Effects of humic acid on the biodegradation of di-n-butyl phthalate in mollisol

Journal Pre-proof Effects of humic acid on the biodegradation of di-n-butyl phthalate in mollisol Yue Tao, Hongtao Shi, Yaqi Jiao, Siyue Han, Modupe S. Akindolie, Yang Yang, Zhaobo Chen, Ying Zhang PII:

S0959-6526(19)34274-X

DOI:

https://doi.org/10.1016/j.jclepro.2019.119404

Reference:

JCLP 119404

To appear in:

Journal of Cleaner Production

Received Date: 8 July 2019 Revised Date:

25 October 2019

Accepted Date: 20 November 2019

Please cite this article as: Tao Y, Shi H, Jiao Y, Han S, Akindolie MS, Yang Y, Chen Z, Zhang Y, Effects of humic acid on the biodegradation of di-n-butyl phthalate in mollisol, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/j.jclepro.2019.119404. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Effects of humic acid on the biodegradation of di-n-butyl phthalate in mollisol

2

Yue Tao1, Hongtao Shi1, Yaqi Jiao1, Siyue Han1, Modupe S Akindolie1, Yang Yang1,

3

Zhaobo Chen2, Ying Zhang1*

4

1

5

Changjiang Road, Harbin, Heilongjiang Province, PR China.

6

2

7

West Road, Jinzhou New District, Dalian, Liaoning Province, PR China.

School of Resources and Environment, Northeast Agricultural University, No.600,

College of Environment and Resources, Dalian Minzu University, No. 18, Liaohe

* Corresponding authors. E-mail: [email protected] Abbreviations PAEs: Phthalate esters DBP: Dibutyl phthalate HA: Humic acid FTIR: Fourier Transform Infrared spectroscopy 2D-FTIR-COS: Two-dimensional FTIR correlation spectroscopy DOM: Dissolved organic matter SEM: Scanning electron microscope 3D-EEM: Three-dimensional excitation-emission matrix MBC: microbial biomass carbon PLFA: Phospholipid fatty acid 1

8

Abstract

9

Soil phthalic contamination has received more and more attention due to the

10

widespread use of plastic mulching films. Humic acid (HA) is a natural antidote. The

11

effects of HA on the biodegradation of dibutyl phthalate (DBP) in mollisol was

12

investigated in this study. Through the calculation by bi-exponential model, the

13

half-life of DBP was effectively shortened after adding HA, from 11.65 days to 3.36

14

days, and soil bulk density decreased. The enhancement mechanism for DBP removal

15

by HA was analyzed by fluorescence spectrometry, two-dimensional FTIR correlation

16

spectroscopy (2D-FTIR-COS) and PLFA analysis. Two major functional groups (aryl

17

C-O and alkyl ester C=O) were found at the binding site between DBP and HA. By

18

mediating the transportation of DBP, HA could provide more time for soil

19

microorganisms to degrade DBP. Meanwhile, HA effectively stabilized the mollisol

20

microbial community composition and promote the growth of aerobic bacteria, which

21

contributes to the degradation of DBP. The results are valuable for relieving DBP

22

pollution caused by agricultural production and promoting sustainable use of mollisol.

23

Keywords: humic acid; di-n-butyl phthalate; two-dimensional FTIR correlation

24

spectroscopy (2D-FTIR-COS); microbial community structure;

25

2

26

1. Introduction

27

Phthalate esters (PAEs), an environmental hormone, are a class of refractory

28

organic compounds, which can affect humans’ health even at low concentrations

29

(Feng et al., 2017). DBP is one of the most widely and frequently identified PAEs

30

compounds

31

carcinogenicity, teratogenicity, and mutagenicity (Matsumoto et al., 2008). Globally,

32

the DBP content in agricultural land varies between regions. Xu et al. (2018) reported

33

0.3-453, 7.9-8, and 6 µg kg-1 DBP contents in the soil of Denmark, the United

34

Kingdom, and the Netherland, respectively. In China, the DBP concentrations reached

35

mg kg-1 in some agricultural soils (Niu et al., 2014). With a hydrolysis half-life of

36

about 20 years, DBP is quite stable in the natural environment and difficult to remove

37

(Huang et al., 2015). Even more, DBP has been linked to the plastic materials through

38

the hydrogen bond or van der Waal forces. Therefore, DBP can migrate from plastic

39

products into the environment (Kong et al., 2012). DBP in the soil can be taken up

40

and accumulated by crops, which affect food production and food safety (Wu et al.,

41

2018). The harmful effects of DBP have attracted worldwide attention and made it be

42

listed among the priority pollutants by the US Environmental Protection Agency.

used

in

different

environments

and

exhibits

hepatotoxicity,

43

Mollisol is a crucial natural resource in the world. It has made significant

44

contributions to global grain production. A lot of studies showed that soil with a

45

higher organic matter content could retain more organic pollutants (Yang et al., 2011;

