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Improvement for isolation of soil bacteria by using common culture media.

To date, microbial cultivation has provided unprecedented advances in understanding of microbial diversity. Still the progress is insufficient considering that only less than 1% of the total microbial species can be recovered by traditional cultivation methods (1). Development of novel approaches to isolation and cultivation of yet unculturalable bacteria is therefore an important target and considerable challenge for microbiological research The main aim of modern methodology is finding the best way to artificially reproduce growth conditions similar to those in natural microbial habitats including complex interactions between bacterial species. Species interdependence, which exists in complex bacterial communities, can be an obstacle for bacterial growth in pure cultures, which is why species should not be separated as we cannot observe at the initial step of cultivation.

Since Grobstein introduced the transfilter culture of metanephric mesenchymal cells in 1953 (2), membrane filters have proven to be an invaluable tool in experimental cell biology. They have been widely used as cell growth substrates to study cellular transport, absorption and secretion; for example porous transwell membranes were used for cultivation of human bronchial epithelial cells at the air/liquid interface (3) and for adhesion, invasion, and migration of SK-Hep1 human hepatoma cells (4). Successful application of transwell membranes in mammalian cell culture prompted their utilization as substrates for growth of bacteria from diverse habitats with the notion that transwell membranes would promote isolation of new microbes recalcitrant to traditional cultivation.

A previous study showed that utilization of soil substrate membrane system (SSMS) allowed for selection of previously uncultured soil bacteria (5). As a result, 8 dominant microcolonies were isolated after 7-day incubation using SSMS; only 1 of them could grow in subculture, confirming that SSMS-microcultivated organisms preferred a slow growth K-strategy (6). This observation was further substantiated in another study showing benefits of a polycarbonate membrane for recovery of microcolony-forming bacteria resistant to traditional growth conditions (7).

Based on these data, the current study presents a novel cultivation method for previously uncultured soil bacteria that is not only very simple but also productive for subsequent subcultivation. This method is based on utilization of a transwell permeable membrane as a growth support, soil as microbial source, and soil extract as the substrate, enabling isolation of previously uncharacterized bacterial species.

MATERIALS AND METHODS

Soil sampling and transwell plates

Soil was collected from the surface around plant roots in the forest at Kyonggi University, Suwon, Gyeonggi-do, South Korea. Samples were dried at room temperature, passed through a 2mm-mesh sieve to remove soil aggregates, gravels, and debris of plant materials, analysed for physic-chemical properties (Table 1), and used as a source of soil nutrients in cultivation medium. A subsample of soil was used as an inoculum, which was added to media in a transwell insert-this strategy ensured recovery of all bacteria present in the environment, as some microorganisms could be lost to sample preparation methods such as sieving. Note that we added medium to each transwell insert when it sank down, which ensured sufficient supply of nutrients for microbial growth, preventing membrane drying and subsequent bacterial death.

Transwell plates (Corning, Lowell, NY, USA) originally designed for cell culture, in this study were used for cultivating soil bacteria, especially strains resistant to traditional cultivation. Each 6-well plate was supplied with 6 24 mm-diameter transwell inserts that at the bottom had a 0.4-pm porous membrane permeable only for water-soluble soil nutrients, but not for soil particles or bacteria (Fig. 1A). Figures 1B and 1C show the schematic diagram and actual setting of transwell experiments, respectively.

Cultivation and isolation of soil bacteria

In the novel cultivation method, 3 g of soil were added to each well of a 6-well plate and covered with an insert; then 3 mL of each medium supplemented with 100 [micro]L of soil suspension (1 g-soil in 10 mL DW) was added to each insert. Plates were incubated in a shaking incubator (Hanbaek Science Co., Bucheon, Korea) at 120 rpm and 28[degrees]C for 2 weeks. During the incubation, the level of medium in each well was monitored every 2 days and restored to original volume as needed. After 2 weeks we collected the culture media and performed a 10-fold serial dilutions up to 10-6 using 15 mL test tubes; 100 [micro]L of each dilution was spread on an agar plate, and incubated at 28[degrees]C in an incubator (Vision Scientific, Daejeon, Korea) until colonies appeared. The number of colonies was counted and all different types of colonies were picked up and streaked on a plate for isolation of pure culture. As a control conventional cultivation method was used in which 3 grams of soil were added to 30 mL of each medium in 50 mL Erlenmeyer fask (the most popular liquid culture method in laboratory); incubation and isolation conditions and processes were the same as for the new method.

