Identification and characterization of acidity-tolerant and aluminum-resistant bacterium isolated from tea soil

An acidity-tolerant, aluminum resistant bacterium was isolated from tea soils in Kagoshima Experimental Station (Japan). Based on the morphological, physiological and biochemical characteristics and 16S rDNA nucleotide sequence analysis, the bacterium was identified as Bacillus sp. An 3 (DQ234657) in Bacillus cereus group. The bacterium was able to grow on S-LB plates (pH 3.7) with 1.0 g/L Al and survived in LB broth even at 10 g/L Al (pH 2.0). While cultured, the growth of the bacterial strain in LB liquid medium containing increasing concentrations of Al (0, 100 and 200 ppm), was inhibited by the presence of Al, especially at concentration of 200 ppm. The pH of culture medium without Al increased steeply and reached pH 7.0 after 10 days, meanwhile it was almost constant in the other cases. The elimination of Al from culture medium by the bacterium was also affirmed and it was more conspicuous at 100 ppm Al. Due to their tolerance to high acidity, resistance to and removal of a substantial amount of Al, the bacterium might be applicable in restoring acidic soils, particularly acidified tea garden soils.


INTRODUCTION
Green tea (Camellia sinensis) is a nitrophilic crop.As a farming practice, large amounts of nitrogenous fertilizers, especially ammonium sulfate fertilizer, have been applied to tea soils in order to increase the amino acid content of tea leaves and produce an attractively colored, tasty tea.When tea plants absorb a large amount of ammonium, sulfate accumulates in the soil.Also, ammonium applied to tea soil is rapidly converted to nitrate by acid-tolerant autotrophic nitrifiers (Hayatsu and Kosuge, 1993).Consequently, a considerable quantity of nitrate and sulfate has gradually accumulated in soil (Nioh et al., 1993), decreasing the pH to 4.0 or even lower and raising remarkably soluble aluminum (Al) levels (Wang et al., 2010).In these conditions, the activity of soil microorganisms decreases and tea plants are considered to accumulate a high level of Al, thus posing a serious threat to the health of consumers (Fung and Wong, 2004).The utilization of soil microorganisms, which are indispensable participants in biogeochemical cycles, should be considered as a potential solution.Following this trend, it is necessary to know what happens to microorganisms living in the extreme environment of the tea garden soil or how microorganisms cope with the unfavorable conditions of this extreme environment.In other words, the study of the microbial ecology of the extremely acidic tea soil has promised to provide insights into environmental and applied aspects of indigenous microorganisms.
Therefore, this study purports to fill the gap of the microbial ecology of the extremely acidic tea soil.It aims at identifying and characterizing acidity-tolerant and Alresistant bacteria isolated from acidic tea garden soil in Kagoshima (Japan).

Soil samples
Samples of Kuroboku (high-humic Andosol), Akahoya (light-colored Andosol), Kuroniga (thick high-humic Andosol), Andesite-derived (weathered soil of Neogene layer in Tertiary Period) and Sedimentary rock-derived (weathered soil of Shimantogawa layer in Jurassic Period) soils were collected from tea gardens at a depth of 0 to 20 cm at the Kagoshima Tea Experimental Station.All the fresh soil samples were passed through a 2 mm mesh sieve (JIS standard), dried for 24 h, passed through a 0.5 mm mesh sieve (JIS standard) and kept in closed glass bottles for storage at 5°C.

Soil properties
Moisture content was based on the gravimetric loss of free water associated with heating to 105°C for 24 h.The pH (H 2 0) was measured with a PB-20 Sartorius Basic pH Meter and expressed as the ratio of air-dried soil to solution: 1:2.5.The amounts of total C and total N were determined using a N-C analyzer.The watersoluble Al in soils was extracted with pure water (1:20), followed by shaking for 2 h (Iwasaki et al., 1993), diluted with 1% nitric acid and then quantified by using Inductively coupled plasma-mass spectrometry (ICP-MS).
The total number of microorganisms was estimated by the dilution agar plate method on NA medium (beef extract 5 g, peptone 10 g, NaCl 5 g, agar 15 g, water up to 1000 ml, pH 4.0 and 7.0), for three to five days at 30°C.The coarse organic substances in the soil samples were dissociated by dispersion using a Waring Blender at 16,000 rpm for 3 min (Kanazawa et al., 1986).Cycloheximide was also spread on the surface of the plates to prevent the overgrowth of several rapidly growing fungi, which would restrict the growth of slower-growing molds.