46

Yang et al., 2013). It is necessary to study the impact of PAEs pollution on mollisol 3

47

where rich organic matter exists. Cheng et al. (2018) found that the half-life of DBP in

48

soil with more OM content was shorter. Therefore, if the binding characteristics of

49

DBP and soil organic components can be clarified, the influence of organic

50

components on DBP degradation will be understood, and external organic substances

51

applied to remediate soil organic pollution maybe possible. Humic acid, a kind of

52

DOM, has good ion exchange, catalytic, chelating and buffering ability due to its

53

aromatic structure, multiple functional groups and microscopic spherical structure

54

(Panettieri et al., 2014). Humic acid is best known for its ability to stimulate plant

55

growth. Khaled and Fawy (2011) found that applying HA to soil increased the N

56

uptake of corn. Also, many studies have shown that HA could improve the soil

57

properties and promote the electron transfer between organic pollutants and degrading

58

bacteria (Ciarkowska, 2017; Hu et al., 2011). If HA can be used to soil organic

59

pollution remediation, it will be beneficial for improving mollisol productivity,

60

sustainable production, and ensuring crop yields.

61

In this study, the effects of HA on the biodegradation of DBP in mollisol were

62

discussed. Scanning electron microscope (SEM), soil bulk density, and FTIR were

63

used to observe soil structure changes. Synchronous fluorescence two-dimensional,

64

three-dimensional

65

spectroscopy (2D-FTIR-COS) and UV-Vis absorption spectrum were used to obtain

66

the binding process of DBP in mollisol. Soil base respiration and microbial biomass

67

carbon (MBC) were used to analyze changes of microbial activity in mollisol. The

excitation-emission

matrix

4

(3D-EEM),

FTIR

correlation

68

phospholipid fatty acid (PLFA) was used to characterize the variation of soil

69

microbial species.

70

2. Methods and materials

71

2.1 Materials

72

DBP (purity above 99%), DBP stock standard solution and thirty-seven fatty acid

73

methyl ester mixed standards were purchased from Tianjin Bodi Chemical Industry

74

Co., Ltd (China), Sigma-Aldrich and NU-CHEK Co., Ltd (USA), respectively. HA

75

was purchased from Shanghai Ryon biological Technology CO., Ltd. The purity of the

76

HA is more than 98% (BR grade).

77

The soils used were randomly collected from the top 20 cm of cultivated land in

78

suburban Harbin China. The soils were air-dried in the laboratory and sieved (2 mm).

79

The total nitrogen, total phosphorus, pH, water content, and organic matter was 1.006

80

g kg-1, 0.562 g kg-1, 7.785, 11.39% and 2.75%, respectively.

81

2.2 Experimental setup

82

To determine the effect of the different addition amount of HA on soil DBP

83

degradation. We blended 20 mg kg-1 DBP with soil as DBP contaminated soil. Then

84

all of the DBP contaminated soil was divided into six groups, which added different

85

quality of HA. The addition HA content was 0, 0.5, 1.0, 1.5, 2.0, and 3.0 g kg-1,

86

respectively. Soil DBP contents were measured at 0, 3, 5, and 7 days. The preliminary

87

experiment results show that 1.5, 2.0, and 3.0 g kg-1 HA could significantly promote

88

the degradation of DBP in contaminated soil (Fig. S1). Considering the economic 5

89

benefits, we chose the HA addition amount of 1.5 g kg-1 in this study.

90

The experiments were divided into four groups with different treatments: (1)

91

Only water was added to the soil (CK). (2) Soil blended with 20 mg kg-1 DBP (DBP).

92

(3) Commercial HA, was added into the soil at an application dosage of 0.15% (HA).

93

(4) The soil was first blended with 20 mg kg-1 DBP, and then HA was added to the

94

soil at an application dose of 0.15% (DBP with HA). Soil respiration intensity was

95

measured at 0, 1, 3, 5, 7, 8, 14, and 21 days after the experiment. SMBC and DBP

96

contents at day 0, 7, 14, 21, 28, 45, and 60 were determined. PLFA contents were

97

tested on the 21st day. One kg soil was used in one pot and the soil water content was

98

kept at 70% of the field water content.

99

2.3 Soil functional groups and microstructure

100

Soil functional groups were measured by FTIR spectroscopy on a Nicolet Avatar

101

370DTGS spectrophotometer (Thermo Fisher Scientific, USA) (Huang et al., 2017).

102

After dried by a freeze dryer, soil samples were measured in KBr pellet at room

103

temperature in the spectra over the wavelength range from 4000-400 cm-1.

104

Observation of soil structures of the four different treatment samples was done by

105

SEM using HITACHI SU8010. Soil samples were fixed with glutaraldehyde and

106

dehydrated with ethanol. Because the soil sample is not electrically conductive, it

107

needs to be ion-sputtered. Ion sputtering treatment was performed by using E-1010

108

(HITACHI, Japan) to coat a Pt film on the surface of the sample. The applied voltage

109

was performed with as 5 kV. Soil bulk density was measured by the cutting-ring 6

110

method (Mbuthia et al., 2015). It was determined by using a stainless cutting-ring

111

(5 cm diameter, 5 cm height, the total volume of 100 cm3). The core samples were

112

immediately weighed, dried at 105℃ for 48 h to a constant weight.