For cultivation we used 5 traditional media such as nutrient broth (NB), Luria Bertani (LB), tryptic soy broth (TSB), mineral salts medium (MSM), and R2A; soil extract (SE) and its mixtures MSMSE (500 mL MSM plus 500 mL SE) and R2ASE (500 mL R2A plus 500 mL SE); and distilled water (DW). SE was completed through the following steps: mixing soil (500 g) and DW (1 L) by stirring overnight, filtration, and centrifugation. Chemical composition of each medium is shown in Table 2.

Among isolates, the potential new species were stored as glycerol stocks and freeze-dry amples, resulting in regrowth afterward.

DNA extraction and 16S rRNA gene amplification

Bacterial genomic DNA was extracted from bacterial cells grown on agar plates using an InstaGene[TM] Matrix (BIO-RAD). Primers 518F-52 CCA GCA GCC GCG GTAATA CG-32 and 800R 52-TAC CAG GGT ATC TAA TCC-32 were used for the PCR amplification (EF-Taq, SolGent, Daejeon, Korea). Reactions were performed using 20 ng of genomic DNA as a template in 30-pl reaction mixture in following conditions: activation of Taq polymerase at 95[degrees]C for 2 min; 35 cycles of 95[degrees]C, 55[degrees]C, and 72[degrees]C for 1 min each; and a final elongation step at 72[degrees]C for 10 min.

Amplified products were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA) and sequenced using the PRISM BigDye Terminator v3.1 Cycle sequencing Kit. DNA samples were added to Hi-Di formamide (Applied Biosystems, Foster City, CA), and mixtures were incubated at 95[degrees]C for 5 min, followed by 5 min on ice and analysed using the ABI Prism 3730 XL DNA analyzer (Applied Biosystems, Foster City, CA).

Phylogenetic tree construction

The EzTaxon server was used to identify phylogenetic neighbours and to calculate the pairwise 16S rRNA gene sequence similarities (8). The related 16S rRNA sequences were obtained from GenBank database and edited with the BioEdit program (9). Multiple alignments were performed with the CLUSTAL_X program (10). A phylogenetic tree was constructed by applying evolutionary distance, parsimony, and bootstrapped parsimony using the neighbour-joining algorithm (11), maximum likelihood, and maximum parsimony method (12) in the MEGA5.03 program (13) with bootstrap value on 1000 replications (14).

RESULTS AND DISCUSSION

Isolation properties of the novel cultivation method

In this study, 68 soil-bacterial strains among total 151 bacterial isolates including many same strains were obtained from the same soil through the new and traditional cultivation methods using several conventional media and distilled water. Among the 68 strains (100%), 20 (29.4%) overlapped in both methods, i.e., only 1 strain (NHI-5) (1.5%) was obtained through the traditional while 47 strains (69.1%) through the new method (Table 3 and Fig. 2A). Thus, the new cultivation method appeared to be much more effective in isolation of soil bacteria. However, strain NHI-5 probably grew well in only synthetic media without the addition of soil nutrients as the new method.

Furthermore, only 5 potentially new species were isolated by the conventional method versus 14 new species obtained by the new method (Table 3). The 5 conventionally isolated species were also selected by the new method, indicating that the additional 9 isolates could be obtained only by using the new method. The potentially new species were identified based on the 16S rRNA gene sequence similarity of less than 98.5% with the closest phylogenetic neighbour. Thus, the new method appeared to be over 3 times more efficient for isolation of new microbial species than the traditional cultivation technique.