Isolation of acidity-tolerant and Al-resistant bacteria
Bacteria tolerant to acidity and resistance to Al was isolated as follows.Autoclaved non-acidic field soil (100 g) was added to 1 L of distilled water and agitated for 30 min at 100 rpm.The mixture was filtered using a 0.50 µm filter.The filtrate was mixed with LB liquid medium (0.05% peptone, 0.025% yeast extract and 1% NaCl) to produce S-LB liquid medium.After this medium was autoclaved at 121°C for 15 min, Al sterilized using a 0.20 µm filter was added to a final concentration of 100 ppm, and the medium was adjusted to pH 3.7.The acidic tea soils (1 g) were added to 10 ml of this, and the culture was performed on a shaker at 150 rpm, 30°C for 7 days.The resulting bacterial strains were purified by incubation on S-LB agar plates (pH 7.0) (Konishi et al., 1994) (Method I).On the other hand, bacterial strains were directly isolated by the dilution agar plate method on S-LB (Method II) or LB agar plates (Method III), containing Al at a concentration of 100 ppm (pH 3.7).All the aboveisolated bacterial strains were then transferred to S-LB agar plates containing Al concentrations of 200 to 1000 ppm (pH 3.7) and incubated at 30°C for seven days in order to screen for resistance to Al.Although small amount of yeast extract (0.2 g/L) and peptone (0.5 g/L) were included in the S-LB agar plates, their effects on the existence of inorganic monomeric Al were negligible (Kawai et al., 2000).The bacterium with higher ability to resist Al was selected for further analysis.

Identification
Identification was based on a morphological, physiological and biochemical characterization and phylogenetic analysis.

Morphological, physiological and biochemical characteristics
The tests were investigated on cultures grown at 30°C for 48 h.The bacterium was examined with an optical microscope for its cell form and size, Gram reaction, spore formation and motility.Colony form was observed on a medium plate.The catalase reaction, oxidase reaction, acid or gas production from glucose and oxidation or fermentation (O/F) of glucose were tested (Barrow and Feltham, 1993).Besides, physiological and biochemical characteristics were also determined using API 50 CHB kit (bioMerieux, Lyon, France) consisting of 49 carbohydrates of API 50 CH strip associated with the API 20 E strip.

Phylogenetic analysis
Colonies which developed on LB agar plates after 48 h at 30°C were harvested for analysis.InstaGene matrix was used for extraction and purification of genomic DNA, following its protocol.The nucleotide sequence (1500 to 1600 bp) of 16S rDNA of the isolate was amplified by PCR.The extracted genomic DNA acted as a template.Primers 9F and 1510R were added to Ready-To-Go PCR beads (Amersham Pharmacia Biotech, NJ, USA) which consist of deoxynucleotides, Taq DNA polymerase and PCR buffer to produce a complete PCR mixture.The nucleotide sequence of the amplified 16S rDNA was determined with an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit.This kit was used with a GeneAmp PCR Systems 9600 thermal cycler and ABI Prism 3100 DNA Sequencer (Applied Biosystems, CA, USA).Eight kinds of sequence primers were used for the cycle sequencing.The sequences were screened for repeats, using an Auto Assembler 2.1 (Applied Biosystems, CA, USA) to rule out overlaps.The nucleotide sequence was analyzed by using MicroSeq Microbial Identification System Software V.1.4.1 (Applied Biosystems, CA, USA).MicroSeq Bacterial Full Gene Library v.0001 (Applied Biosystems, CA, USA) acted as a sequence database in similarity searches using the BLAST system (Saitou and Nei, 1987).Subsequently, a Neighbor-Joining molecular phylogenetic tree was constructed (Altschul et al., 1997).Then, in order to acquire more information, a similarity search with the international nucleotide sequence database offered by U.S. National Center for Biotechnology Information (NCBI) using the BLAST was carried out.The nucleotide sequence data was submitted to GeneBank/DDBJ/EMBL for the accession number.

Bacterial tolerance to acidity and Al
To minimize the possible effect of soil eluate on the initial Al concentrations in the culture medium, the LB liquid medium was used in the following studies.Al 2 (SO 4 ) 3 solution filtered with a sterilized filter (0.20 μm pore size) was added to the LB liquid medium autoclaved at 121°C for 15 min to final concentrations of 0.1 to 50 g/L 1 , and the pH of the medium was adjusted to 2.0, 2.5, 3.0 and 3.5.The bacterial suspension (1 ml) was then inoculated into the medium and cultured by shaking at 150 rpm, 30°C for 7 days.After that, 50 µL of each culture was transferred onto LB plates in the absence of Al and heavy metals (pH 7.0), and continuously cultured at 30°C for three days.A positive test result under given culture conditions was affirmed via the development of colonies on these plates after incubating.

Culture conditions, bacterial growth and changes in medium pH
Cultures of 100 ml LB medium containing various concentrations of Al (pH 3.5) were inoculated with 1 ml of bacterial suspension and incubated by shaking at 150 rpm, 30°C for 10 days.During bacterial growth, the change of medium pH was measured with a PB-20 Sartorius Basic pH meter and the number of bacterial cells was counted by the dilution method on LB agar plates (pH 7.0).

