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C3 fatty acid in upflow anaerobic sludge blanket reactors
Bioresource Technology 193 (2015) 408–414
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Nitrate removal by organotrophic anaerobic ammonium oxidizing bacteria with C2/C3 fatty acid in upflow anaerobic sludge blanket reactors Yuhai Liang a, Dong Li a,⇑, Xiaojing Zhang b, Huiping Zeng a, Yin Yang a, Jie Zhang a,c a b c
Key Laboratory of Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China Henan Collaborative Innovation Center of Environmental Pollution Control and Ecological Restoration, Zhengzhou University of Light Industry, Zhengzhou 450001, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
h i g h l i g h t s Organotrophic AAOB which could reduce nitrate was successfully cultivated. Denitrifiers could not outcompete with organotrophic AAOB in low TOC/N condition. Enriching organotrophic AAOB was profitable for decreasing effluent TN. The dominant species of organotrophic AAOB in UASB was Candidatus Jettenia.
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Article history: Received 1 June 2015 Received in revised form 26 June 2015 Accepted 27 June 2015 Available online 2 July 2015 Keywords: Organotrophic Anammox Granular sludge Nitrate removal High-throughput pyrosequencing
a b s t r a c t In anaerobic ammonium oxidation (Anammox) process, a harsh ratio of nitrite to ammonia in influent was demanded, and the max nitrogen removal efficiency could only achieve to 89%, both of which limited the development of Anammox. The aim of this work was to study the nitrate removal by organotrophic anaerobic ammonium oxidizing bacteria (AAOB) with C2/C3 fatty acid in upflow anaerobic sludge blanket (UASB) reactors. In this study, organotrophic AAOB was successfully enriched by adding acetate and propionate with the total organic carbon to nitrogen (TOC/N) ratio of 0.1. In the condition of low substrate, the TN removal efficiency reached 90%, with the effluent TN of around 11.8 mg L1. After the addition of acetate and propionate, the predominant species in Anammox granular sludge transformed to Candidatus Jettenia that belonging to organotrophic AAOB from the Candidatus Kuenenia relating to general AAOB. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, anaerobic ammonium oxidation (Anammox) process becomes to the research point with the development of energy autarkic wastewater treatment plant (Khiewwijit et al., 2015; Wett et al., 2013; Xu et al., 2015). In anaerobic condition, anaerobic ammonia-oxidizing bacteria (AAOB) could convert ammonia and nitrite to nitrogen gas, with a small production of nitrate, as shown in Eq. (1) (Strous et al., 1998). There are two disadvantages of Anammox process, one was the strict demand of the ratio of nitrite to ammonia in influent, and another was the nitrate production resulting in the TN removal efficiency less than 89%. Both of the two problems hindered the application of Anammox process. ⇑ Corresponding author. Tel.: +86 01067392099 4. E-mail address: [email protected] (D. Li). http://dx.doi.org/10.1016/j.biortech.2015.06.133 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Anammox bacteria
NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:26NO3 þ 0:066CH2 O0:5 N0:15 þ 2:03H2 O
ð1Þ
To resolve these problems, simultaneous Anammox and denitrification process was developed (Du et al., 2014; Kumar and Lin, 2010; Zhong and Jia, 2012). However, although this process could reduce nitrate and then elevate the TN removal efficiency, the introduction of denitrifiers would compete with anaerobic ammonia-oxidizing bacteria (AAOB), the complicated microbial communities brought difficulties for the operation. Thus, a new resolving strategy was needed. AAOB was regarded as an absolute anaerobic bacteria which only consumes ammonia and nitrite without organics. Whereas, the recent study showed that AAOB could also reduce nitrate with the consumption of volatile fatty acid (VFA). Guven et al. (2005) presented that acetate and propionate could enhance the Anammox reaction rate, and Kartal et al. (2007b, 2008) suggested that AAOB had the ability for
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simultaneously reduce acetate, propionate and nitrate. Kartal et al. (2007a) found that the organotrophic AAOB would firstly reduce nitrate to nitrite, and then to ammonia, finally the ammonia and nitrite were converted to N2 via the general Anammox pathway. AAOB consumed ammonia and nitrite via the Anammox pathway when VFA was not available, while AAOB also utilize the VFA to reduce nitrate to nitrite or ammonia besides of the general Anammox reaction with the presence of VFA. Simultaneous nitrate reduction and Anammox could be realized by one single bacteria, which was named as organotrophic AAOB. This not only relieve the strict effluent demand of partial nitrification, but also elevate the effluent quality of Anammox and avoid the competition between denitrifiers and AAOB. In conclusion, enriching organotrophic AAOB in the Anammox process would be a better choice for the development of this process. Few reports were focused on the process operation of organotrophic AAOB nowadays. Winkler (Winkler et al., 2012a,b) adopted organotrophic AAOB into SBR and MBBR reactor with completely autotrophic nitrogen removal over nitrite process, and achieved to 38% and 68% less nitrate production, respectively. Huang (Huang et al., 2014) investigated the effect of C2/C3 VFA on the microbial communities of Anammox granular sludge in batch experiments. All the above studies were carried out in the condition of high substrate concentration, no report was related to the low substrate, and the microbial characteristics still needs to be further analyzed. In this study, Anammox granular sludge was operated in two identical upflow anaerobic sludge blanket (UASB) reactors. Then acetate and propionate were added to the reactors with a total organic carbon to nitrogen (TOC/N) ratio of 0.1, to investigate the enrichment of organotrophic AAOB which could reduce nitrate in the condition of low substrate. High-throughput pyrosequencing was used to analyze the microbial variations in the Anammox granular sludge after the addition of VFA. The results would promote the application of Anammox in engineering practice.
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Youke, China). TOC was detected by TOC analyzer (vario TOC cube, Elementar, Germany). MLSS was determined by weight method. 2.3. Sampling and DNA extraction In this study, the initial granular sludge (named R0) and the granular sludge taken out from the two reactors (named R1 and R2) on day 80 were used to analyze the microbial characteristic. The three samples were stored in 50 mL sterile plastic test tubes at 20 °C. Finally DNA was extracted and purified using a bacterial genomic mini extraction kit (Sangon, China) each sludge sample. The purified DNA was detected by 0.8% (w/V) agarose gel electrophoresis.