Among the 39 strains (including overlapped) obtained through the traditional method, the majority (12 strains) were isolated from the R2A cultures, 8 from LB, 7 from NB, 6 from TSB, 4 from MSM, and 1 from each of MSMSE and SE cultures (Fig. 2B). Among the 10 potentially new species (including overlapped) 3 were isolated from R2A, 2 from each of NB and TSB, and 1 from each of LB, MSMSE, and SE media. Among total of 112 strains (including overlapped) detected by the new method, the largest number of isolates, 31, was similarly obtained from the R2A cultures, while 17 were isolated from NB, 15 from DW, 13 from TSB, 12 from LB, 8 from MSM, 7 from MSMSE, 5 from SE, and 4 from R2ASE cultures (Fig. 2B). Among the 24 potentially new species (including overlapped) 7 were isolated from R2A, 4 from each of NB, TSB and DW, 2 from LB, and 1 from each of MSMSE, R2ASE, and SE media. These results indicate that among all the media tested R2A was the best for cultivation of diverse strains including potentially new species, either by the traditional or by the new method. The reason that R2A provides more diverse colonies seems to be the low concentration of carbon and energy sources (Table 2) as mentioned in the previous study regarding cultivation of uncultivable bacteria (1).

Otherwise, the total colony number was not distinguishable between two methods, but it depended on the kinds of media: 50-80 cfu in 100 [micro]L at [10.sup.-4] dilution in TSB, LB, NB and R2A; 30-60 cfu at [10.sup.-2] dilution in MSM, MSMSE and SE. This indicates that the new method influence only the diversity of colonies, not the number.

The modified new method developed in this study differs from the techniques used in previous studies (5,7) in that it promotes adaptation of cultivation-resistant soil bacteria to traditional culture media with continuous supply of soil micronutrients thereby facilitating continuous cultivation. An increased number of different strains successfully isolated by the new method using traditional media proved that this novel cultivation approach can be a useful tool for isolation and characterization of diverse soil bacteria.

Community structure analysis

All 68 strains are shown in a phylogenetic tree with their closest neighbours and are grouped according to phylum or class level (Fig. 3). They are affiliated with 4 phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. All isolated strains in Proteobacteria fall into three classes: a-Proteobacteria, [alpha]-Proteobacteria, and [??]-Proteobacteria. Most of the strains - 27 out of 68 -belong to Firmicutes, including 18 of 45 strains isolated exclusively by the new cultivation method. Other 24 strains (including 14 isolated by the new method) belong to Proteobacteria as the second most populated phylum; among them [alpha]-Proteobacteria comprises [11.sup.8], [alpha]-Proteobacteria [8.sup.5], and [gamma]-Proteobacteria 51, respectively. Fifteen strains were identified as members of Actinobacteria, among them 13 (87%) were isolated exclusively by the new method constituting the highest proportion among the 4 phyla. The last 2 strains belong to Bacteroidetes; they were isolated by both methods.

The distribution of potentially new species among Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, was 6, 5, 2, and 1, respectively. Among the 6 Proteobacteria strains, 5 belong to [alpha]-Proteobacteria and 1 to [gamma]-Proteobacteria. The 9 potentially new species isolated exclusively by the new method belong to Proteobacteria (4), Firmicutes (4), and Actinobacteria (1). Among the 4 Proteobacteria species, 3 belong to [alpha]-Proteobacteria, and 1 to [gamma]-Proteobacteria. These results indicate that the new method may be particularly useful for isolation of new species of soil bacteria related to Firmicutes and [alpha]-Proteobacteria.

