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After fungal transformation, the contents of humic acid and water-soluble humic material increased from 38.6% to 55.1%, and from less than 4.0% to 28.2%, respectively. Humic acids are polyelectrolytic macromolecules which play an important role in global carbon and nitrogen cycling.
After fungal transformation, the contents of humic acid and water-soluble humic material increased from 38.6% to 55.1%, and from less than 4.0% to 28.2%, respectively. Humic acids are polyelectrolytic macromolecules which play an important role in global carbon and nitrogen cycling.
After fungal transformation, the contents of humic acid and water-soluble humic material increased from 38.6% to 55.1%, and from less than 4.0% to 28.2%, respectively. Humic acids are polyelectrolytic macromolecules which play an important role in global carbon and nitrogen cycling.
Changes of chemical properties of humic acids from crude
and fungal transformed lignite
LianHua Dong, Quan Yuan, HongLi Yuan * College of Biological Science, Key Laboratory of Agro-Microbial Resource and Application, Ministry of Agrio, China Agricultural University, Beijing 100094, PR China Received 27 February 2006; received in revised form 19 May 2006; accepted 19 May 2006 Available online 27 June 2006 Abstract The development of biological processes for fossil energy utilization has received increasing attention in recent years. There are abun- dance of lignite resources in China and the lignite, a low-grade coal, can be transformed by a Penicillium sp. After fungal transformation, the contents of humic acid and water-soluble humic material increased from 38.6% to 55.1%, and from less than 4.0% to 28.2%, respec- tively. The dierences in chemical properties of crude lignite humic acid (aHA), fungal transformed lignite humic acid (bHA) and water- soluble humic material from fungal transformed lignite (WS) were studied. Elemental analysis and size exclusion chromatography showed that the N content of bHA increased by 47.36% compared with aHA, and the molecular mass of bHA was smaller than aHA. And the WS with the smallest molecular mass contained most content of N. The 13 C NMR and FT-IR spectra of aHA and bHA showed that aHA contained more aromatic structure than bHA. 2006 Elsevier Ltd. All rights reserved. Keywords: Humic acids; Water-soluble humic material; Fungal transformation 1. Introduction Humic acids are polyelectrolytic macromolecules which play an important role in global carbon and nitrogen cycling and in the regulation of the mobility and fate of plant nutrients and environmental contaminants [13]. During the past few decades, there have been many researches on humic substances, but their chemical struc- ture is still not fully understood. To study the chemical nat- ure of humic substances, a variety of chemical/physical methods and spectroscopic techniques have been used, such as elemental analysis, Fourier transformed infrared spectrophotometry (FT-IR), 13 C nuclear magnetic reso- nance with cross-polarization/ magic angle spinning (CP/ MAS 13 C NMR), and UV/VIS and uorescence spectro- scopy. Lignite, a low-grade coal, has a lignin-like structure and contains humic acid and water-soluble humic material including fulvic acids. It was reported that some microbes could grow on coal and modify it with both non-enzymatic and enzymatic processes taking place [4,5]. The non-enzy- matic action may be due to the presence of various metab- olites of an alkaline, surfactant and chelating nature [68]. Oxidative and non-oxidative enzymes are considered to be the coal-degrading agents [914]. In recent years, more and more research for producing several kinds of fuel and industrial materials by biodegradation have been taken into account [15,16]. It has been established that treatment of low-rank coals with aerobic coal-solubilizing microor- ganisms results in the production of highly polar, heteroge- neous materials with a relatively high oxygen content [17]. There are abundant lignite resources in China and the lignite with low caloric value and high ash content is piled up as rubbish causing a serious waste of resources as well as environmental pollution [18]. Research into biological processes for the