2.4. High-throughput pyrosequencing and phylogenetic assignment The DNA was qualified by Qubit2.0 DNA detection kit (Sangon, China). The qualified DNA was high-throughput pyrosequencing sequenced by Sangon Company (Sangon, China). The PCR primers were V3–V4 universe primers 341F/805R (341F: CCTACGGGNGGCWGCAG; 805R: GACTACHVGGGTATCTAATCC) (Herlemann et al., 2011; Hugerth et al., 2014). The high-throughput pyrosequencing used Miseq sequencing platform (Illumina, Inc., San Diego, CA, USA). More than 10,000 sequences with 410 bp length was obtained from each sample. Operational taxonomic units (OTUs) were defined at 3% variation in sequences by uclust (uclust v1.1.579), and OTU was regarded as relating to genus. Shannon diversity index was analyzed by mothur (http://www.mothur.org/), and the sequence was compared with the reference microorganisms available in Silva (http://www.arb-silva.de).
3. Results and discussion 3.1. Reactor performance
2. Methods 2.1. Experimental setup Two identical UASB (R1 and R2) were adopted in this study, with the inner diameter and effective volume of 100 mm and 7.8 L, respectively. The initial granular sludge was taken from an Anammox UASB reactor, which was operated for more than one year. The initial mixed liquid suspended solids (MLSS) was 4.5 g L1. The reaction temperature was between 22 and 24 °C without control. In phase I (day 1–11), the influent was simulated as the effluent of partial nitrification treating domestic sewage, as follows (g L1): (NH4)2SO4 (NH+4-N: 50 mg L1), NaNO2 ammonia 1 (NO ), CaCl2 (180 mg L1), NaHCO3 (alkalinity as 2 -N: 69 mg L CaCO3: 500 mg L1) and the trace element solution I and II (1 mL L1). The contents of trace element solution I and II were as follows (g L1): EDTA: 15, H3BO4: 0.014 in solution I and EDTA: 15, H3BO4: 0.014, MnCl24H2O: 0.99, NaMoO42H2O: 0.22, NiCl26H2O: 0.19, ZnSO47H2O: 0.43, NaSeO410H2O: 0.21, CuSO45H2O: 0.25 in solution II (deGraaf et al., 1996). In phase II (after day 11), sodium acetate (40 mg L1) was added to the influent of R1 and sodium propionate (30 mg L1) was added to that of R2, with a TOC/N ratio of 0.1. 2.2. Analytical methods According to Standard Methods (APHA, 1998), the nitrogen concentrations in influent and effluent were daily monitored using colorimetric methods by UV/VIS spectrophotometers (UV755b,
The nitrogen removal performance of R1 with the addition of acetate was depicted in Fig. 1. The general Anammox reaction occurred in R1 during day 1–11, in which the average ammonia nitrogen removal efficiency, nitrite removal efficiency and TN removal efficiency were 94.3%, 93.9% and 76.2%, respectively, with the effluent TN of 31.1 mg L1. Acetate was added to the influent with a TOC/N ratio of 0.1 from day 12. Then the nitrate production immediately decreased, while the nitrogen removal ability became worse. This result perhaps was due to that the acetate accelerated the reaction rate of Anammox (Ali et al., 2014; Dapena-Mora et al., 2007; Guven et al., 2005), which led to the massive gas production and then went up with the Anammox granules. As a result, it outstripped the ability of gas–liquid–sludge separator and sludge was lost. During day 35–57, the inflow rate was decreased, and HRT was prolonged from 1.1 to 3.0 h, to resolve this problem. The MLSS was decreased from 4.5 to 2.9 g L1. During day 58–86, the average ammonia removal efficiency, nitrite removal efficiency and TN removal efficiency were 95.7%, 97.3% and 90%, respectively, with the effluent TN of 11.8 mg L1. The effluent TN concentration was 2.64 times less than that of the effluent in phase I without acetate addition, indicating the profitable effect of the acetate on nitrogen removal of Anammox process. Moreover, this result suggested that the Anammox granular system be stably operated in the low substrate condition. The nitrogen removal performance of R2 with the addition of propionate was depicted in Fig. 2. Same as R1, the general Anammox occurred in R2 during day 1–11, in which the average ammonia removal efficiency, nitrite removal efficiency and TN removal efficiency were 85.6%, 86.6% and 72.3%, respectively, with
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the effluent TN of 35.0 mg L1. Propionate was added to R2 from day 12 with the TOC/N of 0.1. Since the sludge was also significantly lost in R2 with a gradual decreasing of MLSS, inflow rate was decreased to elevate the effluent quality. The MLSS was decreased from 4.5 to 2.0 g L1. The nitrogen removal rose up with the increase of HRT. The HRT was prolonged from 1.1 to 2.7 h. After adding propionate to R2, the average ammonia removal efficiency, nitrite removal efficiency and TN removal efficiency changed to 84.6%, 84.8% and 75.5%, respectively, with the effluent TN of 29.4 mg L1. Even the nitrogen removal ability fluctuated, the TN removal efficiency increased with the decrease of effluent TN. This result suggested that the gas–liquid–sludge separator could not effectively retain all the sludge, and then limited the enhancement of nitrogen removal. However, the addition of propionate was still profitable for nitrogen removal. 3.2. Nitrate removal by organotrophic Anammox bacteria According to Eq. (1), the ratio of nitrate production to ammonia and nitrite removal was 1:1.32:0.26 in Anammox reaction. In phase I of R1 without acetate addition, the ratio in R1 was 1:1.314:0.255, which was very close to the theoretical ratio, indicating the occurrence of general Anammox. The ratio changed to 1:1.346:0.135 after adding the acetate to influent, the nitrite removal was much more than that of general Anammox reaction, while the nitrate production was far less than that of general Anammox. This was because the organotrophic AAOB could reduce nitrate and nitrite to ammonia with the consumption of acetate (Kartal et al., 2007a). In phase I, the average nitrate production was 12.3 mg L1, which was close to the theoretical production. And during day 58–86 with acetate addition, the average nitrate
production was 4.2 mg L1, far less than the theoretical production of 11.6 mg L1. In denitrification process, 3.7 g chemical oxygen demand (COD) would be consumed when 1 g nitrate was reduced (Ahn and Choi, 2006; Wang et al., 2010), so 27.4 mg L1 (7.4 * 3.7 = 27.4) COD would be consumed since 7.4 g (11.6–4.2 = 7.4) nitrate was reduced in other pathway. The TOC/N ratio was 0.1 (equal as 32.0 mg L1 COD, COD/N = 0.27), and the TOC removal efficiency was around 50%. 16.0 mg L1 (32.0 * 50% = 16.0) COD was consumed in R1, indicating the shortage for denitrification. So organotrophic AAOB existed in R1, which reduced the nitrate with COD consumption. For R2, the ratio of nitrate production to ammonia and nitrite removal in phase I was 1:1.305:0.248, closing to the theoretical ratio. And the ratio changed to 1:1.311:0.173 in phase II with the addition of propionate. The average nitrate production in phase I was 11.0 mg L1, while that changed to 7.4 mg L1 in phase II, less than the theoretical production of 10.0 mg L1. As previously reported (Winkler et al., 2012b), the addition of sodium propionate with a COD/N ratio of 0.5 would neither suppress the Anammox bioactivity nor introduce the denitrifying bacteria, and organotrophic AAOB could outcompete with other organisms in this condition. The ratio of TN removal to nitrate production (4TN/4NO 3 -N) was calculated to analyze the effect of addition of acetate and propionate on the nitrogen removal pathway of Anammox granular sludge, as shown in Fig. 3. The ratio of R1 and R2 were both around the theoretical ratio (7.84) without addition of acetate and propionate, which changed to be much higher than the theoretical ratio. The ratio of R1 in phase with addition of acetate was higher than that of R2 with addition of propionate, indicating the less nitrate production of R1 and suggesting the better nitrogen removal ability.
