Using partial nitrification and anammox to remove nitrogen from low

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Using partial nitrification and anammox to remove nitrogen from low-strength wastewater by coimmobilizing biofilm inside a moving bed bioreactor Rong Chen, Yasuyuki Takemura, Yuan Liu, Jiayuan Ji, Satoshi Sakuma, Kengo Kubota, Haiyuan Ma, and Yu-You Li ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05055 • Publication Date (Web): 09 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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ACS Sustainable Chemistry & Engineering

Using partial nitrification and anammox to remove nitrogen from low-strength wastewater by co-immobilizing biofilm inside a moving bed bioreactor

Rong Chen †,‡, Yasuyuki Takemura ‡, Yuan Liu ‡, Jiayuan Ji ‡, Satoshi Sakuma ‡, Kengo Kubota ‡, Haiyuan Ma ‡, Yu-You Li ‡,*

†International S&T Cooperation Center for Urban Alternative Water Resources Development, Key Lab of Environmental Engineering, Shaanxi Province, Xi'an University of Architecture and Technology, No.13 Yanta Road, Xi'an 710055, PR China ‡Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan

*Corresponding author: Yu-You Li, [email protected] Telephone: +81 22 795 7464 Fax: +81 22 795 7465

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ABSTRACT The stable operation of partial nitrification and anammox (PNA) process is a challenge in nitrogen removal from extremely low-strength ammonia wastewater like sewage mainstream. A moving bed reactor with functional carriers (30% filling rate) was developed to treat a synthetic influent with 50 mg/L ammonia. The long-term operation results showed nitrogen removal efficiencies of 71.7±9.1% have been stably obtained under a relatively short hydraulic retention time (HRT) of 2h. Microbial analysis revealed anammox bacteria and ammonium oxidizing bacteria (AOB) with 29.7% and 6.32% abundance were the two most dominant bacteria in the reactor. Carriers largely retained slow-growing anammox bacteria in their hollow space and established a sandwich-like biofilm structure of co-immobilization of anammox bacteria and AOB. The anammox activity was much higher in carrier biofilms than in suspended flocs while for the AOB activity, the situation was reversed. Correspondingly, a fluorescent in situ hybridization analysis illustrated the active cell fractions of anammox bacteria and AOB in carrier biofilms were 63.7% and 4.8%, and 2.7% and 61.4% in suspended flocs. Biofilm formation and dissolved oxygen control were deemed to be the two key factors affecting the optimal co-immobilization of anammox bacteria and AOB, which guaranteed the efficient PNA. Keywords: Anammox; AOB; low-strength wastewater; co-immobilization; biofilm

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Introduction Anaerobic ammonium oxidation (Anammox), a chemolithotropic process, is a completely autotrophic oxidation of ammonium with nitrite as an electron acceptor which therefore does not require organic electron donors 1,2. Recently Anammox has been recognized as a promising nitrogen removal technology for wastewater treatment 3,4

. Partial nitrification and anammox (PNA), an integrated process in which

approximately 57% of the influent ammonia is firstly oxidized to nitrite (shown in Eq. 1) by ammonia oxidizing bacteria (AOB), and then the remaining ammonia and the produced nitrite are converted to N2 (shown in Eq. 2) by anammox bacteria, opened up a new way for biological nitrogen removal for ammonium-containing wastewater. The stoichiometric relationship of PNA process can be expressed as Eq. 3 5. That is, the PNA relies on two microbial consortia: AOB and anammox bacteria. 1.3𝑁𝐻4+ + 1.95𝑂2 → 1.3𝑁𝑂2− + 2.6𝐻 + + 1.3𝐻2 𝑂

(1)

𝑁𝐻4+ + 1.3𝑁𝑂2− → 0.26𝑁𝑂3− + 1.02𝑁2 + 2𝐻2 𝑂

(2)

𝑁𝐻4+ + 0.85𝑂2 → 0.11𝑁𝑂3− + 0.445𝑁2 + 1.13𝐻 + + 1.43𝐻2 𝑂

(3)

Currently, PNA has been applied at full scale to the treatment of high-strength ammonium wastewater (the concentration is larger than 100 mg/L) involving sewage sludge reject water, landfill leachate and industrial wastewaters 6–8. Many researchers have reported the application of PNA process for the treatment of warm and concentrated wastewaters characterized by temperatures exceeding 25 oC and influent nitrogen concentrations over 100 mg/L is nowadays part of the state of the art 4,7. Conceivably, an implementation of PNA in the sewage mainstream could multiply the 3



benefits because sewage is the most abundant type of wastewater 9, hence, research focus has now moved to possible applications to treating sewage mainstream characterized by low-strength ammonium content (20-60 mg NH4+-N/L) 4,10–12. Two recently published works have succeeded in lab-scale PNA in treating low-strength wastewater at low temperature. Gilbert et al. achieved 6-8 mg NH4+‑N/L in the effluent when the influent concentration was set as 50 mg/L and the operating HRT was 1-6 d 12. Lotti et al. obtained 75-85% nitrogen removal efficiencies at an HRT of 0.23-0.3 d when the influent ammonium concentration was in a range of 60-160 mg/L, in a granular-sludge reactor 11. But both the HRT and corresponding nitrogen loading rate (NLR) implemented in these studies were far from the necessary demands towards practical application. Based on current studies, the challenges mainly lie in four aspects: (1) Both AOB and anammox bacteria grow very slowly when fed continuously with low-strength influent 5,13,14; (2) AOB need O2 to convert ammonium to nitrite, but the O2 should be in limited supply to avoid the inhibition on anaerobic anammox bacteria 14; (3) Relatively low temperatures in sewage affect the growth rates of AOB more than nitrite oxidizing bacteria (NOB) 15, hence O2 regulation is also important to control the growth of NOB to limit nitrite oxidation to nitrate under a hypothermal condition; and (4) The slow-growing biomass is likely being washed out due to the impact of both intensified hydraulic loading rate and air-lift mixing, which can deteriorate operation performance. Thus, sufficient AOB and anammox bacteria cells with restricted NOB cells, as well as optimally syntrophic association between AOB and 4


