Subscriber access provided by Kaohsiung Medical University
Environmental Processes
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 25
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
Environmental Science & Technology
1
Abstract
2
Microbial activities play a central role in the global cycling of selenium. Microorganisms can
3
reduce, methylate and assimilate Se, controlling the transport and fate of Se in the environment.
4
However, the mechanisms controlling these microbial activities are still poorly understood. In
5
particular, it is unknown how the negatively-charged Se(IV) and Se(VI) oxyanions that dominate
6
the aqueous Se speciation in oxidizing environments bind to negatively-charged microbial cell
7
surfaces in order to become bioavailable. Here, we show that the adsorption of selenite onto
8
Bacillus subtilis bacterial cells is controlled by cell envelope sulfhydryl sites. Once adsorbed
9
onto the bacteria, selenite is reduced, and forms reduced organo-Se compounds (e.g., R1S-Se-
10
SR2). Because sulfhydryl sites are present within cell envelopes of a wide range of bacterial
11
species, sulfhydryl-controlled adsorption of selenite likely represents a general mechanism
12
adopted by bacteria to make selenite bioavailable. Therefore, sulfhydryl binding of selenite likely
13
occurs in a wide range of oxidized Se-bearing environments, and because it is followed by
14
microbial conversion of selenite to other Se species, the process represents a crucial step in the
15
global cycling of Se.
16 17 18 19 20 21 22 23
2 ACS Paragon Plus Environment
Page 2 of 25
Page 3 of 25
24
Environmental Science & Technology
TOC Art
25 26
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 3 ACS Paragon Plus Environment
Environmental Science & Technology
43
Introduction
44
Microorganisms control the oxidation, reduction and alkylation of Se in many natural and
45
contaminated settings.1-4 In particular, microbial reduction of water-soluble selenite (Se(IV)O32-)
46
and selenate (Se(VI)O42-) to insoluble Se(0) nanoparticles1-2 significantly reduces the
47
bioavailability and toxicity of Se, and has received increasing attention not only in order to
48
understand the global cycling of Se, but also in order to optimize applications such as the
49
bioremediation of seleniferous environments5 and the synthesis of Se nanomaterials.6
50
Microbial reduction of Se(VI) to Se(0) typically follows two sequential steps:1, 7-8 (1)
51
reduction of Se(VI) to Se(IV); and (2) reduction of Se(IV) to elemental Se(0). The reduction of
52
Se(VI) to Se(IV) occurs only in anaerobic1, 8 or microaerobic environments,9 and is promoted
53
only by a limited range of microorganisms.5 In contrast, a wide range of microorganisms can
54
promote reduction of Se(IV) to elemental Se(0) under either anaerobic or aerobic conditions.10-14
55
We focus on Se(IV) bioavailability as it represents the key step in the reduction of both Se(VI)
56
and Se(IV). Whatever the reduction mechanism of Se(IV) to Se(0),12-13, 15-16 electron transfer
57
from molecules in the electron transport chain within microbial cell walls to Se(IV) atoms
58
requires that Se(IV) is in close proximity to the cell wall. Thus, the bioavailability of Se(IV)
59
relies on an initial step of adsorption of Se(IV) onto cell surfaces. Because selenous acid
60
(H2SeO3) has pKa values of 2.7 and 8.5,17 the negatively-charged aqueous species HSeO3- and
61
SeO32- are the dominant Se(IV) species under most geologic conditions. Microbial cells are also
62
strongly negatively-charged under environmental pH conditions due to deprotonation of organic
63
acid functional groups that are located within the cell envelope.18-19 Positively-charged aqueous
64
cations can adsorb onto bacteria through both electrostatic and covalent binding with these
65
functional groups, and important binding site types include carboxyl, phosphoryl, and sulfhydryl
4 ACS Paragon Plus Environment
Page 4 of 25
Page 5 of 25
Environmental Science & Technology
66
sites.20-22 However, due to electrostatic repulsion, adsorption of aqueous anions onto these
67
negatively-charged binding sites is typically difficult.23-24 There is experimental evidence that
68
some bacterial species exhibit a capacity to adsorb selenite and selenate, especially under acidic
69
conditions where the electronegativity of the cell wall is diminished due to protonation.25-26
70
However, Se(IV) reduction can occur under circumneutral pH conditions, meaning that selenite
71
adsorption onto bacteria under these conditions must be possible. The mechanisms responsible
72
for selenite adsorption onto bacteria remain unknown, and must be determined in order to better
73
understand and optimize Se biogeochemical cycles, whether they occur in nature or in
74
engineered systems.
75
In this study, we measure the extent of selenite adsorption onto non-metabolizing
76
bacterial cells, and we explore the mechanisms controlling the adsorption. Because selenite can
77
readily react with sulfhydryl-containing molecules such as cysteine, glutathione and
78
bacillithiol,15, 27-29 we hypothesize that the sulfhydryl sites within bacterial cell envelopes play an
79
important role in the adsorption. Therefore, Bacillus subtilis, a common soil bacterial species that
80
can be grown to contain a high concentration of sulfhydryl sites within its cell envelopes30 and
81
that can reduce selenite to elemental Se(0) aerobically,11 was chosen for our experiments. The
82
role of sulfhydryl sites in the adsorption was determined by comparing selenite adsorption onto
83
the biomass with and without sulfhydryl sites blocked chemically, and the Se speciation on the
84
biomass was analyzed using X-ray absorption near edge structure (XANES) spectroscopy and
85
extended X-ray absorption fine structure (EXAFS) spectroscopy.
