different zones. From Fig. S3 C, the shortest relative Bray-Curtis distance appeared between AN1 and AN2 and could be gathered together. Cluster analysis proved that ANA and O1 were highly similar and O2 that had the farthest Bray-Curtis distance with other samples showed significantly different. This view was also confirmed by PCA (Fig.S4) and NMDS diagrams (Fig.S5). In PCA diagram, AN1 and AN2 could be divided into one category according to any two dimensions and the differences between oxic and anoxic zones were not obvious. However the differences could be clearly seen by NMDS analysis (Fig.S5). Microbial community in two kinds of reaction zones (oxic and anoxic zones) did not show a higher similarity. Both oxic zones exited vary markedly compared with anoxic zones. Therefore, dissolved oxygen posed a significant influence on the changes of microbiome and abundance.
3.3.2 Microbial community richness and biodiversity Rarefaction curves could reflect the richness of microbial community and abundance-based coverage estimation (ACE) index and Chao 1 index (Table 2) are another two important standards to describe the species richness. The higher ACE and Chao 1 index, the more richness in sample. Microorganism in five zones presented a similar richness ranging from 400 to 450, and the first anoxic zone (AN1) owned the most richness, i.e., 442 and 444 for ACE and Chao 1 index, respectively. However, the least richness was 408 and 407, which was observed in the first oxic zone (O1). It is interesting that microbial community in both oxic zones was less richness than two anoxic zones and similar to anaerobic tank. This phenomenon is not consistent with Kim et al. (2013), in whose research the microbial richness in oxic zone was higher than anoxic and anaerobic zones and anoxic was better than anaerobic. In this research, it maybe because that organic (COD or BOD) in influent was utilized in anoxic zones and large number of heterotrophic microorganism bred resulted in high richness. On the contrary, the fact that no enough organic was in oxic zones leaded to a poor nutrition environment, which accelerated some microorganisms lose viability and more autotrophic organisms breeding. Meanwhile, in anaerobic tank, due to the lack of electron acceptors (O2 or NO3--N), anaerobic microbes will grow well and reduces the microbial richness. Microbial community biodiversity could be estimated by Shannon index and Simpson index (Table 2). A higher Shannon index represented more diversity, while Simpson was on the contrary. It was clear that the result of biodiversity was nearly consistent changing trend with ACE and Chao 1. The biodiversity in anoxic zones was higher than two oxic zones and anaerobic zone. The difference is the highest biodiversity (4.34 for Shannon and 0.620 for Simpson) was in the second anoxic zones (AN2). From richness and biodiversity analysis, there were no significant differences among five compartments. The same conclusion was got from A2O process (Kim et al. 2016). 3.3.3 Microbial community composition Dynamic changes of microbial community structures among microbial samples were revealed depicted in Fig. 4. Significant changes of community composition were found according to the relative abundances of different phylum (Fig. 4 A) and genus (Fig. 4 B) in the five compartments. It was obvious that Proteobacteria occupied the leading position in all samples with the relative abundance of 37.27% (ANA), 26.49% (AN1), 47.03% (O1), 34.45% (AN2), and 42.11% (O2). Bacteroidetes (6.99%-29.67%), Chloroflexi (7.34%-15.02%), and Acidobacteria (6.45%-18.18%) were the most dominant phylum behind Proteobacteria. Other phylum like Fimicutes, Deinococcus-Thermus, Actinobacteria, TM6, and Gemmatimonadetes were detected over 1% in five samples. Generally, the main microbial population was quite different at phylum level. The similar conclusion was achieved by Kim et al. (2013) whose research found that Proteobacteria (53.80%), Bacteroidetes (18.80%), and Chloroflexi (6.60%) were the dominant phylum in A2O process. There were reported that Proteobacteria was the most abundant in WWTPs (Hu et al., 2012; Ye et al., 2011). The differences among five samples were apparent at genus level, especially, ANA, O1, and O2. The heatmap (Fig. 4 B) for the 50 abundant genera was constructed to discuss the similarities
and differences among five samples. The results showed that the microorganism in samples at genus level was diverse. Blastocatella, Flavobacterium and Pseudomonaswere dominant and large variations occurred among them. Nevertheless, high level of similarity was maintained in AN1 and AN2 samples. Blastocatella (16.51%), Pseudomonas (16.21%), and TK10_norank (8.71%) were the dominant species in ANA sample. AN1 and AN2 were main represented by genus Blastocatella (12.57% for AN1, 11.26% for AN2), Flavobacterium (10.77% for AN1, 11.82% for AN2), and TK10-norank sp. (9.09% for AN1, 9.35% for AN2), and Meiothermus sp. (9.21% for AN1, 4.00% for AN2). It is interesting that O1 and O2 was on the contrary to what we thought, a small similarity was existed between them. In O1 sample, the most dominant genus was Pseudomonas (20.30%) and the main genus in O2 sample were Flavobacterium (22.79%) and Massilia (19.93%). Therefore, it can be inferred that the dominant genus could shift in two aerobic zones for A-MAO system. Cluster analysis (Fig. 4 B) also confirmed the above discussion that AN1 and AN2 were gathered into one category. Besides, Flavobacterium and Massilia were homologous relationship, and the same with Blastocatella and TK10_norank. Flavobacterium has been reported about its functions with nutrients removal and enhancing the biological phosphate removal (Kong et al., 2007; Zengin et al., 2010; Zou et al., 2014). Yeh et al. (2010) reported that Pseudomonas played an important role in organic matter and nitrogen removal in field-scale constructed wetlands. The nutrients removal function of Pseudomonas was also reported by Bouki et al. (2013) and Zheng et al. (2013). 3.3.4 Dynamic change of functional microorganism Nitrifying and denitrifying bacterial communities that play core role for NH4+-N and TN removal in the A-MAO process was stable. Ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) convert NH4+-N to NO3--N collectively in nitrification process. Previous studies found that Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio were the main AOB genus (Duan et al.,2013) and Nitrospira, Nitrospine, Nitrobacter, and Nitrococcus were the core bacterial for NOB (Rani et al.,2016). In five sludge samples, most AOB belonged to uncultured Nitrosomonadaceae and the abundance changed in the range of 0.14%-0.37% (Fig. 5 A). However, other AOB genus was not found in this process. For NOB, Nitrospine, Nitrobacter, and Nitrococcus were a lot less than Nitrospira, which abundance was more than 0.3%. Thus, in this A-MAO process, Nitrosomonas and Nitrospira occupied a dominant position in AOB and NOB communities, respectively and the stable nitrification was obtained depended on the existence of them. The conclusion was consistent with Chen et al. (2016) who also reported that Nitrosomonas and Nitrospira were the dominant nitrifying bacterial communities occurring in YXM process. It is interesting that the abundance of AOB and NOB in anaerobic compartment (ANA), and anoxic compartments (AN1 and AN2), which owned low DO concentration, was more than aerobic compartments (O1 and O2) with sufficient oxygen supply. However, the phenomenon proved the previous results that the NH4+-N removal efficiencies in AN1 and AN2 were higher than O1 and O2. Therefore, the change trend indicated that the more Nitrosomonas and Nitrospira resulted in higher NH4+-N removal efficiency. This unnatural phenomenon was also revealed by previous study and assumed that Nitrosomonas and Nitrospira lack the sensitivily to low DO which could make contribution to energy saving in waste water treatment (Chen et al., 2016). Liu
