Diversity in the Mechanisms of Humin Formation During Composting

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Environmental Measurements Methods

Diversity in the Mechanisms of Humin Formation During Composting with Different Materials Xintong Gao, Wenbing Tan, Yue Zhao, Junqiu Wu, Qinghong Sun, Haishi Qi, Xin-Yu Xie, and Zimin Wei Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06401 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Environmental Science & Technology

1

Diversity in the Mechanisms of Humin Formation During

2

Composting with Different Materials

3 4

Xintong Gao,†,‡ Wenbing Tan,†,§ Yue Zhao,‡ Junqiu Wu,‡ Qinghong Sun,‡ Haishi

5

Qi,‡ Xinyu Xie,‡ and Zimin Wei*,‡

6 7

‡

College of Life Science, Northeast Agricultural University, Harbin 150030, China

8

§

State key laboratory of environment criteria and risk assessment, Chinese Research

9

Academy of Environmental Sciences, Beijing 100012, China

10 11 12 13 14 15 16

†Gao, Xintong and Tan, Wenbing contributed equally to this work.

17

*Corresponding Author:

18

College of Life Science, Northeast Agricultural University, Harbin 150030, China.

19

Tel/Fax: +86 45155190413

20

E-mail address: [email protected] or [email protected]

ACS Paragon Plus Environment


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21

ABSTRACT

22

Humins (HMs) play a very important role in various environmental processes and are

23

crucial for regulating global carbon and nitrogen cycles in various ecosystems.

24

Composting is a controlled decomposition process accompanied by the stabilization

25

of organic solid waste materials. During composting, active fractions of organic

26

substances can be transformed into HMs containing stable and complex

27

macromolecules. However, the structural heterogeneity and formation mechanisms of

28

HMs during composting with various substrates have not been clarified. Here, the

29

structure and composition of HMs extracted from livestock manure (LM) and straw

30

(SW) during composting were investigated by excitation-emission matrices

31

spectroscopy and Fourier transform infrared spectroscopy. The results showed that the

32

stability and humification of LM-HM were lower than that of SW-HM. The

33

PARAFAC components of the HM in LM composting contained the same fluorescent

34

unit, and the intermediate of cellulose degradation affected the structure of the HM

35

from

36

low-molecular-weight compounds were key factors in humification. On the basis of

37

the structure and key factors impacting HM, we constructed two mechanisms for the

38

formation of HM from different composting processes. The LM-HMs from different

39

humification processes have multiple identical fluorescent structural units, and the

40

high humification of SW is affected by its polysaccharide constituents, which contains

41

a fluorescent component in their skeleton, providing a basis for studying HM in

42

composting.

SW

composting.

Structural

equation

modeling


demonstrated

that

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43 44

Table of Content (TOC)



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46

INTRODUCTION

47

Composting is an attractive and low-environment-risk option for managing organic

48

solid waste, a growing output produced annually worldwide, which affects the

49

environment.

50

remediation of contaminated sites represent a large potential market for organic

51

compost.1,2 The application potential of compost is to large extent dependent on

52

compost quality. The formation of humic substances (HS) is an important process of

53

organic matter conversion during composting.3 Given the dual functionality of HS in

54

nourishing environmental matrices and mediating the redox reactions of pollutants,4,5,6

55

regulating the formation of highly active HS has become the driving factor in

56

determining the quality of compost, thus governing the potential compost markets.

Currently,

applications

in

agricultural/horticultural

lands

and

57

The process through which HS is formed is often called humification, which

58

mainly involves microorganism-dominated biological and biochemical processes and

59

abiotic chemical reactions.7,8 Many different speculations, including lingo-protein

60

theory, phenol-protein theory, micellar hypothesis of Wershaw, supramolecular

61

concept of Piccolo, humo-nanotube-membrane hypothesis and sugar-amine

62

condensation theory, have been used to explain humification.9 Each theory has three

63

common stages: (i) the decomposition of HS precursors into organic compounds with