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anammox bacteria, are essential to maintain stable operation for efficient nitrogen removal in a PNA system. Biomass retention and DO regulation are two key factors. Biofilms have been shown to be effective because of their enhanced capacity to retain biomass within the reactor system, and out-performed the suspended growth process. The formation of biofilms represents a protected mode of bacterial growth that allows cells to survive and multiply in hostile or unfavorable environments 16. It has been reported that anammox bacteria could sustain reasonable activities in biofilm reactors even the temperature down to 15 oC 12. This study focuses on the efficiencies of PNA by co-immobilizing AOB and anammox bacteria in treating extremely low-strength ammonia wastewater using a moving bed biofilm reactor. A kind of hollow cylinder with a size of 4 mm Φ×4 mm L, a true specific gravity of 0.98 g/cm3 and a void ratio of 65% were used as functional carriers for biofilm growth. Special attention is given to the treatment performance of PNA under various operating HRTs which are essential operating parameters in the practical application. In addition, a better understanding of the anammox bacteria-AOB association in the PNA system is necessary for improving process operation, so batch test, 16S rRNA gene technology and fluorescent in situ hybridization (FISH) were carried out to give clear illustration of AOB-anammox co-immobilization, from the perspectives of microbial activities, bacteria community and active cells make-up. Materials and methods Reactor setup and operation As shown in Fig. 1, the PNA process was carried out in an air-lift moving bed 5



biofilm reactor. The reactor, made of acryl, had a 2 L reaction volume and 0.4 L sedimentation volume. A volume of 0.6 L carriers (around 1170 cylinders) were added to the reaction area, resulting in a filling rate of 30%. The carriers (RK 04Z098, Sumitomo Bakelite co.，Ltd, Japan) used for biofilm growth were hollow cylinders made of hydrophobic polypropylene resin, with a size of 4 mm Φ×4 mm L, a specific gravity of 0.98 g/cm3 and a specific surface area of 1500 m2/m3. Air was supplied by a constant-speed diaphragm pump (APN-085 LV-1, Iwaki, Japan) to the bottom of the reaction area through a fixed glass tube, to provide continuously air-lift mixing and oxygen diffusion. A rotameter was used to regulate the aeration rate. A peristaltic pump (FP-100-1515, AS ONE, Japan) was used to transfer synthetic influent to the reactor. The reactor was kept at constant 25oC through water bath and was operated under ordinary daylight/night darkness shifts without any light adjustment.

P Influent pump

Ｍ

Effluent Gas meter

Water bath

Air pump

A

25oC

Fig. 1 Diagram of an air-lift moving bed biofilm reactor with a 30% carrier filling rate for PNA process

The seed sludge was collected from an existing PNA reactor fed continuously by relatively high-ammonia synthetic wastewater containing 250 mgNH4+-N/L for a long 6


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time. After inoculation, the ammonium concentration in the influent was set as 100 mg/L with a purpose to enable the culture to adapt to the decreased ammonium content, which took 43 days before the ammonium concentration was finally changed to 50 mg/L in the influent. As Table 1 shows, the reactor was operated successively for 234 days. When the NH4+-N concentration in the influent was changed to 50 mg/L, the reactor was operated successively under three HRTs including 3, 2 and 1 hours, resulting in NLRs of 0.4, 0.6 and 1.2 gN/(L•d), respectively. The stable performance of the reactor was difficult to sustain under an extremely short HRT of 1h because only very low nitrogen removal efficiencies can be obtained at this HRT. Hence, we turned the HRT back to 2h to investigate the long-term stable performance and the mechanisms under this favorable HRT. The NH4+-N in the influent was made of NH4HCO3. Other compounds were also added, including 21.9 mg/L KH2PO4 (resulting in 5 mg/L PO43--P), 36 mg/L CaCl2, 25 mg/L MgCl2 and 0.5 ml/L trace element solution (containing 8.30 g/L Na2·EDTA·2H2O, 5 g/L FeSO4·7H2O, 0.215 g/L ZnSO4·7H2O, 0.120 g/L CoCl2·6H2O, 0.495 g/L MnCl2·4H2O, 0.125 g/L CuSO4·5H2O, 0.110 g/L Na2MoO4·2H2O, 0.095 g/L NiCl2·6H2O, 0.078 g/L Na2SeO3, 0.007 g/L H3BO4). The DO concentration and pH in the effluent was regularly measured using a portable DO meter (HORIBA OM-70, Japan) and pH meter (HORIBA D-72, Japan), respectively. The pH in the influent ranged between 7.8 and 8.5.

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Table 1 Operating conditions Duration (day)

1-41

42-61

62-102

103-131

132-234

HRT

6h (startup)

3h

2h

1h

2h

NH4+-N (mg/L)

100

50

50

50

50

NLR (gN/(L•d))

0.4

0.4

0.6

1.2

0.6

DNA extraction, PCR amplification and sequencing Carriers and suspended flocs were sampled on Day 200 when the reactor was deemed to have achieved its steady-state operation and the biofilms grown on the inner surface of the carriers were generally reddish and smooth. DNA was extracted and purified with ISOIL for the Beads Beating kit (Nippon gene, Japan) following the manufacturer’s protocol. Both the biofilms and floc samples were physically broken by a Micro Smash (MS-100R, TOMY, Japan) for 45 sec at 5500 rpm for DNA extraction. The extracted DNA was measured by NanoDrop 2000 (Nanodrop Inc., USA) and diluted to approximately 10 ng/µl for PCR. The PCR amplification of prokaryotic 16S rRNA gene was performed with the forward primer 515F and reverse primer 806R 17. The DNA sequencing was performed using MiSeq reagent Kit v2 (Illumina, USA) of the MiSeq system. The sequences were analyzed using QIIME 1.8.0 18. The forward and reverse reads were assembled using the fastq-join method 19. Operational taxonomic units (OTUs) were picked by the de novo strategy at a 97% identity cutoff by using the UCLUST algorithm 20. The most abundant sequences were used to represent each OTU and were aligned using the Greengenes database. The 8


