Appl Microbiol Biotechnol DOI 10.

1007/s00253-011-3669-5

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Biodegradation of Leonardite by an Alkali-producing bacterial community and characterization of the degraded products
Tong-Guo Gao & Feng Jiang & Jin-Shui Yang & Bao-Zhen Li & Hong-Li Yuan

Received: 31 August 2011 / Revised: 2 October 2011 / Accepted: 23 October 2011 # Springer-Verlag 2011

Abstract In this study, three bacterial communities were obtained from 12 Leonardite samples with the aim of identifying a clean, effective, and economic technique for the dissolution of Leonardite, a type of low-grade coal, in the production of humic acid (HA). The biodegradation ability and characteristics of the degraded products of the most effective bacterial community (MCSL-2), which degraded 50% of the Leonardite within 21 days, were further investigated. Analyses of elemental composition, 13 C NMR, and Fourier transform infrared revealed that the contents of C, O, and aliphatic carbon were similar in biodegraded humic acid (bHA) and chemically (alkali) extracted humic acid (cHA). However, the N and carboxyl carbon contents of bHA was higher than that of cHA. Furthermore, a positive correlation was identified between the degradation efficiency and the increasing pH of the culture medium, while increases of manganese peroxidase and esterase activities were also observed. These data demonstrated that both alkali production and enzyme reactions were involved in Leonardite solubilization by MCSL-2, although the former mechanism predominated. No fungus was observed by microscopy. Only four bacterial phylotypes were recognized, and Bacillus licheniformis-related bacteria were identified as the main group in MCSL-2 by analysis of amplified 16S rRNA genes, thus demonstrating that Leonardite degradation ability has a limited distribution in bacteria. Hormone-like bioactivities of bHA were also detected. In this study, a bacterial community capable of Leonardite degradation was
T.-G. Gao : F. Jiang : J.-S. Yang : B.-Z. Li : H.-L. Yuan (*) State Key Lab for Agrobiotechnology, College of Biological Sciences and Center of Biomass Engineering, China Agricultural University, Beijing, Peoples Republic of China e-mail: hlyuan@cau.edu.cn

identified and the products characterized. These data implicate the use of such bacteria for the exploitation of Leonardite as a biofertilizer. Keywords Leonardite . Biodegradation . Humic acid . Bacterial community

Introduction As polyelectrolytic macromoleculars, humic substances are ubiquitous organic materials in terrestrial and aquatic ecosystems which play an important role in global carbon cycling and regulate the absorption of nutrients and metabolites of higher plants (Stevenson 1994; Nardi et al. 2002, 2007; Liu et al. 2010), as well as binding metal ions (Kinniburgh et al. 1996) and supporting microbial respiration (Lovley et al. 1996). Furthermore, products of humic acids (HAs) are widely used as biofertilizers (Nardi et al. 2002; Clapp et al. 2001) and medicines (Vakov et al. 2011). As the predominant fraction of humic substances, previous studies on humic acid have described the properties of crude and microbial-transformed coals (Dong et al. 2006; Conte et al. 2007; Sutton and Sposito 2005). However, the chemical composition of degraded products remains to be elucidated, and research on the degradation of coals by bacterial communities is rare. Low-grade coals with low calorific value and high ash content pollute the environment when they are burned or abandoned. Conversion technologies are available for the transformation of low-grade coals into highly polar, heterogeneous materials with relatively high oxygen content by biotechnological or chemical procedures. Microbial, enzymatic, or enzyme-mimetic technology, which are carried out at moderate temperatures and normal pressures,

