One-Pot Transformation of Technical Lignins into Humic-Like Plant

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Article Cite This: ACS Omega 2018, 3, 7441−7453

One-Pot Transformation of Technical Lignins into Humic-Like Plant Stimulants through Fenton-Based Advanced Oxidation: Accelerating Natural Fungus-Driven Humification Hae Jin Jeong,†,‡,§ Joon-Yung Cha,†,∥ Jung Hoon Choi,⊥ Kyoung-Soon Jang,⊥ Jongkoo Lim,# Woe-Yeon Kim,†,‡,∥,§,▽ Dong-Cheol Seo,‡,§ and Jong-Rok Jeon*,‡,§ †

Division of Applied Life Science (BK21Plus), ‡Department of Agricultural Chemistry and Food Science & Technology, §IALS, RILS, and ∥PMBBRC, Gyeongsang National University, 501, Jinju-daero, Jinju 52828, Republic of Korea ⊥ Biomedical Omics Group, Korea Basic Science Institute, 162, Yeongudanji-ro, Cheongju 28119, Republic of Korea # R&D Center, GS Caltex Corporation, 359, Expo-ro, Daejeon 34122, Republic of Korea

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S Supporting Information *

ABSTRACT: Commercial humic acids mainly obtained from leonardite are in increasing demand in agronomy, and their market size is growing rapidly because these materials act as soil conditioners and direct stimulators of plant growth and development. In nature, fungus-driven nonspecific oxidations are believed to be a key to catabolizing recalcitrant plant lignins, resulting in lignin humification. Here we demonstrated the effective transformation of technical lignins derived from the Kraft processing of woody biomass into humic-like plant fertilizers through one-pot Fenton oxidations (i.e., artificially accelerated fungus reactions). The lignin variants resulting from the Fenton reaction, and manufactured using a few different ratios of FeSO4 to H2O2, successfully accelerated the germination of Arabidopsis thaliana seeds and increased the tolerance of this plant to NaCl-induced abiotic stress; moreover, the extent of the stimulation of the growth of this plant by these manufactured lignin variants was comparable or superior to that induced by commercial humic acids. The results of high-resolution (15 T) Fourier transform-ion cyclotron resonance mass spectrometry, electrostatic force microscopy, Fourier transform-infrared spectroscopy, and elemental analyses strongly indicated that oxygen-based functional groups were incorporated into the lignins. Moreover, analyses of the total phenolic contents of the lignins and their sedimentation kinetics in water media together with scanning electron microscopy- and Brunauer−Emmett− Teller-based surface characterizations further suggested that polymer fragmentation followed by modification of the phenolic groups on the lignin surfaces was crucial for the humic-like activity of the lignins. A high similarity between the lignin variants and commercial humic acids also resulted from autonomous deposition of iron species into lignin particles during the Fenton oxidation, although their short-term effects of plant stimulations were maintained whether the iron species were present or absent. Finally, we showed that lignins produced from an industrial-scale acid-induced hydrolysis of wood chips were transformed with the similar enhancements of the plant effects, indicating that our fungus-mimicking processes could be a universal way for achieving effective lignin humification.

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INTRODUCTION The continual misuse of NPK-based fertilizers has increased the need to develop and use sustainable and ecofriendly materials for increasing soil fertility and hence enhancing crop productivity.1 Humic substances (HSs), which are mainly derived from the decay of plant tissues, are known to stimulate plant growth and development in a multifunctional way.2−4 In addition, the capacity of high-molecular-weight HSs to immobilize inorganic plant nutrients5 and to aggregate soil particles6 suggests that they may serve as excellent soil conditioners. Commercial HSs manufactured in bulk are extracted from coal resources such as lignite and leonardite, whereas natural humic materials are widespread in the environment. The production and quality control of © 2018 American Chemical Society

commercial HSs rely thus on the distribution of coal, which is limited. To overcome this bottleneck, developments of artificial humification pathways, in particular those with ecofriendly materials, have been recently highlighted. For examples, Savy et al. demonstrated that soluble fractions of technical lignins exhibit a humic-like activity toward maize.7 Polymeric products obtained from oxidative polymerization of natural phenols such as catechol and vanillic acid were proven to be effective at stimulating Arabidopsis plants.8 Received: April 10, 2018 Accepted: June 20, 2018 Published: July 6, 2018 7441

DOI: 10.1021/acsomega.8b00697 ACS Omega 2018, 3, 7441−7453

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analytical tools including a scanning electron microscopy (SEM), measurements of specific surface area, high-resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS), sedimentation kinetics, electron paramagnetic resonance (EPR), electrostatic force microscopy (EFM), element analysis, determination of phenol contents, Fourier transform-infrared spectroscopy (FT-IR), and inductively coupled plasma-optical emission spectrometry (ICP-OES) were used to compare the variants with commercial humic acids with respect to both their organic and inorganic structures and to decipher structural aspects key for driving humic-like fertilization. Plant-stimulation activities of lignins before and after Fenton oxidations were evaluated by using Arabidopsis plants.

