Adsorption of Selenite onto Bacillus subtilis: The Overlooked Role of

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Adsorption of selenite onto Bacillus subtilis: the overlooked role of cell envelope sulfhydryl sites in the microbial conversion of Se(IV) Qiang Yu, Maxim I. Boyanov, Jinling Liu, Kenneth M. Kemner, and Jeremy B. Fein Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02280 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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Environmental Science & Technology

Adsorption of selenite onto Bacillus subtilis: the overlooked role of cell envelope sulfhydryl sites in the microbial conversion of Se(IV)

Qiang Yu1,*, Maxim I. Boyanov2,3, Jinling Liu1,4, Kenneth M. Kemner3, Jeremy B. Fein1

1

Department of Civil & Environmental Engineering & Earth Sciences, University of Notre Dame, Notre Dame, IN 46556, USA 2 Bulgarian Academy of Sciences, Institute of Chemical Engineering, Sofia, 1113, Bulgaria 3 Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA 4 School of Earth Science, China University of Geoscience, Wuhan 430074, China

A revised manuscript submitted to Environmental Science & Technology

(July 24th, 2018)

*

Author for correspondence.

Tel: (574) 631-4534; Fax: (574) 631-9236; Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract

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Microbial activities play a central role in the global cycling of selenium. Microorganisms can

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reduce, methylate and assimilate Se, controlling the transport and fate of Se in the environment.

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However, the mechanisms controlling these microbial activities are still poorly understood. In

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particular, it is unknown how the negatively-charged Se(IV) and Se(VI) oxyanions that dominate

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the aqueous Se speciation in oxidizing environments bind to negatively-charged microbial cell

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surfaces in order to become bioavailable. Here, we show that the adsorption of selenite onto

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Bacillus subtilis bacterial cells is controlled by cell envelope sulfhydryl sites. Once adsorbed

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onto the bacteria, selenite is reduced, and forms reduced organo-Se compounds (e.g., R1S-Se-

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SR2). Because sulfhydryl sites are present within cell envelopes of a wide range of bacterial

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species, sulfhydryl-controlled adsorption of selenite likely represents a general mechanism

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adopted by bacteria to make selenite bioavailable. Therefore, sulfhydryl binding of selenite likely

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occurs in a wide range of oxidized Se-bearing environments, and because it is followed by

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microbial conversion of selenite to other Se species, the process represents a crucial step in the

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global cycling of Se.

16 17 18 19 20 21 22 23


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TOC Art

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27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 3 ACS Paragon Plus Environment


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Introduction

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Microorganisms control the oxidation, reduction and alkylation of Se in many natural and

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contaminated settings.1-4 In particular, microbial reduction of water-soluble selenite (Se(IV)O32-)

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and selenate (Se(VI)O42-) to insoluble Se(0) nanoparticles1-2 significantly reduces the

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bioavailability and toxicity of Se, and has received increasing attention not only in order to

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understand the global cycling of Se, but also in order to optimize applications such as the

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bioremediation of seleniferous environments5 and the synthesis of Se nanomaterials.6

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Microbial reduction of Se(VI) to Se(0) typically follows two sequential steps:1, 7-8 (1)

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reduction of Se(VI) to Se(IV); and (2) reduction of Se(IV) to elemental Se(0). The reduction of

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Se(VI) to Se(IV) occurs only in anaerobic1, 8 or microaerobic environments,9 and is promoted

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only by a limited range of microorganisms.5 In contrast, a wide range of microorganisms can

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promote reduction of Se(IV) to elemental Se(0) under either anaerobic or aerobic conditions.10-14

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We focus on Se(IV) bioavailability as it represents the key step in the reduction of both Se(VI)

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and Se(IV). Whatever the reduction mechanism of Se(IV) to Se(0),12-13, 15-16 electron transfer

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from molecules in the electron transport chain within microbial cell walls to Se(IV) atoms

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requires that Se(IV) is in close proximity to the cell wall. Thus, the bioavailability of Se(IV)

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relies on an initial step of adsorption of Se(IV) onto cell surfaces. Because selenous acid

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(H2SeO3) has pKa values of 2.7 and 8.5,17 the negatively-charged aqueous species HSeO3- and

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SeO32- are the dominant Se(IV) species under most geologic conditions. Microbial cells are also

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strongly negatively-charged under environmental pH conditions due to deprotonation of organic

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acid functional groups that are located within the cell envelope.18-19 Positively-charged aqueous

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cations can adsorb onto bacteria through both electrostatic and covalent binding with these

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functional groups, and important binding site types include carboxyl, phosphoryl, and sulfhydryl


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sites.20-22 However, due to electrostatic repulsion, adsorption of aqueous anions onto these

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negatively-charged binding sites is typically difficult.23-24 There is experimental evidence that

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some bacterial species exhibit a capacity to adsorb selenite and selenate, especially under acidic

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conditions where the electronegativity of the cell wall is diminished due to protonation.25-26

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However, Se(IV) reduction can occur under circumneutral pH conditions, meaning that selenite

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adsorption onto bacteria under these conditions must be possible. The mechanisms responsible

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for selenite adsorption onto bacteria remain unknown, and must be determined in order to better

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understand and optimize Se biogeochemical cycles, whether they occur in nature or in

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engineered systems.