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have great advantages compared with physical and chemical coal conversion (Fakoussa and Hofrichter 1999). Moreover, biocatalysts are smaller than conventional catalyst particles, more efficient, and function at normal pressure, resulting in simpler and less expensive technology (PETC 1991). It has been shown that the conversion of coal using microorganisms results in relatively higher oxygen content and lower molecular mass products than chemical conversion (Gupta and Birenda 2000; Helena et al. 2002). Previous studies on the microbial solubilization of lowgrade coals (Cohen and Gabriele 1982; Steffen et al. 2002; Solarska et al. 2009) demonstrated that both enzymatic and non-enzymatic processes were involved in microbial coal degradation/liquefaction. The non-enzymatic action is responsible for the formation of alkaline metabolites and natural chelators (Standberg and Lewis 1987; Yuan et al. 2006b; Yin et al. 2011), while the enzymatic system consists mainly of peroxidases (e.g., manganese peroxidase, lignin peroxidase), phenol oxidases, supporting enzymes, and low-molecular-weight organic acids (Fakoussa and Hofrichter 1999; Wondrack et al. 1989). Low-grade coal resources are abundant, and biological processes for fossil energy utilization have received increasing attention in recent years. Due to the complex structure of coal, only a few groups of microorganisms, the majority of which are fungi, are reported to degrade coals (Yuan et al. 2006a; Lamar et al. 1990; Cohen and Bowers 1987). A number of these have been isolated. Natural cellulosic and lignin materials are degraded rapidly by microbes that have received increasing research attention over recent years (Haruta et al. 2002; Lv et al. 2008). Therefore, it is hypothesized that materials such as low-grade coals that are structurally similar to lignin are also efficiently degraded by microbial activity in the natural environment. However, little information is available on coal biodegradation by bacterial
Table 1 Features of Leonardite samples and isolation of Leonardite-degrading bacterial communities

communities, with the exception of Maka et al. (1989) who reported the use of mixed bacterial and bacterial/fungal cultures to dissolve chemically treated and untreated lignite. Less than 1% of prokaryotic microorganisms in nature environments form visible colonies on agar plates (Amann et al. 1995), and molecular studies based on 16S rRNA gene analysis provide an effective method for the identification of uncultured prokaryotic microorganisms (Kaeberlein et al. 2002; Rappe and Giovannoni 2003). In this study, an effective bacterial community that was capable of Leonardite degradation was enriched, screened out, and evaluated. Furthermore, the chemical properties of biodissolved humic acid (bHA) were investigated by elemental analysis, micro-Fourier transform infrared (FTIR), and solid-state CP/MAS 13C NMR spectroscopy. The bioactivity of bHA was analyzed and the community structure was also analyzed by microscopy observation and analyses of 16S rRNA gene sequences.

Materials and methods Leonardite sampling In total, 12 Leonardite samples (Table 1) were obtained from coal mines in the Provinces of Xinjiang, Inner Mongolia, Shanxi and Yunnan, where the majority of these resources are located in China. Samples were collected at 15- to 20-cm depth beneath the surface, pulverized, and stored at 4C prior to use. The pH of each air-dried Leonardite sample (1 g) suspended in 2.5 mL water was measured (HORIBA B-212 pH meter, Japan); the pH of the samples ranged from 2.7 to 7.5, although most were between 2.5 and 5.0 (Table 1). Ash contents ranged from 4.58% to 40.25%, as determined by the recommendations

Sample no.

HA productiona F0 F1 H H L M M M M L H Fn H H L M M M L H Fa H H L M M M L H

Features of sample Ash (%) 4.580.08 17.750.01 31.070.07 33.320.32 32.890.01 29.911.01 7.280.06 6.670.03 21.660.25 40.250.20 12.000.13 32.950.29 pH 3.350.05 3.70.00 6.70.1 7.50.1 4.40.00 4.20.00 2.70.00 2.70.00 4.450.05 3.750.05 4.950.05 4.400.00

Geographic origin

SL1 SL2 SX1 SX2 AKZO1 AKZO2 BJ1 BJ2 YN1 YN2 TLF1 NM1

H H L M M M H M H

Urumqi, XJ Urumqi, XJ Yangquan, SX Yangquan, SX Akzo, XJ Akzo, XJ Changji, XJ Changji, XJ Kunming, YN Zhaotong, YN Turpan, XJ Huolingele, IM

XJ Xinjiang, SX Shanxi, YN Yunnan, IM Inner Mongolia, F0 original culture, F1 first subculture, Fn 10th subculture, Fa culture after 6 months storage
a