et al. (2016) thought that Nitrosomonas (AOB), Nitrobacter (NOB), and Nitrospira (NOB) mainly contributed to the nitrification under low DO conditions and the waste water treatment system was able to complete nitrification under 0.5 mg L-1. It could provide a reasonable evidence that the effluent from WWTP reaches standard though reducing DO and a strategy for energy saving. The potential denitrifying bacteria were classified into 11 families in five samples (Fig. 5 A). The dominant denitrifying bacteria belonged to Bradyrhizobiaceae, Comamonadaceae, Rhodobacteraceae, Bacillaceae, and Pseudomonadaceae. The high abundances were those of Bacillus (0.37%-8.44%), Pseudomonas (0.41%-20.23%), uncultured Cytophagaceae (0.51%-1.31%), Acinetobacter (0.02%-1.72%), Comamonadaceae (0.40%-6.26%), and Ottowia (1.10%-2.66%). Denitrifying bacteria in Pseudomonadaceae and Bacillaceae were more abundance in aerobic compartments than anoxic compartments because some Pseudomonas and Bacillus is aerobic denitrifying bacteria and able to convert NO3--N to N2 under aerobic environment (Gui et al., 2016; Koike and Hattori, 1975; Kim et al., 2005). This also confirmed why NO3--N was reduce in aerobic zone in this study. Comamonadaceae, Acinetobacter, and Ottowia are typical denitrifying bacteria and have been reported by Spring et al. (2004) and Jahan et al. (2012). There are four main enzymes which are associated with denitrification process, i.e. Nitrate reductase, Nitrite reductase, Nitric oxide reductase, and Nitrous oxide reductase (Averill, 1996). Nitrate reductase is sensitive to oxygen and inhibited in aerobic condition (Mendel et al., 1981). Hence, higher abundance was occurred in anoxic compartments. On the whole, denitrifying bacteria and aerobic denitrifying bacteria formed a combined effect on TN removal. In order to discuss the the mechanism of phosphorus removal, dominant microorganisms associated with phosphorus removal was shown in Fig. 5 B. Beijerinckiaceae, Bacillaceae, Sphingomonadaceae, Rhizobiales_Incertae_Sedis, Rhodospirillaceae, and Rhodocyclaceae were the six families, which contained the core PAO. They were relatively stable in five compartments. Rhodospirillaceae (0.27%-1.24%) and Rhodocyclaceae (0.22%-0.74%) occupied a considerable proportion and their abundance was higher than other families. Jiang et al. (2012) found that uncultured Rhodocyclaceae bacterium dominated the granule-based for enhancing biological phosphorus removal (EBPR) sludge in SBR reactor． Zhang et al. (2012) reported a new finding that denitrification and phosphorus removal were simultaneously achieved in an EBPR system, and Azospirillum that belonged to Rhodospirillaceae was 52.54%. This study once again proved that Azospirillum is a PAO genus. Bacillaceae, Rhodocyclaceae, Hyphomicrobiaceae, Rhodobacteraceae and Pseudomonadaceae were typical denitrifying phosphorus accumulating organisms (DPAOs) that have been reported in previous research (Lee et al. 2008; Kim et al. 2013; Aguilar-May et al. 2009; Barak and van Rijn, 2000). Therefore, three main processes existed in the A-MAO process, i.e., anaerobic phosphorus release, aerobic phosphorus absorption, and denitrifying phosphorus accumulating process. The phosphorus removal bacteria could utilize O2, NO2--N, and NO3--N as electron acceptor and be catalyzed by polyphosphate kinase under aerobic and anoxic conditions (Jung et al. 2012). 3.4 Correlation of microbial community with environmental variables Environment variables played a cooperative role on affecting the microbial components and the expression of function. Heatmap analysis of the correlation between environment variables (TN, MLSS, NH4+-N, COD, TP, pH, NO3--N, and DO) and community composition was
illustrated in Fig. 6. It was obviously that some community composition was significantly affected by environment variables. JG30-KF-CM45, Pseudomonas Clostridium, Blastocatella ， Methylocystaceae，and Iamia were closely positive correlated to TN, NH4+-N, MLSS, COD, and TP. The six genus had a significant response to nitrogen, phosphorus and organic matter removal process and gather into a category. On the other hand, Terrimonas, unculured Blastocatella, Xanthomonadales nobank, unculured TK10, Ferruginibacter, Saccharibacteria, and Geodematophilaceae were closely negative correlated to DO, and a significant positive correlation with Massilia.. JG30-KF-CM45 belongs to Chloroflexi. It accounted for 1.09%-1.50% in five samples. Wang et al. (2010) reported Chloroflexi was the predominant bacteria group in nitrosation and 11.67% was assigned to this phylum. 0.45%-20.27% of total bacteria fell into Pseudomonas, which could degrade many organic pollutants. Besides, it is also an important microorganism in aerobic denitrification process. Therefore, NO3--N obviously decreased and negative correlation trend was shown in the modified process. Lin et al. (2008) found that the coefficients of the heterotrophic bacterial quantity had good correlations with TP, total organic carbon (TOC), and TN (the correlation coefficient R2 were 0.8229, 0.9143, and 0.7954, respectively), and dominant heterotrophic bacteria belonged to Pseudomonas. Meanwhile, Clostridium and Methylocystaceae are anaerobic bacteria, DO inhibited their growth .Besidese, Methylocystaceae is a typical methane-oxidizing bacteria. The increase of COD promoted Methylocystaceae quantity (Sundh et al., 2005). Prior to this study, Blastocatella was rarely reported and described in the activated sludge, Foesel et al. (2013) first isolated the bacteria and supposed that some species of genus Blastocatella are aerobic, chemoorganotrophic bacterium with strictly respiratory type of metabolism. In this study, however, some species of Blastocatella were negative correlation with DO, and they may grow in anaerobic conditions. Yang et al. (2015) reported that Blastocatella was one of predominant genera in microaerobic hydrolysis–acidification (MHA), anoxic and oxic reactors. Cao et al. (2016) demonstrated Blastocatella was relatively high abundances in sludge samples from two WWTPs in China. Xanthomonadales nobank is a denitrifying phosphorus removal bacteria and utilize NO3--N, instead of O2 to absorb phosphorus (Martín et al., 2006). 4. Conclusions The A-MAO system in this study demonstrated a high potential for nutrient removal from municipal wastewater. The effluent concentrations of COD, NH4+-N, TN, and TP from the modified process were lower than the class A discharge standard. High-throughput sequencing revealed the pollutant removal mechanisms, microbial community, functional microbial characteristics, and correlation of microbial community with environmental variables in five compartments. Blastocatella, Flavobacterium and Pseudomonas were the dominant genus. Besides, Nitrosomonas, Nitrospira, Rhodospirillaceae and Rhodocyclaceae occupied dominant position in nitrogen and phosphorus removal communities, respectively. DO and COD played significant roles on affecting the microbial components. Acknowledgment This work was supported by a project grant from the National Major Project of Water
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Figure captions Fig. 1 Schematic diagram of the modified multistage AO process (1) anaerobic zone (2) first anoxic zone (3)first oxic zone (4) second anoxic zone (5)second oxic zone.
