Role of Extracellular Polymeric Substances in a Methane Based

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Role of Extracellular Polymeric Substances in a Methane Based Membrane Biofilm Reactor Reducing Vanadate Chun-yu Lai, Qiu-Yi Dong, Jiaxian Chen, Quan-Song Zhu, Xin Yang, Wen-Da Chen, He-Ping Zhao, and Liang Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02374 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Environmental Science & Technology

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Role of Extracellular Polymeric Substances in a Methane

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Based Membrane Biofilm Reactor Reducing Vanadate

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∥ † † † † Chun-Yu Lai†, , Qiu-Yi Dong , Jia-Xian Chen , Quan-Song Zhu , Xin Yang , Wen-Da

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Chen†, He-Ping Zhao†, ,

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‡ §,

†

*

, Liang Zhu†, , * ‡

College of Environmental and Resource Science, Zhejiang University, Hangzhou,

China. ‡

Zhejiang Prov Key Lab Water Pollut Control & Envi, Zhejiang University, Hangzhou,

Zhejiang, China. §

MOE Key Lab of Environmental Remediation and Ecosystem Health, College of

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Environmental and Resource Science, Zhejiang University, Hangzhou, China,

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310058.

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∥

Advanced Water Management Centre, The University of Queensland, St Lucia,

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Queensland 4072, Australia

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* Correspondance to Dr. Liang Zhu, Email: [email protected];

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and Dr. He-Ping Zhao. E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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For the first time, we demonstrated vanadate (V(V)) reduction in a membrane biofilm

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reactor (MBfR) using CH4 as the sole electron donor. V(V)-reducing capability of the

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biofilm kept increasing, with complete removal of V(V) achieved when the influent

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surface loading of V(V) was 363 mg m-2 day-1. Almost all V(V) was reduced to V(IV)

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precipitates, which is confirmed by scanning electron microscope coupled to energy

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dispersive X-ray spectroscopy (SEM-EDS) and X-ray photoelectron spectroscope

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(XPS). Microbial community analysis revealed that denitrifiers Methylomonas and

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Denitratisoma might be the main genera responsible for V(V) reduction. The constant

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enrichment of Methylophilus suggests that the intermediate (i.e., methanol) from CH4

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metabolism was used as the electron carriers for V(V) bioreduction.

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V(V) (2-5 mg/L, at the surface loading of 150-378 mg m-2 day-1) into the biofilm

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stimulated the secretion of extracellular polymeric substances (EPS), but high loading

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of V(V) (10 mg/L, at the surface loading of 668 mg m-2 day-1) decreased the amount

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of EPS.

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the secretion of EPS and the microbial metabolism associated with V(V) reduction,

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tricarboxylic acid cycle (TCA) cycle, methane oxidation and ATP production, and

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EPS might relieve the oxidative stress induced by high loading of V(V). Colorimetric

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determination and three-dimensional excitation–emission matrix (3D-EEM) showed

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that tryptophan and humic acid-like substances might play important roles in

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microbial cells protection and V(V) binding. Fourier transform infrared (FTIR)

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spectroscopy identified hydroxyl (-OH), and carboxyl (COO-) groups in EPS as the

Intrusion of

Metagenomic prediction analysis established the strong correlation between

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candidate functional groups for binding V(V).

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Keywords:

vanadate, methane, MBfR, extracellular polymeric substances

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TOC

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Introduction

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Vanadium (V), a widely spreading transition metal in the world, is associated with a

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variety of natural resources, e. g., rocks1, minerals2, as well as industries such as

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metallurgy3, petroleum refining4, and manufactures of oxidation catalysis and alloys.5

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Although V is an essential element that benefits organisms at trace levels, it becomes

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toxic when the concentration is > 1 mg/L.6

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and phosphate, V can interfere with the phosphate-related enzymes and negatively

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affect the phosphate metabolism.7 Besides, V is mutagenic and cytotoxic, and can

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interfere with gene expression and cellular differentiation.8

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maximum permitted V concentration at 0.05 mg/L in drinking water (Standard of

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China GB 5749-20, 2015).

