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Fulvic acid-like substance and its characteristics, an innovative waste recycling material from pulp black liquor
Journal of Cleaner Production 243 (2020) 118585
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Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Fulvic acid-like substance and its characteristics, an innovative waste recycling material from pulp black liquor Zhonghua Wang a, 1, Tianlin Shen a, 1, Yuechao Yang a, c, *, Bin Gao b, Yongshan Wan c, Yuncong C. Li c, Yuanyuan Yao a, Lu Liu a, Yafu Tang a, Jiazhuo Xie a, Fangjun Ding d, Jianqiu Chen e a National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, National Engineering & Technology Research Center for Slow and Controlled-Release Fertilizers, College of Resources and Environment, Shandong Agricultural University, Daizong Road No. 61, Taian, Shandong, 271018, China b Agricultural and Biological Engineering, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611, United States c Department of Soil and Water Science, Tropical Research and Education Center, University of Florida, Homestead, FL, 33031, United States d Key Laboratory of Humic Acid Fertilizer of Ministry of Agriculture, Shandong Agricultural University Fertilizer Technology Co. Ltd, Feicheng, Shandong, 271600, China e State Key Laboratory of Nutrition Resources Integrated Unitilization, Kinggenta Ecological Engineering Group Co., Ltd, Linshu, Shandong, 276700, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2019 Received in revised form 6 September 2019 Accepted 23 September 2019 Available online 24 September 2019
The pulp black liquor accounts for about 90 percent of pollution of the whole paper and pulp industry. The clean production technology of extracting useful bio-based substances from pulp black liquor can increase wastewater utilization and benefit the environment. This paper examines chemical characteristics and biological activities of fulvic acid-like substance extracted from leonardite (FA1), which serves as the benchmark, and from pulp black liquor (FA2). Instrumental analyses with Fourier transform infrared (FT-IR) spectroscopy, 13C-nuclear magnetic resonance (13C-NMR) spectroscopy and 1H-nuclear magnetic resonance (1H-NMR) spectroscopy indicated that FA1 and FA2 shared common functional groups existed in typical fulvic acid. A series of rice seed germination bioassays proved that FA2 had the same growth promotion function as FA1. The optimal concentration for water absorption and seed germination was 5 mg/L for FA1 and 60 mg/L for FA2. FA1 and FA2 treatments also increased the activity of a-amylase under the action of hydrolytic enzymes and promoted root development by increasing cell size and cell division. The study implied that pulp black liquor can be used for bio-renewable and ecofriendly production of fulvic acid for wide applications in agriculture. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: Prof. S Alwi Keywords: Fulvic acid Functional groups Pulp black liquor Seed germination
1. Introduction
Abbreviations: FA1, fulvic acid-like substance extracted from leonardite; FA2, fulvic acid-like substance from pulp black liquor; FT-IR, Fourier transform infrared spectroscopy; 13C-NMR, 13C-nuclear magnetic resonance spectroscopy; 1H-NMR, 1 H-nuclear magnetic resonance spectroscopy; COD, Chemical oxygen demand; NH3eN, Ammonia nitrogen; TOC, Total organic carbon; RRG, Relative root growth. * Corresponding author. National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources; National Engineering & Technology Research Center for Slow and Controlled-Release Fertilizers, College of Resources and Environment, Shandong Agricultural University, Daizong Road No. 61, Taian, Shandong, 271018, China. E-mail address: [email protected] (Y. Yang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jclepro.2019.118585 0959-6526/© 2019 Elsevier Ltd. All rights reserved.
Bio-renewable and eco-friendly management and utilization of industrial wastes have received increasing attention owing to energy crisis and growing concerns about environmental pollution (Koutinas et al., 2014). The environmental effect of wastewater effluents from pulp and paper industry has been extensively studied (Lin and Zheng, 2016). The black liquor is the major pollution source in the whole paper industry (Sun et al., 2014) due to its high carbon and ammonia contents (Gao et al., 2013). The conventional methods for treatment of pulp black liquor include recovering lignin by acid separation, agglomeration and separation, superfiltration and recovering alkali by combustion (Stoica et al., 2009). However, these methods have the disadvantages of large equipment investment and high operating cost. Alternative technology of
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wastewater treatment is strongly needed for paper and pulp enterprises. Humic substances, formed by biological and chemical transformations of animal and plant matter from microbial metabolism (Li, 2013), can be divided into humic acid (HA), fulvic acid (FA) and humin (HU) according to their behaviors of dissolution in acid or alkaline solution. Fulvic acid is a typical humic substance of comparatively low molecular weight (Yang et al., 2004). It is soluble under all pH conditions while humic acid only dissolves in alkali (Amir et al., 2008). Fulvic acid consists of small hydrophilic molecules which contain abundant functional groups, while humic acid is comprised of predominantly hydrophobic compounds (Piccolo, 2002). Fulvic acid is regarded as a plant growth regulator, with multi-functionalities in increasing cell membrane permeability and photosynthesis, enhancing secondary metabolites, and controlling hormone levels (C ̧ I_Mri_N et al., 2010). Compared with humic acid, fulvic acid contains more oxygen and carboxyl functional groups, and its pH value is closer to neutral (Müller and Gasser, 2006). The specific properties of fulvic acid products enable its wide applications in environmental remediation, biomedicine, industry and agriculture (Penamendez et al., 2005). For example, fulvic acid can increase seed germination rates and penetration, shoot development, root initiation, enhance plant resistance to environmental stresses, and improve the quality and quantity of agricultural products (Marosz, 2009). It has the repair function for heavy metal and organic polluted soils (Paolis and Kukkonen, 1997). It also enhances the immunity of human body and promotes the absorption of vitamins (Sun et al., 2012). Fulvic acid can promote the growth and development of animals and prevent the common diseases of livestock. The conventional raw materials to extract fulvic acid include mainly lignite and leonardite (Hemati et al., 2012), and these materials are non-renewable resources. The practical application of fulvic acid as a plant growth regulator in agriculture is restricted due to the low caloric value and extraction rate of the raw materials (Liu et al., 2016). In addition to the high cost and technological difficulties in the extraction process, a large amount of strong acids and alkali have to be used, thereby inducing potential environmental concerns and corrosion of machinery and equipment (Li et al., 2016). The low percentage of small bioactive molecules in fulvic acid extracted from leonardite is another limiting factor for agricultural applications. In light of the high contents of intermediate FA-like substance (fulvic acid-like substance), lignin, pectin and hemicellulose in paper mill black liquor (Mema et al., 2006), it can be hypothesized that FA-like substances could be extracted from pulp black liquor and they would have comparable functionalities with traditional fulvic acid. Moreover, the original source of pulp black liquor such as wheat straw is renewable resources, suggesting that extraction of FA-like substance from pulp black liquor is a sustainable production process. To test this hypothesis, FA-like substance was extracted from leonardite (FA1) which serves as the benchmark, and the pulp black liquor (FA2) and their similarities and differences in chemical characterization and biological activities were determined. Instrumental analyses were conducted with Fourier transform infrared (FT-IR) spectroscopy, 13C-nuclear magnetic resonance (13C-NMR) spectroscopy, and 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. To determine whether FA2 has a similar growth promoting effect on plants as FA1, rice seed germination bio-assays were also conducted. Another objective involved in the bioassay experiment is to search for the optimum growth promotion concentrations of FA1 and FA2. This study provides the needed technical basis for resourceful management of pulp black liquor as an alternative to traditional fulvic acid production for agricultural applications.