Fig. 2 Removal performance of the modified multistage AO system (A) COD, (B) TN, (C)NH4+-N, (D)TP. Gray area was sudden cooling (94th -100th day).
Fig. 3 Evolution of organics and nutrients in A-MAO process (A) concentration change of pollutions; (B) pollutions mass balance in each compartment.
Fig. 4 Relative abundances of different (A) phylum level and (B) genus level in the five compartments, and less than 1% of total bacterial community composition were classified as “others”.
Fig. 5 Abundance of the potential (A) nitrifying and denitrifying bacterial and (B) phosphorus removing bacteria in the five compartments.
Fig. 6 The responds of microbial community to environmental variables.
Fig. 1
Fig. 1 Schematic diagram of the A-MAO process (1) anaerobic zone (2) first anoxic zone (3)first oxic zone (4) second anoxic zone (5)second oxic zone
Fig. 2
Fig.2 Removal performance of the modified multistage AO system (A) COD, (B) TN, (C)NH4+-N, (D)TP. Gray area was sudden cooling (94th -100th day)
Fig. 3
Fig.3 Evolution of organics and nutrients in A-MAO process (A) concentration change of pollutions; (B) pollutions mass balance in each compartment.
Fig.4
Fig.4. Relative abundances of different (A) phylum level and (B) genus level in the five compartments, and less than 1% of total bacterial community composition were classified as “others”
Fig. 5
Fig. 5 Abundance of the potential (A) nitrifying and denitrifying bacterial and (B) phosphorus removing bacteria in the five compartments.
Fig. 6
Fig. 6 The responds of microbial community to environmental variables
Table captions Table 1 Main characteristics of influent wastewater
Table 2 Bacterial diversity of five samples
Table 1 Main characteristics of influent wastewater Parameter -1
COD/(mg L ) (0-160d) COD/( mg L-1) (160-220d) NH4+-N/( mg L-1) NO3--N/( mg L-1) TN/( mg L-1) TP/( mg L-1) pH T/℃
Range
Average
315.6-660.7 323.1-448.3 39.3-55.9 0.0-4.6 44.4-78.1 3.8-8.4 6.5-8.1 8-30
513.3 377.2 49.7 2.1 60.2 5.5 6.6 22
Table 2 Bacterial diversity of five samples Sample ID
Level a
ANA AN1 O1 AN2 O2
97%
Sequences
OTUs
ACE b
Chao
Shannon d
c
34004 34889 34871 40857 33107
396 417 386 426 385
415 442 408 426 416
423 444 407 426 428
4.19 4.13 3.85 4.34 3.77
Simpson
Coverage
e
f
0.0426 0.0411 0.0620 0.0351 0.0735
0.998236 0.997884 0.998104 0.998677 0.997663
Note: a Degrees of similarity. b, c
Community richness. A higher number represented more richness Community diversity. A higher Shannon index represented more diversity, Simpson was on the contrary. f Sampling depth. d, e
Highlights
The A-MAO process treated municipal wastewater with an effluent superior first-A standard. Temperature plunge and C/N decrease have a certain impact on the modified process. Microbial community exited differences in five compartments. DO and COD played significant roles on affecting the microbial components.
S0960-8524(17)30829-5 http://dx.doi.org/10.1016/j.biortech.2017.05.161 BITE 18187
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
22 March 2017 19 May 2017 25 May 2017
Please cite this article as: Huang, X., Dong, W., Wang, H., Jiang, S., Biological nutrient removal and molecular biological characteristics in an anaerobic- multistage anaerobic/oxic (A-MAO) process to treat municipal wastewater, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.161
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Biological nutrient removal and molecular biological characteristics in an anaerobicmultistage anaerobic/oxic (A-MAO) process to treat municipal wastewater
Xiao Huang a,
Wenyi Dong a,b,
Hongjie Wang a,b *, Shilong Jiang
a.
School of Civil and Environmental Engineering, Harbin Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China b. Shenzhen Key Laboratory of Water Resource Utilization and Environmental Pollution Control, Shenzhen, 518055, China * Corresponding author: Hongjie Wang School of Civil and Environmental Engineering, Harbin Institute of Technology Shenzhen Graduate School Shenzhen 518055, China Tel. / Fax: +86 0755 26032718 E-mail: [email protected] (H. Wang).
Abstract This study aimed to present an anaerobic- multistage anaerobic/oxic (A-MAO) process to treat municipal wastewater. The average COD, NH4+-N, TN, and TP removal efficiency were 91.81%, 96.26%, 83.73% and 94.49%, respectively. Temperature plunge and C/N decrease have a certain impact on the modified process. Characteristics of microbial community, function microorganism, and correlation of microbial community with environmental variables in five compartments were carried out by Illumina Miseq high-throughput sequencing. The differences of microbial community were observed and Blastocatella, Flavobacterium and Pseudomonas were the dominant genus. Nitrosomonas and Nitrospira occupied a dominant position in AOB and NOB, respectively. Rhodospirillaceae and Rhodocyclaceae owned a considerable proportion in phosphorus removal bacteria. DO and COD played significant roles on affecting the microbial components. The A-MAO process in this study demonstrated a high potential for nutrient removal from municipal wastewater. Keyword: A-MAO process, Biological nutrient removal, Molecular biological characteristics, High-throughput sequencing，Microbial community 1. Introduction Eutrophication, as one of the most urgent issue to limit the economic development and pose a threat to the safety of drinking water resources, has been widely concerned in recent years. The nutrients (nitrogen and phosphorus) in effluent of waste water treatment plants (WWPTs) have been confirmed a main reason that cases receiving surface water eutrophication (Kowalkowski et al., 2012). A large number of WWPTs have been built up for several decades to meet the population and economic development in china. Currently, the standard for municipal sewage discharge is getting more and more stringent. The discharge has increased from standards first class B to class A. Nitrogen and phosphorus are the important control parameters and their control has become the urgent problem for sewage treatment. Biological nutrient removal (BNR) processes are effective for nitrogen and phosphorus removing and own economic advantages compared with other ways. Some biological treatment processes, such as anoxic/oxic (AO), anaerobic/anoxic/oxic (A 2O), multistage AO and 5-stage Bardenpho process (phoredox process), et al., have been developed for nitrogen and phosphorus removal. For these traditional biological nitrogen and phosphorus removal processes, carbon sources play a restrictive role due to the competitive effect on carbon sources was occurred between denitrifying bacteria and phosphorus removal bacteria (Wang et al., 2006; Wang et al., 2015; Barker and Dold, 1996). In China, especially in the south, the ratio of chemical oxygen demand (COD) to total nitrogen (TN) in influent is lower. Therefore, it is difficult to achieve stable removal of nitrogen and phosphorus and meet discharge standards. For low carbon to nitrogen ratio (C/N) wastewater, one of the most effective ways was adding additional carbon sources. However, this method increases the operating cost. Besides, some advanced wastewater treatment methods followed BNR processes, such as post-denitrification biofilter (Li et al., 2016; Shi et al., 2015), chemical phosphorus removal (Kim