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and vanadyl (V(IV)).

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less toxic and usually exists as precipitates in neutral or alkaline condition.9

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reduction of V(V) to V(IV) is a feasible way to detoxify V in wastewaters.10

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Due to the simplicity and low costs, bio-reduction of V(V) to V(IV) is deemed a

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promising strategy for V(V) removal.

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diverse, including bacteria Shewanella oneidensis5, Acidithiobacillus thiooxidans11,

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and Geobacter metallireducens10, and archea Methanosarcina mazei and

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Methanothermobacter thermautotrophicus.12 These microorganisms perform V(V)

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reduction depending on cytochromes located in the cytoplasmic and outer membranes,

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menaquinone, or nitrate reductase.13,14

Due to the similar structure between V(V)

China has set the

The common oxidation states of V are vanadate (V(V))

While V(V) has high solubility and toxicity2, V(IV) is much Thus,

Vanadate-reducers are phylogenetically

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Various of pure strains or mixed cultures have been studied for bio-reduction of V(V).

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In these studies, soluble organics, e., g. methanol, acetate, lactate, glucose, citrate or

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soluble starch was supplied as the electron donor and carbon sources.5,12,15 All of

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them have the potential to leave electron donor residual in the treated water, which

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adds the cost and leads to biomass clogging.

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methane (CH4) was able to serve as the electron donor to support microbial

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bio-reduction of oxidized contaminants.16,17

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greenhouse gas that is readily available from large fossil reserves and anaerobic

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digestion of biomass.

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water.

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potential of CO2/CH4 (-0.24V) suggests the possibility of bio-reduction of V(V) to

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V(IV) using CH4 as the electron donor.18

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evaluate V(V) reduction in a CH4-based membrane biofilm reactor (MBfR) and

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investigate the functional microbial groups involved in CH4 oxidation and V(V)

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reduction.

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Applying biofilm based technologies for remediation of wastewater which contains

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toxic metals like V has great advantage since biofilm has strong resistance and

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acclimation to toxic metals compared with free-swimming cells.19,20 Reported

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mechanisms that contribute to biofilm’s resistance and acclimation are sequestration,

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reduction, and binding of toxic metal by extracellular polymeric substances (EPS).21,22

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EPS, which mainly consists of polysaccharides, proteins, and nucleic acids, is usually

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secreted by microbial cells in the biofilms.23,24

In our recent studies, we reported that

CH4 is an inexpensive and potent

It does not leave electron residue due to its low solubility in

The high redox potential of V(V)/V(IV) (0.99 V) and the negative redox

Thus, the first objective of this study is to

EPS may play an important role in

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metal reduction via redox active components or extracellular enzymes.22,25,26

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also play an important role in binding of toxic metals via its functional groups such as

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hydroxyl, acetamido or amino groups.27,28 In addition, EPS is recognized as an

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alternative to chemical polymers due to its non-toxicity and biodegradability.29

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Therefore, it is essential to understand the production and properties of EPS in this

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study.

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chromate (Cr(VI)), cadmium(II), lead(II), nickel(II) have been studied, the response

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of EPS on V(V) removal (especially in the CH4 based system) has never been

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explored.

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between the secreted ESP and the V(V) removal in the CH4-based MBfR.

It may

Although the interactions between EPS and toxic heavy metals such as

Thus, the second objective of this study is to investigate the relationship

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Materials and Methods

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MBfR Startup and continuous operation

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The MBfR system was set up and the medium were prepared in the similar way as

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described by Lai et al., except that the number of coupon fibers was increased to 32.16

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The total volume of the MBfR was 65 mL, and the total surface area of all fibers was

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100 cm2.

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the speed of 100 mL/min, and anther pump was used to continuously feed the influent

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into the reactor at 0.5 mL/min.

