effective soil extraction method for cultivating ...oct 05, 2018 · 62 diffusion chamber s (27,...

Effective soil extraction method for cultivating previously uncultured 1

soil bacteria 2

3

Tuan Manh Nguyena,d

, Chan Seoc, Moongi Ji

c, Man-Jeong Paik

c, Seung-Woon Myung

b, and Jaisoo 4

Kima*

5

aDepartment of Life Science, College of Natural Sciences and Engineering, Kyonggi University, 6

Suwon, Gyeonggi-Do 16227, Republic of Korea 7

bDepartment of Chemistry, College of Natural Sciences and Engineering, Kyonggi University, Suwon, 8

Gyeonggi-Do 16227, Republic of Korea 9

cCollege of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National 10

University, Suncheon, Jeollanam-do 57922, Republic of Korea 11

dThai Nguyen University of Agriculture and Forestry, Quyet Thang, Thai Nguyen, Vietnam 12

13

Running title: Cultivation of uncultured soil bacteria 14

15

Author for correspondence: Jaisoo Kim 16

Tel: +82-31-249-9648 17

Fax: +82-31-253-1165 18

E-mail: [email protected] 19

20

21

AEM Accepted Manuscript Posted Online 5 October 2018Appl. Environ. Microbiol. doi:10.1128/AEM.01145-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.

on June 23, 2020 by guesthttp://aem

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ABSTRACT Here, a new medium named as intensive soil extract medium (ISEM) based on new soil 22

extract (NSE) using 80% methanol was used to efficiently isolate previously uncultured bacteria and 23

new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n=258), 24

respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, 25

Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of 26

chemical analysis showed that NSE included more diverse components of low-molecular-weight 27

organic substances than two conventional soil extracts made using distilled water. Cultivation of 28

previously uncultured bacteria is expected to extend knowledge through the discovery of new 29

phenotypic, physiological and functional properties, and even roles of unknown genes. 30

IMPORTANCE Either metagenomics or single-cell sequencing can detect unknown genes from 31

uncultured microbial strains in environments and may find their significant potential metabolites and 32

roles. However, such gene/genome-based techniques do not allow detailed investigations that are 33

possible with cultures. To solve this problem, various approaches for cultivation of uncultured 34

bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture. 35

KEYWORDS uncultured bacteria, cultivation, new soil extract (NSE), intensive soil extract medium 36

(ISEM), new taxonomic candidates, low-molecular-weight organic substances (LMWOS), isolation, 37

subculture 38

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Molecular tools revealed that prokaryotic species are very diverse and abundant in soil, and contain 44

numerous unexplored potential metabolites (1-4). Although these tools enable analysis of the broad 45

range of metabolic diversity of microorganisms without the need to isolate species, many bacterial 46

characteristics are unknown because of limitations of cultivation (5, 6). Since the establishment of 47

solid culture media, secondary metabolites have been isolated from microorganisms cultured in 48

laboratories. The lack of complex factors/conditions in the laboratory has contributed to the inability 49

to isolate various species (7, 8). Since the concept of “uncultured bacteria” was published in 1990 (9) 50

to refer these bacteria as not yet cultured in laboratories, several methods have been developed in an 51

attempt to culture these bacteria so far. These methods involved transporting bacteria from their 52

natural environment to the laboratory for growth in artificial media/conditions similar to those in the 53

natural environment by modifying growth media components (7) or growth conditions such pH and 54

salt concentrations (9, 10), adding inorganic compounds or metals lacking electron donors/acceptors 55

(11, 12), using various factors (13), coculture with helper bacteria (14, 15), soil extracts using water 56