H = OD450 > 2; M=1<OD450 < 2; L=0.1<OD450 <1; -=OD450 < 0.1; the growth was evaluated after 7 days of cultivation

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in the national standards GB/212-2001 of China (Table 1). For chemical analysis, the sample was sieved with a 70-mesh sieve and dried at 60C. Enrichment of bacterial community To enrich the Leonardite-degrading bacterial communities, each sample (1.2 g, non-sterilized) was inoculated into 150 mL LuriaBertani (LB) broth in 150-mL flasks (designated F0) and incubated under static conditions at 37C. After 7 days, 5 mL of each culture was centrifuged at 8000g for 15 min and the supernatant obtained was used for the estimation of the humic acid concentration by measuring the absorption at 450 nm (OD450; Hlker et al. 1997). The cell density of bacterial communities was measured simultaneously using the colony forming units (CFU) method on LB plates; the CFU were counted after 2 days of incubation (Dasari and Hwang 2010). A further sample of each culture (5 mL, approximately 5108 cells/ mL) was transferred into fresh medium with 1.2 g sterilized Leonardite (F1). This procedure was repeated until the degradation ability was stable; the final culture was designated as Fn. Thereafter, the bacterial community was stored in 25% glycerol (v/v) at 20C and 70C. After 6 months, the residual biodegradation ability of all stored bacterial communities (identified as Fa) was analyzed as previously described for F0. Growth conditions and biodegradation rate A single bacterial community, MCSL-2, associated with relatively high solubilization of HA was selected for further investigation. To evaluate the degradation ability of MCSL-2, 5 mL of the seeding culture (approximately 5108 CFU/mL) and 1.2 g sterilized Leonardite were added to 150 mL LB medium and the cultures were incubated under static conditions at 37C. An aliquot (2 mL) of the culture was sampled daily, centrifuged, and the content of bHA estimated by the measurement of OD450 using a Shimadzu UV-1800 spectrophotometer (Shimadzu Scientific Instrument, USA) compared with a humic acid standard curve. The biodegradation rate of Leonardite was calculated according to the formula: % of remotion 100 Wb =Wo , where Wo represents the original weight of Leonardite in the medium and Wb is the weight of biodissolved humic acid. Preparation of humic acid samples for characterization Chemically extracted humic acid (cHA) derived from Leonardite sample (SL-2) was obtained as described by Dong et al. (2006). Briefly, 2 g of Leonardite powder was suspended in 100 mL 0.1 M NaOH and stirred at 20C for 24 h and then centrifuged at 6,000g for 15 min. The supernatant was

filtered through Whatman no. 1 paper and the pH was adjusted to 2.0 with 6.0 M HCl. The solution was precipitated for at least 12 h; humic acid was precipitated by centrifugation at 8,000g for 5 min. The HA pellet was washed with distilled water three times and dried at 60C. bHA was obtained from the culture of MCSL-2. The culture was centrifuged at 8,000g for 15 min to remove cells and residual Leonardite after 21 day of incubation. The supernatant was filtered through Whatman no. 1 paper and the bHA precipitated as described for cHA. Elemental analysis The elemental composition (C, H, N, and S) of HA samples was analyzed in triplicate using a Vario MICRO CUBE (Elementar Analysensysteme, Germany). The original Leonardite and cHA were included as references. Comparison of elemental contents was performed to provide information about the chemical modification of HA dissolved by bacterial communities. Solid-state CP/MAS
13

