Efficient Bacterial Inactivation by Transition Metal Catalyzed Auto

Oct 9, 2017 - Hubei Key Lab of Biomass Resource Chemistry and Environmental Biotechnology, School of Resources and Environmental Science, Wuhan ... tr...
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Efficient Bacterial Inactivation by Transition Metal Catalyzed Auto-oxidation of Sulfite Long Chen, Min Tang, Chuan Chen, Mingguang Chen, Kai Luo, Jing Xu, Danna Zhou, and Feng Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03705 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Environmental Science & Technology

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Efficient Bacterial Inactivation by Transition Metal

2

Catalyzed Auto-oxidation of Sulfite

3 4

Long Chen†, ‡, Min Tang‡, Chuan Chen†, Mingguang Chen†, Kai Luo§, Jing Xu‡, Danna

5

Zhou*∥, Feng Wu*‡, ⊥

6



7

Riverside, Riverside, California, 92521, USA

8



9

School of Resources and Environmental Science, Wuhan University, Wuhan, 430079, P.

Department of Chemical and Environmental Engineering, University of California,

Hubei Key Lab of Biomass Resource Chemistry and Environmental Biotechnology,

10

R. China

11

§

12

MN 55455, USA

13



14

430074, P. R. China

15



16

Institute of Eco-Environmental Science & Technology, Guangzhou, 510650, P. R. China

Masonic Cancer Center, University of Minnesota, 2231 6th Street SE, Minneapolis,

Faculty of Material Science and Chemistry, China University of Geosciences, Wuhan,

Guangdong Key Laboratory of Agro-Environmental Integrated Control, Guangdong

17 18 19

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Abstract

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Disinfection is an indispensable process in wastewater treatment plants. New bacterial

22

inactivation technologies are of increasing interest and persistent demand. A category

23

of simple and efficient bactericidal systems have been established in this study, i.e., the

24

combination of divalent transition metal (Mn(II), Co(II), Fe(II), or Cu(II)) and sulfite. In

25

these systems, metal catalyzed auto-oxidation of sulfite was manifested to generate

26

reactive intermediary SO4•- that played the major role in Escherichia coli inactivation at

27

pH 5-8.5. Increasing concentrations of metal ion or sulfite, and lower pH, led to higher

28

bacterial deaths. Bacterial inactivation by Me(II)/Sulfite systems was demonstrated to be

29

a surface-bound oxidative damage process through destructing vital cellular

30

components, such as NADH and proteins. Additionally, the developed Me(II)/Sulfite

31

systems also potently killed other microbial pathogens, i.e. Pseudomonas aeruginosa,

32

Bacillus subtilis, and Cu(II)-/antibiotic-resistant E. coli. The efficacy of Me(II)/Sulfite in

33

treating real water samples was further tested with two sewages from a wastewater

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treatment plant and a natural lake water body, and Cu(II)/Sulfite and Co(II)/Sulfite

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rapidly inactivated viable bacteria regardless of bacteria species and cell density,

36

therefore holding great promises for wastewater disinfection.

37 38 39 40 41

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Introduction

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Waterborne pathogenic microbes are of great concern to public health. Two methods

44

are conventionally employed for bacteria removal, i.e. membrane filtration and bacterial

45

disinfection. Membrane filtration, taking advantage of the small pore size of synthetic

46

sheets, however, has the drawbacks of easy fouling, incomplete removal of tiny

47

organisms such as virus, and heavy demand of high pressure.1,2 Hence, a secondary

48

disinfection process is necessary for post-membrane treatment. Up to now, an

49

expanding range of disinfection processes or their combinations have been widely

50

applied. Traditionally used chlorine and ozone are powerful oxidants disinfecting

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bacterial virulence, but the large-scale production, storage, and transportation are

52

challenging owing to their acute toxicities.3,4 Moreover, certain fraction of chlorination

53

byproducts are carcinogenic and their presences in potable water may secondarily

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impair human health.5 Alternatively, advanced oxidation processes (AOPs) generating

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various oxidative radicals have been considered for bacterial inactivation purposes.

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Hydroxyl radical, HO•, derived from Fenton reaction has been verified to be potent in

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killing bacteria via degrading vital cellular components.6,7 Another emerging strong

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radical, sulfate radical (SO4•-), has gained a lot of attractions as well due to its selective

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degradation towards electron-rich recalcitrant contaminants.8,9 Recently, it has been

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reported that SO4•- is especially effective at bacterial/viral inactivation.10-13

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Previous SO4•- generating processes heavily relied on peroxymonosulfate (PMS) or

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persulfate (PS) through a one-step activation,8,9,14-16 while another intricate sulfite-

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associated radical chain reaction capable of deriving SO4•- has been largely ignored.

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Sulfite auto-oxidation catalyzed by transition metal ions with the presence of molecular

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oxygen is a key part of the sulfur cycle that mediates the transformation of S(IV) to

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S(VI).17-19 Briefly, at first SO3•- is generated after the instant decomposition of transient

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complex between high-valence metal ion and sulfite via internal electron transfer, but it

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rapidly

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disproportionally oxidizes sulfite producing SO4•- and sulfate anion (Table S1). The most

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intriguing property of the sulfite oxidation process is that, plethoral SO4•- is effectively

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generated. As a result, without exogenous competing substrates, SO4•- exclusively

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oxidizes parent sulfite, leading to the kinetically favored auto-oxidation of sulfite; on the

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other hand, the pool of total SO4•- is supposed to be re-assigned with the simultaneous

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presences of sulfite and other heterologous substrates. We have thus utilized this

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attractive

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decontamination purpose.20-23 However, up to date, the application of sulfite-based

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AOPs for waterborne bacteria disinfection still remains unexplored, and there were

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lacking

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decontamination/disinfection.

