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Weak magnetic field: A powerful strategy to enhance partial nitrification
Accepted Manuscript Weak magnetic field: A powerful strategy to enhance partial nitrification Zhibin Wang, Xiaolin Liu, Shou-Qing Ni, Jian Zhang, Xu Zhang, Hafiz Adeel Ahmad, Baoyu Gao PII:
S0043-1354(17)30326-3
DOI:
10.1016/j.watres.2017.04.058
Reference:
WR 12857
To appear in:
Water Research
Received Date: 21 November 2016 Revised Date:
24 March 2017
Accepted Date: 18 April 2017
Please cite this article as: Wang, Z., Liu, X., Ni, S.-Q., Zhang, J., Zhang, X., Ahmad, H.A., Gao, B., Weak magnetic field: A powerful strategy to enhance partial nitrification, Water Research (2017), doi: 10.1016/j.watres.2017.04.058. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The existence of 5 mT magnetic field increased the activity of AOB while
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Weak magnetic field: A powerful strategy to enhance partial nitrification
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ZhibinWanga, b, Xiaolin Liu a, Shou-Qing Ni a,∗, Jian Zhang a, Xu Zhang a, Hafiz Adeel
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Ahmad a, Baoyu Gao a
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a
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Reuse,School of Environmental Science and Engineering, Shandong University, Jinan
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250100, China
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b
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Shandong Provincial Key Laboratory of Water Pollution Control and Resource
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Institute of Marine Science and Technology, Shandong University, 250000, China
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Abstract
Partial nitrification (PN) combined with anaerobic ammonium oxidation process
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has been recognized as a promising technology for the removal of nitrogenous
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contaminants from wastewater. This research aimed to investigate the potential of
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external magnetic field for enhancing the PN process in short and long term
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laboratory-scale experiments. Different strength magnetic fields (0, 5, 10, 15, 20 and
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25 mT) were evaluated in short-term batch tests and 5 mT magnetic field was found
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to have better ability to increase the activities of aerobic ammonium oxidizing
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bacteria (AOB) of PN consortium. Long-term effect of magnetic field on PN
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consortium was studied with 5 mT magnetic field. The results demonstrated that the
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∗
Corresponding author. Tel.: +86-531-88365660;fax: +86-531-88364513; E-mail: [email protected]. Author contributions: Zhibin Wang and Xiaolin Liu performed theresearch; Shou-Qing Ni and Jian Zhang designed the research; Xu Zhang analyzed the data; Zhibin Wang wrote the paper; Hafiz Adeel Ahmad and BaoyuGao revised the paper. The authors declare no conflict of interest. 1
ACCEPTED MANUSCRIPT positive effect of magnetic field on PN process could also be testified at all of the four
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stages. Furthermore, a decrease of bacterial diversity was noted with the increase of
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magnetic field strength. Relative abundance of Nitrosomonadaceae decreased
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significantly (p < 0.01) from 13.9% in RCK to 12.9% in R5mT and 5.5% in R25mT.
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Functional genes forecast based on KEGG database indicated that the expressions of
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functional genes related to signal transduction and cell motility in 5 mT environment
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were higher expressed compared with no magnetic field addition and high magnetic
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field addition. The existence of 5 mT magnetic field didn’t increase the abundance of
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AOB but increased the activity of AOB by increasing the rate of free ammonia into
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the interior of microbial cells. Addition of magnetic field couldn’t change the final
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state of PN process according to the hypothesis proposed in this article. These
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findings indicated that the weak magnetic field was useful and reliable for the fast
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start-up of PN process since it was proved as a simple and convenient approach to
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enhance AOB activity.
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Keywords: Free ammonia; high-throughput sequencing; magnetic field; partial
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nitrification; PICRUSt
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1. Introduction High concentration of ammonium nitrogen and stubborn organics usually present
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in wastewater of food products factory (Cristian, 2010), pharmaceutical factory
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(Stackelberg et al., 2004) and landfill leachate (Kjeldsen et al., 2002; Justin and
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Zupančič, 2009) et al.. A large number of ammonia-nitrogen wastewater discharged
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into the water body result in not only water eutrophication (Conley et al., 2009), black
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and malodorous (Wei et al., 2011), increasing the cost of water treatment, but also
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produce toxic effects on people and other biology (Ono et al., 2000).
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Ammonia-nitrogen removal processes include biological and physicochemical
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method (Wiszniowski et al., 2006; Mook et al., 2012). Processes of traditional
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biological nitrogen removal included nitrification of ammonium to nitrate under
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aerobic conditions (Schmidt, 1982) and denitrifying of nitrate to nitrogen gas under
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anaerobic conditions by using biodegradable organics as electron acceptors (Kuai and
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Verstraete, 1998). While, there are some drawbacks when conventional biological
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techniques are applied to treat the high concentration ammonium and insufficient
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carbon sources wastewater (Du et al., 2015; Bonomo et al., 1997), such as low total
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nitrogen removal efficiency, unstable running conditions, and high consumption of
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energy (Khin and Annachhatre, 2004).
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Anaerobic ammonium oxidation (anammox) is an innovative technique
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established in 1990s (Dalsgaard et al., 2005). Anammox is technically superior and
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economically beneficial to treat high concentration ammonium and insufficient
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carbon sources wastewater compared to traditional process of biological nitrogen 3
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(Kotay et al., 2013). In anammox process, ammonium is directly oxidized to nitrogen
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gas by nitrite under anoxic conditions. In theory, the consumption ratio of nitrite to
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ammonium is about 1.3 (Van de Graaf et al., 1996; Strous et al., 1998). Therefore, if
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nitrite does not present in the wastewater, a prior partial nitrification (PN) step is
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required to converts half or more of the ammonium to nitrite (Hao et al., 2002).
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However, the realization and control of PN process is difficult (Guo et al. 2009a).
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Firstly, there must be prevention of the activity of nitrite oxidizing bacteria (NOB)
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which oxidize nitrite to nitrate. Secondly, 57% conversion efficiency of NH4+-N to
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NO2−-N must be ensured on the basis of the nitrite/ammonium stoichiometric ratio of
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anammox process (Liang et al., 2007; Qiao et al., 2010). Some other operational
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difficulties such as incoming solids, aeration control and nitrate built-up were also
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reported (Lackner et al. 2014). Joss et al. (2011) evaluated conditions resulting in
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instability and strategies to regain efficient operation of full-scale and lab-scale PN
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process. To startup a PN process, the activity of NOB must be inhibited without
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affecting ammonium oxidizing bacteria (AOB) by establishing conditions that favor
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AOB development (Feng et al., 2007). The inhibition of the NOB growth could be
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achieved by controlling aeration duration to leave no extra time for NOB to convert
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the accumulated nitrite (Guo et al. 2009b). Liang et al. suggested that PN could be
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achieved by stepwise increasing of influent NH4+-N at pH of 7.8±0.2, temperature of
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30±1o C, dissolved oxygen (DO) of 0.5-0.8mg l-1, and the HCO3-/NH4+ molar ratio of
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1:1 (Liang et al., 2011). The high NH4+-N concentration, high pH, high temperature,
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nitrite accumulation (Lemaire et al., 2008). To achieve stability and efficiency of the
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PN process, some strategies have been applied. Feng et al. applied a membrane
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bioreactor (MBR) to enrich the AOB in a partial nitrification reactor (Feng et al.,
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2007). Stable nitrite accumulation was realized for more than 300 days in a nitrifying
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granular sludge reactor (Vázquez-Padín et al., 2010). The enhancement of AOB and
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the suppression of NOB activity were realized by 0.09 kJ/mg VSS ultrasonic
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treatment (Zheng et al. 2015).
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Usually, organisms have magnetism. The external magnetic field and magnetic
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field exist inside the organisms can bring positive or negative effect to the metabolism
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of living organisms and organization, which is named magnetic biological effect
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(Moore, 1979). Since 1960s, the effect of magnetic field on the microorganisms has
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been investigated (Moore, 1979). For example, the magnetic field with the strength of
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370 mT resulted in an increase of 35% phenolic degradation (Křiklavová et al., 2014).