Lignin and HSs have been suggested to be structurally similar.8,9 Aromatic portions of HSs that persist in the environment are believed to be derived from lignin because lignins with multiple aromatic rings supplied from plant biomass tend to be recalcitrant compounds against most microbes.9 Fungi, however, have been shown to actively oxidize lignin under aerobic conditions,10 presumably resulting in the transformations of its aromatic architectures. Although a detailed relationship between humification and microbe-driven metabolism of lignin has not yet been demonstrated, some reports have suggested that fungal metabolism with ligninrelated biomaterials can contribute to humification.11,12 Nonspecific oxidations resulting from Fenton chemistry and ligninolytic enzymes are mainly involved in the biotic transformation of lignin due to the structural diversity and three-dimensional structure of lignin.10 Through these reactions, lignin fragmentation concomitant with addition of oxygen-based functional groups occurs,13 and it is not unreasonable to suggest that such structural changes give rise to the enhancement of the cationic exchange capacity of HSs, which is one of their main beneficial characteristics.14 In addition, phenols and carboxylic acids in aromatic polymers have been proven to directly stimulate plants, indicating that oxygen incorporation leading to the formation of such hydrophilic functional groups allows lignins to directly boost the stimulation of plant growth and development.8 Production of technical lignins from woody biomass has been increasing steadily due to the industrialization of cellulose-based fermentation as well as due to the traditional pulp industry.15 Effective valorization strategies for lignins are thus required to maximize the cost-effectiveness of the biomass refinery. Toward this end, some methods have been introduced to isolate humic-like molecules from lignin biomass; note that such methods are also important because humic-like molecules are applicable to crop production and alternative to commercial humic substances.7,16,17 However, their plantstimulation action relies heavily on their high water solubility, suggesting that low-molecular-weight lignin-derived phenolics exhibiting strong phytotoxicity8,18 become mixed with each other during the separation processes. Moreover, little attention has been paid to the inorganic portion of humic materials despite humic−Fe complexes being readily identifiable in the environment19 and lignite, one of the major coal sources for commercial humic acids (HAs), also containing significant amounts of iron.20 It is thus not unreasonable to suggest that humic substances showing hydroxyphenyl groups persist as organic−Fe complexes in the environment. Several biomolecules containing polyphenolic chains (i.e., the structures readily found in HSs) have been shown to recruit Fe ions to modulate their physicochemical and biological functionalities through the formation of polyphenol−Fe complexes21,22 and Fe uptake, which is essential for healthy plant growth, has been shown to be significantly affected by the presence of organic matter in soils.23 These results strongly indicated that the Fe-associated inorganic portion of humic acids must be considered for optimizing artificial humification of plant materials. The aim of this study was to develop a novel ecofriendly methodology for effective humification of technical lignins that are used in Kraft and sugar fermentation processes. A Fentonbased one-pot advanced oxidation was employed not only to mimic fungus-driven lignin humification, but also to induce the formation of Fe-based complexes with lignin variants. Several

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RESULTS AND DISCUSSION Fenton-Induced Changes in Kraft Lignin Structures. Kraft lignins have been produced as a byproduct in pulp industries, but their applicability has not yet been extended to agronomy.24 To induce an accelerated humification, Fenton agents were mixed with Kraft lignin particles. Because the reactions were based on water matrices, lignins were partitioned into two phases (i.e., water-soluble and waterinsoluble phases). Here, we employed Fenton lignin variants belonging to the water-insoluble phases for further experiments because low-molecular-weight phenolics derived from Fentonbased lignin depolymerization are known to exhibit strong phytotoxicity.25,26 The insoluble portion was harvested through the same centrifugal force with both Fenton-treated and nontreated samples, thus assuring that lignin-related molecules showing similar solubility were collected (see Materials and Methods). The collected particles after freezedrying were colloidally dispersed in distilled water as did commercial Sigma humic acids. The intensity of broad peaks ranging from 3200 to 3400 cm−1 clearly increased in the IR spectra of the samples subjected to Fenton oxidations (Figure 1A). The peak positions corresponded to the O−H of the phenolic groups. In addition, the intensities of the peaks in the vicinity of 1705 cm−1 corresponding to CO of the carboxylic acids increased with increasing concentration of Fe2+, suggesting that carboxylic acids were incorporated into the lignin particles (Figure 1C). To observe the morphological changes in lignin particles, the lyophilized powders were visualized using SEM. As shown in Figure 2A, globular structures in Kraft lignins were frequently identified, but the structures mostly appeared degraded, presumably due to the Fenton-based advanced oxidations. The globular structures are also identifiable in supramolecular structure of melanin involving π−π stacking.27 Several hydrophilic groups introduced through Fenton oxidation would facilitate the disassembly of π−π stacking, thus making the polymeric architecture much irregular. In addition, other factors such as polymer length and metal chelating would be involved in the destruction of the globular structures. Such a morphological change would link to a change in the surface areas of the lignin powders. As shown in Table 1, the Fenton reactions were observed to yield significant increases in the Brunauer−Emmett−Teller (BET) surface areas of the lignin particles. This increase would be also attributable to the physical opening of a tightly packed π−π stacking. The significant increases in the total phenolic contents derived from particle surfaces of the lignin variants (Figure 2B) suggest that the particle fragmentation leading to 7442


