World J Microbiol Biotechnol DOI 10.1007/s11274-014-1763-4
ORIGINAL PAPER
Purification of high ammonia wastewater in a biofilm airlift loop bioreactor with microbial communities analysis Chunsheng Qiu • Dandan Zhang • Liping Sun Jianping Wen
•
Received: 31 August 2014 / Accepted: 20 October 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A 70 m3 gas–liquid–solid three-phase flow airlift loop bioreactor, in which biofilm attached on granular active carbon carriers, was used for purification of the high ammonia wastewater from bioethanol production. Under the optimum operating conditions, COD and NH4?-N average removal rate of 89.0 and 98.6 % were obtained at hydraulic retention time of 10 h. Scanning electron microscopy was applied for observation of the biofilm formation. High contaminants removal efficiency was achieved by holding high biomass concentration in the reactor due to the attached biofilm over the carriers. The 16S rRNA gene clone library analysis indicated that 68.6 % of the clones were affiliated with the two phyla Bacteroidetes and Proteobacteria, and residual clones clustered with various sequences from uncultured bacteria. The presence of various anoxic/anaerobic bacteria indicated that the oxygen gradient inside the biofilm could provide appropriate micro-environment for nitrogen removal through simultaneous nitrification and denitrification. Keywords Airlift loop bioreactor Biofilm High ammonia wastewater Microbial community C. Qiu D. Zhang L. Sun School of Environmental and Municipal Engineering, Tianjin Chengjian University, Tianjin 300384, People’s Republic of China C. Qiu (&) D. Zhang L. Sun Tianjin Key Laboratory of Aqueous Science and Technology, Tianjin 300384, People’s Republic of China e-mail: [email protected] J. Wen Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China
Abbreviations ALR Airlift loop bioreactor BPS Baffle plate separator COD Chemical oxygen demand DO Dissolved oxygen GAC Granular active carbon HRT Hydraulic retention time ID Internal diameter NLR Nitrogen loading rate OTU Operational taxonomic unit SAA Soaking in aqueous ammonia SEM Scanning electron microscopy
Introduction To reduce the negative environmental impacts by the use of fossil fuels, many studies have been carried out on ethanol production from lignocellulosic materials, such as agricultural residues, forest products and dedicated energy crops (Sanchez and Cardona 2008). Corn stover and corncob are abundant agricultural residues in Northern China that could be used as feedstock for bioethanol. For efficient hydrolysis of the carbohydrate fractions in the raw materials by enzymes, a pretreatment process is necessary (ElMekawy et al. 2013). Soaking in aqueous ammonia (SAA) has been shown to be one of the most feasible methods for corn stover pretreatment (Isci et al. 2008; Kim et al. 2009). However, large amounts of pretreatment wastewater with high ammonia content are generated (Qiu et al. 2011), which need to be solved in order to make bioethanol production process more environmental-friendly. The airlift loop bioreactor (ALR), characterized by several advantages, such as better mixture, faster mass
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and oxygen transfer rate, higher operational flexibility, greater processing capability and so on (Wang et al. 2011), has been investigated to remove nutrients (N and P) in the wastewater (Liu et al. 2005; Wen et al. 2005; Dhamole et al. 2009; Walters et al. 2009). By using carriers with elevated specific surface area (Wen et al. 2005; Walters et al. 2009) or culturing granular sludge to hold high biomass concentration (Jin et al. 2008; Fu et al. 2011) or combining anaerobic, anoxic and aerobic conditions in a single reactor (Jiang et al. 2013), ALR is a valuable alternative for nitrogen and COD removal. Wen et al. (2005) presented a gas–liquid–solid three-phase flow multiple ALR to remove ammonia nitrogen from chemical fertilizer industrial wastewater, in which biofilm replaced the activated sludge. Walters et al. (2009) introduced a biofilm airlift suspension reactor with biodegradable carrier material to achieve simultaneous nitrification and denitrification. Such studies indicated that it is possible to obtain nitrification and denitrification in a biofilm reactor. Biofilm is especially useful for slow growing organisms like nitrifiers that have to be kept in a wastewater treatment process (Guo et al. 2005), and the form of biofilm is critical to the performance of the ALR. However, previous studies mainly focused on hydrodynamics and mass transfer characteristics of the ALR (Luo et al. 2011; Wang et al. 2011; Luo et al. 2013; Fakhari et al. 2014), information is still highly limited on microbial community structure of the biofilm formed in this reactor. Further investigation of the microbial communities in the biofilm is essential for improving reactor performance. 16S rRNA/DNA-based molecular methods with high degrees of precision and specificity have been widely used for environmental microbial studies (Stackebrandt and Goebel 1994; Bosshard et al. 2000; Fu et al. 2010). In this study, a pilot-scale biofilm ALR, with effective volume of 70 m3, was established and applied for carbon and nitrogen removal of the pretreatment wastewater produced from bioethanol production, with granular active carbon (GAC) as carriers. Furthermore, the community structures of biofilm microflora were analyzed using clone library analysis.
Materials and methods Wastewater The pretreatment wastewater from SAA process during bioethanol production, after primary settling, was used as influent in this study. Table 1 shows the fluctuating ranges of the compositions of the real wastewater.
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Table 1 Characters of the pretreatment wastewater and effluents Parameter
Pretreatment wastewater
pH
7.8 ± 0.32 -1
Effluent 7.5 ± 0.26
Discharge standards 6–9
COD (mg L )
655 ± 56
73.1 ± 5.8
BOD (mg L-1)
312 ± 43
20 ± 2.3
NH4?-N (mg L-1)
365 ± 22
4.5 ± 0.5
B15
TN (mg L-1)
395 ± 23
183 ± 16
–
SS (mg L-1)
122 ± 16
26 ± 2.6
B70
29 ± 5.5
B50
Color
–
B100 B30
Analytical methods Levels of COD, BOD, ammonia nitrogen (NH4?-N), total nitrogen (TN), suspended solids (SS), volatile suspended solids (VSS) and color were determined in accordance with APHA standard methods (APHA 1998). Dissolved oxygen (DO) was measured by a RSS-5100 DO meter (Leici Instrument Plant, Shanghai, China). Liquor pH was analyzed using PHS-2 acidometers (Leici Instrument Plant, Shanghai, China). All samples were measured in triplicate. Configuration of the ALR The ALR (70 m3) used in this study has a similar structure as it has been described in previous studies (Liu et al. 2005; Wen et al. 2005). As shown in Fig. 1, seven 9.5 m high draft tubes with 0.70 m internal diameter (ID) were fixed inside the main 10 m high reactor tube of 2.8 m in diameter. Seven jet nozzles of 6 mm ID were designed and located concentrically in the bottom of each draft tube. The bioreactor was ended by a disengaging cap with 4.2 m ID and a height of 2.5 m as the expanded section. The wastewater, stored in reservoir, was pumped into the bottom of the bioreactor at a certain flow rate. Air influx produced by the compressor was introduced into the draft tube through the jet nozzles for sufficient DO supply and mixing. Dissolved oxygen was measured by a DO meter fixed in the expanded section. The pH was maintained at 7.0–8.0 by adding NaHCO3 solution by a control system. The role of baffle plate separator (BPS) with an effective volume of 25 m3 was to separate the carriers as well as the activated sludge from the liquid in the effluent. After the sediment process, the liquid was analyzed and reused for washing and soaking lignocellulosic materials. The reactor and the separator were made of stainless steel lined with rubber. Granular activated carbon was used as carrier and its physical characteristics were offered by the supplier as follows: size range, 1.0–1.5 mm; surface area, 5,500 m2 m-3; bulk density, 1,100 kg m-3 (Guangfu Chemical Institute, Tianjin, China).