et al., 2015), and membrane technology (Yang et al., 2010) were applied to decrease the effluent concentration of nitrogen and phosphorus further. Nevertheless, these methods not only need additional carbon sources, increase the construction cost and operating cost, but also result in wasting occupied area. For some existing WWTPs, there is no enough area to increase the treatment structures. Thus, modifying the conventional treatment process (e. g. A2O or 5-stage Bardenpho process) and excavating their treatment efficiency fully are necessary. In the previous studies, there were many modifying ways to remedy the disadvantage of conventional activated sludge system. Zeng et al. (2011) set up a pre-anoxic zone before A2O process and a better biological nutrients removal was obtained than the conventional A 2O process. Zhao et al. (2016) put forward a pre-denitrification process of anaerobic/anoxic/aerobic nitrification, which are operated as a two sludge system and exhibited an excellent phosphorus and nitrogen removal performance. In this study, an A-MAO process was put forward to remedy the defects of 5-stage Bardenpho process. Two important adjustments were conducted to improve the process performance, i.e., a varying internal recycle and distribution of influent. (i) The influent was divided into two parts, The carbon source in influent was sufficiently used for nitrogen and phosphorus removal, which reduces treatment cost. (ii) Adding influent into the 2nd anoxic zone brought about a problem that NO3--N will be out of limit in effluent. For reducing NO 3--N concentration, an adjustment of internal recycle was carried out to improve denitrification. For revealing the relationships between composition of microbial communities and pollutants removal high throughput sequencing method was conducted in this study. There were a lot of researches on microbial community characteristics, which have been studied. However, most biochemical characteristics, microbial community, and functional microorganisms in wastewater treatment have focused on different wastewater treatment plants (Hu et al. 2012), environmental conditions (Zou et al. 2014), and operating conditions (Duan et al. 2013; Liu et al. 2016) rather than different units (or compartments) in one complex wastewater treatment process. Many microbial communities involve in the removal of nitrogen, phosphorus and organic matter and the generation of many heterotrophic bacteria are short to many minutes or hours. How the microbial communities change when pollutants are removed in the different units? Whether a necessary connection of environmental factors and microbial communities in different units is existent or not? Therefore, it is necessary to discuss the composition of the microbial community, and then explain the pollutant degradation mechanism from the angle of microbial community. In this study, the performance of an anaerobic- multistage anaerobic/oxic (A-MAO) process was monitored and demonstrated its ability to remove TN and TP efficiently with an effluent superior to Chinese integrated wastewater discharge standard first-A standard, even under low C/N. In addition, microbial composition, diversity and abundance in different compartments were analyzed by high throughput sequence technology. It was found that there were some differences of microbial diversity and abundance among five compartments and the change of microbial community had significant association with environmental factors. Thus, this study is aiming to present an A-MAO process and reveal the functional species that are about TN and TP removal during the operation process.
2. Materials and Methods
2.1 Experimental system and operational conditions A lab-scaled A-MAO system and 5-stage Bardenpho process (shown in Fig. 1and Fig. 1S), was consisted of three parts: an influent tank, an A-MAO reactor (with a working volume 192 L) and a settler. The A-MAO reactor(shown in Fig. 1), which was made of colorless Plexiglas, was separated into five compartments in sequence to create anaerobic (13.6 L)/first anoxic (30.6 L)/first oxic (54 L)/ second anoxic (37.4 L)/second oxic zones (56.4 L). The influent was pumped into anaerobic and the second anoxic zone with 60% and 40% distribution ratio by peristaltic pumps. To keep sludge suspension, mechanical stirrers were installed at the top of the anaerobic and two anoxic zones. Besides, sand bubble diffusers which play roles in supplying air and mixing, were installed at the bottom in two oxic zones. Sewage mixed liquor returned from second oxic zone to first anoxic zone to improve the nutrients removal and the sludge was from secondary settler to anaerobic. The compartments of 5-stage Bardenpho process was same with A-MAO process, the influent was only pumped into anaerobic and mixed liquor returned from first oxic zone to first anoxic zone. Two systems were operated at natural temperature and treated actual sewage. The mean influent was maintained 240 L d-1, the total hydraulic retention time (HRT) was 19.2 h and each zone was 1.36, 3.06, 5.4, 3.74 and 5.64 h in sequence. The sludge was discharged from secondary settler every day and kept sludge retention time (SRT) 16 d. The sewage mixed liquor and sludge recycle ratio was at 100% and 85%, respectively. Mixed liquor suspended solids (MLSS) concentration was 3400 mg/L. The dissolved oxygen (DO) concentration was controlled at 5-6 mg/L in both oxic zones. 2.2 Seed sludge and wastewater characteristics The seed sludge used in this study was collected from the secondary sedimentation tank at the Luofang Municipal wastewater Treatment Plant (first phase project) (Shenzhen, China). Where Adsorption (aerobic aeration)-Biodegradation (anaerobic/anoxic/oxic, A2O) process (AB process) was employed to treat 100,000 m3/d wastewater. The wastewater source was collected from the drain well of Harbin Institute of Technology Shenzhen Graduate School (Shenzhen, China) to an intermediate tank, and then pumped into an influent tank. The main characteristics of influent wastewater were summarized in Table 1. 2.3 Pollutant mass balance The pollutants balance is shown in Fig. 2S and calculated according to Eqs. (1) to Eqs. (5). Q1·SINF C,N,P +Qr·Sset C,N,P =(Q1+Qr)·S(ANA-AN1)+SANA C,N,P (1) (Q1+Qr)·S(ANA-AN1) + q·SO2 C,N,P =(Q1+Qr+q)·S(AN1-O1) C,N,P +SAN1 C,N,P (2) (Q1+Qr+q)·S(AN1-O1) C,N,P =(Q1+Qr+q)·S(O1-AN2) C,N,P +SO1 C,N,P (3) (Q1+Qr+q)·S(O1-AN2) C,N,P + Q2·Sinf C,N,P =(Q1+Qr+ Q2+q)·S(AN2-O2) C,N,P +SAN2 C,N,P (4)
(Q1+Qr+ Q2+q)·S(AN2-O2) C,N,P =(Q1+Qr+ Q2+ q)·SO2 C,N,P +SO2 C,N,P (5) Where, Q1, Q2, Qr, and q present the flow of two step-feed, return sludge, and mixed liquid return; SINF C,N,P and Sset C,N,P are the concentration of pollutants of influent and effluent; S(ANA-AN1) S(AN1-O1) C,N,P, S(O1-AN2) C,N,P, S(AN2-O2) C,N,P, SANA C,N,P, are pollutant concentrations of anaerobic to first anoxic, first anoxic to first oxic, first oxic to second anoxic, and second anoxic to second oxic, respectively. SO2 C,N,P is pollutant concentration after second oxic zone; SAN1 C,N,P, SO1 C,N,P, SAN2 C,N,P, and SO2 C,N,P denote the removal amount of pollutants changed in anaerobic, first anoxic , first oxic, second anoxic, second oxic zones, respectively; C, N, P present the concentration of COD, NH4+-N, TN, and TP, respectively. C,N,P,