C NMR

Solid-state CP/MAS 13C NMR spectroscopy was used to investigate the chemical structure of humic acid and to distinguish aliphatic carbon (C), aromatic C, and carbonyl C (Conte et al. 2004). Samples were analyzed with a Bruker av-300 spectrometer (Bruker BioSpin AG, Switzerland) at a frequency of 75.47 MHz with magic angle spinning at 4 kHz, a contact time of 3 ms, and a pulse delay of 5 s. Approximately 2,290 scans were performed for each spectrum. The C chemical shifts were related to tetramethylsilane (0 ppm) as an external standard. For quantification, the spectra were divided into different chemical shift regions assigned to specific carbon groups, as shown in Table 2. Micro-FTIR spectroscopy The relative peak intensities of micro-FTIR spectra reflect the proportion of functional groups in samples (Tognotti et al. 1991). The micro-FTIR spectra of local areas of sliced specimens were measured using a NICOLET iN10 MX spectrophotometer (Thermo Scientific, USA) connected to a Nicolet NicPlan IR microscope and a MCT/ A detector. The resolution was 4 cm1 and the spectral range was 4,000650 cm1. Enzyme activities and pH changes in culture media For these analyses, 21-day cultures of MCSL-2 in LB broth supplied with 0.8% Leonardite (described in Growth conditions and biodegradation rate) were sampled for the

Appl Microbiol Biotechnol Table 2 Elemental and chemical composition (%) of bHA in comparison with Leonardite and cHA Sample N C H S Oa Ash H/Cb O/Cb Distribution (%) of carbon Aliphatic Leonardite bHA cHA 1.61 3.72 1.68 46.74 52.18 52.79 3.26 3.65 3.35 0.39 0.30 0.19 30.25 40.15 41.99 17.75 ND ND 0.84 0.84 0.76 0.49 0.58 0.60 51.0 37.6 37.4 Aromatic 34.4 41.0 44.1 Carboxyl 14.6 21.4 18.5 0.67 1.09 1.18 Carom/Calip

Results are presented as the mean of three analyses ND not detected

a b

Calculated as difference compared with 100% control Atomic ratio

determination of the activity of the main lignin degradation enzymeslignin peroxidase, manganese peroxidase, laccase, and esteraseusing the methods described by Lee and Moon (2003), Heinfling et al. (1998), Pickard et al. (1999), and Torres et al. (2009), respectively. The pH of the culture medium was determined daily during the incubation of MCSL-2 with Leonardite. A culture inoculated with MCSL2 alone and another supplied with Leonardite in the absence of MCSL-2 inoculation served as controls. Analysis of the community structure of MCSL-2 In this study, the existence of fungi in MCSL-2 was examined microscopically following methyl blue staining. To investigate the composition of the bacterial population, metagenomic DNA was extracted according to the method described by Zhou et al. (1996) and further purified using Silver Beads DNA Recoverage Kit (Sangon, Shanghai, China). The extracted DNA was separated by 1.0% (w/v) agarose gel electrophoresis and used as a template for 16S rRNA gene amplification by PCR using the universal primers for bacteria, 27F and 1495R (Bianciotto et al. 1996). Ten independent PCR amplifications were performed and the resulting DNA fragments were mixed and purified using Tiangen Microcolumns (Tiangen, Beijing, China) according to the manufacturers instructions. Purified PCR products were ligated into the pEGM-T easy vector (Promega, USA) and transformed into competent Escherichia coli DH5 cells (Takara Bio Inc., Japan) according to the manufacturers instructions. Recombinant cells were selected by ampicillin selection and blue/white screening (Sambrook et al. 1989). The plasmid-targeted primers T7 and SP6 were used to amplify cloned DNA fragments from positive colonies and the fragments were screened by comparison of HinfI/Csp6 restriction endonuclease cleavage patterns. Three clones of each amplified ribosomal DNA restriction analysis (ARDRA) pattern were chosen for sequencing with an ABI 3730 XL 96-capillary sequencer (Applied Biosystems, Foster City, CA, USA).