86 87
Materials and Methods
88
Biomass preparation
5 ACS Paragon Plus Environment
Environmental Science & Technology
89
In this study, all of the biomass mass values are reported in terms of wet mass, and the
90
ratio of wet mass to dry mass is 4.7 for Bacillus subtilis biomass.31 The preparation procedures
91
for B. subtilis biomass samples were similar to those described previously,30-31 and previous
92
study shows that most cells in the prepared biomass samples are intact and alive, although not
93
actively metabolizing in our experiments due to a lack of electron donor or C source.32 Briefly,
94
bacteria were first cultured aerobically in 3 mL of pre-sterilized TSB medium at 32 ºC for 24 h.
95
The TSB medium contains 30 g of trypticase soy broth and 5 g of yeast extract per L of ultrapure
96
18 MΩ cm water. The initial culture was then transferred to 1 L of the TSB medium
97
supplemented with 50 g/L of glucose in order to induce a higher concentration of sulfhydryl
98
sites,30 and was cultured aerobically for 24 h at 32 ºC in order for the bacterial cell suspensions to
99
reach early stationary phase. Prior to use, the TSB medium was autoclaved at 121 oC for 30 min,
100
and the glucose stock solution was filtered using a Nalgene 0.22 µm nylon filtration membrane
101
for sterilization. After incubation, the bacterial cells were harvested by centrifugation at 10,970 ×
102
g for 5 min. The biomass pellets were rinsed with a 0.1 M NaCl solution, followed by
103
centrifugation at 8,100 × g for 5 min, and the same process was repeated three times in order to
104
remove adsorbed growth medium components from the bacterial cells that may interfere in the
105
adsorption experiments. The biomass pellets were then transferred into pre-weighed centrifuge
106
tubes and centrifuged for two 30-minute intervals at 8,100 × g. After decanting the supernatant,
107
the biomass was measured for wet mass and then was either subjected to a qBBr treatment to
108
block cell envelope sulfhydryl sites (see below), or was used without further treatment in the
109
adsorption experiments. The untreated B. subtilis biomass samples have a total binding site
110
concentration of 275±10 µmol/g, of which 93±8 µmol/g are sulfhydryl sites.30
6 ACS Paragon Plus Environment
Page 6 of 25
Page 7 of 25
Environmental Science & Technology
111
Monobromo(trimethylammonio)bimane bromide (qBBr), purchased from Toronto
112
Research Chemical, Inc., was used to block cell envelope sulfhydryl sites for selenite adsorption.
113
qBBr effectively blocks cell envelope sulfhydryl sites (Figure S1),31, 33 but does not react with
114
bacterial carboxyl or phosphoryl sites.31 The treatment involved the suspension of the bacterial
115
pellets in a freshly prepared qBBr solution in 0.1 M NaCl with pH buffered to 7.0 ± 0.1 using
116
Na2HPO4/NaH2PO4, with a qBBr:biomass ratio of approximately 180 µmol/g, and the
117
suspension was allowed to react for 2 h at room temperature under continuous shaking on a
118
rotating plate at 60 rpm. Our previous study demonstrated that the reaction between qBBr and
119
the cell envelope sulfhydryl sites is complete after 2 h of reaction time.31 After reaction, the
120
qBBr-treated cells were separated from the qBBr solution by centrifugation at 8,100 × g for 5
121
min, and then were washed following the same washing procedure that we used for harvesting
122
bacterial cells from the TSB medium. After decanting the supernatant, the wet cells were used in
123
the subsequent experiments.
124
Adsorption Experiments
125
Se(IV) adsorption experiments were conducted using both the untreated and qBBr-treated
126
B. subtilis biomass, which represent biomass samples with and without sulfhydryl sites available
127
for binding with Se, respectively. Prior to the adsorption experiments, a freshly prepared 158
128
ppm Se(IV) stock solution using sodium selenite was diluted to 1 ppm using 0.1 M NaCl, and the
129
pH values of the resulting solutions were adjusted to 7.0 ± 0.2 using 1 M NaOH and 1M HCl.
130
The pre-weighed bacterial cell pellet was then suspended into the dilute Se(IV) solution with a
131
vortex mixer to achieve a biomass concentration of 20 g/L in each experimental system. The well
132
mixed Se(IV)-biomass suspensions were then transferred into test tubes with 10 mL of
133
suspension in each, and the initial pH values were adjusted to 3.0 - 8.0 using 0.1 M NaOH or 0.1
7 ACS Paragon Plus Environment
Environmental Science & Technology
134
M HCl. The tubes were slowly rotated for 4 h at room temperature, after which the final pH of
135
the solution was measured and used as the reported pH values. Finally, the bacterial suspensions
136
were filtered using 0.22 µm nylon membranes, and the concentrations of Se in the aqueous phase
137
were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES).
138
Biomass-free control experiments were also conducted using 1 ppm or 158 ppm Se(IV) solutions
139
under similar conditions except that no biomass was added to the systems. The biomass-free
140
controls using the 1 ppm Se(IV) solution showed that the decrease in aqueous Se(IV) after the
141
experiments was negligible within the pH range of 3-8 (with Se(IV) losses of