2.4Microbial community analysis The total activity sludge DNA from five different compartments (Sample named as ANA, AN1, O1, AN2, and O2 and represented samples are taken from anaerobic, first anoxic, first oxic, second anoxic, and second-oxic compartments, respectively) are extracted using the Power Soil DNA kit (Mobio, CA, USA), and following the method described in Simister et al. (2015). Nanodrop Spectrophotometer (2000c, Thermo, USA) was used to checked DNA density and 5 µL DNA sample was taken to electrophoresis detection by 1% agarose gel electrophoresis and Polymerase chain reaction (PCR) amplification was conducted by PCR instrument (9700, GeneAmp® ABI, USA) using the DNA extracts as a template, and the V4 region of 16S rDNA was amplified with universal primers 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACCAGGGTATCTAAT-3’). All PCR reactions were carried out in 20 μL reactions with 4 μL 5×FastPfu Buffer, 2.5mM dNTPs, 0.4μL FastPfu Polymerase, 0.2μL BSA, 0.8 μL of forward and reverse primers, and about 10 ng template DNA .Thermal cycling consisted of initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 45 s. Finally 72 °C for 5 min. PCR products were monitored with 2% agarose gel electrophoresis. After PCR products were purified and fluorescence quantitative detection, sequencing was determined by using two-terminal sequencing (Paired-End) method and constructing a small fragment library on an Illumina MiSeq plantform by Majorbio company (Shanghai, China). Paired-end reads from the original DNA fragments were merged with the software of Trimmomatic and FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/) (Magoč et al., 2011) and sequencing reads were assigned to each sample according to the unique barcodes. The richness and diversity of microbial communities which include ACE index, Chao 1 index, Simpson index, and Shannon index, were calculated and analyzed using Mothur version v.1.30.1 (http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity) (Schloss et al., 2011). In order to study the species diversity of information in different samples, the sequences were assigned to operational taxonomic units (OTUs) by Usearch software (vsesion 7.1 http://drive5.com/uparse/) and all the sequence column similarity within the scope of the threshold (> 97%) sequence were grouped together and regarded as one OTU. The rarefaction curves, principle component analysis and Venn diagram were generated basic on OTUs. Graphical representation of the relative abundance of microbial diversity at phylum and genus levels was counted basic on the taxonomic data. The sequences of main functional microorganisms were analyzed by BioEdit software. A phylogenetic tree was constructed using Mega 4.0 software.
2.5 Analytical methods Samples were collected from the influent tank and secondary settler once three days. Besides, samples in anaerobic, first-anoxic, first-oxic, second-anoxic and second-oxic zones were collected once a week. COD, NH4+-N, NO3--N, TN, TP, MLSS and MLVSS were analyzed according to Standard Methods (APHA, 2005). DO, pH, temperature were monitored online by using WTW pH/Oxi 340i meter with DO and pH probes (WTW, Germany). 2.6 Statistical analysis Three replicates were conducted for each sample and average values were calculated and showed in charts. Origin 8.6 software was used for drawing figures 3. Results and discussion
3.1 Overall performance of organics and nutrients removal In this study, the lab-scale reactor was operated for a period of 220 days. The start-up process took about 50 days to reach steady (Fig. 2). When the removal efficiencies reached a satisfactory hill, it was identified reaching steady-state period. On the other hand, the overall system, after 50 days cultured, performed steadily with reference to the pollution removal. It is interesting that a sudden cooling which was appeared one week (94th to 100th day) and C/N changed from 8-10 to 5-6 (166th -172th day) were observed during the operation period. Under unsteady-state and steady-state periods, the concentration of COD, TN, NH4+-N and TP in influent and effluent as well as removal efficiencies are shown in Fig. 2 COD, NH4+-N and TN removal efficiencies were lower compared to steady-state, but removal efficiencies were also highly satisfactory during the unsteady-state. TP removal efficiency, however, during the unsteady-state was range from 50% to 80% and not satisfactory. In the steady-state the effluent concentration of COD, TN, NH 4+-N and TP from the A-MAO system all met the integrated wastewater discharge standard (class A discharge standards, GB18918-2002, China) which were lower than 50.0, 15.0, 5.0 and 0.5 mg L-1, respectively. The results from the lab-scale study suggest that the A-MAO system can be used efficiently for organics and nutrient removal for municipal wastewater. The COD removal of the lab-scale A-MAO system during the whole operation period (220 days) is presented in Fig. 2 A. The influent COD concentration was in the range of 315.6 - 660.7 mg L-1 with an average value of 513.3 mg/L in the first 160 d. Afterwards, The COD concentration decreased to 323.1 - 448.3 mg L-1 (average value was 377.2 mg L-1) from 160th to 220th days and the C/N of two periods were 8-10 and 5-6, respectively. During the start-up process, the removal efficiency rose from 80.1% to 97.5% gradually and effluent concentration were steady with the influent concentration increasing. Besides, the COD removal efficiency in this A-MAO system kept as high as 80% and effluent was lower than discharge standard despite of the variation of COD concentration in common situation. The effluent, however, rapidly worsened when sudden cooling (94th to 100th day) and C/N droped from 8-10 to 5-6 (166th -217th day),
and then quickly returned to a stable state. The influent COD load fluctuated from 0.4 to 0.8 kg COD (m3·d)-1, the removal efficiency remained around 90%. According to the 220 days research, the system reflected a good effect for COD removal. Fig. 2 B and Fig. 2 C show the NH4+-N and TN concentration in influent and effluent as well as the removal efficiencies during the 220 days experiment period. The excellent NH4+-N removal was achieved in the whole treatment process, with the average effluent concentration and removal efficiency (without sudden cooling and C/N drop) of NH4+-N was 2.75 mg L-1 and 94.6%, respectively. No matter start-up and steady-state periods, the effluent NH4+-N remained a favorable concentration and a weak effect for C/N. In contrast, the effluent and removal efficiency of TN were unsatisfactory during start-up period. The effluent concentration varies 20 to 50 mg L-1, higher than the discharge standard of 15 mg L-1 obviously. After 50 d start-up period, the average effluent concentration 9.66 mg L-1 and removal efficiency 84.3% were obtained. However, with the C/N decrease from 8-10 to 5-6, the effluent TN showed a large fluctuations in the first 20 days, and the removal efficiency range from 74.57% to 94.27%, and remained the level of discharge standard (15 mg L-1). Results indicated C/N decrease did not inhibit the TN removal, enough carbon sources were offered for denitrification. TP removal efficiency was shown in Fig. 2 D，as we can see, TP removal efficiency maintained rising trend at initial operation period and reached stability until 40th day. The effluent concentration was below 0.5 mg L-1 at steady-state period and exceeded 90% removal efficiency was achieved. The comparative study of Bardenpho process and A-MAO process was conducted to demonstrate the advantages of modified process. From Table S1, there were no changes for COD and NH4+-N removing among three processes, the mean removal efficiency was higher than 93% and 97%, respectively. The removal efficiencies of TN and TP were significantly increase by A-MAO process compared with 5-stage Bardenpho process. Hence, the A-MAO, which was modified depending on 5-stage Bardenpho process, was effective for denitrification. 