Quantification of Al eliminated from culture medium
The spent culture supernatant was separated by centrifugation at 12,000 rpm for 10 min, then filtered with a sterilized filter (0.20 μm pore size), diluted with 1% nitric acid and subjected to ICP-MS analysis to determine the amount of Al remaining in the spent culture medium.
All the values represented the means of three independent experiments and were plotted along with their respective standard deviations.Differences of means were tested with Turkey-Kramer's method.

Soil properties
Several soil properties were determined (Table 1).The pH of tea soils varied in the range of 2.69-4.18.Soluble Al levels were significantly higher in the Kuroboku and Akahoya soil samples than in the other samples.The numbers of acidity tolerant microorganisms in the Kuroboku and Kuroniga samples probably increased due to the high acidity.

Isolation of acidity-tolerant and Al-resistant bacteria
Based on the differences in colony form, 41 bacterial strains which were able to tolerate pH 3.7 and 100 ppm Al was initially isolated.The result of the subsequent screening shows that two of these strains, namely Kb 1 and An 3, were able to grow on S-LB plates in the presence of 1000 ppm Al (Table 2).Therefore, strain An 3 derived from the Andesite-derived soil sample (pH 4.18) was selected for further research.

Morphological, physiological and biochemical characteristics
A photograph of strain An 3 is shown in Figure 1.The strain was a Gram-positive rod, 1.0 x 2.0-3.0 µm in cell size.This strain had motility and spore formation and was positive for both catalase and oxidase reactions.The characteristics of strain An 3 presented in Table 3 seemed to be in agreement with those of Bacillus genus.However, it was unlikely that this strain belonged to B. mycoides or B. anthracis which is included in the B. cereus group because they have no motility (Barrow and Feltham, 1993;Sneath et al., 1984).This is different from the above-mentioned suggestion based on the result of the nucleotide sequence analysis.
Besides, in physiological and chemical tests using the API 50 CHB kit, fermentation by strain An 3 of carbohydrate substrates such as ribose, glucose, fructose, arabinose, salicin, cellobiose, etc was detected, whereas that of others such as xylose, galactose, mannose, melibiose, raffinose, etc was not detected (Table 4).These characteristics of strain An 3 appeared to be similar to those of B. cereus and B. thuringiensis which were also contained in the B. cereus group.Although strain An 3 was considered to be closely related to B. cereus based on positivity for urease activity, however, their negativity of acetoin reaction (VP) was different.
In addition, in supplementary tests, strain An 3 was found to be positive in hemolysis, lecithinase and anaerobiosis, and negative in crystalline inclusion (Table 5).Based on these results, the possibility that strain An 3 belongs to B. cereus was greatest.

16S rDNA nucleotide sequence analysis
The nucleotide sequence of 16S rDNA of the bacterium was determined and presented in Figure 2. The result of the homology search with the MicroSeq Bacterial Full Gene Library using the BLAST system showed that the 16S rDNA base sequence of strain An 3 had more than 99% homology with that of B. thuringiensis, B. cereus and B. mycoides (Table 6).The result with the International Nucleotide Sequence Database using BLAST indicated 99.8% homology in 16S rDNA sequence with B. cereus H1439.Moreover, the first 20 hits in this  (Skerman et al., 1980) (anthrax, Bio Safety Level 3), are assigned to the B. cereus group with close relationships.In the Neighborjoining phylogenetic tree constructed using MicroSeq (Figure 3), the cluster formed by strain An 3, B. thuringiensis, B. cereus and B. mycoides was considered to be the cluster of the B. cereus group (B.weihenstephanensis and B. anthracis were not registered in MicroSeq).
The 16S rDNA nucleotide sequence of strain An 3 has been deposited in the DDBJ/EMBL/GenBank database with the accession number DQ234657.
To sum up, the isolate may be identified as Bacillus sp.An 3 (with accession number DQ234657), part of the B. cereus group and related to B. cereus, B. weihenstephanensis or B. thuringiensis.

Tolerance to acidity and resistance to Al
Bacterial acidity tolerance and aluminum resistance in LB liquid medium were investigated.Bacillus sp.An 3 could survive in the presence of Al and low pH.As shown in Table 8, it could survive in the presence of Al up to 10 g/L at pH 2.0.This suggested that the strain was markedly tolerant to high acidity and resistant to Al.