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pressure was maintained at 15 psig.

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A culture that was able to perform micro-aerobic methane oxidation coupled to nitrate

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reduction was inoculated into the MBfR system.

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containing 10 mg/L V(V) for 48 h to enhance microbial cells attachment to the fibers.

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We degassed the medium with argon (Ar), and then varied the influent V(V)

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concentration as 2, 5, 10 and 5 mg V/L in Stage 1, 2, 3 and 4, respectively. We

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operated each stage for at least 14 days until a steady state was achieved (the variation

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of the concentrations of V(V) and total soluble V in the effluent were < 10%).

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Chemical Analysis

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We sampled the liquids from the influent and effluent of the MBfR using syringes,

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and filtered them instantly using 0.22-µm membrane filters.

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samples to remove V(IV) precipitates (15000g, 10 min). We determined the V(V)

One peristaltic pump was applied to recirculate the liquid in the MBfR at

The temperature was kept at 35±1 0C, and the CH4

We recirculated the medium

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We then centrifuged

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and V(IV) concentration by spectrophotometric reaction rate method described by

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Ensafi et al. and Safavi et al., respectively.30,31 We determined the dissolved oxygen

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(O2) using a dissolved oxygen probe (Starter, Ohaus Instruments Company, Germany),

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and found the concentrations were below 0.2 mg/L for the influent and effluent.

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range of pH values in the influent and effluent was 7.0-7.5 measured by a pH meter

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(Seven Easy, Mettler Toledo, Switzerland).

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Flux Calculation

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The V(V) and O2 removal fluxes (g m−2 day−1) were calculated according to

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J = (S0 − S)Q/A

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in which S0 and S are influent and effluent concentrations of V(V) and oxygen (g/L),

127

respectively; Q is the influent flow velocity of the MBfR system (L/day), and A is the

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membrane surface area (m2).

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removal flux of electron acceptors (equation 1) and chemical stoichiometry (equation

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2 and 3).18

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VO2+ + 0.338CH4 + 0.059NO3- + 1.058H+ → VO2+ + 0.059C5H7O2N + 0.032CO2 +

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0.994H2O (2)

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O2 + 1.25CH4 + 0.214NO3 + 0.214H → 0.179CO2 + 1.86H2O + 0.214C5H7O2N (3)

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We calculated the maximum CH4 delivery capacity (e− meq/m2day) according to Lai

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et al.16,17, 32

The

(1)

-

We also calculated the consumed CH4 flux based on

+

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Biofilm Samples Collecting and Imaging, and DNA Extraction

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We cut a coupon fiber of 8 cm in length for observation by scanning electron

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microscope and energy dispersive X-ray spectroscopy (SEM-EDS), characterization

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by X-ray photoelectron spectroscope (XPS) and DNA extraction for each stage as

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described by Lai et al.16,17 We performed polymerase chain reaction (PCR) targeting

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V4-V5 regions of the bacterial 16S rRNA gene by using the primer pair 515F

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(5’-GTGCCAGCMGCCGCGG-3’ and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’).

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We purified the amplicons using the AxyPrep DNA Gel Extraction Kit (Axygen

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Biosciences, Union City, CA, U.S.A.) and sent them to Novogene Technology

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(Beijing, China) for library construction and Illumina MiSeq sequencing with

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standard protocols.

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as described by Lai et al.33 We also applied PICRUSt to predict the metagenomic

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information of the biofilm for each stage based on the mapping of sequenced 16S

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rRNA gene data and the KEGG database.16

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EPS Extraction, quantification, and characterization

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We cut 5 coupon fibers of 15 cm in length for each stage, and detached the biofilms as

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described by Luo et al.34 We centrifuged the biofilms at 3000 g by a centrifuge

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(3K18, Sigma, Germany) for 5 min and removed the supernatant.