3.2 Nutrients and organics removal efficiency in different compartments For full understanding of the organics and nutrients removal mechanism in the A-MAO system, pollutions concentration and utilization performance were tested in different treatment compartments. The evolution of COD pollutions in the A-MAO system is depicted in Fig. 3. It could be seen that great proportion (75.69%) of COD was removed in anaerobic zone. At the same time, 12.72% and 5.98% COD was utilized in the 1st anoxic and oxic compartments, respectively. 40% influent was fed into the 2nd anoxic compartment, and resulted in the increasing of COD concentration of effluent in the 2nd anoxic and oxic compartments. In fact, 29.20 g COD d-1 and 7.40 g COD d-1 were removed in the two compartments, respectively. The removal amounts of COD in 2nd anoxic and oxic compartments were higher than 1st anoxic and oxic compartments. NH4+-N in the influent was about 41.83-57.98 mg L-1, which was the mainly component of TN (about 95-98%) in the entire experiment period. NH4+-N concentration was decreased to 21.15 and 0.88 mg L-1 fleetly in anaerobic and anoxic compartments due to the dilution of sludge return and internal return. However, small part of ammonia in influent was removed (1.00 g d -1). The concentrations in 1st anoxic and oxic changed slightly. It is interesting that 1.35g d-1 and 1.65 g d-1 NH4+-N were removed in 1st and 2nd anoxic compartments, but only 1.61 g d-1 and 0.57 g d-1
NH4+-N were removed in 1st and 2nd oxic compartments. It may be because that the distribution of functional population was relatively similar in five units. When the oxygen was carried from 2nd oxic to 1st anoxic compartment, nitrifying flora utilized the dissolved oxygen (0.5 mg L-1) to convert NH4+-N to NO2--N or NO3--N in 1st anoxic unit. The change of TN was similar with NH4+-N that most of them were removed in anaerobic and anoxic compartments based on denitrification. TP in influent was 6.62 mg L-1 and rose to 19.29 mg L-1 in anaerobic compartment. Phosphorus-accumulating bacteria (PAO) absorbed small molecular organic to compound poly-β-hydroxybutyrate (PHB), then 5.75 g L-1 phosphorus was released and generated a phenomenon of concentration increasing in anaerobic compartment. Then a continuously decreasing trend was observed in the first anoxic (4.14 g L-1) and oxic compartments (0.99 g L-1). So some denitrifying phosphorus remove bacteria utilized NO3--N as the electron acceptor for denitrifying phosphorus removing. 3.3 Molecular biological characteristics in different compartments
3.3.1 Microbial community similarity analysis A total of 177728 effective sequence reads (34004, 34889, 34871, 40857, and 33107 for ANA, AN1, O1, AN2, and O2, respectively) were obtained from activated sludge samples in five activated sludge by the Illumina MiSeq plantform. Downstream analysis based on the assigning of gene sequences was conducted and 396, 417, 386, 414, and 385 OTUs were divided at a threshold scope of 97%. Rarefaction curves were used to standardize and compare the microbial community richness and sequencing quality (Amato, et al., 2013). As can be seen from Fig. S3 A, The cures of five samples nearly reached gentle which indicated that the sequencing numbers of five samples are reasonable and most microorganisms had been detected. The result could be proved by coverage index (the coverage indexes of all samples exceeded 99.76%) in Table 2. Venn diagram (Fig. S3 B), the cluster analysis (Fig. S3 C) of OTUs, principal component analysis (PCA) (Fig. S4) and non-metric multidimensional scaling (NMDS) (Fig.S5) could effectively evaluate the similarity and difference of microbial communities. From Venn diagram, 75.87% of total OTUs were shared among all samples. In two oxic zones nearly 90% microbial communities were similar and only about 5% was changed in both anoxic zones. A high microbial community similarity was detected in five different zones. From Fig. S3 C, the shortest relative Bray-Curtis distance appeared between AN1 and AN2 and could be gathered together. Cluster analysis proved that ANA and O1 were highly similar and O2 that had the farthest Bray-Curtis distance with other samples showed significantly different. This view was also confirmed by PCA (Fig.S4) and NMDS diagrams (Fig.S5). In PCA diagram, AN1 and AN2 could be divided into one category according to any two dimensions and the differences between oxic and anoxic zones were not obvious. However the differences could be clearly seen by NMDS analysis (Fig.S5). Microbial community in two kinds of reaction zones (oxic and anoxic zones) did not show a higher similarity. Both oxic zones exited vary markedly compared with anoxic zones. Therefore, dissolved oxygen posed a significant influence on the changes of microbiome and abundance.
3.3.2 Microbial community richness and biodiversity Rarefaction curves could reflect the richness of microbial community and abundance-based coverage estimation (ACE) index and Chao 1 index (Table 2) are another two important standards to describe the species richness. The higher ACE and Chao 1 index, the more richness in sample. Microorganism in five zones presented a similar richness ranging from 400 to 450, and the first anoxic zone (AN1) owned the most richness, i.e., 442 and 444 for ACE and Chao 1 index, respectively. However, the least richness was 408 and 407, which was observed in the first oxic zone (O1). It is interesting that microbial community in both oxic zones was less richness than two anoxic zones and similar to anaerobic tank. This phenomenon is not consistent with Kim et al. (2013), in whose research the microbial richness in oxic zone was higher than anoxic and anaerobic zones and anoxic was better than anaerobic. In this research, it maybe because that organic (COD or BOD) in influent was utilized in anoxic zones and large number of heterotrophic microorganism bred resulted in high richness. On the contrary, the fact that no enough organic was in oxic zones leaded to a poor nutrition environment, which accelerated some microorganisms lose viability and more autotrophic organisms breeding. Meanwhile, in anaerobic tank, due to the lack of electron acceptors (O2 or NO3--N), anaerobic microbes will grow well and reduces the microbial richness. Microbial community biodiversity could be estimated by Shannon index and Simpson index (Table 2). A higher Shannon index represented more diversity, while Simpson was on the contrary. It was clear that the result of biodiversity was nearly consistent changing trend with ACE and Chao 1. The biodiversity in anoxic zones was higher than two oxic zones and anaerobic zone. The difference is the highest biodiversity (4.34 for Shannon and 0.620 for Simpson) was in the second anoxic zones (AN2). From richness and biodiversity analysis, there were no significant differences among five compartments. The same conclusion was got from A2O process (Kim et al. 2016). 3.3.3 Microbial community composition Dynamic changes of microbial community structures among microbial samples were revealed depicted in Fig. 4. Significant changes of community composition were found according to the relative abundances of different phylum (Fig. 4 A) and genus (Fig. 4 B) in the five compartments. It was obvious that Proteobacteria occupied the leading position in all samples with the relative abundance of 37.27% (ANA), 26.49% (AN1), 47.03% (O1), 34.45% (AN2), and 42.11% (O2). Bacteroidetes (6.99%-29.67%), Chloroflexi (7.34%-15.02%), and Acidobacteria (6.45%-18.18%) were the most dominant phylum behind Proteobacteria. Other phylum like Fimicutes, Deinococcus-Thermus, Actinobacteria, TM6, and Gemmatimonadetes were detected over 1% in five samples. Generally, the main microbial population was quite different at phylum level. The similar conclusion was achieved by Kim et al. (2013) whose research found that Proteobacteria (53.80%), Bacteroidetes (18.80%), and Chloroflexi (6.60%) were the dominant phylum in A2O process. There were reported that Proteobacteria was the most abundant in WWTPs (Hu et al., 2012; Ye et al., 2011). The differences among five samples were apparent at genus level, especially, ANA, O1, and O2. The heatmap (Fig. 4 B) for the 50 abundant genera was constructed to discuss the similarities