According to distribution of the strains on the genus-level, the new cultivation method appears to be selective for specific genera (Fig. 4). It may improve those members to adapt synthetic media more than other genera by supply of soil nutrients in the beginning. The conventional method detected isolates of the following 16 genera: Bacillus (17.4%), Lysinibacillus (13.0%), Bosea (8.7%), Cupriavidus (8.7%), Achromobacter (4.3%), Arthrobacter (4.3%), Dyella (4.3%), Kitasatospora (4.3%), Mesorhizobium (4.3%), Niabella (4.3%), Pedobacter (4.3%), Pelomonas (4.3%), Pseudomonas (4.3%), Sporosarcina (4.3%), Staphylococcus (4.3%), and Stenotrophomonas (4.3%). The new method identified isolates belonging to Bacillus (19.7%), Lysinibacillus (7.6%), Arthrobacter (6.1%), Paenibacillus (6.1%), Mesorhizobium (4.5%), Pseudomonas (4.5%), Citrobacter (3.0%), Cupriavidus (3.0%), Dyella (3.0%), Methylobacterium (3.0%), Microbacterium (3.0%), Rhodococcus (3.0%), Serratia (3.0%), Staphylococcus (3.0%), Streptomyces (3.0%), Achromobacter (1.5%), Bosea (1.5%), Brevibacillus (1.5%), Burkholderia (1.5%), Enterobacter (1.5%), Kitasatospora (1.5%), Leucobacter (1.5%), Micrococcus (1.5%), Mycobacterium (1.5%), Niabella (1.5%), Nitrobacter (1.5%), Pedobacter (1.5%), Pelomonas (1.5%), Sporosarcina (1.5%), Stenotrophomonas (1.5%), and Tsukamurella (1.5%).

The strains of Bacillus and Lysinibacillus were isolated in relatively high proportion by both methods, while 4 strains of Paenibacillus and 1 or 2 strains in each of other 14 genera: Brevibacillus, Burkholderia, Citrobacter, Enterobacter, Leucobacter, Methylobacterium, Microbacterium, Micrococcus, Mycobacterium, Rhodococcus, Serratia, Streptomyces, Nitrobacter, and Tsukamurella could be obtained exclusively by the new method.

Many microbiologists have successfully cultivated many uncultivable bacteria by using modified or diluted media, and long incubation times; strains thus isolated belonged to 11 phyla such as Acidobacteria, Actinobacteria, Bacteroidetes, Rubrobacteridae, Chloroflexus, Firmucutes, Fusobacteria, Gemmatimonadetes, Planctomydetes, Proteobacteria, and Verrucomicrobia (1). Here, we succeeded in isolation representatives of only 4 of those phyla, suggesting that our new method can be complemented by incorporation of the aforementioned approaches in future investigations of phylogenetic diversity in soil bacterial communities.

CONCLUSION

This study was based on application of transwell membranes previously used in mammalian cell culture, for isolation and growth of soil bacteria resistant to traditional cultivation. This novel method proved efficient for cultivation of uncultivable and identification of new, bacterial species through adaptation to laboratory culturing conditions. The transwell membrane liquid culture technique provided isolation of higher number of strains including potentially new species than the traditional method suggesting that the novel method can be successfully utilized to study phylogenetic diversity of complex bacterial populations. This study also showed that R2A medium was more suitable for isolation of soil bacteria than other media such as LB, NB, TSB, MSM, and SE whether by the conventional or the new method. We believe that the use of modified and diluted media with the transwell membrane system would pave the way for more diverse bacterial isolates and new species in the future.

ACKNOWLEDGEMENT

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2011-0010144), and by the GAIA project (RE201202062) funded by Korea Environmental Industry & Technology Institute and Korean Ministry of Environment.

REFERENCES

(1.) Pham, V.H.T., Kim, J. Cultivation of unculturable soil bacteria. Trends Biotechnol., 2012; 30: 475-484.

(2.) Grobstein, C. Morphogenetic interaction between embryonic mouse tissues separated by a membrane filter. Nature, 1953; 172: 869-871.

(3.) Aufderheide, M., Mohr, U. CULTEX - an alternative technique for cultivation and exposure of cells of the respiratory tract to airborne pollutants at the air/liquid interface. Exp. Toxic. Pathol., 2000; 52: 265-270.

(4.) Hwang, E., Lee, H. Inhibitory effects of lycopene on the adhesion, invasion, and migration of SK-Hep1 human hepatoma cells. Exp. Biol. Med., 2006; 3: 322-327.