(16, 17) or aqueous buffers (18, 19), diluted medium or serial dilution culture (20, 21), long 57

incubation time (10, 22), etc. Furthermore, sophisticated techniques were developed such as ichip for 58

in situ cultivation (23), micro-bioreactor (24), optical tweezers (25), or micro-manipulator (26), 59

which allowed analysis of individual cells in soil samples. However, new artificial media to maintain 60

these cultures are needed. Although scientists can enrich slow-growing microorganisms using 61

diffusion chambers (27, 28) or soil substrate membranes (29), most enriched bacteria do not grow on 62

agar plates for isolation and further cultivation. Without successful cultivation, it is difficult to detect 63

and identify novel organisms, obtain phenotypic and functional information, and determine the 64

functions of unknown genes (30). The most important factors affecting the cultivation of uncultured 65

bacteria and the most appropriate media conditions remain unclear. 66


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Here, we developed a simple culture method based on new soil extract (NSE) using 80% methanol 67

without special equipment(s) and successfully cultured many previously uncultured bacterial strains. 68

To evaluate our method, we checked the proportion of uncultured strains among isolates as well as 69

that of new taxa, and analysed chemical components of NSE to compare with two traditional soil 70

extracts (TSEs) commonly used. 71

72

RESULTS 73

Composition of soil extracts utilized as culture supplements. For nutrition and bacterial growth, 74

heterotrophic soil bacteria depend on low-molecular-weight organic substances (LMWOS) and 75

inorganic compounds (31). Thus, in this study, we compared the major LMWOS such as amino acids, 76

fatty acids, organic acids, and inorganic ions in three different extraction methods (NSE: new 77

extraction developed in this study; TSE1: traditional extraction without autoclaving; TSE2: 78

traditional extraction with autoclaving) (Table 1). To obtain more diverse LMWOS, we used a 79

mixture of methanol:water to be consistent with 4:1 (80%) to extract bacterial nutrients from soil 80

rather than water or aqueous buffers and named this extract as new soil extract (NSE). Briefly, 500 g 81

dry soil was prepared and shaken at 150 rpm with 1.3 L 80% methanol overnight at room temperature. 82

The supernatant was transferred to a new flask and fresh 1.3 L 80% methanol was added to the 83

remaining soil and mixed well for 1 h. The two supernatants were combined, filtered, and evaporated. 84

The NSE was stored at 4°C until use. For amino acids, NSE showed a total yield of 18.50 mgL-1

, 85

which is much higher than for the other two methods which had values of 4.84 and 5.87 mgL-1

, 86

respectively (P<0.004). Additionally, 21 amino acids were extracted, including four more amino 87

acids (valine, pipecolic acid, serine, and threonine) and a very high concentration of tyrosine (9.29 88

mgL-1

) compared to the other methods. This higher concentration and diversity of amino acids in the 89

NSE than in other two methods may cause better cultivability of uncultured soil bacteria. However, 90


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there was little difference between methods TSE1 and TSE2 (PTSE1 vs TSE2 <0.03), indicating that 91

autoclaving at 121C had no significant effect to extract more components of amino acids in soil. The 92

results of fatty acid analysis can be compared because method NSE extracted a much higher total 93

concentration (13.10 mgL-1

vs. 0.79 mgL-1

) and greater number (n=16 vs. n=8) of fatty acids than 94

other two (Table 1). For organic acids, the results showed that method NSE had lower total yield of 95

organic acids (25.14 mgL-1

vs. 62.69–89.00 mgL-1

; P <0.03) but a greater total number (n=16 vs. 96

n=11) including lactic acid, glycolic acid, 2-hydroxybuyric acid, fumaric acid, and -ketoglutaric 97

acid (Table 1). The total amount of organic acids obtained by methods TSE1 and TSE2 were 98

significantly influenced by two major components, acetoacetic acid and oxaloacetic acid, but these 99

did not significantly affect the total amount for method NSE. 100

The total yields of inorganic compounds for each extraction were 748.03, 965.00, and 946.91 101

mg per liter of extract, respectively (Table 1). Although method NSE gave a lower concentration of 102

total inorganic compounds than the other methods (P NSE vs TSE1 or TSE2 < 0.002), the methanol-water 103

mixture appeared to dissolve substances similarly to water and recovered the same inorganic ions 104

extracted with water (methods TSE1 & TSE2). In particular, NO3- and PO4

2- were higher in NSE than 105

TSEs and SO42-

was much lower in NSE than TSEs. Autoclaving did not significantly affect the 106

dissolution of inorganic or organic compounds in water between methods TSE1 and TSE2 (P>0.3). 107