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In this study, we tested four divalent transition metals, i.e. Mn(II), Co(II), Fe(II), and

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Cu(II), catalyzed sulfite oxidation (denoted as Me(II)/Sulfite reactions) and therefore the

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proliferation of SO4•- bactericide. The main aims of this work were to comparatively

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investigate the potential of Me(II)/Sulfite systems in killing various microbial pathogens

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and to reveal the associated bactericidal mechanisms. Our work has vital environmental

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implications as both components of metal elements and sulfite are abundantly present

86

in various environmental media, and thus the associated cost is economically effective.

87

Materials and Methods

reacts

with

feature,

dissolved

and

oxygen

managed

comparisons

on

to

metal

to

drive

produce

SO4•-

catalyzed

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SO5•-.

Followingly,

generation

sulfite

for

SO5•-

wastewater

oxidation

for

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Materials

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Bacteria culture LB (Luria-Bertani) medium, disposable γ-ray sterilized 1-mL deep 96-

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well plates for bactericidal assays, and PBS buffer (pH 7.2) were purchased from

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Thermo Fisher Scientific Inc. (Shanghai, PRC). Antibiotics (kanamycin, tetracyclin,

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ampicillin, and chloramphenicol) were bought from Aladdin Industrial Corporation

93

(Shanghai, PRC). Other chemicals including sodium sulfite (Na2SO3), manganese

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chloride (MnCl2), cobalt chloride (CoCl2), ferrous sulfate (FeSO4), copper sulfate

95

(CuSO4), tert-butanol (TBA), thiourea, and 2,2’-dipyridyl were obtained from Sinopharm

96

Chemical Reagent Co. Ltd. (Shanghai, PRC). Bovine Serum Albumin (BSA) was from

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Shanghai Boao Biochemical Technology (Shanghai, China). All chemicals were of

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reagent grade and used without further purification. Throughout the assays, Milli-Q

99

water was used (18 MΩ•cm, Millipore Co., USA). All chemicals used to assess cellular

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NADH

content

(ethanol,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

101

bromide, phenazine ethosulfate, EDTA, and Bicine buffer) were bought from Sigma

102

(Shanghai, China). HClO4 or NaOH was used to adjust the pH of PBS.

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Methods

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Cell culturing and bactericidal assay quantification. Single colony of Escherichia

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coli BL21(DE3) strain was cultured in 5 mL liquid LB medium at 37 °C and 250 rpm for

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overnight incubation. 1% (50 µL) of saturated bacterial culture was then added into

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fresh 5 mL LB medium, followed by constant shaking at 37 °C and 250 rpm for another

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2 h until the cells reached exponential phase (OD600 ~ 0.4-0.6). Cells were harvested

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by centrifugation at 3000 g for 3 min. Pelleted cells were then resuspended and washed

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with PBS buffer of desired pHs for three times to remove residual nutrients. The freshly

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obtained cells were immediately used without storage. Bactericidal assays were initiated

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by addition of metal ions and/or sodium sulfite at indicated concentrations. 200 µL liquid

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samples were taken at pre-determined intervals, 10-fold serially diluted with PBS (pH

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7.2), and eventually 5 µL liquid of each sample was dropped on LB-agar plates and

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incubated at 37 °C for 12 h. Bacterial survival rate was quantified by counting the CFU

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(Colony Forming Unit) on the plates. Each assay was repeated for at least three times.

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Bactericidal Assays of Me(II)/Sulfite in Sewages and Natural Lake Water. Sewages

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of biological treatment tank and secondary sedimentation tank from Erlangmiao

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wastewater treatment plant (Wuhan, China) were sampled. The sampling sites were set

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at the well-mixed regions that were representative of the whole tank. For each tank, we

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chose at least two sampling points. Natural lake water of a nearby lake was also

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sampled to test the bactericidal effect of Me(II)/Sulfite (Scheme S1). Samples were

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stored in sterilized brown bottles to avoid light irradiation, and bactericidal assays were

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performed within the same day of sampling. PCA (plate count agar) was used to

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quantify the survival rate of total bacteria, while Endo agar was used to monitor the

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survival rate of coliforms. For the bactericidal assays of added E. coli, sewages or

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natural lake water were centrifuged at 1,000 g for 3 min followed by filtration with 0.45

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µm membranes, in order to remove original bacteria. Then exponential phase E. coli

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cultured in LB media was washed with desired water bodies (i.e. sewages or natural

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lake water), and then spiked into the same volume of corresponding sewages or natural

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lake water for Me(II)/Sulfite bactericidal assays (0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM

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Fe(II), 0.1 mM Cu(II), 2 mM sulfite).

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Radical-quenching

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Me(II)/Sulfite reactions (0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM Cu(II), 2

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mM sulfite, pH 6), either of 400 mM TBA (HO• scavenger), 400 mM thiourea (SO4•-/HO•

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scavenger), or 2 mM dipyridyl (metal ions chelator) was added into the solution at the

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beginning of the reactions. The individual effect of TBA, thiourea, or dipyridyl was also

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investigated in the same condition without the presence of metal ions or sulfite.