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to affect the extent of lignin coagulations. Coordination of metals with lignin architecture has been shown to coincide with physical coagulation.28 Clear evidence for the incorporation of oxygen into the lignins was also provided by elemental and ultrahigh-resolution (15 T) electrospray ionization (ESI) (−) FT-ICR MS analyses. The relative amount of oxygen in the lignins increased as a result of the Fenton reactions, whereas those of carbon and nitrogen decreased, suggesting that bleaching of the preexisting organics and addition of oxygen-based functional groups occurred simultaneously (Table 2). The total number of ions detected in the negative ESI mode dramatically increased as a result of the Fenton oxidations (Table 3). This ESI result was attributed to the presence of oxygen-based functional groups, which were readily transformed into negative dipoles and ions. Furthermore, based on the molecular assignments of the detected m/z values, we were able to decipher in detail the structural modifications of the lignin components that resulted from the Fenton reactions (Figure 3). First, the relative number of molecules showing high O/C ratios increased, and this result was comparable with the IR peaks supporting oxygen incorporation. Second, the relative number of molecules showing high H/C ratios (i.e., lipid-like molecules) also increased. Similarly, molecular distribution with respect to double-bond equivalents (DBEs) showed that the DBE values for carbon numbers at about 20 decreased during the course of the Fenton reactions, suggesting that aromatic portions of lignins became saturated (Figure 4). Fenton-based oxidations have been shown to facilitate benzene-ring openings and the resulting formation of aliphatic structures.29 Solid-state NMR analyses have clearly demonstrated heterogeneous mixtures of aromatic and aliphatic groups to constitute one of the key structural features of commercial humic acids.8 Thus, the use of Fenton-based oxidations of lignin powders would be a scalable way to effectively transform aromatic lignins into humic-like structures containing aliphatic groups. This method also overcame our previous humification method,8 which hardly generated aliphatic structures. To characterize the electrostatic interactions occurring on the lignin surfaces, EFM scanning was employed. As shown in Figure 5, the electrostatic roughness was directly proportional to the amount of ferrous ions used for the Fenton reactions. This direct proportionality may have resulted from (i) the addition of oxygen-based hydrophilic groups to lignin surfaces or (ii) with the presence of iron-related inorganics deposited in lignin during Fenton reactions. The formation of iron oxides during Fenton reactions for pollutant degradation has been well characterized.30 Fenton reactions with lignins would thus also be expected to lead to the formation of the oxides. We hypothesized that polyphenolic structures in lignins would recruit the oxides by chelating them, thus retaining them during water-based washing (see Materials and Methods). To test this hypothesis, ICP-OES analyses were performed before and after Fenton oxidations. As shown in Figure 6, much higher amounts of iron-related inorganics were observed in the Fenton-based lignin variants than in the untreated lignins. Interestingly, the iron content in some commercial humic acids manufactured in different countries was also much higher than the iron content in untreated lignins. These results strongly indicated that Fenton-based modifications resulted in lignins displaying inorganic as well as organic structural features. In addition, our results indicated the potential commercial benefit

Figure 1. (A) FT-IR-attenuated total reflection (ATR) spectra of untreated Kraft lignins and their Fenton variants. (B, C) Magnification of the spectra (A) in a specific range. Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O.

the increases in the BET surface areas is initiated with phenolbased surface modifications. It is also noticeable that the total phenolic contents could be achieved, similar to that of commercial humic acids (Figure 2B). However, unexpectedly, their ζ-potential values were slightly decreased, although the IR peaks suggested that the Fenton reactions resulted in the lignin surfaces becoming modified with oxygen-based functional groups. Moreover, the hydrodynamic sizes of some of the Fenton samples were greater than those of the untreated lignins (Table 1). This result was in contrast to changes in the particle morphologies and the surface areas. Both ζ-potential values and hydrodynamic sizes were measured in aqueous media and indicated that water molecules were actively incorporated into macro-organic components of the lignin powders. In addition, iron-related inorganics capable of being cross-linked with lignins after Fenton treatments were expected 7443


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Figure 2. (A) SEM images and (B) total phenolic contents of Kraft lignins and their Fenton variants. Data in the phenolic contents are mean ± standard deviation (SD) (n = 2). Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O.

Table 1. Specific Surface Areas, ζ-Potentials, and Hydrodynamic Sizes of Kraft Lignins and Their Fenton Variantsa sample

BET surface area (m2/g)

ζ-potential (mV)

hydrodynamic size (d, nm)

Kraft Kraft-0.1 Kraft-0.5 Kraft-1.0

0.5 4.2 6.0 4.7

−51.5 −44.5 −42.4 −44.4

1157 1706 2256 1144

of using inorganic compounds in humic acids to stimulate the growth of plants. In fact, iron belongs to one of the essential elements for plant growth and development.31 The EPR spectra we obtained for the lignin and Fenton variants showed similar g values of about 2.00, and this result suggested that the lignin and Fenton variants contained significant amounts of semiquinone-type free radicals (Table 4 and Figure 7A). In fact, such types of radicals are readily identifiable in lignins.32 Overall, the peak-to-peak signals decreased as the concentration of Fe2+ used to treat lignin particles was increased. This kind of signal reduction was consistent with the previous results showing the chelation of metal by polydopamine, the similar polyaromatics.33 The deposition of iron oxide into lignin matrices during Fenton

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Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O.

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ACS Omega Table 2. Elemental Analyses of Kraft Lignins and Their Fenton Variantsa sample (%)

N


0.35 0.19 0.15 0.15

± ± ± ±

C 0.00 0.00 0.00 0.00

63.90 62.14 61.18 61.94

± ± ± ±

H 0.07 0.16 0.09 0.06

5.86 5.50 5.24 5.23

± ± ± ±

S 0.04 0.00 0.02 0.02

1.04 0.94 0.86 0.94

± ± ± ±

O 0.00 0.00 0.00 0.00

26.54 28.63 29.69 28.19

± ± ± ±

0.14 0.13 0.11 0.20

Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4· 7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O.