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were 1174, 1710 and 1230 OTUs, respectively, as shown in Fig. 4. This result suggested that the OTUs and the biodiversity of the two reactors both increased with the addition of organics. Additionally, 483 OTUs simultaneously existed in R0 and R1, while 413 existed in R0 and R2, 336 OTUs existed in all the three samples.
Fig. 3. The 4TN/4NO 3 value of R1 and R2.
3.3. High-throughput pyrosequencing and microbial community The Venn of seed sludge (R0), granular sludge with addition of acetate (R1) and granular sludge with addition of propionate (R2) were shown in Fig. 4. And 13,196, 14,904 and 13,959 effective sequences were obtained from the above three sludge samples, respectively. OTUs for community analysis were defined at 3% variation in nucleic acid sequences, and the OTUs of the three samples
Fig. 4. Venn diagram of R1 and R2.
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Table 1 Taxonomic identification of the sequences of anammox granular sludge. R0
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38.80 15.64 15.10 8.76 7.26 6.55 4.44 2.22 0.60 0.50
Unclassified Proteobacteria Planctomycetes Bacteroidetes Chloroflexi Chlorobi Acidobacteria Firmicutes Verrucomicrobia Gemmatimonadetes
29.56 28.33 11.68 10.87 7.35 4.34 3.94 1.65 1.54 0.62
Proteobacteria Unclassified Bacteroidetes Planctomycetes Chloroflexi Chlorobi Acidobacteria Firmicutes Verrucomicrobia Gemmatimonadetes
38.02 25.83 8.63 6.86 6.30 5.55 5.08 1.76 1.01 0.82
Fig. 5. The taxonomic identities of sequences retrieved from R1 and R2 at the Genus level.
The population succession was obvious, while the dominant OTUs were retained. 606 OTUs both existed in R1 and R2, indicating the similar effect of acetate and propionate on the population succession of Anammox granular sludge. The Shannon index of R0, R1 and R2 were 4.923, 5.369 and 5.037, respectively, indicating the biodiversity increasing of both the two reactors with the addition of VFA. The higher biodiversity of R1 than that of R2 perhaps was due to the shorter carbon chain of acetate more easily consumed, or the preferable sludge retaining ability of R1 that resulting in the better nitrogen removal ability. Blasted with Silva database, the first ten microbial phylums of R0, R1 and R2 were same, as shown in Table 1, including Proteobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, Chlorobi, Acidobacteria, Firmicutes, Verrucomicrobia, Verrucomicrobia, Gemmatimonadetes and unclassified bacteria. The quantity of Proteobacteria was the most, unclassified bacteria was the second, and the Planctomycetes was the third in R0, while those of R1 were unclassified bacteria,
Proteobacteria, and Planctomycetes, respectively. And those of R2 were Proteobacteria, unclassified bacteria and Bacteroidetes, respectively, the Planctomycetes decreased to the fourth one. In conclusion, the share of unclassified bacteria increased with the addition of VFA while that of Planctomycetes decreased. This result perhaps was due to that the AAOB were still uncultured, some kinds of AAOB were not collected in the database. Compared R1 with R2, the decrease extent of Planctomycetes in R2 was higher than R1, correspondingly with worse nitrogen removal ability of R2. The result indicated that the addition of acetate was more profitable for enriching organotrophic AAOB. The genus result was shown in Fig. 5, uncultured_Rhodocyclaceae were detected in all the three samples, which had the ability of denitrifying (Ishii et al., 2009). The share of uncultured_Rhodocyclaceae in R0, R1 and R2 were 12.19%, 6.72% and 14.29%, respectively. This result proved the presence of endogenous denitrification in the reactors. In R1, the quantity of uncultured_Rhodocyclaceae
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decreased due to the competition of organotrophic AAOB in R1. And for R2, since the sludge was significantly lost, the organotrophic AAOB with longer growth cycle than uncultured_Rhodocyclaceae showed a worse competition. The quantity of uncultured_ Rhodocyclaceae was similar to that of seed sludge, indicating that endogenous denitrification had not been enhanced with the addition of organics. AAOB was the functional organism in Anammox process. Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia that relating to AAOB were detected in R0, R1 and R2. 1709 sequences related to AAOB were detected in R0, including 1350 Candidatus Kuenenia, 355 Candidatus Brocadia and 4 Candidatus Jettenia. The share of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia in R0 were 10.23%, 2.69% and 0.03%, respectively. 1166 sequences related to AAOB were detected in R1, including 198, 288 and 680 of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia, respectively. The share of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia in R1 were 1.33%, 1.93% and 4.56%, respectively. And 720 relating sequences were detected in R2, the sequences belonging to the three species were 164, 20 and 536, respectively. The share of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia in R2 were 1.17%, 0.19% and 3.84%, respectively. These results suggested that Candidatus Kuenenia was the dominant species in the Anammox granular sludge without VFA addition. This was because the Candidatus Kuenenia survived in K-strategy (Pynaert et al., 2003; van der Star et al., 2008), which preferred to the low substrate concentration. In contrary, Candidatus Brocadia survived in r-strategy (van der Star et al., 2007, 2008), which was suitable for the high substrate concentration. With the addition of VFA, Candidatus Jettenia increased, indicating the profitable effect of both acetate and propionate on the survival of Candidatus Jettenia, which then became to the dominant AAOB in R1 and R2. This result was consistent with the previous study by Huang (Huang et al., 2014). The quantity of Candidatus Kuenenia in R1 decreased significantly, while that of Candidatus Brocadia decreased less, this was because Candidatus Brocadia had stronger ability for degrade acetate (Kartal et al., 2008). Candidatus Kuenenia and Candidatus Brocadia both decreased significantly, and Candidatus Brocadia presented a small amount in R2, indicating the adverse effect of propionate on the survival of Candidatus Brocadia. No Candidatus Anammoxoglobus propionicus was detected in R2, which was suggested having the ability to degrade propionate (Kartal et al., 2007b), while Candidatus Jettenia that reported by Huang was detected. The reason perhaps was due to the study by Kartal was carried out in the semi-cultured environment, while the present study and study by Huang were both carried out in actual lab-scale reactor. The different condition led to the different dominant species. Combining the nitrate removal and microbial results, it could be concluded that organotrophic AAOB with the ability of degrading nitrate could be enriched by adding acetate and propionate. Candidatus Jettenia was the dominant organotrophic AAOB in UASB.
