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Start-up and Nitrogen Removal Performance of DAMOanammox Inoculated with Anaerobic Granular Sludge in MBfR

This study investigated the rapid start-up of the DAMO-anammox strategy using inoculum of anaerobic granular sludge in MBfR. The results showed that the reactor containing DAMO microorganisms was rapidly and successfully started up after 73 days with only feeding nitrate. Anammox microorganisms were obtained after 1.5-month of ammonium addition. The nitrogen removal rate achieved 603.9 mg N/L/d in this study. Microbial analysis results suggested that the newly discovered DAMO archaea, DAMO bacteria and anammox bacteria were not detected in this enriched culture. The DAMO-anammox process may be carried out by other microbes. This deduction remains to be further established in the future.


Introduction
Nitrogen pollution resulting from excessive discharge of domestic and municipal wastewater to the water bodies has incurred a great concern of society, thus increasing demand for more effective and economical technology for nitrogen removal. Recently, more attention has been focused on a novel and cost-effective biological nitrogen removal process namely anaerobic ammonium oxidation (anammox) integrating with denitrifying anaerobic methane oxidation(DAMO)process [1][2][3].With methane as the sole electron donor, DAMO archaea reduced the nitrate produced by anammox to the nitrite, the nitrite generated by DAMO archaea was utilized by anammox and DAMO bacteria under anoxic condition. As a result, NO 3 -/NO 2 were reduced to N 2 and CH 4 was oxidised to CO 2 . This largely save the operational costs for wastewater treatment due to no requirement of aeration, additional carbon sources supply compared to conventional microbial nitrogen removal processes [4,5].To date, several cultures containing DAMO-anammox microorganisms were obtained from freshwater sediment [6], anammox granules [7], enriched culture of DAMO microorganisms or co-culture of DAMO-anammox microorganisms [1,8], a mixture of methanogenic sludge and activated sludge [9]. However, the enrichment of DAMO-anammox consortia is a lengthy process(4.5-9months), which restricted its application to wastewater treatment. In this case, an alternative seed source should be used for shortening enrichment time of DAMO-anammox microorganisms. The previous work has showed that membrane biofilm reactor (MBfR) was a powerful way to enrich co-culture of DAMO-anammox microorganisms for its developing and retaining high concentrations of extremely slow growing microorganisms [1,6]. The aim of this study was to hasten the start-up of DAMO-anammox process by inoculating anaerobic granular sludge in the MBfR.

Reactor set-up
The MBfR system for enrichment of co-culture of DAMO and anammox microorganisms was set up (Fig.1). The total volume of the membrane module was 60 mL with 1mL of hollow fibres,13 mL interior space for gas supply and 45 mL external space outside the fibres for liquid, respectively. Feeding gas (99.99%, CH 4 ) was supplied into the interior of hollow fibres. Liquid in the reactor was recirculated by a peristaltic pump and gas pressure was maintained by a pressure stabilizing valve.

Operations
The operation of the reactor consisted of four stages: stage1 (Day 0-193), stage 2 (Day 194-292), stage 3 (Day 293-340), and stage 4 (Day341-603). During stage 1, no medium was exchanged into the system to make the biomass attached to the surface of membrane biofilms for initial two weeks. Subsequently, the MBfR was operated under SBR and only nitrate feeding was added to the medium for enrichment of DAMO microorganisms. During stage 3, the medium of the influent contained 200 mg N/L nitrate and gradually increased to 300 mg N/L nitrate. During stage 3, 200 mg N/L ammonium and 200 mg N/L nitrate was added to reactor for enrichment of anammox bacteria. During stage 4, the MBfR was operated as SBR with cycle time of 3.5d (Day 341 -576) and 2.5d (Day 577-603). The temperature was maintained at 25ºC. pH value was controlled at 7.0-7.5 by adding 1M HCL and 1M NaOH. To prevent the air into the system, gas (99.99% N 2 ) was used to sparge the reactor regularly. The partial pressure of CH 4 maintained at approximately 20 KPa.