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Another research demonstrated that the anammox activity was first increased at weak
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magnetic field strength, and reached the maximum at 75 mT, but then declined with
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further increase of magnetic field (Liu et al., 2008). Enhancement of phenol oxidation
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activity under 0.15 and 0.35 T magnetic field was also demonstrated (Jung and Sofer,
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1997). The magnetic biological impact related to biomass metabolism, enzyme
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activity and cell membrane permeability has been proved (Barnothy, 2013). Yet the
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physiology of magnetic biological effect on the microorganisms is not completely
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understood. To the best of literature review, the effect of magnetic field on partial
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nitrification is still in blind. In this study, the possibility of PN enhancement by using static magnetic field
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was first evaluated. The short-term and long-term effects of additional magnetic field
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on PN process were both investigated. Optimum magnetic field strength for AOB was
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ascertained by monitoring the water quality index under different magnetic field
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conditions. SOUR was monitored to verify the effect of optimum magnetic field on
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the activity of AOB and NOB. Finally high-throughput sequencing approach was used
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to elucidate the composition and the change of the microbial community in the PN
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reactors during the long term exposure to magnetic field.
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2. Materials and Methods
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2.1. Inocula and feeding media
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Activated sludge collected from aerobic basin of Guangda municipal wastewater
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treatment plant of Jinan city was used as the seed in this study. To enrich AOB and
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eliminate NOB, high NH4+-N, slightly alkaline medium was used, and the
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composition was as follows: NH4HCO3 (2.82 g L-1), KH2PO4 (0.81 g L-1), CaCl2 (0.2
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g L-1), MgSO4·7H2O (0.03 g L-1) and 1mL trace element solution. Trace element
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solution was prepared and added as described by Kong et al. (2013). The final pH of
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the medium was 8.0 ± 0.1, and no pH control equipment was applied.
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2.2. Batch tests
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Batch tests were performed by using a specially designed bioreactor system. All
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the 6 reactors with working volume of 400mL were incubated in a plastic pool
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(20cm*50cm*100cm) for PN process with a heating rod providing a 35 o C water bath. 6
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The magnetic field strength of the reactor center in the range of 5-25 mT were
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supplied by adjusting the distance of reactors and magnets. The treatment of the
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magnetic field in this study was always continuous. An assembly of air pump,
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electromotor and pottery aerator was used to provide oxygen and blend microbes and
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waste water.
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2.3. Long-term reactor operation
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The long-term experiments were performed in two parallel laboratory-scale
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sequencing batch reactors (SBR). Two columnar reactors made of plexiglas with a
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diameter of 10 cm and height of 30 cm were used. The maximum working volume of
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each reactor was 6L. Temperature of the system was controlled at 35 ◦C by a water
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bath. Oxygen was provided by an air pump. Aeration volume was steadied at 1.0L
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min-1 and under this aeration volume, the dissolved oxygen (DO) was fluctuated from
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0.1 mg L-1 to about 7.8 mg L-1.SRT was controlled at 30 days by withdrawing the
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sludge every two days (Guo et al. 2009a). The whole experiment was separated into
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four stages. Influent ammonium concentration of the first two stages was 500 mg N/L
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and the last two stages was 1000 mg N/L. Hydraulic retention time (HRT) of the first
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and third stages was 12 hours and the second and fourth stages was 8 hours. Therefore
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the nitrogen loading rates (NLR) was increased during the fourth stages. Two pairs of
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permanent magnets were installed to provide a static magnetic field of 5 mT. The
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permanent magnets were attached to the exterior reactor body. In the control system,
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the experiment was carried out using the identical reactor parameters without the
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installation of permanent magnets.
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2.4. Analysis The concentration of nitrogen compounds were measured according to the
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standard method (Apha, 1995). The pH measurement was done using a digital,
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portable pH meter, and DO measurement was done using a digital, portable DO meter
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(YSI, Model 55, USA). The volatile suspended solids (VSS) were determined to
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calculate the biomass concentration according to the standard method (Apha, 1995).
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The magnetic strength was measured by Gauss instrument (HT100S) with
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measurement accuracy of ±5%.
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2.5. Biochemical analysis
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A simple method was developed to characterize the nitrification activity by
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specific oxygen uptake rate (SOUR). The method was based on the subsequent
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addition of allylthiorea (ATU) and NaClO3, selective inhibitors of NOB and AOB,
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respectively (Zhang et al., 2015), to the mixed liquid samples of activated sludge in a
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closed batch respirometer. The effects of nitrification inhibitors on the endogenous
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respiration of activated sludge and the heterotrophic substrate oxidation were
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investigated. The triplicate tests were carried out and the results indicated that the
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average oxidation rates of NH4+-N and NO2--N were 12.6 ± 0.3 mg O2/(L·h) and 2.8 ±
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0.5 mg O2/(L·h), respectively. The nitrification activity measured by SOUR was
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validated by determining the variation of nitrate in the running reactors. By means of
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simple measurement of SOUR combined with the subsequent addition of two
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selective nitrification inhibitors, the oxidation rates of NH4+-N, NO2--N and COD can
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be obtained, which is helpful for control and optimization of nitrification process.
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2.6. DNA extraction, high-throughput sequencing and data analysis Total DNA was extracted from 1.5 mL reactor sludge with power soil DNA
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isolation kit (MO BIO Laboratories, USA) following the manufacturer’s
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recommendation. V4 of 16S rRNA were PCR amplified from microbial genome DNA
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using bar-coded fusion primers (forward primers: AYTGGGYDTAAAGNG, reverse
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primers: TACNVGGGTATCTAATCC) (Li et al., 2014). Bar-coded V4 amplicons
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were sequenced using the pair-end method by Illumina Miseqat Personal
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Biotechnology Co., Ltd. (Shanghai, China). Database referenced was greengene. The
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SPSS13.0 (IBM, USA) software package was used for general statistical analysis and
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to conduct a chi-square test. The chi-square values were converted into p values using
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Excel (Microsoft Office) to indicate the significance (Kong et al., 2015).
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2.7. Symbols defined in this study
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To determine the preferable magnetic field strength for PN consortium, six
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reactors under 0 mT, 5 mT, 10 mT, 15 mT, 20 mT and 25 mT magnetic fields were
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described as r0, r5, r10, r15, r20 and r25. To validate the long-term effect of magnetic
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field on PN process, reactors without extra magnetic field and with 5 mT magnetic
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field were described as CK group and M group respectively. Lastly, to describe the
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change of microbial community occurred among reactor exposed to 0 mT, 5 mT and
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25 mT magnetic field, three reactors were defined as RCK, R5mT and R25mT.
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3. Result
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3.1. Determination of preferable magnetic field strength for PN consortium
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investigated. Six magnetic field strengths were selected for this experiment. The
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relationship of the tested magnetic field strength values versus ratios of NO2-/NH4+
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was presented in Fig.1.
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The existence of magnetic field had significant effects on PN start-up. Compared
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with the control experiment in which no magnetic field was applied, there was an
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observable shortening of PN startup time as magnetic field strength increased to 5 mT.
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While when the magnetic field strength increased to 20 and 25 mT, the PN startup
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time seemed longer than that of the control one. Ratios of nitrite to ammonium of the
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effluent with 20 mT and 25 mT magnetic field were 0.40 and 0.45, lower than that of
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with no extra magnetic field addition 0.55, indicating that high magnetic field strength
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had a negative impact on PN process.
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3.2. Long-term effects of magnetic field on PN process
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In order to validate the long-term effects of magnetic field on PN process, two
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parallel SBRs were operated. NLR was increased gradually by decreasing HRT from
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12 h to 8 h and increasing influent ammonium concentrations from 500 mg N/L to
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1000 mg N/L. Nitrogen removal with or without the application of magnetic field was
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recorded and illustrated in Fig. 2.