(5.) Svenning, M.M., Wartianinen, I., Hestnes, A. G., Binnerup, S. J. Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system. FEMS Microbiol. Ecol., 2003; 44: 347-354.

(6.) Ferrari, B. C., Binnerup, S. J., Gillings, M. Microcolony Cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria. Appl. Environ. Microbiol., 2005; 71: 8714-8720.

(7.) Ferrari, B. C., Winsley, T., Gillings, M., Binnerup, S. J. Cultivating previously uncultured soil bacteria using a soil substrate membrane system. Nat. Protoc., 2008; 3: 1261 1269.

(8.) Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H., Yi, H., Won, S., Chun, J. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol., 2012; 62: 716-721

(9.) Hall, T. A.: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic Acids Symposium Series No. 41. Oxford: Oxford University Press. 1999; pp 98-98.

(10.) Thomson, J. D. The CLUSTAL_X windows interface: flexible strategies multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res., 1997; 25: 4876-4882.

(11.) Saitou, N., Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol, 1987; 4: 406-425.

(12.) Fitch, W. M. Toward defining the course of evolution: minimum change for a specific tree topology. Syst. Zool., 1971; 20: 406-416.

(13.) Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. Molecular evolutionary genetics analysis using maximum likehood, evolutionar distance, and maximum parsimony methods.Mol. Biol. Evol, 2011; 28: 2731-2739.

(14.) F elsenstein, J. Confidence limit on phytogenies: an approach using the bootstrap. Evolution, 1985; 39: 783-791.

Van H. T. Pham and Jaisoo Kim *

Department of Life Science, Graduate School of Kyonggi University, 154-42 Gwanggyosan-Ro, Youngtong-Gu, Suwon, Gyeonggi-Do 443-760, Republic of Korea.

(Received: 13 November 2015; accepted: 19 January 2016)

* To whom all correspondence should be addressed. Tel.: 31-249-9648; fax: 31-253-1165; E-mail: jkimtamu@kgu.ac.kr

Caption: Fig. 1. Transwell plate system.

Caption: Fig. 3. Evolutionary phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic distribution of bacteria isolated from forest soil and their closest neighbors. Bootstrap percentages were based on 1000 replications and are shown at the branch points. Bar, 0.05 substitutions per nucleotide.

Caption: Fig. 4. Genetic diversity of microorganisms isolated from forest soil by the old and new cultivation methods.
Table 1. Physico-chemical properties of the soil sample
used in this study

Anions (mg/kg)

Cl    S[O.sub.4]    Moisture     Temperature
                   content (%)   ([degrees]C)

19        9           14.5            20

Anions (mg/kg)      Soil Texture (%)

Cl    S[O.sub.4]   Sand   Silt   Clay   Texture Class

19        9         76     20     4      Loamy Sand

Table 2. Compositions of media used in the new cultivation method

                                          Medium type (g/L distilled
                                                    water-DW)

No.    Components                          NB    LB    TSB   R2A

1      Beef extract                         3
2      Peptone
3      Proteose Peptone                                      0.5
4      Tryptone                                  10
5      Acid digest of Casein                                 0.5
6      Yeast Extract                              5          0.5
7      Enzymatic Digest of Gelatin          5
8      Enzymatic Digest of Casein                      17
9      Enzymatic Digest of Soybean Meal                 3
10     Soluble Starch                                        0.5
11     Sodium Pyruvate                                       0.3
12     Dextrose                                        2.5   0.5
13     Ca[Cl.sub.2] * 2[H.sub.2]O
14     K[H.sub.2]P[O.sub.4]
15     [K.sub.2]HP[O.sub.4]                            2.5   0.3
16     MgS[O.sub.4] * 7[H.sub.2]O                            0.05
17     NaCl                                      10     5
18     KN[O.sub.3]
19     Fe[Cl.sub.3] * 7[H.sub.2]O
20     [(N[H.sub.4]).sub.2]S[O.sub.4]
21     Trace element solution SL-6 (a)
22     Vitamin solution (b)
23     Soil extract