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were collected for volatile suspended solids (VSS) measurement according to

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Standard Methods.35

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et al.36

Raw sequences were processed using the QIIME (version 1.9.1)

Parts of biofilms

We performed EPS extraction by the updated method from Yu

The remaining pellets were mixed with 20 mL of phosphate buffer saline, 10


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and vortexed (Vortex- Genie® 2, Mo Bio laboratories, Inc., USA) for 15 min.

The

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mixtures were then ultrasonically treated, and incubated in a water bath (CU-420,

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Blue Pard, China) at 80 0C for 20 min.

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15 min, and filtered the supernatants by 0.45-µm membrane filters.

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were collected as EPS. Polysaccharide in the collected EPS was determined by

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anthrone method, and glucose was used for standard curve construction. 37

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the collected EPS was measured by Coomassie brilliant blue G-250 dye-binding

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method as described by Pierce＆Suelter38, and humic acid substances in the collected

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EPS were determined by modified Lowry method as described by Frolund et al. using

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humic acid as the standard.39

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Part of the EPS extractions were dried by a freeze dryer (Alpha 1-2 LD, Martin Christ,

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Germany).

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1:100) and homogenized it using an agate grinder.

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analyzed it using a fourier transform infrared spectrum (FTIR, Nicolet 6700, Thermo,

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USA) between 400-4000 cm-1. We also analyzed the three-dimensional excitation–

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emission matrix (3D-EEM) spectra of the EPS extractions using a luminescence

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spectrometry (F-4500 FL Spectrophotometer, Hitachi, Japan), with ultra-pure water as

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the blank.

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We also took biofilm samples at the end of each stage to observe the distributions of

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proteins and polysaccharides in the biofilms.

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and proteins were stained by Con A, Calcofluor white and fluoresceinisothiocyanate

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(FITC), respectively.40 Laser scanning microscopy (CLSM, Zeiss, LSM710 NLO,

We centrifuged the mixtures at 10000 g for The filtrates

Protein in

We mixed the dried EPS with potassium bromide regent (the ratio is We compressed the mixture and

We processed the EEM data by using Origin (version 9.1) software.

α-polysaccharides, β-polysaccharides

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180

Germany) was applied to investigate the EPS distribution, and α-polysaccharides,

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β-polysaccharides and proteins were detected via excitation at 543 nm, 400 nm, and

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488 nm, and emission at 550-600 nm, 410-480 nm, and 500-540 nm, respectively.

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The details are described in Chen et al.41

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Results and Discussion

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Bio-reduction of V(V) in the CH4-based MBfR

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Figure 1 shows the measured influent and effluent concentration of V(V), effluent

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total V, as well as the removal percentage, flux and surface loading of V(V) during

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Stage 1-4.

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experiment.

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In Stage 1 (0-34 days), when the influent concentration of V(V) was 2 mg/L (at the

192

surface loading of 150 mg m-2 day-1), V(V) was reduced to V(IV) within 4 days, and

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the removal percentage and removal flux of V(V) continually increased up to 73%

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and 109 mg m-2 day-1, respectively, at the steady state.

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V(V) increased to 5 mg/L (at the surface loading of 378 mg m-2 day-1), the removal

196

percentage of V(V) didn’t change too much, but the removal flux of V(V) increased

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to 267 mg m-2 day-1.

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introduced into the MBfR (10 mg/L of V(V) at the surface loading of 668 mg m-2

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day-1), the removal percentage of V(V) slightly decreased to 63%, but the V(V) flux

200

(420 mg m-2 day-1) reached the highest.

201

was returned to 5 mg/L (at the surface loading of 363 mg m-2 day-1), the removal

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percentage of V(V) was 100%, much higher than that in Stage 2, though the operating

203

conditions were the same in the two stages.

