Study on Long-term Effect of Oxytetracycline on ANAMMOX Nitrogen Removal Performance in the Treatment of Salt-containing Wastewater

. The effects of oxytetracycline on anaerobic ammonium oxidation (ANAMMOX) were analyzed in this study. Meanwhile, the change of specific ANAMMOX activity (SAA) and the content of heme c were also revealed. The performance of ANAMMOX reactor decreased gradually when the increase of OTC concentration from 2mg/L to 10mg/L under 10‰ salinity. The nitrogen removal performance almost collapsed until the elevation to 10 mg/L OTC, observed with a huge decrease of SAA of 77.71% and heme c content of 88.04%. After no addition of OTC into the ANAMMOX reactor, the system performance recovered within 20 days. At the end of this phase, NH 4+ -N was completely removed, NO 2--N was removed above 99%, and total nitrogen removal efficiency recovered to 85.33%.


Introduction
In 1990s, ANAMMOX process was discovered in Delft college laboratory [1] .As one of the most concerned research hotspot in wastewater treatment technologies, ANAMMOX has been given much attention because of many advantages in both energy and cost, such as no need for more energy and low cost [2] . Meanwhile, there is no need for addition of organics during nitrogen conversion process. Different from traditional nitrification and denitrification process, ammonia is directly oxidized to dinitrogen by nitrites under anaerobic condition [3] .
Although ANAMMOX has a series of superiority, the anaerobic ammonia oxidizing bacteria(AnAOB) is sensitive to several elements, such as temperature, heavy mental, organics and salinity. Most nitrogen-rich wastewater have the properties of high salinity. High salinity can increase the osmotic pressure of wastewater and destroy the cell membrane and enzyme system of microorganism, leading to the collapse of microorganism activity. Previous researchers confirmed that high salt(30g/L NaCl) inhibited the granulation and aggregation of ANAMMOX granules [4] . Therefore, how to treat saline nitrogen-rich wastewater efficiently has become the focus of nitrogen removal in industrial wastewater.
The discovery of antibiotic is one of the most valuable milestones in the 20th century. The abuse of antibiotics brought a series of challenges to the application of traditional process for wastewater treatment. Numerous convincing research results have shown that antibiotics inhibit ANAMMOX activity. Considering in terms of the use number of antibiotics, the use of tetracyline antibiotics ranks second. Oxytetracycline (OTC) is representative, because of many superiority, it was widely used in the medical field. OTC has negative influence to Gram-positive and Gramnegative bacteria [5] . Because of its property and potential effect on microorganisms, the effect of OTC on bioreactor became a research hotspot for numerous researchers. As a promising process, it is important to research the influences of antibiotic on ANAMMOX.
Consequently, the research was conducted to reveal the nitrogen removal capacity, SAA and heme c by typical antibiotic(OTC) under saline condition. The main purpose is to supply experimental basis and technical support in order to promote the use of ANAMMOX.

Experimental configuration and operational strategies
An UASB reactor was used in experiment. The diameter was 60mm and the height was 500mm. The effective volume was 1.4L. The reactor was shown in Figure.1. NH 4 + -N and NO 2 --N in influent were set at 220mg/L and 250mg/L. The HRT was set at 4 h.The reactor was operated at (35±1)℃.In order to exclude the effect of light, the reactor was operated in dark condition.
The reactor operation were divided into three phase. In phase Ⅰ, there was OTC-free in UASB reactor. In phase Ⅱ, the concentration of OTC was added and increased gradually until the nitrogen removal performance deteriorated seriously or even collapsed. In phase Ⅲ, the addition of OTC was ceased in order to recover the ANAMMOX performance.

Sludge and wastewater
Sludge were taken from an ANAMMOX-UASB reactor stably operating under 10‰ salinity for one year in our laboratory. In this study, synthetic wastewater which consist of substrates and trace elements was used as a substrate for the reactor. NH 4 + -N and NO 2 --N were supplied by NH 4 Cl and NaNO 2 . The salt was prepared by dissolving the sea crystal. The composition of trace element solution Ⅰ: EDTA 5g/L,FeSO 4 ꞏ7H 2 O 5g/L, trace element solution Ⅱ: EDTA 15g/L, MnCl 2 ꞏ4H 2 O 0.99g/L, The pH was adjusted between 7.5 and 8.0 by 2% of HCl and NaOH solution The OTC added was supplied by oxytetracycline hydrochloride.

Determination methods
Three forms of nitrogen were measured by traditional methods. The SAA was determined by the method supplied by Chen [6] .Heme c was determined by the way supplied by Berry [7] .