2. Materials and methods 2.1. Materials Leonardite was taken from Shanxi, China. Pulp black liquor samples were provided by Shandong Quanlin Jiayou Fertilizer Co., Ltd. (Shandong, China). All the chemicals were of analytical grade. Deionized water was used throughout the experiment. 2.2. Extraction and purification of FA-like substance Extraction and purification of FA-like substance followed the recommended method by the International Humic Substances Society (D’Andrilli et al., 2013). Briefly, 100 g leonardite (100 ml pulp black liquor) was mixed with 1,000 ml of NaOH (0.1 M) under continuous shake for 4 h. The mixture was left overnight and centrifuged at 12,000 rpm for 10 min to get the supernatant. The pH of the supernatant was adjusted to 2.0. There was no precipitate after centrifuging, indicating that this pulp black liquor sample didn’t contain humic acid. Then the supernatant (crude FA fraction) was filtered with a 0.45 mm pore-size filter membrane (Xinya, Shanghai, China) before pumping with a column of XAD-8 (Amberlite, U.S.A) and cation exchange resin (IR-120, Amberlite, U.S.A). 2.3. Characterization of FA1 and FA2 The samples were freeze-dried (Eyela FDU-210) for characterization. Elemental analysis was determined by the ECS4024 (Costech Inc., Italy) (Zhang et al., 2015). FT-IR spectroscopic characterization was conducted (Nicolet IS10, U.S.A) at the wavenumber range of 4,000-400 cm 1 (Dang et al., 2016). The NMR spectra were obtained by a Bruker 600 MHz AVANCE III (Germany) spectrometer (Amir et al., 2010). 2.4. Determination of potential contamination indicators in FA2 and pulp black liquor NH3eN was determined by Nessler’s reagent spectrophotometry (HJ 535e2009). Chemical oxygen demand (COD) was obtained by the dichromate method (HJ/T 399e2007) (Chen et al., 2017). The concentration of cadmium, lead and copper (Cd, Pb, and Cu) in FA2 was measured based on the protocols of Shen et al. (2019). 2.5. Rice seed germination and root analysis Rice seeds were surface-sterilized by dipping in 10% hydrogen peroxide for 20 min, followed by three rinses with deionized water (Arite et al., 2012). The seeds were immersed in different concentrations and sources of FA-like substance for 48 h without light in an artificial climate chamber; the seeds immersed in deionized water were used as the control. The concentrations ranged from 2.5 to 40 mg/L for FA1 and 10e100 mg/L for FA2. After 48 h, the liquid was drained, and the seeds were then sown on germination disks with double-layer filter paper in Petri dish. The disks were placed at 25 C without light for germination. When the shoot length in the control group reached half of the grain length, all the seeds (including treated) were transferred to 25 C with a 16-h photoperiod under cool white fluorescent lamps. Fifty seeds were sown in each treatment with three replications. The rice seeds were weighed every 12 h, and the water absorption rate was calculated by the difference subtraction method. Germination rates were measured daily up to the 4th day. To further explore why FA1 and FA2 can promote germination, the sliced transverse sections of rice seeds were done on the second day after incubation. The activity of
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a-amylase was determined on the third day at 40 C by measuring the release of maltose following starch hydrolysis at 540 nm, using 3, 5-dinitrosalicylic acid (DNS) as a reductant. The method of making root tips for microscopic analysis was based on Qi et al. (2012) on the fourth day. Rice seedlings were transferred to sand culture after one week. Finally, the root systems were scanned and the relative root growth (RRG) was calculated on the tenth day (Hussain et al., 2018). 3. Results and discussion 3.1. Elemental analyses The elemental contents of FA1 (extracted from leonardite) and FA2 (extracted from the pulp black liquor) fall into the values reported in the literature for fulvic acid (Farzadnia et al., 2018). The difference between them (Table 1) lies mainly in that FA2 contained slightly more C and less O than FA1 (Allard, 2006). The N and S content of FA2 was about three times that of FA1. The H content of FA2 almost doubled that of FA1. High values of S and N were suggestive of high contents of biomolecules (polypeptides and polysaccharides) (Baddi et al., 2004). The fact that FA2 contained more C, N, H and less O than FA1 indicates that high humification occurred in FA2 due to the chemical process of paper making (Xiaoli et al., 2008).
3.2. FT-IR spectroscopy The infrared spectra of FA1 and FA2 (Fig. 1) showed similar patterns to the standard spectra of fulvic acid with characteristic peaks of major functional groups (Yang et al., 2008). In general, a broad band around 3,400 cm 1 in the infrared spectra corresponded to OeH stretching of hydroxyl groups from phenol and/or to those from the carboxylic groups. Bands around 2,920 cm 1 were resulted from asymmetric and symmetric stretching vibrations of aliphatic CeH bonds in CH2 and CH3 groups. The band at 1,7251,710 cm 1 was mainly caused by the C]O stretching vibrations caused by carboxyl groups and to other carbonyl compounds (Xie et al., 2017). An intense and broad band in the region of 1,6601,600 cm 1 corresponded to C]C vibration of aromatic structure conjugated with C]O. Other bands around 1,360 cm 1, caused by C]O stretching of phenolic groups, COO antisymmetric stretching. There were alcoholic group vibrations around 1,170-1,120 cm 1 while CeO stretching of polysaccharides at 1,080-1,030 cm 1. Compared with FA1, FA2 had a higher aromatic degree and N content as shown in the peaks caused by aromatic compounds and amides (1,725-1,710 cm 1 and 1,660-1,600 cm 1). The more intense peaks at 1,420 cm 1 demonstrate that FA2 has higher contents of phenolic OH and COO . FA2 had two weak bands at 1,1701,120 cm 1 and 1,080-1,030 cm 1, suggesting that FA2 contained more polysaccharides and alcoholic group. According to the above analyses, FA2 had more hydroxyl, carboxyl groups and small molecular active groups than FA1.
Fig. 1. Infrared spectra of FA1and FA2 in leonardite and pulp black liquor.
3.3.
13
C-NMR spectroscopy
All major spectral bands of FA1 and FA2 (Fig. 2) were consistent with those observed in FA by other authors (Keeler and Maciel, 2003). The total area of spectrum consists of four regions (Cao et al., 2016). The peak at 55.8 ppm in the spectra of FA2 and a band at 40e60 ppm in the spectra of FA1 indicated that FA2 exhibited complex structural characteristics of aliphatic carbons, especially the methoxy C of the 55.8 ppm displacement segment and the amino acids as well as carbohydrates near 65e71 ppm (Amir et al., 2005). It was in agreement with the element analysis result (Table 1). A weak resonance at 150 ppm suggested the presence of a small amount of phenolic compounds in FA2. 3.4.
1
H-NMR spectroscopy
In general, the spectrum can be divided into the following regions: (1) protons on methyl and methylene carbon designated to bind to other carbon between 0 and 1.8 ppm; (2) protons on methyl, methylene, and methene carbons as electronegative functional groups from 1.8 to 3.0 ppm; (3) protons on methyl, methylene, and methene carbons bonded to oxygen atoms from 3.0 to 4.5 ppm; (4)
Table 1 Elemental composition of FA1 and FA2 extracted and purified from leonardite and the pulp black liquid. Values reported are means of three replicates ± standard deviation. Sample
FA1 FA2
Elemental composition C (%)
N (%)
H (%)
S (%)
O (%)
38.59 ± 1.13 40.67 ± 1.02
0.57 ± 0.04 2.01 ± 0.15
2.79 ± 0.24 4.53 ± 0.11
1.93 ± 0.13 6.38 ± 0.14
56.12 ± 1.32 46.41 ± 1.09
Fig. 2.
13
C-NMR spectroscopy of FA1 and FA2 in leonardite and pulp black liquor.