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In this study, we measure the extent of selenite adsorption onto non-metabolizing

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bacterial cells, and we explore the mechanisms controlling the adsorption. Because selenite can

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readily react with sulfhydryl-containing molecules such as cysteine, glutathione and

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bacillithiol,15, 27-29 we hypothesize that the sulfhydryl sites within bacterial cell envelopes play an

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important role in the adsorption. Therefore, Bacillus subtilis, a common soil bacterial species that

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can be grown to contain a high concentration of sulfhydryl sites within its cell envelopes30 and

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that can reduce selenite to elemental Se(0) aerobically,11 was chosen for our experiments. The

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role of sulfhydryl sites in the adsorption was determined by comparing selenite adsorption onto

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the biomass with and without sulfhydryl sites blocked chemically, and the Se speciation on the

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biomass was analyzed using X-ray absorption near edge structure (XANES) spectroscopy and

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extended X-ray absorption fine structure (EXAFS) spectroscopy.

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Materials and Methods

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Biomass preparation



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In this study, all of the biomass mass values are reported in terms of wet mass, and the

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ratio of wet mass to dry mass is 4.7 for Bacillus subtilis biomass.31 The preparation procedures

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for B. subtilis biomass samples were similar to those described previously,30-31 and previous

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study shows that most cells in the prepared biomass samples are intact and alive, although not

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actively metabolizing in our experiments due to a lack of electron donor or C source.32 Briefly,

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bacteria were first cultured aerobically in 3 mL of pre-sterilized TSB medium at 32 ºC for 24 h.

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The TSB medium contains 30 g of trypticase soy broth and 5 g of yeast extract per L of ultrapure

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18 MΩ cm water. The initial culture was then transferred to 1 L of the TSB medium

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supplemented with 50 g/L of glucose in order to induce a higher concentration of sulfhydryl

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sites,30 and was cultured aerobically for 24 h at 32 ºC in order for the bacterial cell suspensions to

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reach early stationary phase. Prior to use, the TSB medium was autoclaved at 121 oC for 30 min,

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and the glucose stock solution was filtered using a Nalgene 0.22 µm nylon filtration membrane

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for sterilization. After incubation, the bacterial cells were harvested by centrifugation at 10,970 ×

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g for 5 min. The biomass pellets were rinsed with a 0.1 M NaCl solution, followed by

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centrifugation at 8,100 × g for 5 min, and the same process was repeated three times in order to

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remove adsorbed growth medium components from the bacterial cells that may interfere in the

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adsorption experiments. The biomass pellets were then transferred into pre-weighed centrifuge

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tubes and centrifuged for two 30-minute intervals at 8,100 × g. After decanting the supernatant,

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the biomass was measured for wet mass and then was either subjected to a qBBr treatment to

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block cell envelope sulfhydryl sites (see below), or was used without further treatment in the

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adsorption experiments. The untreated B. subtilis biomass samples have a total binding site

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concentration of 275±10 µmol/g, of which 93±8 µmol/g are sulfhydryl sites.30


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Monobromo(trimethylammonio)bimane bromide (qBBr), purchased from Toronto

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Research Chemical, Inc., was used to block cell envelope sulfhydryl sites for selenite adsorption.

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qBBr effectively blocks cell envelope sulfhydryl sites (Figure S1),31, 33 but does not react with

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bacterial carboxyl or phosphoryl sites.31 The treatment involved the suspension of the bacterial

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pellets in a freshly prepared qBBr solution in 0.1 M NaCl with pH buffered to 7.0 ± 0.1 using

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Na2HPO4/NaH2PO4, with a qBBr:biomass ratio of approximately 180 µmol/g, and the

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suspension was allowed to react for 2 h at room temperature under continuous shaking on a

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rotating plate at 60 rpm. Our previous study demonstrated that the reaction between qBBr and

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the cell envelope sulfhydryl sites is complete after 2 h of reaction time.31 After reaction, the

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qBBr-treated cells were separated from the qBBr solution by centrifugation at 8,100 × g for 5

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min, and then were washed following the same washing procedure that we used for harvesting

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bacterial cells from the TSB medium. After decanting the supernatant, the wet cells were used in

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the subsequent experiments.

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Adsorption Experiments

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Se(IV) adsorption experiments were conducted using both the untreated and qBBr-treated

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B. subtilis biomass, which represent biomass samples with and without sulfhydryl sites available

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for binding with Se, respectively. Prior to the adsorption experiments, a freshly prepared 158

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ppm Se(IV) stock solution using sodium selenite was diluted to 1 ppm using 0.1 M NaCl, and the

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pH values of the resulting solutions were adjusted to 7.0 ± 0.2 using 1 M NaOH and 1M HCl.

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The pre-weighed bacterial cell pellet was then suspended into the dilute Se(IV) solution with a

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vortex mixer to achieve a biomass concentration of 20 g/L in each experimental system. The well

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mixed Se(IV)-biomass suspensions were then transferred into test tubes with 10 mL of

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suspension in each, and the initial pH values were adjusted to 3.0 - 8.0 using 0.1 M NaOH or 0.1



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M HCl. The tubes were slowly rotated for 4 h at room temperature, after which the final pH of

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the solution was measured and used as the reported pH values. Finally, the bacterial suspensions

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were filtered using 0.22 µm nylon membranes, and the concentrations of Se in the aqueous phase

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were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES).

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Biomass-free control experiments were also conducted using 1 ppm or 158 ppm Se(IV) solutions

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under similar conditions except that no biomass was added to the systems. The biomass-free

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controls using the 1 ppm Se(IV) solution showed that the decrease in aqueous Se(IV) after the

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experiments was negligible within the pH range of 3-8 (with Se(IV) losses of

Adsorption of Selenite onto Bacillus subtilis: The Overlooked Role of

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