utilization of fossil energy has received 0016-2361/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fuel.2006.05.027 * Corresponding author. Tel.: +86 10 62733464; fax: +86 10 62731332. E-mail address: hlyuan@cau.edu.cn (H. Yuan). www.fuelrst.com Fuel 85 (2006) 24022407 more and more attention in recent years. Microbial treat- ment has been considered as an economically eective and environmentally safe way of processing coal via degra- dation of the macromolecular network into simpler, low molecular mass products [1921]. In previous work on microbial degradation of Chinese lignite, we isolated a strain of Penicillium sp. P6 which is capable of degrading lignite eectively. After fungal transformation, the contents of humic acid and water-soluble humic material (WS) in the lignite greatly increased and the products of lignite degraded by microbes had a better bioactivity than the ori- ginal extractant from crude lignite [22,23]. It is well known that humic acids have widespread utilization in agriculture, for example, they have been used as organic fertilizer and as a feed additive. By fungal transformation, we can get much more and better bioactivity humic acids. The present work was intended to study the dierences in chemical properties among aHA, bHA, and WS. The humic acids and WS were quantied and characterized using elemental analysis, UV/VIS spectrophotometry, size exclusion chromatography (SEC), micro-FT-IR and solid- state CP/MAS 13 C NMR spectroscopy. 2. Materials and methods 2.1. Fungal transformation The Penicillium sp. P6 was isolated from a coal soil mix- ture collected from the Qiantong coal-processing factory, Liaoning Province, northeast China, and deposited in the China General Microbiological Culture Collection Center, Beijing, China (CGMCCNO. 0866). It is maintained on a yeast extract-malt agar [24]. Slants inoculated with P6 were incubated at 28 C for activation for three days. Both the spore and mycelia of Penicillium P6 were suspended in dis- tilled water and the homogenate was then inoculated into 500 mL asks containing 100 mL culture uid (sucrose, 20.00 g; peptone, 5.00 g; KH 2 PO 4 , 0.10 g; K 2 HPO 4 , 0.10 g; CaCO 3 , 0.10 g; NaCl, 0.10 g; MgSO 4 7H 2 O, 0.04 g; (NH 4 ) 2 SO 4 , 0.25 g; distilled water, 1000 mL; pH 6.57.0) and shaking at 28 C for three days at 150 rpm. Then inoculated with 10% of mycelia into the lignite cul- ture (powder of bean cake, 5.00 g; bran, 2.50 g; (NH 4 ) 2 SO 4, 0.50 g; lignite, 15.00 g) and incubated at 28 C for one week. 2.2. Sample preparation of lignite The crude lignite was collected from the Huolingele Minerals Administration Coalmine, Inner Mongolian Autonomous Region, northwest China. Air-dried crude lignite or biodegraded lignite was pulverized and sieved with a 70-mesh sieve. Two grams of lignite powder were suspended in 100 mL 0.1 M NaOH and stirred at 20 C for 24 h, then centrifuged at 6000g for 15 min. The super- natant was ltered through Whatman no. 1 paper and the pH was adjusted to 2.0 with 6.0 M HCl. The solution was allowed to settle for at least 12 h and was centrifuged at 8000g for 5 min to precipitate the humic acid (aHA) which was washed with distilled water three times and dried at 60 C. Biodegraded lignite were suspended in 100 mL dis- tilled water and then stirred and centrifuged at 8000g for 15 min. The precipitate was collected to extract humic acid (bHA; the extract procedure was same as that for crude lig- nite humic acid). The supernatant was ltered and the l- trate was vacuum-ltered through 0.45 lm pore-size cellulose nitrate and then the ltrate was transferred to a dialysis tube (800012000 Da) to be dialysed for 24 h. The dialyzed water-soluble humic material (WS) was dried at 60 C, weighed and stored in a vacuum-dried chamber. The aHA, bHA and WS samples obtained from the above processes were treated with dilute HF to remove Fe and other paramagnetic materials. Briey, each sample, weighting 5 g, was placed in a 50 mL polyethylene beaker. The sample was shaken with 40 mL 10% (v/v) HF for 30 s and the suspension was allowed to settle for at least 12 h. The rise of temperature that was by exothermic reaction of HF with minerals did not exceed 40 C. The supernatant was removed with a tube attached to a