113

2.4 Fluorescence characteristics and UV-Vis absorption spectra

114

DOM extraction: Weighted 10 g soil in the 250 mL conical flask containing 100

115

mL Milli-Q water and 0.01 M CaCl2, shaken at 25℃ and 150 rpm for 24 h, then

116

centrifuged at 5000 rpm for 10 min and filtration through a 0.45 µm membrane filter.

117

3D-EEM: To characterize the effect of DBP and exogenous HA on soil, DOM

118

was extracted from four different treatment soils (CK, DBP, HA, and DBP with HA)

119

firstly. 3D-EEM spectra were obtained using a fluorescence luminescence

120

spectrometer (F-7000; Hitachi, Japan) by scanning emission at 250-600 nm in 1 nm

121

increments, by varying the excitation wavelength from 200 to 450 nm in 5 nm

122

increments. Excitation and emission slit widths were of 5 nm, and the fluorescence

123

data were recorded at a scan rate of 1200 nm min-1.

124

Synchronous fluorescence spectra: DOM was extracted from uncontaminated

125

mollisol and combined with a series of DBP concentrations to probe the mechanism

126

by which DBP binds with soil DOM. The DOC of the DOM was 10.68 mg L-1. Five

127

mL of DOM was added into different 10 mL glass tubes, then DBP concentrations

128

between 0 to 0.36 mM were added into each glass tube containing DOM. To ensure

129

equilibrium, all solutions were shaken for 16 h before spectral detection. The

130

synchronous fluorescence spectra analyses were performed according to the method 7

131

of Chen et al (2014) under 25℃ and 35℃, respectively. The excitation and emission

132

slits were both adjusted to 5 nm, and the excitation wavelengths ranging from 250 to

133

580 nm were used with a constant offset (∆λ = 60 nm). Due to the special chemical

134

structure of DBP, it contains a benzene ring, which also produces fluorescence.

135

Therefore, an inner-filter correction was used to analyze the result. The method of

136

inner-filter correction followed Steiner (eq S1) (Steiner, 2012).

137

UV-Vis absorption spectrum: Since DBP provides fluorescence intensity at 270

138

nm, which coincides with the position where the protein in the DOM produces

139

fluorescence intensity the peak at 270 nm in synchronous fluorescence spectra of

140

DOM was attributed to protein-like substance. This majorly composed of

141

tryptophan-like fluorophores (Bai et al., 2017). Thus, the UV-Vis spectra were used to

142

survey the interaction between DBP and tryptophan-like fluorophores. In general, the

143

absorption peak of tryptophan ranged from 230 to 310 nm, derived from the

144

absorption of light due to the amino acid side chain groups (Bi et al., 2016). The

145

UV-Vis absorption spectrum was measured by UV-1800 (Shimadzu, Japan) with 1 nm

146

intervals. Milli-Q water was used as a control in the reference cell.

147

2.5 2D-FTIR-COS analysis

148

To investigate the binding of DBP and DOM, we measured the FTIR spectra

149

before and after the combination of DOM and various DBP concentrations. After the

150

addition of 5 mL DOM into 10 mL tube, 0 to 0.36 mM DBP were mixed in the tube

151

for examination. To ensure equilibrium, all solutions were shaken for 16 h before 8

152

freeze drying. For FTIR analysis, 1.0 mg DOM was ground and homogenized with

153

100 mg KBr and pressed under 15 MPa for 2 min. FTIR spectra (FTIR 370DTGS

154

Nicolet USA) was scanned range from 4000-400 cm-1 (Huang et al., 2017).

155

The 1750-750 cm-1 FTIR spectrum was synchronized with various concentrations

156

of DBP using 2D-COS to demonstrate the presence of DBP-HA binding behavior.

157

Synchronous and asynchronous correlation spectroscopy was generated using

158

2D-COS and mathematically was written by Noda et al. (eq S2, eq S3 & eq S4) (Noda

159

et al., 2000).

160

2.6 Soil respiration and SMBC

161

The soil microbial respiration was determined by alkali absorption titration, and

162

the amount of CO2 exhaled per hour was expressed as (mg kg-1 h-1) (Gilsotres et al.,

163

2005). Soil microbial biomass carbon was determined by the fumigation-extraction

164

method (Vance et al., 1987).