Each cloned DNA sequence was compared with sequences available in the National Center for Biotechnology Information (NCBI) database by BLAST analysis. Sequences with high homology were downloaded and a phylogenetic tree was constructed using the neighborjoining method and Kimuras two-parameter model available in MEGA version 4.0.2 (Kumar et al. 2008) after multiple alignments of the data using Clustal W (Thompson et al. 1994). The topology of the tree was evaluated based on 1,000 replicates. The nucleotide sequence data reported in this study were submitted to NCBI, and the accession numbers are indicated in the generated phylogenetic tree. Effect of bHA on lettuce seed germination This analysis was performed to estimate the bioactivity of bHA. Lettuce seeds were surface-sterilized by 95% ethanol for 30 s and 0.1% HgCl2 for 3 min. After washing six times, the seeds were germinated in Petri dish with two layers of sterile filter paper impregnated with 5 mL bHA solution (0, 100, 200, 300, 600, 900, and 1,200 mg/kg). Sterilized water was supplied during germination. After 7 days, the lengths of roots and shoots and the fresh and dry weights of 15 lettuces were measured (Piccolo et al. 1993).

Results Leonardite samples and isolation of bacterial communities From 12 Leonardite samples, effective Leonardite-degrading bacterial communities were enriched in samples SL-1 and SL2 from Urumqi of Xinjiang, NM-1 of Inner Mongolia and YN-1 (OD450 >2.0 after 7 days at 37C). However, only the bacterial communities obtained from SL-1, SL-2, and NM-1 maintained a steady biodegradation efficiency after continuous sub-culturing (Table 1). Four samplesBJ-1, BJ-2, AKZO-2, and YN-2exhibited intermediate degrading

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activity (OD450 between 1.0 and 2.0). Bacterial communities obtained from SL-1, SL-2, and NM-1 maintained high degradation efficiency after storage for 6 months at 20C or 70C. These data indicate that HA-dissolving microbes are rare or absent in Leonardite samples at pH values >4.9, while no apparent relationship was observed with the ash content. Biodegradation rate Leonardite SL-2 was collected from Xinjiang, which represents one of the largest Leonardite resources and is a major source of humic acid in China. Therefore, the MCSL2 bacterial community was selected for further investigation. The coal-degrading ability of microbes was evaluated by measuring the optical density at 450 nm (OD450). After the incubation of MCSL-2 in LB broth supplemented with 0.8% Leonardite for 25 days, the OD450 was increased to 30 and then to 45 on day 41, which implied that approximately 78% of the added Leonardite was solubilized (17.75% ash, Fig. 1). The most effective degradation occurred during the first 21 days in culture as approximately 29% and 50% of Leonardite was dissolved at 14 and 21 days, respectively. Complete Leonardite degradation by MCSL-2 was observed, although only 35% was extracted by alkali (0.1 M NaOH) from Leonardite SL-2. Elemental analysis To analyze the chemical properties of biosolubilized humic acid and to avoid the interference of the medium, the HClinsoluble fraction of bHA was investigated by elemental analysis, micro-FTIR, and solid-state CP/MAS 13C NMR spectroscopy (Table 2 and Figs. 2 and 3). The contents of

N, C, H, S, and ash were obtained from the determination and the oxygen content was calculated based on the difference between the original weight of Leonardite and the sum of the elements N, C, H, S, and ash in bHA (Table 2). The contents of C and O in the HA extracts (bHA and cHA) were apparently greater than those in Leonardite, while the N content in bHA was greater than that in cHA and Leonardite. The content of S in bHA was 23% lower than that in the original Leonardite, and alkali decreased the content of sulfur (cHA). These differences demonstrated that the relative contents of elements in bHA were modified by the bacterial community. The H/C ratios were 0.84 for bHA and Leonardite and 0.76 for cHA, which indicated that cHA contained more aromatic structures (Dong et al. 2006). Analysis of
13

C NMR spectra

The solid-state CP/MAS 13C NMR spectra of the original Leonardite, bHA, and cHA are presented in Fig. 2, and the relative intensities of the chemical shift regions corresponding to aliphatic carbon (0110 ppm), aromatic C (110160 ppm), and carbonyl C (160185 ppm) are shown in Table 2. The results suggested that the overall properties of the carbon functionalities of each sample were similar. The peak at 30 ppm was assigned to aliphatic carbons in alkyl chains (Schnitzer and Preston 1983). In the 53- to 63-ppm regions, peaks recognized as OCH3 or N-alkyl C were observed (Montoneri et al. 2008). Several small peaks at this region were more obvious in the bHA spectrum, which was consistent with the elemental data that indicated that bHA contained more nitrogen (Table 2). The peaks at 128 and 173 ppm were assigned to aromatic C in lignin and carboxyl C, respectively.
Carbonyl C (160-185) Aromatic C (110-160) Aliphatic C (0-110)