World J Microbiol Biotechnol Fig. 1 Schematic diagram of the ALR system
Operation of the ALR
Scanning electron microscopy (SEM)
The startup strategy of the ALR was based on our previous studies (Liu et al. 2005; Wen et al. 2005; Qiu et al. 2011). For the batch adaptation, 20 m3 activated sludge with MLSS of 2.2 g L-1 (20 % V/V), which was obtained from a local municipal wastewater treatment plant (Tianjin, China), was placed in the ALR. Then the reactor was filled with 25 % (v/v) aerobic influent diluted by tap water. Compressed air was introduced into the reactor at the flow rate of 230 m3 h-1 (amounts to the superficial gas velocity of 10 mm s-1), and the batch culturing was then carried out. The supernatant in the reactor after precipitation for 2 h was replaced every day by the mixed wastewater with increased concentration of 10–15 % every time. After 10 days’ preculture, the undiluted mixed wastewater was introduced into the bioreactor at hydraulic retention time (HRT) of 24 h, which meant the continuous culture had begun. Then 10 t of activated carbon was put into the reactor for film forming culture, and the air flow rate was increased to 280 m3 h-1 (amounts to the superficial gas velocity of 12 mm s-1). This process continued until the steady state biomass loading on the carriers was achieved.
Samples of biofilm-GAC particles were fixed in 5 % glutaraldehyde. Then the samples were dehydrated in an ethanol-deionized water series (50, 60, 70, 80 and 100 %v/v) buffered with 1 mL sodium phosphate (pH 7.4). A critical point dryer, using liquid carbon dioxide as the transitional fluid, was used to dry the samples. Samples were sputter coated with gold to a thickness of 20 nm and examined via SEM (Hitachi S-4800, Japan). Bacterial community structure analysis of the biofilm To confirm bacteria species within the biofilm, a general 16S rRNA gene sequence analysis was carried out in this study. Total DNA of the biofilm (day 47) was extracted with a Soil DNA Isolation kit (Soil gDNA kit, Biomiga, USA), and a clone library was constructed by PCR and the primers 27F and 1492R. The PCR conditions used for the general bacteria were as follows: 4 min of initial denaturation at 94 °C, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 90 s, with final extension 72 °C for 5 min. The amplifiedPCR products were purified with a TIANprep Midi
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constant with COD and NH4?-N removal rate of 82.4 and 94.4 % (average value of the last 3 days), respectively. Then the reactor was switched to continuous operating state. Performance of the ALR at various HRTs
Fig. 2 Process performance during batch operation for sludge adaptation
Purification Kit (TIANGEN, China) and cloned into apMD19-T vector (TaKaRa, Japan) by a TA cloning method, according to the manufacturer’s instruction. The recombinant plasmids were transformed into competent Escherichia coli (strain DH5a), and then colonies containing the plasmid were screened on LB-based agar plates with ampicillin, X-gal, and IPTG. Cells from randomly selected white colonies were assayed on a LB medium at 37 °C for 8 h. A commercial kit (TIANprep Mini Plasmid Kit, TIANGEN, China) was used to isolate the plasmid DNA from the screened colonies. Finally, PCR products were sent to a genetic analysis service company (BGI, China) for analyzing DNA sequences. The full-length sequences of the 16S rRNA were compared with similar sequences in National Center for Biotechnology Information data using the BLAST program. All clones having a sequence similarity of more than 97 % with each other were grouped into an operational taxonomic unit (OTU) consistent (Stackebrandt and Goebel 1994).
Results and discussion Performance of the ALR during sludge acclimation state In this study, after the activated sludge placed, the ALR was operated at batch mode for adaptation. The performance of the ALR at batch state is presented in Fig. 2. During the initial 3 days, the reactor the ALR exhibited poor performance, with an average NH4?-N removal rate of 39.6 % due to the low activity of nitrifying bacteria in the sludge. From the fourth day, gradually increase of NH4?-N removal rate was observed. Undiluted wastewater was pumped into the reactor on the eighth day, it could be seen in the Fig. 2 that removal of contaminants tend to be
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In order to determine the maximum treatment capacity of the ALR and form biofilm on the carriers, the loading rate was progressively increased by decreasing the operating HRT, with the relatively stable influent NH4?-N and COD. As indicated in Table 2 and Fig. 3, the continuous operation was carried out under various HRTs. It could be seen in Fig. 3 that COD and NH4?-N removal was unstable due to the combined effect of biodegradation and adsorption of GAC during the biofilm-forming period (1–10 days). After 10 days’ continuous operation, it could be observed that brown biofilm had gradually formed on the carriers and pollutants removal tended to stabilize gradually. Then the HRT of ALR was gradually decreased from 20 h to 10 h, with a concomitant increase in nitrogen loading rate (NLR) from 0.279 to 1.117 kgN m-3 d-1, attaining the NH4?-N removal rate higher than 98 %. However, when the HRT was further reduced to 8 and 6 h, obvious increase of COD (over 100 mg L-1) concentration in the effluent could be observed. So the HRT of 10 h was selected as the optimum value when taking the treatment capacity and efficiency into consideration, with NH4?-N and COD removal efficiencies of 98.6 and 89.0 % (Table 2), and average effluent concentration of 4.5 and 73.0 mg L-1, satisfying the national primary discharging standard of China (Table 1). Microbial attachment SEM images on the surface of the GAC from ALR are presented in Fig. 4, evidence of microbial colonization over GAC in the reactor was found. After the addition of GAC, the dispersed seed sludge tended to attached on the carriers to form biofilm due to the large surface area of the GAC (Fig. 4a). Forming of the biofilm occurred within 20 days (Fig. 4b), and removal efficiency of the reactor tended to be stable thereafter. Moreover, the biofilm structure tended to be denser on day 47 (Fig. 4c) due to the growth of attached microorganisms. As shown in Fig. 4, it was postulated that anoxic zones would form both in the deeper layers of the biofilm as well as in the porous structures of the carriers. Simultaneous nitrification and denitrification could be observed in the reactor with TN treatment efficiency of 53.7 % (Table 1). This phenomenon has been reported in the previous study (Fu et al. 2010).