3.4. Profitable effect of organotrophic AAOB in wastewater treatment The effluent quality could be improved by organotrophic AAOB, since it could degrade nitrate to decrease the effluent TN, and then resolve the problem in Anammox of the limited TN removal efficiency (89%). Moreover, the introduction of organotrophic AAOB could slow down the strict ratio of ammonia to nitrite (1:1.32) in influent, which significantly reduce the operational difficulty of partial nitrification. In addition, VFA could be produced by the sludge from the sewage plant (Jie et al., 2014), which could be used to enrich the organotrophic AAOB. The introduction of organotrophic AAOB was not only profitable for reusing resource, but
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also useful for the application of Anammox process treating sewage in mainstream with low ammonia and high effluent demand. 4. Conclusions Organotrophic AAOB could be enriched by adding acetate or propionate with the TOC/N ratio of 0.1 in the condition of low substrate and room temperature. The low TOC/N ratio would not lead to the excess growth of denitrifying bacteria. Part of the nitrate was reduced by organotrophic AAOB, making for the decrease of effluent TN and improvement of effluent quality. After the addition of acetate and propionate, the dominant species of Anammox bacteria transformed to Candidatus Jettenia relating to organotrophic AAOB from Candidatus Kuenenia relating to general AAOB in the UASB with low substrate concentration. Acknowledgement This work was supported by water project of National Science and Technology Major Project (Grant No. 2012ZX07202-005). References Ahn, Y.-H., Choi, H.-C., 2006. Autotrophic nitrogen removal from sludge digester liquids in upflow sludge bed reactor with external aeration. Process Biochem. 41 (9), 1945–1950. Ali, M., Oshiki, M., Awata, T., Isobe, K., Kimura, Z., Yoshikawa, H., Hira, D., Kindaichi, T., Satoh, H., Fujii, T., Okabe, S., 2014. Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’. Environ. Microbiol. 79 (13), 4145–4148. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC, USA. Dapena-Mora, A., Fernández, I., Campos, J.L., Mosquera-Corral, A., Méndez, R., Jetten, M.S.M., 2007. Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production. Enzyme Microb. Technol. 40 (4), 859–865. deGraaf, A., deBruijn, P., Robertson, L., Jetten, M., Kuenen, J., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142 (8), 2187–2196. Du, R., Peng, Y., Cao, S., Wu, C., Weng, D., Wang, S., He, J., 2014. Advanced nitrogen removal with simultaneous Anammox and denitrification in sequencing batch reactor. Bioresour. Technol. 162, 316–322. Guven, D., Dapena, A., Kartal, B., Schmid, M.C., Maas, B., van de Pas-Schoonen, K., Sozen, S., Mendez, R., Op den Camp, H.J.M., Jetten, M.S.M., Strous, M., Schmidt, I., 2005. Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria. Appl. Environ. Microbiol. 71 (2), 1066–1071. Herlemann, D.P., Labrenz, M., Jurgens, K., Bertilsson, S., Waniek, J.J., Andersson, A.F., 2011. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME J. 5 (10), 1571–1579. Huang, X.-L., Gao, D.-W., Tao, Y., Wang, X.-L., 2014. C2/C3 fatty acid stress on anammox consortia dominated by Candidatus Jettenia asiatica. Chem. Eng. J. 253, 402–407. Hugerth, L., Wefer, H., Lundin, S., Jakobsson, H., Lindberg, M., Rodin, S., Engstrand, L., Andersson, A., 2014. DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies. Appl. Environ. Microbiol. 80 (16), 5116–5123. Ishii, S., Yamamoto, M., Kikuchi, M., Oshima, K., Hattori, M., Otsuka, S., Senoo, K., 2009. Microbial populations responsive to denitrification-inducing conditions in rice paddy soil, as revealed by comparative 16S rRNA gene analysis. Appl. Environ. Microbiol. 75 (22), 7070–7078. Jie, W., Peng, Y., Ren, N., Li, B., 2014. Volatile fatty acids (VFAs) accumulation and microbial community structure of excess sludge (ES) at different pHs. Bioresour. Technol. 152, 124–129. Kartal, B., Kuypers, M.M., Lavik, G., Schalk, J., Op den Camp, H.J., Jetten, M.S., Strous, M., 2007a. Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ. Microbiol. 9 (3), 635–642. Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb, R.I., Schouten, S., Fuerst, J.A., Damsté, J.S., Jetten, M.S.M., Strous, M., 2007b. Candidatus ‘‘Anammoxoglobus propionicus’’ a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30 (1), 39–49. Kartal, B., Van Niftrik, L., Rattray, J., Van De Vossenberg, J.L.C.M., Schmid, M.C., Damste, J.S.S., Jetten, M.S.M., Strous, M., 2008. Candidatus ‘Brocadia fulgida’: an autofluorescent anaerobic ammonium oxidizing bacterium. FEMS Microbiol. Ecol. 63 (1), 46–55. Khiewwijit, R., Temmink, H., Rijnaarts, H., Keesman, K.J., 2015. Energy and nutrient recovery for municipal wastewater treatment: how to design a feasible plant layout? Environ. Modell. Softw. 68, 156–165.