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aromatic and heteroaromatic protons with the contribution of unsaturated groups from 6.0 to 8.0 ppm. The spectra of FA1 and FA2 show the strongest peak in at 4.8 ppm (Fig. 3). In the first region, there are 2e3 minor peaks for FA2 but there are no peaks for FA1. The peaks of FA2 are concentrated in the second, third and fourth region, while the peaks of FA1 are concentrated in the third and fourth region. Protons on unsaturated, carboxyl, and carbonyl groups between 1.6 and 3.0 ppm, were indicative of more of these compounds in FA2. The data were in agreement with those reported in 1H NMR studies on natural fulvic acid (Fujitake et al., 2012). 3.5. NH3eN, COD and heavy metals NH3eN and COD are important water quality parameters in environmental monitoring and assessment (Zhang et al., 2016). The contents of NH3eN and COD in pulp black liquor were 2.58 105 mg/L and 1.92 105 mg/L (Table 2), which were detrimental to aquatic ecosystems if released without treatment. The contents of NH3eN and COD in FA2 treatment solutions obtained by the treatment process were 2.67 mg/L and 5.76 mg/L. The content of NH3eN and COD in FA2 were 2% and 5% of that in pulp black liquor at the same carbon concentration. They are within EPA standards for wastewater discharge and can be applied to agricultural production (Lv et al., 2009). Meanwhile, the concentrations of Cd, Pb, and Cu were 5 10 4 mg/L, 7.6 10 4 mg/L and 6.9 10 4 mg/L. They are lower than the levels of the WHO standards for drinking water. 3.6. Influence of FA1 and FA2 on the water absorption rate of rice seeds Water absorption rate is an important index to measure rice seed germination rate (Shafaei and Masoumi, 2014). For FA1 treatment (Fig. 4A), water absorption rate was the highest at the
Table 2 NH3eN and COD were measured in the pulp black liquid and FA2. Values reported are means of three replicates ± standard deviation. COD (mg/L)
NH3eN (mg/L)
FA2 (60 mg-C/L) FA2 (1 mg-C/L)
5.76 ± 0.14 0.096 ± 0.002
2.67 ± 0.12 0.046 ± 0.002
Pulp black liquor (100,000 mg-C/L) Pulp black liquor (1 mg-C/L)
192,000 ± 577 1.92 ± 0.006
258,000 ± 866 2.58 ± 0.009
concentration of 5 mg/L, about 4 times higher than that of the control. Further increase in FA1 concentration resulted in decrease in water adsorption rate. Beyond 20 mg/L, the water absorption rate was even lower than that of the control, suggesting a suppressive effect at that high concentration. For FA2 treatment, the water absorption rate increased with increasing FA2 concentration until 60 mg/L (Fig. 4B). Further increase in concentrations (up to 100 mg/ L) reduced water adsorption rate but was still higher than the control. Although both FA1 and FA2 promoted water absorption of rice seeds, their optimal concentration was different, and this may reflect the influence of their chemical structure (Figs. 1e3), especially the different functional groups in FA1 and FA2 (Mullin and Xu, 2001). The results suggested that FA2 can be used as a seed coating agent to promote seeds germination. 3.7. Influence of FA1 and FA2 on germination rate of rice seeds The effect of FA1 and FA2 on germination rate was similar to that of water absorption (Fig. 5). FA1 and FA2 promoted seed germination in low concentrations and inhibited germination in high concentrations. The optimal concentration of FA1 and FA2 were 5 mg/L and 60 mg/L. The germination rate was 96.8% treated by FA1 in 5 mg/L, 5.6% higher than the control (Fig. 5A). The germination rate was 98.5% treated by FA2 at 60 mg/L, 9.5% higher than the control (Fig. 5B). Improved germination of rice seeds by FA2 might be
Fig. 3. 1H -NMR spectroscopy of FA1and FA2 in leonardite and pulp black liquor.
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Fig. 5. Germination rate of rice seeds in FA1and FA2 treatments. Fig. 4. Rate of water absorption of rice seeds in FA1 (A) and FA2 (B) treatments. Results are means of three replicates with error bars as the standard deviation.
related to small molecule fractions which may promote the respiration of seeds, accelerate the hydrolysis of nutrients, and increase the energy for germination. 3.8. Effect of FA1 and FA2 on degradation of starchy endosperm To further explore why FA1 and FA2 can promote germination, the sliced transverse sections of rice seeds was taken on the second day after incubation to examine the endosperm, which has high mechanical strength as a barrier to germination. Hence, endosperm weakening is a prerequisite for germination (Wang et al., 2016). The endosperm began to degrade with amyloplast being hydrolyzed during seed germination (Fig. 6). Consumption of endosperm started from the center, adjacent to the embryo, and expanded to the dorsal side near the embryo along the ventral groove. The endosperm initially had a compact structure but it became loose as hydrolysis progressed. Gaps appeared between the endosperm and the aleurone layer on the second day. The large pebble-shaped amyloplast was hydrolyzed into fragments, and then became a small spherical amyloplast. Starch degradation rates were observably influenced by FA1 and FA2. Lots of amyloplast were hydrolyzed in FA1 and FA2 groups, probably because they enhanced the activity of amylase and accelerated the hydrolysis of starch (Fig. 6B and C). In contrast, the amyloplast remained in the rice seeds for the
Fig. 6. Light microscopy images of transverse sections of rice seeds at second day after incubation. Seeds were treated with Control 0 mg/L (A), FA1 5 mg/L (B) and FA2 60 mg/ L (C); Scale bars: 50 mm.
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control treatment (Fig. 6A). The endosperm and amyloplast were hydrolyzed to provide energy for seed germination (Gayral et al., 2015). They are preferentially degraded and transformed; further validating that FA2 can promote germination. 3.9. Effect of FA1 and FA2 on a-amylase activity FA can influence the amylase activity in seed germination, thereby affecting the growth of the seed (Qin et al., 2016). Compared with the control, the a-amylase activity of rice seeds was improved significantly by FA1 and FA2 (Fig. 7), consistent with result of the slices (Fig. 6). FA1 with concentrations from 2.5 mg/L to 30 mg/L increased the activity of the a-amylase over the control. The maximum increase of 73.4% was at 5 mg/L (Fig. 7A). At the concentration of 40 mg/L, the index was less than the control. For FA2, the activity of the a-amylase was higher than the control treatment in all the tested concentration range. When the concentration was 60 mg/L, a maximum increase of 64.36% was reached (Fig. 7B). Beyond 60 mg/L, the index showed a downward trend but still higher than the control. The a-amylase hydrolyzed starch to monose and increased the concentration of monose needed by respiration (Kaczmarska et al., 2017). The results revealed that FA1 and FA2 affect the amylase activity in the process of germination. Under the action of hydrolytic enzymes, amyloplast in endosperm cells became loose and are
Fig. 7. a-Amylase activity of rice seeds following FA1 (A) and FA2 (B) treatments.
hydrolyzed to provide nutrients for seeds germination, thereby enhancing water absorption and germination of the seeds. 3.10. Influence of FA1 and FA2 on root system of rice FA-induced elongating of root tips was related to an increase in cell number or size. To better understand the response of root tip cells to FA, the root tip cells from the control and FA-treated seedlings were observed by microscopy. During the development of root, cells in proximal meristem zone, which are generated by the root apical meristem, experience a certain number of cell divisions, and subsequently they differentiate into mature cells after elongating in the transition zone. The volume of each mature cell remains relatively constant. Therefore, the elongation and division rates of cell determined the elongation of root. The change in cell size was observed in longitudinal sections. In FA1 and FA2 treated seedling root tips, cortical cells were uniformly arranged as rectangles (Fig. 8). In the control seedlings, root tip cortical cells shrank in size, and became irregularly shaped. In addition, in FA-treated roots, the cells were more closely packed and larger in number than in the control. The cross-sections of FA1 and FA2 seedling root tips also showed that the epidermal and ectodermal cells adjoined closely with each other and their boundary was indiscernible. The cortical cells in FA1 and FA2 were aligned and formed regular ellipses, but the cells in control were shaped irregularly and less organized. The increased size and accelerated division of cells in FA1 and FA2 root tips promoted the elongation of root. However, the mechanism of FA2 in promotion of root growth is still unknown. Previous research indicated that FA had a similarity with plant hormones in promoting the growth of root (Priya et al., 2014).