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Samples were taken and analyzed after 2 h.

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Scanning Electron Microscopy analysis. 5 mL exponential phase bacteria after

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treatment for 2 h were collected and resuspended into an Eppendorf vial containing 1

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mL PBS buffer (pH 7.2). 2.5% glutaraldehyde was used to fix the bacterial cells at room

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temperature for 1.5 h. Samples were then collected and exposed for 10 min to a series

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of ethanol concentration solutions (30, 50, 70, 80, 95, or 100% ethanol/PBS) for

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dehydration. The 100% ethanol treatment was repeated for twice, and the solutions

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were then further dehydrated with anhydrous sodium sulfate. 10 µL of bacterial solution

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in 100% ethanol was dropped on glass substrate, and then a uniform layer of 10 nm Au

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was coated on the bacterial samples with an E-beam evaporator (QPrep400, Mantis,

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England). Samples were observed under 10-5 Torr vacuum in SEM (JSM-6510, JEOL,

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Japan).

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Protein Degradation Assay with Me(II)/Sulfite. The oxidative degradation capability

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towards polypeptide (0.1 mg/mL BSA) of Me(II)/Sulfite (0.8 mM Mn(II), 0.4 mM Co(II),

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0.4 mM Fe(II), 0.1 mM Cu(II), and 2 mM sulfite) was investigated in PBS buffer (pH 6).

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Reactions were carried out in a 96-well plate with constant shaking at 37 °C. After 2 h,

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samples were boiled at 98 °C for 5 min, and 40 µL boiled samples were then loaded on

Assay

of

Me(II)/Sulfite.

During bactericidal assays with

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10% SDS-PAGE for resolution. Protein gels were run at 200 V for 60 min with a Bio-rad

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electrophoresis cell.

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NADH Measurement. Conventional cofactor recycling assay was used to determine

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intracellular NADH concentrations.24 5mL cells after treatment were at first collected via

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centrifugation at 8000 g for 2 min, then 300 µL of 0.2 M NaOH was added to the cell

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pellets, followed by incubation at 55 °C for 10 min. The resulting extracts were then

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neutralized with 300 µL of 0.1 M HCl. Samples were then centrifuged at 12000 g for 5

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min, and supernatants were collected for further analysis. NADH recycling assay was

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performed with 50 µL neutralized supernatant, 50 µL of yeast ADH with 25 units activity,

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and 0.9 mL reagents mixture (11% ethanol, 4.4 mM EDTA, 0.47 mM 3-(4,5-dimethyl-2-

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thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and 3.7 mM phenazine ethosulfate in

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100 mM Bicine buffer, pH 8). The absorbance changes at 570 nm after 2 min reactions

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were recorded. The same amount of cells without any treatment was used as a control.

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Results and Discussion

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Inactivation of Escherichia coli with Me(II)/Sulfite systems

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Divalent metal ions, i.e. Mn(II), Co(II), Fe(II), Cu(II), and sulfite combinatorically killed

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exponential phase E. coli cells at pH 6. In control experiments with single component of

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metal ion or sulfite, negligible cell deaths were observed (Figure 1a). The bactericidal

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effect of Me(II)/Sulfite systems exhibited a time- and metal species-dependent manner.

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Me(II)/Sulfite systems, except Cu(II)/Sulfite system, resulted in less than 0.5 log

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inactivation at 1 h while more than 1.2 log inactivation at 3 h. Although lower

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concentration of cupric ion (0.1 mM) was used, Cu(II)/Sulfite exhibited higher degree of

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bactericidal effect than the other three metal ions combined with sulfite. Cu(II)/Sulfite

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inactivated 1.3 log of E. coli at 1 h, and more than 2.2 log of cells at 3 h.

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Auto-oxidation of (bi)sulfite catalyzed by metal ions has been widely recognized through

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radical chain reactions, involving several intermediary short-lived oxysulfur radicals, i.e.

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SO3•-, SO5•-, and SO4•-.17-19 During the radical chain reactions, SO5•- and SO4•- were

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secondarily derived from initially generated SO3•- (Table S1). As expected, evident

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signals corresponding to DMPO/SO3•- adduct (aHβ = 16.0 G, aN = 14.7 G)25,26 were

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visualized in the ESR spectra of Me(II)/Sulfite systems during the first 1 min reaction

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(Figure 1b). Moreover, consistent with above observation, Cu(II)/Sulfite produced the

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most pronounced signals indicating the highest abundance of oxidative radicals (Figure

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1b). Among the three oxysulfur radicals, SO4•- possesses a standard reduction potential

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of 2.5-3.1 V (NHE),27 comparable to that of HO• (1.8-2.7 V, NHE).28,29 SO4•- owned

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preeminent oxidative hydrogen abstraction capability, and Fe(II)/Sulfite system has

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been tentatively used for wastewater decontamination.20-22 Hence the most possible

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effective bactericidal species was SO4•-.

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Effect of metal ions or sulfite concentrations

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E. coli survival rates were determined after inactivation by Me(II)/Sulfite systems for 2 h

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with varying concentrations of metal ions or sulfite at pH 7. Figure 2 showed significant

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enhancements of bactericidal effect with increasing concentrations of sulfite (0-8 mM) or

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metal ions (for Mn(II), Co(II) and Fe(II): 0.05-0.8 mM; for Cu(II): 0.0125-0.2 mM).