       Medium type (g/L distilled
               water-DW)

No.    MSM    SE    MSMSE    R2ASE

1                   500 mL   500 mL
                    of MSM   of R2A
2
3
4
5
6
7
8
9
10
11
12
13     0.02
14      2
15      2
16     0.2
17     0.4
18      1
19     0.01
20      2
21     1 mL
22     1 mL
23            1 L    500      500
                      mL       mL

(a) ZnS[O.sub.4]*7[H.sub.2]O, 0.1 g; Mn[Cl.sub.2] * 4[H.sub.2]O,
0.03 g; [H.sub.3]B[O.sub.3], 0.3 g; Co[Cl.sub.2]*6[H.sub.2]O, 0.2
g; Cu[Cl.sub.2]*2[H.sub.2]O, 0.01 g; Ni[Cl.sub.2]*6[H.sub.2]O, 0.02
g; [Na.sub.2]Mo[O.sub.4]*2[H.sub.2]O, 0.03 g; DW, 1000 ml.

(b) biotin, 10 mg; nicotiamide, 35 mg; thiamine dicloride, 30 mg;
p-aminobenzoic acid, 20 mg; pyridoxal chloride, 10 mg;
Ca-pantothenate, 10 mg; vitamin B12, 5 mg; DW, 100 ml.

Table 3. Bacterial strains isolated from forest soil by using the
traditional and new cultivation methods using various
traditional media.