Bacterial response to increasing concentrations of Al in culture medium
In this study, the growth of Bacillus sp.An 3 was influenced by the presence of Al in the culture medium, especially at an initial concentration of 200 ppm (Figure 5).In addition, during the growth, the pH of the culture medium without Al increased steeply and reached about 7.0 after 10 days, meanwhile it was almost constant at Al concentrations of 100 and 200 ppm (Figure 4).The result of the investigation on microbial elimination of Al from the culture medium showed that Al was removed by Bacillus sp.An 3 and it was more significantly conspicuous in the presence of 100 ppm Al than in the presence of 100 ppm Al (Figure 6).

DISCUSSION
Aluminum comprises 8.3% of the earth crust and is the most abundant metal and the third most abundant element after oxygen (45.5%) and silicon (25.7%).Aluminum appears in the Al 3+ oxidation state and aluminum minerals are almost insoluble at neutral pH.As the pH drops below 5.5, however, Al-containing materials begin to dissolve.High levels of soluble Al in soils become toxic to plants and microorganisms (Mossor-Pietraszewska, 2001;Slattery et al., 2001).In order to deal with this, some microorganisms have developed mechanisms to tolerate high acidity and resistance to Al-stress conditions.In fact, a number of microorganisms tolerant to high acidity and resistant to Al from acidic soils have been isolated and identified (Konishi et al., 1994;Kanazawa and Kunito, 1996;Kawai et al., 2000;Nguyen et al., 2001;Kanazawa et al., 2005).However, it is remarkable that most of the microorganisms isolated were fungi and yeasts.This may be ascribed to the fact that fungi and  yeasts are generally more tolerant to acidity than bacteria (Myrold and Nason, 1992;Pina and Cervantes, 1996).In addition to the acid-and Al-tolerant bacterial strain which was isolated and identified as Flavobacterium sp.(Konishi et al., 1994), in the present study, Bacillus sp.An 3 was able to survive in LB liquid medium containing a concentration of 10 g L -1 Al at pH 2.0.However, because of the various culture media, incubation conditions and assessment methods employed, it is difficult to make comparisons of Al resistance among bacteria from different studies.
It was reported that the bacterial adaptation to changes of medium pH may refer to the synthesis of an array of new proteins as part of what has been called their acidic tolerance response (Lansing et al., 2001).Furthermore, it was also proposed that either a high internal buffering capacity or reduced membrane permeability might play a role in pH homeostasis (Ian, 1985).
When pH decreases to 5.0 or lower, Al becomes soluble and toxic to microorganisms.The toxic effect of Al may be due to the substitution of essential metal ions at critical sites in the cell (Ganrot, 1986).However, the molecular mechanism of the toxicity has not been clarified.Here, the growth of Bacillus sp.An 3 was  influenced by the presence of Al, especially at a concentration of 200 ppm.This may be ascribed to Al's toxic effect at high concentration to the bacterium.In order to deal with the toxicity, some microorganisms have developed mechanisms to tolerate metal-stress conditions.Mechanisms for metal detoxification include export, chelation and metabolism.The export and metabolism of Al have not been reported, while the tolerance of plants to Al is related to the secretion of organic acids, which chelate inorganic monomeric Al (Kochian, 1995).Additionally, acid-and Al-tolerant root nodule bacteria produce a larger amount of exopolysaccharides (EPS) than sensitive strains under stress (Appanna, 1988).It has been indicated that the production of EPS is a strategy to neutralize the toxic effects of Al (Appanna, 1989), since an EPS capable of chelating Al may substantially decrease the activity of toxic ions on the cell surface (Cunningham and Munns, 1984).Besides, it has been suggested that the acid-and Al-tolerant isolate, Flavobacterium sp.ST-3991, released certain substances, perhaps protein and chelators, during  its growth, which might mask ionic Al and increase the pH of the medium.The masked Al appeared to form Al complexes because the culture medium became turbid and very viscous during the growth (Konishi et al., 1994).However, in the present study, during the growth of Bacillus sp.An 3, the pH of the medium without Al increased steeply and was neutral after 10 days, but that of the culture medium with Al was almost gradually decreased.This difference suggested the existence of a mechanism of responding to an increasing concentration of Al in the culture medium.However, elucidation of the precise mechanisms requires further study.
From acidic tea soils in Kagoshima in Japan, an aciditytolerant and Al-resistant bacterium was isolated.The

Figure 2 .
Figure 2. The 16 S rDNA nucleotide sequence of An 3 strain.

Figure 3 .
Figure 3.The Neighbor-joining molecular phylogenetic tree of strain An 3.

Table 1 .
Some properties of tea garden soil samples.

Table 2 .
Resistance to Al of acidity-tolerant bacteria.

Table 4 .
Physiological and biochemical tests using API 50CHB Kit for the An 3 strain.

Table 5 .
Supplementary tests for bacterial identification.

Table 6 .
Homology search to MicroSeq Bacterial Full gene Library using BLAST.

Table 7 .
Homology search to international nucleotide sequence database using BLAST.