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Cu(II)/Sulfite system mediated the most evident E. coli inactivation. For instance,

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addition of 8 mM sulfite and 0.1 mM Cu(II) resulted in 31-fold enhancement of bacterial

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death compared with the mere presence of Cu(II). Further, 2 mM sulfite combined with

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0.2 mM Cu(II) killed bacteria 40-fold more efficient than that combined with 0.0125 mM

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Cu(II). The sole presence of either sulfite or metal ions at all tested concentrations did

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not kill bacteria more than 0.6 log of cells (Figure S1). Cu(II) was obviously more toxic

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than the other three metal ions, as 0.2 mM Cu(II) inactivated over 0.6 log of cells, higher

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than the inactivation efficiencies by other metal ions at 0.8 mM (Figure S1). The

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molecular mechanism of Cu(II) toxicity involved enhancement of cell membrane

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permeability and cellular protein denaturation.30,31 In contrast to previously observed

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inhibitive growth in nutritious media,32 the presence of 0.05-0.8 mM Co(II) surprisingly

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led to around 2-fold growth of E. coli in PBS buffer without any carbon source (Figure

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S1), indicating its pivotal role of participating in the synthesis of metalloprotein with poor

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nutrients.33

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Effect of pH

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pH mainly affected the metal ion species distribution, and thus possibly affected the

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initiation of catalyzed oxidation of sulfite.21 Aside from the bactericidal assays, we also

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used a triphenylmethane dye (Coomassie Brilliant Blue) to probe the amount of

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generated oxidative radicals by Me(II)/Sulfite at varying pHs (Figure S2). Through all

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the assays, near-neutral pHs (5-8.5) were chosen as other pHs adversely inactivated

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cells by destabilizing cell membrane and denaturing inherent functional enzymes34,35

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and therefore may cause false positive observations with Me(II)/Sulfite systems.

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Mn(II)/Sulfite and Fe(II)/Sulfite systems were potent at lower pH, in terms of both dye

221

removal and bacterial inactivation (Figure 3a, Figure S2). At pH 5, Mn(II)/Sulfite

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inactivated over 3.2 log of cells, but its potency rapidly decayed with increasing pH; at

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pHs above 8, negligible bacterial inactivation was observed. Fe(II)/Sulfite inactivated 0.9

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log of cells at pH 8.5, while its bactericidal efficiency modestly increased to 1.9 log at pH

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5.

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Consistent with our previous results,23 Co(II)/Sulfite was more capable in dye removal at

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higher pHs (Figure S2), whereas its bactericidal effect exhibited a reverted pattern

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(Figure 3). At pH 8.5, Co(II)/Sulfite inactivated 0.5 log of cells, but at pH 5 inactivated

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1.8 log of cells, resulting in a 22-fold enhancement. In sharp contrast, dye removal at pH

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5 was significantly lower than that at pH 8.5. The mechanism for this contradictory

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observation was not clear, and one plausible explanation was that higher pHs increased

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negative charges of bacterial surface and thus efficiently repulsing sulfite anion away

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from surface-bound catalytically active metal species.

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Cu(II)/Sulfite at lower pH values tended to mediate higher bacterial deaths, while a

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notable exception was that Cu(II)/Sulfite at pH 8.5 inactivated 9.6-fold cells of that at pH

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8 (Figure 3a), resulting in a bell-shaped bacterial inactivation pattern against pH.

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Different from other metals, Cu(II) was a severely toxic metal, in particular at higher pHs

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(Figure 3b), because 1) formed Cu(II)-OH complex readily adsorbed on bacterial

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surface,36,37 and 2) moreover Cu(II)-OH complex was highly toxic and effective at

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disrupting membrane structure than the other Cu(II) species.38,39 Therefore, at lower

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pHs the bactericidal effects mainly came from Cu(II)/Sulfite as evidenced by efficient

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dye removals (Figure S1), while the toxicity of Cu(II)-OH complex played the major role

243

at higher pHs.

244

Morphological and Biochemical Characterizations of E. coli

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Morphological changes of E. coli in response to Me(II)/Sulfite treatments were examined

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by SEM (Figure 4a). Bacterial samples were coated with a uniform layer of 10 nm

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atomic Au to improve the resolution of SEM by increasing the conductivity of the

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samples and thus reducing the accumulation of charges upon irradiation of electron

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beams. As a result, intensive darkness within the cytoplasmic region was observed for

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treated cells indicating severe cellular components disruption, but untreated cells

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exhibited normal homogeneity. In addition, the Mn(II)/Sulfite and Fe(II)/Sulfite

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treatments altered a fraction of E. coli with rod shape into fragmented irregular shape.

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The contents of cellular reduced nicotinamide adenine dinucleotide, NADH, an

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important biochemical indicator of the reducing power in cytoplasmic environment, was

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quantified through a cofactor cycling method.24 NADH was especially important for

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respiration-associated electron transport chain (ETC) and synthesis of certain

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metabolites.40,41 Remarkable decreases of NADH contents of cells treated by

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Me(II)/Sulfite were observed, wherein Cu(II)/Sulfite treatment particularly eliminated

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93.7% of total cellular NADH (Figure 4b).