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comprehensive research of EPS in the next two sections since EPS usually contribute

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significantly to protecting microbial cells against harsh conditions (e. g., antibiotic

Table 1 shows the CH4 supply was sufficient throughout the whole

In Stage 2, when the input

In Stage 3, when a higher concentration of V(V) was

In Stage 4, when the influent V(V) loading

This difference can be explained by

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interference, heavy metal poison, and water deprivation).42

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The effluent soluble V(IV) was not detected through the whole experiments (the

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lowest detection limit was 2.5 µg V(IV)/L by spectrophotometry).

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that a large amount of mineral precipitates accumulated in the biofilm, and EDS

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analysis (Figure 2B) demonstrated that V were the main elemental composition of the

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precipitates.

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valent state of V for the precipitates, indicating that precipitated vanadyl (V(IV)) was

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the main V(V) reduction product (Figure 2C).

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first time, that bio-reduction of V(V) was feasible using CH4 as the sole electron

215

donor and carbon source.

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Changes of EPS composition in response to V(V)

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Table 2 shows that the amounts of all analyzed constituents (including proteins,

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polysaccharides and humic substances) in EPS for Stages 1-4 were higher than that in

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the inoculum, suggesting the secretion of EPS was stimulated by the reduction of

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V(V).

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(except for Stage 3).

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between V(V) removal flux and the quantities of proteins, polysaccharides and humic

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substances through Stage 1, 2 and 4.

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observation that the polysaccharides and proteins in the biofilms increased from Stage

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1, 2 to 4 (Figure S2).

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the amounts of the constituents in EPS decreased sharply.

Figure 2A shows

Furthermore, XPS spectra demonstrated V(IV) was the predominant

These results demonstrate, for the

The amounts of these constituents increased from Stage 1, 2 to Stage 4 Figure S1 shows that there were good linear relationships

This was further supported by CLSM

However, when 10 mg/L of V(V) was introduced in Stage 3,

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The role of EPS in V(V) binding and reduction

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The intrusion of heavy metals usually stimulates microbial cells to secrete more

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EPS.43 Proteins and polysaccharides in EPS have been reported to strengthen the

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resistance of bacterial cells and biofilms to heavy metals or strong oxidizing

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substances42,43, as these substances usually contain functional groups which serve as

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permeability barrier to the toxic heavy metals.44 Moreover, the presence of

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humic acid-like substances implies their possible roles in V(V) binding at neutral pH,

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which has been demonstrated by Lu et al.45

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protective and reductive mechanisms of EPS, 3D-EEM and FTIR were employed to

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analyze the specific composition and functional groups of EPS in all stages.

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Three chemical components in EPS were identified by the 3D-EEM: tryptophan (Peak

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A, Ex/Em of 280-290/305-345 nm), humic acid (Peak B, Ex/Em of 355-365/440-445

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nm), and aromatic protein-like substances (Peak C, Ex/Em of 230/325) (Figure 3＆

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Table S1).

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that tryptophan might be the main constituent of proteins in EPS.

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variation trends of the peak intensities of tryptophan and humic acid substances in

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stage 1, 2 and 4 were similar to the trend of the flux of V(V).

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tryptophan protein-like substances could be oxidized to aromatic ring substances by

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strong oxidants such as sodium hypochlorite, ferrous iron or peroxydisulfate, thus

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forming a protective shield for the microbial cells.42,46

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tryptophan-like substances in dissolved organic matter which was able to bind heavy

248

metals.

In order to further understand the

The high intensities of peak A assigned to tryptophan in all stages imply Besides, the

It was reported that

The presence of

Therefore, the potential V(V) mechanisms due to tryptophan might be 15



249

shielding and complexation.

In addition, humic substances have been reported to

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bind V(V) using their hydrosulphonyl group.45

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We employed FTIR to further reveal the response of functional groups of EPS to V(V)

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in the four stages (Figure 4＆Table S2).

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biofilms include OH-, O–C–O, C=O, and COO- groups of proteins, polysaccharides or

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humic acid substances.47-51 The peak intensities of these functional groups in EPS

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were in similar trend as that in the component analysis by colorimetric method and

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3D-EEM.