Method validation based on isolation rate of uncultured or new taxonomic bacteria. To compare 108

the three cultivation methods, the newly developed method for isolation of previously uncultured soil 109

bacteria using ISEM (intensive soil extract medium: simply new method), traditional soil extract 110

culture medium (simply traditional method), and modified transwell culture method (simply modified 111

method), the different bacterial strains (258, 243, and 252 for each method) were isolated from three 112

soil samples and identified as described in Materials and Methods. The ratio of previously uncultured 113

bacterial strains was significantly increased by 49% (126 isolated strains/total 258 isolated strains) for 114

Table 1


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the new method, with values of 5% (11 isolated strains/total 243 isolated strains) for the traditional 115

method and 15% (39 isolated strains/total 252 isolated strains) for the modified method (Fig. 1a; 116

Tables S1–S3). 117

Taxonomic analysis also showed that this new method was much better than the two other methods in 118

terms of new taxa isolation. In the new method, 142 new species candidates were found among 119

isolates, including 13 genus level and 1 family level, showing 55% (142/258) efficiency compared to 120

13% (32/243) and 26% (65/252) for the traditional and modified methods, respectively (Tables S1–121

S3). For the isolation efficiency at the genus level or higher candidates, the new method showed a 122

value of 5.4% (14/258), which is much higher than 0.0% (0/243) and 0.4% (1/252) obtained for the 123

other two methods (Fig. 1b). The new method isolated a family level candidate, while the other two 124

methods did not. Furthermore, the new method showed the highest ratio and largest number of new 125

taxa candidates (at least species level) among uncultured isolated strains (75.4%: 95/126) compared 126

to the other two methods, which showed values of 36.4% (4/11) and 48.7% (19/39), respectively (Fig. 127

1c). In addition, our method can directly isolate bacterial strains from a soil suspension without an 128

enrishment culture step, saving time and labour. 129

Method validation through taxonomic analysis. As a standard reference, this study tried to identify 130

possible strains present in the same soil samples through a molecular technique, which can give some 131

information important to evaluate the method developed. 16S amplicon sequencing data as a last 132

generation method were analysed according to Chao1 at a 3% evolutionary distance and revealed the 133

diversity of microbial community genomics in the soil samples, with 1744–2402 operational 134

taxonomic units (Fig. S1a). Pyrosequencing analysis suggested that nine identified bacterial phyla 135

commonly present in all soil samples were Chloroflexi, Planctomycetes, Verrucomicrobia, 136

Bacteroidetes, Gemmatimonadetes, Actinobacteria, Acidobacteria, Proteobacteria, and 137

Parcubacteria_OD1 and three unidentified phyla were Nitrospirae, Saccharibacteria_TM7 and AD3 138

Fig. 1


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(Fig. 2a). Proteobacteria and Acidobacteria were the most dominant phyla. Minor phyla (<1%) 139

involved in 3.1% ETC (et cetera) were major lineages that have cultured representatives (Chlorobi, 140

Elusimicrobia, Armatimonadetes, Firmicutes, Chlamydiae, Tenericutes, Latescibacteria, 141

Omnitrophica, and Hydrogenedentes), candidate phyla without cultured representatives (Kazan, 142

Gracilibacteria, Berkelbacteria), and other phyla. Additionally, the distribution and relative 143

abundance of species identified by pyrosequencing in each soil sample were determined, showing 144

that abundant species (more reads within a species) would be a few and rare species (less reads within 145

a species would be many (Fig. 2b). Phylogenetically, the new method achieved successful cultivation 146

of strains from seven phyla among all bacteria present in the soils (Fig. 3): Proteobacteria (α, β, and γ) 147