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The whole experiment was separated into four stages. Influent ammonium
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concentration of the first two stages was 500 mg N/L and the last two stages was 1000
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mg N/L. HRT of the first and third stages was 12 hours and the second and fourth
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stages was 8 hours. PN process successfully started up in 30 days with nitrite 10
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M group. The HRT was decreased to 8 hours at the second stage. PN process was
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successfully recovered in 10 days in M group while this time was about 18 days for
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CK group. Concentration of the influent ammonium was increased to 1000 mg N/L
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from the 65th day. As expected, PN successfully recovered in a short period of time
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with weak magnetic field compared with no extra magnetic field addition.
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Moreover, there is another important parameter, the ratio of effluent nitrite to
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ammonium which was much higher than 1.3 in both CK group and M group at the
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end of the third stage. Therefore we shortened the HRT into 8 hours on the 95th day.
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Both CK group and the M group cannot startup PN process at the last stage mostly
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because of the short HRT. But we have to clarify that effluent nitrite concentration of
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the M group was higher than that in the CK group. Effluent nitrite concentration of
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the M group and CK group were 450 and 425 mg N/L respectively. What’s more, ratio
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of effluent nitrite to ammonium in M group was 0.9313 and this value in CK group
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was 0.8654. Though the effluent of both groups at the fourth stage unquantified for
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PN process, the effluent of M group was better since the effluent ammonia and nitrite
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were closer. It became clear that the application of magnetic field was an effective
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method to improve the performance of PN reactor and could shorten the start-up time
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of PN process.
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3.3. Nitrogen transformation during partial nitrification process under different
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magnetic field
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The cyclical data which include pH, DO, concentration of ammonium and nitrite 11
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ammonium (RFA) - percentage of free ammonium from the sum of free ammonium
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(FA) and ammonium ion (AI) - under different temperatures were calculated
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simultaneously according the following expression (Ford et al., 1980). Relationship
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among pH, temperature and RFA was shown by Fig.3. RFA was very sensitive to the
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change of pH. Less than 0.05% RFA existed when pH dropped down to 6.1 ± 0.2 at
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28 o C. The amount of free ammonia in the environment is minimal when the pH is
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below 6.5. This is the direct reason that AOB suddenly lost activity in the last period.
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FA FA + AI
As seen in the Fig.4, when the reaction came to 200 minutes, pH and RFA have a
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sharp decline while DO has a rapid rise. At the same time, concentrations of
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ammonium and nitrite have no change after 200 minutes. While it is also clear that the
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accommodation of R5mT to operational parameters was ahead of the CK group. DO
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rise up at 175 minutes in R5mT and the time for CK group was 210 minutes.
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3.4. SOUR of AOB and NOB under different magnetic condition
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In order to further explain the effect of magnetic field on AOB and NOB
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properties of the sludge, SOUR of AOB and NOB in each SBR were tested and
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calculated, as shown in Table 1. The SOUR of AOB in R5 was 2.38 times higher than
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that in R0 and, correspondingly, SOUR of NOB in R5 was lower than that in R0.
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3.5. Effect of magnetic field on the bacterial community of PN reactor 12
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(RCK), and the reactors exposed to 5 mT (R5mT) and 25 mT (R25mT) magnetic field,
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which could be attributed to the magnetic field. A total of 202,443 sequences reads
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(67,476 for RCK, 67,600 for R5mT and 67,367 for R25mT) were obtained after the quality
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control. Among 202,443 sequences reads, a total of 546 OTUs were obtained based on
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identity level at 97%. The three samples shared 264 OTUs and there were 53, 55 and
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13 unique OTUs in the RCK, R5mT and R25mT communities.
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Based on Chao index, the species richness for bacteria was 475.6 and 476.1 in
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RCK and R5mT, and decreased to 430.6 in R25mT. The Shannon index of diversity
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results also demonstrated that the bacteria diversity decreased from 3.35 to 2.99 and
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2.87 with the increased of magnetic field strength to 5 mT and 25 mT.
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The taxonomic classification of effective bacterial sequences from activated
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sludge samples in RCK, R5mT and R25mT at four different taxonomic levels (phylum,
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class, family and genus) is summarized in Fig. 4 which demonstrated that the bacterial
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community compositions were significant distinct. From the phylum assignment
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result (Fig. 4a), it was found that the relative abundance of Bacteroidetes decreased
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from 53.6% in RCK to 40.1% in R5mT and increased to 59.1% in R25mT. On the contrary,
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the relative abundance of Proteobacteria increased from 27.6% in RCK to 35.4% in
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R5mT and decreased to 15.5% in R25mT. Proteobacteria was the second dominant
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phylum in R5mT which include many of the bacteria responsible for nitrogen
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transformation, such as Betaproteobacteria and Gammaproteobacteria belonging to
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AOB (Hommes et al., 1998; Kong et al., 2016). From the class assignment result (Fig.
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4b), the relative abundance of Sphingobacteria increased from 22.8% in RCK to 31.6%
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in R5mT and 30.5% in R25mT. The relative abundance of Betaproteobacteria increased
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from 18.6% in RCK to 29.6% in R5mT and decreased to 11% in R25mT. Bacteria which have a foundation of oxiding ammonium were taxonomically
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belonging to Proteobacteria phylum, Betaproteobacteria class, Nitrosomonadales
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order and Nitrosomonadaceae family. Influence of magnetic field to AOB at different
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taxonomic category level was summarized into table 2. From the family assignment
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result, the relative abundance of Nitrosomonadaceae changed significantly from 13.9%
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in RCK to 12.9% in R5mT and 5.5% in R25mT (p < 0.01). From the class assignment
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result, the relative abundance of Betaproteobacteria changed from 18.6% in RCK to
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29.6% in R5mT and 15.5% in R25mT (p < 0.01).
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To explain the effects of magnetic field on AOB at gene level, species and
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abundances of functional genes were predicted, and statistical analyzed based on 16S
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rRNA gene abundance using the software of PICRUSt (Kanehisa et al., 2016).
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Abundance of functional genes was forecasted based on KEGG database. As shown in
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Fig. 7, the majority of functional gene contents were declined with the increase of
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magnetic field strength, while some functional gene related to signal transduction
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(Herring et al., 2015), cell motility (Guo et al., 2008) and membrane transport
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(Reuscher et al., 2016) were higher expressed compared with no magnetic field and
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high magnetic field strength. Additionally, functional genes abundance forecasted
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based on COG database showed that the ammonia monooxygenase (EC 1.13.12.-) has
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enriched in R5mT compared with RCK and R25mT at q<0.05. Further information about
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metagenome difference among three reactors was supplied in supplementary material.
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4. Discussion The effect of magnetic field to environmental microorganisms has been
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evaluated at the laboratory level. At proper strength, magnetic field may improve the
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activity of bacteria. Otherwise it may deteriorate bacterial performance (Kohno et al.,
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2000). However, the optimum magnetic field intensity is not the same for different
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environmental microorganisms (Filipič et al., 2012). The effect of magnetic field on
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AOB was studied in this paper. Magnetic field inhibited the function of AOB after
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promoting it with the optimum magnetic field intensity of 5 mT. The study has a
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similar conclusion as the reference of Liu et al, in which the anammox activity was
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the highest of 50% increased with the magnetic field strength of 75.0 mT, while the
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activity at higher magnetic field sstrength over 95.0 mT was comparatively low (Liu
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et al., 2008). The insensitive of anammox to magnetic field was mostly caused by its
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anammoxosome, an intracytoplasmic compartment bounded by a single ladderane
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lipid-containing membrane, which decreased the effect of extracellular physical or
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chemical factors. To explore the biological mechanisms of magnetic field to AOB,
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AOB were studied at ammonia oxidation
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expression three levels in this paper.
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microbial community and functional genes
At the first 40 days, wastewater with a high ammonia concentration of 500 mg/L
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was used to inoculate AOB, and no pH control instrument was adopted. HRT of this
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stage was 12 hours. At the end of this stage, PN was achieved. While from fig.4 we
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could see that not the entire 6 hours have ammonia oxidizing activities. When the 15
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while DO has a rapid rise. RFA was lower than 0.05% when pH dropped down to 6.1
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± 0.2 at 28 o C. A hypothesis was therefore been proposed: when the pH of a SBR for
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PN reaction was controlled by pH buffer solutions rather than pH control instrument,
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pH will be decrease with the consumption of ammonia by AOB within a cycle. Ratio
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of free ammonia was dropped down respectively. While only free ammonia could pass
326
the cytomembrane into AOB cells by the way of passive diffusion (Knepper and Agre
327
2004; Zheng et al. 2004). There existed a point where the pH was low enough and
328
ratio of free ammonia was small enough that AOB cannot use. AOB was inactive after
329
that point and therefore dissolved oxygen increased as they cannot be used by AOB.
330
pH and nitrogen concentration didn’t change respectively. PN got into the end and
331
there was useless to prolong the reaction time. According to this hypothesis, magnetic
332
field cannot change the end point compared with the CK group. The cyclical data also
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proved that magnetic field enhanced the activity of AOB as the accommodation of
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R5mT to operational parameters was ahead of the CK group.
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AOB exposed to 5 mT magnetic field expressed higher SOUR than that of the
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CK group, suggesting that AOB had higher abundance or higher activity at 5 mT
337
magnetic field. On the contrary, SOUR of NOB at 5 mT magnetic field was lower
338
than that of the control, indicating lower abundance or activity of NOB at 5 mT
339
magnetic field. The results suggested that weak magnetic field had positive effect on
340
AOB and negative effect on NOB.
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On the community level, magnetic bacteria population increased with the 16
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increase of magnetic field strength. However, AOB declined at order and family level
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with the increase of magnetic field strength. So, activity rather than abundance of
344
AOB lead to the better effect of M group. A necessary step to enhance the effect of PN is to increase the rate of free
346
ammonia into the interior of microbial cells. Abundance of functional genes was
347
forecast based on KEGG database. Majority of functional gene content declined with
348
the rise of the magnetic field strength, while functional gene related to signal
349
transduction, cell motility and membrane transport were higher expressed compared
350
with no magnetic field and high magnetic field strength. And the higher express of
351
those genes may have a directly relationship to the positive effect of magnetic field.
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Functional genes abundance forecasted based on COG database showed that the
353
ammonia monooxygenase (EC 1.13.12.-) was enriched in R5mT compared with RCK
354
and R25mT at q<0.05. Ammonia monooxygenase oxidizing ammonia to hydroxylamine
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is the key enzyme of AOB. Enrichment of ammonia monooxygenase in R5mT was the
356
direct evidence that 5 mT magnetic field enriched AOB.
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The static magnetic field can be generated from two methods. First one is
358
inserting a coil with iron core inside. Direct current can be generated when charging
359
the coil. Magnetic field can also be provided by permanent magnets as the way
360
proposed in this article or other magnetic carriers like magnetically loaded beads.
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Electromagnet is easy to control while it is costly compared with permanent magnets.
362
Despite it is costly to construct magnetic-system, the magnetic field leads to
363
remarkably increased biological activity of PN process. The authors believe that the
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application of magnetic field on PN process could be an alternative choice to enhance
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PN activity and shorten its reaction time.
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5. Conclusion In this study, positive effect of magnetic field on PN process was testified.
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Bacterial diversity decreased under magnetic field exposed condition. Relative
369
abundance of Nitrosomonadaceae changed highly significantly from 13.9% in RCK to
370
12.9% in R5mT and 5.5% in R25m. Functional genes forecast indicated that functional
371
genes related to signal transduction, cell motility and signal transduction of microbes
372
in 5 mT environment were higher expressed compared with no magnetic field
373
addition and high magnetic field addition. Addition of 5 mT magnetic field increased
374
the activity of AOB by increasing the rates of free ammonia into the interior of
375
microbial cells rather than the abundance of AOB. While, magnetic field could not
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change the final state of PN process according to the hypothesis proposed in this
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article.
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Acknowledgement
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The authors thank the support from the National Basic Research Program of
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China (2013CB934301), National Natural Science Foundation of China (21477063
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and 21177075), Taishan Scholar Program (ts201511003), Key R & D project of
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Shandong Province (2016GSF117032), Jinan Science and Technology Project
383
(201401364) and Young Scholars Program of Shandong University.
384
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Table 1 The SOUR of AOB and NOB (mgO2/(gMLSS.min),n=3)
Table 2 Relative contents of AOB at different taxonomic category level in
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RCK, R5mT and R25mT
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Table 1 The SOUR of AOB and NOB (mg O2/(g MLSS.min), n=3) AOB
NOB
R0
0.392 ± 0.0166
0.086 ± 0.018
R5
0.936 ± 0.0809
0.027 ± 0.010
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Table 2 Relative contents of AOB at different taxonomic category level in RCK, R5mT and R25mT RCK
R5mT
R25mT
p__Proteobacteria
27.6 %
35.4 %**
15.5 %**
c__Betaproteobacteria
18.6 %
29.6 %**
15.5 %**
o__Nitrosomonadales
13.9 %
12.9 %**
5.5 %**
f__Nitrosomonadaceae
13.9 %
12.9 %**
5.5 %**
Total reads number
67,476
67,600
67,367
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Taxonomic category of AOB
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**: Statistically significant at p < 0.01 compared with RCK
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Fig.1. Relationships of magnetic field strength and ratios of nitrite to
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ammonium during PN start-up.
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Fig.2.Long-term nitrogenremoval performance without (CK) and with (M) the application of magnetic field.
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Fig. 3. Relationship among pH, ratio of free ammonium and temperature.
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10
3.0
9 2.5
DO(mg/L)
7 6 5 4
M CK
3 2
2.0
1.5
1.0
M CK
0.5
1 0
0.0 0
50
100
150
200
250
300
350
400
0
50
100
Time (min)
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Ratio of free ammonia (%)
8
150
200
250
300
350
400
Time (min)
8.0
450
Nitrogen concentration (mg/L)
7.0
6.5
6.0
5.5
350 300 250
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pH
M CK
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ammunia(M) nitrite (M) ammonium (CK) nitrite (CK)
200 150 100
0
50
100
150
200
250
300
Time (min)
350
400
0
50
100
150
200
250
300
350
400
Time (min)
Fig.4. Time profiles of DO concentration, ratio of free ammonium, pH
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and nitrogen compounds, during typical cycles of SBRs under different
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Fig. 5. Venn diagram of OTUs in RCK, R5mT and R25mT
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Fig. 6. Taxonomic classification of the bacterial communities in RCK, R5mT and R25mT at (A) phylum, (B) class, (C) order and (D) family levels.
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Fig. 7. Prediction of abundance of functional gene contents.
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1. The best Magnetic field for PN process was 5 mT. 2. Weak magnetic field increased the activity of ammonium oxidizing bacteria.
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3. MF increased the rate of free ammonia into cells rather than bacteria abundance.
4. Magnetic field cannot change the final state of partial nitrification
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sludge samples in RCK, R5mT and R25mT at four different taxonomic levels (phylum,
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class, family and genus) is summarized in Fig. 6 which demonstrated that the bacterial
269
community compositions were significant distinct. From the phylum assignment
270
result (Fig. 6A), it was found that the relative abundance of Bacteroidetes decreased
271
from 53.6% in RCK to 40.1% in R5mT and increased to 59.1% in R25mT. On the
272
contrary,the relative abundance of Proteobacteria increased from 27.6% in RCK to
273
35.4% in R5mT and decreased to 15.5% in R25mT. Proteobacteriawas the second
274
dominant phylum in R5mT which include many of the bacteria responsible for nitrogen
275
transformation, such as Betaproteobacteria and Gammaproteobacteria belonging to
276
AOB (Hommes et al., 1998; Kong et al., 2016). From the class assignment result (Fig.
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6B), the relative abundance of Sphingobacteria increased from 22.8% in RCK to 31.6%
278
in R5mT and 30.5% in R25mT. The relative abundance of Betaproteobacteria increased
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from 18.6% in RCK to 29.6% in R5mT and decreased to 11% in R25mT.
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S0043-1354(17)30326-3
DOI:
10.1016/j.watres.2017.04.058
Reference:
WR 12857
To appear in:
Water Research
Received Date: 21 November 2016 Revised Date:
24 March 2017
Accepted Date: 18 April 2017
Please cite this article as: Wang, Z., Liu, X., Ni, S.-Q., Zhang, J., Zhang, X., Ahmad, H.A., Gao, B., Weak magnetic field: A powerful strategy to enhance partial nitrification, Water Research (2017), doi: 10.1016/j.watres.2017.04.058. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The existence of 5 mT magnetic field increased the activity of AOB while
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Weak magnetic field: A powerful strategy to enhance partial nitrification
2
ZhibinWanga, b, Xiaolin Liu a, Shou-Qing Ni a,∗, Jian Zhang a, Xu Zhang a, Hafiz Adeel
4
Ahmad a, Baoyu Gao a
5
a
6
Reuse,School of Environmental Science and Engineering, Shandong University, Jinan
7
250100, China
8
b
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Shandong Provincial Key Laboratory of Water Pollution Control and Resource
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Institute of Marine Science and Technology, Shandong University, 250000, China
9 10
Abstract
Partial nitrification (PN) combined with anaerobic ammonium oxidation process
12
has been recognized as a promising technology for the removal of nitrogenous
13
contaminants from wastewater. This research aimed to investigate the potential of
14
external magnetic field for enhancing the PN process in short and long term
15
laboratory-scale experiments. Different strength magnetic fields (0, 5, 10, 15, 20 and
16
25 mT) were evaluated in short-term batch tests and 5 mT magnetic field was found
17
to have better ability to increase the activities of aerobic ammonium oxidizing
18
bacteria (AOB) of PN consortium. Long-term effect of magnetic field on PN
19
consortium was studied with 5 mT magnetic field. The results demonstrated that the
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∗
Corresponding author. Tel.: +86-531-88365660;fax: +86-531-88364513; E-mail: [email protected]. Author contributions: Zhibin Wang and Xiaolin Liu performed theresearch; Shou-Qing Ni and Jian Zhang designed the research; Xu Zhang analyzed the data; Zhibin Wang wrote the paper; Hafiz Adeel Ahmad and BaoyuGao revised the paper. The authors declare no conflict of interest. 1
ACCEPTED MANUSCRIPT positive effect of magnetic field on PN process could also be testified at all of the four
21
stages. Furthermore, a decrease of bacterial diversity was noted with the increase of
22
magnetic field strength. Relative abundance of Nitrosomonadaceae decreased
23
significantly (p < 0.01) from 13.9% in RCK to 12.9% in R5mT and 5.5% in R25mT.
24
Functional genes forecast based on KEGG database indicated that the expressions of
25
functional genes related to signal transduction and cell motility in 5 mT environment
26
were higher expressed compared with no magnetic field addition and high magnetic
27
field addition. The existence of 5 mT magnetic field didn’t increase the abundance of
28
AOB but increased the activity of AOB by increasing the rate of free ammonia into
29
the interior of microbial cells. Addition of magnetic field couldn’t change the final
30
state of PN process according to the hypothesis proposed in this article. These
31
findings indicated that the weak magnetic field was useful and reliable for the fast
32
start-up of PN process since it was proved as a simple and convenient approach to
33
enhance AOB activity.
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Keywords: Free ammonia; high-throughput sequencing; magnetic field; partial
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nitrification; PICRUSt
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1. Introduction High concentration of ammonium nitrogen and stubborn organics usually present
39
in wastewater of food products factory (Cristian, 2010), pharmaceutical factory
40
(Stackelberg et al., 2004) and landfill leachate (Kjeldsen et al., 2002; Justin and
41
Zupančič, 2009) et al.. A large number of ammonia-nitrogen wastewater discharged
42
into the water body result in not only water eutrophication (Conley et al., 2009), black
43
and malodorous (Wei et al., 2011), increasing the cost of water treatment, but also
44
produce toxic effects on people and other biology (Ono et al., 2000).
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Ammonia-nitrogen removal processes include biological and physicochemical
46
method (Wiszniowski et al., 2006; Mook et al., 2012). Processes of traditional
47
biological nitrogen removal included nitrification of ammonium to nitrate under
48
aerobic conditions (Schmidt, 1982) and denitrifying of nitrate to nitrogen gas under
49
anaerobic conditions by using biodegradable organics as electron acceptors (Kuai and
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Verstraete, 1998). While, there are some drawbacks when conventional biological
51
techniques are applied to treat the high concentration ammonium and insufficient
52
carbon sources wastewater (Du et al., 2015; Bonomo et al., 1997), such as low total
53
nitrogen removal efficiency, unstable running conditions, and high consumption of
54
energy (Khin and Annachhatre, 2004).
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Anaerobic ammonium oxidation (anammox) is an innovative technique
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established in 1990s (Dalsgaard et al., 2005). Anammox is technically superior and
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economically beneficial to treat high concentration ammonium and insufficient
58
carbon sources wastewater compared to traditional process of biological nitrogen 3
ACCEPTED MANUSCRIPT removal as organic source for nitrogen removal is not required in this technique
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(Kotay et al., 2013). In anammox process, ammonium is directly oxidized to nitrogen
61
gas by nitrite under anoxic conditions. In theory, the consumption ratio of nitrite to
62
ammonium is about 1.3 (Van de Graaf et al., 1996; Strous et al., 1998). Therefore, if
63
nitrite does not present in the wastewater, a prior partial nitrification (PN) step is
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required to converts half or more of the ammonium to nitrite (Hao et al., 2002).
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However, the realization and control of PN process is difficult (Guo et al. 2009a).
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Firstly, there must be prevention of the activity of nitrite oxidizing bacteria (NOB)
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which oxidize nitrite to nitrate. Secondly, 57% conversion efficiency of NH4+-N to
68
NO2−-N must be ensured on the basis of the nitrite/ammonium stoichiometric ratio of
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anammox process (Liang et al., 2007; Qiao et al., 2010). Some other operational
70
difficulties such as incoming solids, aeration control and nitrate built-up were also
71
reported (Lackner et al. 2014). Joss et al. (2011) evaluated conditions resulting in
72
instability and strategies to regain efficient operation of full-scale and lab-scale PN
73
process. To startup a PN process, the activity of NOB must be inhibited without
74
affecting ammonium oxidizing bacteria (AOB) by establishing conditions that favor
75
AOB development (Feng et al., 2007). The inhibition of the NOB growth could be
76
achieved by controlling aeration duration to leave no extra time for NOB to convert
77
the accumulated nitrite (Guo et al. 2009b). Liang et al. suggested that PN could be
78
achieved by stepwise increasing of influent NH4+-N at pH of 7.8±0.2, temperature of
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30±1o C, dissolved oxygen (DO) of 0.5-0.8mg l-1, and the HCO3-/NH4+ molar ratio of
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1:1 (Liang et al., 2011). The high NH4+-N concentration, high pH, high temperature,
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nitrite accumulation (Lemaire et al., 2008). To achieve stability and efficiency of the
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PN process, some strategies have been applied. Feng et al. applied a membrane
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bioreactor (MBR) to enrich the AOB in a partial nitrification reactor (Feng et al.,
85
2007). Stable nitrite accumulation was realized for more than 300 days in a nitrifying
86
granular sludge reactor (Vázquez-Padín et al., 2010). The enhancement of AOB and
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the suppression of NOB activity were realized by 0.09 kJ/mg VSS ultrasonic
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treatment (Zheng et al. 2015).
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Usually, organisms have magnetism. The external magnetic field and magnetic
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field exist inside the organisms can bring positive or negative effect to the metabolism
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of living organisms and organization, which is named magnetic biological effect
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(Moore, 1979). Since 1960s, the effect of magnetic field on the microorganisms has
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been investigated (Moore, 1979). For example, the magnetic field with the strength of
94
370 mT resulted in an increase of 35% phenolic degradation (Křiklavová et al., 2014).
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Another research demonstrated that the anammox activity was first increased at weak
96
magnetic field strength, and reached the maximum at 75 mT, but then declined with
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further increase of magnetic field (Liu et al., 2008). Enhancement of phenol oxidation
98
activity under 0.15 and 0.35 T magnetic field was also demonstrated (Jung and Sofer,
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1997). The magnetic biological impact related to biomass metabolism, enzyme
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activity and cell membrane permeability has been proved (Barnothy, 2013). Yet the
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physiology of magnetic biological effect on the microorganisms is not completely
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understood. To the best of literature review, the effect of magnetic field on partial
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nitrification is still in blind. In this study, the possibility of PN enhancement by using static magnetic field
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was first evaluated. The short-term and long-term effects of additional magnetic field
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on PN process were both investigated. Optimum magnetic field strength for AOB was
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ascertained by monitoring the water quality index under different magnetic field
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conditions. SOUR was monitored to verify the effect of optimum magnetic field on
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the activity of AOB and NOB. Finally high-throughput sequencing approach was used
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to elucidate the composition and the change of the microbial community in the PN
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reactors during the long term exposure to magnetic field.
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2. Materials and Methods
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2.1. Inocula and feeding media
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Activated sludge collected from aerobic basin of Guangda municipal wastewater
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treatment plant of Jinan city was used as the seed in this study. To enrich AOB and
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eliminate NOB, high NH4+-N, slightly alkaline medium was used, and the
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composition was as follows: NH4HCO3 (2.82 g L-1), KH2PO4 (0.81 g L-1), CaCl2 (0.2
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g L-1), MgSO4·7H2O (0.03 g L-1) and 1mL trace element solution. Trace element
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solution was prepared and added as described by Kong et al. (2013). The final pH of
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the medium was 8.0 ± 0.1, and no pH control equipment was applied.
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2.2. Batch tests
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Batch tests were performed by using a specially designed bioreactor system. All
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the 6 reactors with working volume of 400mL were incubated in a plastic pool
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(20cm*50cm*100cm) for PN process with a heating rod providing a 35 o C water bath. 6
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The magnetic field strength of the reactor center in the range of 5-25 mT were
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supplied by adjusting the distance of reactors and magnets. The treatment of the
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magnetic field in this study was always continuous. An assembly of air pump,
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electromotor and pottery aerator was used to provide oxygen and blend microbes and
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waste water.
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2.3. Long-term reactor operation
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The long-term experiments were performed in two parallel laboratory-scale
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sequencing batch reactors (SBR). Two columnar reactors made of plexiglas with a
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diameter of 10 cm and height of 30 cm were used. The maximum working volume of
135
each reactor was 6L. Temperature of the system was controlled at 35 ◦C by a water
136
bath. Oxygen was provided by an air pump. Aeration volume was steadied at 1.0L
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min-1 and under this aeration volume, the dissolved oxygen (DO) was fluctuated from
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0.1 mg L-1 to about 7.8 mg L-1.SRT was controlled at 30 days by withdrawing the
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sludge every two days (Guo et al. 2009a). The whole experiment was separated into
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four stages. Influent ammonium concentration of the first two stages was 500 mg N/L
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and the last two stages was 1000 mg N/L. Hydraulic retention time (HRT) of the first
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and third stages was 12 hours and the second and fourth stages was 8 hours. Therefore
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the nitrogen loading rates (NLR) was increased during the fourth stages. Two pairs of
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permanent magnets were installed to provide a static magnetic field of 5 mT. The
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permanent magnets were attached to the exterior reactor body. In the control system,
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the experiment was carried out using the identical reactor parameters without the
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installation of permanent magnets.
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2.4. Analysis The concentration of nitrogen compounds were measured according to the
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standard method (Apha, 1995). The pH measurement was done using a digital,
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portable pH meter, and DO measurement was done using a digital, portable DO meter
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(YSI, Model 55, USA). The volatile suspended solids (VSS) were determined to
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calculate the biomass concentration according to the standard method (Apha, 1995).
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The magnetic strength was measured by Gauss instrument (HT100S) with
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measurement accuracy of ±5%.
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2.5. Biochemical analysis
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A simple method was developed to characterize the nitrification activity by
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specific oxygen uptake rate (SOUR). The method was based on the subsequent
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addition of allylthiorea (ATU) and NaClO3, selective inhibitors of NOB and AOB,
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respectively (Zhang et al., 2015), to the mixed liquid samples of activated sludge in a
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closed batch respirometer. The effects of nitrification inhibitors on the endogenous
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respiration of activated sludge and the heterotrophic substrate oxidation were
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investigated. The triplicate tests were carried out and the results indicated that the
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average oxidation rates of NH4+-N and NO2--N were 12.6 ± 0.3 mg O2/(L·h) and 2.8 ±
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0.5 mg O2/(L·h), respectively. The nitrification activity measured by SOUR was
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validated by determining the variation of nitrate in the running reactors. By means of
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simple measurement of SOUR combined with the subsequent addition of two
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selective nitrification inhibitors, the oxidation rates of NH4+-N, NO2--N and COD can
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be obtained, which is helpful for control and optimization of nitrification process.
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2.6. DNA extraction, high-throughput sequencing and data analysis Total DNA was extracted from 1.5 mL reactor sludge with power soil DNA
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isolation kit (MO BIO Laboratories, USA) following the manufacturer’s
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recommendation. V4 of 16S rRNA were PCR amplified from microbial genome DNA
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using bar-coded fusion primers (forward primers: AYTGGGYDTAAAGNG, reverse
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primers: TACNVGGGTATCTAATCC) (Li et al., 2014). Bar-coded V4 amplicons
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were sequenced using the pair-end method by Illumina Miseqat Personal
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Biotechnology Co., Ltd. (Shanghai, China). Database referenced was greengene. The
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SPSS13.0 (IBM, USA) software package was used for general statistical analysis and
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to conduct a chi-square test. The chi-square values were converted into p values using
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Excel (Microsoft Office) to indicate the significance (Kong et al., 2015).
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2.7. Symbols defined in this study
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To determine the preferable magnetic field strength for PN consortium, six
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reactors under 0 mT, 5 mT, 10 mT, 15 mT, 20 mT and 25 mT magnetic fields were
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described as r0, r5, r10, r15, r20 and r25. To validate the long-term effect of magnetic
185
field on PN process, reactors without extra magnetic field and with 5 mT magnetic
186
field were described as CK group and M group respectively. Lastly, to describe the
187
change of microbial community occurred among reactor exposed to 0 mT, 5 mT and
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25 mT magnetic field, three reactors were defined as RCK, R5mT and R25mT.
189
3. Result
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3.1. Determination of preferable magnetic field strength for PN consortium
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investigated. Six magnetic field strengths were selected for this experiment. The
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relationship of the tested magnetic field strength values versus ratios of NO2-/NH4+
194
was presented in Fig.1.
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The existence of magnetic field had significant effects on PN start-up. Compared
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with the control experiment in which no magnetic field was applied, there was an
197
observable shortening of PN startup time as magnetic field strength increased to 5 mT.
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While when the magnetic field strength increased to 20 and 25 mT, the PN startup
199
time seemed longer than that of the control one. Ratios of nitrite to ammonium of the
200
effluent with 20 mT and 25 mT magnetic field were 0.40 and 0.45, lower than that of
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with no extra magnetic field addition 0.55, indicating that high magnetic field strength
202
had a negative impact on PN process.
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3.2. Long-term effects of magnetic field on PN process
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In order to validate the long-term effects of magnetic field on PN process, two
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parallel SBRs were operated. NLR was increased gradually by decreasing HRT from
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12 h to 8 h and increasing influent ammonium concentrations from 500 mg N/L to
207
1000 mg N/L. Nitrogen removal with or without the application of magnetic field was
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recorded and illustrated in Fig. 2.
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The whole experiment was separated into four stages. Influent ammonium
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concentration of the first two stages was 500 mg N/L and the last two stages was 1000
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mg N/L. HRT of the first and third stages was 12 hours and the second and fourth
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stages was 8 hours. PN process successfully started up in 30 days with nitrite 10
ACCEPTED MANUSCRIPT concentration in effluent wastewater over ammonium concentration in CK group and
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M group. The HRT was decreased to 8 hours at the second stage. PN process was
215
successfully recovered in 10 days in M group while this time was about 18 days for
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CK group. Concentration of the influent ammonium was increased to 1000 mg N/L
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from the 65th day. As expected, PN successfully recovered in a short period of time
218
with weak magnetic field compared with no extra magnetic field addition.
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Moreover, there is another important parameter, the ratio of effluent nitrite to
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ammonium which was much higher than 1.3 in both CK group and M group at the
221
end of the third stage. Therefore we shortened the HRT into 8 hours on the 95th day.
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Both CK group and the M group cannot startup PN process at the last stage mostly
223
because of the short HRT. But we have to clarify that effluent nitrite concentration of
224
the M group was higher than that in the CK group. Effluent nitrite concentration of
225
the M group and CK group were 450 and 425 mg N/L respectively. What’s more, ratio
226
of effluent nitrite to ammonium in M group was 0.9313 and this value in CK group
227
was 0.8654. Though the effluent of both groups at the fourth stage unquantified for
228
PN process, the effluent of M group was better since the effluent ammonia and nitrite
229
were closer. It became clear that the application of magnetic field was an effective
230
method to improve the performance of PN reactor and could shorten the start-up time
231
of PN process.
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3.3. Nitrogen transformation during partial nitrification process under different
233
magnetic field
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The cyclical data which include pH, DO, concentration of ammonium and nitrite 11
ACCEPTED MANUSCRIPT of the two reactors were monitored and calculated on day 40. Ratios of free
236
ammonium (RFA) - percentage of free ammonium from the sum of free ammonium
237
(FA) and ammonium ion (AI) - under different temperatures were calculated
238
simultaneously according the following expression (Ford et al., 1980). Relationship
239
among pH, temperature and RFA was shown by Fig.3. RFA was very sensitive to the
240
change of pH. Less than 0.05% RFA existed when pH dropped down to 6.1 ± 0.2 at
241
28 o C. The amount of free ammonia in the environment is minimal when the pH is
242
below 6.5. This is the direct reason that AOB suddenly lost activity in the last period.
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As seen in the Fig.4, when the reaction came to 200 minutes, pH and RFA have a
244
sharp decline while DO has a rapid rise. At the same time, concentrations of
245
ammonium and nitrite have no change after 200 minutes. While it is also clear that the
246
accommodation of R5mT to operational parameters was ahead of the CK group. DO
247
rise up at 175 minutes in R5mT and the time for CK group was 210 minutes.
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3.4. SOUR of AOB and NOB under different magnetic condition
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In order to further explain the effect of magnetic field on AOB and NOB
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properties of the sludge, SOUR of AOB and NOB in each SBR were tested and
251
calculated, as shown in Table 1. The SOUR of AOB in R5 was 2.38 times higher than
252
that in R0 and, correspondingly, SOUR of NOB in R5 was lower than that in R0.
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3.5. Effect of magnetic field on the bacterial community of PN reactor 12
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(RCK), and the reactors exposed to 5 mT (R5mT) and 25 mT (R25mT) magnetic field,
256
which could be attributed to the magnetic field. A total of 202,443 sequences reads
257
(67,476 for RCK, 67,600 for R5mT and 67,367 for R25mT) were obtained after the quality
258
control. Among 202,443 sequences reads, a total of 546 OTUs were obtained based on
259
identity level at 97%. The three samples shared 264 OTUs and there were 53, 55 and
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13 unique OTUs in the RCK, R5mT and R25mT communities.
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Based on Chao index, the species richness for bacteria was 475.6 and 476.1 in
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RCK and R5mT, and decreased to 430.6 in R25mT. The Shannon index of diversity
263
results also demonstrated that the bacteria diversity decreased from 3.35 to 2.99 and
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2.87 with the increased of magnetic field strength to 5 mT and 25 mT.
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The taxonomic classification of effective bacterial sequences from activated
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sludge samples in RCK, R5mT and R25mT at four different taxonomic levels (phylum,
267
class, family and genus) is summarized in Fig. 4 which demonstrated that the bacterial
268
community compositions were significant distinct. From the phylum assignment
269
result (Fig. 4a), it was found that the relative abundance of Bacteroidetes decreased
270
from 53.6% in RCK to 40.1% in R5mT and increased to 59.1% in R25mT. On the contrary,
271
the relative abundance of Proteobacteria increased from 27.6% in RCK to 35.4% in
272
R5mT and decreased to 15.5% in R25mT. Proteobacteria was the second dominant
273
phylum in R5mT which include many of the bacteria responsible for nitrogen
274
transformation, such as Betaproteobacteria and Gammaproteobacteria belonging to
275
AOB (Hommes et al., 1998; Kong et al., 2016). From the class assignment result (Fig.
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4b), the relative abundance of Sphingobacteria increased from 22.8% in RCK to 31.6%
277
in R5mT and 30.5% in R25mT. The relative abundance of Betaproteobacteria increased
278
from 18.6% in RCK to 29.6% in R5mT and decreased to 11% in R25mT. Bacteria which have a foundation of oxiding ammonium were taxonomically
280
belonging to Proteobacteria phylum, Betaproteobacteria class, Nitrosomonadales
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order and Nitrosomonadaceae family. Influence of magnetic field to AOB at different
282
taxonomic category level was summarized into table 2. From the family assignment
283
result, the relative abundance of Nitrosomonadaceae changed significantly from 13.9%
284
in RCK to 12.9% in R5mT and 5.5% in R25mT (p < 0.01). From the class assignment
285
result, the relative abundance of Betaproteobacteria changed from 18.6% in RCK to
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29.6% in R5mT and 15.5% in R25mT (p < 0.01).
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To explain the effects of magnetic field on AOB at gene level, species and
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abundances of functional genes were predicted, and statistical analyzed based on 16S
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rRNA gene abundance using the software of PICRUSt (Kanehisa et al., 2016).
290
Abundance of functional genes was forecasted based on KEGG database. As shown in
291
Fig. 7, the majority of functional gene contents were declined with the increase of
292
magnetic field strength, while some functional gene related to signal transduction
293
(Herring et al., 2015), cell motility (Guo et al., 2008) and membrane transport
294
(Reuscher et al., 2016) were higher expressed compared with no magnetic field and
295
high magnetic field strength. Additionally, functional genes abundance forecasted
296
based on COG database showed that the ammonia monooxygenase (EC 1.13.12.-) has
297
enriched in R5mT compared with RCK and R25mT at q<0.05. Further information about
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metagenome difference among three reactors was supplied in supplementary material.
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4. Discussion The effect of magnetic field to environmental microorganisms has been
301
evaluated at the laboratory level. At proper strength, magnetic field may improve the
302
activity of bacteria. Otherwise it may deteriorate bacterial performance (Kohno et al.,
303
2000). However, the optimum magnetic field intensity is not the same for different
304
environmental microorganisms (Filipič et al., 2012). The effect of magnetic field on
305
AOB was studied in this paper. Magnetic field inhibited the function of AOB after
306
promoting it with the optimum magnetic field intensity of 5 mT. The study has a
307
similar conclusion as the reference of Liu et al, in which the anammox activity was
308
the highest of 50% increased with the magnetic field strength of 75.0 mT, while the
309
activity at higher magnetic field sstrength over 95.0 mT was comparatively low (Liu
310
et al., 2008). The insensitive of anammox to magnetic field was mostly caused by its
311
anammoxosome, an intracytoplasmic compartment bounded by a single ladderane
312
lipid-containing membrane, which decreased the effect of extracellular physical or
313
chemical factors. To explore the biological mechanisms of magnetic field to AOB,
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AOB were studied at ammonia oxidation
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expression three levels in this paper.
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microbial community and functional genes
At the first 40 days, wastewater with a high ammonia concentration of 500 mg/L
317
was used to inoculate AOB, and no pH control instrument was adopted. HRT of this
318
stage was 12 hours. At the end of this stage, PN was achieved. While from fig.4 we
319
could see that not the entire 6 hours have ammonia oxidizing activities. When the 15
ACCEPTED MANUSCRIPT reaction came to 200 minutes, pH and ratio of free ammonium have a sharp decline
321
while DO has a rapid rise. RFA was lower than 0.05% when pH dropped down to 6.1
322
± 0.2 at 28 o C. A hypothesis was therefore been proposed: when the pH of a SBR for
323
PN reaction was controlled by pH buffer solutions rather than pH control instrument,
324
pH will be decrease with the consumption of ammonia by AOB within a cycle. Ratio
325
of free ammonia was dropped down respectively. While only free ammonia could pass
326
the cytomembrane into AOB cells by the way of passive diffusion (Knepper and Agre
327
2004; Zheng et al. 2004). There existed a point where the pH was low enough and
328
ratio of free ammonia was small enough that AOB cannot use. AOB was inactive after
329
that point and therefore dissolved oxygen increased as they cannot be used by AOB.
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pH and nitrogen concentration didn’t change respectively. PN got into the end and
331
there was useless to prolong the reaction time. According to this hypothesis, magnetic
332
field cannot change the end point compared with the CK group. The cyclical data also
333
proved that magnetic field enhanced the activity of AOB as the accommodation of
334
R5mT to operational parameters was ahead of the CK group.
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AOB exposed to 5 mT magnetic field expressed higher SOUR than that of the
336
CK group, suggesting that AOB had higher abundance or higher activity at 5 mT
337
magnetic field. On the contrary, SOUR of NOB at 5 mT magnetic field was lower
338
than that of the control, indicating lower abundance or activity of NOB at 5 mT
339
magnetic field. The results suggested that weak magnetic field had positive effect on
340
AOB and negative effect on NOB.
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increase of magnetic field strength. However, AOB declined at order and family level
343
with the increase of magnetic field strength. So, activity rather than abundance of
344
AOB lead to the better effect of M group. A necessary step to enhance the effect of PN is to increase the rate of free
346
ammonia into the interior of microbial cells. Abundance of functional genes was
347
forecast based on KEGG database. Majority of functional gene content declined with
348
the rise of the magnetic field strength, while functional gene related to signal
349
transduction, cell motility and membrane transport were higher expressed compared
350
with no magnetic field and high magnetic field strength. And the higher express of
351
those genes may have a directly relationship to the positive effect of magnetic field.
352
Functional genes abundance forecasted based on COG database showed that the
353
ammonia monooxygenase (EC 1.13.12.-) was enriched in R5mT compared with RCK
354
and R25mT at q<0.05. Ammonia monooxygenase oxidizing ammonia to hydroxylamine
355
is the key enzyme of AOB. Enrichment of ammonia monooxygenase in R5mT was the
356
direct evidence that 5 mT magnetic field enriched AOB.
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The static magnetic field can be generated from two methods. First one is
358
inserting a coil with iron core inside. Direct current can be generated when charging
359
the coil. Magnetic field can also be provided by permanent magnets as the way
360
proposed in this article or other magnetic carriers like magnetically loaded beads.
361
Electromagnet is easy to control while it is costly compared with permanent magnets.
362
Despite it is costly to construct magnetic-system, the magnetic field leads to
363
remarkably increased biological activity of PN process. The authors believe that the
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application of magnetic field on PN process could be an alternative choice to enhance
365
PN activity and shorten its reaction time.
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5. Conclusion In this study, positive effect of magnetic field on PN process was testified.
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Bacterial diversity decreased under magnetic field exposed condition. Relative
369
abundance of Nitrosomonadaceae changed highly significantly from 13.9% in RCK to
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12.9% in R5mT and 5.5% in R25m. Functional genes forecast indicated that functional
371
genes related to signal transduction, cell motility and signal transduction of microbes
372
in 5 mT environment were higher expressed compared with no magnetic field
373
addition and high magnetic field addition. Addition of 5 mT magnetic field increased
374
the activity of AOB by increasing the rates of free ammonia into the interior of
375
microbial cells rather than the abundance of AOB. While, magnetic field could not
376
change the final state of PN process according to the hypothesis proposed in this
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article.
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Acknowledgement
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The authors thank the support from the National Basic Research Program of
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China (2013CB934301), National Natural Science Foundation of China (21477063
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and 21177075), Taishan Scholar Program (ts201511003), Key R & D project of
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Shandong Province (2016GSF117032), Jinan Science and Technology Project
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(201401364) and Young Scholars Program of Shandong University.
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Table 1 The SOUR of AOB and NOB (mgO2/(gMLSS.min),n=3)
Table 2 Relative contents of AOB at different taxonomic category level in
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RCK, R5mT and R25mT
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Table 1 The SOUR of AOB and NOB (mg O2/(g MLSS.min), n=3) AOB
NOB
R0
0.392 ± 0.0166
0.086 ± 0.018
R5
0.936 ± 0.0809
0.027 ± 0.010
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Table 2 Relative contents of AOB at different taxonomic category level in RCK, R5mT and R25mT RCK
R5mT
R25mT
p__Proteobacteria
27.6 %
35.4 %**
15.5 %**
c__Betaproteobacteria
18.6 %
29.6 %**
15.5 %**
o__Nitrosomonadales
13.9 %
12.9 %**
5.5 %**
f__Nitrosomonadaceae
13.9 %
12.9 %**
5.5 %**
Total reads number
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67,600
67,367
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Taxonomic category of AOB
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Fig.1. Relationships of magnetic field strength and ratios of nitrite to
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ammonium during PN start-up.
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Fig.2.Long-term nitrogenremoval performance without (CK) and with (M) the application of magnetic field.
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Fig. 3. Relationship among pH, ratio of free ammonium and temperature.
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10
3.0
9 2.5
DO(mg/L)
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M CK
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Fig.4. Time profiles of DO concentration, ratio of free ammonium, pH
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Fig. 5. Venn diagram of OTUs in RCK, R5mT and R25mT
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Fig. 6. Taxonomic classification of the bacterial communities in RCK, R5mT and R25mT at (A) phylum, (B) class, (C) order and (D) family levels.
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Fig. 7. Prediction of abundance of functional gene contents.
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1. The best Magnetic field for PN process was 5 mT. 2. Weak magnetic field increased the activity of ammonium oxidizing bacteria.
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4. Magnetic field cannot change the final state of partial nitrification
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sludge samples in RCK, R5mT and R25mT at four different taxonomic levels (phylum,
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class, family and genus) is summarized in Fig. 6 which demonstrated that the bacterial
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community compositions were significant distinct. From the phylum assignment
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result (Fig. 6A), it was found that the relative abundance of Bacteroidetes decreased
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from 53.6% in RCK to 40.1% in R5mT and increased to 59.1% in R25mT. On the
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contrary,the relative abundance of Proteobacteria increased from 27.6% in RCK to
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35.4% in R5mT and decreased to 15.5% in R25mT. Proteobacteriawas the second
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dominant phylum in R5mT which include many of the bacteria responsible for nitrogen
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transformation, such as Betaproteobacteria and Gammaproteobacteria belonging to
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AOB (Hommes et al., 1998; Kong et al., 2016). From the class assignment result (Fig.
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6B), the relative abundance of Sphingobacteria increased from 22.8% in RCK to 31.6%
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in R5mT and 30.5% in R25mT. The relative abundance of Betaproteobacteria increased
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from 18.6% in RCK to 29.6% in R5mT and decreased to 11% in R25mT.
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