World J Microbiol Biotechnol Table 2 Performance of ALR with different operating HRTs (average value)
Time (d)
HRT (h)
NH4?-N (mg L-1) Influent
Effluent
NH4?-N removal efficiency (%)
COD (mg L-1) Influent
Effluent
COD removal efficiency (%)
0–9
24
298.5
19.4
93.5
635.2
66.7
10–16
20
315.2
14.2
95.5
656.6
78.1
89.5 88.1
17–23
16
313.5
9.1
97.1
606.5
67.3
88.9
24–29
12
308.8
5.1
98.3
660.4
75.9
88.5
30–34
10
326.4
4.8
98.5
665.1
71.8
89.2
35–39
8
302.1
5.1
98.2
645.3
96.1
85.1
40–42
6
319.5
4.9
98.4
630.5
129.2
79.5
43–48
10
335.5
4.5
98.6
664.5
73.0
89.0
which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Bacteroidetes
Fig. 3 Process performance during continuous operation of the ALR
The attached biomass concentrations in terms of VSS were 21.3 g L-1 on day 20 and 31.6 g L-1 on day 47, which were superior to other immobilized-biomass process and even a process using granular sludge (Jin et al. 2008). For this reason, high NLR was achieved by holding slow growing nitrifiers in the rector. Composition of bacterial communities Total DNA was extracted from the mixed culture attached on the carrier (day 47). Clone libraries of full-length 16S rDNA were constructed with the primer set (27F and 1492R). A total of 100 clones were isolated, after excision of 4 chimeric sequences, these clones were classified into 34 OTUs as shown in Table 3. The results indicated that the bacterial community of the biofilm was highly diverse. All 34 OTUs were affiliated with sequences from two major known lineages of the domain bacteria, namely Bacteroidetes (12 OTUs, 36 clones), and Proteobacteria (10 OTUs, 30 clones, including a-, b- and c-subdivisions), as well as some ungrouped species (12 OTUs, 30 clones),
The largest fractions were class Sphingobacteria (6 OTUs, 15 clones), which accounted for 41.7 % of the total clones belonged to the phylum Bacteroidetes. The portions of class Flavobacteriia, Cytophagia and unclassified group were 5.6, 5.6 and 41.7 %, respectively. The residual clones (15 clones of 4 OTUs) clustered with various sequences from uncultured bacteria retrieved from aerated submerged membrane bioreactor treating domestic wastewater (Du et al. 2008), rivers, freshwater sediment, and so on. OTU 1 was identified as Runella sp. P.slu-06, which was isolated from environmental water samples and showed the ability to form biofilm and degrade carbohydrates (Furuhata et al. 2008). OTU 5 and OTU 6 were identified to be close relatives of Ferruginibacter lapsinanis isolated from freshwater sediment (Lim et al. 2009). Ferruginibacter was reported to be one of the dominant genera in bio-denitrification sludge for high ammonia leachate treatment (Sun 2013). The other uncultured Bacteroidetes were speculated to be related with organic degradation, just as OTU 11 and OTU 12 (11 clones) that were found in aerated submerged membrane bioreactor (Du et al. 2008). Proteobacteria As for the Proteobacteria, a- (11 clones, 7 OTUs) and cProteobacteria (13 clones, 2 OTUs) were the dominant bacteria, the relatively lower portions were 6 clones of bProteobacteria (1 OTU). OTU 13, OTU 15 and OTU 18 were identified to be close relatives of Rhodobacter sp. OTU 18 was closely related to Rhodobacter vinaykumaraii that could grow with organic compounds as carbon sources and electron donors (Srinivas et al. 2007), moreover, genus Rhodobacter was
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OTU 19 was closely related with Rhizobium, which was able to reduce nitrate under anaerobic conditions (Daniel et al. 1982). It should be responsible to the denitrification performance in the ALR biofilm. That also supports the hypothesis that there would be anoxic/ anaerobic environments in the deeper layers of the biofilm in the gas–liquid–solid three-phase ALR with relatively high DO concentration. Six clones (OTU 20) affiliated with b-Proteobacteria had highest sequence similarities to Zoogloea oryzae, a nitrogen-fixing bacterium isolated from rice paddy soil (Xie and Yokota 2006). Zoogloea is known to have the ability to form cell aggregates embedded in gelatinous matrices and is commonly found in activated sludge (Fu et al. 2010), it may contribute to the form of biofilm in the ALR. OTU 21 and OTU 22 were identified to be close relatives of Plasticicumulans acidivorans, which can accumulate polyhydroxybutyrate as carbon source from organic substance in the wastewater (Jiang et al. 2011). Other bacterial divisions
Fig. 4 Scanning electron micrograph of biofilm developed in the ALR: a GAC carrier without attached biofilm (day 1), b GAC carrier with attached biofilm in the ALR (day 20), c GAC carrier with attached biofilm in the ALR (day 47)
also known to utilize organic compounds under anaerobic conditions for heterotrophic growth (Sun et al. 2011), which indicated that there may be anaerobic zone inside the biofilm.
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The residual 32 clones could be divided into 12 OTUs. The closest relatives in NCBI database belonged to uncultured bacteria retrieved from various environmental samples. OTU 25, which included ten clones, was identified to be close relatives of uncultured bacterium clone 29 (DQ413088.1), phylogenetic analysis showed that it had closest affinity with Rhodobacter sphaeroides (Lu et al. 2006). Moreover, the other clones clustered with various sequences from uncultured bacteria retrieved from wastewater treatment process with limited oxygen supply (OTU 23) (Sadaie et al. 2007), hybrid biofilm-activated sludge reactor for N and P removal (OTU 30) (Feng et al. 2013), upflow microaerobic sludge blanket reactor with reduced air supply (OTU 34) (Zheng et al. 2013), and so on, which indicated that there could be microorganism resource with abundant diversity to be developed. The community structure of the biomass depends on the operational conditions, including HRT, C/N, and so on. For the biofilm system, HRT has more obvious effect on biomass concentration, due to the relatively stable microbial community structure formed on the carriers (Muda et al. 2011, Xia et al. 2008). In this study, samples for the analysis of biomass were taken on day 47, 4 days after a new HRT (10 h). Although relatively stable microbial species for COD and N removal could be cultured for 16S rRNA gene clone library analysis, much longer adaption time should be selected for the microbial community analysis, when investigate how the structure of microbial communities depend on the operational conditions using more accurate tools of molecular biology.
World J Microbiol Biotechnol Table 3 Clone library analyses of the bacteria species within the biofilm OTU No.