Fig. 1 Biodegradation rate of MCSL-2 in culture with Leonardite. OD450 reflects the concentration of HA produced by biodegradation. Biodegradation rate (%) was estimated by comparing with an HA standard curve analyzed by lineal regression

Fig. 2 Solid-state cross-polarization magic angle spinning 13C nuclear magnetic resonance spectra of bHA showing intensity as a function of chemical shift (in parts per million)

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Enzyme activities and pH change in medium It has been demonstrated that wood, in particular lignin, is the main parent material in the formation of low-grade coal and therefore that low-grade coals exhibit structural similarities with lignin. To elucidate the potential mechanism of Leonardite degradation by MCSL-2, activities of these ligninolytic enzymes (MnP, Lip, laccases) and esterases were measured during the highest degradation period (21 days) and the pH of the medium was measured daily. In this study, only the activities of MnP and esterase were detected. These results indicated that Leonardite could greatly enhance MnP activity. The MnP activities in the supernatants of cultures in the presence and absence of Leonardite were 15.54 and 1.70 U/L, respectively. However, Leonardite did not much affect the esterase activity significantly as the activities of culture supernatants in the presence and absence of Leonardite were 74.86 and 65.53 U/L, respectively. The pH changes in culture media are presented in Fig. 4. The original pH in the medium was 7.0. This decreased to below 5.5 after 3 days of incubation in the presence of Leonardite and subsequently remained stable. The pH in cultures of MCSL-2 in the presence and absence of Leonardite increased to 8.7 by 21 days and subsequently remained stable. Comparisons of these data (Figs. 1 and 4) revealed that MCSL-2 mediated a relatively high degradation within 21 days and was associated with increased pH from 7.0 to 8.7, which indicated a positive correlation (R2 =0.87) between the degradation rate and increased pH. Community structure of MCSL-2 In this study, fungus was not identified in MCSL-2 by microscope, and only four bacterial phylotypes were

Fig. 3 Fourier transform infrared spectra of humic acids and original coal. 1,709 cm1: C=O stretches of COOH and ketones; 1,265 cm1: COC stretches of aromatic esters and ethers; 1,107 cm1: SiOSi stretches; 2,974 cm1: aliphatic CH stretches

Variations in the relative intensities of different carbon shifts were observed in the three samples (Table 2). The spectrum data were analyzed quantitatively by division into three regions according to Montoneri et al. (2008) and David and Johnson (2003). The existence of more carboxyl C, aromatic C, and less aliphatic C in bHA than in the original Leonardite was in accordance with the elemental data showing that bHA contained equivalent H/C and higher O/C compared with the original Leonardite. Compared with cHA, bHA contained more carboxyl C and less aromatic C, which was in accordance with the elemental data of H/C ratios. The Carom/Calip values for the samples revealed that the aliphatic C dominated in original Leonardite and that the content of aromatic C was increased in bHA and cHA. Micro-FTIR spectroscopy analysis The micro-FTIR spectra of the three samples are shown in Fig. 3. All samples exhibited absorption bands typical of humic material (Inbar et al. 1990). The relative peak intensities reflected the proportion of each functional group in samples. The peak of bHA at 1,709 cm1 (C=O stretches of COOH and ketones) was stronger than those in Leonardite and cHA, which was consistent with the results of NMR for the increase in carboxyl C. The peaks at 1,265 cm1 (COC stretches of aromatic esters and ethers) of both bHA and cHA were stronger than that of Leonardite, although the peaks at 1,107 cm1 (SiOSi stretching) were weaker. Leonardite showed an obvious peak at 2,974 cm1 (aliphatic CH stretches), which was in accordance with the results of NMR in which more aliphatic C was detected in Leonardite than in bHA and cHA.