and differences among five samples. The results showed that the microorganism in samples at genus level was diverse. Blastocatella, Flavobacterium and Pseudomonaswere dominant and large variations occurred among them. Nevertheless, high level of similarity was maintained in AN1 and AN2 samples. Blastocatella (16.51%), Pseudomonas (16.21%), and TK10_norank (8.71%) were the dominant species in ANA sample. AN1 and AN2 were main represented by genus Blastocatella (12.57% for AN1, 11.26% for AN2), Flavobacterium (10.77% for AN1, 11.82% for AN2), and TK10-norank sp. (9.09% for AN1, 9.35% for AN2), and Meiothermus sp. (9.21% for AN1, 4.00% for AN2). It is interesting that O1 and O2 was on the contrary to what we thought, a small similarity was existed between them. In O1 sample, the most dominant genus was Pseudomonas (20.30%) and the main genus in O2 sample were Flavobacterium (22.79%) and Massilia (19.93%). Therefore, it can be inferred that the dominant genus could shift in two aerobic zones for A-MAO system. Cluster analysis (Fig. 4 B) also confirmed the above discussion that AN1 and AN2 were gathered into one category. Besides, Flavobacterium and Massilia were homologous relationship, and the same with Blastocatella and TK10_norank. Flavobacterium has been reported about its functions with nutrients removal and enhancing the biological phosphate removal (Kong et al., 2007; Zengin et al., 2010; Zou et al., 2014). Yeh et al. (2010) reported that Pseudomonas played an important role in organic matter and nitrogen removal in field-scale constructed wetlands. The nutrients removal function of Pseudomonas was also reported by Bouki et al. (2013) and Zheng et al. (2013). 3.3.4 Dynamic change of functional microorganism Nitrifying and denitrifying bacterial communities that play core role for NH4+-N and TN removal in the A-MAO process was stable. Ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) convert NH4+-N to NO3--N collectively in nitrification process. Previous studies found that Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio were the main AOB genus (Duan et al.,2013) and Nitrospira, Nitrospine, Nitrobacter, and Nitrococcus were the core bacterial for NOB (Rani et al.,2016). In five sludge samples, most AOB belonged to uncultured Nitrosomonadaceae and the abundance changed in the range of 0.14%-0.37% (Fig. 5 A). However, other AOB genus was not found in this process. For NOB, Nitrospine, Nitrobacter, and Nitrococcus were a lot less than Nitrospira, which abundance was more than 0.3%. Thus, in this A-MAO process, Nitrosomonas and Nitrospira occupied a dominant position in AOB and NOB communities, respectively and the stable nitrification was obtained depended on the existence of them. The conclusion was consistent with Chen et al. (2016) who also reported that Nitrosomonas and Nitrospira were the dominant nitrifying bacterial communities occurring in YXM process. It is interesting that the abundance of AOB and NOB in anaerobic compartment (ANA), and anoxic compartments (AN1 and AN2), which owned low DO concentration, was more than aerobic compartments (O1 and O2) with sufficient oxygen supply. However, the phenomenon proved the previous results that the NH4+-N removal efficiencies in AN1 and AN2 were higher than O1 and O2. Therefore, the change trend indicated that the more Nitrosomonas and Nitrospira resulted in higher NH4+-N removal efficiency. This unnatural phenomenon was also revealed by previous study and assumed that Nitrosomonas and Nitrospira lack the sensitivily to low DO which could make contribution to energy saving in waste water treatment (Chen et al., 2016). Liu
et al. (2016) thought that Nitrosomonas (AOB), Nitrobacter (NOB), and Nitrospira (NOB) mainly contributed to the nitrification under low DO conditions and the waste water treatment system was able to complete nitrification under 0.5 mg L-1. It could provide a reasonable evidence that the effluent from WWTP reaches standard though reducing DO and a strategy for energy saving. The potential denitrifying bacteria were classified into 11 families in five samples (Fig. 5 A). The dominant denitrifying bacteria belonged to Bradyrhizobiaceae, Comamonadaceae, Rhodobacteraceae, Bacillaceae, and Pseudomonadaceae. The high abundances were those of Bacillus (0.37%-8.44%), Pseudomonas (0.41%-20.23%), uncultured Cytophagaceae (0.51%-1.31%), Acinetobacter (0.02%-1.72%), Comamonadaceae (0.40%-6.26%), and Ottowia (1.10%-2.66%). Denitrifying bacteria in Pseudomonadaceae and Bacillaceae were more abundance in aerobic compartments than anoxic compartments because some Pseudomonas and Bacillus is aerobic denitrifying bacteria and able to convert NO3--N to N2 under aerobic environment (Gui et al., 2016; Koike and Hattori, 1975; Kim et al., 2005). This also confirmed why NO3--N was reduce in aerobic zone in this study. Comamonadaceae, Acinetobacter, and Ottowia are typical denitrifying bacteria and have been reported by Spring et al. (2004) and Jahan et al. (2012). There are four main enzymes which are associated with denitrification process, i.e. Nitrate reductase, Nitrite reductase, Nitric oxide reductase, and Nitrous oxide reductase (Averill, 1996). Nitrate reductase is sensitive to oxygen and inhibited in aerobic condition (Mendel et al., 1981). Hence, higher abundance was occurred in anoxic compartments. On the whole, denitrifying bacteria and aerobic denitrifying bacteria formed a combined effect on TN removal. In order to discuss the the mechanism of phosphorus removal, dominant microorganisms associated with phosphorus removal was shown in Fig. 5 B. Beijerinckiaceae, Bacillaceae, Sphingomonadaceae, Rhizobiales_Incertae_Sedis, Rhodospirillaceae, and Rhodocyclaceae were the six families, which contained the core PAO. They were relatively stable in five compartments. Rhodospirillaceae (0.27%-1.24%) and Rhodocyclaceae (0.22%-0.74%) occupied a considerable proportion and their abundance was higher than other families. Jiang et al. (2012) found that uncultured Rhodocyclaceae bacterium dominated the granule-based for enhancing biological phosphorus removal (EBPR) sludge in SBR reactor． Zhang et al. (2012) reported a new finding that denitrification and phosphorus removal were simultaneously achieved in an EBPR system, and Azospirillum that belonged to Rhodospirillaceae was 52.54%. This study once again proved that Azospirillum is a PAO genus. Bacillaceae, Rhodocyclaceae, Hyphomicrobiaceae, Rhodobacteraceae and Pseudomonadaceae were typical denitrifying phosphorus accumulating organisms (DPAOs) that have been reported in previous research (Lee et al. 2008; Kim et al. 2013; Aguilar-May et al. 2009; Barak and van Rijn, 2000). Therefore, three main processes existed in the A-MAO process, i.e., anaerobic phosphorus release, aerobic phosphorus absorption, and denitrifying phosphorus accumulating process. The phosphorus removal bacteria could utilize O2, NO2--N, and NO3--N as electron acceptor and be catalyzed by polyphosphate kinase under aerobic and anoxic conditions (Jung et al. 2012). 3.4 Correlation of microbial community with environmental variables Environment variables played a cooperative role on affecting the microbial components and the expression of function. Heatmap analysis of the correlation between environment variables (TN, MLSS, NH4+-N, COD, TP, pH, NO3--N, and DO) and community composition was