plastic syringe to prevent the loss of ne materials by decanting. The HF treatment as described above was repeated twice, after which the remaining sediment was washed with distilled water and vacuum-ltered on a 0.45 lm pore-size cellulose nitrate to remove HF. Finally, the isolated pellet was freeze-dried. All samples were stored at 4 C until used. 2.3. Size exclusion chromatography (SEC) SEC was performed on solutions of the humic sub- stances with a concentration of 500 mg L 1 in 0.414 M TrisHCl buer solution at pH 9.0 using a Sephadex G- 100 medium gel (Code No. 17-0060-02 Pharmacia Biotech AB). The diameter of the column and the height of the gel were 16 mm and 90 cm, respectively, and the ow rate was approximately 18 mL h 1 . The elution proles were moni- tored using UV absorbance at 400 nm. 2.4. Chemical analysis Elemental analysis (C, H, N and S) of the three samples was carried out using a Vario-EL Elemental Analyzer (Germany). Each treatment was performed in triplicate and then mixed well to analysis three times. The light absorbance of samples in 0.05 M NaHCO 3 was measured in the range from 200 to 800 nm with a Perkin Elmer Lambda 35 UV/VIS Spectrometer (USA). The E 4 /E 6 ratio (the absorbance at 465 nm divided by that at 665 nm) was calculated [25]. 2.5. Solid-state CP/MAS 13 C NMR The solid-state CP/MAS 13 C NMR spectra of the three demineralized samples were obtained with a Bruker av-300 L. Dong et al. / Fuel 85 (2006) 24022407 2403 spectrometer (Switzerland) at a frequency of 75.47 MHz with magic angle spinning at 4 kHz with a contact time of 3 ms and a pulse delay of 5 s. Approximately 2290 scans were performed for each spectrum. The C chemical shifts were related to tetramethylsilane (0 ppm) as external stan- dard. For quantication, the spectra were divided into dif- ferent chemical shift regions assigned to specic carbon groups: the assignments are shown in Table 2. 2.6. Micro-FT-IR spectroscopy As the samples are too hard to pulverize, we use local micro-FT-IR of sliced specimen instead of KBr wafer with the homogeneous and representative samples. Micro-FT- IR spectra of local areas of sliced specimen were measured using Nicolet Magna-IR 750 spectrophotometer (USA), connected to a Nicolet NicPlan IR microscope and a MCT detector. The resolution was 4 cm 1 and the spectral range was 4000650 cm 1 . 3. Results 3.1. Extraction and purication of humic acids and water-soluble humic material The humic acid content of the crude lignite was 38.6% and that of the water-soluble humic material was much lit- tle (less than 4.0%). However, after fungal transformation, the content of humic acid and water-soluble humic mate- rial reached 55.1% and 28.2%, respectively. 3.2. Analysis by SEC SEC was used to separate the single sample of humic material by size. The Sephadex G-100 has a fractionation range of 40001500000 Da for globular proteins and 1000150000 Da for dextran. Since humic substances are more spherical than dextran, the fractionation range for humic materials is possibly between dextran and globular proteins, with a greater similarity to dextran. As no exter- nal calibration standard was used, the SEC results in Fig. 1 were only qualitative. However, relatively high and low average molecular mass (MM) fractions from each sample were recognized by normalizing to the total peak area [26]. For the humic acids two well-separated peaks were observed. For aHA, there was a high MM elution peak and a low MM elution peak, and the eluent volume of low average MM peak was 142 mL. In contrast, the eluent volume of low average MM peak for bHA was 162 mL. The SEC of the WS was dierent: only WS displayed the low MM elution peak. Qualitative evaluation of the average MM derived from the SECs are as expected: WS < bHA < aHA. As the curve of WS had no high aver- age MM elution peak, it was considered that WS had the lowest average MW compared to the other humic samples. 