Fig. 4 pH change of medium during Leonardite degradation by bacterial communities showing MCSL-2 alkali production. The lower pH associated with MCSL-2 + Leonardite treatment compared with that of MCSL-2 alone demonstrated that the alkali production by MCSL-2 involved the release of HA

Appl Microbiol Biotechnol

recognized from 48 clones by ARDRA, which gave 97.9% coverage of this clone library. In this analysis, identical sequences were obtained from the three clones of each ARDRA pattern, which supported the definition of four ARDRA types. In the constructed phylogenetic tree (Fig. 5), the ARDRA types SL45 (representing 31 clones), SL418 (representing 9 clones), and SL78 (representing 7 clones), which represent 97.9% of the clones, were closely related to Bacillus licheniformis (9899% similarity). Only one clone (ARDRA type SL417) was related to Stenotrophomonas maltophilia, which is an uncommon pathogenic bacterium in Gamaproteobacteria. Effect of bHA on lettuce seed germination and growth It is known that humic acids increase growth in higher plants (Nardi et al. 2002; Clapp et al. 2001). In this study, bHA significantly stimulated concentrationdependent growth of roots and shoots and increased dry matter accumulation (Table 3); the germination rates of lettuce treated with bHA were not altered. The stimulation of lettuce root growth was greatest (3.77 cm) following treatment with 200 mg/kg bHA. However, treatment with concentrations higher than 200 mg/kg resulted in decreased root growth. The optimal bHA concentration for the stimulation of shoot growth was 600 mg/kg, which resulted in 37.4% increase in shoot length compared with the control, thus indicating that shoot growth was less sensitive to bHA concentrations than roots. The greatest increase in the total length of lettuce seedlings occurred following treatment with 300 and 600 mg/kg bHA, although the root/shoot ratio was highest at 200 mg/kg. Furthermore, the fresh and dry

weights of lettuce seedlings were highest at 900 mg/kg. These data demonstrated the hormone-like bioactivity of bHA (Nardi et al. 2002), thus implicating the use of this product as a biofertilizer.

Discussion In nature, the complex structure of Leonardite exhibits similarities with lignin which is resistant to degradation. There are few reports of the solubilization of low-grade coal by bacterial communities (Maka et al. 1989). In this study, the identification of three stable bacterial communities demonstrated that mixed cultures are valuable resources for the biodegradation of polymers (Table 1). Only 35% humic acid was isolated from Leonardite SL-2 by alkaline extraction (0.1 M NaOH). However, MCSL-2 was shown to achieve almost complete degradation (Fig. 1), indicating that bioconversion is more effective than chemical conversion, although the former method may be more time-consuming. OD450 is considered to be a simple method for the measurement of coal solubilization (Hlker et al. 1997). Compared with the two lignite degrading bacterial communities reported by Maka et al. (1989), which increased the OD425 to 0.1 in 25 days of incubation with untreated lignite, MCSL-2 increased OD450 to 30 in an equivalent time. This result indicated that the MCSL-2 bacterial community efficiently degraded untreated Leonardite. The efficiencies of MCSL-2 for the other 11 Leonardite samples were also studied, and the degradation rates were lower than that of the SL-2 sample, which may have been related to the structure of the coals. Furthermore, the use of bacterial communities might avoid

Fig. 5 Neighbor-Joining 16S rRNA gene phylogenetic tree showing relationships of most homologous bacteria. Numbers at the nodes indicate percentage occurrence in 100 bootstrapped trees (only values >50% are shown)

Appl Microbiol Biotechnol Table 3 Effect of biodegraded humic acid on lettuce germination (7 days) Conc. (mg/L) Length (cm) Root 0 100 200 300 600 900 1200 2.750.11a 3.040.14ab 3.770.16d 3.560.14cd 3.370.12bc 3.200.12bc 2.770.12a Shoot 1.870.76a 2.030.44ab 2.050.18ab 2.380.06cd 2.570.10d 2.260.77bc 2.270.75bc Total 4.620.17a 5.060.15b 5.810.18cd 5.940.16d 5.940.15d 5.460.12bc 5.040.19b 1.490.06a 1.510.08a 2.350.41b 1.520.07a 1.350.06a 1.460.08a 1.260.09a Ratio of root/shoot Weight (mg) of 15 seedlings Fresh 208.90.00a 219.60.00a 266.50.00b 275.80.02bc 295.60.01cd 309.20.01d 292.00.01cd Dry 5.200.7b 3.750.3a 5.800.3bc 5.750.00bc 6.450.1cd 7.300.5d 7.200.1d