Fig. 8. Longitudinal (B) and Transverse (C) sections of root tips in control 0 mg/L (Control), FA1 5 mg/L (FA1-5) or FA2 60 mg/L (FA2-60) treated seedlings of rice at fourth day after incubation. Scale bar: 200 mm (B), 100 mm (C).
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Table 3 Root scans data of rice following FA1 and FA2 treatments. Treatments
Length (cm)
Area (cm2)
Surface area (cm2)
Volume (cm3)
Average diameter (cm)
Nodes
Root numbers
Control FA1-2.5 FA1-5 FA1-10 FA1-20 FA1-30 FA1-40
103.1e 132.2cd 177.3a 128.8cd 114.7de 98.21e 74.79f
6.66cd 8.11bc 8.26b 6.47d 5.47de 5.11de 4.02e
25.43d 33.03bc 30.59cd 23.06de 21.14e 18.51ef 14.64 fg
0.62e 1.5bcd 1.78a 0.6e 0.64e 0.54e 0.44e
0.54cd 0.69bc 1.33a 0.72bc 0.74bc 0.51cd 0.39d
249ef 289de 519c 317d 321d 216f 202f
241e 318d 568a 330d 307d 229e 156f
FA2-10 FA2-20 FA2-40 FA2-60 FA2-80 FA2-100
134.1cd 147.6bc 160.2 ab 171.0a 165.4 ab 159.2 ab
8.54bc 8.86 ab 10.27a 10.30a 9.58 ab 9.17 ab
33.66bc 35.53bc 41.66a 41.19a 38.87 ab 35.45abc
1.39cd 1.43cd 1.82a 1.65 ab 1.57bc 1.25d
0.68bcd 0.67bcd 0.74bc 0.91b 0.71bc 0.64bcd
501c 549bc 574b 658a 602b 596b
325d 357cd 399bc 435b 393bc 427b
a, b, c, d, e
Values followed by a different letter were significantly different based on one-way ANOVAs analyses followed by Duncan tests for mean separation (p < 0.05).
Future research can be geared to further verify the growth promotion mechanism of FA. The root system plays a crucial role in the growth of plants; especially with nutrient absorption (Felizeter et al., 2014). The rice root physiological activity was significantly increased by FA1 and FA2 (Table 3, Fig. 9). The optimal concentration of FA1 was 5 mg/L. Compared with the control, the length, area and surface area increased 72.0%, 24.0% and 20.3%. Similarly, root indicators were greater than other treatments when the concentration of FA2 was 60 mg/L. Compared with the control, the length, area and surface area increased 65.9%, 54.7% and 63.8%. Moreover, relative root growth (RRG) in rice under FA1 (5 mg/L) and FA2 (60 mg/L) was 1.72 and 1.65 times greater than that of the control (Fig. 10). It revealed that FA1 and FA2 promoted the growth of the rice roots, enhancing the absorption capacity of nutrients and water. Therefore, FA2 can be used as a stimulant or additive in agriculture. It can also be used with fertilizer as a new type of functional fertilizer synergist to promote plant roots development and uptake of nutrients and water from soil. This offers ample opportunities of broad applications in the future.
and germination rate by fulvic acid-like substance were through softening seed coat and promoting water absorption into the seed. They also enhanced the activity of a-amylase which could loosen seed endosperm cells and hydrolyze their amyloplast. This study provides the needed technical basis of using pulp black liquor as a promising alternative to conventional non-renewable materials for producing fulvic acid, thereby promoting eco-friendly utilization of black liquor in the paper and pulp industries.
4. Conclusions The treatment of pulp black liquor is becoming an important issue with the increasing demand for paper. Eco-friendly and biorenewable fulvic acid-like substance can be extracted from black liquor. The fulvic acid-like substance was similar to the traditional fulvic acid extracted from leonardite. They contained similar elemental compositions and functional groups; and both significantly enhanced rice seed germination. The optimal concentrations of the fulvic acid-like substance extracted from leonardite and pulp black liquor were 5 mg/L and 60 mg/L. The increased water uptake
Fig. 9. Roots stem scan of rice in FA1 (A) and FA2 (B) treatments.
Fig. 10. Relative root growth of rice in FA1 (A) and FA2 (B) treatments.
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Acknowledgments This research was funded by Shandong Province Key R&D Program (2017CXGC0306); Taishan industrial experts programme (LJNY201609), Shandong Agricultural Innovation Team (SDAIT-1704); Great Innovation Projects in Agriculture of Shandong Province (Grant No. 2013 136). References Allard, B., 2006. A comparative study on the chemical composition of humic acids from forest soil, agricultural soil and lignite deposit: bound lipid, carbohydrate and amino acid distributions. Geoderma 130 (1e2), 77e96. Amir, S., Benlboukht, F., Cancian, N., Winterton, P., Hafidi, M., 2008. Physicochemical analysis of tannery solid waste and structural characterization of its isolated humic acids after composting. J. Hazard Mater. 160 (2e3), 448e455. Amir, S., Hafidi, M., Merlina, G., Revel, J.C., 2005. Structural characterization of fulvic acids during composting of sewage sludge. Process Biochem. 40 (5), 1693e1700. Amir, S., Jouraiphy, A., Meddich, A., El, G.M., Winterton, P., Hafidi, M., 2010. Structural study of humic acids during composting of activated sludge-green waste: elemental analysis, FTIR and 13C NMR. J. Hazard Mater. 177 (1e3), 524e529. Arite, T., Kameoka, H., Kyozuka, J., 2012. Strigolactone positively controls crown root elongation in rice. J. Plant Growth Regul. 31 (2), 165e172. , Antonio, Jose , Gonz Baddi, G.A., Hafidi, M., Cegarra, J., Alburquerque, Jose alvez, ronique, Gilard, 2004. Characterization of fulvic acids by elemental and Ve 13 spectroscopic (FTIR and C-NMR) analyses during composting of olive mill wastes plus straw. Bioresour. Technol. 93 (3), 285e290. Cao, X., Drosos, M., Leenheer, J.A., Mao, J., 2016. Secondary structures in a freezedried lignite humic acid fraction caused by hydrogen-bonding of acidic protons with aromatic rings. Environ. Sci. Technol. 50 (4), 1663e1669. Chen, J., Liu, Y.S., Zhang, J.N., Yang, Y.Q., Ying, G.G., 2017. Removal of antibiotics from piggery wastewater by biological aerated filter system: treatment efficiency and biodegradation kinetics. Bioresour. Technol. 238, 70e77. € Turan, M., Tuncer, B., 2010. Phosphorus and humic acid C ̧ i_Mri_n, K.M., Türkmen, O., application alleviate salinity stress of pepper seedling. Afr. J. Biotechnol. 9 (36), 5845e5851. D’Andrilli, J., Foreman, C.M., Marshall, A.G., Mcknight, D.M., 2013. Characterization of IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and fluorescence spectroscopy. Org. Geochem. 