No    Name of    Closest type strains
      isolates

1     TSB11      Achromobacter spanius LMG [5911.sup.T]
2     EU-2       Arthrobacter oxydans DSM [20119.sup.T]
3     NHI-27     Arthrobacter parietis LMG [22281.sup.T]
4     NHI-19     Arthrobacter ramosus CCM [1646.sup.T]
5     EU-8       Arthrobacter ramosus CCM [1646.sup.T]
6     NHI-15     Bacillus anthracis ATCC [14578.sup.T]
7     R2ASE9     Bacillus anthracis ATCC [14578.sup.T]
8     R2A1       Bacillus aryabhattai [B8W22.sup.T]
9     NHI-1      Bacillus cereus ATCC [14579.sup.T]
10    SE2        Bacillus horikoshii DSM [8719.sup.T]
11    TSB1       Bacillus fordii [R-7190.sup.T]
12    Aii-TSB    Bacillus fordii [R-7190.sup.T]
13    LB1        Bacillus fortis [R-6514.sup.T]
14    NHI-37     Bacillus licheniformis DSM [13.sup.T]
15    NHI-38     Bacillus methanolicus NCIMB [13113.sup.T]
16    AR-II-1    Bacillus methylotrophicus [CBMB205.sup.T]
17    NHI-10     Bacillus mycoides DSM [12442.sup.T]
18    NHI-16     Bacillus thuringiensis ATCC [10792.sup.T]
19    NHI-5      Bosea robittiae [LMG2638.sup.T]
20    NHI-8      Bosea thiooxidans [AJ25079.sup.T]
21    R2A2       Brevibacillus reuszeri NRRL NRS-[1206.sup.T]
22    EU-1       Burkhoideria stabils LMG [14294.sup.T]
23    NB2        Citrobacter famteri CDC 2991-[81.sup.T]
24    NB1        Citrobacter famteri CDC 2991-[81.sup.T]
25    NHI-6      Cupriavidus basilettsis CCUG [49340.sup.T]
26    NHI-14     Cupriavidus necator [N-1.sup.T]
27    NHI-48     Dyella japonica [XD53.sup.T]
28    NHI-12     Dyella terrae [JS14-6.sup.T]
29    R2A        Enterobacter asburiae JCM [6051.sup.T]
30    NHI-20     Kitasatospora saccharophila [SK15.sup.T]
31    TSB3       Leucobacter iaritis [40.sup.T]
32    NHI-46     Lysinibacillus boronitolerans [10a.sup.T]
33    NB6        Lysinibacillus fusiformis NBRC [15717.sup.T]
34    TSB13      Lysinibacillus macroides LMG [18474.sup.T]
35    NB9        Lysinibacillus sphaericus C3-[41.sup.T]
36    NB11       Lysinibacillus xylanilyticus [XDB9.sup.T]
37    TSB12      Lysinibacillus xylanilyticus [XDB9.sup.T]
38    NHI-23     Mesorhizobium chacoense [Pr5.sup.T]
39    NB4        Mesorhizobium robiniae CCNWYC [115.sup.T]
40    LB10       Mesorhizobium shangrilense
                   CCBAU [65327.sup.T]
41    NHI-34     Methylobacterium komagatae 002-[079.sup.T]
42    NHI-33     Methylobacterium oryzae [CBMB20.sup.T]
43    EU-7       Microbacterium natoriense TNJL143-[2.sup.T]
44    SEM-I-3    Microbacterium oleivorans [DSM16091.sup.T]
45    SEM-II-5   Micrococcus yunnanensis YIM [65004.sup.T]
46    SEM-II-6   Mycobacterium obuense ATCC [27023.sup.T]
47    NHI-24     Niabella tibetensis 15-[4.sup.T]
48    ET-1       Nitrobacter alkalicus [AN1.sup.T]
49    NHI-39     Paenibacillus alvei DSM [29.sup.T]
50    NB5        Paenibacillus nanensis MX2-[3.sup.T]
51    NHI-28     Paenibacillus pabuli JCM [9074.sup.T]
52    R2ASE5     Paenibacillus terrae AM141T
53    NHI-13     Pedobacterpanaciterrae Gsoil [042.sup.T]
54    NHI-21     Pelomonas puraquae CCUG [52769.sup.T]
55    TSB5       Pseudomonas beteli ATCC [19861.sup.T]
56    R2A7       Pseudomonas geniculata ATCC [19374.sup.T]
57    NHI-42     Pseudomonas koreensis Ps 9-[14.sup.T]
58    EU-6       Rhodococcus equi DSM [20307.sup.T]
59    NHI-47     Rhodococcus erythropolis [PR4.sup.T]
60    TSB8       Serratia marcescens subsp.
                   sahtensis [KRED.sup.T]
61    TSB7       Serratia tiematodiphila [DZ0503SBS1.sup.T]
62    NHI-3      Sporosarcina koreensis [F73.sup.T]
63    NHI-11     Staphylococcus warneri ATCC [27836.sup.T]
64    II-5-3     Staphylococcus epidemidis ATCC [14990.sup.T]
65    NHI-22     Stenotrophomonas maltophilia
                   ATCC [13637.sup.T]
66    NHI-49     Streptomyces althioticus NRRL B-[3981.sup.T]
67    NHI-35     Streptomyces xanthocidicus NBRC [13469.sup.T]
68    NHI-25     Tsukamurella pulmonis DSM [44142.sup.T]