3.3. Elemental analysis and UVVIS The elemental analysis results for aHA, bHA and WS are shown in Table 1. Since the ash contents of the puried humic acids and water-soluble humic material were negligi- ble, the dierence to 100% was assigned to the oxygen con- tent. The results for the three samples showed a clear trend: N and O contents increased from aHA to WS indicating that the contents of N or O functional groups increased during the process of fungal transformation. The H/C ratios were 0.78 for aHA, 0.83 for bHA and 0.89 for WS, which was consistent with previous reports: the lower H/C ratio of humic acids, the higher the MM and the amount of aromatic structures [18,25]. Generally, the E 4 /E 6 ratio is expected to decrease with increasing MM and content of condensed aromatic rings [27]. In addition, the ratio increases with the increase in oxygen content [25]. In Table 1, the E 4 /E 6 ratios for aHA, bHA and WS increased in this order. The E 4 /E 6 mea- surements also revealed that WS should have the lowest MM range and bHA had smaller MM than aHA, in agree- ment with the SEC analysis. 3.4. The analysis of 13 C NMR spectra More detailed information about the structure of the humic substances was obtained using solid-state CP/MAS 13 C NMR spectroscopy. The spectra of aHA, bHA and WS are presented in Fig. 2 and the relative intensities of the chemical shift regions are summarized in Table 2. In general, every sample showed peaks in the resonance areas of aliphatic carbon (0110 ppm), aromatic C (110160 ppm), and carbonyl C (160220 ppm). This 0 50 100 150 200 250 300 350 400 Elution volume (ml) aHA bHA WS 200 kDa 80 kDa 50 kDa Fig. 1. Size exclusion chromatograms of aHA (humic acids of crude lignite), bHA (humic acids of degraded lignite) and WS (water-soluble humic substances of degraded lignite), expressed as the relative absor- bances at 400 nm as function of elution volume given in mL. 2404 L. Dong et al. / Fuel 85 (2006) 24022407 suggested that the bulk properties of the carbon function- alities of each sample were similar to one another. The spectra had peaks at 30 ppm in the alkyl C (050 ppm) range. This peak was likely due to aliphatic carbons in alkyl chains [28,29]. In the O-alkyl C (50110 ppm) region, signals for aliphatic carbon substituted by oxygen and nitrogen are usually observed [30,31]. In this region, we observed a weak signal at 73 ppm only in the spectrum of WS, which was consistent with the elemental data, where WS contained the most oxygen and nitrogen. In the aro- matic region (110160 ppm), there were strong peaks at 128 ppm in the spectra of all three samples, which was assigned to aromatic units in lignin [32,33]. The peak at 173 ppm in the carbonyl C region was attributed to car- boxyl C (160190 ppm). However, the relative intensities of the dierent carbon shifts diered for the three samples. The spectra data for the humic acids and WS were analyzed quantitatively, according to the literature [31,34,35], which divided the spectra into three regions as in Table 2. A comparison indi- cated that WS contained more carboxyl carbon and less aromatic carbon in agreement with the elemental data which showed that WS contained more oxygen and less carbon. The C arom /C alip values for aHA and bHA revealed the change occurring during in the process of fungal trans- formation: the spectrum of aHA (with the large MM) was dominated by aromatic carbon, while in the spectra of bHA (lower MM), the percentage of aromatic carbon decreased and the percentage of aliphatic and carboxyl car- bon increased. Table 1 Elemental composition (%), atomic ratios, and some spectroscopic characteristic of aHA, bHA and WS Samples Elemental analysis UV/VIS Element (%) Atomic ratio C H N O b S Ash H/C c O/C c E 4 /E 6 aHA 56.1 a 3.7 1.5 38.0 0.6 0.1 0.79 0.51 6.41 bHA 53.9 3.7 2.2 39.7 0.5 ND 0.82 0.55 6.98 WS 49.7 3.7 2.5 43.6 0.5 ND 0.89 0.66 8.81 a Results are presented as mean of three analyses. b Calculated as dierence to 100%. c The atomic ratio. 250 200 150 100 50 0 - 50 ppm WS bHA aHA Fig. 2. The solid-state cross-polarization magic-angle spinning 13 C nuclear magnetic resonance spectra of the humic substances showing intensity as function of chemical shift, given in parts per million (ppm). Table 2 Distribution of carbon in aHA, bHA and WS determined by solid-state CP/MAS 13 C NMR spectroscopy Samples Percentage distribution of carbon 090 ppm 110160 ppm 160190 ppm (110160)/(090) Aliphatic C Aromatic C Carboxyl C C arom /C alip aHA 26.8 a 56.9 16.3 2.13 bHA 31.2 50.2 18.6 1.61 WS 31.3 43.2 24.9 1.38 a Results are presented as mean of three analyses. 