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Plant-Stimulation Effects of Kraft Lignins and Their Fenton Variants. The Kraft lignins were concluded, based on the above-described results, to be oxygenated and then their polymers fragmented when the Fenton-based modifications occurred. In addition, significant amounts of iron-related species were shown to be deposited with lignin particles, thus resembling commercial humic acids with respect to inorganic sides. The commercial value of humic acids in agronomy lies in their plant-stimulation activity. It has been reported several times that the multifunctional effects of humic acids allow plants to take up macroelements more efficiently, to increase their biomass more quickly, and to endure abiotic stresses effectively.36−38 The extents of plant stimulations for Kraft lignins and their variants must be thus examined to assess the effectiveness of our artificial humification strategy. First, as lignin−iron complexes themselves, variants of Arabidopsis thaliana seedlings were examined for their effects. Interestingly, specific conditions used to produce the complexes (i.e., Kraft0.5 and Kraft-1.0) induced a faster germination than did those of untreated lignins and commercial humic acids (Figure 8A). The rate of germination at these conditions increased with increasing concentration of the variants treated (Figure 8A). Photographic images of A. thaliana showing longer radicles for the Fenton variants were also consistent with the observed germination kinetics (Figure 8B). To determine the extents of any effects of iron deposits derived from the Fenton processes on plant stimulations, acid washing was employed to remove the iron deposits from the lignin particles. The ICP analyses indicated that acid washing resulted in a dramatic decrease in the amount of iron (Figure 9). The washed lignin particles still induced accelerated A. thaliana germination, suggesting that the iron particles hardly affected the germination process (Figure 10). However, the association of iron species with humic acids was previously shown to be correlated with plant growth.23,39 Different kinds of plant experiments aimed at determining long-term effects of iron-related inorganics are planned. In addition, experiments using this strategy and involving the assessment of soil conditions where most crops are cultivated are also planned because the dynamics of soil microorganisms affecting plant growth can be modulated with iron, which is one of the ubiquitous electron donors/acceptors.40 The physiological effects of HSs on higher plants are multifunctional and several mechanisms related with ion absorption, metabolism, and hormone-like activity have been so far suggested to explain these effects.41 The capacity of the lignin variants to tolerate salt-induced stress was also evaluated to determine whether the multifunctional effects of humic acids on plants also occur. The chlorophyll contents demonstrated that the Fenton-based transformation of lignins showed a better plant-stimulation activity than did both untreated and commercial humic acids (Figure 11). Our previous results strongly support that the salt tolerance is associated with gene induction such as high-affinity

Table 3. Number of Molecular Compositions Assigned through FT-ICR-MS Analyses of Kraft Lignins and Their Fenton Variantsa composition

Kraft

Kraft-0.1

Kraft-0.5

Kraft-1.0

CH CHO CHON CHOS CHONS total

2 676 570 205 1 1454

3 1031 1264 254 12 2564

1 993 1389 202 6 2591

1 859 1100 169 6 2135

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oxidations was thus hypothesized to lead to a decrease in the relative amount of semiquinone-type free radicals. We also studied the sedimentation kinetics of the lignins to determine whether the extent of supramolecular interaction of the lignin molecules under water matrices was modified. As shown in Figure 7B, commercial humic acids sedimented at the slowest rate. Much slower kinetics for the sedimentation in aqueous solutions was observed for all of the Fenton variants than for the untreated lignin. Interestingly, the specific mass ratio of FeSO4 and H2O2 (i.e., Kraft-0.5) for Fenton oxidations exhibited kinetics similar to that of humic acids. These patterns indicate that the hydrophobic region of lignin contributing to self-aggregation was destroyed via Fentonbased nonspecific oxidation. Solid-state 13C NMR spectra were acquired from the lignins before and after being subjected to the Fenton oxidations, and the carbon signals were clearly diminished as a result of the Fenton oxidations, suggesting that the carbon−hydrogen located at the surfaces of the particles were replaced with carbon−oxygen (Table 5). In addition, the paramagnetic impurities derived from iron oxide deposition during Fenton reactions would also contribute to the lower signals. According to the ratios of the intensities of the aliphatic carbon to aromatic carbon peaks,34 the aromaticity levels of the samples subjected to Fenton-based modifications were slightly less than those not subjected to oxidation. This result was consistent with the other results including FT-ICR MS, IR, and sedimentation (Figures 1, 3, and 7B). The Kraft lignin transformation did not yield any significant changes in the intensities or positions of the NMR peaks corresponding to the oxygen-based functional and aliphatic groups (Figure S1). Note, in this regard, that the oxygen-containing organics have been shown to chelate metal oxides.35 The formation of oxygen-based functional groups followed by their chelating of iron-based inorganics would hinder the detection of carbonbased shifts using NMR. Indeed, the chelation of ferric ions by lignins made 31P NMR-based structural elucidation not possible.25 7445


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Figure 3. van Krevelen plots showing the distribution of chemical classes based on the molar H/C and O/C ratios of the assigned elemental compositions from Kraft lignins and their Fenton variants, analyzed by ESI (−) FT-ICR MS (color code: CH, red; CHO, blue; CHON, orange; CHOS, green; CHONS, purple). (A) Untreated Kraft lignin, (B) lignin treated with 0.05 g of FeSO4·7H2O, (C) lignin treated with 0.25 g of FeSO4·7H2O, and (D) lignin treated with 0.50 g of FeSO4·7H2O.