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groups make a great contribution to binding of metal species with high valent state.52

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Besides, Wang et al found O–C–O group in polysaccharides were the main

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constituents for complexation with chromate (Cr(VI)).53

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It should be notable that the peak intensities in 3D-EEM and FTIR analysis in Stage 3

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were lower compared with that in Stage 2 and 4, in agreement with the EPS

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determination by colorimetric method.

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Microbial community structure

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To elucidate the microbial mechanisms involved in this biological phenomenon, we

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analyzed the relative abundances of the phylotypes at the class and genus levels in the

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biofilms for all stages (Figure 5).

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(γ-Gammaproteobacteria) and Denitratisoma (β-Betaproteobacteria), a methanotroph

268

and an oestradiol-degrading denitrifier, has strong correlation with V(V) removal flux

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(Table S1).54,55

The main functional groups of EPS in the

It has been reported that chemical reactive groups such as -OH, -COO

The relative abundance of Methylomonas

Considering the nitrate reductase from denitrifiers such as 16


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Rhodocyclus and Pseudomonas played an important role in V(V) reduction,

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Denitratisoma might actively participate in V(V) reduction.56,57

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The relative abundance of Methylophilus (β- Proteobacteria), a methanol-utilizing

273

genus,58 continuously increased throughout the experiments.

274

methanol might be released during the oxidation of CH4 by methanotrophs

275

(Methylomonas).

276

might be the electron carriers for synergism between methanotrophs and

277

vanadate-reducing bacteria in the CH4 based biofilm, as methanol is a typical product

278

from CH4 oxidation59 and a versatile electron donor available for V(V)-reducing

279

microorganisms.12,60

280

Based on these findings, we proposed the synergy in the CH4-fed biofilm:

281

Methylomonas oxidized CH4 to methanol using O2, which served as the electron

282

donor for Denitratisoma to perform V(V) reduction.

283

regulated by the oxygen content available in the system, as methanotrophs are aerobic,

284

while most denitrifiers (V(V) reducers) prefer anoxic conditions.

285

these two groups created an excellent symbiotic environment for each other.

286

Considering ~ 0.2 mg/L of O2 was consumed in the MBfR system, we calculated the

287

maximum V(V) reduced by aerobic CH4 oxidation was 1.3 mg/L of V.

Thus,

288

anaerobic V(V) reduction might also be responsible for CH4 oxidation.

For instance,

289

Kits et al. reported a Methylomonas strain could use nitrate as the electron acceptor to

290

oxidize CH4 and alleviate the low oxygen stress in anoxic environment.54

This implies that

This further supports that intermediate metabolites (e.g., methanol)

This process should be highly

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The co-existing of


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Changes of functional genes by metagenomic prediction

292

PICRUSt was applied to predict the functional genes in biofilms of all stages and

293

results are shown in Figure 6.

294

The abundances of predictive functional genes associated with the TCA cycle (citrate

295

synthase, aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate

296

dehydrogenase, succinate dehydrogenase, fumarate hydratase, and malate

297

dehydrogenase)61 and ATP production (adenylate kinase)62 in Stage 3 and 4 were

298

higher than that in Stage 1 and 2, suggesting the increased metabolic activities of

299

biofilms.

300

monooxygenase, formate dehydrogenase and formaldehyde dehydrogenase)59 and the

301

potential genes associated with V(V) reduction (nitrate reductase, enzymes associated

302

with cytochrome c metabolism, glutathione synthase)13,14,63 were enriched in the latter

303

stages.

304

These results allow further understanding of the protective mechanisms of EPS on

305

microbial cells in the CH4 fed biofilms.

306

the biofilm was susceptible to the direct oxidative stress and toxicity of V(V) to

307

microbial cells, leading to the lower metabolic activities of the biofilms.

308

and 4, the “adaption” of the biofilms caused secretion of much more EPS, which

309

restrained the diffusion of the V(V) into microbial cells, thus maintaining the normal

310

metabolisms of biofilms.