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chimeric sequences were removed by ChimeraSlayer 21. After assigning taxonomy to each OTU by BLAST, the relative abundance of each OTU, and its alpha diversity was calculated. Specific activity batch tests Specific anammox activity (SAA) by anammox bacteria, ammonia oxidation activity (SAOA) by AOB, nitrite oxidation activity (SNOA) by NOB and denitrification activity (SDA) were tested for the carriers and suspended flocs on Day 220. Regarding the tests for suspended flocs, 40 mL mixed liquor were used for inoculation, while regarding those for carriers, we used approximately 12 mL carriers, which were deemed to be equal to 40 mL mixed liquor in the reactor because of the 30% filling rate of carriers within the reactor. The procedures of batch tests were introduced in the Supporting Information (SI). Fluorescence in situ hybridization and confocal laser scanning microscope observation Both the carriers and the suspended flocs samples were first washed by a freshly prepared phosphate buffer solution and then fixed in 4% paraformaldehyde solution for 24 h at 4 oC. After fixation, a dehydration series with 50, 80 and 96% ethanol was conducted. The carrier samples were embedded in an OCT compound (Sakura Finetek, Torrance, CA) overnight to ensure OCT infiltrated the biofilm. The frozen carriers were fixed on a stage of a cryostat microtome (OSK 97LF509, Ogawa Seiki Co., LTD, Japan), and then cut vertically into 30 μm thick slices using a steel razor blade. Slices were placed on a piece of tape which was then pasted to microscopic slides. Floc 9



samples were applied to 8-well glass slides and air-dried. All the slides were immersed in 50, 80 and 96% ethanol series for dehydrating. The 16S rRNA-targeted oligonucleotide probes (Table S1, SI) were used for FISH. To detect the most bacteria, probes EUB338, EUB338II, EUB338III and EUB338IV were used in an equimolar mixture 22–24. Probe Amx820 was used to detect anammox bacteria 25,26 and probe Nso190 was used to detect AOB 27. The probes were purchased from Japan Bio Service Co., LTD (Saitama, Japan). All in situ hybridizations were performed according to the procedure described by Manz et al. (1992) 28. Analytical methods The NH4+-N, NO2--N and NO3--N concentration was measured by capillary electrophoresis (Agilent 7100, Agilent Technologies, USA). During measurements, a mixed solution (10mM C3H4N2, 5mM (CH3)2C(OH)COOH, 2mM C12H24O6 and 0.2w% CH3COOH, ≥99% purity) made according to the manufacturer's instructions was used as running buffer for NH4+-N detection, while the other solution (30 mM C7H5NO4 and 0.5 mMC19H43NO at pH 9.0 by Tris adjustment) was used as running buffer for NO2--N and NO3--N detection. All the chemicals were from Wako, Japan. Results and discussion Reactor performance The continuous experiment was operated for 234 days. The entire operation period can be divided into 4 stages: the start-up period (Stage I), the HRT decreasing period (Stage II), the recovery period (Stage III), and the stable operation period (Stage IV). As shown in Fig. 2, the startup was performed for 43 days in Stage I by continuously 10


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feeding the influent with 100 mg NH4+-N/L at a HRT of 6h to provide an adaptive phase for the seed sludge from treating high-strength wastewater (250 mg NH4+-N/L) to a low-strength one. From Stage II on, the NH4+-N concentration in the influent was lowered to 50 mg/L. In this stage, the HRT was shortened stepwise from 3h to 1h with the NLR increased from 0.4 up to 1.2 gN/(L•d). The stable performance can be sustained at HRT 3h and 2h. As shown in Fig. 2A, a relatively low aeration rate of 0.1 L/min resulted in a DO range of 0.12-0.22 mg/L at HRT 3h and an increased aeration rate of 0.15 L/min resulted in a similar DO range of 0.1-0.23 mg/L. An increased aeration rate resulting in an almost unchanged DO concentration in the mixed liquor may indicate additionally supplied DO was consumed by increased oxygen-demand bacteria which were likely to be AOB. This hypothesis can be proved by Fig. 2B, in which NH4+-N was the main remaining nitrogen form at HRT 3h while it was greatly reduced at HRT 2h. It means the AOB converting NH4+ to NO2- was promoted by the increased aeration rate at HRT 2h, resulting in a decrease in the NH4+-N concentration in the effluent. In addition, anammox bacteria were not impacted by the increased DO supplement because AOB formed a protective layer on the clusters of anammox bacteria (see Fig. 6), but instead the anammox activities were greatly motivated due to the largely promoted AOB activities which provided abundant NO2- for anammox reaction. Therefore, at this HRT, the total nitrogen (TN) concentration in the effluent averaged 13.5mg/L and was maintained in a range of 7.23–17.5 mg/L (Fig. 2C), which brought about an average nitrogen removal efficiency of 72.7% within a range 11



of 63.4-85.6% (Fig. 2D). The stable performance was suddenly broken when the HRT was decreased to an extremely short HRT of 1h. The main problem is the large amount of NH4+-N which remained in the effluent without being consumed (Fig. 2B). In order to enhance NH4+-N oxidization, we increased the aeration rate continuously up to 0.8 L/min and the DO tended to increase accordingly up to 0.8 mg/L in the solution (Fig. 2A), however the remaining NH4+-N concentrations were still more than 20 mg/L. Furthermore, the NO3+-N concentrations in the effluent were largely increased to approach 15 mg/L. There were four factors which contributed to this deteriorated performance: (1) The oxygen transfer efficiency is thought to have been lowered under a condition of a high hydraulic loading rate (HLR) caused by short HRT, impeding efficient oxygen consumption by AOB; (2) A large portion of the AOB was washed out (Table S2, SI) when the HRT was extremely short because most of the AOB tended to be in the suspended state instead of attached to the carrier biofilms, as indicated by the results of the FISH investigation; (3) The increase in the aeration strength intensified the friction among moving carriers, resulting in the observable detachment of biofilms when the aeration rate was greater than 0.4 L/min; and (4) The growth rate of AOB and NOB was quite different under a similar DO concentration, and NOB may overgrow and outperform AOB for oxygen utilization when the DO concentration is relatively high. Therefore, TN in the effluent increased to an average 36.2 mg/L (Fig. 2C) and the nitrogen removal efficiencies were down to an average 28.1% (Fig. 2D) in this HRT. We failed to recover the performance even within 30 12