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Furthermore, inspired by strong potencies of Me(II)/Sulfite systems in depleting NADH,

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we attempted to investigate their abilities in degrading proteins. A model protein, bovine

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serum albumin (BSA), was chosen as our target for polypeptide degradation assays. 0.1

263

mg/mL BSA was spiked into PBS buffer (pH = 6) together with metal ion and sulfite, and

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40 µL samples were subjected to protein gel electrophoresis. The most evident

265

decrease in band intensity was observed with Cu(II)/Sulfite, whereas the other

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Me(II)/Sulfite systems did not show detectable weakenings (Figure 4c). Densitometry

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analysis assisted by ImageJ software showed that around 56% of BSA was degraded

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by Cu(II)/Sulfite. Although the other three Me(II)/Sulfite systems (i.e. Mn(II)/Sulfite,

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Co(II)/Sulfite, and Fe(II)/Sulfite) did not exhibit polypeptide degradation capabilities,

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however, it was possible for them to inactivate cells through deactivating functional

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enzymes.25,42 During the course of running SDS-PAGE, denatured linearized BSA

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polypeptide was resolved based on its molecular weight. The denaturation of BSA due

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to Me(II)/Sulfite prior to SDS-PAGE thus cannot be told. A recent example supportive of

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protein denaturation by SO4•- was disease-associated prion protein inactivation.11

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Roles of Radicals in Bactericidal Effect of Me(II)/Sulfite Systems

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Me(II)/Sulfite reactions rapidly depleted dissolved oxygen (Figure S3a) accompanied by

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proton generation (Table S1). However, oxygen deprivation did not cause detectable

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bacterial death without Me(II)/Sulfite treatments (Figure S4), and generated proton did

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not acidify the buffered solution (Figure S3b) and was thus not responsible for bacterial

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inactivation (Figure 3). Moreover, three oxysulfur radicals, i.e. SO3•-, SO4•-, and SO5•-,

281

were directly produced during sulfite oxidation catalyzed by metal ions (Table S1). SO3•-

282

is fairly inert towards organic chemicals such as alcohols, but is extremely prone to

283

oxidation by oxygen into SO5•- with an estimated second-order rate constant of 109 M-1

284

s-1.43 It was thus concluded that SO3•- did not likely cause bacterial death. In addition,

285

because SO5•- was scarcely generated and less oxidative than SO4•-,43,44 the possibility

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of its bactericidal action was also excluded. Hence, SO4•- could be the most possible

287

bactericide responsible for cell inactivation during Me(II)/Sulfite treatments.

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Besides SO4•-, another plausible possibility is that formed hydroxyl radical (HO•), due to

289

the electron-scavenging of H2O by SO4•- (eq 1),8,9 inactivated bacterial cells. We

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therefore used a scavenging method to distinguish the roles of SO4•- and HO• in

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Me(II)/Sulfite systems at pH 6.8,9 Tert-butanol (TBA) quenched HO• with a 1000-fold

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selectivity higher than SO4•-, while thiourea non-selectively quenched both radicals. For

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all Me(II)/Sulfite systems, addition of SO4•-/HO•-scavenging thiourea greatly suppressed

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bacterial inactivation, more evidently for Mn(II)/Sulfite and Cu(II)/Sulfite, while the

295

addition of HO•-scavenging TBA exhibited less potent inhibition (Figure 5a). It was

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therefore concluded that SO4•- played the major role in inactivating cells, in agreement

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with previous report.21 We further used a strong metal ion chelator, 2,2’-dipyridyl, to fully

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stall the catalyzed sulfite oxidation. As expected, since there was no intermediary

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oxidative species generated, negligible bacterial deaths were observed (Figure 5a). It

300

should be noted that, conventionally used ethanol (SO4•-/HO• scavenger) and EDTA

301

(metal ions chelator) were avoided because they were reported to be detrimental to

302

bacteria.45,46 But TBA, thiourea, and dipyridyl at used concentrations did not affect cell

303

survival rates (Figure S4).

304

SO4•- + H2O  SO42- + HO• + H+

(k2 = (103-104) s-1)

(1)

305 306

Possible Mechanisms of Bactericidal Effect of Me(II)/Sulfite Systems

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We reasoned that, generated reactive species, such as oxidative short-lived SO4•-, had

308

to act on cellular components for bacterial inactivation purpose. Accordingly, the

309

catalytically active metal ion species had to be bound onto cell surface. We hence

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ultrasonically cracked the cells treated by either metal ions alone or Me(II)/Sulfite

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together, centrifuged and collected the supernatants, and subsequently quantified

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cellular metal ions with AAS (Figure 5b). As expected, significant cellular uptakes were

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found for all four metal ions. Among them, cells treated by 0.1 mM Cu(II) mediated a

314

most evident 134-fold increasement in cellular Cu(II) content by comparison with

315

untreated cells, presumably because Cu(II) readily complexed with cellular polypeptide

316

through highly affinitive Cu(II)-thiol group interactions.47,48 Additionally, we also found

317

that cellular metal ion uptakes by Me(II)/Sulfite treatments were generally less effective

318

than the treatments of metal ions alone (Figure 5b), likely because the presence of

319

sulfite complexed with metal ions and thus reducing the chances of metal ions adsorbed

320

onto bacterial surface. The observed less Cu(II) adsorption in the presence of sulfite

321

also supportively explained the fact that bacterial inactivation efficiencies of

322

Cu(II)/Sulfite were even lower than that of the single role of Cu(II) at pH 8 and 8.5

323

(Figure 3). We concluded that the toxicity of Cu(II)/Sulfite at higher pHs was mainly

324

from Cu(II), and therefore less surface-bound Cu(II) due to sulfite complexation led to

325

attenuated bacterial deaths. Hence, we believed the bacterial inactivations by

326

Me(II)/Sulfite systems were a surface-bound in situ oxidative damage process.

327

Cu(II)/Sulfite was an especially attractive system as it owned the most potent

328

bactericidal effects. During the initiation of radical chain reactions, Cu(I) was generated

329

via reduction of Cu(II) by sulfite (Table S1), and the presence of Cu(I) was

330

experimentally verified (Figure S5). More importantly, Cu(I) has been reported to be an

331

effective bactericide.49 We thus assessed the bactericidal role of Cu(I) in an anaerobic

332

chamber purged with nitrogen. The exclusion of oxygen did not allow the propagation of

333

radical chain reactions for Cu(II)/Sulfite system (Table S1), and the effect of Cu(I) could

334

be specifically investigated. We surprisingly found that Cu(I) formed by Cu(II)/Sulfite

335

with the absence of oxygen absolutely inactivated all the cells (~ 8 log), while the

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presence of oxygen led to 1.8 log of bacterial inactivation (Figure 5c). Since oxygen

337

played inverse roles in the generation of Cu(I) (Figure S5) and SO4•- (Table S1), the net

338

effect of oxygen on Cu(II)/Sulfite bactericidal performance was still vague, and therefore

339

the role of oxygen was investigated in more details (Figure S6). Oxygen ratio of 0, 0.01,

340

0.05, 0.2, and 0.4 in oxygen/nitrogen mixing gas at fixed flow rate of 400 mL/min were

341

constantly purged into 200 mL of Cu(II)/Sulfite reaction solution. When oxygen ratio

342

slightly increased from 0 to 0.01, Cu(II)/Sulfite killing efficiency dramatically decreased

343

from 3.7-log to 0.8-log after reaction for 1 h, indicating the strong bactericidal potency of

344

Cu(I). The increase of oxygen ratio 0.01 up to 0.4 led to the improvement of

345

Cu(II)/Sulfite bactericidal efficiency from 0.8-log to 2.1-log. While the increase of oxygen

346

ratio reduced Cu(I) amount, it improved the SO4•- production, and therefore can further

347

enhance bactericidal effect of Cu(II)/Sulfite. As expected, without molecular oxygen, no

348

reactive species were generated for Mn(II)/Sulfite, Co(II)/Sulfite, or Fe(II)/Sulfite, and

349

hence bacterial deaths were greatly suppressed (Figure 5c). Conclusively, based on

350

the obtained results, triple components majorly contributed to the bactericidal effect of

351

Cu(II)/Sulfite, i.e. 1) oxidative SO4•-, 2) toxic Cu(II)-OH complex at higher pHs, and 3)

352

monovalent Cu(I) (Scheme 1). The triply synergistic actions may also explain the fact

353

that Cu(II)/Sulfite system was the most efficient system among the Me(II)/Sulfite

354

systems investigated in this work.

355

Disinfection of Sewages and Natural Lake Water with Me(II)/Sulfite Systems

356

Application of Me(II)/Sulfite systems in inactivation of other bacteria genres, e.g. various

357

antibiotic-resistant E. coli strains (Figure S7), Cu(II)-resistant E. coli (Figure S8),

358

Pseudomonas aeruginosa and Bacillus subtilis (Figure S9) were proved to be valid

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(Text S1). Despite all this, our aim was to apply the established Me(II)/Sulfite systems

360

into real wastewater treatment plants. Sewage samples were taken at two

361

representative sites from Erlangmiao wastewater treatment plant, i.e. biological

362

treatment tank and secondary sedimentation tank. The samples had typical sewage

363

characters, such as near-neutral pH, and high contents of ammonium and phosphate

364

ions (Table S2). Bactericidal assays were performed immediately after sampling, in

365

order to avoid possible bacterial inactivation during storage. Moreover, to mimic real

366

condition, sewage samples were directly used without filtration, and reaction solutions

367

were subjected to minimum stirring. Furtherly, we used PCA (plate count agar), a rich

368

and non-selective medium, to recover total viable bacteria. And Endo agar, a selective

369

medium, was used to isolate coliform colonies that formed green metallic sheen on the

370

colorless plate, such as E. coli. Initially, total bacteria CFUs per mL of biological

371

treatment tank and secondary sedimentation tank were 5 × 105 and 5 × 103,

372

respectively. In both tanks, coliforms were around 10% of total bacteria. In biological

373

treatment tank, Cu(II)/Sulfite inactivated 2 log of both total bacteria and coliforms after

374

reaction for 3 h, while the other three Me(II)/Sulfite systems did not show detectable

375

bacterial inactivation (Figure 6a, 6b). In secondary sedimentation tank, both

376

Cu(II)/Sulfite and Co(II)/Sulfite rapidly and absolutely inactivated total bacteria and

377

coliforms within 2 h, whereas Mn(II)/Sulfite and Fe(II)/Sulfite did not mediate significant

378

bactericidal effects (Figure 6d, 6e). Additionally, it has been observed that Cu(II)/Sulfite

379

and Co(II)/Sulfite killed coliforms more efficiently than total bacteria, likely due to the

380

complex bacterial species within total bacteria.

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381

The upper limit of bacterial density in aqueous solution is around 108 CFU/mL, even in

382

the case of nutritious media. We therefore attempted to examine the bactericidal

383

efficiencies of Me(II)/Sulfite systems with the presence of maximum bacterial density.

384

Exponential phase E. coli with OD600 around 0.4-0.6, corresponding to 108 CFU/mL,

385

was collected and added into the same volume of biological treatment tank and

386

secondary sedimentation tank sewage samples. Consistent with above results,

387

Cu(II)/Sulfite absolutely inactivated bacteria within 1 h, and Co(II)/Sulfite killed all

388

bacteria at 2 h. The preeminent performance of Co(II)/Sulfite may associate with the

389

toxicity of Co(II) with the presence of sewage carbon source.32

390

Disinfection of lake water is important as it is a possible source of drinking water. Hence,

391

besides sewage samples, we also tested bacterial inactivation efficiencies of

392

Me(II)/Sulfite systems towards natural lake water (Figure S10). Cu(II)/Sulfite could

393

rapidly inactivate total bacteria (4 log) in lake water within 1 h. Co(II)/Sulfite also potently

394

killed bacteria but slower than Cu(II)/Sulfite, and Co(II)/Sulfite inactivated all bacteria

395

after 2 h. Neither Mn(II)/Sulfite nor Fe(II)/Sulfite showed significant bacterial inactivation

396

(Figure S10a). Consistently, Cu(II)/Sulfite and Co(II)/Sulfite inactivated 8 log and 3.2 log

397

of added E. coli (~ 108 CFU/mL) in natural lake water after reactions for 3 h (Figure

398

S10b). Moreover, CT (concentration [mg/L] × time [min]) value of Cu(II)/Sulfite to

399

inactivate 4-log E. coli varied between 1600 and 9600 in terms of different water bodies

400

(Figure 6c, 6f, Figure S10), comparable to the CT values of chlorine (i.e. 1575) and

401

ozone (i.e. 1440).50 Considering the costs/risks associated with production, storage,

402

and/or carcinogenic byproducts of chlorine and ozone, the cost-effectiveness of

403

Cu(II)/Sulfite is especially attractive. In conclusion, Cu(II)/Sulfite exhibited especially

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potent bactericidal performances in all tested water bodies, and therefore it ought to be

405

considered as a promising disinfection method in real practice.

406

A major finding of this study was that SO4•- generated from sulfite autoxidation catalyzed

407

by divalent metal ion, i.e. Mn(II), Co(II), Fe(II) or Cu(II), was potent in killing expanding

408

varieties of bacteria regardless of their antibiotic resistance. Cu(II)-resistant E. coli

409

persisters were especially sensitive to Cu(II)/Sulfite treatment, highlighting its significant

410

environmental implication as proliferative waterborne bacteria usually develop

411

resistance to aqueous toxic species. Concerns of oxygen-dependence and organic

412

matter interference may be raised towards Me(II)/Sulfite systems in treating real sewage

413

samples with complex components, and we therefore attempted the bactericidal

414

performance of Me(II)/Sulfite systems into sewage disinfection. As a result, Cu(II)/Sulfite

415

and Co(II)/Sulfite remarkably inactivated total bacteria and coliforms without additional

416

aeration, validating the feasibility of these two systems in real practice. Notably,

417

catalysts for sulfite oxidation are not limited to the four herein investigated metal ions,

418

and other heavy metals such as Cr(III/VI) or Ni(II) also own powerful catalysis

419

capabilities,17,51 expanding the application scope of our methods. Hereby, we propose

420

two ways of applying Me(II)/Sulfite into real wastewater disinfection. At first, free metal

421

ions can be added into wastewater together with sulfite. Tandem post-Me(II)/Sulfite

422

treatment units, including coagulation/sedimentation, sludge disposal, and adsorption,

423

can eliminate most of present metal ions. Alternatively, efficient heterogeneous

424

catalysts, such as cobalt ferrite nanoparticles,23 can be used to activate sulfite oxidation

425

in wastewater. Heterogeneous catalysts own the advantages of minimum metal ion

426

leaching, stable performance, and high catalytic activity,23,52 therefore holding more

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427

promises. The most important aspect of environmental implications is that catalytically

428

active metal ions and sulfite can be readily obtained from industrial wastewater and

429

combustion flue gas respectively, and eventually multiple purposes could be achieved,

430

i.e. flue gas desulfurization, bacterial inactivation, and organics decontamination if

431

present.

432 433

■ ASSOCIATED CONTENT

434

Supporting Information

435

Methods, reaction equations, water quality characterizations, and additional figures

436

depicting bacteria survival rate under various treatments. This material is available free

437

of charge via the Internet athttp://pubs.acs.org.

438 439

■ AUTHOR INFORMATION

440

Corresponding Authors

441

Danna Zhou, e-mail: [email protected].

442

Feng Wu, e-mail: [email protected].

443

Notes

444

The authors declare no competing financial interest.

445

■ ACKNOWLEDGMENTS

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446

This work was supported by the National Natural Science Foundation of China (NSFC

447

No. 21477090 and 41103066), the NSFC-CNRS_PRC Cooperation Project (No.