165

2.7 PLFA analysis

166

PLFAs were extracted using the method of Roosendaal et al. (2016). The FAMEs

167

were quantified using gas chromatography-mass spectrometry (GC-MS) (Shimadzu

168

QP-20120SE) with an HP-5 column. The temperature program started at 100℃

169

followed by a heating rate of 30℃ min-1 to 160℃, followed by a final heating rate of 5℃

170

min-1 to 280℃. The fatty acids were identified by retention time and confirmed by

171

mass spectrometry. Concentrations of FAMEs were calculated from peak areas and

172

reported as nmol g-1 soil. 9

173

2.8 Extraction and measurement of DBP

174

Five grams of soil sample (dry weight) was ultrasound extracted in 10 mL of

175

dichloromethane for 10 min and then centrifuged at 3000 rpm for 5 min to obtain

176

supernatants. This process was then repeated twice for a total of three extractions. The

177

supernatants were pooled and then evaporated to near dryness. The residue was

178

redissolved in 2 mL dichloromethane for GC-MS analysis. The detection conditions

179

of GC-MS were deployed according to Zhang et al. (2015). According to Beulke and

180

Brown (2001), the degradation dynamics of DBP in the soil correspond with the

181

bi-exponential model (eq.S5).

182

2.9 Statistical analysis

183

All experiments were performed in triplicate. The experimental data were

184

processed using Origin Pro 9.1, SPSS 24, and MATLAB R2016a statistical software

185

(version 19.0).

186

3 Results and discussion

187

3.1 Soil structure and FTIR analysis

188

Soil structure is a vital physical property, which can affect biological and physical

189

processes in soil. Fig.1a showed the microstructure of untreated mollisol. The soil had

190

good porosity with almost no polymerization. The surface of the soil particles being

191

uneven, rich soil pores form a large specific surface area of soil particles. The layered

192

structure of mollisol particles can be seen through the soil pores. After the soil was

193

contaminated by DBP (Fig.1b), the soil began to agglomerate; the porosity was 10

194

reduced, making it difficult to observe the layered structure. After the addition of

195

exogenous HA (Fig.1c), soil micro-morphology was still similar to CK treatment. In

196

DBP with HA treatment (Fig.1d), the soil reappeared in a dispersed state with an

197

uneven surface. Soil bulk density is related to soil texture, soil particle density, soil

198

organic matter content, and various soil management measures. The soil bulk density

199

changes of different treatments were shown in Table 1. Compared with the CK

200

treatment, the soil bulk density in HA treatment was slightly decreased while DBP

201

significantly increased the soil bulk density by 0.15 g cm-3 (8.71% increment). In the

202

DBP and HA treatment, the soil bulk density showed a significant decrease compared

203

with DBP treatment but still higher than that of CK treatment.

204

The decrease in the porosity of the soil particles and the specific surface area may

205

probably be caused by DBP retention. Pollutants are adsorbed to organic matter after

206

entering the soil and spillages of DBP do not tend to infiltrate deeply into the subsoil

207

unless it presents preferential flow paths. Also, the retention by capillarity in the fine

208

delicate pores will form an adhered film in the soil (Carrara et al., 2011). The

209

interfacial tension between DBP and air, as well as DBP and water, and by the

210

wettability of the DBP on the surface of the soil will make it limited for infiltration

211

into the soil. These changes in soil produced by DBP contamination will lead to a

212

deterioration in the water permeability and air permeability of the soil. HA can

213

improve soil aggregation, aeration and water holding capacity (Nan et al., 2016). It

214

proves that exogenous HA can loosen the soil. HA is less dense than soil and the 11

215

volume increases after water absorption, which reduces the amount of soil per unit

216

volume and causes a decrease in soil bulk density.

217

Several functional groups were detected in the soil by FTIR (Fig.2), the most

218

prominent finding was the shift of the functional group at 1877 cm-1. The absorption

219

peak at 1877 cm-1 indicates the H-O bond (Hadjar et al., 2008). FTIR data showed

220

that the peak at 1877 cm-1 which belongs to the H-O bond has a redshift in the soil

221

containing 20 mg kg-1 DBP. This result may be due to the conjugative effects of DBP

222

and H-O functional groups in the soil. It may also be due to the increase of H-O bond

223

length or bond strength of DBP. The addition of HA will cause more electron

224

transport in the soil, making the conjugate effect on the H-O bond larger than the

225

induced effect. Finally, the H-O absorption peak at 1870 cm-1 in the

226

DBP-contaminated soil recovered and eventually returned to the position of 1876 cm-1.

227

The broadband at about 3000-3800 cm-1 was exclusively associated with sorbed H-O

228

and the absorption peaks at 3621 cm-1 and 3426 cm-1 were the behaviors of mollisol

229

adsorbing water (Saikia and Parthasarathy, 2010). Near 1636 cm-1 was the protein

230

absorption peak. Near 1030 cm-1 was the stretching vibration absorption peak of the

231

C-O bond of carbohydrate. The absorption peak from less than 778 cm-1 represented

232

various mineral elements in the soil, including quartz absorption peaks and Si-O

233

bonds.