Closest relatives in NCBI database
Similarity (%)
Accession number
1
Runella sp. P.slu-06
100
AB249681.1
2
Bacteroidetes
2
Uncultured Bacteroidetes clone VC5
100
AY211071.1
1
Bacteroidetes
3
Flavobacteriaceae TDMA-34
100
AB264128.1
2
Bacteroidetes
4
Uncultured Chitinophaga sp. clone 4.6h39
97
JN679126.1
3
Bacteroidetes
5
Uncultured Ferruginibacter sp. clone SB51
99
JQ723682.1
2
Bacteroidetes
6
Ferruginibacter lapsinanis strain HU1-HG42
100
NR044589.1
1
Bacteroidetes
7 8
Uncultured Sphingobacteriales bacterium clone SB25 Sphingobacteriaceae bacterium enrichment culture clone hao06
99 100
JQ723677.1 FJ386545.1
2 4
Bacteroidetes Bacteroidetes
9
Sphingobacteriales bacterium HU1-IH3
100
FJ177531.1
3
Bacteroidetes
10
Uncultured Bacteroidetes bacterium clone IRD18F11
100
AY947961.1
3
Bacteroidetes
11
Uncultured Bacteroidetes bacterium clone AS56
99
EU283377.1
4
Bacteroidetes
12
Uncultured Bacteroidetes bacterium clone AS138
99
EU283407.1
7
Bacteroidetes
13
Rhodobacter sp. AP-10
100
AB079681.1
1
a-Proteobacteria
14
Roseovarius sp. AMV6
100
FN376425.1
1
a-Proteobacteria
15
Rhodobacter sp. enrichment culture clone AOCRB-EC-1
100
GU557150.1
1
a-Proteobacteria
16
Uncultured Alphaproteobacteria clone QEDR2DH06
94
CU922491.1
4
a-Proteobacteria
17
Alpha proteobacterium HINUF007
100
AB470417.1
1
a-Proteobacteria
18
Rhodobacter vinaykumaraii strain JAJA249
100
AM600642.1
1
a-Proteobacteria
19
Rhizobium etli strain SEMIA 6409
100
FJ025117.1
2
a-Proteobacteria
20
Zoogloea oryzae
99
AB201044.1
6
b-Proteobacteria
21
Plasticicumulans acidivorans strain TUD-YJ37
100
NR117458.1
4
c-Proteobacteria
22 23
Plasticicumulans lactativorans strain YD Uncultured bacterium clone 0749
99 100
NR118276.1 AB286466.1
9 1
c-Proteobacteria Ungrouped
24
Uncultured bacterium clone SludgeA_bottom_13
97
AB516002.1
1
Ungrouped
25
Uncultured bacterium clone 29
99
DQ413088.1
10
Ungrouped
26
Uncultured bacterium clone 2C228563
91
EU800455.1
1
Ungrouped
27
Uncultured bacterium clone A194
100
FJ660602.1
4
ungrouped
28
Uncultured bacterium clone 1_2-E6
100
FN824940.1
2
Ungrouped
29
Uncultured bacterium clone DR301
99
JF429308.1
1
ungrouped
30
Uncultured bacterium clone ASSO-20
99
JN391631.1
4
Ungrouped
31
Uncultured bacterium clone Q7725-HYBO
89
JN391830.1
2
Ungrouped
32
Uncultured bacterium clone BSB0302-09
99
JN397772.1
1
Ungrouped
33
Uncultured bacterium clone a-124
96
JX040400.1
2
Ungrouped
34
Uncultured bacterium clone F11
99
JX272032.1
3
Ungrouped
Conclusions Results from the present study showed that the ALR displayed excellent performance for treating high ammonia wastewater with NH4?-N and COD removal efficiency of 98.6 and 89.0 %, and average effluent concentration of 4.5 and 73.0 mg L-1, respectively, satisfying the national primary discharging standard of China. It was demonstrated that the large scale gas–liquid–solid three-phase system is able to treat the high ammonia wastewater. The SEM images on the surface of the GAC indicated that high
Clones
Phylogenetic group
biomass concentration could be hold in the ALR due to the formation of biofilm. The 16S rRNA gene clone library analysis shows that phyla Bacteroidetes and Proteobacteria were the dominant bacteria. The presence of denitrifying bacteria Rhizobium and other anoxic/anaerobic species has indicated that there would be anoxic/anaerobic environments in the deeper layers of the biofilm, which may be responsible for the TN removal in the aerobic ALR. Acknowledgments The authors wish to acknowledge the financial support provided by the National Natural Science Foundation of
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World J Microbiol Biotechnol China (No. 21206106), the High School Science & Technology Fund Planning Project of Tianjin education committee, China (No. 20130515), and Research Fund of Tianjin Key Laboratory of Aquatic Science and Technology.
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Kim TH, Nghiem NP, Hicks KB (2009) Pretreatment and fractionation of corn stover by soaking in ethanol and aqueous ammonia. Appl Biochem Biotechnol 153:171–179 Lim JH, Baek SH, Lee ST (2009) Ferruginibacter alkalilentus gen. nov., sp nov and Ferruginibacter lapsinanis sp nov., novel members of the family ‘Chitinophagaceae’ in the phylum Bacteroidetes, isolated from freshwater sediment. Int J Syst Evol Micrbiol 59:2394–2399 Liu XL, Wen JP, Yuan Q, Zhao XM (2005) The pilot study for oil refinery wastewater treatment using a gas-liquid-solid three phase flow airlift loop bioreactor. Biochem Eng J 27:40–44 Lu S, Park M, Ro HS, Lee DS, Park W, Jeon CO (2006) Analysis of microbial communities using culture-dependent and cultureindependent approaches in an anaerobic/aerobic SBR reactor. J Microbiol 44:155–161 Luo LJ, Liu FN, Xu YY, Yuan JQ (2011) Hydrodynamics and mass transfer characteristics in an internal loop airlift reactor with different spargers. Chem Eng J 175:494–504 Luo LJ, Yuan JQ, Xie P, Sun JW, Guo W (2013) Hydrodynamics and mass transfer characteristics in an internal loop airlift reactor with sieve plates. Chem Eng Res Des 91:2377–2388 Muda K, Aris A, Salim RS, Ibrahim Z, van Loosdrecht MCM, Ahmad A, Nawahwi MZ (2011) The effect of hydraulic retention time on granular sludge biomass in treating textile wastewater. Water Res 45:4711–4721 Qiu CS, Jia XQ, Wen JP (2011) Purification of high strength wastewater originating from bioethanol production with simultaneous biogas production. World J Microb Biotech 27:2711–2722 Sadaie T, Sadaie A, Takada M, Hamano K, Ohnishi J, Ohta N, Matsumoto K, Sadaie Y (2007) Reducing sludge production and the domination of Comamonadaceae by reducing the oxygen supply in the wastewater treatment procedure of a foodprocessing factory. Biosci Biotechnol Biochem 71:791–799 Sanchez OJ, Cardona CA (2008) Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour Technol 99:5270–5295 Srinivas TNR, Kumar PA, Sasikala C, Ramana CV, Imhoff JF (2007) Rhodobacter vinaykumarii sp nov., a marine phototrophic alphaproteobacterium from tidal waters, and emended description of the genus Rhodobacter. Int J Syst Evol Micrbiol 57:1984–1987 Stackebrandt E, Goebel BM (1994) A place for DNA-DNA reassociation and 16S rRNA sequence-analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849 Sun FQ (2013) Nitrogen removal from landfill leachate by a combined ex situ and in situ biological processes. Ph.D Thesis, Zhejiang University, 81–84 Sun WJ, Sierra-Alvarez R, Field JA (2011) Long term performance of an arsenite-oxidizing-chlorate-reducing microbial consortium in an upflow anaerobic sludge bed (UASB) bioreactor. Bioresour Technol 102:5010–5016 Walters E, Hille A, He M, Ochmann C, Horn H (2009) Simultaneous nitrification/denitrification in a biofilm airlift suspension (BAS) reactor with biodegradable carrier material. Water Res 43:4461–4468 Wang X, Jia XQ, Wen JP (2011) Transient CFD modeling of toluene waste gas biodegradation in a gas–liquid–solid three-phase airlift loop reactor by immobilized Pseudomonas putida. Chem Eng J 172:735–745 Wen JP, Jia XQ, Pan L, Wang CL, Mao GZ (2005) Nitrifying treatment of wastewater from fertilizer production in a multiple airlift loop bioreactor. Biochem Eng J 25:33–37 Xia S, Li J, Wang R (2008) Nitrogen removal performance and microbial community structure dynamics response to carbon nitrogen ratio in a compact suspended carrier biofilm reactor. Ecol Eng 32:256–262