64

a simple structure, followed by (ii) the metabolism and repeated cycles of

65

microorganisms on these compounds and ending with (iii) the formation of

66

recalcitrant macromolecular organic products through microbial synthesis.9 Thus,

67

precursors from different sources can cause HS produced by composting to have


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68

different molecular composition and formation pathways,9 due to their different

69

organic compositions and different effects on the community and structure of

70

microorganisms.10,11

71

Generally, HS can be operationally categorized into extractable humic and fulvic

72

acids (HA and FA, respectively) and unextractable humin (HM) according to what

73

can be extracted with an alkaline solution.12 HM that has a relatively large molecular

74

weight and a high degree of polymerization often lacks of ionizable functional

75

groups.13 In addition, HM in various matrices is always adsorbed to or occluded

76

within an inorganic solid-phase fraction,14 which could create a good environment for

77

the stability of HM and thus allows HM functionality, such as electron shuttle, to last

78

for a long time. We speculate that these special properties of HM and its unique

79

spatial location in matrices may cause the molecular composition and formation

80

mechanism of HM to be different from those of HA and FA during composting.

81

According to the theory of HS formation, the effects of microbial metabolism and

82

repetitive cycling on the “C core” of HM and its bound functional groups may be

83

unique. Although previous work has provided evidence on the formation processes of

84

HA and FA during composting,15,16 the molecular composition and formation

85

mechanism of compost-derived HM and their associations with compost precursors

86

during composting are presently unknown because the fact that the task of extracting

87

and purifying HM is relatively difficult. Therefore, such association mechanisms

88

warrant investigation given their expected importance in filling the knowledge gaps in

89

HM-formation

processes

during

composting


and

in

developing

a


90

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classification-oriented approach for composting with different precursor materials.

91

Fourier transform infrared (FTIR) and fluorescence spectroscopy techniques are

92

relatively easy and cost-efficient approaches to characterize organic matter. However,

93

the use of FTIR or fluorescence spectra alone often fails to provide information for

94

molecular-level understanding of organic matter due to the overlap of many molecular

95

signals.17 Two-dimensional correlation spectroscopy (2DCOS) can distinguish

96

overlapped peaks by extending spectra along the second dimension and providing

97

information about the relative directions and sequential orders of molecular structure

98

variations.18 In essence, 2DCOS aims to estimate the correlation coefficients between

99

two spectral variables. A high correlation indicates that the associated bands may

100

have the same origin. Consequently, the burgeoning use of 2DCOS in studies on

101

organic matter can tremendously advance our understanding of the interaction and

102

evolution mechanisms of organic molecular composition. FTIR and fluorescence

103

spectroscopy techniques are the major probes used in 2DCOS analysis to provide

104

particular

105

2D-heterospectral correlation (hetero-2DCOS) analysis can compare two spectral data

106

for a system obtained by using spectral probes under the same external

107

perturbation.20,21 Owing to these appealing features, 2DCOS and hetero-2DCOS have

108

been utilized to provide an in-depth understanding of the molecular composition and

109

formation mechanisms of organic matter.

molecular

information

about

the

binding

process.19

Moreover,

110

In this study, different stages of composts produced by composting different

111

precursor materials were sampled, and their HM was correspondingly extracted and


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112

purified. 2DCOS of FTIR spectra and hetero-2DCOS based on FTIR spectra and

113

fluorescence spectra of parallel factor analysis (PARAFAC) components were

114

combined with structural equation modeling (SEM) to reveal differences in the

115

molecular composition and formation mechanisms of HM formed by different

116

precursor materials. The main objectives of this study were to (i) explore the

117

dynamics of HM molecular composition during composting, (ii) identify the

118

mechanisms of HM formation during composting, and (iii) evaluate the effect of

119

compost precursors on the molecular composition and formation pathway of HM.

120

MATERIALS AND METHODS

121

Composting Experiment and HM Preparation. Solid piles of two types of

122

livestock manure (chicken manure (CM) and cow dung (CD)) and three types of straw

123

(corn straw (CS), rice straw (RS), and soybean straw (SS)) were composted in a

124

reactor.22 To achieve sufficient composting, the initial moisture content of the solid

125

waste mixture was maintained in the range of 60–65%, the C/N ratio was 20:1, and

126

the ventilation rate of the reactor was 0.5 L/min. According to the changes in the

127

composting temperature, the samples were regraded in the warming phase (WP),

128

thermophilic phase (TP), cooling phase (CP) and mature phase (MP). The temperature

129

changes and sampling days corresponding to various stages during composting are

130

shown in Table S1. The basic physical and chemical indicators in the composting

131

process are shown in Table S2. The piles were turned over at each sampling time to

132

ensure a stable oxygen supply. All samples were freeze dried and stored at −20 °C

133

prior to testing.