Microbial analysis
Fast DNA SPIN Kit for Soil was used to extract total DNA. Universal primers(341b4F-806R) was used for the PCR amplifications with high-throughput Illumina MiSeq PE 300.The methods were described in previous study [11].

Fast start-up of DAMO process
As is shown in Fig.2, during the stage 1, nitrate was only supplied into the reactor. The nitrate removal rate closed to zero for initial two weeks. For the next one month, nitrate concentration was fluctuated, and nitrate removal rate increased gradually. From Day 50-72, the nitrate concentration stepwise decreased and nitrate removal rate reached 42.9 mg N/L/d. Onwards Day 73, obvious removal rate of nitrate was obtained. During stage 2, the reactor was operated as fed-batch mode at an HRT of 3.5d with the influent only containing nitrate. Stable performance was achieved after Day 208.On Day 245, the influent nitrate concentration increased to 300 mg N/L/d. The nitrate remained in the range of 53.6-71.3 mg N/L/d, giving rise to increasing improvement of nitrate removal rate. Nitrite concentration was undectable level during the operation.
To verify the occurrence of DAMO process, a batch test on Day 227 to Day 230 without methane feeding was performed with the results shown in Fig.3. Without CH 4 supply, the nitrate reduction rate was about 2.7 mg N/L/d compared to 36.3 mg N/L/d with CH 4 supply, suggesting that the DAMO microorganisms were responsible for nitrate reduction.

Enrichment of co-culture DAMO-anammox microorganisms
As is shown in Fig.4, during stage 3, ammonium and nitrate were supplied into the reactor for enrichment of anammox bacteria with a constant HRT of 3.5d. The effluent ammonium concentration was fluctuated and even higher than the influent ammonium concentration for initial 10 days. The impossible reasons may be attributed to cell lysis and breakdown of organic nitrogen to ammonium, causing high effluent concentration of ammonium. After Day 333, the ammonium concentration in the effluent decreased slowly to 181.

Microbial analysis
As was shown in Fig.6 [12], were detected in this biofilm. Furthermore, the Phycisphaerae class containing Phycisphaerales order have been found in the anammox enrichment and several DAMO cultures [2].

Discussion
The DAMO process occurred after the enrichment of 73 days with nitrate removal rate of 42.9 mg N/L/d. The enrichment time of DAMO microorganisms was relatively short compared to the previous studies (3-18month). This indicated that the MBfR was a good system to enrich DAMO microorganisms. After 1.5 months of feeding ammonium, the decrease of ammonium concentration was obviously observed. This suggested that the formation of DAMO microorganisms could promote the development of anammox bacteria. In this study, simultaneous decrease of the nitrate and ammonium concentrations and undectable level of nitrite in the effluent showed that the partial nitrite generated from nitrate reduction by DAMO archaea was consumed by anammox bacteria. The high-throughout sequencing analysis showed that the currently known DAMO archaea, DAMO bacteria and anammox bacteria were not found in this enrichment culture. But the Methanosarcinales, which have been enriched in several DAMO culture under feeding nitrate and methane [12], were detected in this reactor. Furthermore, the Phycisphaerae Class containing Phycisphaerales Order were found in the anammox enrichment and several DAMO cultures. Based on the experimental data and high-throghout sequencing results, we deduced that the DAMO-anammox process may be accomplished by other microbes. This deduction remains to be further elucidated in the future.

Conclusion
The MBfR inoculated with anaerobic granular sludge showed an excellent performance for its fast startup DAMO-anammox process and obtaining high TN removal rate. The DAMO process occurred only after 73 days. By adding ammonium, anammox microorganisms were obtained after 1.5-month. The nitrogen removal rate achieved 603.9 mg N/L/d in this study. Microbial analysis suggested that the discovered DAMO archaea, DAMO bacteria and anammox bacteria were not detected in this enriched culture. The DAMO-anammox process may be carried out by other microbes. This deduction remains to be further established in the future.