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Kumar, M., Lin, J.-G., 2010. Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal—strategies and issues. J. Hazard. Mater. 178 (1–3), 1–9. Pynaert, K., Smets, B.F., Wyffels, S., Beheydt, D., Siciliano, S.D., Verstraete, W., 2003. Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor. Appl. Environ. Microbiol. 69 (6), 3626–3635. Strous, M., Heijnen, J., Kuenen, J., Jetten, M., 1998. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50 (5), 589–596. van der Star, W.R., Abma, W.R., Blommers, D., Mulder, J.W., Tokutomi, T., Strous, M., Picioreanu, C., van Loosdrecht, M.C., 2007. Startup of reactors for anoxic ammonium oxidation: experiences from the first full-scale anammox reactor in Rotterdam. Water Res. 41 (18), 4149–4163. van der Star, W.R., Miclea, A.I., van Dongen, U.G., Muyzer, G., Picioreanu, C., van Loosdrecht, M.C., 2008. The membrane bioreactor: a novel tool to grow anammox bacteria as free cells. Biotechnol. Bioeng. 101 (2), 286–294. Wang, C.C., Lee, P.H., Kumar, M., Huang, Y.T., Sung, S., Lin, J.G., 2010. Simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD)
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Nitrate removal by organotrophic anaerobic ammonium oxidizing bacteria with C2/C3 fatty acid in upflow anaerobic sludge blanket reactors Yuhai Liang a, Dong Li a,⇑, Xiaojing Zhang b, Huiping Zeng a, Yin Yang a, Jie Zhang a,c a b c
Key Laboratory of Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China Henan Collaborative Innovation Center of Environmental Pollution Control and Ecological Restoration, Zhengzhou University of Light Industry, Zhengzhou 450001, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
h i g h l i g h t s Organotrophic AAOB which could reduce nitrate was successfully cultivated. Denitrifiers could not outcompete with organotrophic AAOB in low TOC/N condition. Enriching organotrophic AAOB was profitable for decreasing effluent TN. The dominant species of organotrophic AAOB in UASB was Candidatus Jettenia.
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Article history: Received 1 June 2015 Received in revised form 26 June 2015 Accepted 27 June 2015 Available online 2 July 2015 Keywords: Organotrophic Anammox Granular sludge Nitrate removal High-throughput pyrosequencing
a b s t r a c t In anaerobic ammonium oxidation (Anammox) process, a harsh ratio of nitrite to ammonia in influent was demanded, and the max nitrogen removal efficiency could only achieve to 89%, both of which limited the development of Anammox. The aim of this work was to study the nitrate removal by organotrophic anaerobic ammonium oxidizing bacteria (AAOB) with C2/C3 fatty acid in upflow anaerobic sludge blanket (UASB) reactors. In this study, organotrophic AAOB was successfully enriched by adding acetate and propionate with the total organic carbon to nitrogen (TOC/N) ratio of 0.1. In the condition of low substrate, the TN removal efficiency reached 90%, with the effluent TN of around 11.8 mg L1. After the addition of acetate and propionate, the predominant species in Anammox granular sludge transformed to Candidatus Jettenia that belonging to organotrophic AAOB from the Candidatus Kuenenia relating to general AAOB. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, anaerobic ammonium oxidation (Anammox) process becomes to the research point with the development of energy autarkic wastewater treatment plant (Khiewwijit et al., 2015; Wett et al., 2013; Xu et al., 2015). In anaerobic condition, anaerobic ammonia-oxidizing bacteria (AAOB) could convert ammonia and nitrite to nitrogen gas, with a small production of nitrate, as shown in Eq. (1) (Strous et al., 1998). There are two disadvantages of Anammox process, one was the strict demand of the ratio of nitrite to ammonia in influent, and another was the nitrate production resulting in the TN removal efficiency less than 89%. Both of the two problems hindered the application of Anammox process. ⇑ Corresponding author. Tel.: +86 01067392099 4. E-mail address: [email protected] (D. Li). http://dx.doi.org/10.1016/j.biortech.2015.06.133 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Anammox bacteria
NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:26NO3 þ 0:066CH2 O0:5 N0:15 þ 2:03H2 O
ð1Þ
To resolve these problems, simultaneous Anammox and denitrification process was developed (Du et al., 2014; Kumar and Lin, 2010; Zhong and Jia, 2012). However, although this process could reduce nitrate and then elevate the TN removal efficiency, the introduction of denitrifiers would compete with anaerobic ammonia-oxidizing bacteria (AAOB), the complicated microbial communities brought difficulties for the operation. Thus, a new resolving strategy was needed. AAOB was regarded as an absolute anaerobic bacteria which only consumes ammonia and nitrite without organics. Whereas, the recent study showed that AAOB could also reduce nitrate with the consumption of volatile fatty acid (VFA). Guven et al. (2005) presented that acetate and propionate could enhance the Anammox reaction rate, and Kartal et al. (2007b, 2008) suggested that AAOB had the ability for
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simultaneously reduce acetate, propionate and nitrate. Kartal et al. (2007a) found that the organotrophic AAOB would firstly reduce nitrate to nitrite, and then to ammonia, finally the ammonia and nitrite were converted to N2 via the general Anammox pathway. AAOB consumed ammonia and nitrite via the Anammox pathway when VFA was not available, while AAOB also utilize the VFA to reduce nitrate to nitrite or ammonia besides of the general Anammox reaction with the presence of VFA. Simultaneous nitrate reduction and Anammox could be realized by one single bacteria, which was named as organotrophic AAOB. This not only relieve the strict effluent demand of partial nitrification, but also elevate the effluent quality of Anammox and avoid the competition between denitrifiers and AAOB. In conclusion, enriching organotrophic AAOB in the Anammox process would be a better choice for the development of this process. Few reports were focused on the process operation of organotrophic AAOB nowadays. Winkler (Winkler et al., 2012a,b) adopted organotrophic AAOB into SBR and MBBR reactor with completely autotrophic nitrogen removal over nitrite process, and achieved to 38% and 68% less nitrate production, respectively. Huang (Huang et al., 2014) investigated the effect of C2/C3 VFA on the microbial communities of Anammox granular sludge in batch experiments. All the above studies were carried out in the condition of high substrate concentration, no report was related to the low substrate, and the microbial characteristics still needs to be further analyzed. In this study, Anammox granular sludge was operated in two identical upflow anaerobic sludge blanket (UASB) reactors. Then acetate and propionate were added to the reactors with a total organic carbon to nitrogen (TOC/N) ratio of 0.1, to investigate the enrichment of organotrophic AAOB which could reduce nitrate in the condition of low substrate. High-throughput pyrosequencing was used to analyze the microbial variations in the Anammox granular sludge after the addition of VFA. The results would promote the application of Anammox in engineering practice.
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Youke, China). TOC was detected by TOC analyzer (vario TOC cube, Elementar, Germany). MLSS was determined by weight method. 2.3. Sampling and DNA extraction In this study, the initial granular sludge (named R0) and the granular sludge taken out from the two reactors (named R1 and R2) on day 80 were used to analyze the microbial characteristic. The three samples were stored in 50 mL sterile plastic test tubes at 20 °C. Finally DNA was extracted and purified using a bacterial genomic mini extraction kit (Sangon, China) each sludge sample. The purified DNA was detected by 0.8% (w/V) agarose gel electrophoresis.