134

The close association of inorganic minerals is the main reason for the poor

135

solubility of HM.14 Therefore, a demineralized HM was obtained according to the

136

method of Song et al.23 with some modifications. Briefly, approximately 100 g of the

137

freeze-dried raw compost sample was passed through a 200-mesh sieve to obtain a

138

homogeneous sample. Then, compost samples (2 g each, more than ten samples from

139

each stage of composting) were transferred to centrifuge tubes and 0.1 M HCl was

140

added to the samples three times to remove water-soluble organic matter. Then, 0.1 M

141

NaOH and 0.1 M Na4P2O7 were added to remove the HA and FA. These steps were

142

repeated several times until the supernatant was colorless or pale yellow. The solid

143

residue was thoroughly washed with deionized water to remove excess base. Then,

144

the samples were treated with a mixture (6 M HF + 10 M HCl) at 50 °C for 12 h to

145

remove minerals from the matrix and rinsed with distilled water until the aqueous

146

phase was neutral. Finally, some of the HM was extracted by adding 0.1 M NaOH.

147

The pH was adjusted to 7 with 6 M HCl and let stand for 12 h. The precipitate was

148

washed with deionized water until no precipitation was detected with silver nitrate,

149

and the obtained solid was freeze -dried (centrifugal speed of 12000 r/min,

150

solid-liquid ratio of 1:15). All sample extracts were obtained performed under the

151

same conditions and using the same method of extracting the HM from the

152

composting samples. In this case, the structure and extraction recovery of HM should

153

be consistent among the different composting samples. We defined the extraction

154

recovery of HM from the composting samples as the percentage of organic carbon in

155

the HM extracted from the composting samples relative to the amount of organic


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156

carbon in the residual composting samples after extraction of the humic acid and

157

fulvic acid. The result showed that there were no significant differences in the

158

recoveries of HM among the different composting samples (Figure S1).

159

FTIR Spectroscopy. The HMs extracted from the 2-g powdered samples were

160

ground into homogeneous powders, and 1 mg of the powder was used for FTIR

161

analysis. This 1 mg of powdered HM sample is representative of the HM extracted

162

from the 2-g powdered compost samples (more than ten samples from each stage of

163

composting and three repetitions were conducted). The FTIR spectra were obtained

164

with a Tensor II FTIR spectrometer (the spectral resolution was 4 cm-1, and the range

165

was 4000-400 cm-1) at room temperature. Each spectrum was used to reduce the level

166

of noise (number of iterations 1-3) and baseline corrected. The dominant band at 3430

167

cm-1 can be assigned to O-H stretching in carbohydrates or N-H stretching in amides,

168

and there is a weak shoulder at 3230 cm-1 due to the presence of amide and other

169

substances.24 The two minor peaks at 2900 cm−1 and 2840 cm−1 can be assigned to

170

aliphatic C-H stretching.24 Previous reports indicate that these peaks might be due to

171

the presence of lipids.25 The absorption bands at 1730 cm−1 and 1650 cm−1 are typical

172

of C=O stretching of COOH or ketones and aromatic C=C stretching,

173

respectively.24,26 In general, the absorption band at 1500 cm−1 is also indicative of

174

aromatic C=C stretching. A broad peak at 1040 cm−1 indicates C=O stretching of

175

polysaccharides and carbohydrates.25 A continuous absorption band from 1100 cm−1

176

to 1500 cm−1 can be attributed to various low-molecular-weight compounds, such as

177

phenols, carboxylic acids, aromatic ethers, and nitrates.26



178

EEM Fluorescence Spectroscopy and PARAFAC Modeling. In this study, the

179

HMs from all samples were diluted to 10 mg/L with 0.1 M NaOH (Figure S2). We

180

were recorded EEM maps by a luminescence spectrophotometer (Hitachi model

181

F-7000). The emission wavelengths (250–550 nm) and excitation wavelength (220 to

182

490 nm) over the range of were observed in 5 nm and 10 nm increments, respectively.