234

3.2 Soil DBP residues

235

Fig.3 showed the trend of soil DBP content for 60 days. The DBP concentrations 12

236

decreased in two treatment soils, indicating that the soil contains indigenous

237

microorganisms that degrade DBP. Degradation curves of the two treatment soils had

238

a good relationship with the biexponential models, which the R2 value were 0.9957

239

and 0.9896, respectively (Table 2). The half-lives derived from the biexponential

240

equations were 11.65 and 3.36 days for the DBP treatment and DBP with HA

241

treatment, respectively. The addition of HA greatly enhanced the DBP degradation in

242

mollisol. The DBP contents in the two treatments decreased slowly after an initial

243

rapid decline. DBP contents in the soil with and without HA decreased to 3.55 and

244

7.72 mg kg-1 respectively after 60 days incubation. The shorter DBP half-life in the

245

HA-amended soil may be due to the improvement of microbial habitat conditions.

246

Because decreased soil bulk density in HA-amended soil will be beneficial to the

247

aerobic degradation of organic pollutants by the degrading bacteria. It's worth noting

248

that DBP could not be degraded completely in either treatment and it may be related

249

to the transport of DBP in mollisol.

250

3.3 Fluorescence characteristics and UV-Vis absorption spectra

251

In order to clarify the transport process of DBP in mollisol, the interaction between

252

DBP and HA in mollisol was investigated. Two characteristic peaks in the 3D-EEM

253

plots were observed at excitation/emission (Ex/Em) of 270/433 nm (peak A) and

254

320/423 nm (peak B) (Fig.4). As indicated by past reports, the two peaks have been

255

portrayed as humic acid-like substance, respectively (Wang et al., 2017). As shown in

256

Table S1, DBP contamination and the addition of exogenous HA affected both peaks 13

257

A and peak B to varying degrees. Both peak heights of the HA treatment were higher

258

than those of the CK treatment. In contrast, DBP caused a decrease in the

259

fluorescence intensity of the two peaks.

260

The results of 3D-EEM spectra indicate that there was a strong complexation

261

between DBP and humic acid-like substances in mollisol. Wu et al. (2011) have

262

already proven that soil humic-like substances affect the migration and removal of

263

heavy metals during soil adsorption with a quenching effect between dissolved

264

organic matter components and four heavy metals. This is also in line with the report

265

by Wang et al. (2017) who studied the characterization of spectral responses of DOM

266

for atrazine binding during the sorption process onto mollisol.

267

Fig.5a showed the synchronous fluorescence spectra of DOM with different added

268

concentrations of DBP at 25℃. Two distinct peaks can be observed. Usually, peak A

269

and peak B belong to protein-like substances and humic acid-like substances,

270

respectively. However, due to the special chemical structure of DBP, it contains a

271

benzene ring. DBP itself produces fluorescence intensity at peak A (270 nm), which

272

coincides with the position where the protein in the DOM produces fluorescence

273

intensity. Therefore, UV-Vis spectra was used to analyze the interaction between DBP

274

and protein-like substances. With DBP addition, a prominent absorption appeared at

275

280 nm (Fig.S2). The result indicates that interaction exist between DBP and the

276

protein in the DOM. This interaction will lead to conformational changes of

277

protein-like substance. For peak B, with the increase of DBP concentration (0-0.36 14

278

mM), the fluorescence intensity of peak B decreased.

279

The modes of interaction between fluorophores and quenchers were different

280

(collision or complexation), so the fluorescence quenching process was divided into

281

two mechanisms: dynamic and static quenching. The Stern-Volmer equation (eq.S5)

282

under 25℃ and 35℃ demonstrated that the binding process between DBP and HA fit

283

the quenching extinguishing information condition (Song et al., 2010). As shown in

284

Fig.5b, both of (F0/F)-1 under 25℃ and 35℃ has a linear relationship (R2=0.9276,

285

R2=0.9478) with [DBP]. The quenching rate constants of Ksv for 25℃ and 35℃ were

286

247 L mol-1 and 206 L mol-1, respectively. To exclude the inner-filter effect,

287

inner-filter correction was used to analyze the result. The obtained rate constant of Kq

288

under 25℃ and 35℃ were 2.48 and 2.06×1010 L mol-1 s-1, which were greater than the

289

maximum scatters collision-quenching constant of the quencher to macromolecule

290

(2.0×1010 L mol-1 s-1). When the temperature rises from 25℃ to 35℃, the KSV value

291

decreases. Therefore, the fluorescence quenching of humic acid-like substances by

292

DBP was mainly caused by static quenching.

293

To know more about the mechanism of static quenching, fluorescence decay curves

294

for the solutions of humic acid-like substances data were used to obtain the binding

295

constants and the number of binding sites for the complex. The number of binding

296

constants and binding sites was inferred by the equation S6 (Zhang et al., 2017).

297

Fig.5c showed the plots of log [(F0-F)/F0] as a function of log [DBP], which explains

298

the binding of humic acid-like substances with DBP. The binding sites (n) for humic 15

299

acid-like substances under 25℃ and 35℃ were around 0.317±0.021 and 0.213±0.021,

300

respectively. The binding sites (n) for humic acid-like substances were smaller than

301

one showing that there was one binding site of fluorophores between humic acid-like

302

substances and DBP.