K+ transporter 1 via humic-like polyphenolic structure.42 Detailed differences in the gene induction between commercial humic acids and lignin variants should be assessed to fully understand the exact molecular mechanisms. Plant-Stimulation Effects of Acid Hydrolysis-Derived Lignins and Their Fenton Variants. Recently, acid hydrolysis-based fermentations of celluloses from woody biomass have been shown to give rise to lignin byproducts. The detailed structures of lignins are not easily determined because the materials are highly cross-linked and coagulated. In addition, the detailed structures vary depending on the sources of the lignins and methods used to extract them.43 Advanced oxidations including Fenton reactions have been shown to induce nonspecific oxidations, strongly suggesting that lignins can be transformed regardless of their structural diversity.29,30 To determine whether our strategy for lignin humification is universal, lignins of a different type, specifically derived from

acid hydrolysis of woody biomass, were transformed using Fenton reactions. Here, the extent of plant stimulations differed from those with Kraft lignins. However, Fenton variants of the lignin from acid hydrolysis exhibited a much more rapid germination than did both commercial humic acids and the untreated lignin. A similar trend was also observed in the cases of salt-induced abiotic stresses (Figure 12). FT-ICR MS-based molecular assignments of the lignins derived from the acid treatment were also made, and the results indicated that the overall distribution of their molecular compositions differs from that of the Kraft lignins (Figure S2), supporting the previous results regarding the structural diversity of lignins.43 The atomic ratios derived from these results corresponding to protein- and lipid-like molecules showed the acid lignins to have a greater diversity of molecules than did the Kraft lignins. Moreover, the lignins subjected to the Fenton oxidations displayed higher O/C ratios than did 7446


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Figure 4. DBE and carbon number plots of (A) untreated Kraft lignin, (B) lignin treated with 0.05 g of FeSO4·7H2O, (C) lignin treated with 0.25 g of FeSO4·7H2O, and (D) lignin treated with 0.50 g of FeSO4·7H2O. The size of the circle indicates the relative intensity of the assigned composition.

ratio of hydrogen peroxide to iron(II) sulfate for lignin humification.

the untreated lignins, suggesting the occurrence of efficient oxygenations during the Fenton reactions (Figure S2 and Table S1). Carbon numbers with same the DBE values significantly decreased during the course of the Fenton reactions (Figure S3). This pattern may have been due to the bond cleavage through the Fenton-induced nonspecific oxidations. Morphological changes of the acid lignins were similarly achieved with those of Kraft lignins (Figures 2A and S4). Efficacy of Fenton oxidation is known to depend on the organic structures of oxidation targets.29 The similar humification results compared with those of Kraft lignins suggest that key structural aspects of polyaromatics for plantstimulation are inducible with the overall control of amphiphilic groups, especially oxygen-related functional groups. Further optimization of the Fenton processes based on a response surface methodology is planned to find the best

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CONCLUSIONS Here, we designed a scalable and lignin structure-independent strategy to convert technical lignins into humic-like plant fertilizers using Fenton-based advanced oxidations. Several analyses including FT-ICR mass spectrometry, sedimentation, FT-IR, BET, determination of total phenolic content, and elemental analysis showed that the oxygen groups were incorporated into the lignin particles concomitant with polymer fragmentation. Such changes in amphiphilic groups and aggregation patterns led to accelerated rates of seed germination and the tolerance of A. thaliana to salt-induced abiotic stress. The extent of plant-stimulation here was comparable to that of commercial humic acids, implying that the Fenton reaction for technical lignin humification could be a 7447


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Figure 5. (A) EFM images and (B) Signal averages of Kraft lignins and their Fenton variants. Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4· 7H2O.

heptahydrate, humic acid, and hydrogen peroxide (35%) were obtained from Junsei, Sigma-Aldrich, and Duksan, respectively. Additional humic acids used in the ICP-OES analyses for determining the Fe content originated from China and the United States and were purchased from MR Innovation Corporation (Daegu, Korea). Sulfuric acid and 60% perchloric acid were purchased from Samchun Chemical and J. T. Baker, respectively. Both Folin and Ciocalteu’s phenol reagent and gallic acid were obtained from Sigma-Aldrich. Fenton Oxidation and Plant-Stimulation Tests. After crushing Kraft and acid lignins with a mortar and pestle, a volume of 20 mL of hydrogen peroxide (35%) was mixed with 1 g of each lignin powder followed by stirring each of these two mixtures at 130 rpm with a magnetic bar. Each of three different amounts of iron(II) sulfate heptahydrate (i.e., 0.05, 0.25, or 0.50 g for Kraft lignins; 0.05, 0.25, or 0.35 g for acid hydrolysis-related lignins) were then added to different samples of these stirred mixtures to initiate Fenton reactions at room temperature. The ratios of hydrogen peroxide to iron(II) sulfate were based on the previous study showing

promising lignin valorization strategy. Interestingly, analytical tools such as ICP-OES and EFM suggested iron-related inorganics to be widespread in commercial humic acids. The Fenton reactions allowed lignin particles to mimic the inorganic aspects of commercial humic acids because some of the oxidized iron-based inorganics spontaneously deposited into the lignin matrices during the Fenton reactions. Further tests of the long-term effects of iron-based inorganics on plant stimulations are planned to confirm the necessity of humic mimic−iron complexes.