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days. Hence, in Stage III, the HRT was turned back to 2h. In Stage III, the aeration rate was lowered to 0.3 L/min, resulting in an average DO concentration of 0.24 mg/L (Fig. 2A). It took around 60 days to recover the performance and the TN in the effluent was down to approximately 15 mg/L and kept stable from the 190th day on (Fig. 2C), resulting in nitrogen removal efficiencies of 71.7±9.1% (Fig. 2D) in Stage IV. Regarding the nitrogen form in the effluent, The NH4+-N, NO2--N and NO3--N concentrations in the effluent averaged 7.35, 0.44 and 5.83 mg/L, respectively (Fig. 2B). It can be found the NH4+-N was not utilized fully in Stage III and IV. Referring to the stoichiometric reaction of PNA process, approximately 57% of the influent ammonia is firstly oxidized to nitrite by AOB, and then the remaining ammonium and the produced nitrite are converted to N2 by anammox bacteria. Hence, that NH4+-N was not utilized fully was likely attributed to two aspects: (1) insufficient activity of AOB failed to convert 57% of NH4+ to NO2-, and (2) some amount of produced NO2- were further oxidized into NO3- by NOB, so that the remaining NO2- amount were not enough to match the appropriate NO2--toNH4+ ratio for anammox reaction. Based on the stable performance in Stage IV, we analyzed the ratio of produced NO3- to consumed NH4+ (NO3--to-NH4+ ratio) (Fig. S1, SI) to clarify the difference of nitrogen conversion by NOB and anammox. Referring to the stoichiometric reactions of PNA (Eq. 3) and nitrification, the NO3--to-NH4+ ratio in PNA and nitrification should be 0.11 和 1. The average NO3--to-NH4+ ratio (Fig. S1, SI) is 0.14 which is quite close to 0.11. It can be inferred NO3- production by NOB has been well limited, 13



that is to say, the majority of NH4+ were consumed by first AOB nitritation and then anammox converting to N2 rather than by NOB nitratation into NO3-. Stage I

+-N

Influent NH4

100 mg/L

HRT

Aeration rate (L/min)

Stage II

Stage IV

2h

2h

50 mg/L

6h

2h

3h

1h

1.5

Aeration rate

A

1.2

Stage III

0.9

DO

0.6

0.9 0.6

0.3

0.3

N in effluent (mg/L)

0.0 50

TN in effluent (mg/L)

DO (mg/L)

Stage

0 NH₄⁺-N

B

40

NO₂⁻-N

NO₃⁻-N

30 20 10 0 50

TN in the effluent

C

40 30 20 10 0 100

TN removal (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80

60

D

40

TN removal efficiency

20

0 0

20

40

60

80

100

120

140

160

180

200

220

240

Time (days)

Fig. 2 Reactor performance. (A) Aeration rate and DO content. (B) Remaining NH4+-N, NO2--N and NO3--N in the effluent. (C) TN in the effluent. (D) TN removal efficiencies.

Biofilm formation Fig. 3 shows the biofilm formation to the carriers. The inner surface of the carriers 14


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provided favorable space for creating biofilms. As shown, the biofilms on Day 200 at HRT 2h in Stage IV were much thicker than those at Day 90 at a same HRT in Stage II. However, as shown in Fig. 2D, the nitrogen removal efficiencies in these two phases (HRT 2h in Stage II and HRT 2h in Stage IV) were similar (72.7±12.9% vs 71.7±9.1%). Therefore, the activities of bacteria in the biofilms and suspended flocs in the two phases were deemed to be quite different: while the suspended flocs surely had higher activity than the biofilms in the former phase, those of biofilms seemed to be largely enhanced in the later phase. According to our observation, the biofilm thickness tended to be stabilized from Day 200 on without any obvious variation. It should be noted that this result is very different from the observed results in our previous study regarding a PNA process treating high-strength wastewater. In that study, the hollow spaces of many carriers were crowded by the biofilms without any gaps 29. The relatively thin biofilms in this study were likely the result of the trade-off between biofilm formation and deformation, which enables the bacteria in the biofilms to capture the substrate in an easy way when being fed with the low-strength influent. New carriers

Day 90 at HRT 2h in Stage II

Day 200 at HRT 2h in Stage IV

5 mm Fig. 3 Biofilm formations in the hollow space of functional carriers 15



Microbial composition The phylogenetic trees of the AOB and Anammox bacteria lineages are shown in Fig. 4A and 4B. In Fig. 4A, OTUs with a relative abundance more than 1% belonging to the family Nitrosomonadaceae (i.e., OTU1964 and OTU373) were selected for analysis. Both these two OTUs were closely related to the genus Nitrosomonas which are widely found in wastewater treatment process, and have been clearly identified as AOB with chemoautotrophic nature 30. The OTU1964 was closely related to Nitrosomonas sp. Nm47 and Nitrosomonas oligotropha. The OTU373 was very closely related to Nitrosomonas europaea which is most common in sewage treatment plants and in eutrophic waters 31. For anammox bacteria in Fig. 4B, OTUs determined to belong to the family Brocadiaceae in phylum Planctomycetes were considered, because the family Brocadiaceae are composed of five candidate genera including “Candidatus Brocadia”, “Candidatus Anammoxoglobus”, “Candidatus Jettenia”, “Candidatus Kuenenia” and “Candidatus Scalindua” 32, and all up-to-date determined 10 species of anammox bacteria belong to these five genera 33. As shown in Fig. 4B, all the clone sequences grouped into two OTUs including OTU3040 and OTU852. The OTU3040 was closely related to the “Candidatus Brocadia fulgida”. The other OTU852 was much more abundant than the OTU3040, and they were closely related to the “Candidatus Kuenenia stuttgartiensis”.