(46.9%), Actinobacteria (43.4%), Bacteroidetes (4.7%), Firmicutes (3.9%), Acidobacteria (0.4%), 148

Verrucomicrobia (0.4%), and Planctomycetes (0.4%) (Fig. S1b). In contrast, the two other methods 149

did not recover strains in three phyla: Acidobacteria, Verrucomicrobia, and Planctomycetes. Thus, the 150

new method extended the taxonomic range of cultivation at the phylum level. Pyrosequencing 151

analysis indicated that the three soil samples included 120 identified families, excluding 10% 152

unclassified sequences, and 38, 22, and 29% of the identified families were recovered by the new, 153

traditional, and modified methods, respectively (Fig. S2a). The isolates obtained using the new 154

method represented 100 genera (86 known and 14 novel genera), which are compared with the 50 and 155

60 genera isolated using the traditional and modified methods, respectively (Fig. S2b). For the 156

comparison at the species level, the new method independently cultivated soil bacteria as compared 157

to the other two methods, as only one species among the 258 species overlapped with the traditional 158

method (none with the modified method), while the two other methods showed 31 overlapping 159

species with each other (Fig. S2c). This result indicated that the new method was more specific for 160

the growth of uncultured bacteria than cultured bacteria. Hitherto, uncultured isolates from the new 161

method were distributed in 53 genera of six phyla, while the other two methods showed a very 162

limited taxonomic distribution: 5 genera in two phyla and 20 genera in three phyla, respectively (Fig. 163


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S2d). 164

Method validation through subculture of novel isolates. We tested 7 different media to compare 165

their ability to support subculture of newly-isolated bacteria: basic salts (BS as a negative control), 166

BS plus micro-nutrients, BS plus vitamin B, BS plus D-amino acids, BS plus NSE, NSE only, and full 167

ISEM (as a positive control). Here, 131 bacterial isolates (= 131 species) including 126 previously 168

uncultured bacteria and 5 novel genus candidates (Table 2; S4), obtained using our developed culture 169

method and three different soil samples, were used to determine the effectiveness of the various 170

nutrient components by forming visible colonies on agar plates after streaking. While NSE and BS 171

plus NSE showed 100% growth recovery (n = 131/131), BS plus D-amino acids showed a value of 8% 172

(n = 11/131) and other components had a value of 0% (n = 0/131). Therefore, NSE and NSE-173

containing media can be more effective for isolating and subculturing uncultured soil bacteria than 174

other media. 175

176

DISCUSSION 177

Soil contains elements necessary for living organisms. An aqueous soil extraction method was 178

established previously (for example see ref. 36 or ATCC medium 654 or DSMZ medium 80) and 179

remains widely used. Most mineral or organic ingredients such as ionic salts, vitamins, antibiotics, 180

plant hormones, and plant-promoting growth factors, among others, can be dissolved in distilled 181

water, while some organic components such as non-polar compounds cannot be sufficiently dissolved 182

in distilled water or aqueous buffers. Thus, we used 80% methanol to overcome this problem and 183

achieved higher concentrations and more different types of organic ingredients compared to using 184

distilled water (Table 1). Although methanol and water are polar protic solvents that easily solubilize 185

polar molecules, methanol is less polar than water based on their polarity values of 5.1 and 10.2, 186

respectively. Therefore, methanol may more easily dissolve or extract a greater amount of 187