No    Similarity   Accession
      (%)          number

1     99.93        AY170848
2     99.32        X83408
3     99.93        AJ639830
4     98.86        AM039435
5     99.06        AM039435
6     98.69        AB190217
7     100.00       AB190217
8     100.00       EF114313
9     99.86        AE017333
10    98.99        X76443
11    98.29        AY443039
12    99.48        AY443039
13    98.38        AY443038
14    100.00       AE017333
15    96.15        AB112727
16    99.90        EU194 897
17    99.02        ACMX01000133
18    99.78        ACNF010000156
19    99.64        FR774994
20    93.19        AJ250796
21    99.71        D78464
22    100          AF148554
23    98.45        AF025371
24    99.32        AF025371
25    99.93        FN597608
26    98.89        CP002878
27    99.86        AB110498
28    99.02        EU604273
29    99.78        AB004744
30    99.58        AB278568
31    99.79        AM040493
32    99.53        AB199591
33    99.32        AB271743
34    99.05        AJ628749
35    100          CP000817
36    98.44        FJ477040
37    99.26        FJ477040
38    97.79        AJ278249
39    97.34        EU849582
40    98.06        EU074203
41    100          AB252201
42    99.36        AY683045
43    99.93        AY566291
44    99.85        AJ698725
45    99.76        FJ214355
46    99.76        X55597
47    96.74        GU291295
48    96.66        AF069956
49    99.13        AJ320491
50    96.85        AB265206
51    100.00       AB073191
52    99.73        AF391124
53    98.26        AB245368
54    100.00       AM501439
55    99.79        AB021406
56    99.08        AB021404
57    99.66        AF468452
58    98.88        X80614
59    100.00       AP008957
60    99.87        AB061685
61    99.66        EU036987
62    99.72        DQ073393
63    100.00       L37603
64    99.86        L37605
65    99.29        AB008509
66    98.39        AY999791
67    99.71        AB184427
68    99.65        X92981

No    Used Media
      Traditional             New
      culture                 culture
      method                  method

1     LB, R2A                 LB. R2A. MSM
2                             R2A
3                             LB. NB. SE
4                             MSM
5                             SE. MSM
6     LB.TSB                  TSB. LB. MSM. NB. R2A
7                             R2ASE
8                             MSMSE
9     LB.TSB                  TSB. LB. DW(NA)
10                            MSM
11                            TSB
12                            TSB
13                            TSB
14                            R2A. MSMSE
15                            R2A
16                            R2A
17    NB. R2A                 DW(R2A)
18    R2A                     R2A
19    LB. NB. TSB. R2A. MSM
20    NB. R2A                 NB. DW(R2A)
21                            LB
22                            R2A
23                            R2A
24                            R2A
25    LB                      DW(LB). TSB
26    R2A                     R2A. NB
27                            R2A
28    R2A                     MSM. R2A
29                            R2A
30    MSM                     DW(MSM), SE
31                            LB
32                            NB. R2A
33                            NB. R2A
34                            NB
35                            NB
36    LB, TSB.R2A.NB          TSB. LB. NB. R2A
37    LB. R2A, NB             TSB. LB. NB. R2A
38    MSMSE, SE               MSMSE. SE. R2A. NBLB
39                            R2A
40                            TSB
41                            DW(MSM). SE
42                            DW(MSM). R2A
43                            R2A
44                            MSMSE
45                            MSMSE. R2A
46                            MSMSE
47    TSB                     DW(TSA). DW(R2A)
48                            R2A
49                            TSB
50                            R2A
51                            NB. R2A
52                            MSM
53    R2A                     NB. DW(R2A)
54    MSM                     MSM
55                            TSB
56    R2A                     R2A. NB
57                            R2A. NB
58                            R2A
59                            R2ASE
60                            LB
61                            LB
62    LB. NB. TSB, R2A        TSB. LB. NB. R2A
63    NB                      TSB. NB. DW(R2A). DW(LB)
64                            MSMSE
65    MSM                     R2ASE
66                            R2ASE
67                            R2A
68                            DW(TSA).DW(LB).
                              DW(MSM)

Note: distilled water as a medium was added into one transwell
insert, and soil samples were cultured for 2 weeks as described in
Materials and Methods. The cultivated inoculum was transferred to
agar plates containing TSA (tryptic soy agar), NA (nutrient agar),
LB, MSM, and R2A media. Visible colonies obtained on these plates
were named as DW(TSA), DW(NA), DW(LB), DW(MSM), and DW(R2A),
respectively. The potential new species are bolded to be
distinguished.

Fig. 2. Comparison of bacteria isolation efficiency
between the new and traditional cultivation methods
entirely (A) and by each medium (B).

Old culture method only    1.5%
New culture method only    69.1%
Both                       29.4%

Note: Table made from pie chart.
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Article Details
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Author:Pham, Van H.T.; Kim, Jaisoo
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Mar 1, 2016
Words:4635
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