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MATERIALS AND METHODS Chemicals and Materials. Kraft lignin was purchased from Sigma-Aldrich, whereas lignin from the acid-induced hydrolysis of wood chips was kindly donated by the GS Caltex Corporation. The acid lignin containing approximately 1% H2SO4 was directly used without further washing. Plantstimulation tests were based on A. thaliana wild-type (Col-0 background) cultivated on Murashige and Skoog (MS) medium purchased from Duchefa Biochemie. Iron(II) sulfate 7448


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Figure 6. Fe contents in Kraft lignins and their Fenton variants analyzed by ICP-OES. Data are mean ± SD (n = 3). Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O; HA, commercial humic acids.

Table 4. EPR Parameters of Kraft Lignins and Their Fenton Variantsa Kraft Kraft-0.1 Kraft-0.5 Kraft-1.0

line width (g)

g value

peak-to-peak

35 39 33 32

2.003 2.003 2.003 2.003

82.6185 80.7596 47.2729 40.9867


Figure 7. (A) EPR spectra and (B) sedimentation kinetics of Kraft lignins and their Fenton variants. Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O; HA, humic acids.

Fenton-based oxidation of aromatics.29 A fifth of the tested mass iron(II) sulfate heptahydrate completely dissolved in 5 mL of distilled water was injected every 10 min into the mixture. Each oxidation reaction was terminated after 2 h by subjecting the respective mixture to centrifugation at 3000 rpm for 15 min. The pellets were then washed two times with distilled water by repeated centrifugation (3000 rpm for 15 min). The final powders for the plant experiments were obtained by carrying out lyophilization. The lignin used as a control was subjected to the same methods except that neither hydrogen peroxide nor iron(II) sulfate heptahydrate was included as control. Commercial Sigma humic acids were colloidally dispersed in distilled water for further use. Seed germination and NaCl-related salt tolerance tests were based on the methodologies previously described.8 Briefly, the seeds were germinated on the MS media, each of them containing commercial humic acids, lignin, and lignin variants. Radicle and cotyledon emergence after 2 and 4 days of the incubation were monitored and photographed with a microscope (Olympus). Arabidopsis seedlings grown on the MS media for 5 days were transferred onto new MS media, each containing commercial humic acids, lignin, and lignin variants in the absence or presence of NaCl (250 mM). After a further 7 day incubation, the plants were photographed with a conventional digital camera and then their chlorophyll was

extracted by using 80% (v/v) acetone and then quantitatively measured by using a visible spectrophotometer.8 Characterizations of Technical Lignins and Their Fenton Variants. Lyophilized lignins and their Fenton variants were treated with gold sputtering after they were attached to a carbon tape (Tedpella). The field-emission SEM (Philips, XL30S FEG) images of the powders were then obtained to visualize their ultrastructures. The electrostatic interactions made by the functional groups of lignins and their Fenton variants were monitored using EFM scanning (+8 V tip, Nanoscope IV, Veeco Instruments, Inc.). Flat plates of lignins for EFM analyses were manufactured with a conventional compressor. The specific surface areas (i.e., Brunauer− Emmett−Teller (BET)) of the particles were measured with N2 gas (ASAP 2010 system, Micromeritics Corp.). The relative amounts of C, H, O, N, and S in the samples were determined using a Flash EA 2000 series instrument (Thermo Fisher). The solid-state 13C NMR spectra were acquired using a 400 MHz NMR spectrometer (Avance III HD, Bruker, Germany) with the use of tetramethylsilane as a chemical shift reference. The operating conditions used for the NMR experiments were described in detail previously.8 All of the powders were scanned 6000 times. The X-band continuous-wave EPR spectra were obtained with a Bruker EMX Plus 6/1 spectrometer equipped with a dual-mode cavity (ER 4116DM). The detailed experimental parameters used were

a

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ACS Omega Table 5. Percent of 13C Distribution in 13C NMR Spectra of Kraft Lignins and Their Fenton Variantsa peak intervals (ppm) samples

0−52

52−96

97−162

162−188

188−262

aromaticityb

total peak area comparison (relative %)


18.7 18.1 17.2 16.5

20.2 21.4 24.0 22.4

55.3 54.8 54.5 53.4

1.1 1.6 1.7 2.1

4.6 4.0 2.6 5.6

58.7 58.1 56.9 57.9

100 77.6 69.2 79.9

Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4· 7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O. bAromaticity (%) = [Aromatic C peak area (97−162 ppm)]100/[Total peak area (0−162 ppm)].34 a

Figure 9. Fe contents in Kraft lignins and their Fenton variants before and after HCl-based acid washing. Abbreviation: Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O.