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A

B

Fig. 4 Phylogenetic tree of Nitrosomonadaceae (A) and Brocadiaceae (B) showing the position of the OTUs obtained from the reactor after 200 days operation.

Based on the amplicon sequencing analysis, all the dominant bacteria with a relative abundance of more than 1% were listed in Fig. 5. In total, 21 identified bacteria were involved and they mainly belonged to 7 phyla including Planctomycetes, Proteobacteria, Chloroflexi, Chlorobi, Bacteroidetes, Nitrospirae, and Acidobacteria. Anammox bacteria of “Candidatus Kuenenia stuttgartiensis” were determined to be the most abundant species in the reactor with a relative abundance of 26.7%, much higher than that of other bacteria. Apart from this, the other anammox bacteria “Candidatus Brocadia” have also been detected to have a relative abundance of 3.0%. Hence, nearly 30% of the reads could be attributed to anammox bacteria. As reported in our previously published work, “Ca. Kuenenia” and “Ca. Brocadia” covered a 17



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respective abundance of 4.95% and 0.32% in the sludge 29 which was used as seed sludge in this study. It can be found that “Ca. Kuenenia” has been largely enhanced after long-term operation. Actually, both “Ca. Brocadia” and “Ca. Kuenenia stuttgartiensis” have been previously reported to be enriched in sludge from wastewater treatment plants. A recently published review article summarized the physiological characteristics of the determined 10 species of anammox bacteria. It mentioned the half-saturation constant (Ks) for NH4+ of “Ca. Kuenenia stuttgartiensis” is much smaller than that of “Ca. Brocadia”, indicating “Ca. Brocadia” would prefer NH4+-rich environments because this anammox bacterium has low affinities for NH4+, conversely, “Ca. Kuenenia stuttgartiensis” has higher affinity constant for NH4+ 33. The kinetic difference between these two bacteria can reasonably explain the occurrence of “Ca. Kuenenia stuttgartiensis” prevailing “Ca. Brocadia” to be predominant after the long-term operation by feeding low-strength NH4+ influent. Anammox “Candidatus Kuenenia stuttgartiensis” (Planctomycetes) AOB

26.7 6.3

Nitrosomonadaceae (Proteobacteria) envOPS12 (Chloroflexi) Ignavibacteriaceae (Chlorobi) Rhodocyclaceae (Proteobacteria) OPB56 (Chlorobi) Phycisphaerales (Planctomycetes ) Deltaproteobacteria (Proteobacteria)

Anammox “Candidatus Brocadia” (Planctomycetes)

3.0

Phycisphaerae (Planctomycetes ) Cytophagaceae (Bacteroidetes) DRC31 (Chloroflexi) Saprospiraceae (Bacteroidetes) NOB

Nitrospira (Nitrospirae)

1.9

Chloracidobacteria (Acidobacteria) Chitinophagaceae (Bacteroidetes) Betaproteobacteria (Proteobacteria) Pirellulaceae (Planctomycetes ) Sediminibacterium (Bacteroidetes) SBR1031 (Chloroflexi) Sphingobacteriales (Bacteroidetes) 0

5

10 15 20 Relative abundance (%)

Fig. 5 Dominant bacteria with a relative abundance more than 1% 18


25

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Regarding AOB, Nitrosomonas-like bacterium covered a 6.3% abundance in the total bacteria, which was found to be slightly increased comparing with their abundance (5.66%) in the seed sludge 29 and were the second most dominant microorganism in the reactor. Therefore, the co-existence of anammox bacteria and AOB, i.e. “Ca. Kuenenia stuttgartiensis” and Nitrosomonas-like AOB, was expected to have formed, enabling anammox bacteria to be fed NO2- produced by AOB oxidizing NH4+. In addition, 1.9% of Nitrospira, a common genus of NOB in the wastewater treatment process 15, was detected. Trade-off between NOB and AOB is always a challenge in a PNA process 12. Basically, a reasonable concentration of nitrate in the effluent (Fig. 2B) indicated the growth of NOB was efficiently controlled. Probably, NOB can be outcompeted either by AOB consuming dissolved oxygen and anammox bacteria consuming nitrite. In addition, temperature affects growth rates of AOB more than those of NOB 34, that is to say, AOB have higher growth rates than NOB when reactors were operated under high temperature e.g. more than 30oC 35, but lower growth rates than NOB under low temperature e.g. less than 20oC 12. Therefore, DO regulation was reported to be more effective than temperature adjustment in the inhibition of NOB growth because of the definitely different affinities of AOB and NOB to the oxygen 36,37. Ex-situ specific activities Table 2 shows the results of SAA, SAOA, SNOA and SDA tests on Day 220 at HRT 2h in Stage IV. The SDA was rarely detected in both carrier biofilms and suspended flocs, indicating heterotrophic denitrification provided quite a limited 19



contribution to nitrogen removal. Therefore, chemoautotrophic de-nitrogen by anammox bacteria was the predominant pathway of nitrogen removal. The SAA was 0.32 gN/(L•d) in the carrier biofilms, but it was only 0.05 gN/(L•d) in the suspended flocs. This meant the overwhelming majority of anammox bacteria preferred to inhabit the biofilms rather than the suspended flocs. The value of SAOA and SNOA was expected to be much higher than the actual activities in the reactor because the aeration was not controlled during the tests and the DO was much higher than that in the reactor. However, the SNOA-to-SAOA ratios in the biofilms and suspended flocs averaged only 0.17 and 0.15, respectively. These low ratios may imply the NOB growth was well controlled in the reactor. Moreover, the SAOA in the suspended flocs was approximately 2 times that in the biofilms. These results can likely be attributed to the remarkably different distribution of anammox bacteria and AOB in the biofilms and flocs: rich anammox bacteria and rare AOB in the biofilms, and the opposite case in the suspended flocs. Table 2 Specific activities of anammox bacteria, AOB, NOB and denitrification bacteria Specific