World J Microbiol Biotechnol Xie CH, Yokota A (2006) Zoogloea oryzae sp. nov., a nitrogen-fixing bacterium isolated from rice paddy soil, and reclassification of the strain ATCC 19623 as Crabtreella saccharophila gen. nov., sp. nov. Int J Syst Evol Microbiol 56:619–624
Zheng SK, Cui CC, Quan Y, Sun J (2013) Microaerobic DO-induced microbial mechanisms responsible for enormous energy saving in upflow microaerobic sludge blanket reactor. Bioresour Technol 140:192–198
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ORIGINAL PAPER
Purification of high ammonia wastewater in a biofilm airlift loop bioreactor with microbial communities analysis Chunsheng Qiu • Dandan Zhang • Liping Sun Jianping Wen
•
Received: 31 August 2014 / Accepted: 20 October 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A 70 m3 gas–liquid–solid three-phase flow airlift loop bioreactor, in which biofilm attached on granular active carbon carriers, was used for purification of the high ammonia wastewater from bioethanol production. Under the optimum operating conditions, COD and NH4?-N average removal rate of 89.0 and 98.6 % were obtained at hydraulic retention time of 10 h. Scanning electron microscopy was applied for observation of the biofilm formation. High contaminants removal efficiency was achieved by holding high biomass concentration in the reactor due to the attached biofilm over the carriers. The 16S rRNA gene clone library analysis indicated that 68.6 % of the clones were affiliated with the two phyla Bacteroidetes and Proteobacteria, and residual clones clustered with various sequences from uncultured bacteria. The presence of various anoxic/anaerobic bacteria indicated that the oxygen gradient inside the biofilm could provide appropriate micro-environment for nitrogen removal through simultaneous nitrification and denitrification. Keywords Airlift loop bioreactor Biofilm High ammonia wastewater Microbial community C. Qiu D. Zhang L. Sun School of Environmental and Municipal Engineering, Tianjin Chengjian University, Tianjin 300384, People’s Republic of China C. Qiu (&) D. Zhang L. Sun Tianjin Key Laboratory of Aqueous Science and Technology, Tianjin 300384, People’s Republic of China e-mail: [email protected] J. Wen Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China
Abbreviations ALR Airlift loop bioreactor BPS Baffle plate separator COD Chemical oxygen demand DO Dissolved oxygen GAC Granular active carbon HRT Hydraulic retention time ID Internal diameter NLR Nitrogen loading rate OTU Operational taxonomic unit SAA Soaking in aqueous ammonia SEM Scanning electron microscopy
Introduction To reduce the negative environmental impacts by the use of fossil fuels, many studies have been carried out on ethanol production from lignocellulosic materials, such as agricultural residues, forest products and dedicated energy crops (Sanchez and Cardona 2008). Corn stover and corncob are abundant agricultural residues in Northern China that could be used as feedstock for bioethanol. For efficient hydrolysis of the carbohydrate fractions in the raw materials by enzymes, a pretreatment process is necessary (ElMekawy et al. 2013). Soaking in aqueous ammonia (SAA) has been shown to be one of the most feasible methods for corn stover pretreatment (Isci et al. 2008; Kim et al. 2009). However, large amounts of pretreatment wastewater with high ammonia content are generated (Qiu et al. 2011), which need to be solved in order to make bioethanol production process more environmental-friendly. The airlift loop bioreactor (ALR), characterized by several advantages, such as better mixture, faster mass
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and oxygen transfer rate, higher operational flexibility, greater processing capability and so on (Wang et al. 2011), has been investigated to remove nutrients (N and P) in the wastewater (Liu et al. 2005; Wen et al. 2005; Dhamole et al. 2009; Walters et al. 2009). By using carriers with elevated specific surface area (Wen et al. 2005; Walters et al. 2009) or culturing granular sludge to hold high biomass concentration (Jin et al. 2008; Fu et al. 2011) or combining anaerobic, anoxic and aerobic conditions in a single reactor (Jiang et al. 2013), ALR is a valuable alternative for nitrogen and COD removal. Wen et al. (2005) presented a gas–liquid–solid three-phase flow multiple ALR to remove ammonia nitrogen from chemical fertilizer industrial wastewater, in which biofilm replaced the activated sludge. Walters et al. (2009) introduced a biofilm airlift suspension reactor with biodegradable carrier material to achieve simultaneous nitrification and denitrification. Such studies indicated that it is possible to obtain nitrification and denitrification in a biofilm reactor. Biofilm is especially useful for slow growing organisms like nitrifiers that have to be kept in a wastewater treatment process (Guo et al. 2005), and the form of biofilm is critical to the performance of the ALR. However, previous studies mainly focused on hydrodynamics and mass transfer characteristics of the ALR (Luo et al. 2011; Wang et al. 2011; Luo et al. 2013; Fakhari et al. 2014), information is still highly limited on microbial community structure of the biofilm formed in this reactor. Further investigation of the microbial communities in the biofilm is essential for improving reactor performance. 16S rRNA/DNA-based molecular methods with high degrees of precision and specificity have been widely used for environmental microbial studies (Stackebrandt and Goebel 1994; Bosshard et al. 2000; Fu et al. 2010). In this study, a pilot-scale biofilm ALR, with effective volume of 70 m3, was established and applied for carbon and nitrogen removal of the pretreatment wastewater produced from bioethanol production, with granular active carbon (GAC) as carriers. Furthermore, the community structures of biofilm microflora were analyzed using clone library analysis.