4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers (cm -1 ) WS bHA aHA Fig. 3. Fourier-transform infrared spectra of the humic substances. L. Dong et al. / Fuel 85 (2006) 24022407 2405 3.5. Micro-FT-IR spectroscopy analysis Fig. 3 shows the micro-FT-IR spectra of the three sam- ples. All samples exhibited the typical absorption bands of humic material [18,36]. Although absorption magnitudes of FT-IR spectra cannot be compared directly, the dierences between the micro-FT-IR spectra of the humic acids and WS can easily be seen. The relative peak intensities, which were dierent in the samples, reected the relative amount of each functional group. The aliphatic, carboxyl and polysaccharide-like character represented by absorption bands at 2932 cm 1 (aliphatic CH stretching), 1720 cm 1 (C@O stretching of COOH and ketones) and 1037 cm 1 (CO stretch of polysaccharide-like components) became stronger with decreasing MM which was also consistent with the NMR results: more aliphatic and carboxyl carbon in bHA and WS. 4. Discussion The SEC results showed that the MM of the humic acid from degraded lignite (bHA) was smaller than that of the humic acid from crude lignite (aHA). The abscissa of the SEC spectra in Fig. 1, reected the relative absorbance with the elution volume, and as no external standard was applied in the study, it was impossible to quantify absolute size ranges of the two peak eluents. Furthermore, a calcu- lation of the absolute concentration would require known absorptivities of the two size fractions, as the capacity for light absorbance generally diers for humic materials according to the nature of chromophores present within the dierent size fractions. In spite of the diculties in the application of size exclusion chromatography for humic substances, so far it is the eective and general method to estimate the MM of them and the measured peak elution volumes relate to the MM for humic acids [3739]. It was reported that the smaller humic acid size fractions contained more functional groups than the larger ones [3,40]. This might be caused by the less amount of aromatic carbon and a lower degree of condensation in the smaller ones, as indicated by the E 4 /E 6 ratio, which increased with decreasing MM. The E 4 /E 6 ratio has also been shown to increase with increase in the oxygen content [25,41]. It is interesting to note that the smaller humic acid size fraction (bHA) contained more N and O than aHA, based on the elemental data in Table 1. And more aliphatic and carboxyl carbon structure for bHA, as determined with 13 C NMR spectroscopy, corresponded more closely to these results. The comparison of micro-FT-IR spectroscopy for aHA and bHA showed similar trends as those revealed by the NMR results. All the results indicate that the lignite struc- ture might have changed in the process of transformation by Penicillium sp. P6. The structure of lignite could be destroyed and transformed by the lignin peroxidase [42], and maybe a part of the alkyl moiety of the aliphatic chain structure was transformed into carboxyl or COH groups by enzymatic-oxidation. This may be why the content of WS, with the smallest MM and most abundant carboxyl groups, increased in the degraded lignite. Our previous experiment showed that the products of lignite by micro- bial degradation had a better bioactivity than the extract- ant from crude lignite [22]. There are abundant lignite resources in China, with reserves reaching 130 million tons [43], but their utiliza- tion is very limited. Since the products of degraded lignite have better bioactivity, the use of them is widespread. Also fungal transformation is proved to be an eective way in enhancing the content of lignite humic acid and water-sol- uble humic material. The elemental data also showed that the nitrogen con- tent of bHA and WS was much higher than for aHA. It is interesting to note that this was also found in earlier report [12]. However, the mechanism of increasing nitrogen content is still unknown. As we added some nitrogen nutri- ments to the culture medium, either the microbes or the humic acids themselves play an important role in nitrogen xation. 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