Gao Et Al 2011 Bio Degradation of Leonardite by an Alkali-Producing Bacterial Community and Characterization of the Degraded Products

8/3/2019 Gao Et Al 2011 Bio Degradation of Leonardite by an Alkali-Producing Bacterial Community and Characterization of t

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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 ofidentifying 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,13C 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

identified and the products characterized. These data

implicate the use of such bacteria for the exploitation ofLeonardite 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 respira-

tion (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 con-

tent by biotechnological or chemical procedures. Microbial,

enzymatic, or enzyme-mimetic technology, which are

carried out at moderate temperatures and normal pressures,

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: [email protected]

Appl Microbiol Biotechnol

DOI 10.1007/s00253-011-3669-5


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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 microorgan-

isms results in relatively higher oxygen content and lower

molecular mass products than chemical conversion (Guptaand Birenda 2000; Helena et al. 2002).

Previous studies on the microbial solubilization of low-

grade 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 andHofrichter 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

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

Table 1 Features of Leonardite

samples and isolation of

Leonardite-degrading bacterial

communities

XJ Xinjiang, SX Shanxi,

YN Yunnan, IM Inner Mongolia,

F0 original culture, F1 first

subculture, Fn 10th subculture,

Fa culture after 6 months

storageaH = OD450 > 2; M=1


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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 into150 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 sterilizedLeonardite (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 biodegra-

dation rate of Leonardite was calculated according to the

formula: % of remotion 100Wb=Wo, where Wo repre-sents 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 Leo-

nardite 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 13C 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 tetrame-

thylsilane (0 ppm) as an external standard. For quantifica-

tion, 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



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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 ofMCSL-2 with Leonardite. A culture inoculated with MCSL-

2 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 per-

formed and the resulting DNA fragments were mixed and

purified using Tiangen Microcolumns (Tiangen, Beijing,

China) according to the manufacturers instructions. Puri-

fied 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 endonu-

clease 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 Biotech-

nology Information (NCBI) database by BLAST analysis.

Sequences with high homology were downloaded and a

phylogenetic tree was constructed using the neighbor-

joining method and Kimuras two-parameter modelavailable 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 SL-

2 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 continu-

ous sub-culturing (Table 1). Four samplesBJ-1, BJ-2,

AKZO-2, and YN-2exhibited intermediate degrading

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 Carom/Calip

Aliphatic Aromatic Carboxyl

Leonardite 1.61 46.74 3.26 0.39 30.25 17.75 0.84 0.49 51.0 34.4 14.6 0.67

bHA 3.72 52.18 3.65 0.30 40.15 ND 0.84 0.58 37.6 41.0 21.4 1.09

cHA 1.68 52.79 3.35 0.19 41.99 ND 0.76 0.60 37.4 44.1 18.5 1.18

Results are presented as the mean of three analyses

ND not detectedaCalculated as difference compared with 100% controlb Atomic ratio



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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 MCSL-

2 bacterial community was selected for further investiga-

tion. 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 solubi-lized (17.75% ash, Fig. 1). The most effective degradation

occurred during the first 21 days in culture as approxi-

mately 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 HCl-

insoluble 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,andash 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 theoriginal 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 of13

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

re la tiv e in te ns itie s o f th e c he mic a l s h ift re gio nscorresponding 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.

Aliphatic C

(0-110)

Aromatic C

(110-160)

Carbonyl C

(160-185)

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)

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



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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. TheCarom/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.

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 Leonar-

dite degradation by MCSL-2, activities of these ligninolytic

enzymes (MnP, Lip, laccases) and esterases were measuredduring 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 below5.5 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

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



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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 closelyrelated to Bacillus licheniformis (9899% similarity). Only

one clone (ARDRA type SL417) was related to Stenotro-

phomonas 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 concentration-

dependent growth of roots and shoots and increased dry

matter accumulation (Table 3); the germination rates oflettuce treated with bHA were not altered. The stimula-

tion 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 commu-

nities 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 methodmay 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)



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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 distribu-

tion 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 struc-

tures, some of them have similar structures with acenaph-

thylene 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.

Table 3 Effect of biodegraded humic acid on lettuce germination (7 days)

Conc. (mg/L) Length (cm) Ratio of root/shoot Weight (mg) of 15 seedlings

Root Shoot Total Fresh Dry

0 2.750.11a 1.870.76a 4.620.17a 1.49 0.06a 208.90.00a 5.200.7b

100 3.040.14ab 2.030.44ab 5.060.15b 1.51 0.08a 219.60.00a 3.750.3a

200 3.770.16d 2.050.18ab 5.810.18cd 2.35 0.41b 266.50.00b 5.800.3bc300 3.560.14cd 2.380.06cd 5.940.16d 1.52 0.07a 275.80.02bc 5.750.00bc

600 3.370.12bc 2.570.10d 5.940.15d 1.35 0.06a 295.60.01cd 6.450.1cd

900 3.200.12bc 2.260.77bc 5.460.12bc 1.46 0.08a 309.20.01d 7.300.5d

1200 2.770.12a 2.270.75bc 5.040.19b 1.26 0.09a 292.00.01cd 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.



9/10

Acknowledgments This research was partly supported by the

Ministry of Science and Technology of China (the 863 program, no.

2003AA241170).

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Gao Et Al 2011 Bio Degradation of Leonardite by an Alkali-Producing Bacterial Community and Characterization of the Degraded Products