Fig. 2&3

Table 2


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hydrophobic or amphipathic molecules in soil than water. In contrast, based on their dielectric 188

constants according to Harris (32), water (approximately 80) is more likely to dissolve inorganic 189

compounds than methanol (approximately 30). 190

NSE contained 21 amino acids, which was greater than that obtained using water extraction methods 191

because of 17 in this study (Table 1) and even less in previous studies: 16 amino acids were obtained 192

using 6 N HCl (33) and 14 amino acids were obtained using MOPS buffer (31). Fatty acids contain a 193

polar carboxylic group and non-polar hydrocarbon group of 4–36 carbons, making only short-chain 194

fatty acids more or less water-soluble. Thus, a combination of methanol and water improved their 195

dissolution. This led to significant differences in the total number and amount of fatty acids between 196

methods NSE and TSE1 (P<0.002), but the total fatty acids obtained for the two comparative 197

methods (method TSE1, and method TSE2) were similar each other (P>0.9) (Table 1). Greater 198

amounts and larger numbers of fatty acids may improve the cultivability of uncultured soil bacteria. 199

Organic acids are widely present in soil (31) and low-molecular weight organic acids are typically 200

miscible in water. Thus, the three extraction methods were relatively effective. Although methanol 201

extracted lower concentrations than water, more diverse organic acids were extracted, increasing the 202

spectrum of either carbon or electron donors/acceptors for microorganisms. Because inorganic 203

substances typically dissolve well in water and even in pure methanol (34), 80% methanol can extract 204

large amounts of inorganic compounds (5.41–489.67 mg/L) from soil, although lower amounts than 205

the two water extraction methods. We supposed that the components present only in NSE, or present 206

at greater concentrations than in the two TSEs, might stimulate growth of uncultured bacteria. 207

Overall, NSE was superior compared to the two traditional soil extracts (TSEs) and other extraction 208

methods because greater concentrations and types of LMWOS were obtained, which may be required 209

to support most soil bacteria including uncultured bacteria. 210

Although an enhanced medium (traditional soil extract culture medium) derived from soil extract 211


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(TSE) containing yeast extract, tryptone, and salts was designed to support various soil bacteria (17), 212

necessary elements for many uncultured bacteria may be absent, so that most isolates (~96%) seemed 213

to belong to previously cultured groups (Fig. 1a; Table S2). Additionally, the modified method with 214

R2A, a complex efficient artificial media for cultivating heterotrophic bacteria including fast-growing 215

and slow-growing bacteria (35), showed better results and isolated a greater number of bacteria and 216

new taxa candidates than the traditional method did (Fig. 1a; Fig. 2a, b; Table S3). Although this 217

method is better than the traditional method, the isolation step shows limited recovery of various 218

enriched uncultured soil bacteria. Particularly, ISEM developed in this study allowed cultivation of 219

large numbers of isolates of various uncultured bacteria and new taxa candidates compared to the 220

numbers obtained using the other two methods. Thus, the new method more effectively isolated 221

uncultured bacteria and new bacterial taxa present in soil. The most important aspect of this new 222

method (ISEM) is inclusion of NSE (described above). Results, the direct isolation from a soil 223

suspension using ISEM agar plates can be an effective way because, during the enrichment step, fast-224

growing microorganisms may overcome slow-growing bacteria. For any new cells to be formed, 225

substrate complexes including nutrients, supporting growth factors, etc. are required. Under in vitro 226

conditions, artificial nutrient-limited, most of the energy may be consumed by fast-growing bacteria, 227

while slow-growing bacteria need longer times for cell division process. When the fast-growing 228

bacteria reach the highest growth rate, nutrient concentrations in culture media may shortly be too 229

low or completely consumed. This leads to a nutritional physiological stress (starvation) for slow-230

growing species (36). In addition, antibiotic produced by some fast-growing species may also be a 231

growth-inhibiting factor for slower growing species (36). As a result, the diversity of bacterial species 232

can be reduced. 233

Among recently introduced methods, Kakumanu & Williams (37) developed a soil diffusion system 234

and found uncultured bacteria in the phyla Proteobacteria, Bacteroidetes, Verrucomicrobia, 235


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Planctomycetes, and OP10, but only 8 uncultured bacteria at the species level. Furthermore, the study 236

suggested conducting only enrichment culture, with no method for isolation of pure bacterial strains. 237