Materials and methods Wastewater The pretreatment wastewater from SAA process during bioethanol production, after primary settling, was used as influent in this study. Table 1 shows the fluctuating ranges of the compositions of the real wastewater.
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Table 1 Characters of the pretreatment wastewater and effluents Parameter
Pretreatment wastewater
pH
7.8 ± 0.32 -1
Effluent 7.5 ± 0.26
Discharge standards 6–9
COD (mg L )
655 ± 56
73.1 ± 5.8
BOD (mg L-1)
312 ± 43
20 ± 2.3
NH4?-N (mg L-1)
365 ± 22
4.5 ± 0.5
B15
TN (mg L-1)
395 ± 23
183 ± 16
–
SS (mg L-1)
122 ± 16
26 ± 2.6
B70
29 ± 5.5
B50
Color
–
B100 B30
Analytical methods Levels of COD, BOD, ammonia nitrogen (NH4?-N), total nitrogen (TN), suspended solids (SS), volatile suspended solids (VSS) and color were determined in accordance with APHA standard methods (APHA 1998). Dissolved oxygen (DO) was measured by a RSS-5100 DO meter (Leici Instrument Plant, Shanghai, China). Liquor pH was analyzed using PHS-2 acidometers (Leici Instrument Plant, Shanghai, China). All samples were measured in triplicate. Configuration of the ALR The ALR (70 m3) used in this study has a similar structure as it has been described in previous studies (Liu et al. 2005; Wen et al. 2005). As shown in Fig. 1, seven 9.5 m high draft tubes with 0.70 m internal diameter (ID) were fixed inside the main 10 m high reactor tube of 2.8 m in diameter. Seven jet nozzles of 6 mm ID were designed and located concentrically in the bottom of each draft tube. The bioreactor was ended by a disengaging cap with 4.2 m ID and a height of 2.5 m as the expanded section. The wastewater, stored in reservoir, was pumped into the bottom of the bioreactor at a certain flow rate. Air influx produced by the compressor was introduced into the draft tube through the jet nozzles for sufficient DO supply and mixing. Dissolved oxygen was measured by a DO meter fixed in the expanded section. The pH was maintained at 7.0–8.0 by adding NaHCO3 solution by a control system. The role of baffle plate separator (BPS) with an effective volume of 25 m3 was to separate the carriers as well as the activated sludge from the liquid in the effluent. After the sediment process, the liquid was analyzed and reused for washing and soaking lignocellulosic materials. The reactor and the separator were made of stainless steel lined with rubber. Granular activated carbon was used as carrier and its physical characteristics were offered by the supplier as follows: size range, 1.0–1.5 mm; surface area, 5,500 m2 m-3; bulk density, 1,100 kg m-3 (Guangfu Chemical Institute, Tianjin, China).
World J Microbiol Biotechnol Fig. 1 Schematic diagram of the ALR system
Operation of the ALR
Scanning electron microscopy (SEM)
The startup strategy of the ALR was based on our previous studies (Liu et al. 2005; Wen et al. 2005; Qiu et al. 2011). For the batch adaptation, 20 m3 activated sludge with MLSS of 2.2 g L-1 (20 % V/V), which was obtained from a local municipal wastewater treatment plant (Tianjin, China), was placed in the ALR. Then the reactor was filled with 25 % (v/v) aerobic influent diluted by tap water. Compressed air was introduced into the reactor at the flow rate of 230 m3 h-1 (amounts to the superficial gas velocity of 10 mm s-1), and the batch culturing was then carried out. The supernatant in the reactor after precipitation for 2 h was replaced every day by the mixed wastewater with increased concentration of 10–15 % every time. After 10 days’ preculture, the undiluted mixed wastewater was introduced into the bioreactor at hydraulic retention time (HRT) of 24 h, which meant the continuous culture had begun. Then 10 t of activated carbon was put into the reactor for film forming culture, and the air flow rate was increased to 280 m3 h-1 (amounts to the superficial gas velocity of 12 mm s-1). This process continued until the steady state biomass loading on the carriers was achieved.
Samples of biofilm-GAC particles were fixed in 5 % glutaraldehyde. Then the samples were dehydrated in an ethanol-deionized water series (50, 60, 70, 80 and 100 %v/v) buffered with 1 mL sodium phosphate (pH 7.4). A critical point dryer, using liquid carbon dioxide as the transitional fluid, was used to dry the samples. Samples were sputter coated with gold to a thickness of 20 nm and examined via SEM (Hitachi S-4800, Japan). Bacterial community structure analysis of the biofilm To confirm bacteria species within the biofilm, a general 16S rRNA gene sequence analysis was carried out in this study. Total DNA of the biofilm (day 47) was extracted with a Soil DNA Isolation kit (Soil gDNA kit, Biomiga, USA), and a clone library was constructed by PCR and the primers 27F and 1492R. The PCR conditions used for the general bacteria were as follows: 4 min of initial denaturation at 94 °C, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 90 s, with final extension 72 °C for 5 min. The amplifiedPCR products were purified with a TIANprep Midi
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constant with COD and NH4?-N removal rate of 82.4 and 94.4 % (average value of the last 3 days), respectively. Then the reactor was switched to continuous operating state. Performance of the ALR at various HRTs
Fig. 2 Process performance during batch operation for sludge adaptation
Purification Kit (TIANGEN, China) and cloned into apMD19-T vector (TaKaRa, Japan) by a TA cloning method, according to the manufacturer’s instruction. The recombinant plasmids were transformed into competent Escherichia coli (strain DH5a), and then colonies containing the plasmid were screened on LB-based agar plates with ampicillin, X-gal, and IPTG. Cells from randomly selected white colonies were assayed on a LB medium at 37 °C for 8 h. A commercial kit (TIANprep Mini Plasmid Kit, TIANGEN, China) was used to isolate the plasmid DNA from the screened colonies. Finally, PCR products were sent to a genetic analysis service company (BGI, China) for analyzing DNA sequences. The full-length sequences of the 16S rRNA were compared with similar sequences in National Center for Biotechnology Information data using the BLAST program. All clones having a sequence similarity of more than 97 % with each other were grouped into an operational taxonomic unit (OTU) consistent (Stackebrandt and Goebel 1994).