SAA

SDA

SAOA

SNOA

activities

(gN/(L•d))

(gN/(L•d))

(gN/(L•d))

(gN/(L•d))

Carrier biofilms

0.32±0.03

0.09±0.02

0.64±0.05

0.11±0.03

Suspended flocs

0.05±0.01

0.02±0.00

1.27±0.06

0.19±0.03

Spatial distribution of Anammox bacteria and AOB Fig. 6 shows the spatial distribution of AOB and anammox bacteria in the biofilm. 20


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As shown in Fig. 6A, almost all the bacteria (detected with EUB338mix (EUB338+EUB338II+EUB338III+EUB338IV) probe) were distributed in the inner surface of the carrier. Comparing Fig. 6A with B, most of the EUB338mix probe-hybridized cells were simultaneously hybridized with either Amx820 or Nso190, which means anammox bacteria (detected with Amx820) and AOB (detected with Nso190) were the two dominant bacteria in the biofilm. Strong signals colored in red in Fig. 6B indicated anammox cells occupied a large proportion of the total bacteria in the biofilm. Over 40 FISH images were analyzed to determine the percentage of anammox bacteria and AOB in the total bacteria. The results showed anammox bacteria covered an average 63.7±9.2% of the total bacteria, while AOB covered 4.8±1.9%. In addition, as shown in Fig. 6C and D, the co-immobilization of anammox bacteria and AOB established a specific biofilm structure: (1) Anammox bacteria were sandwiched between the carrier internal surface and AOB forming layer; (2) A majority of the anammox cells agglomerated in clusters close to the AOB layer; and (3) Many fissures can be clearly observed and go deeply inward into the internal biofilm, and these fissures may function as the tunnels through which substrates and waste flow. It has been suggested that when co-enrichment of syntrophic microorganisms in biofilm environments, they function at different zone depths in the biofilm 38. With this specific structure, a portion of influent NH4+ was oxidized into NO2- by AOB at the surface layer of the biofilm and the remaining NH4+ and produced NO2- were then used by the anammox in deeper parts of the biofilm. Furthermore, the surface coverage and O2 consumption by AOB provided protection 21



Page 22 of 29

for anammox bacteria to be inhibited by O2 and other environmental factors.

A

B

500 μm

C

D

20 μm

Fig. 6 FISH analysis of AOB and anammox bacteria in the carrier biofilm on the day 200. (A) Carrier phase contrast image with labeled EUB probe mixture (blue), Amx820 probe (red) and Nso190 probe (green). Purple signals result from binding EUB probe mixture and Amx820 probe into one cell and bright blue signals result from binding EUB probe mixture and Nso190 probe. (B) Carrier image with Amx820 probe (specific to anammox bacteria) and Nso190 probe (specific to AOB). (C) Biofilm image with EUB probe mixture, Amx820 probe and Nso190 probe. (D) Biofilm image with Amx820 probe and Nso190 probe. For (C) and (D), carrier inner surface is at the left margin and biofilm surface at the right margin.

22


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Fig. 7 shows the distribution of AOB and anammox bacteria in the suspended flocs. The green signals of AOB cells were much more abundant than the red ones of anammox cells. Only a small amount anammox cells were scattered within the flocs without any clusters. Over 40 FISH images were analyzed to determine the percentage of anammox bacteria and AOB in the total bacteria. The results showed anammox cells covered only an average fraction of 2.7±1.5% of the total bacteria, but AOB cells covered a large fraction of 61.4±10.6%.

A

B

20 μm

Fig. 7 FISH analysis of AOB and anammox bacteria in the suspended flocs on the day 200. (A) Flocs image with labeled EUB probe mixture (blue), Amx820 probe (red) and Nso190 probe (green). Bright blue signals result from binding EUB probe mixture and Nso190 probe into one cell and purple signals result from binding EUB probe mixture and Amx820 probe. (B) Flocs image with Amx820 probe (specific to anammox bacteria) and Nso190 probe (specific to AOB)

A comparison of Fig. 6 and 7 leads to the conclusion that the growth of anammox bacteria mainly occurred in the biofilms, while the AOB growth occurred mainly in the suspended flocs. These results provided evidence for the performance in the 23



ex-situ specific activity tests which revealed the SAA was much higher in the biofilms than in the suspended flocs whereas the situation for SAOA was the opposite. It is possible that the reason for the failure of the reactor at HRT 1h in Stage II (Fig. 2) was that the high hydraulic washing strength derived from short HRT inevitably resulted in the continuous washout of suspended flocs: this would have resulted in the loss of a large portion of AOB in the reactor, and therefore most of the NH4+ would have remained in the effluent despite the increased aeration rate designed to enhance the DO in the solution. Conclusions We succeeded in efficient treatment of low-strength ammonia wastewater using a moving bed biofilm reactor for PNA process. Stable operation and high nitrogen removal efficiencies of 75±10% were achieved at a short HRT of 2h. Retaining anammox bacteria through the formation of biofilms and DO control by regulating aeration rate were deemed to be the two key factors affecting the optimal co-immobilization of anammox bacteria and AOB. A microbial analysis revealed that the anammox bacteria and AOB were the two most dominant bacteria after long-term operation. A FISH illustration unveiled the spatial co-immobilization of anammox bacteria and AOB in the biofilms and suspended flocs. Most anammox cells presented in the biofilms and agglomerated in clusters, and AOB formed a thin layer on them. However, the cells in the suspended flocs were predominated by AOB. These results provide insight into the optimal operation of PNA based moving bed biofilm reactors.