448

21711530144) and Science and Technology Project of Guangdong Province (No.

449

2014B030301055). Comments from anonymous reviewers are also appreciated.

450 451

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452

Reference:

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2018, 221, 380-392.

599

.

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Table of Content

600

O2

Me(IIa/IIIb)

SO32-

Me(Ia/IIb)

a. Cu b. Mn, Co, Fe

O2

SO32-

SO3● SO5●-

SO32-

SO4●-

601 602

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Page 31 of 38

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603 604

Figure 1. E. coli inactivation efficiencies (a) and ESR spectra (b) of Me(II)/Sulfite. (0.8

605

mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM Cu(II), 2 mM sulfite, PBS, pH 6).

606

ACS Paragon Plus Environment

Environmental Science & Technology

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

100 10 1 0

607

0.5

1

2

4

8

Sulfite concentration (mM)

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

b100 Survival rate (%)

Survival rate (%)

a

Page 32 of 38

10 1

Mn(II)/Co(II)/Fe(II)0.05 0.1 Cu(II) 0.0125 0.025

0.2 0.05

0.4 0.1

0.8 0.2

Metal ion concentration (mM)

608

Figure 2. Effects of sulfite (a) and metal ion (b) concentration on the E. coli inactivation

609

efficiencies of Me(II)/Sulfite. (a) 0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM

610

Cu(II), sulfite concentrations varied between 0 and 8 mM, PBS, pH 7, reaction for 2 h.

611

(b) 2 mM sulfite, Mn(II) (0.05, 0.1, 0.2, 0.4, 0.8 mM), Co(II) (0.05, 0.1, 0.2, 0.4, 0.8 mM),

612

Fe(II) (0.05, 0.1, 0.2, 0.4, 0.8 mM), Cu(II) (0.0125, 0.025, 0.05, 0.1, 0.2 mM). PBS, pH 7,

613

incubation for 2h.

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a

Survival rate (%)

Survival rate (%)

100 10 1 0.1 5

616

b

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

6

7

8

8.5

Sulfite Mn(II) Co(II) Fe(II) Cu(II)

100

1 0.1

5

6

7

8

8.5

pH

pH

617

Figure 3. Effect of pH on the E. coli inactivation efficiencies of Me(II)/Sulfite (a) and

618

sulfite or Me(II) (b). Magenta curve in panel a represented the trendline of Cu(II)/Sulfite

619

bactericidal performance against pH. 0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1

620

mM Cu(II), 2 mM sulfite, incubation in PBS for 2h.

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a No treatment

Mn(II)/Sulfite

Fe(II)/Sulfite

Cu(II)/Sulfite

c Residual NADH (%)

b

622

Co(II)/Sulfite

120

kD 95 72 55

80 40

43 0

t en ulfite ulfite ulfite ulfite m t a /S /S /S /S tre n(II) o(II) e(II) u(II) o F C C M N

34

623

Figure 4. Morphological characterization of E. coli with SEM (a), cellular NADH

624

depletion (b), and BSA degradation (c) after treatments with Me(II)/Sulfite. 0.8 mM

625

Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM Cu(II), 2 mM sulfite. PBS, pH 6, incubation

626

for 2h.

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629

Figure 5. (a) Me(II)/Sulfite bactericidal performances on E. coli after quenching with

630

TBA, thiourea, or dipyridyl. (b) Cellular metal content comparative quantifications by

631

treatments of Me(II) alone or Me(II)/Sulfite towards E. coli. (c) Effect of oxygen on E. coli

632

inactivation efficiencies of Me(II)/Sulfite, 2h. 0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II),

633

0.1 mM Cu(II), 2 mM sulfite. PBS, pH 6, incubation for 2 h. (n.d., not detectable).

634

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O2

Me(IIa/IIIb)

O2

SO3

636

2-

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite

SO32-

Me(Ia/IIb)

a. Cu b. Mn, Co, Fe

635

SO32-

SO4●-

SO3

●-

Me(II)-OH complex

SO5●SO4●-

SO32-

Cu(I)

Cu(II)/Sulfite

SO4●Cu(II)-OH SO 23

Scheme 1. Bactericidal mechanisms of Me(II)/Sulfite.

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10 Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

0

1

2

10 Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

1

3

0

d100

e100

Survival rate (%)

Survival rate (%)

10 Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

0

638

1

2

2

3

10 1

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

0.1 0.01 0

Time (h)

Time (h)

1

1

Added E. coli

c 100

3

f

10 Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

1 0.1 0

Time (h)

1

2

1

2

3

Time (h)

Survival rate (%)

1

Coliforms

b100

Survival rate (%)

Total bacteria

Survival rate (%)

Survival rate (%)

a100

Page 38 of 38

3

Time (h)

100 1 0.01

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

1E-4 1E-6

0

1

2

3

Time (h)

639

Figure 6. Bactericidal performance of Me(II)/Sulfite on sewage samples. Samples from

640

biological treatment tank (a-c) and secondary sedimentation tank (d-f) in Erlangmiao

641

wastewater treatment plant were tested. Bactericidal efficiencies with total bacteria (a,

642

d), coliforms (b, e), and added E. coli (c, f) were monitored. Mn(II) 0.8mM, Co(II) 0.4mM,

643

Fe(II) 0.4mM, Cu(II) 0.1mM, 2mM sulfite.

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