183

Finally, the scattering was calibrated to the area by the Milli-Q water Raman peak

184

acquired in the meantime.27

185

We used the procedure described of Stedmon and Markager to EEM-PARAFAC

186

analysis.28 The clear composition was obtained by decrease the impact of scatter lines

187

in the EEM maps using MATLAB 2016a (Mathworks, Natick, MA) with a

188

DOMFluor toolbox (www.models.life. ku.dk). We were separated the fluorescence

189

components by EEM-PARAFAC and no abnormal components were found. For

190

EEM-PARAFAC modeling, the EEMs of the emission and excitation wavelengths

191

were the same as those of the fluorescence spectra. More than one hundred samples

192

were evaluated for each set of data.

193

2D Correlation Spectroscopy. We are according to the mothed of Noda to

194

acquire the 2D correlation spectra employing 2Dshige, v.1.0.0.0 software.29 In this

195

study, compost samples were divided into two categories, manure and straw, and the

196

2DCOS of FRIT and the hetero-2DCOS of FRIT/EEM fluorescence were obtained for

197

both categories. The 2D spectral changes of 𝑦𝑗 can be by a function of an external

198

variable and a spectral variable at n equally spaced points, can be expressed as

199

follows:


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200 201 202 203 204 205


(1)

𝑦𝑗(𝑥) = 𝑦(𝑥,𝐶𝑗) A clearly defined of dynamic spectra y(x,C) as follows: 𝑦𝑗(𝑥) =

1≤𝑗≤𝑚 {𝑦 (𝑥) ― 𝑦(𝑥)0 𝑓𝑜𝑟𝑜𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒 𝑗

(2)

Two maps (The synchronous (Ф) and asynchronous (Ψ) correlation spectra) was generated by the discrete Hilbert-Noda transform: 𝑚

𝑧𝑗(𝑥) = ∑𝑘 = 1𝑁𝑗𝑘 ∙ 𝑦𝑘(𝑥),𝑁𝑗𝑘 =

{

0 1 𝜋(𝑘 ― 𝑓)

𝑖𝑓 𝑗 = 𝑘 𝑜𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒

(3)

1

𝑚

(4)

1

𝑚

(5)

206

Φ(𝑥1,𝑥2) = 𝑚 ― 1∑𝑗 = 1𝑦𝑗(𝑥1) ∙ 𝑦𝑗(𝑥2)

207

Ψ(𝑥1,𝑥2) = 𝑚 ― 1∑𝑗 = 1𝑦𝑗(𝑥1) ∙ 𝑧𝑗(𝑥2)

208

Two different maps were provided by the 2D correlation analysis. The synchronous

209

map shows correlations of changes in each band that change during the experiment

210

and whether the correlation between them increase or decrease. The asynchronous

211

correlation map relates that information to the sequence of events taking place.29

212

SEM Analysis. The SEM was used to clarify the direct and indirect relationships

213

of the HM functional groups. SEM is an effective method to study relationships

214

between latent and observed variables.30 It has been widely used to interpret and

215

predict interplay in multivariate data sets.31 Before SEM analysis, autoregressive

216

correlation structures to identify potential autocorrelations in the IBM SPSS AMOS

217

23.0 data were studied. Subsequently, we tested two assumptions regarding internal

218

structural changes in the HM, each time removing the corresponding arrow from the

219

complete SEM and using SPSS to compare the resulting model to the complete

220

model. We used the chi-square test in SPSS software to verify the quality of the fit,

221

such as, if the P value >0.05, then this model is true. Finally, we iteratively removed



222

all nonsignificant missing paths, and retested the model's adequacy each time. In

223

addition, we use standardized total effects from SEM to verification indirect and

224

direct relationships in the model.