Effect of OTC on ANAMMOX system
In 1-36 days(Phase Ⅰ), the NLR was maintained at 2.82kgN/(m 3 ꞏd). In this phase, the ratio of NO 2 --N consumed/NH 4 + -N consumed was 1.13, which slightly lower than theoretical value for typical ANAMMOX reaction [8] . The removal efficiency of NH 4 + -N and NO 2 --N reached to 99.79% and 99.05%, and TN removal efficiency was 83.04%. It indicated that the reactor was operated stably before the addition of OTC.In the following experiment, the concentration of OTC in influent was increased to observe the nitrogen removal performance of the system.
During 37-109 days(Phase Ⅱ), a series concentration level of OTC were added into reactor. Compared to P1,The removal efficiency were declined to 90.85% and 86.62% after the addition of 2mg/L of OTC for 18 days(37-55) . It was reported that the ANAMMOX process was significantly inhibited by 2mg/L of OTC. In this study, the reactor performance maintained well under the effect of 2mg/L OTC. It was hypothesized the existence of 10‰ salinity may alleviated the inhibition of OTC to AnAOB activity. As OTC increased to 5mg/L (56-77 days), the removal efficiency further decreased to 82.93% and 72.99%. The average concentration of effluent NO 3 --N reached to 72.63mg/L. When OTC increased to 7 mg/L(78-101 days), they were continued to decrease to 67.09% and 49.18%, and ANAMMOX process was seriously deteriorated. At the end of Phase Ⅱ(102-108 days), the concentration of OTC added reached to 10mg/L, the removal efficiency of NH 4 + -N and NO 2 --N were abruptly aggravated to 46.21% and 10.07%.Compared to Phase Ⅰ, under the effect of a series of OTC concentrations, the performance of the system became worse and worse. To research the inhibition could be mitigated or not, a recovery experiment was carried out after this phase.
During 109-128 days(Phase Ⅲ), the system was operated for recovery by the removal of the addition of OTC, at the same time, HRT was changed to 10h (109-118). In this period, the concentration of effluent NH 4 + -N and NO 2 --N decreased quickly, and the removal efficiency of NH 4 + -N and NO 2 --N was increased from 46.21% to 97.42% and 10.07% to 83.44%, respectively. During 119-128 days, the HRT was gradually changed to 4h(119-128 days) the NH 4 + -N was all removed and the removal efficiency of NO 2 --N was restored above 99% revealing that the ANAMMOX-UASB reactor can quickly recover from the OTC inhibition under 10‰ salinity.

Effect of OTC on SAA
The SAA usually be used to estimated the performance of AnAOB [9] . In this study, the SAA under the effect of OTC at different concentration levels were mesured and shown at Figure 3. In Phase Ⅰ, the SAA was 4.71 mgN/(gVSSꞏh). The reactor was operated a period of time without addition of OTC. After exposed to 2 mg/L OTC for 18 days, it decreased to 2.99 mgN/(gVSSꞏh). Then, from 55 days to 77 days, under the effect of 5 mg/L OTC ,it decreased to 1.73 mgN/(gVSSꞏh). Compared to 0 mg/L , it was reduced by 63.4%. Then, the SAA was 1.25 mgN/(gVSSꞏh) under 7 mg/L OTC. Compared to no addition of OTC, the SAA was reduced by 73.5%. Under the effect of 10mg/L OTC, the SAA decreased to 1.05 mgN/(gVSSꞏh). The tendency was similar to the change of system performance under stress of different OTC concentrations. In Phase Ⅱ, the SAA declined gradually, indicating that OTC had a complete inhibition on ANAMMOX activity.

Effect of OTC on Heme c
Heme c is regard as an important part for some enzymes, such as HZS and HZO [10] . High content of heme c is one of the special characteristics of AnAOB. It is relative to the color of ANAMMOX sludge and ANAMMOX activity [11] . In this reasearch, the content of heme c was used to reveal the activity of reactor system and enzymes. Heme c under the effect of OTC at different concentration levels were measured. The change of heme c under different OTC concentrations was depicted in Figure 4. Without the addition of OTC, the content of heme c was 4.18 μmol/g VSS. It declined to 3.78 μmol/g VSS under 2mg/L OTC , about 9.5% lower than the upper phase. Then, the OTC concentration increased to 5mg/L, the it further declined to 2.96 μmol/g VSS. Next,the content of heme c were 1.74 μmol/g VSS and 0.5 μmol/gVSS under the stress of 7mg/L and 10mg/L OTC. The tendency was similar to the change of SAA under stress of different OTC concentrations. Results showed that the activity of ANAMMOX was inhibited under the effect of OTC at different concentration levels.

Conclusions
This research analyzed the effect of OTC on ANAMMOX process. Meanwhile, the change of SAA and Heme c were also revealed. The system performance decreased gradually when the concentration of OTC increase from 2mg/L to 10mg/L under 10‰ salinity. The nitrogen removal performance almost collapsed until the elevation to 10 mg/L OTC, observed by a huge decreased of SAA and heme c content. With no addition of OTC into reactor, the ANAMMOX performance improved within 20 days. At the end of this phase, NH 4 + -N was completely removed, NO 2 --N was removed above 99%, and total nitrogen removal efficiency recovered to 85.33%. Three indexes of inhibition period and recovery period were analyzed in this study, which makes the study more complete.Meanwhile,they indicated the effect of OTC on the reactor at different operating stages. The study was carried out under the condition of the ANAMMOX process with salinity and antibiotic, which will supply basis and data for the anaerobic ammonia oxidation system to treat high salinity antibiotic wastewater.