illustrated in Fig. 6. It was obviously that some community composition was significantly affected by environment variables. JG30-KF-CM45, Pseudomonas Clostridium, Blastocatella ， Methylocystaceae，and Iamia were closely positive correlated to TN, NH4+-N, MLSS, COD, and TP. The six genus had a significant response to nitrogen, phosphorus and organic matter removal process and gather into a category. On the other hand, Terrimonas, unculured Blastocatella, Xanthomonadales nobank, unculured TK10, Ferruginibacter, Saccharibacteria, and Geodematophilaceae were closely negative correlated to DO, and a significant positive correlation with Massilia.. JG30-KF-CM45 belongs to Chloroflexi. It accounted for 1.09%-1.50% in five samples. Wang et al. (2010) reported Chloroflexi was the predominant bacteria group in nitrosation and 11.67% was assigned to this phylum. 0.45%-20.27% of total bacteria fell into Pseudomonas, which could degrade many organic pollutants. Besides, it is also an important microorganism in aerobic denitrification process. Therefore, NO3--N obviously decreased and negative correlation trend was shown in the modified process. Lin et al. (2008) found that the coefficients of the heterotrophic bacterial quantity had good correlations with TP, total organic carbon (TOC), and TN (the correlation coefficient R2 were 0.8229, 0.9143, and 0.7954, respectively), and dominant heterotrophic bacteria belonged to Pseudomonas. Meanwhile, Clostridium and Methylocystaceae are anaerobic bacteria, DO inhibited their growth .Besidese, Methylocystaceae is a typical methane-oxidizing bacteria. The increase of COD promoted Methylocystaceae quantity (Sundh et al., 2005). Prior to this study, Blastocatella was rarely reported and described in the activated sludge, Foesel et al. (2013) first isolated the bacteria and supposed that some species of genus Blastocatella are aerobic, chemoorganotrophic bacterium with strictly respiratory type of metabolism. In this study, however, some species of Blastocatella were negative correlation with DO, and they may grow in anaerobic conditions. Yang et al. (2015) reported that Blastocatella was one of predominant genera in microaerobic hydrolysis–acidification (MHA), anoxic and oxic reactors. Cao et al. (2016) demonstrated Blastocatella was relatively high abundances in sludge samples from two WWTPs in China. Xanthomonadales nobank is a denitrifying phosphorus removal bacteria and utilize NO3--N, instead of O2 to absorb phosphorus (Martín et al., 2006). 4. Conclusions The A-MAO system in this study demonstrated a high potential for nutrient removal from municipal wastewater. The effluent concentrations of COD, NH4+-N, TN, and TP from the modified process were lower than the class A discharge standard. High-throughput sequencing revealed the pollutant removal mechanisms, microbial community, functional microbial characteristics, and correlation of microbial community with environmental variables in five compartments. Blastocatella, Flavobacterium and Pseudomonas were the dominant genus. Besides, Nitrosomonas, Nitrospira, Rhodospirillaceae and Rhodocyclaceae occupied dominant position in nitrogen and phosphorus removal communities, respectively. DO and COD played significant roles on affecting the microbial components. Acknowledgment This work was supported by a project grant from the National Major Project of Water
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Figure captions Fig. 1 Schematic diagram of the modified multistage AO process (1) anaerobic zone (2) first anoxic zone (3)first oxic zone (4) second anoxic zone (5)second oxic zone.
Fig. 2 Removal performance of the modified multistage AO system (A) COD, (B) TN, (C)NH4+-N, (D)TP. Gray area was sudden cooling (94th -100th day).
Fig. 3 Evolution of organics and nutrients in A-MAO process (A) concentration change of pollutions; (B) pollutions mass balance in each compartment.
Fig. 4 Relative abundances of different (A) phylum level and (B) genus level in the five compartments, and less than 1% of total bacterial community composition were classified as “others”.
Fig. 5 Abundance of the potential (A) nitrifying and denitrifying bacterial and (B) phosphorus removing bacteria in the five compartments.
Fig. 6 The responds of microbial community to environmental variables.
Fig. 1
Fig. 1 Schematic diagram of the A-MAO process (1) anaerobic zone (2) first anoxic zone (3)first oxic zone (4) second anoxic zone (5)second oxic zone
Fig. 2
Fig.2 Removal performance of the modified multistage AO system (A) COD, (B) TN, (C)NH4+-N, (D)TP. Gray area was sudden cooling (94th -100th day)
Fig. 3
Fig.3 Evolution of organics and nutrients in A-MAO process (A) concentration change of pollutions; (B) pollutions mass balance in each compartment.
Fig.4
Fig.4. Relative abundances of different (A) phylum level and (B) genus level in the five compartments, and less than 1% of total bacterial community composition were classified as “others”
Fig. 5
Fig. 5 Abundance of the potential (A) nitrifying and denitrifying bacterial and (B) phosphorus removing bacteria in the five compartments.
Fig. 6
Fig. 6 The responds of microbial community to environmental variables
Table captions Table 1 Main characteristics of influent wastewater
Table 2 Bacterial diversity of five samples
Table 1 Main characteristics of influent wastewater Parameter -1
COD/(mg L ) (0-160d) COD/( mg L-1) (160-220d) NH4+-N/( mg L-1) NO3--N/( mg L-1) TN/( mg L-1) TP/( mg L-1) pH T/℃
Range
Average
315.6-660.7 323.1-448.3 39.3-55.9 0.0-4.6 44.4-78.1 3.8-8.4 6.5-8.1 8-30
513.3 377.2 49.7 2.1 60.2 5.5 6.6 22
Table 2 Bacterial diversity of five samples Sample ID
Level a
ANA AN1 O1 AN2 O2
97%
Sequences
OTUs
ACE b
Chao
Shannon d
c
34004 34889 34871 40857 33107
396 417 386 426 385
415 442 408 426 416
423 444 407 426 428
4.19 4.13 3.85 4.34 3.77
Simpson
Coverage
e
f
0.0426 0.0411 0.0620 0.0351 0.0735
0.998236 0.997884 0.998104 0.998677 0.997663
Note: a Degrees of similarity. b, c
Community richness. A higher number represented more richness Community diversity. A higher Shannon index represented more diversity, Simpson was on the contrary. f Sampling depth. d, e
Highlights
The A-MAO process treated municipal wastewater with an effluent superior first-A standard. Temperature plunge and C/N decrease have a certain impact on the modified process. Microbial community exited differences in five compartments. DO and COD played significant roles on affecting the microbial components.