65 (6), 19e28. Dang, Y., Lei, Y., Liu, Z., Xue, Y., Sun, D., Wang, L.Y., Holmes, D.E., 2016. Impact of fulvic acids on bio-methanogenic treatment of municipal solid waste incineration leachate. Water Res. 106, 71e78. Farzadnia, S., Nimmagadda, R.D., Mcrae, C., 2018. A comparative structural study of nitrogen-rich fulvic acids from various antarctic lakes. Environ. Chem. 14 (8), 502e514. Felizeter, S., Mclachlan, M.S., Voogt, P.D., 2014. Root uptake and translocation of perfluorinated alkyl acids by three hydroponically grown crops. J. Agric. Food Chem. 62 (15), 3334e3342. Fujitake, N., Tsuda, K., Aso, S., Kodama, H., Maruo, M., Yonebayashi, K., 2012. Seasonal characteristics of surface water fulvic acids from Lake Biwa and Lake Tankai in Japan. Limnology 13 (1), 45e53. Gao, Y., Yue, Q., Gao, B., Sun, Y., Wang, W., Li, Q., Wang, Y., 2013. Preparation of high surface area-activated carbon from lignin of papermaking black liquor by KOH activation for Ni (II) adsorption. Chem. Eng. J. 217 (2), 345e353. Gayral, M., Bakan, B., Dalgalarrondo, M., Elmorjani, K., Delluc, C., Brunet, S., Linossier, L., Morel, M.H., Marion, D., 2015. Lipid partitioning in maize (Zea mays L.) endosperm highlights relationships between starch lipids, amylose and vitreousness. J. Agric. Food Chem. 63 (13), 3551e3558. Hemati, A., Alikhani, H.A., Marandi, G.B., 2012. Extractants and extraction time effects on physicochemical properties of humic acid. Int. J. Agric. Res. Rev. 2, 975e984. Hussain, N., Das, S., Goswami, L., Das, P., Sahariah, B., Bhattacharya, S.S., 2018. Intensification of vermitechnology for kitchen vegetable waste and paddy straw employing earthworm consortium: assessment of maturity time, microbial community structure, and economic benefit. J. Clean. Prod. 182, 414e426. Kaczmarska, K.T., Chandrahioe, M.V., Zabaras, D., Frank, D.C., Arcot, J., 2017. Effect of germination and fermentation on carbohydrate composition of Australian sweet lupin and soybean seeds and flours. J. Agric. Food Chem. 65 (46), 10064e10073. Keeler, C., Maciel, G.E., 2003. Quantitation in the solid-state 13C NMR analysis of soil and organic soil fractions. Anal. Chem. 75 (10), 2421e2432. Koutinas, A.A., Vlysidis, A., Pleissner, D., Kopsahelis, N., Lopez Garcia, I., Kookos, I.K., Papanikolaou, S., Kwan, T.H., Lin, C.S.K., 2014. Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and
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Dendrobiology 62 (1), 47e53. Mema, V., Chimphango, A., Lorenzen, L., Gorgens, J.F., 2006. Effect of extraction method on the quantity, molecular structure and chemical composition of humic and fulvic acids from black liquor. Endeavour 5 (1), 181e187. Müller, H.G., Gasser, T., 2006. Changes of chemical properties of humic acids from crude and fungal transformed lignite. Fuel 85 (17e18), 2402e2407. Mullin, W.J., Xu, W., 2001. Study of soybean seed coat components and their relationship to water absorption. J. Agric. Food Chem. 49 (11), 5331e5335. Paolis, F.D., Kukkonen, J., 1997. Binding of organic pollutants to humic and fulvic acids: influence of pH and the structure of humic material. Chemosphere 34 (8), 1693e1704. Penamendez, E.M., Havel, J., Patocka, J., 2005. Humic substances-compounds of still unknown structure: applications in agriculture, industry, environment, and biomedicine. J. Appl. Biomed. 33 (2), 279e288. Piccolo, A., 2002. 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Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Fulvic acid-like substance and its characteristics, an innovative waste recycling material from pulp black liquor Zhonghua Wang a, 1, Tianlin Shen a, 1, Yuechao Yang a, c, *, Bin Gao b, Yongshan Wan c, Yuncong C. Li c, Yuanyuan Yao a, Lu Liu a, Yafu Tang a, Jiazhuo Xie a, Fangjun Ding d, Jianqiu Chen e a National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, National Engineering & Technology Research Center for Slow and Controlled-Release Fertilizers, College of Resources and Environment, Shandong Agricultural University, Daizong Road No. 61, Taian, Shandong, 271018, China b Agricultural and Biological Engineering, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611, United States c Department of Soil and Water Science, Tropical Research and Education Center, University of Florida, Homestead, FL, 33031, United States d Key Laboratory of Humic Acid Fertilizer of Ministry of Agriculture, Shandong Agricultural University Fertilizer Technology Co. Ltd, Feicheng, Shandong, 271600, China e State Key Laboratory of Nutrition Resources Integrated Unitilization, Kinggenta Ecological Engineering Group Co., Ltd, Linshu, Shandong, 276700, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2019 Received in revised form 6 September 2019 Accepted 23 September 2019 Available online 24 September 2019
The pulp black liquor accounts for about 90 percent of pollution of the whole paper and pulp industry. The clean production technology of extracting useful bio-based substances from pulp black liquor can increase wastewater utilization and benefit the environment. This paper examines chemical characteristics and biological activities of fulvic acid-like substance extracted from leonardite (FA1), which serves as the benchmark, and from pulp black liquor (FA2). Instrumental analyses with Fourier transform infrared (FT-IR) spectroscopy, 13C-nuclear magnetic resonance (13C-NMR) spectroscopy and 1H-nuclear magnetic resonance (1H-NMR) spectroscopy indicated that FA1 and FA2 shared common functional groups existed in typical fulvic acid. A series of rice seed germination bioassays proved that FA2 had the same growth promotion function as FA1. The optimal concentration for water absorption and seed germination was 5 mg/L for FA1 and 60 mg/L for FA2. FA1 and FA2 treatments also increased the activity of a-amylase under the action of hydrolytic enzymes and promoted root development by increasing cell size and cell division. The study implied that pulp black liquor can be used for bio-renewable and ecofriendly production of fulvic acid for wide applications in agriculture. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: Prof. S Alwi Keywords: Fulvic acid Functional groups Pulp black liquor Seed germination
1. Introduction
Abbreviations: FA1, fulvic acid-like substance extracted from leonardite; FA2, fulvic acid-like substance from pulp black liquor; FT-IR, Fourier transform infrared spectroscopy; 13C-NMR, 13C-nuclear magnetic resonance spectroscopy; 1H-NMR, 1 H-nuclear magnetic resonance spectroscopy; COD, Chemical oxygen demand; NH3eN, Ammonia nitrogen; TOC, Total organic carbon; RRG, Relative root growth. * Corresponding author. National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources; National Engineering & Technology Research Center for Slow and Controlled-Release Fertilizers, College of Resources and Environment, Shandong Agricultural University, Daizong Road No. 61, Taian, Shandong, 271018, China. E-mail address: [email protected] (Y. Yang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jclepro.2019.118585 0959-6526/© 2019 Elsevier Ltd. All rights reserved.