311

However, when high loading of V(V) (10 mg/L, at the surface loading of 668 mg m-2

Accordingly, genes responsible for methane oxidation (methane

In stage 1, the relative low amount of EPS in

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In Stage 2

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day-1) was introduced, all of the constituents in EPS decreased obviously, although the

313

biofilm retained its high V(V) reducing activity.

314

V(V) imposed to proteins, humic acids and polysaccharides.

315

that during V(V) reduction process, reactive oxygen species (ROS) such as reactive

316

oxygen (O2-·), hydroxyl radical (OH·) and hydrogen peroxide would be produced, 64

317

leading to the modifications of amino acid side chains, alteration of protein structure,

318

and oxidation of polysaccharides.42,65

319

by the PIRUSt results that the predictive genes associated with defense system to the

320

ROS, e.g., glutathione peroxidase, thioredoxin and catalase were more enriched in

321

Stage 3 compared with other stages.65

322

might serve as the barrier at the same time, consuming these ROS, protecting cells in

323

the biofilm from being attacked.42

324

Possible protect mechanisms of EPS

325

These above-mentioned results indicate two protective mechanisms of EPS in the CH4

326

based MBfR, as illustrated in Figure 8.

327

and humic acid substances in EPS were able to bind V(V) using functional groups

328

such as –OH and –COO-, alleviating the damage of V(V) to microbial physiological

329

metabolisms including TCA cycle, ATP production, methane oxidation and V(V)

330

reduction.

331

from bio-reduction of V(V), especially when the loading of V(V) was high.

332

interaction between EPS and ROS alleviated the oxidative stress on microbial key

333

metabolisms, and maintaining the high activities of the biofilm.

This might be due to the damage It has been reported

This understanding can be further supported

The proteins and polysaccharides in EPS then

Tryptophan-like proteins, polysaccharides,

EPS also triggered a sacrificial reaction with ROS, which were formed

19


The


334

In summary, we demonstrated V(V) reduction in a CH4-based membrane biofilm

335

reactor.

336

in V(V) reduction.

337

corresponded to the increased production of EPS which consisted of proteins,

338

polysaccharides and humic acid substances as the vital constituents for V(V)

339

resistance and binding, although high loading of V(V) lowered the these constituents

340

in EPS.

341

V(V), relieving the toxicity and oxidizing stress of V(V) to microbial cells, as well as

342

maintaining the normal metabolisms.

343

the biofilm by sonication and centrifugation, transform them into a dialysis bag, and

344

suspend the bag into a container having certain concentrations of V(V), to confirm the

345

binding between EPS and V(V).

346

bag and then identify the binding V(V) and the functional groups by using

347

characterizing technologies including SEM, XPS, XRD, EDS, XANES, and 13C

348

NMR.

349

concentrated stress imparting biofilm, and reduce the availability of V(V) for

350

reductive respiration. Biofilm management, such as backwashing, can be applied to

351

remove excess of EPS as well as the binding V(V).

Two denitrifiers, Methylomonas and Denitratisoma, might play crucial roles The increasing V(V)-reducing capability of the biofilm

Functional groups including –OH and -COO might play a role in binding

In the future work, we will detach the EPS in

We will also freeze dry the solution in the dialysis

However, the binding of too large amount of V(V) to EPS might create a

352

20


Page 20 of 36

Page 21 of 36


353

Acknowledgments

354

Authors greatly thank “The National Key Technology R&D Program

355

(2017ZX07206-002)”, “National Natural Science Foundation of China (Grant No.

356

21577123)”, the “Natural Science Funds for Distinguished Young Scholar of Zhejiang

357

Province (LR17B070001)”, and the “Fundamental Research Funds for the Central

358

Universities (2017XZZX010-03)” for their financial support.

359

Supporting Information Available

360

The Supporting Information is available free of charge via the Internet at

361

http://pubs.asc.org.