Length: mean SD (n=30 seedlings). Values with different alphabets in the same column are significantly (p < 0.05) different from each other, according to LSD test.

the sterilization process required prior to biodegradation by pure bacteria or fungi. In this study, the culture was incubated statically, which is a simple industry process for the utilization of coals. The elemental data showed that the nitrogen content of bHA was much higher than Leonardite and cHA (Table 2), which is in accordance with previous reports (Dong et al. 2006; Dong and Yuan 2009). However, the mechanism by which the proportion of nitrogen is increased is unknown, although it is speculated that either the microbes or humic acid plays an important role (Dong and Yuan 2009). The decrease of S in bHA indicated that the microorganisms in MCSL-2 possess desulfurization activity, as reported by Nawaz et al. (2006). The contents of N, C, O, the carboxyl carbon, and aromatic carbon were all increased in bHA compared with the original coal; aliphatic carbon was decreased (Figs. 2 and 3 and Table 2). The contents of N and carboxyl carbon were higher in bHA compared with cHA. All results revealed that the chemical groups and structure of bHA were modified during the process of transformation by bacterial communities. It should be noted that bHA includes two fractions and that the chemical characteristics were determined only for the HCl-insoluble fraction. The HCl-soluble humic acid fraction has a relatively low molecular mass, indicating a higher carboxyl C content (Dong et al. 2006). Ligninolytic enzyme systems and esterases are regarded as important degrading enzymes involved in Leonardite solubilization (Fakoussa and Hofrichter 1999). The production of Mn peroxidase was induced by the addition of Leonardite, although lignin peroxidase and laccase activities were not detected. These observations were similar to those reported by Willmann and Fakoussa (1997). Furthermore, the positive correlation identified between pH and the biodegradation rate (Figs. 1 and 4) demonstrated that both enzymatic and non-enzymatic

reactions (alkali extraction) were involved in Leonardite degradation/liquefaction and that alkali extraction was confirmed as the predominant mechanism underlying Leonardite solubilization by MCSL-2 (Maka et al. 1989). However, the chemical differences in bHA and cHA (Table 2 and Figs. 2 and 3) also provided evidence of ligninolytic enzyme involvement in this process. It should be noted that the pH was prohibitively low for the growth of microorganisms at higher Leonardite concentrations. This observation provided a rational basis for the selection of 0.8% (w/v) Leonardite (1.2 g in 150 mL LB broth) for the isolation and culture of bacterial communities in this study. Fungi constitute the majority of coal-degrading microorganisms; few bacteria have been identified. In this study, B. licheniformis predominated in the MCSL-2 population, which was similar to previous reports of the bacteria in the mixed cultures isolated by Maka et al. (1989) identified on the basis of morphological and biochemical analyses. These observations indicate that low-grade coal degradation ability has a limited distribution in bacteria. Stenotrophomonas sp. has also been isolated from coals (Nayak et al. 2009), and these are reported to mediate the degradation of acenaphthylene and five-ring compounds (Nayak et al. 2009; Juhasz et al. 2002). Also, coals contain polynuclear aromatic structures, some of them have similar structures with acenaphthylene or five-ring compounds, which means that S. maltophilia-related bacteria may play an important role in the biodegrading ability of MCSL-2. Taken together, these results suggest that MCSL-2 is an effective bacterial community for Leonardite degradation, which can modify the structure of HA by non-enzymatic and enzymatic processes. The production of bHA has a hormone-like bioactivity, which reveals a potential procedure to explore the use of Leonardite as a biofertilizer by bacterial degradation.

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