Supplementary Information

The following information is provided to the article in

JOURNAL OF HAZARDOUS MATERIALS

on

“A novel manganese oxidizing bacterium-Aeromonas

hydrophila strain DS02: Mn(II) oxidization and biogenic Mn-

oxides generation ”Yue Zhang1, Yankui Tang*1,2, Zhiyi Qin1, Penghong Luo1, Zhou Ma1, Mengying

Tan1, Houyao Kang1

1. School of Resources, Environment and Materials, Guangxi University, 530004,

Nanning, China

2. Guangxi Key Laboratory of Petrochemical Resource Processing and Process

Intensification Technology, Guangxi University, 530004, Nanning, China.

*Corresponding author: School of Resources, Environment and Materials, Guangxi

University, 530004, Nanning, China

E-mail address: cindyyktang@gxu.edu.cn

Tel: +86(13977187116)

Fax: +86(0771-3273440)

Detection of Mn(II) inhibitory concentrations (ICs)

The experiments were carried out in 300-mL Erlenmeyer flasks (Duran) in a

reciprocating shaker at 35 and 200 rpm. All the parameters above were determined℃

by series of preliminary experiments (data not shown).

The inhibitory concentrations (ICs) of Mn(II) for strain DS02 were determined.

The strain was inoculated into sterilized liquid LB culture medium (10%) with different

initial concentrations of Mn(II) (1-50 mM), and cultivated for 24 h. The inhibitory

concentrations (ICs) were estimated using Eq. (1):

IC = (OD0 − ODt) / OD0 (1)

where ODt is the OD600 at 24 h. As for blank control group, the synchronous OD600 of

inoculated Mn(II)-free media is OD0. All the experiments were repeated in triplicate and

the reported data represented the average of the triplicates with a standard deviation less

than 5%.

Mn(II) oxidizing activity of the DS02 at different pH values

The initial pH values of LB medium (L-1) were adjusted to 6.0, 7.0, 8.0, 9.0 by

adding different buffers, respectively. The corresponding buffers were as follows: MES,

pH 6; HEPES, pH 7 and 8, Tris–HCl, pH 9. The OD600 and Mn(II) oxidation of strain

DS02 at different time intervals were detected.

Scanning Electron Microscope (SEM) analysis

The morphology features of strain DS02 was observed by SEM

analysis as follows:

Specimen collection: the colony was inoculated into a LB medium

and cultured for 24 h, centrifuged at 11, 000 rpm for 10 min, and the

supernatant was discarded. The specimen was washed 2-3 times with

ultrapure water to remove the impurity.

Double fixation: the specimen was first fixed with 2.5%

glutaraldehyde in phosphate buffer (pH7.0) for more than 4hours;

washed three times with ultrapure water.

Dehydration: the specimen was first dehydrated by a graded

series of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for

about 15 to 20 min at each step, transferred to absolute acetone for

20 minutes.

Coating and Observation: the specimen was coated with gold-

palladium in Eiko Model IB5 ion coater for 4-5 min and then observed

in Hitachi Model SU8020 SEM.

Fourier Transform Infrared Spectroscopy (FTIR) analysis

In order to analyze the functional groups of the BioMnOx

produced by strain DS02, FTIR were carried out by a FT Infrared

Spectroscope (Nicolet iS 50, Thermo Fisher Scientific, USA) in the range 4,000-400

cm-1 at a resolution of 4 cm-1 by making the KBr thin pellet with the samples.

Raman analysis

The Raman spectrum was recorded on a Renishaw inVia Raman Microscope with

resolution of 2 cm−1, the excitation source was an argon–ion laser at the wavelength of

532 nm.

Fig.S1. Satellite image of sampling location.

Fig. S2. Colony morphologies of strain DS02 on LB medium agar plate (A).

Colony morphologies of strain DS02 on LB medium agar plate with Mn(Ⅱ), a

drop of 10 mM hydroxylamine hydrochloride was added to spot 1, and a drop of

0.004% LBB solution was added to spot 2 (B). Strain DS02 in Mn(II)-free LB

medium (left) and LB medium with Mn(II) (right) after cultivation for 144 h (C).

LBB assay of bacterial suspensions cultivating with and without Mn(II) for 144 h

(D).

Fig. S3. OD600 of strain DS02 under different Mn( ) concentrations in 24 h. (ICⅡ 50 (shown in black dashed line) represented the OD600 of half growth inhibition)

Fig. S4. (a) growth curves and (b) manganese oxidation of strain DS02 at different pH values with initial Mn(II) concentration of 10 mM within 144 h.

Fig. S5. SEM (A, B) micrographs of Aeromonas hydrophila strain DS02 with different magnifications.

(B)(A)

Fig. S6. EDX result of the selected area in the freeze-dried powdered sample.

Fig. S7. Raman and FT-IR spectra of the BioMnOx.

Tab. S1. Colony morphology of strain DS02.

Character trait Result

Colony shape Round

Colony edge Neat

Colony color Creamy-white

Surface morphology Smooth

Bump condition Plano convex

Transparency Opaque

Moisture Content Semihumid

Tab. S2. Physiological and biochemical properties of strain DS02.

Character trait Result

Strain length (μm) 1.0-1.5

Gram staining -

Motility test +

Indole test +

Starch hydrolysis +

Glucose hydrolysis -

Catalase test +

Oxidase test -

+ Positive; - Negative.

Tab. S3. The Mn AOS and the percentages of three Mn valence states in the as-prepared BioMnOx samples under different conservation times.

Sample number

Conservation time/d

Mn(II)/%

Mn(III)/%

Mn(IV)/%

Mn AOS

a 30 10.9 7.1 82.0 3.71

a 60 7.5 9.8 82.7 3.75

b 30 13.7 5.9 80.4 3.67

b 60 11.2 7.6 81.2 3.70

Appendix S1. Corresponding nucleotide sequence (1,292 bp) of strain DS02.>DS02CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAACATTCTGATTTGCGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCGCTTTTTGGGATTCGCTCACTATCGCTAGCTTGCAGCCCTCTGTACGCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCCTTGAGTTCCCACCATTACGTGCTGGCAACAAAGGACAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCTGATTCCCGAAGGCACTCCCGTATCTCTACAGGATTCCAGACATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGTCTCAAGGACACAGCCTCCAAATCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCTGGACAGTTTTAAATGCAATTCCCAGGTTGAGCCCGGGGCTTTCACATCTAACTTATCCAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCACAGTTGATACGTATTAGGCATCAACCTTTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGATCATCCTCTCAGACCAGCTAGGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCCACCTGGGCATATCCAATCGCGCAAGGCCCGAAGGTCCCCTGCTTTCCCCCGTAGGGCGTATGCGGTATTAGCAGTCGTTTCCAACTGTTATCCCCCTCGACTGGGCAATTTCCCAGGCATTACTC