2.4. High-throughput pyrosequencing and phylogenetic assignment The DNA was qualified by Qubit2.0 DNA detection kit (Sangon, China). The qualified DNA was high-throughput pyrosequencing sequenced by Sangon Company (Sangon, China). The PCR primers were V3–V4 universe primers 341F/805R (341F: CCTACGGGNGGCWGCAG; 805R: GACTACHVGGGTATCTAATCC) (Herlemann et al., 2011; Hugerth et al., 2014). The high-throughput pyrosequencing used Miseq sequencing platform (Illumina, Inc., San Diego, CA, USA). More than 10,000 sequences with 410 bp length was obtained from each sample. Operational taxonomic units (OTUs) were defined at 3% variation in sequences by uclust (uclust v1.1.579), and OTU was regarded as relating to genus. Shannon diversity index was analyzed by mothur (http://www.mothur.org/), and the sequence was compared with the reference microorganisms available in Silva (http://www.arb-silva.de).
3. Results and discussion 3.1. Reactor performance
2. Methods 2.1. Experimental setup Two identical UASB (R1 and R2) were adopted in this study, with the inner diameter and effective volume of 100 mm and 7.8 L, respectively. The initial granular sludge was taken from an Anammox UASB reactor, which was operated for more than one year. The initial mixed liquid suspended solids (MLSS) was 4.5 g L1. The reaction temperature was between 22 and 24 °C without control. In phase I (day 1–11), the influent was simulated as the effluent of partial nitrification treating domestic sewage, as follows (g L1): (NH4)2SO4 (NH+4-N: 50 mg L1), NaNO2 ammonia 1 (NO ), CaCl2 (180 mg L1), NaHCO3 (alkalinity as 2 -N: 69 mg L CaCO3: 500 mg L1) and the trace element solution I and II (1 mL L1). The contents of trace element solution I and II were as follows (g L1): EDTA: 15, H3BO4: 0.014 in solution I and EDTA: 15, H3BO4: 0.014, MnCl24H2O: 0.99, NaMoO42H2O: 0.22, NiCl26H2O: 0.19, ZnSO47H2O: 0.43, NaSeO410H2O: 0.21, CuSO45H2O: 0.25 in solution II (deGraaf et al., 1996). In phase II (after day 11), sodium acetate (40 mg L1) was added to the influent of R1 and sodium propionate (30 mg L1) was added to that of R2, with a TOC/N ratio of 0.1. 2.2. Analytical methods According to Standard Methods (APHA, 1998), the nitrogen concentrations in influent and effluent were daily monitored using colorimetric methods by UV/VIS spectrophotometers (UV755b,
The nitrogen removal performance of R1 with the addition of acetate was depicted in Fig. 1. The general Anammox reaction occurred in R1 during day 1–11, in which the average ammonia nitrogen removal efficiency, nitrite removal efficiency and TN removal efficiency were 94.3%, 93.9% and 76.2%, respectively, with the effluent TN of 31.1 mg L1. Acetate was added to the influent with a TOC/N ratio of 0.1 from day 12. Then the nitrate production immediately decreased, while the nitrogen removal ability became worse. This result perhaps was due to that the acetate accelerated the reaction rate of Anammox (Ali et al., 2014; Dapena-Mora et al., 2007; Guven et al., 2005), which led to the massive gas production and then went up with the Anammox granules. As a result, it outstripped the ability of gas–liquid–sludge separator and sludge was lost. During day 35–57, the inflow rate was decreased, and HRT was prolonged from 1.1 to 3.0 h, to resolve this problem. The MLSS was decreased from 4.5 to 2.9 g L1. During day 58–86, the average ammonia removal efficiency, nitrite removal efficiency and TN removal efficiency were 95.7%, 97.3% and 90%, respectively, with the effluent TN of 11.8 mg L1. The effluent TN concentration was 2.64 times less than that of the effluent in phase I without acetate addition, indicating the profitable effect of the acetate on nitrogen removal of Anammox process. Moreover, this result suggested that the Anammox granular system be stably operated in the low substrate condition. The nitrogen removal performance of R2 with the addition of propionate was depicted in Fig. 2. Same as R1, the general Anammox occurred in R2 during day 1–11, in which the average ammonia removal efficiency, nitrite removal efficiency and TN removal efficiency were 85.6%, 86.6% and 72.3%, respectively, with
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the effluent TN of 35.0 mg L1. Propionate was added to R2 from day 12 with the TOC/N of 0.1. Since the sludge was also significantly lost in R2 with a gradual decreasing of MLSS, inflow rate was decreased to elevate the effluent quality. The MLSS was decreased from 4.5 to 2.0 g L1. The nitrogen removal rose up with the increase of HRT. The HRT was prolonged from 1.1 to 2.7 h. After adding propionate to R2, the average ammonia removal efficiency, nitrite removal efficiency and TN removal efficiency changed to 84.6%, 84.8% and 75.5%, respectively, with the effluent TN of 29.4 mg L1. Even the nitrogen removal ability fluctuated, the TN removal efficiency increased with the decrease of effluent TN. This result suggested that the gas–liquid–sludge separator could not effectively retain all the sludge, and then limited the enhancement of nitrogen removal. However, the addition of propionate was still profitable for nitrogen removal. 3.2. Nitrate removal by organotrophic Anammox bacteria According to Eq. (1), the ratio of nitrate production to ammonia and nitrite removal was 1:1.32:0.26 in Anammox reaction. In phase I of R1 without acetate addition, the ratio in R1 was 1:1.314:0.255, which was very close to the theoretical ratio, indicating the occurrence of general Anammox. The ratio changed to 1:1.346:0.135 after adding the acetate to influent, the nitrite removal was much more than that of general Anammox reaction, while the nitrate production was far less than that of general Anammox. This was because the organotrophic AAOB could reduce nitrate and nitrite to ammonia with the consumption of acetate (Kartal et al., 2007a). In phase I, the average nitrate production was 12.3 mg L1, which was close to the theoretical production. And during day 58–86 with acetate addition, the average nitrate
production was 4.2 mg L1, far less than the theoretical production of 11.6 mg L1. In denitrification process, 3.7 g chemical oxygen demand (COD) would be consumed when 1 g nitrate was reduced (Ahn and Choi, 2006; Wang et al., 2010), so 27.4 mg L1 (7.4 * 3.7 = 27.4) COD would be consumed since 7.4 g (11.6–4.2 = 7.4) nitrate was reduced in other pathway. The TOC/N ratio was 0.1 (equal as 32.0 mg L1 COD, COD/N = 0.27), and the TOC removal efficiency was around 50%. 16.0 mg L1 (32.0 * 50% = 16.0) COD was consumed in R1, indicating the shortage for denitrification. So organotrophic AAOB existed in R1, which reduced the nitrate with COD consumption. For R2, the ratio of nitrate production to ammonia and nitrite removal in phase I was 1:1.305:0.248, closing to the theoretical ratio. And the ratio changed to 1:1.311:0.173 in phase II with the addition of propionate. The average nitrate production in phase I was 11.0 mg L1, while that changed to 7.4 mg L1 in phase II, less than the theoretical production of 10.0 mg L1. As previously reported (Winkler et al., 2012b), the addition of sodium propionate with a COD/N ratio of 0.5 would neither suppress the Anammox bioactivity nor introduce the denitrifying bacteria, and organotrophic AAOB could outcompete with other organisms in this condition. The ratio of TN removal to nitrate production (4TN/4NO 3 -N) was calculated to analyze the effect of addition of acetate and propionate on the nitrogen removal pathway of Anammox granular sludge, as shown in Fig. 3. The ratio of R1 and R2 were both around the theoretical ratio (7.84) without addition of acetate and propionate, which changed to be much higher than the theoretical ratio. The ratio of R1 in phase with addition of acetate was higher than that of R2 with addition of propionate, indicating the less nitrate production of R1 and suggesting the better nitrogen removal ability.