225

RESULTS AND DISCUSSION

226

Variations in the chemical constituents of HM during different composting

227

types. FTIR spectroscopy was used to identify the functional groups present in the

228

HM. The intensities of the characteristic absorption bands were used to determine the

229

relative contents of the functional groups within the HM (Figure S3). In LM and SW

230

(Figure 1), the contents of amide and polysaccharide-like substances decreased by

231

approximately 14% (64%-49%) and 9% (57%-48%), respectively. This may be

232

because the amide and polysaccharide substances in the HM are used as a carbon

233

sources by microorganisms. In contrast, the contents of bound amide and

234

polysaccharide-like substances showed the opposite trend, and one of the reasons for

235

this is the carbonamide condensation (Maillard reaction) between the amides and the

236

polysaccharide-like substances. The Maillard reaction32 is an important pathway in

237

natural humification processes. Although the Maillard reaction is kinetically sluggish

238

under normal environmental conditions,33,34 its reaction rate can increase during

239

composting. This reaction may be one of the main mechanisms of HM formation

240

during composting. Moreover, polysaccharide-like substances dominate the SW

241

compost due to the structural differences in the materials. Polysaccharides increase

242

the humification of polyphenols through the Maillard reaction.35 Therefore, the

243

content of aromatic compounds in SWHM can rapidly increase, and this is why the

244

aromatic content in the SW increased significantly (8%-14%) and was stable in the

245

LM (10%-11%).

246


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247 248

Figure 1. Variations in the FTIR spectra of HM of LM (livestock manure

249

composting) and SW (straw composting). The content of each component is

250

expressed as the ratio of the individual fluorescence intensity to the total fluorescence

251

intensity. WP, TP, CP and MP indicate the warming phase, thermophilic phase,

252

cooling phase and mature phase, respectively.

253 254

Unlike in SW, the contents of heteropolysaccharide-like substances, carboxylic

255

acids, aromatic ethers, and a series of low-molecular-weight compounds in LM

256

tended to increase, probably because amides are the dominant components of LM, and

257

free amides exhibited substantial activity and could provide many binding sites for



258

low-molecular-weight compounds. The formation of HS is a typical polymerization

259

process carried out by various organisms;36 low-molecular-weight substances can be

260

incorporated into the HM structure in a variety of ways, and the addition of these

261

substances affects the material composition and spatial structure of the HM.

262

Compared with LM, the composition of SW is more complicated. Therefore, LM-HM

263

may have an unstable chemical-structure due to the relatively lower degree of

264

humification. The above result showed that the degree of humification of HM in SW

265

was higher than that in LM by the end of composting.

266

Trend in the Conversion of HM Functional Groups. Due to the complex

267

structure of HM, it is difficult to judge the changes in the contents of certain

268

functional groups based on the changes in the absorption peaks obtained by simple

269

infrared spectroscopy. Therefore, detailed changes in HM structure during different

270

composting stages were investigated using 2DCOS (Figure 2). Six autopeaks, at 3430,

271

2900, 1700, 1530, 1250, and 1030 cm−1 were observed in the synchronous 2DCOS of

272

LM, and positive cross-peaks were observed at (1700, 2900), (1030, 2900), (1500,

273

1700), (1250, 1700), (1030, 1700), and (1030, 1250) nm. Unlike LM, in addition to

274

the four autopeaks at 3430, 1500, 1100, and 1010 nm, four positive cross-peaks at

275

(1500, 1700), (1250, 3430), (1100, 3430), and (1030, 3430) nm and three negative

276

cross-peaks at (1030, 1500), (1700, 3430), and (1500, 3430) nm were also detected in

277

SW. The changes in these absorption peaks are consistent with the changes in the

278

contents shown in Figure 1. However, for LMHM, a continuous negative cross-peak

279

appeared at 1030–3200 nm and the absorption peaks, including those at 1450, 1320,


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Page 15 of 36


280

and 1110 nm, were not easily observed via FTIR spectroscopy. These substances may

281

be immobilized in the HM structure, or the changes may be caused by a random

282

combination of substances causing the absorption peaks to shift. No new absorption

283

peaks were observed for SWHM. These results suggest that SWHM has a relatively

284

simple and stable structure due to the stability of this intermediate of cellulose

285

degradation.

286 287

Figure 2. Synchronous and asynchronous 2D FTIR correlation maps generated from

288

the 400-4000 cm-1 region of the spectra of HM in the LM and SW. Red and blue

289

represent positive and negative correlations, respectively. A more intense color

290

indicates a stronger correlation.