362

Fluorescence spectral parameters of EPS in the biofilm of each stage determined by

363

3D-EEM (Table S1); Absorbance of EPS in the biofilms of each stage measured by

364

FTIR (Table S2); Pearson Correlation analysis of V(V) flux and predominant genus in

365

the biofilms of all stages (Table S3); Linear relationship between V(V) flux and

366

proteins, polysaccharides, and humic acid substances in Stages 1, 2 and 4 (Figure S1);

367

CLSM images targeting biofilms of Stage 1, 2 and 4 (Figure S2).

368

21



369

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27



Table 1.

The V(V) and O2 removal fluxes, and methane-supply fluxes for each stage. V(V)

O2

Stage

Surface Loading (mmol/m2-day)

Flux (mmol/m2-day)

Stage1 Stage2 Stage3 Stage4

2.94 7.41 13.09 7.12

2.14 5.24 8.24 7.12

a:

Page 28 of 36

a

Calculated from equation 1. b:

Electron donor consumedb (mmol CH4 /m2-day) 0.72 1.77 2.79 2.41

Surface Loading (mmol/m2-day)

Flux (mmol/m2-day)

0.45 0.45 0.45 0.45

0.45 0.45 0.45 0.45

Calculated from equations 2 and 3.

c:

Electron donor consumed (mmol CH4 /m2-day) 0.56 0.56 0.56 0.56

Actual CH4 flux (mmol CH4 /m2-day) 1.28 2.33 3.35 2.97

Calculated according to Luo et al (2015).


Maximum CH4 fluxc (mmol CH4 /m2-day) 86.80 86.80 86.80 86.80

1 bar = 14.5 psig.

Page 29 of 36


Figure 1.

Performance profile during whole experiments of the CH4-based MBfR.



Figure 2.

V(IV) precipitates characterization and EPS observation.

Page 30 of 36

Cells and

precipitates (shown by red arrows) accumulated in the biofilm observed at 300 times magnification (A); EDS spectrum for V(IV) precipitates (B); XPS spectra of precipitates in the biofilm. The two peaks located at 516.38 eV and 523.98 eV were in good agreement with the value of precipitated V(IV) obtained by Wang et al (2017) (C); Cells and a large amount of EPS in biofilm samples of Stage 4 at 10000 times magnification (D).


Page 31 of 36


Figure 3.

EPS contents of inoculum and biofilm samples of each stage.



Figure 4.

3D-EEM fluorescence spectra of EPS extracted from inoculum (A), and

biofilms of Stage 1 (B), Stage 2 (C), Stage 3 (D), and Stage 4 (E).


Page 32 of 36

Page 33 of 36


Figure 5.

FTIR spectra of EPS extracted from inoculum and biofilms of all stages.

The predominant functional groups of EPS in the biofilms were OH- groups (3387-3425 cm−1) of polysaccharides or humic acid substances44, O–C–O groups (1079-1081 cm-1) of polysaccharides45, C=O (1637-1648 cm−1)46, and COO- groups (1403-1406 cm−1) of proteins or humic acids47, and C-H groups of aliphatic chains of protein, polysaccharides, or humic acids (2931-2937 cm−1).48



Figure 6.

Phylogenetic profiling of the biofilms at the levels of class (A) and genus

(B).


Page 34 of 36

Page 35 of 36


Figure 7.

Relative abundance of predicted functional genes involved in key metabolisms in biofilms for all stages by PICRUSt analysis.



Figure 8.

Schematic representation of the possible protect mechanisms of EPS in

the CH4-fed biofilm reducing V(V).

Mechanism 1: Proteins (tryptophan),

polysaccharides and humic acid substances in the EPS were able to bind V(V) using functional groups. Mechanism 2: Proteins and polysaccharides consumed ROS which were produced from bio-reduction of V(V), especially when the loading of V(V) is high.


Page 36 of 36

Role of Extracellular Polymeric Substances in a Methane Based

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