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were 1174, 1710 and 1230 OTUs, respectively, as shown in Fig. 4. This result suggested that the OTUs and the biodiversity of the two reactors both increased with the addition of organics. Additionally, 483 OTUs simultaneously existed in R0 and R1, while 413 existed in R0 and R2, 336 OTUs existed in all the three samples.
Fig. 3. The 4TN/4NO 3 value of R1 and R2.
3.3. High-throughput pyrosequencing and microbial community The Venn of seed sludge (R0), granular sludge with addition of acetate (R1) and granular sludge with addition of propionate (R2) were shown in Fig. 4. And 13,196, 14,904 and 13,959 effective sequences were obtained from the above three sludge samples, respectively. OTUs for community analysis were defined at 3% variation in nucleic acid sequences, and the OTUs of the three samples
Fig. 4. Venn diagram of R1 and R2.
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Table 1 Taxonomic identification of the sequences of anammox granular sludge. R0
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Unclassified Proteobacteria Planctomycetes Bacteroidetes Chloroflexi Chlorobi Acidobacteria Firmicutes Verrucomicrobia Gemmatimonadetes
29.56 28.33 11.68 10.87 7.35 4.34 3.94 1.65 1.54 0.62
Proteobacteria Unclassified Bacteroidetes Planctomycetes Chloroflexi Chlorobi Acidobacteria Firmicutes Verrucomicrobia Gemmatimonadetes
38.02 25.83 8.63 6.86 6.30 5.55 5.08 1.76 1.01 0.82
Fig. 5. The taxonomic identities of sequences retrieved from R1 and R2 at the Genus level.
The population succession was obvious, while the dominant OTUs were retained. 606 OTUs both existed in R1 and R2, indicating the similar effect of acetate and propionate on the population succession of Anammox granular sludge. The Shannon index of R0, R1 and R2 were 4.923, 5.369 and 5.037, respectively, indicating the biodiversity increasing of both the two reactors with the addition of VFA. The higher biodiversity of R1 than that of R2 perhaps was due to the shorter carbon chain of acetate more easily consumed, or the preferable sludge retaining ability of R1 that resulting in the better nitrogen removal ability. Blasted with Silva database, the first ten microbial phylums of R0, R1 and R2 were same, as shown in Table 1, including Proteobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, Chlorobi, Acidobacteria, Firmicutes, Verrucomicrobia, Verrucomicrobia, Gemmatimonadetes and unclassified bacteria. The quantity of Proteobacteria was the most, unclassified bacteria was the second, and the Planctomycetes was the third in R0, while those of R1 were unclassified bacteria,
Proteobacteria, and Planctomycetes, respectively. And those of R2 were Proteobacteria, unclassified bacteria and Bacteroidetes, respectively, the Planctomycetes decreased to the fourth one. In conclusion, the share of unclassified bacteria increased with the addition of VFA while that of Planctomycetes decreased. This result perhaps was due to that the AAOB were still uncultured, some kinds of AAOB were not collected in the database. Compared R1 with R2, the decrease extent of Planctomycetes in R2 was higher than R1, correspondingly with worse nitrogen removal ability of R2. The result indicated that the addition of acetate was more profitable for enriching organotrophic AAOB. The genus result was shown in Fig. 5, uncultured_Rhodocyclaceae were detected in all the three samples, which had the ability of denitrifying (Ishii et al., 2009). The share of uncultured_Rhodocyclaceae in R0, R1 and R2 were 12.19%, 6.72% and 14.29%, respectively. This result proved the presence of endogenous denitrification in the reactors. In R1, the quantity of uncultured_Rhodocyclaceae
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decreased due to the competition of organotrophic AAOB in R1. And for R2, since the sludge was significantly lost, the organotrophic AAOB with longer growth cycle than uncultured_Rhodocyclaceae showed a worse competition. The quantity of uncultured_ Rhodocyclaceae was similar to that of seed sludge, indicating that endogenous denitrification had not been enhanced with the addition of organics. AAOB was the functional organism in Anammox process. Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia that relating to AAOB were detected in R0, R1 and R2. 1709 sequences related to AAOB were detected in R0, including 1350 Candidatus Kuenenia, 355 Candidatus Brocadia and 4 Candidatus Jettenia. The share of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia in R0 were 10.23%, 2.69% and 0.03%, respectively. 1166 sequences related to AAOB were detected in R1, including 198, 288 and 680 of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia, respectively. The share of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia in R1 were 1.33%, 1.93% and 4.56%, respectively. And 720 relating sequences were detected in R2, the sequences belonging to the three species were 164, 20 and 536, respectively. The share of Candidatus Kuenenia, Candidatus Brocadia and Candidatus Jettenia in R2 were 1.17%, 0.19% and 3.84%, respectively. These results suggested that Candidatus Kuenenia was the dominant species in the Anammox granular sludge without VFA addition. This was because the Candidatus Kuenenia survived in K-strategy (Pynaert et al., 2003; van der Star et al., 2008), which preferred to the low substrate concentration. In contrary, Candidatus Brocadia survived in r-strategy (van der Star et al., 2007, 2008), which was suitable for the high substrate concentration. With the addition of VFA, Candidatus Jettenia increased, indicating the profitable effect of both acetate and propionate on the survival of Candidatus Jettenia, which then became to the dominant AAOB in R1 and R2. This result was consistent with the previous study by Huang (Huang et al., 2014). The quantity of Candidatus Kuenenia in R1 decreased significantly, while that of Candidatus Brocadia decreased less, this was because Candidatus Brocadia had stronger ability for degrade acetate (Kartal et al., 2008). Candidatus Kuenenia and Candidatus Brocadia both decreased significantly, and Candidatus Brocadia presented a small amount in R2, indicating the adverse effect of propionate on the survival of Candidatus Brocadia. No Candidatus Anammoxoglobus propionicus was detected in R2, which was suggested having the ability to degrade propionate (Kartal et al., 2007b), while Candidatus Jettenia that reported by Huang was detected. The reason perhaps was due to the study by Kartal was carried out in the semi-cultured environment, while the present study and study by Huang were both carried out in actual lab-scale reactor. The different condition led to the different dominant species. Combining the nitrate removal and microbial results, it could be concluded that organotrophic AAOB with the ability of degrading nitrate could be enriched by adding acetate and propionate. Candidatus Jettenia was the dominant organotrophic AAOB in UASB.