291



Page 16 of 36

292

The asynchronous 2DCOS analyses of LM and SW showed significant differences

293

(Figure 2). In LM, three positive cross-peaks were observed at (1500, 3430), (1250,

294

3430), and (1030, 3430) nm and seven negative cross-peaks appeared at (1700, 3430),

295

(2900, 3430), (1700, 2900), (1500, 2900), (1030, 1500), (1030, 1700), and (1500,

296

1700) nm. In SW, three positive cross-peaks were detected at (1600, 3430), (1500,

297

3430), and (1030, 1500) nm and four negative cross-peaks were observed at (2900,

298

3430), (1700, 3430), (1250, 3430), and (1030, 3430) nm. According to Noda’s rules,

299

the color and intensity represent correlations and increase or decrease with the

300

strength of the correlation. The reactions of the peaks observed during composting

301

were in the following order: 2900 nm > 1700 nm > 3430 nm > 1500 nm, 1250 nm >

302

1030 nm for LM and 3430 nm > 2900 nm > 1700 nm, 1600 nm, 1500 nm > 1250

303

nm > 1030 nm for SW. Therefore, the HM components in LM changed in the

304

following sequence: aliphatic-like substances > carboxylic acids > amides > aromatic

305

compounds, aryl ethers > heteropolysaccharide-like substances. The components in

306

SW varied in the following order: polysaccharide-like substances > aliphatic-like

307

substances > carboxylic acids, aromatic compounds > phenols, aryl ethers >

308

heteropolysaccharide-like

309

polysaccharide-like substances are the main components of LM and SW, respectively,

310

and are also the preferred C sources of microorganisms in the compost. These

311

compounds are also the first to form in the HM. Hence, the formation of HM is

312

closely related to the order of organic matter degradation and content during

313

composting. The amide and polysaccharide-like substances were confirmed to be

substances.

Aliphatic-like


substances

and

Page 17 of 36


314

precursors to HM formation, even though they form the basic structure of the HM

315

carbon skeleton. Moreover, carboxylic acids are incorporated into two HM structures

316

simultaneously. We suspect that the combination of other substances formed may be

317

closely related to the presence of substances such as carboxylic acids, especially

318

aromatic compounds.

319

HM Fluorescence Component Analysis. Various aromatic compounds are

320

present in different concentrations in HM, one of the fractions of natural organic

321

matter. Analyzing the contents and variations of each aromatic constituent by FTIR is

322

difficult. Therefore, the EEM spectra of the HM were analyzed by PARAFAC. The

323

results of split-half and residual analyses and visual inspection could be used to

324

determine the correct number of components (Table S3). The accuracy of the HM

325

components modeled by PARAFAC was ensured using the split validation procedure

326

(Figure S5 and S6).37 In this study, three independent components could be identified

327

in each sample (Figure S4). At each source component, the excitation spectra of HM

328

showed two maxima at different wavelengths and the emission spectra showed a

329

single peak. The increase in molecular complexity and structural condensation were

330

indicated by the increase in wavelength in the fluorescence spectra.38 Moreover, the

331

fluorescence intensities of the PARAFAC components of HM from different compost

332

sources were significantly different (Table S5). Previous studies rarely analyzed the

333

differences between the overlapping peaks of the PARAFAC components. Here, we

334

used hetero-2DCOS to further analyze the chemical structure of HM.

335

Relationship between the HM Fluorescence Component and Functional



336

Group. This method was used to elucidate the relationship between the fluorescence

337

components and the FTIR-active functional groups in LMHM and SWHM, and the

338

bonding mode and mechanism of formation of the aromatic compounds in HM were

339

analyzed. In the synchronous maps of LMHM (Figure 3A), the FTIR spectra showed

340

the two main bands at 2700–3250 and 920–1320 nm remained in the same positions.

341

In the three components, these two continuous positive bands included aliphatic-like

342

substances, amides and polysaccharide-like substances, carboxylic acids, and phenols.

343

The bands in the EEM spectrum maps indicated various types of functional groups. (i)

344

The band at 270–310 nm was assigned to C1, (ii) the band at 300–370 nm was

345

assigned to C2, and (iii) the bands at 300–340 and 350–450 nm were assigned to C3.

346

In C1, aliphatic-like substances, amides and polysaccharide-like substances,

347

carboxylic acids, and phenols are tightly bound to amino acids with fluorescent

348

structures, such as tyrosine and tryptophan. In the other two components, these

349

substances are closely bound to fulvic-like acids and humin-like acids. The absorption

350

band of C3 showed a significant negative correlation at 300–340 nm, suggesting the

351

fluorescence component may be dependent on other structures in the HM, and

352

fluorescence quenching is occurring. Moreover, three new positive peaks appeared in

353

C2 and C3 with the FTIR bands at 1450, 1550, and 1600 cm-1, which represented

354

aromatic compounds and nitrate containing substances. This result shows that during

355

the synthesis of HM, a large number of aromatic C and nitrate-containing substances

356

were generated. In other words, the aromatization of HM occurs mainly in the middle

357

and late stages of composting. In the asynchronous map, the absorption peaks


Page 18 of 36

Page 19 of 36


358

appeared in the same position as they were in the synchronous spectrum, but in the

359

opposite direction; a new positive absorption peak appeared at 3340 nm. These results

360

indicate that fluorescence reactions occurred before the functional groups changed

361

and were closely related to the presence of tyrosine and tryptophan. Through chemical

362

reactions and the action of microorganisms, small molecules are continuously

363

polymerized to form macromolecules with complex 3D structures. The growth and

364

development of HM follows the same procedure.39



366 367

Figure 3. Synchronous and asynchronous maps were obtained via 2D hetero-spectral

368

correlation analysis of the FTIR and EEM spectra of HM in LM (A) and SW (B). Red

369

represents positive correlations and blue represents negative correlations. More

370

intense colors indicate stronger positive or negative correlations.


Page 20 of 36

Page 21 of 36


371 372

The synchronous maps of SWHM changed because its composition is more

373

complicated than that of LMHM (Figure 3B). In C1, six cross-peaks at (3310, 310),

374

(1550, 270), (1410, 270), (3310, 270), (1010, 270), and (1310, 270) nm could be

375

observed. The three first and the three last cross-peaks were positive and negative

376

absorption peaks, respectively. In the FTIR analysis, C2 and C3 were in exactly the

377

same position as in C1. In the EEM of C2 and C3, bands were present at 260–360 and

378

310–460 nm, respectively, indicating that in the initial stage, SWHM mainly contains

379

polysaccharides and has only a small amount of aromatic species. As SWHM

380

progresses, a small amount of polysaccharide-like substances containing aromatic

381

species are combined with the HM, however, polysaccharide-like substances that do

382

not fluoresce are still the major constituent of SWHM. On one hand, the

383

lignocellulose biomass actively degrades in the cooling phase. During the degradation

384

of lignocellulose biomass, some carbohydrates may undergo acid-catalyzed

385

hydrolysis or dehydration reactions which lead to the production of the majority of the

386

HM. The intermediate product of lignocellulose degradation contains some aromatic

387

C itself, which may be an important source of the aromatic C in SWHM. On the other

388

hand, a large number of intermediates from cellulose degradation combine in different

389

ways, forming the main structure of SWHM.

390

In the asynchronous map of C1, four positive peaks at (3310, 290), (2790, 290),

391

(1100, 290), and (1030, 290) nm and one negative absorption peak at (1420, 290) nm

392

could be observed. These results indicated that the fluorescence reaction occurred



393

before polymerization of the polysaccharides, aliphatic compounds, and carboxylic

394

acid compounds. In C2, two positive peaks at (3300, 280) and (1410, 320) nm and

395

five negative absorption peaks at (3300, 320), (1400, 280), (1140, 280), (1040, 280),

396

and (900, 280) nm were observed. These findings suggested that the reactions of the

397

polysaccharides, aromatics, and carboxylic acids occurred before the fluorescence

398

reactions. In C3, the cross-peaks were located at the same places as they were in the

399

synchronous fluorescence, but the directions of their changes were reversed. This

400

phenomenon showed that the fluorescence reaction occurred first and was followed

401

by the condensations of the other substances, which is consistent with our previous

402

understanding that lignocellulose substances have a high degree of aromatization and

403

can directly participate in the formation of SWHM through acid-catalyzed hydrolysis.

404

Key Factors Influencing HM Formation. Generally, microbial activity is a key

405

factor affecting HS formation.40 SEM was conducted to detect the causal relationships

406

among the microbial and HM components (Figure 4). The results show that

407

microorganisms are significantly correlated with the formation of amides (λ=-0.38,

408

P aromatic compounds, aryl

455

ethers > heteropolysaccharide-like substances. This result also indicates the basic

456

condensation process of the fluorescent unit. Based on the low-molecular-weight

457

compounds present, fluorescent substances condensed to form fluorescent peaks with

458

characteristic humic-like components. As the organic matter degraded, small

459

molecules became attached to the C skeleton of HM by condensation reactions, such

460

as those between hydroxyl and carboxyl-rich groups of aromatic compounds and

461

quinine compounds. Finally, small-molecule HM species aggregated to form

462

macromolecules with complex and uneven structures that could satisfy the

463

composition and the ratio of the functional groups of the HM components indicated

464

by the FTIR and EEM spectra.



466 467 468

Figure 5. The basic fluorescence unit and PARAFAC component space structure of

469

HM in livestock manure (A) and straw (B) composting.

470 471

The formation of SWHM is more complicated than that of LMHM (Figure 5B). In

472

the initial stage of composting, the intermediates from cellulose degradation are rich

473

in carboxyl and hydroxyl moieties and serve as the precursor of HM, promoting the


Page 26 of 36

Page 27 of 36


474

formation

475

polysaccharide-like substances combine with the precursors of HM to form a tight

476

polymer that can also be regarded as the “core” of SWHM. Consequently, the content

477

of aliphatic compounds in the HM noticeably increased during the cooling period.

478

Simultaneously, a large amount of lignocellulose degradation intermediates with a

479

fluorescent structure combine with the core of the HM. With the formation of

480

fluorescent substances and their reactions with low-molecular-compounds, fluorescent

481

substances gradually exhibit characteristics of humic-like components. However, the

482

intermediates of cellulose degradation, namely, polysaccharide-like substances,

483

dominate and mask the fluorophores (Figure 3B). The addition of a large number of

484

carbon-containing structures with regular shapes suggests that SWHM has a compact

485

structure and even forms spheres. Jin et al. also showed that HM comprises solid,

486

spherical agglomerates and that the fluorescence intensity of aromatic compound

487

increases at short wavelengths as humus structures are destroyed.42 This principle

488

explains why the SWHM structure is compact and its degree of humification is high.

489

During maturation, the aromatics species and small-molecule organic acids are

490

attached to the surface of the HM spheres forming mature and stable HM

491

macromolecules. HM was formed because these low-molecular-weight compounds

492

continued, causing to achieve growth and development. Even with increasing time,

493

the spherical structures of SWHM condensed with each other to form a complex 3D

494

structure. Based on the above conclusion, we could clearly deduce the molecular

495

composition and 3D configuration of the two kinds of HM. Therefore, the combined

of

polyphenols.

As

composting

continues,


the

aliphatic-

and


Page 28 of 36

496

application of hetero-2DCOS and SEM provided theoretical support and evidence for

497

the mechanism of the formation of complex, macromolecular organic compounds.

498

Significance of This Work. Understanding the mechanism of HM formation is

499

essential for elucidating the mechanisms of humification in different soil

500

environments and realizing their profound roles in environmental issues. In the

501

present study, the formation mechanisms of HM from different composting sources

502

were determined by FTIR spectroscopy with EEM technology. The HM structures

503

from the two composting materials showed notable differences. LM was rich in

504

protein-like components, and intermediates from cellulose degradation dominated the

505

SW. In particular, the aromatization of SW was significantly faster than that of LM,

506

resulting

507

heteropolysaccharide-like substances were the key factors affecting the degree of HM.

508

Therefore, we can tune the HM and its 3D structure for application in various fields

509

according to its formation mechanism. This finding is of great significance for

510

assessing the value of composting products.

in

high

aromatization.

Moreover,

aliphatic-like

compounds

and

511 512 513

ASSOCIATED CONTENT

514

Supporting Information

515

Additional details are presented about supplementary text, figures and tables. This

516

material is available free of charge via the Internet at http://pubs.acs.org.

517 518

Note


Page 29 of 36


519

The authors declare no competing financial interest.

520

ACKNOWLEDGEMENTS

521

This work was financially supported by the National Natural Science Foundation of

522

China (No. 51778116, No. 51178090, No. 51878132, No. 51378097). Moreover, we

523

are very grateful to Mr. Ben Lohr and three anonymous reviewers for contribution in

524

the revision of the paper.



526

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Diversity in the Mechanisms of Humin Formation During Composting

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