Remediation performance and mechanism of hexavalent chromium

in alkaline soil using multi-layer loaded nano-zero-valent iron

Siyu Hou1, Bin Wu1, Dinghua Peng1, Ziru Wang1, Yiyang Wang1, Heng Xu1*

1Key Laboratory of Bio-Resource and Eco-Evironment of Ministry of Education,

College of Life Sciences, Sichuan Univ ersity, Chengdu 610065, P.R.China

CONTENTS

Soil mixing and aging methods

Detailed method for soil enzyme and pH analysis

Table S1 Immobilization rate of different CNH applied amounts.

Figure S1 Characterizations of CN and CNH. EDS characterization of CN (a) and

CNH (b), FTIR analysis map of CN and CNH (c), XRD analysis map of CN and

CNH(d).

Figure S2 The HOAc-extractable changes of different treatments within 240 d (a) and

species distribution of Cr in different treatments on the 240th d (b). Error bars

represent standard deviations, and means with different letters are significantly

different from each other (P < 0.05) according to the LSD test (n=3). The “-” between

the letters represents a hyphen. The significant labels of HOAc-extractable were

1*Corresponding author. Tel: +86 28 85414644; Fax: +86 28 85418262.E-mail address: xuheng64@sina.com(H.Xu)The first two authors contributed equally to this work and should be considered co-first authors.

general.

Figure S3 pH of different treatments on the 240th d. Error bars represent standard

deviations, and means with different letters are significantly different from each other

(P < 0.05) according to the LSD test (n=3). NS: natural soil.

Figure S4 Soil Eh on d 240 of all treatments. Error bars represent standard deviations,

and means with different letters are significantly different from each other (P < 0.05)

according to the LSD test (n=3).

Figure S5 Comparisons of soil microbial counts (a) and four enzyme activities (b)

with natural soil (NS) on d 90. Error bars represent standard deviations, and means

with different letters are significantly different from each other (P < 0.05) according to

the LSD test (n=3). Significant labels of each enzyme activity were individual.

Soil mixing and aging methods:To prepare Cr-spiked soil samples, 1 L 2.5 g L-1 K2Cr2O7 solution and 25 kg soil

were carefully mixed at 37 on a clean plastic film to constant weight and then℃

placed in a plastic bucket. Thereafter, the soil was aged for 3 months and was mixed

again every half month in a constant temperature greenhouse at 37 and 60% air℃

humidity for subsequent experiments.

Detailed method for soil enzyme and pH analysis

1. Soil enzyme analysis

Urease activity was measured at 578 nm after culturing 1 g soil, 200 μL toluene,

2 mL 10% urea solution and 4 mL of citrate buffer (pH = 6.7) for 24 hours with a 150

rpm 37°C constant temperature shaker.

invertase activity was evaluated at 508 nm after culturing a mixture of 1 g soil, 3

mL 8% sucrose solution and 1 mL phosphate buffer (pH = 5.5) at 150 rpm for 24

hours at 37 .℃

As to acid phosphatase activity, 1 g air-dried soil, 4 mL phosphate buffer (pH

6.5), 0.2 mL toluene and disodium 4-nitrophenyl phosphate (pnpp) were incubated for

1 h at 37 . Then 1 mL 0.5 M calcium chloride and 4 mL 0.5 M sodium hydroxide℃

were supplemented and mingled well. The mixture was centrifuged at 2500 rpm for 5

min and the supernatant was immediately measured at 420 nm.

1 g soil, 7.5 mL phosphate buffer (pH 7.6) and 200 μL FDA solution (1 mg mL-1)

were mixed and incubated at 30 °C for 40 min. Then 7.5 mL chloroform methanol

mixture (v: v = 2: 1) was added into above mixture and the absorbance of the

supernatant was measured at 490 nm to measure the FDA hydrolase activity.

2. soil pH measurement

Soil and water were mixed and oscillated on the oscillator at a ratio of 1: 9 of

soil: water (w: w) for ten minutes. The pH of the supernatant was measured with a pH

meter (METTLER-S220).

Table S1 Immobilization rate of different CNH applied amounts Applied amount (mg g-1) Immobilization rate (significant level)

5 44.27% a10 51.62%b15 58.93% c30 69.46% d60 73.79% e

mg g-1 meant the mg of CNH per gram of soil.

a

b

c

Figure S1 EDS characterization of CN (a) and CNH (b), FTIR analysis map of CN

and CNH (c), XRD analysis map of CN and CNH(d).

a

d

Figure S2 The HOAc-extractable changes of different treatments within 240 d (a) and

species distribution of Cr in different treatments on the 240th d (b). Error bars

represent standard deviations, and means with different letters are significantly

different from each other (P < 0.05) according to the LSD test (n=3). The “-” between

the letters represents a hyphen. The significant labels of available Cr were general.

b

Figure S3 pH of different treatments on the 240th d. Error bars represent standard

deviations, and means with different letters are significantly different from each other

(P < 0.05) according to the LSD test (n=3). NS: natural soil.

Figure S4 Soil Eh on d 240 of all treatments. Error bars represent standard deviations,

and means with different letters are significantly different from each other (P < 0.05)

according to the LSD test (n=3).

Figure S5 Comparisons of soil microbial counts (a) and four enzyme activities (b)

a

b

with uncontaminated soil (NS) on d 90. Error bars represent standard deviations, and

means with different letters are significantly different from each other (P < 0.05)

according to the LSD test (n=3). Significant labels of each enzyme activity were

individual.