Bio-renewable and eco-friendly management and utilization of industrial wastes have received increasing attention owing to energy crisis and growing concerns about environmental pollution (Koutinas et al., 2014). The environmental effect of wastewater effluents from pulp and paper industry has been extensively studied (Lin and Zheng, 2016). The black liquor is the major pollution source in the whole paper industry (Sun et al., 2014) due to its high carbon and ammonia contents (Gao et al., 2013). The conventional methods for treatment of pulp black liquor include recovering lignin by acid separation, agglomeration and separation, superfiltration and recovering alkali by combustion (Stoica et al., 2009). However, these methods have the disadvantages of large equipment investment and high operating cost. Alternative technology of
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wastewater treatment is strongly needed for paper and pulp enterprises. Humic substances, formed by biological and chemical transformations of animal and plant matter from microbial metabolism (Li, 2013), can be divided into humic acid (HA), fulvic acid (FA) and humin (HU) according to their behaviors of dissolution in acid or alkaline solution. Fulvic acid is a typical humic substance of comparatively low molecular weight (Yang et al., 2004). It is soluble under all pH conditions while humic acid only dissolves in alkali (Amir et al., 2008). Fulvic acid consists of small hydrophilic molecules which contain abundant functional groups, while humic acid is comprised of predominantly hydrophobic compounds (Piccolo, 2002). Fulvic acid is regarded as a plant growth regulator, with multi-functionalities in increasing cell membrane permeability and photosynthesis, enhancing secondary metabolites, and controlling hormone levels (C ̧ I_Mri_N et al., 2010). Compared with humic acid, fulvic acid contains more oxygen and carboxyl functional groups, and its pH value is closer to neutral (Müller and Gasser, 2006). The specific properties of fulvic acid products enable its wide applications in environmental remediation, biomedicine, industry and agriculture (Penamendez et al., 2005). For example, fulvic acid can increase seed germination rates and penetration, shoot development, root initiation, enhance plant resistance to environmental stresses, and improve the quality and quantity of agricultural products (Marosz, 2009). It has the repair function for heavy metal and organic polluted soils (Paolis and Kukkonen, 1997). It also enhances the immunity of human body and promotes the absorption of vitamins (Sun et al., 2012). Fulvic acid can promote the growth and development of animals and prevent the common diseases of livestock. The conventional raw materials to extract fulvic acid include mainly lignite and leonardite (Hemati et al., 2012), and these materials are non-renewable resources. The practical application of fulvic acid as a plant growth regulator in agriculture is restricted due to the low caloric value and extraction rate of the raw materials (Liu et al., 2016). In addition to the high cost and technological difficulties in the extraction process, a large amount of strong acids and alkali have to be used, thereby inducing potential environmental concerns and corrosion of machinery and equipment (Li et al., 2016). The low percentage of small bioactive molecules in fulvic acid extracted from leonardite is another limiting factor for agricultural applications. In light of the high contents of intermediate FA-like substance (fulvic acid-like substance), lignin, pectin and hemicellulose in paper mill black liquor (Mema et al., 2006), it can be hypothesized that FA-like substances could be extracted from pulp black liquor and they would have comparable functionalities with traditional fulvic acid. Moreover, the original source of pulp black liquor such as wheat straw is renewable resources, suggesting that extraction of FA-like substance from pulp black liquor is a sustainable production process. To test this hypothesis, FA-like substance was extracted from leonardite (FA1) which serves as the benchmark, and the pulp black liquor (FA2) and their similarities and differences in chemical characterization and biological activities were determined. Instrumental analyses were conducted with Fourier transform infrared (FT-IR) spectroscopy, 13C-nuclear magnetic resonance (13C-NMR) spectroscopy, and 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. To determine whether FA2 has a similar growth promoting effect on plants as FA1, rice seed germination bio-assays were also conducted. Another objective involved in the bioassay experiment is to search for the optimum growth promotion concentrations of FA1 and FA2. This study provides the needed technical basis for resourceful management of pulp black liquor as an alternative to traditional fulvic acid production for agricultural applications.
2. Materials and methods 2.1. Materials Leonardite was taken from Shanxi, China. Pulp black liquor samples were provided by Shandong Quanlin Jiayou Fertilizer Co., Ltd. (Shandong, China). All the chemicals were of analytical grade. Deionized water was used throughout the experiment. 2.2. Extraction and purification of FA-like substance Extraction and purification of FA-like substance followed the recommended method by the International Humic Substances Society (D’Andrilli et al., 2013). Briefly, 100 g leonardite (100 ml pulp black liquor) was mixed with 1,000 ml of NaOH (0.1 M) under continuous shake for 4 h. The mixture was left overnight and centrifuged at 12,000 rpm for 10 min to get the supernatant. The pH of the supernatant was adjusted to 2.0. There was no precipitate after centrifuging, indicating that this pulp black liquor sample didn’t contain humic acid. Then the supernatant (crude FA fraction) was filtered with a 0.45 mm pore-size filter membrane (Xinya, Shanghai, China) before pumping with a column of XAD-8 (Amberlite, U.S.A) and cation exchange resin (IR-120, Amberlite, U.S.A). 2.3. Characterization of FA1 and FA2 The samples were freeze-dried (Eyela FDU-210) for characterization. Elemental analysis was determined by the ECS4024 (Costech Inc., Italy) (Zhang et al., 2015). FT-IR spectroscopic characterization was conducted (Nicolet IS10, U.S.A) at the wavenumber range of 4,000-400 cm 1 (Dang et al., 2016). The NMR spectra were obtained by a Bruker 600 MHz AVANCE III (Germany) spectrometer (Amir et al., 2010). 2.4. Determination of potential contamination indicators in FA2 and pulp black liquor NH3eN was determined by Nessler’s reagent spectrophotometry (HJ 535e2009). Chemical oxygen demand (COD) was obtained by the dichromate method (HJ/T 399e2007) (Chen et al., 2017). The concentration of cadmium, lead and copper (Cd, Pb, and Cu) in FA2 was measured based on the protocols of Shen et al. (2019). 2.5. Rice seed germination and root analysis Rice seeds were surface-sterilized by dipping in 10% hydrogen peroxide for 20 min, followed by three rinses with deionized water (Arite et al., 2012). The seeds were immersed in different concentrations and sources of FA-like substance for 48 h without light in an artificial climate chamber; the seeds immersed in deionized water were used as the control. The concentrations ranged from 2.5 to 40 mg/L for FA1 and 10e100 mg/L for FA2. After 48 h, the liquid was drained, and the seeds were then sown on germination disks with double-layer filter paper in Petri dish. The disks were placed at 25 C without light for germination. When the shoot length in the control group reached half of the grain length, all the seeds (including treated) were transferred to 25 C with a 16-h photoperiod under cool white fluorescent lamps. Fifty seeds were sown in each treatment with three replications. The rice seeds were weighed every 12 h, and the water absorption rate was calculated by the difference subtraction method. Germination rates were measured daily up to the 4th day. To further explore why FA1 and FA2 can promote germination, the sliced transverse sections of rice seeds were done on the second day after incubation. The activity of
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a-amylase was determined on the third day at 40 C by measuring the release of maltose following starch hydrolysis at 540 nm, using 3, 5-dinitrosalicylic acid (DNS) as a reductant. The method of making root tips for microscopic analysis was based on Qi et al. (2012) on the fourth day. Rice seedlings were transferred to sand culture after one week. Finally, the root systems were scanned and the relative root growth (RRG) was calculated on the tenth day (Hussain et al., 2018). 3. Results and discussion 3.1. Elemental analyses The elemental contents of FA1 (extracted from leonardite) and FA2 (extracted from the pulp black liquor) fall into the values reported in the literature for fulvic acid (Farzadnia et al., 2018). The difference between them (Table 1) lies mainly in that FA2 contained slightly more C and less O than FA1 (Allard, 2006). The N and S content of FA2 was about three times that of FA1. The H content of FA2 almost doubled that of FA1. High values of S and N were suggestive of high contents of biomolecules (polypeptides and polysaccharides) (Baddi et al., 2004). The fact that FA2 contained more C, N, H and less O than FA1 indicates that high humification occurred in FA2 due to the chemical process of paper making (Xiaoli et al., 2008).