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Acknowledgements This work was supported by the Shaanxi Provincial Key Program for Science and Technology Development (No. 2018KWZ-06) and the Shaanxi Program for Overseas Returnees (No. 2018012). Supporting Information Procedure of specific activity batch tests; The 16S rRNA-targeted oligonucleotide probes used for FISH (Table S1); Variation of mixed liquor volatile suspended solids (MLVSS) (Table S2); And the ratio of NO3- produced to NH4+ removed in the Stage IV (Fig. S1).

References (1) Chu, Z. rui; Wang, K.; Li, X. kun; Zhu, M. ting; Yang, L.; Zhang, J. Microbial Characterization of Aggregates within a One-Stage Nitritation-Anammox System Using High-Throughput Amplicon Sequencing. Chem. Eng. J. 2015, 262, 41–48. (2) Ni, B. J.; Ruscalleda, M.; Smets, B. F. Evaluation on the Microbial Interactions of Anaerobic Ammonium Oxidizers and Heterotrophs in Anammox Biofilm. Water Res. 2012, 46 (15), 4645–4652. (3) Kuenen, J. G. Anammox Bacteria: From Discovery to Application. Nat. Rev. Microbiol. 2008, 6 (4), 320–326. (4) Lackner, S.; Gilbert, E. M.; Vlaeminck, S. E.; Joss, A.; Horn, H.; van Loosdrecht, M. C. M. Full-Scale Partial Nitritation/Anammox Experiences An Application Survey. Water Res. 2014, 55 (0), 292–303. (5) Qiao, S.; Tian, T.; Duan, X.; Zhou, J.; Cheng, Y. Novel Single-Stage Autotrophic Nitrogen Removal via Co-Immobilizing Partial Nitrifying and Anammox Biomass. Chem. Eng. J. 2013, 230, 19–26. (6) Joss, A.; Salzgeber, D.; Eugster, J.; Konig, R.; Rottermann, K.; Burger, S.; Fabijan, P.; Leumann, S.; Mohn, J.; Siegrist, H. Full-Scale Nitrogen Removal from Digester Liquid with Partial Nitritation and Anammox in One SBR. Environ. Sci. Technol. 2009, 43 (14), 5301–5306. (7) Hulle, S. W. H. Van; Vandeweyer, H. J. P.; Meesschaert, B. D.; Vanrolleghem, P. A.; Dejans, P.; Dumoulin, A. Engineering Aspects and Practical Application of Autotrophic Nitrogen Removal from Nitrogen Rich Streams. Chem. Eng. J. 2010, 162 (1), 1–20. 25



(8)

(9)

(10)

(11)

(12)

(13)

(14) (15)

(16)

(17)

(18)

(19) (20) (21)

Meng, H.; Yang, Y. C.; Lin, J. G.; Denecke, M.; Gu, J. D. Occurrence of Anammox Bacteria in a Traditional Full-Scale Wastewater Treatment Plant and Successful Inoculation for New Establishment. Int. Biodeterior. Biodegrad. 2017, 120, 224–231. Chen, R.; Nie, Y.; Tanaka, N.; Niu, Q.; Li, Q.; Li, Y. Y. Enhanced Methanogenic Degradation of Cellulose-Containing Sewage via Fungi-Methanogens Syntrophic Association in an Anaerobic Membrane Bioreactor. Bioresour. Technol. 2017, 245, 810–818. Reino, C.; Carrera, J. Low-Strength Wastewater Treatment in an Anammox UASB Reactor: Effect of the Liquid Upflow Velocity. Chem. Eng. J. 2017, 313, 217–225. Lotti, T.; Kleerebezem, R.; Hu, Z.; Kartal, B.; Jetten, M. S. M.; van Loosdrecht, M. C. M. Simultaneous Partial Nitritation and Anammox at Low Temperature with Granular Sludge. Water Res. 2014, 66, 111–121. Gilbert, E. M.; Agrawal, S.; Karst, S. M.; Horn, H.; Nielsen, P. H.; Lackner, S. Low Temperature Partial Nitritation/Anammox in a Moving Bed Biofilm Reactor Treating Low Strength Wastewater. Environ. Sci. Technol. 2014, 48 (15), 8784–8792. Ma, Y.; Yuan, D.; Mu, B.; Zhou, J.; Zhang, X. Reactor Performance, Biofilm Property and Microbial Community of Anaerobic Ammonia-Oxidizing Bacteria under Long-Term Exposure to Elevated Cu (II). Int. Biodeterior. Biodegrad. 2018, 129 (February), 156–162. Jin, R.-C.; Yang, G.-F.; Yu, J.-J.; Zheng, P. The Inhibition of the Anammox Process: A Review. Chem. Eng. J. 2012, 197, 67–79. Yao, Q.; Peng, D. C. Nitrite Oxidizing Bacteria (NOB) Dominating in Nitrifying Community in Full-Scale Biological Nutrient Removal Wastewater Treatment Plants. AMB Express 2017, 7 (1). Boltz, J. P.; Smets, B. F.; Rittmann, B. E.; Van Loosdrecht, M. C. M.; Morgenroth, E.; Daigger, G. T. From Biofilm Ecology to Reactors: A Focused Review. Water Sci. Technol. 2017, 75 (8), 1753–1760. Caporaso, J. G.; Lauber, C. L.; Walters, W. A.; Berg-Lyons, D.; Huntley, J.; Fierer, N.; Owens, S. M.; Betley, J.; Fraser, L.; Bauer, M.; et al. Ultra-High-Throughput Microbial Community Analysis on the Illumina HiSeq and MiSeq Platforms. ISME J. 2012, 6 (8), 1621–1624. Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.; Costello, E. K.; Fierer, N.; Peña, A. G.; Goodrich, J. K.; Gordon, J. I.; et al. QIIME Allows Analysis of High-Throughput Community Sequencing Data. Nat. Methods 2010, 7, 335. Aronesty, E. Comparison of Sequencing Utility Programs. Open Bioinforma. J. 2013, 7 (1), 1–8. Edgar, R. C. Search and Clustering Orders of Magnitude Faster than BLAST. Bioinformatics 2010, 26 (19), 2460–2461. Haas, B. J.; Gevers, D.; Earl, A. M.; Feldgarden, M.; Ward, D. V.; Giannoukos, G.; Ciulla, D.; Tabbaa, D.; Highlander, S. K.; Sodergren, E.; et al. Chimeric 26