303

3.4 2D-FTIR-COS analysis

304

By converting the spectrum into a two-dimensional form, 2D-COS could enhance

305

the spectral resolution and could more clearly observe the peaks detail. The primary

306

infrared spectral characteristics of HA in DOM occurred in the range of 1750-750

307

cm-1, where almost all the vibrational information on HA backbones could be

308

identified (Chen et al., 2015). As the DBP concentration increased, the infrared

309

spectrum of the DOM changed as shown in Fig.S2. In the synchronous map (Fig.6a),

310

nine auto-peaks on the diagonal of the sync pattern were observed, which centered at

311

744, 941, 1074, 1122, 1286, 1728 cm-1. In the asynchronous map (Fig.6b), there were

312

several observable positive and negative peaks below the diagonal line. The details of

313

the spectrum were shown in Table.S3. The fluorophores of HA are mainly related to

314

aryl and phenolic substances. The positive peak zone was showed up at the 744, 941,

315

1074, 1122, 1286 and 1728 cm-1 in the synchronous map demonstrating that the

316

spectral changes occur in the same direction along with the corresponding areas. The

317

asynchronous map provided additional useful information about the sequence of DBP

318

binding to HA. According to Noda’s rule (Noda, 2012), the sign of an asynchronous

319

cross peak becomes positive if the intensity change at X-axis wavelength occurs 16

320

predominantly before the Y-axis wavelength in the sequential order of the external

321

variable. It becomes negative, on the other hand, if the change occurs after Y-axis

322

wavelength. Therefore, the sequence of the binding affinities followed the order aryl

323

C-O > alkyl ester C=O > aliphatic C-C > phenyl ring ortho disubstituted >

324

polysaccharide C-O > carboxylic acid and aromatic moieties C-O (1286 > 1728 >

325

941 > 744 > 1076 > 1122 cm-1).

326

When DBP enters the soil, it will combine with organic or inorganic surfaces,

327

non-aqueous liquids, and rubbery non-rigid structures of soil organic matter (Yang et

328

al., 2017). Then DBP will distribute into the small pores which are inaccessible to soil

329

microorganisms, tissues, and other organisms, or into the glassy rigid structure like

330

humin (Yu et al., 2017). In addition, due to the strong adsorption by clay minerals,

331

organic matter, and other environment element, organic pollutants will be more

332

refractory in the aged contaminated soils. That is to say, DBP's extraction and

333

microbial utilization will reduce when pollutant enters into a blockade stage. HA has a

334

complex structure, which containing fatty acids, polymethylenic chains, and a lot of

335

aromatic rings with -COOH and -OH groups. According to the results of

336

2D-FTIR-COS analysis, the functional groups of HA can bind to DBP. It means that

337

HA can compete with soil minerals to adsorb organic matter. Furthermore, HA has

338

surfactant-like micelle microstructures that can increase the solubility of organic

339

compounds and have the potential for enhancing the degradation of hydrophobic

340

organic compounds (Holman et al., 2002). Increased release of bound organic matter 17

341

in the soil may delay the blockade of DBP in rigid structures, and strive for more time

342

for aerobic microorganisms to degrade DBP (Liu et al., 2019).

343

3.5 Soil respiration

344

Soil respiration is often used to represent total soil microbial activity and is also

345

used to assess soil fertility (Ge et al., 2010). Fig.7a showed that the highest respiration

346

intensities were found on the 5th and 8th day. At the beginning of the experiment,

347

both DBP and HA enhanced soil respiration and DBP had a more significant

348

enhancement than HA. The promotive effect of HA became higher than DBP after 7

349

days and eventually higher than that in CK treatment. The respiration intensities of

350

DBP with HA treatment in the first seven days were significantly lower than the DBP

351

treatment but slightly higher than the HA treatment. After 7 days, the respiration

352

intensity of the DBP with HA treatment became weaker than that of the CK treatment

353

but stronger than that in the DBP treatment.

354

DBP could stimulate the physiological activity of degrading bacteria and activate

355

the respiration of such microbes (Gao and Chen, 2008). With an increase in culture

356

time, the contents of DBP decrease, the amount of specifically available substrate for

357

microorganisms decreases, and the stimulating effect became weaker. In DBP with

358

HA treatment, HA contributed to reduce the effect of DBP on soil respiration by

359

reduced DBP residues in the soil. HA elevate soil physical property and nutrition,

360

which is helpful to microbial growth in soil (Liu et al., 2019). HA also has the positive

361

effects of detoxification resulting from their binding properties, forming less 18

362

bioavailable complexes and adducts, and bioaccumulation of metals and/organic

363

compounds (Kulikova et al., 2005). Meanwhile, HA itself is a kind of organic matter

364

which can be used by microorganisms. The increase of the substrate in the soil will

365

also lead to the enhancement of soil respiration.