3.2. FT-IR spectroscopy The infrared spectra of FA1 and FA2 (Fig. 1) showed similar patterns to the standard spectra of fulvic acid with characteristic peaks of major functional groups (Yang et al., 2008). In general, a broad band around 3,400 cm 1 in the infrared spectra corresponded to OeH stretching of hydroxyl groups from phenol and/or to those from the carboxylic groups. Bands around 2,920 cm 1 were resulted from asymmetric and symmetric stretching vibrations of aliphatic CeH bonds in CH2 and CH3 groups. The band at 1,7251,710 cm 1 was mainly caused by the C]O stretching vibrations caused by carboxyl groups and to other carbonyl compounds (Xie et al., 2017). An intense and broad band in the region of 1,6601,600 cm 1 corresponded to C]C vibration of aromatic structure conjugated with C]O. Other bands around 1,360 cm 1, caused by C]O stretching of phenolic groups, COO antisymmetric stretching. There were alcoholic group vibrations around 1,170-1,120 cm 1 while CeO stretching of polysaccharides at 1,080-1,030 cm 1. Compared with FA1, FA2 had a higher aromatic degree and N content as shown in the peaks caused by aromatic compounds and amides (1,725-1,710 cm 1 and 1,660-1,600 cm 1). The more intense peaks at 1,420 cm 1 demonstrate that FA2 has higher contents of phenolic OH and COO . FA2 had two weak bands at 1,1701,120 cm 1 and 1,080-1,030 cm 1, suggesting that FA2 contained more polysaccharides and alcoholic group. According to the above analyses, FA2 had more hydroxyl, carboxyl groups and small molecular active groups than FA1.
Fig. 1. Infrared spectra of FA1and FA2 in leonardite and pulp black liquor.
3.3.
13
C-NMR spectroscopy
All major spectral bands of FA1 and FA2 (Fig. 2) were consistent with those observed in FA by other authors (Keeler and Maciel, 2003). The total area of spectrum consists of four regions (Cao et al., 2016). The peak at 55.8 ppm in the spectra of FA2 and a band at 40e60 ppm in the spectra of FA1 indicated that FA2 exhibited complex structural characteristics of aliphatic carbons, especially the methoxy C of the 55.8 ppm displacement segment and the amino acids as well as carbohydrates near 65e71 ppm (Amir et al., 2005). It was in agreement with the element analysis result (Table 1). A weak resonance at 150 ppm suggested the presence of a small amount of phenolic compounds in FA2. 3.4.
1
H-NMR spectroscopy
In general, the spectrum can be divided into the following regions: (1) protons on methyl and methylene carbon designated to bind to other carbon between 0 and 1.8 ppm; (2) protons on methyl, methylene, and methene carbons as electronegative functional groups from 1.8 to 3.0 ppm; (3) protons on methyl, methylene, and methene carbons bonded to oxygen atoms from 3.0 to 4.5 ppm; (4)
Table 1 Elemental composition of FA1 and FA2 extracted and purified from leonardite and the pulp black liquid. Values reported are means of three replicates ± standard deviation. Sample
FA1 FA2
Elemental composition C (%)
N (%)
H (%)
S (%)
O (%)
38.59 ± 1.13 40.67 ± 1.02
0.57 ± 0.04 2.01 ± 0.15
2.79 ± 0.24 4.53 ± 0.11
1.93 ± 0.13 6.38 ± 0.14
56.12 ± 1.32 46.41 ± 1.09
Fig. 2.
13
C-NMR spectroscopy of FA1 and FA2 in leonardite and pulp black liquor.
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aromatic and heteroaromatic protons with the contribution of unsaturated groups from 6.0 to 8.0 ppm. The spectra of FA1 and FA2 show the strongest peak in at 4.8 ppm (Fig. 3). In the first region, there are 2e3 minor peaks for FA2 but there are no peaks for FA1. The peaks of FA2 are concentrated in the second, third and fourth region, while the peaks of FA1 are concentrated in the third and fourth region. Protons on unsaturated, carboxyl, and carbonyl groups between 1.6 and 3.0 ppm, were indicative of more of these compounds in FA2. The data were in agreement with those reported in 1H NMR studies on natural fulvic acid (Fujitake et al., 2012). 3.5. NH3eN, COD and heavy metals NH3eN and COD are important water quality parameters in environmental monitoring and assessment (Zhang et al., 2016). The contents of NH3eN and COD in pulp black liquor were 2.58 105 mg/L and 1.92 105 mg/L (Table 2), which were detrimental to aquatic ecosystems if released without treatment. The contents of NH3eN and COD in FA2 treatment solutions obtained by the treatment process were 2.67 mg/L and 5.76 mg/L. The content of NH3eN and COD in FA2 were 2% and 5% of that in pulp black liquor at the same carbon concentration. They are within EPA standards for wastewater discharge and can be applied to agricultural production (Lv et al., 2009). Meanwhile, the concentrations of Cd, Pb, and Cu were 5 10 4 mg/L, 7.6 10 4 mg/L and 6.9 10 4 mg/L. They are lower than the levels of the WHO standards for drinking water. 3.6. Influence of FA1 and FA2 on the water absorption rate of rice seeds Water absorption rate is an important index to measure rice seed germination rate (Shafaei and Masoumi, 2014). For FA1 treatment (Fig. 4A), water absorption rate was the highest at the
Table 2 NH3eN and COD were measured in the pulp black liquid and FA2. Values reported are means of three replicates ± standard deviation. COD (mg/L)
NH3eN (mg/L)
FA2 (60 mg-C/L) FA2 (1 mg-C/L)
5.76 ± 0.14 0.096 ± 0.002
2.67 ± 0.12 0.046 ± 0.002
Pulp black liquor (100,000 mg-C/L) Pulp black liquor (1 mg-C/L)
192,000 ± 577 1.92 ± 0.006
258,000 ± 866 2.58 ± 0.009
concentration of 5 mg/L, about 4 times higher than that of the control. Further increase in FA1 concentration resulted in decrease in water adsorption rate. Beyond 20 mg/L, the water absorption rate was even lower than that of the control, suggesting a suppressive effect at that high concentration. For FA2 treatment, the water absorption rate increased with increasing FA2 concentration until 60 mg/L (Fig. 4B). Further increase in concentrations (up to 100 mg/ L) reduced water adsorption rate but was still higher than the control. Although both FA1 and FA2 promoted water absorption of rice seeds, their optimal concentration was different, and this may reflect the influence of their chemical structure (Figs. 1e3), especially the different functional groups in FA1 and FA2 (Mullin and Xu, 2001). The results suggested that FA2 can be used as a seed coating agent to promote seeds germination. 3.7. Influence of FA1 and FA2 on germination rate of rice seeds The effect of FA1 and FA2 on germination rate was similar to that of water absorption (Fig. 5). FA1 and FA2 promoted seed germination in low concentrations and inhibited germination in high concentrations. The optimal concentration of FA1 and FA2 were 5 mg/L and 60 mg/L. The germination rate was 96.8% treated by FA1 in 5 mg/L, 5.6% higher than the control (Fig. 5A). The germination rate was 98.5% treated by FA2 at 60 mg/L, 9.5% higher than the control (Fig. 5B). Improved germination of rice seeds by FA2 might be
Fig. 3. 1H -NMR spectroscopy of FA1and FA2 in leonardite and pulp black liquor.
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5
Fig. 5. Germination rate of rice seeds in FA1and FA2 treatments. Fig. 4. Rate of water absorption of rice seeds in FA1 (A) and FA2 (B) treatments. Results are means of three replicates with error bars as the standard deviation.
related to small molecule fractions which may promote the respiration of seeds, accelerate the hydrolysis of nutrients, and increase the energy for germination. 3.8. Effect of FA1 and FA2 on degradation of starchy endosperm To further explore why FA1 and FA2 can promote germination, the sliced transverse sections of rice seeds was taken on the second day after incubation to examine the endosperm, which has high mechanical strength as a barrier to germination. Hence, endosperm weakening is a prerequisite for germination (Wang et al., 2016). The endosperm began to degrade with amyloplast being hydrolyzed during seed germination (Fig. 6). Consumption of endosperm started from the center, adjacent to the embryo, and expanded to the dorsal side near the embryo along the ventral groove. The endosperm initially had a compact structure but it became loose as hydrolysis progressed. Gaps appeared between the endosperm and the aleurone layer on the second day. The large pebble-shaped amyloplast was hydrolyzed into fragments, and then became a small spherical amyloplast. Starch degradation rates were observably influenced by FA1 and FA2. Lots of amyloplast were hydrolyzed in FA1 and FA2 groups, probably because they enhanced the activity of amylase and accelerated the hydrolysis of starch (Fig. 6B and C). In contrast, the amyloplast remained in the rice seeds for the
Fig. 6. Light microscopy images of transverse sections of rice seeds at second day after incubation. Seeds were treated with Control 0 mg/L (A), FA1 5 mg/L (B) and FA2 60 mg/ L (C); Scale bars: 50 mm.