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30) (31) (32) (33) (34)

16S RRNA Sequence Formation and Detection in Sanger and 454-Pyrosequenced PCR Amplicons. Genome Res. 2011, 21 (3), 494–504. Schmid, M. C.; Maas, B.; Dapena, A.; de Pas-Schoonen, K. V; de Vossenberg, J. V; Kartal, B.; van Niftrik, L.; Schmidt, I.; Cirpus, I.; Kuenen, J. G.; et al. Biomarkers for in Situ Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria. Appl. Environ. Microbiol. 2005, 71 (4), 1677–1684. Daims, H.; Brühl, A.; Amann, R.; Schleifer, K. H.; Wagner, M. The Domain-Specific Probe EUB338 Is Insufficient for the Detection of All Bacteria: Development and Evaluation of a More Comprehensive Probe Set. Syst. Appl. Microbiol. 1999, 22 (3), 434–444. Appl, S.; Feeds, R. S. S. Combination of 16S RRNA-Targeted Oligonucleotide Probes with Flow Cytometry for Analyzing Mixed Microbial Populations . Combination of 16S RRNA-Targeted Oligonucleotide Probes with Flow Cytometry for Analyzing Mixed Microbial Populations. 1990, 56 (6), 1919– 1925. Schmid, M.; Twachtmann, U.; Klein, M.; Strous, M.; Juretschko, S.; Jetten, M.; Metzger, J. W.; Schleifer, K. H.; Wagner, M. Molecular Evidence for Genus Level Diversity of Bacteria Capable of Catalyzing Anaerobic Ammonium Oxidation. Syst. Appl. Microbiol. 2000, 23 (1), 93–106. Schmid, M.; Schmitz-Esser, S.; Jetten, M.; Wagner, M. 16S-23S RDNA Intergenic Spacer and 23S RDNA of Anaerobic Ammonium-Oxidizing Bacteria: Implications for Phylogeny and in Situ Detection. Environ. Microbiol. 2001, 3 (7), 450–459. Mobarry, B. K.; Wagner, M.; Urbain, V.; Rittmann, B. E.; Stahl, D. a; Mobarry, B. K.; Wagner, M.; Urbain, V.; Rittmann, B. E.; Stahl, D. a. Phylogenetic Probes for Analyzing Abundance and Spatial Organization of Nitrifying Bacteria . These Include : Phylogenetic Probes for Analyzing Abundance and Spatial Organization of Nitrifying Bacteria. Appl. Environ. Microbiol. 1996, 62 (6), 2156–2162. Manz, W.; Amann, R.; Ludwig, W.; Wagner, M.; Schleifer, K. H. Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions. Syst. Appl. Microbiol. 1992, 15 (4), 593–600. Liu, Y.; Niu, Q.; Wang, S.; Ji, J.; Zhang, Y.; Yang, M.; Hojo, T.; Li, Y. Y. Upgrading of the Symbiosis of Nitrosomanas and Anammox Bacteria in a Novel Single-Stage Partial Nitritation–anammox System: Nitrogen Removal Potential and Microbial Characterization. Bioresour. Technol. 2017, 244 (August), 463–472. Pommerening-Röser, A.; Rath, G.; Koops, H.-P. Phylogenetic Diversity within the Genus Nitrosomonas. Syst. Appl. Microbiol. 1996, 19 (3), 344–351. Green, P. N. Genus I. Methylobacterium; 2005. Firmicutes, T. Systematic Bacteriology; 2009. Oshiki, M.; Satoh, H.; Okabe, S. Ecology and Physiology of Anaerobic Ammonium Oxidizing Bacteria. Environ. Microbiol. 2016, 18 (9), 2784–2796. Qin, Y. Y.; Zhang, X. W.; Ren, H. Q.; Li, D. T.; Yang, H. Population 27



(35)

(36)

(37)

(38)

Dynamics of Ammonia-Oxidizing Bacteria in an Aerated Submerged Biofilm Reactor for Micropolluted Raw Water Pretreatment. Appl. Microbiol. Biotechnol. 2008, 79 (1), 135–145. Gabarró, J.; Ganigué, R.; Gich, F.; Ruscalleda, M.; Balaguer, M. D.; Colprim, J. Effect of Temperature on AOB Activity of a Partial Nitritation SBR Treating Landfill Leachate with Extremely High Nitrogen Concentration. Bioresour. Technol. 2012, 126, 283–289. Ciudad, G.; Werner, A.; Bornhardt, C.; Muñoz, C.; Antileo, C. Differential Kinetics of Ammonia- and Nitrite-Oxidizing Bacteria: A Simple Kinetic Study Based on Oxygen Affinity and Proton Release during Nitrification. Process Biochem. 2006, 41 (8), 1764–1772. Ma, B.; Bao, P.; Wei, Y.; Zhu, G.; Yuan, Z.; Peng, Y. Suppressing Nitrite-Oxidizing Bacteria Growth to Achieve Nitrogen Removal from Domestic Wastewater via Anammox Using Intermittent Aeration with Low Dissolved Oxygen. Sci. Rep. 2015, 5 (September), 1–9. Wu, G.; Nielsen, M.; Sorensen, K.; Zhan, X.; Rodgers, M. Distributions and Activities of Ammonia Oxidizing Bacteria and Polyphosphate Accumulating Organisms in a Pumped-Flow Biofilm Reactor. Water Res. 2009, 43 (18), 4599–4609.

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Graphic abstract

P Anammox

P

dominate

NH4+

O2

N2

NO2-

NH4+

NO3-

dominate

Synopsis The application of PNA process to treating low-ammonium wastewater achieves low energy consumption, no carbon demand and small excess sludge production.

29


Using partial nitrification and anammox to remove nitrogen from low

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