366

3.6 Soil microbial biomass carbon

367

The variation of SMBC contents is shown in Fig.7b. Similar to soil respiration, in

368

the first 7 days of the experiment, the SMBC of the DBP treated soil was higher than

369

that of the other three treatments and reached 165.73 mg kg-1 on the 7th day. But 7

370

days later, DBP showed a significant inhibitory effect on SMBC. HA had always

371

promoted SMBC contents, at 21 days HA had the most potent effect, compared with

372

the CK treatment it increased the MBC content by 13.42% reaching 160.36 mg kg-1.

373

For DBP with HA treatment, in addition to the promotion of SMBC on the 7th day

374

(increased by 23.00%), SMBC was almost maintained at the same level as the CK

375

treatment.

376

Chen et al., (2018) have shown that soil microbial biomass is affected by soil

377

organic carbon content, the higher the organic carbon contents, the greater the soil

378

microbial biomass. In this study, SMBC in HA treatment was higher than that in the

379

CK group. The result was consistent with the changes in soil respiration intensity. We

380

observed that DBP promoted soil MBC during the first 7 days of the experiment,

381

confirming the short-term influence of DBP in the soil microbial population, which in

382

turn promoted the proliferation of some microorganisms capable of using DBP as a 19

383

substrate. However, it was a transient promotion; this indicated that DBP has an

384

overall inhibitory effect on the quantity and activity of soil microorganisms (Gao and

385

Chen, 2008). HA could form a microenvironment around the cell, and the hydrophilic

386

part combines with the cell membrane, making the hydrophobic part away from the

387

surface of the cell. This prevents the hydrophobic DBP molecules from contacting the

388

cell membrane of the soil microbial cells directly, thus preventing the hydrophobic

389

damage of the cells and reducing the death of soil microbes (Xie et al., 2017). Thus,

390

using HA can effectively alleviate the impact of DBP on soil microbes.

391

3.7 PLFA analysis

392

Thirty-four PLFAs ranging from C1 to C34 were identified in the soil samples on

393

day 21 (Table S4). The PCA for the profiles of PLFAs was conducted to analyze the

394

changes in the microbial communities in different treatments (Fig.8). The cumulative

395

contribution rate of the first two principal components (PC1 and PC2) was 88.13%.

396

Representative fatty acids most relevant to PC1 were C12 and C16. The most

397

representative fatty acids associated with PC2 were C11 and C24. DBP had an

398

adverse effect on most soil microorganisms except C11 and C12. However, HA had

399

positive effects on C6, C9, C10, C11, C13, C16, C18, C31, C32. This means that

400

exogenous HA can effectively alleviate DBP pollution in soil by protecting the

401

stability of the microbial community structure. A decreased fungi/bacteria values and

402

an increased MUFA/STFA ratio were found in all treatments. Although the indicators

403

of the DBP with HA treatment were still slightly different from the CK treatment, 20

404

exogenous HA made the contents of each microorganism in the soil returned to a level

405

similar to that in CK treatment (Table.3). The research by Fan et al. (2016) showed

406

that the higher the soil organic carbon, the more abundant the soil microbial biomass,

407

and the PLFA content of various soil microorganisms also increases. This may be one

408

of the reasons for the different effects of DBP and HA on PLFA in the soil. Exogenous

409

HA can use its characteristics to improve soil properties (porosity) to reduce the

410

damage of DBP together with poisonous secondary metabolites to the soil

411

environment. Fungi/Bacteria can reflect the relative abundance of two microbial

412

populations. The stronger the buffer capacity of the soil ecosystem, the higher the

413

ratio of fungi/bacteria (Treseder, 2010). DBP pollution leads to the weakening of the

414

buffer capacity of the soil ecosystem. However, the decrease in the ratio of HA

415

treatment group is due to the increase in bacterial PLFA contents. MUFA/STFA ratio

416

reflects the relative advantages of aerobic and anaerobic bacteria. The higher value

417

indicates aerobic bacteria predominate. Carrara et al., (2011) showed that phthalic

418

esters are degraded under aerobic conditions by a wide range of bacteria. After the

419

soil was contaminated with DBP, the aerobic bacteria degrading DBP multiplied. HA

420

can also promote the growth of aerobic bacteria by increasing the amount of oxygen

421

in the soil, creating favorable growth conditions for aerobic bacteria.

422

4 Conclusions

423

In this study, HA was used as an exogenous additive to repair DBP contaminated

424

soil. The result elucidated that HA promotes DBP degradation. Adding HA to 21

425

DBP-contaminated soil can shorten the half-life of DBP from 11.65 to 3.36 day. Static

426

quenching confirmed the character of binding process between DBP and HA and one

427

binding site was found. The aryl C-O and alkyl ester C=O in HA are the two major

428

functional groups to bind with DBP. HA can mediate the transportation of DBP,

429

reduce soil bulk density, improve soil porosity and provide an enabling environment

430

and more time for aerobic degradation of DBP. Meanwhile, HA can stabilize

431

microbial activity in contaminated soil. The change of PLFA content in different

432

treatment soils further indicates that HA can protect microbial community structure

433

and promote the growth of aerobic bacteria in contaminated soil. Overall, HA has

434

shown its potential in soil DBP remediation, which contribute to mollisol sustainable

435

production and agricultural product safety.