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control treatment (Fig. 6A). The endosperm and amyloplast were hydrolyzed to provide energy for seed germination (Gayral et al., 2015). They are preferentially degraded and transformed; further validating that FA2 can promote germination. 3.9. Effect of FA1 and FA2 on a-amylase activity FA can influence the amylase activity in seed germination, thereby affecting the growth of the seed (Qin et al., 2016). Compared with the control, the a-amylase activity of rice seeds was improved significantly by FA1 and FA2 (Fig. 7), consistent with result of the slices (Fig. 6). FA1 with concentrations from 2.5 mg/L to 30 mg/L increased the activity of the a-amylase over the control. The maximum increase of 73.4% was at 5 mg/L (Fig. 7A). At the concentration of 40 mg/L, the index was less than the control. For FA2, the activity of the a-amylase was higher than the control treatment in all the tested concentration range. When the concentration was 60 mg/L, a maximum increase of 64.36% was reached (Fig. 7B). Beyond 60 mg/L, the index showed a downward trend but still higher than the control. The a-amylase hydrolyzed starch to monose and increased the concentration of monose needed by respiration (Kaczmarska et al., 2017). The results revealed that FA1 and FA2 affect the amylase activity in the process of germination. Under the action of hydrolytic enzymes, amyloplast in endosperm cells became loose and are
Fig. 7. a-Amylase activity of rice seeds following FA1 (A) and FA2 (B) treatments.
hydrolyzed to provide nutrients for seeds germination, thereby enhancing water absorption and germination of the seeds. 3.10. Influence of FA1 and FA2 on root system of rice FA-induced elongating of root tips was related to an increase in cell number or size. To better understand the response of root tip cells to FA, the root tip cells from the control and FA-treated seedlings were observed by microscopy. During the development of root, cells in proximal meristem zone, which are generated by the root apical meristem, experience a certain number of cell divisions, and subsequently they differentiate into mature cells after elongating in the transition zone. The volume of each mature cell remains relatively constant. Therefore, the elongation and division rates of cell determined the elongation of root. The change in cell size was observed in longitudinal sections. In FA1 and FA2 treated seedling root tips, cortical cells were uniformly arranged as rectangles (Fig. 8). In the control seedlings, root tip cortical cells shrank in size, and became irregularly shaped. In addition, in FA-treated roots, the cells were more closely packed and larger in number than in the control. The cross-sections of FA1 and FA2 seedling root tips also showed that the epidermal and ectodermal cells adjoined closely with each other and their boundary was indiscernible. The cortical cells in FA1 and FA2 were aligned and formed regular ellipses, but the cells in control were shaped irregularly and less organized. The increased size and accelerated division of cells in FA1 and FA2 root tips promoted the elongation of root. However, the mechanism of FA2 in promotion of root growth is still unknown. Previous research indicated that FA had a similarity with plant hormones in promoting the growth of root (Priya et al., 2014).
Fig. 8. Longitudinal (B) and Transverse (C) sections of root tips in control 0 mg/L (Control), FA1 5 mg/L (FA1-5) or FA2 60 mg/L (FA2-60) treated seedlings of rice at fourth day after incubation. Scale bar: 200 mm (B), 100 mm (C).
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Table 3 Root scans data of rice following FA1 and FA2 treatments. Treatments
Length (cm)
Area (cm2)
Surface area (cm2)
Volume (cm3)
Average diameter (cm)
Nodes
Root numbers
Control FA1-2.5 FA1-5 FA1-10 FA1-20 FA1-30 FA1-40
103.1e 132.2cd 177.3a 128.8cd 114.7de 98.21e 74.79f
6.66cd 8.11bc 8.26b 6.47d 5.47de 5.11de 4.02e
25.43d 33.03bc 30.59cd 23.06de 21.14e 18.51ef 14.64 fg
0.62e 1.5bcd 1.78a 0.6e 0.64e 0.54e 0.44e
0.54cd 0.69bc 1.33a 0.72bc 0.74bc 0.51cd 0.39d
249ef 289de 519c 317d 321d 216f 202f
241e 318d 568a 330d 307d 229e 156f
FA2-10 FA2-20 FA2-40 FA2-60 FA2-80 FA2-100
134.1cd 147.6bc 160.2 ab 171.0a 165.4 ab 159.2 ab
8.54bc 8.86 ab 10.27a 10.30a 9.58 ab 9.17 ab
33.66bc 35.53bc 41.66a 41.19a 38.87 ab 35.45abc
1.39cd 1.43cd 1.82a 1.65 ab 1.57bc 1.25d
0.68bcd 0.67bcd 0.74bc 0.91b 0.71bc 0.64bcd
501c 549bc 574b 658a 602b 596b
325d 357cd 399bc 435b 393bc 427b
a, b, c, d, e
Values followed by a different letter were significantly different based on one-way ANOVAs analyses followed by Duncan tests for mean separation (p < 0.05).
Future research can be geared to further verify the growth promotion mechanism of FA. The root system plays a crucial role in the growth of plants; especially with nutrient absorption (Felizeter et al., 2014). The rice root physiological activity was significantly increased by FA1 and FA2 (Table 3, Fig. 9). The optimal concentration of FA1 was 5 mg/L. Compared with the control, the length, area and surface area increased 72.0%, 24.0% and 20.3%. Similarly, root indicators were greater than other treatments when the concentration of FA2 was 60 mg/L. Compared with the control, the length, area and surface area increased 65.9%, 54.7% and 63.8%. Moreover, relative root growth (RRG) in rice under FA1 (5 mg/L) and FA2 (60 mg/L) was 1.72 and 1.65 times greater than that of the control (Fig. 10). It revealed that FA1 and FA2 promoted the growth of the rice roots, enhancing the absorption capacity of nutrients and water. Therefore, FA2 can be used as a stimulant or additive in agriculture. It can also be used with fertilizer as a new type of functional fertilizer synergist to promote plant roots development and uptake of nutrients and water from soil. This offers ample opportunities of broad applications in the future.
and germination rate by fulvic acid-like substance were through softening seed coat and promoting water absorption into the seed. They also enhanced the activity of a-amylase which could loosen seed endosperm cells and hydrolyze their amyloplast. This study provides the needed technical basis of using pulp black liquor as a promising alternative to conventional non-renewable materials for producing fulvic acid, thereby promoting eco-friendly utilization of black liquor in the paper and pulp industries.
4. Conclusions The treatment of pulp black liquor is becoming an important issue with the increasing demand for paper. Eco-friendly and biorenewable fulvic acid-like substance can be extracted from black liquor. The fulvic acid-like substance was similar to the traditional fulvic acid extracted from leonardite. They contained similar elemental compositions and functional groups; and both significantly enhanced rice seed germination. The optimal concentrations of the fulvic acid-like substance extracted from leonardite and pulp black liquor were 5 mg/L and 60 mg/L. The increased water uptake
Fig. 9. Roots stem scan of rice in FA1 (A) and FA2 (B) treatments.
Fig. 10. Relative root growth of rice in FA1 (A) and FA2 (B) treatments.
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