Results and discussion Performance of the ALR during sludge acclimation state In this study, after the activated sludge placed, the ALR was operated at batch mode for adaptation. The performance of the ALR at batch state is presented in Fig. 2. During the initial 3 days, the reactor the ALR exhibited poor performance, with an average NH4?-N removal rate of 39.6 % due to the low activity of nitrifying bacteria in the sludge. From the fourth day, gradually increase of NH4?-N removal rate was observed. Undiluted wastewater was pumped into the reactor on the eighth day, it could be seen in the Fig. 2 that removal of contaminants tend to be
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In order to determine the maximum treatment capacity of the ALR and form biofilm on the carriers, the loading rate was progressively increased by decreasing the operating HRT, with the relatively stable influent NH4?-N and COD. As indicated in Table 2 and Fig. 3, the continuous operation was carried out under various HRTs. It could be seen in Fig. 3 that COD and NH4?-N removal was unstable due to the combined effect of biodegradation and adsorption of GAC during the biofilm-forming period (1–10 days). After 10 days’ continuous operation, it could be observed that brown biofilm had gradually formed on the carriers and pollutants removal tended to stabilize gradually. Then the HRT of ALR was gradually decreased from 20 h to 10 h, with a concomitant increase in nitrogen loading rate (NLR) from 0.279 to 1.117 kgN m-3 d-1, attaining the NH4?-N removal rate higher than 98 %. However, when the HRT was further reduced to 8 and 6 h, obvious increase of COD (over 100 mg L-1) concentration in the effluent could be observed. So the HRT of 10 h was selected as the optimum value when taking the treatment capacity and efficiency into consideration, with NH4?-N and COD removal efficiencies of 98.6 and 89.0 % (Table 2), and average effluent concentration of 4.5 and 73.0 mg L-1, satisfying the national primary discharging standard of China (Table 1). Microbial attachment SEM images on the surface of the GAC from ALR are presented in Fig. 4, evidence of microbial colonization over GAC in the reactor was found. After the addition of GAC, the dispersed seed sludge tended to attached on the carriers to form biofilm due to the large surface area of the GAC (Fig. 4a). Forming of the biofilm occurred within 20 days (Fig. 4b), and removal efficiency of the reactor tended to be stable thereafter. Moreover, the biofilm structure tended to be denser on day 47 (Fig. 4c) due to the growth of attached microorganisms. As shown in Fig. 4, it was postulated that anoxic zones would form both in the deeper layers of the biofilm as well as in the porous structures of the carriers. Simultaneous nitrification and denitrification could be observed in the reactor with TN treatment efficiency of 53.7 % (Table 1). This phenomenon has been reported in the previous study (Fu et al. 2010).
World J Microbiol Biotechnol Table 2 Performance of ALR with different operating HRTs (average value)
Time (d)
HRT (h)
NH4?-N (mg L-1) Influent
Effluent
NH4?-N removal efficiency (%)
COD (mg L-1) Influent
Effluent
COD removal efficiency (%)
0–9
24
298.5
19.4
93.5
635.2
66.7
10–16
20
315.2
14.2
95.5
656.6
78.1
89.5 88.1
17–23
16
313.5
9.1
97.1
606.5
67.3
88.9
24–29
12
308.8
5.1
98.3
660.4
75.9
88.5
30–34
10
326.4
4.8
98.5
665.1
71.8
89.2
35–39
8
302.1
5.1
98.2
645.3
96.1
85.1
40–42
6
319.5
4.9
98.4
630.5
129.2
79.5
43–48
10
335.5
4.5
98.6
664.5
73.0
89.0
which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Bacteroidetes
Fig. 3 Process performance during continuous operation of the ALR
The attached biomass concentrations in terms of VSS were 21.3 g L-1 on day 20 and 31.6 g L-1 on day 47, which were superior to other immobilized-biomass process and even a process using granular sludge (Jin et al. 2008). For this reason, high NLR was achieved by holding slow growing nitrifiers in the rector. Composition of bacterial communities Total DNA was extracted from the mixed culture attached on the carrier (day 47). Clone libraries of full-length 16S rDNA were constructed with the primer set (27F and 1492R). A total of 100 clones were isolated, after excision of 4 chimeric sequences, these clones were classified into 34 OTUs as shown in Table 3. The results indicated that the bacterial community of the biofilm was highly diverse. All 34 OTUs were affiliated with sequences from two major known lineages of the domain bacteria, namely Bacteroidetes (12 OTUs, 36 clones), and Proteobacteria (10 OTUs, 30 clones, including a-, b- and c-subdivisions), as well as some ungrouped species (12 OTUs, 30 clones),
The largest fractions were class Sphingobacteria (6 OTUs, 15 clones), which accounted for 41.7 % of the total clones belonged to the phylum Bacteroidetes. The portions of class Flavobacteriia, Cytophagia and unclassified group were 5.6, 5.6 and 41.7 %, respectively. The residual clones (15 clones of 4 OTUs) clustered with various sequences from uncultured bacteria retrieved from aerated submerged membrane bioreactor treating domestic wastewater (Du et al. 2008), rivers, freshwater sediment, and so on. OTU 1 was identified as Runella sp. P.slu-06, which was isolated from environmental water samples and showed the ability to form biofilm and degrade carbohydrates (Furuhata et al. 2008). OTU 5 and OTU 6 were identified to be close relatives of Ferruginibacter lapsinanis isolated from freshwater sediment (Lim et al. 2009). Ferruginibacter was reported to be one of the dominant genera in bio-denitrification sludge for high ammonia leachate treatment (Sun 2013). The other uncultured Bacteroidetes were speculated to be related with organic degradation, just as OTU 11 and OTU 12 (11 clones) that were found in aerated submerged membrane bioreactor (Du et al. 2008). Proteobacteria As for the Proteobacteria, a- (11 clones, 7 OTUs) and cProteobacteria (13 clones, 2 OTUs) were the dominant bacteria, the relatively lower portions were 6 clones of bProteobacteria (1 OTU). OTU 13, OTU 15 and OTU 18 were identified to be close relatives of Rhodobacter sp. OTU 18 was closely related to Rhodobacter vinaykumaraii that could grow with organic compounds as carbon sources and electron donors (Srinivas et al. 2007), moreover, genus Rhodobacter was
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OTU 19 was closely related with Rhizobium, which was able to reduce nitrate under anaerobic conditions (Daniel et al. 1982). It should be responsible to the denitrification performance in the ALR biofilm. That also supports the hypothesis that there would be anoxic/ anaerobic environments in the deeper layers of the biofilm in the gas–liquid–solid three-phase ALR with relatively high DO concentration. Six clones (OTU 20) affiliated with b-Proteobacteria had highest sequence similarities to Zoogloea oryzae, a nitrogen-fixing bacterium isolated from rice paddy soil (Xie and Yokota 2006). Zoogloea is known to have the ability to form cell aggregates embedded in gelatinous matrices and is commonly found in activated sludge (Fu et al. 2010), it may contribute to the form of biofilm in the ALR. OTU 21 and OTU 22 were identified to be close relatives of Plasticicumulans acidivorans, which can accumulate polyhydroxybutyrate as carbon source from organic substance in the wastewater (Jiang et al. 2011). Other bacterial divisions
Fig. 4 Scanning electron micrograph of biofilm developed in the ALR: a GAC carrier without attached biofilm (day 1), b GAC carrier with attached biofilm in the ALR (day 20), c GAC carrier with attached biofilm in the ALR (day 47)
also known to utilize organic compounds under anaerobic conditions for heterotrophic growth (Sun et al. 2011), which indicated that there may be anaerobic zone inside the biofilm.