436

Acknowledgments

437

This research was supported by the National Natural Science Foundation of

438

China (41877128), the MOA Modern Agricultural Talents Support Project, the

439

National Science Fund for Distinguished Young Scholars (41625002), "Young

440

Talents" Project of Northeast Agricultural University (18QC13).

441

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594 595

Figure captions Fig.1 SEM micrographic image of soil samples: (a) CK treatment; (b) DBP treatment; (c) HA treatment; (d) DBP with HA treatment

596

Fig.2 FTIR spectra of soils under different treatments

597

Fig.3 Soil DBP residues

598

Fig.4 Three-dimensional EEM fluorescence spectra of DOM with different

599

treatment: (a) CK; (b) DBP treatment; (c) HA treatment; (d) DBP with HA treatment

600

Fig.5 (a) Synchronous fluorescence spectra of interaction between soil DOM and

601

DBP with increasing concentration (0-0.36 mM); (b) Stern-Volmer plot of humic-like

602

substances with increased dosages of DBP under 25℃ and 35℃ ; (c) plot of

603

log[(F0-F)/F0] as a function of log[DBP] for the binding of soil humic-like substances

604

with DBP under 25℃ and 35℃.

605 606

Fig.6 (a) Synchronous and (b) asynchronous 2D FTIR correlation maps generated from 750 to 1750 cm-1 region of the FTIR spectra

607

Fig.7 (a) Soil respiration and (b) SMBC of different treatment

608

Fig.8

Principal

component

analysis

(PCA)

for

different

treatment

609

soils.(Individual PLFAs from the PLFA analysis of soil samples were subjected to

610

principal component analysis (PCA) after standardisation for equal unit variance)

30

Table 1. Soil bulk density. Treatment

CK

DBP

HA

DBP with HA

Bulk Density (g·cm-3)

1.7208 ± 0.0176bc

1.8706 ± 0.0692a

1.6737 ± 0.0185c

1.7824 ± 0.02034b

a–c: Means with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E.

Table 2. Biexponential modelling parameters for the degradation of DBP in soils under the general incubation conditions. Treatment

A

K1

B

K2

R2

Half-life (days)

DBP

9.26

0.0031

11.23

0.1952

0.9957

11.65

DBP with HA

4.99

0.0058

15.27

0.3195

0.9896

3.36

Table. 3. PLFA profiles under different treatments.

CK

DBP

HA

DBP with HA

(nmol·g-1)

(nmol·g-1)

(nmol·g-1)

(nmol·g-1)

Total fatty acid

249.25±10.11

217.11±5.42

262.72±7.34

235.54±6.28

Bacteria

139.37±4.21

132.58±3.87

159.67±5.44

145.21±4.11

Fungi

97.64±3.34

70.71±2.82

92.60±3.41

80.41±2.97

Actinomycetes

5.68±0.44

10.84±0.69

4.95±0.27

6.44±0.36

fungi/bacteria (%)

0.70

0.53

0.58

0.55

MUFA/STFA ratio

1.20

1.41

1.30

1.27

Fig.1 SEM micrographic image of soil samples: (a) CK treatment; (b) DBP treatment; (c) HA treatment; (d) DBP with HA treatment.

Fig.2 FTIR spectra of soils under different treatments.

Fig.3 Soil DBP residues.

Fig.4 Three-dimensional EEM fluorescence spectra of DOM with different treatment: (a) CK; (b) DBP treatment; (c) HA treatment; (d) DBP with HA treatment.

Fig.5 (a) Synchronous fluorescence spectra of interaction between soil DOM and DBP with increasing concentration (0-0.36 mM); (b) Stern-Volmer plot of humic-like substances with increased dosages of DBP under 25℃ and 35℃; (c) plot of log[(F0-F)/F0] as a function of log[DBP] for the binding of soil humic-like substances with DBP under 25℃and 35℃.

Fig.6 (a) Synchronous and (b) asynchronous 2D FTIR correlation maps generated from 750 to 1750 cm-1 region of the FTIR spectra.

Fig.7 (a) Soil respiration and (b) SMBC of different treatment a–c: Means with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E.

Fig. 8 Principal component analysis(PCA) for different treatment soils.

(Individual PLFAs from the PLFA analysis of soil samples were subjected to principal component analysis (PCA) after standardisation for equal unit variance).

HA shorten the half-life of DBP in black soil HA and DBP are bound by static quenching and have only one binding site HA stabilizes microbial activity and promotes the growth of aerobic microorganisms

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: