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Bromate and Nitrate Bio-reduction Coupled with Poly-#-Hydroxybutyrate Production in a Methane-based Membrane Biofilm Reactor Chun-Yu Lai, Pan-Long Lv, Qiu-Yi Dong, Shi Lei Yeo, Bruce E. Rittmann, and He-Ping Zhao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00152 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018
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Bromate and Nitrate Bio-reduction Coupled with
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Poly-β-Hydroxybutyrate Production in a Methane-based
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Membrane Biofilm Reactor
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Chun-Yu Lai1, #, Pan-Long Lv1, Qiu-Yi Dong1, Shi Lei Yeo1, Bruce E. Rittmann2,
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He-Ping Zhao1, *
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1. College of Environmental and Resource Science, Zhejiang University, Hangzhou,
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China.
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2. Biodesign Swette Center for Environmental Biotechnology, Arizona State
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University, P.O. Box 875701, Tempe, Arizona 85287-5701, USA.
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# Current address: Advanced Water Management Centre, The University of Queensland, St
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Lucia, Queensland 4072, Australia
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* Correspondence to Dr. He-Ping Zhao. Tel (Fax): 0086-571-88982739
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E-mail:
[email protected] 15
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Abstract
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This work demonstrates bromate (BrO3-) reduction in a methane (CH4)-based
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membrane biofilm reactor (MBfR), and it documents contrasting impacts of nitrate
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(NO3-) on BrO3- reduction, as well as formation of poly--hydroxybutyrate (PHB), an
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internal C- and electron-storage material.
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ample supply, NO3- enhanced BrO3- reduction by stimulating the growth of
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denitrifying bacteria (Meiothermus, Comamonadaceae, and Anaerolineaceae) able to
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reduce BrO3- and NO3- simultaneously. This was supported by increases in
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denitrifying enzymes (e. g., nitrate reductase, nitrite reductase, nitrous-oxide reductase
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and nitric-oxide reductase) through quantitative polymerase chain reaction (qPCR)
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analysis and metagenomic prediction of these functional genes. When the electron
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donor was in limited supply, NO3- was the preferred electron acceptor over BrO3- due
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to competition for the common electron donor; this was supported by the significant
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oxidation of stored PHB when NO3- was high enough to cause electron-donor
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limitation.
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Comamonadaceae) were implicated as the main PHB producers in the biofilms, and
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their ability to oxidize PHB mitigated the impacts of competition for CH4.
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Key Words: methane, poly-β-hydroxybutyrate, bromate, nitrate, membrane-biofilm
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reactor, microorganism
When the electron donor, CH4, was in
Methanotrophs (e. g., Methylocystis, Methylomonas, and genera within
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Introduction
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Bromate (BrO3-) is a disinfection by-product mainly formed from bromide ion (Br-)
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during advanced oxidation, such as ozonation.1
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of natural sources (e. g., rock dissolution and saltwater intrusion)2 and human
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activities (e.g., a gasoline additive),3 BrO3- is common in drinking-water sources that
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are treated by advanced oxidation.1
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contamination can cause a high BrO3- concentration (>1 mg/L)1.
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suspected human carcinogen due to its strong ability to damage DNA.4, 5 Drinking
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BrO3- may lead to renal tumors and peritoneal mesothelioma.6, 7
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contaminant level (MCL) for BrO3- is established at 10 µg/L by Environmental
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Protection Agency in USA (2015).8
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A frequent co-contaminant is nitrate (NO3-), which is associated with a variety of
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human activities, e.g., manufacturing, agriculture, and domestic sewage.9-11 Butler et
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al. reported that the groundwater obtained from a contaminated aquifer in the UK had
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a BrO3- concentration of 1.1-1.4 mg/L and a NO3--N concentration of 8.6-30.7 mg/L.12
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NO3- and its bioreduction intermediate nitrite (NO2-) can cause methemoglobinemia in
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infants;13 the MCLs for NO3- and NO2- are 10 and 1 mg N/L, respectively.8
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Bio-reduction of BrO3- and NO3- to innocuous bromide (Br-) and nitrogen gas (N2)
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holds promise for their removal due to its simplicity, sustainability, and low cost. In
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recent years, the application of methane (CH4) as the electron donor for bio-reduction
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has drawn attention, because CH4 gas has extensive availability, is low cost, and does
Since Br- is associated with a variety
Butler et al. reported that industrial
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BrO3- is a
The maximum
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not leave a residual due to its low solubility.14-16 The CH4-based membrane biofilm
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reactor (MBfR) is a novel technology for delivering CH4 as the electron donor. CH4
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diffuses through the wall of gas-transfer membranes and supports growth of a biofilm
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of bacteria able to respire the oxidized contaminants.17-19
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NO3- in the CH4-based MBfR has been demonstrated,18 CH4-based bio-reduction of
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BrO3- hardly has been explored.20
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We hypothesize that BrO3- also can be respired using CH4 as the sole electron donor
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and carbon source.
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bromate-reducing bacteria should gain substantial energy by BrO3- respiration
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coupled with CH4 oxidation (the CO2/CH4 standard reduction potential is −240 mV).
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Furthermore, bromate-reducing bacteria are common and phenotypically diverse,
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including denitrifiers,22 chlorate-reducing bacteria,23 and sulfate-reducing bacteria.24
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All of these functional microbes have been enriched in the CH4-based MBfR.17-19, 25
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NO3- could have multiple effects on BrO3- reduction. NO3- might promote BrO3-
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reduction, since nitrate reductases in bacteria are reported to reduce BrO3-.2, 26
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may act as the primary electron acceptor to support the accumulation of bacteria also
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able to respire BrO3-.22, 27
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competition for a scarce electron donor.28, 29 Downing et al. showed that NO3- was
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reduced prior to BrO3- in a hydrogen (H2)-based MBfR when H2 was limiting.30
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The first objective of this study was to document BrO3- reduction using CH4 as the
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sole electron donor and carbon source. The second objective was to investigate the
Although respiration of
BrO3-/Br- has a high redox potential (1440 mV),21 meaning the
NO3-
NO3- might also slow BrO3- reduction due to the
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interactions between NO3- on BrO3- reductions in the MBfR.
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mechanisms involved in the interactions between NO3- on BrO3-, we analyzed the
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microbial community structure in the biofilms by high-throughput Illumina
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sequencing of the 16S rRNA gene, tracked the changes of key functional genes using
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Phylogenetic Investigation of Communities by Reconstruction of Unobserved States
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(PICRUSt) pipeline, and quantified the denitrifying genes by quantitative polymerase
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chain reaction (qPCR).
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We also tracked the accumulation of poly (3-hydroxybutyrate) (PHB), which many
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methanotrophs use as an intracellular electron- and carbon-storage material when the
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CH4 supply is in excess of the supply of electron acceptors.31
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et al.33 reported that PHB can be hydrolysed and oxidized when the donor is in short
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supply, but acceptors are plentiful.
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Thus, the third objective of this study was to quantify the effect of BrO3- and NO3-
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reduction on PHB synthesis and oxidation in the CH4-fed biofilms.
In order to explain the
Baric et al.32 and Third
In addition, PHB is a feedstock for bioplastics.34
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Materials and Methods
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MBfR Operation.
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The CH4-MBfR system, the same as use by Lai et al.17, contained 42 hollow fibers
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manufactured by Mitsubishi Rayon (model MHF-200TL, Mitsubishi, Ltd., Japan).
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The total volume and total membrane surface area of the MBfR were 65 mL and 58
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cm2, respectively. The liquid recirculation rate was 100 mL/min, and the CH4
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pressure was 10 psig (1.68 atm) throughout the experiments.
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operated in a thermostatic chamber having a temperature of 35 ± 1 °C throughout the
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experiments.
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The MBfR was inoculated with 5 mL of a mixed culture able to perform anaerobic
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oxidation of methane coupled to nitrate and perchlorate reduction. 19
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for the MBfR was an inorganic medium deoxygenated with argon (Ar).17
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introduced into the MBfR medium containing 5 mg/L BrO3- and recirculated the
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medium for 48 h. We then kept the influent BrO3- at 0.8 mg/L (corresponding to a
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surface loading of 100 mg BrO3-/m2-d) throughout the experiments. In order to
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evaluate the effect of NO3- on BrO3- reduction, we then applied NO3- in the influent at
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0, 1, 0, 5, and 0 mg NO3--N/L through Stages 1-5, respectively. We moved to a new
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stage when the effluent concentrations of BrO3- and NO3- varied by