Figure 8. (A) Accelerated rates and (B) representative photographical images of A. thaliana germination in the presences of commercial humic acids, Kraft lignins and their Fenton variants (86 or 860 mg/ L). Data are mean ± standard error (SE) (n = 3). Significant differences are shown as asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). Abbreviation: HA, commercial humic acids; Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O. The control samples without any treatments and the samples treated with humic acids were also used to compare the germination rates in Figure 12.

as described previously.8 The hydrodynamic size distribution and ζ-potentials of the lignins and their Fenton variant particles dispersed in distilled water were measured with an electrophoretic light-scattering spectrophotometer (ELS 8000, Otsuka, Japan). The IR spectra of the powders were acquired in attenuated total reflection (ATR) mode (iS50, ThermoFisher). ICP-OES (OPTIMA 5300DV) was employed to quantify Fe-containing inorganics in commercial humic acids (i.e., Sigma, United States, and China), lignins, and Fenton variants of lignins. The procedures for digesting the organic materials used here have been described in detail previously.33 The

Figure 10. Accelerated rates of A. thaliana germination in the presence of commercial humic acids, acid-washed Kraft lignins, acidwashed Fenton variants, and nonwashed Fenton variant (1060 mg/ L). Data are mean ± SE (n = 3). Significant differences are shown as asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). Abbreviation: HA, commercial humic acids; Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O; HCl, HCl-washed lignins and their Fenton variants.

organic materials were digested while heating them up for 6 h up to 300 °C, diluted with distilled water (i.e., 100 mL), and 7450


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Figure 12. (A) Accelerated germination rates and (B) salt stressrelated chlorophyll contents and related photographical images of A. thaliana in the presence of commercial humic acids, acid hydrolysisrelated lignins, and their Fenton variants (86 or 860 mg/L for the germination and 860 mg/L for the salt stress). NaCl (250 mM) was treated to induce salt-related abiotic stresses for the chlorophyll measurements. Data are mean ± SE (n = 3). Significant differences are shown as asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). Abbreviation: HA, commercial humic acids; acid, untreated acid lignin; acid-0.1, lignin treated with 0.05 g of FeSO4·7H2O; acid-0.5, lignin treated with 0.25 g of FeSO4·7H2O; acid-0.7, lignin treated with 0.35 g of FeSO4·7H2O.

Figure 11. (A) Induction of A. thaliana salt (NaCl; 250 mM) tolerance in the presence of commercial humic acids, Kraft lignins, and their Fenton variants (860 mg/L) revealed by chlorophyll contents and (B) representative photographical images of salt-stressed A. thaliana. Data are mean ± SE (n = 3). Significant differences are shown as asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). Abbreviation: HA, commercial humic acids; Kraft, untreated Kraft lignin; Kraft-0.1, lignin treated with 0.05 g of FeSO4·7H2O; Kraft-0.5, lignin treated with 0.25 g of FeSO4·7H2O; Kraft-1.0, lignin treated with 0.50 g of FeSO4·7H2O. The control samples without any treatments and the samples treated with humic acids were also used to compare the extent of the salt tolerance in Figure 12.

sedimentation kinetics. To remove the Fe-containing inorganics after Fenton treatments, the lignin particles were washed three times with 0.1 N HCl and then three times with distilled water to achieve neutralization of the pH. The final powders were lyophilized for further A. thaliana germination and ICP-OES analyses. FT-ICR MS Analysis and Elemental Composition Assignments. To obtain the elemental compositions for lignin and Fenton variant samples, ultrahigh-resolution mass spectra were acquired using a 15 T FT-ICR MS (solariXR system, Bruker Daltonics, Billerica, MA) equipped with the standard electrospray ionization (ESI) interface. The samples dissolved in methanol including 7% NH 4OH for pH adjustment to 8 were directly infused into the MS at a flow rate of 3 μL/min using a syringe pump and analyzed in negative ion mode at a capillary voltage of 4.5 kV. The lower and upper mass limits were set to the mass-to-charge (m/z) value of 150 and 1000, respectively. The drying gas flow rate was held at 1.5 L/min, drying gas temperature at 200 °C, an ion accumulation time at 0.001 s, and a transient length at 2.796 s for all the experiments. Two hundreds scans with 8 M

subsequently passed through a filter paper (AVANTEC, no. 2, 9 cm diameter); these digested filtered organic materials were then analyzed using ICP-OES analyses. The total amounts of phenolic compounds in the lignins and their Fenton variants were determined as described previously.44,45 The lignin particles (0.5 mg) were added to a mixture of Folin and Ciocalteu’s phenol reagent (0.25 mL) and distilled water (2.25 mL). The resulting mixture was incubated for 5 min at room temperature, and we mixed a 7% Na2CO3 (w/v) solution (2.5 mL) into this incubated mixture. The resulting mixture was incubated for 90 min at room temperature, and the intensity of the blue color generated in this mixture was monitored at a wavelength of 550 nm with a visible spectrophotometer. The absorbance of lignins and their Fenton variants totally dispersed in distilled water samples (0.5 mg/mL) were monitored at 600 nm every 10 s for 5 min to evaluate the 7451


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(5) Eyheraguibel, B.; Silvestre, J.; Morard, P. Effect of humic substances derived from organic waste enhancement on the growth and mineral nutrition of maize. Bioresour. Technol. 2008, 99, 4206− 4212. (6) Piccolo, A.; Pietramellara, G.; Mbagwu, J. S. C. Use of humic substances as soil conditioners to increase aggregate stability. Geoderma 1997, 75, 267−277. (7) Savy, D.; Cozzolino, V.; Nebbioso, A.; Drosos, M.; Nuzzo, A.; Mazzei, P.; Piccolo, A. Humic-like bioactivity on emergence and early growth of maize (Zea mays K.) of water-soluble lignins isolated from biomass for energy. Plant Soil 2016, 402, 221−233. (8) Cha, J. Y.; Kim, T. W.; Choi, J. H.; Jang, K. S.; Khaleda, L.; Kim, W. Y.; Jeon, J. R. Fungal laccase-catalyzed oxidation of naturally occurring phenols for enhanced germination and salt tolerance of Arabidopsis thaliana: A green route for synthesizing humic-like fertilizers. J. Agric. Food Chem. 2017, 65, 1167−1177. (9) Shevchenko, S. M.; Bailey, G. W. Life after death: lignin-humic relationship reexamined. Crit. Rev. Environ. Sci. Technol. 1996, 26, 95− 153. (10) Datta, R.; Kelkar, A.; Baraniya, D.; Molaei, A.; Moulick, A.; Meena, R. S.; Formanek, P. Enzymatic degradation of lignin in soil: A Review. Sustainability 2017, 9, No. 1163. (11) Chefetz, B.; Chen, Y.; Hadar, Y. Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl. Environ. Microbiol. 1998, 64, 3175−3179. (12) Haider, K.; Martin, J. P. Synthesis and transformation of phenolic compounds by Epicoccum nigrum in relation to humic acid formation. Soil Sci. Soc. Am. J. 1967, 31, 766−772. (13) Bajpai, P.; Anand, A.; Bajpai, P. K. Bleaching with ligninoxidizing enzymes. Biotechnol. Annu. Rev. 2006, 12, 349−378. (14) Van Dijk, H. Cation binding of humic acids. Geoderma 1971, 5, 53−67. (15) Calvo-Flores, F. G.; Dobado, J. A. Lignin as renewable raw material. ChemSusChem 2010, 3, 1227−1235. (16) Savy, D.; Canellas, L.; Vinci, G.; Cozzolino, V.; Piccolo, A. Humic-like water-soluble lignins from giant reed (Arundo donax L.) display hormone-like activity on plant growth. J. Plant Growth Regul. 2017, 995−1001. (17) Savy, D.; Mazzei, P.; Drosos, M.; Cozzolino, V.; Lama, L.; Piccolo, A. Molecular characterization of extracts from biorefinery wastes and evaluation of their plant biostimulation. ACS Sustainable Chem. Eng. 2017, 5, 9023−9031. (18) Muscolo, A.; Panuccio, M. R.; Sidari, M. The effect of phenols on respiratory enzymes in seed germination. Plant Growth Regul. 2001, 35, 31−35. (19) Pinton, R.; Cesco, S.; De Nobili, M.; Santi, S.; Varanini, Z. Water- and pyrophosphate-extractable humic substances fractions as a source of iron for Fe-deficient cucumber plants. Biol. Fertil. Soils 1997, 26, 23−27. (20) Kermer, R.; Hedrich, S.; Bellenberg, S.; Brett, B.; Schrader, D.; et al. Lignite ash: Waste material or potential resourceInvestigation of metal recovery and utilization options. Hydrometallurgy 2017, 168, 141−152. (21) Haas, K. L.; Franz, K. J. Application of metal coordination chemistry to explore and manipulate cell biology. Chem. Rev. 2009, 109, 4921−4960. (22) Werneke, S. W.; Swann, C.; Farquharson, L. A.; Hamilton, K. S.; Smith, A. M. The role of metals in molluscan adhesive gels. J. Exp. Biol. 2007, 210, 2137−2145. (23) Zeng, F.; Ali, S.; Zhang, H.; Ouyang, Y.; Qiu, B.; Wu, F.; Zhang, G. The influence of pH and organic matter content in paddy soil on heavy metal availability and their uptake by rice plants. Environ. Pollut. 2011, 159, 84−91. (24) Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; et al. Lignin valorization: Improving lignin processing in the biorefinery. Science 2014, 344, No. 1246843. (25) Zeng, J.; Yoo, C. G.; Wang, F.; Pan, X.; Vermerris, W.; Tong, Z. Biomimetic Fenton-catalyzed lignin depolymerization to high-value aromatics and dicarboxylic acids. ChemSusChem 2015, 8, 861−871.

words of data were collected per each sample, resulting in a mass resolving power of greater than 900 000 (at m/z 400). The instrument was externally calibrated using an arginine solution (10 μg/mL in methanol) before the sample analysis. The data processing for elemental composition assignments was conducted as described previously.8 The molecular formulas considering the elements C, H, O, N, and S were deduced from the raw data by using Composer software (Sierra Analytics, Modesto, CA). The formulas showing the assignment errors >0.3 ppm were ruled out for further interpretation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00697. Solid-state 13C NMR spectra of Kraft lignins and their Fenton variants and the number of molecular composition, van Krevelen plots, DBE/carbon number plots, and morphological images of acid hydrolysisrelated lignins and their Fenton variants derived from FT-ICR-MS and SEM analyses (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-55-772-1962. ORCID

Jong-Rok Jeon: 0000-0002-5815-7815 Notes

The authors declare the following competing financial interest(s): Jongkoo Lim is a full-time employee of GS Caltex Corporation.

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ACKNOWLEDGMENTS This study was carried out with the support of “R&D Program for Forest Science Technology (2017041B10-1819-BA01)” provided by Korea Forest Service (Korea Forestry Promotion Institute) and the Next-Generation BioGreen 21 Program (SSAC, grant No. PJ013195012018) of Rural Development Administration, Republic of Korea. Further support from GS Caltex is appreciated. Solid-state 13C NMR, EFM, EPR, FTICR-MS, and elemental analyses were conducted at the Korea Basic Science Institute.

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REFERENCES

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