3.4. Profitable effect of organotrophic AAOB in wastewater treatment The effluent quality could be improved by organotrophic AAOB, since it could degrade nitrate to decrease the effluent TN, and then resolve the problem in Anammox of the limited TN removal efficiency (89%). Moreover, the introduction of organotrophic AAOB could slow down the strict ratio of ammonia to nitrite (1:1.32) in influent, which significantly reduce the operational difficulty of partial nitrification. In addition, VFA could be produced by the sludge from the sewage plant (Jie et al., 2014), which could be used to enrich the organotrophic AAOB. The introduction of organotrophic AAOB was not only profitable for reusing resource, but
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also useful for the application of Anammox process treating sewage in mainstream with low ammonia and high effluent demand. 4. Conclusions Organotrophic AAOB could be enriched by adding acetate or propionate with the TOC/N ratio of 0.1 in the condition of low substrate and room temperature. The low TOC/N ratio would not lead to the excess growth of denitrifying bacteria. Part of the nitrate was reduced by organotrophic AAOB, making for the decrease of effluent TN and improvement of effluent quality. After the addition of acetate and propionate, the dominant species of Anammox bacteria transformed to Candidatus Jettenia relating to organotrophic AAOB from Candidatus Kuenenia relating to general AAOB in the UASB with low substrate concentration. Acknowledgement This work was supported by water project of National Science and Technology Major Project (Grant No. 2012ZX07202-005). References Ahn, Y.-H., Choi, H.-C., 2006. Autotrophic nitrogen removal from sludge digester liquids in upflow sludge bed reactor with external aeration. Process Biochem. 41 (9), 1945–1950. Ali, M., Oshiki, M., Awata, T., Isobe, K., Kimura, Z., Yoshikawa, H., Hira, D., Kindaichi, T., Satoh, H., Fujii, T., Okabe, S., 2014. Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’. Environ. Microbiol. 79 (13), 4145–4148. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC, USA. Dapena-Mora, A., Fernández, I., Campos, J.L., Mosquera-Corral, A., Méndez, R., Jetten, M.S.M., 2007. Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production. Enzyme Microb. Technol. 40 (4), 859–865. deGraaf, A., deBruijn, P., Robertson, L., Jetten, M., Kuenen, J., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142 (8), 2187–2196. Du, R., Peng, Y., Cao, S., Wu, C., Weng, D., Wang, S., He, J., 2014. Advanced nitrogen removal with simultaneous Anammox and denitrification in sequencing batch reactor. Bioresour. Technol. 162, 316–322. Guven, D., Dapena, A., Kartal, B., Schmid, M.C., Maas, B., van de Pas-Schoonen, K., Sozen, S., Mendez, R., Op den Camp, H.J.M., Jetten, M.S.M., Strous, M., Schmidt, I., 2005. Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria. Appl. Environ. Microbiol. 71 (2), 1066–1071. Herlemann, D.P., Labrenz, M., Jurgens, K., Bertilsson, S., Waniek, J.J., Andersson, A.F., 2011. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME J. 5 (10), 1571–1579. Huang, X.-L., Gao, D.-W., Tao, Y., Wang, X.-L., 2014. C2/C3 fatty acid stress on anammox consortia dominated by Candidatus Jettenia asiatica. Chem. Eng. J. 253, 402–407. Hugerth, L., Wefer, H., Lundin, S., Jakobsson, H., Lindberg, M., Rodin, S., Engstrand, L., Andersson, A., 2014. DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies. Appl. Environ. Microbiol. 80 (16), 5116–5123. Ishii, S., Yamamoto, M., Kikuchi, M., Oshima, K., Hattori, M., Otsuka, S., Senoo, K., 2009. Microbial populations responsive to denitrification-inducing conditions in rice paddy soil, as revealed by comparative 16S rRNA gene analysis. Appl. Environ. Microbiol. 75 (22), 7070–7078. Jie, W., Peng, Y., Ren, N., Li, B., 2014. Volatile fatty acids (VFAs) accumulation and microbial community structure of excess sludge (ES) at different pHs. Bioresour. Technol. 152, 124–129. Kartal, B., Kuypers, M.M., Lavik, G., Schalk, J., Op den Camp, H.J., Jetten, M.S., Strous, M., 2007a. Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ. Microbiol. 9 (3), 635–642. Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb, R.I., Schouten, S., Fuerst, J.A., Damsté, J.S., Jetten, M.S.M., Strous, M., 2007b. Candidatus ‘‘Anammoxoglobus propionicus’’ a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30 (1), 39–49. Kartal, B., Van Niftrik, L., Rattray, J., Van De Vossenberg, J.L.C.M., Schmid, M.C., Damste, J.S.S., Jetten, M.S.M., Strous, M., 2008. Candidatus ‘Brocadia fulgida’: an autofluorescent anaerobic ammonium oxidizing bacterium. FEMS Microbiol. Ecol. 63 (1), 46–55. Khiewwijit, R., Temmink, H., Rijnaarts, H., Keesman, K.J., 2015. Energy and nutrient recovery for municipal wastewater treatment: how to design a feasible plant layout? Environ. Modell. Softw. 68, 156–165.
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