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The residual 32 clones could be divided into 12 OTUs. The closest relatives in NCBI database belonged to uncultured bacteria retrieved from various environmental samples. OTU 25, which included ten clones, was identified to be close relatives of uncultured bacterium clone 29 (DQ413088.1), phylogenetic analysis showed that it had closest affinity with Rhodobacter sphaeroides (Lu et al. 2006). Moreover, the other clones clustered with various sequences from uncultured bacteria retrieved from wastewater treatment process with limited oxygen supply (OTU 23) (Sadaie et al. 2007), hybrid biofilm-activated sludge reactor for N and P removal (OTU 30) (Feng et al. 2013), upflow microaerobic sludge blanket reactor with reduced air supply (OTU 34) (Zheng et al. 2013), and so on, which indicated that there could be microorganism resource with abundant diversity to be developed. The community structure of the biomass depends on the operational conditions, including HRT, C/N, and so on. For the biofilm system, HRT has more obvious effect on biomass concentration, due to the relatively stable microbial community structure formed on the carriers (Muda et al. 2011, Xia et al. 2008). In this study, samples for the analysis of biomass were taken on day 47, 4 days after a new HRT (10 h). Although relatively stable microbial species for COD and N removal could be cultured for 16S rRNA gene clone library analysis, much longer adaption time should be selected for the microbial community analysis, when investigate how the structure of microbial communities depend on the operational conditions using more accurate tools of molecular biology.
World J Microbiol Biotechnol Table 3 Clone library analyses of the bacteria species within the biofilm OTU No.
Closest relatives in NCBI database
Similarity (%)
Accession number
1
Runella sp. P.slu-06
100
AB249681.1
2
Bacteroidetes
2
Uncultured Bacteroidetes clone VC5
100
AY211071.1
1
Bacteroidetes
3
Flavobacteriaceae TDMA-34
100
AB264128.1
2
Bacteroidetes
4
Uncultured Chitinophaga sp. clone 4.6h39
97
JN679126.1
3
Bacteroidetes
5
Uncultured Ferruginibacter sp. clone SB51
99
JQ723682.1
2
Bacteroidetes
6
Ferruginibacter lapsinanis strain HU1-HG42
100
NR044589.1
1
Bacteroidetes
7 8
Uncultured Sphingobacteriales bacterium clone SB25 Sphingobacteriaceae bacterium enrichment culture clone hao06
99 100
JQ723677.1 FJ386545.1
2 4
Bacteroidetes Bacteroidetes
9
Sphingobacteriales bacterium HU1-IH3
100
FJ177531.1
3
Bacteroidetes
10
Uncultured Bacteroidetes bacterium clone IRD18F11
100
AY947961.1
3
Bacteroidetes
11
Uncultured Bacteroidetes bacterium clone AS56
99
EU283377.1
4
Bacteroidetes
12
Uncultured Bacteroidetes bacterium clone AS138
99
EU283407.1
7
Bacteroidetes
13
Rhodobacter sp. AP-10
100
AB079681.1
1
a-Proteobacteria
14
Roseovarius sp. AMV6
100
FN376425.1
1
a-Proteobacteria
15
Rhodobacter sp. enrichment culture clone AOCRB-EC-1
100
GU557150.1
1
a-Proteobacteria
16
Uncultured Alphaproteobacteria clone QEDR2DH06
94
CU922491.1
4
a-Proteobacteria
17
Alpha proteobacterium HINUF007
100
AB470417.1
1
a-Proteobacteria
18
Rhodobacter vinaykumaraii strain JAJA249
100
AM600642.1
1
a-Proteobacteria
19
Rhizobium etli strain SEMIA 6409
100
FJ025117.1
2
a-Proteobacteria
20
Zoogloea oryzae
99
AB201044.1
6
b-Proteobacteria
21
Plasticicumulans acidivorans strain TUD-YJ37
100
NR117458.1
4
c-Proteobacteria
22 23
Plasticicumulans lactativorans strain YD Uncultured bacterium clone 0749
99 100
NR118276.1 AB286466.1
9 1
c-Proteobacteria Ungrouped
24
Uncultured bacterium clone SludgeA_bottom_13
97
AB516002.1
1
Ungrouped
25
Uncultured bacterium clone 29
99
DQ413088.1
10
Ungrouped
26
Uncultured bacterium clone 2C228563
91
EU800455.1
1
Ungrouped
27
Uncultured bacterium clone A194
100
FJ660602.1
4
ungrouped
28
Uncultured bacterium clone 1_2-E6
100
FN824940.1
2
Ungrouped
29
Uncultured bacterium clone DR301
99
JF429308.1
1
ungrouped
30
Uncultured bacterium clone ASSO-20
99
JN391631.1
4
Ungrouped
31
Uncultured bacterium clone Q7725-HYBO
89
JN391830.1
2
Ungrouped
32
Uncultured bacterium clone BSB0302-09
99
JN397772.1
1
Ungrouped
33
Uncultured bacterium clone a-124
96
JX040400.1
2
Ungrouped
34
Uncultured bacterium clone F11
99
JX272032.1
3
Ungrouped
Conclusions Results from the present study showed that the ALR displayed excellent performance for treating high ammonia wastewater with NH4?-N and COD removal efficiency of 98.6 and 89.0 %, and average effluent concentration of 4.5 and 73.0 mg L-1, respectively, satisfying the national primary discharging standard of China. It was demonstrated that the large scale gas–liquid–solid three-phase system is able to treat the high ammonia wastewater. The SEM images on the surface of the GAC indicated that high
Clones
Phylogenetic group
biomass concentration could be hold in the ALR due to the formation of biofilm. The 16S rRNA gene clone library analysis shows that phyla Bacteroidetes and Proteobacteria were the dominant bacteria. The presence of denitrifying bacteria Rhizobium and other anoxic/anaerobic species has indicated that there would be anoxic/anaerobic environments in the deeper layers of the biofilm, which may be responsible for the TN removal in the aerobic ALR. Acknowledgments The authors wish to acknowledge the financial support provided by the National Natural Science Foundation of
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World J Microbiol Biotechnol China (No. 21206106), the High School Science & Technology Fund Planning Project of Tianjin education committee, China (No. 20130515), and Research Fund of Tianjin Key Laboratory of Aquatic Science and Technology.
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