Another study found that 27% of species belonged to 20 unnamed family-level groupings among 350 238

isolates (38), but these species were isolated from many different artificial media, not including soil 239

extracts, and the effectiveness of each media on the cultivability of uncultured soil bacteria was not 240

determined. Various methods for cultivating uncultured soil bacteria have been developed: 241

modification of growth media, modification of growth conditions, community culture, coculture, 242

trans-well plates with membranes, micromanipulator, optical tweezers, laser microdissection, high-243

throughput microbioreactor, simulated natural environments using diffusion chambers, single cell 244

encapsulation combined with flow cytometry, multiwell microbial culture chip or iChip, and 245

entrapped gelating agent coated with polymer (30). However, these methods exhibit low isolation 246

efficiency of uncultured bacteria or lack strategies for subsequent pure culture. The new method 247

developed in this study showed high isolation efficiency (49%), a 100% recovery rate of isolated 248

uncultured soil bacteria, and easier application in laboratories compared to most previously 249

developed methods. 250

Although Acidobacteria were the second most abundant phylum in pyrosequencing analysis for three 251

soil samples, only one isolate was obtained by our new method since the medium (ISEM) is not 252

acidic (pH 6.8), while Acidobacteria subgroups 1, 2, 3, 12, 13, and 15 exhibit most abundance at pH 253

of soils < 6.5 (39-41). As a future work, if using ISEM with low pH or low/high temperature, more 254

uncultured or novel Acidobacteria or other bacteria may successfully be isolated. 255

In summary, our new method showed a much higher isolation rate of new taxa candidates among 256

uncultured isolates and greater isolation rate of uncultured soil bacteria and new taxa candidates than 257

traditional and modified methods tested in this study. Additionally, isolation was simpler and did not 258

require enrichment culture, and could be directly subcultured to obtain more uncultured bacterial pure 259


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cultures for further experiments. Further, the variety of uncultured soil bacteria or new taxa 260

candidates can be extended with ISEM by altering the cultivation conditions such as incubation 261

temperature, pH, salt concentration, anaerobic conditions or using various soil samples and other 262

samples. 263

264

MATERIALS AND METHODS 265

Soil sample used for making soil extracts. Rhizosphere soil where Robinia pseudoacacia L. 266

dominated was collected at Kyonggi University (154-42 Gwanggyosan-ro, Iui-dong, Yeongtong-gu, 267

Suwon, Gyeonggi-do, South Korea; 37°30'04'' N, 127°03'58'' E) during June 2016. Fresh soil was 268

dried at room temperature for 48 h by spreading soil sample on surface of aluminum foil and using an 269

air conditioner in dry mode (25~30°C), and then any plant debris, gravels and rocks were removed by 270

a 0.2 mm sieve. To determine physicochemical properties of soil, the soil was dried at 110°C for 24 h, 271

and cooled at room temperature. Soil contained approximately 78% sand, 17% silt and 5% clay. Its 272

pH (5.7) was measured directly from fresh soil. 273

Preparing new soil extract (NSE) in 1 L medium. Approximately 1000 g of the dry soil sieved at 274

room temperature was divided into two equal parts (500 g each) in a 2-L flask and then mixed with 275

1.3 L of 80% methanol (#494291, methanol (HPLC grade of purity: ≥99.9%), Sigma Aldrich, St. 276

Louis, MO, USA) in deionized water and shaken at 150 rpm overnight at room temperature (below 277

25°C). After settling for 30 min, the supernatant was transferred to a new flask. Next, 1.3 L of 80% 278

methanol was added to the soil and mixed well for 1 h. The two supernatants were combined and 279

filtered through WhatmanTM

paper (#1001-150, 150 mm, GE Healthcare, Little Chalfont, UK). 280

Methanol was removed by a general rotary evaporator (~40°C). The NSE was adjusted to a final 281

volume of 200 mL with deionized water, sterilized through a 0.22 µm nitrocellulose filter 282

(#GSWP04700, Merck Millipore Ltd., Billerica, MA, USA) using a vacuum pump, stored in a dark 283


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Schott Duran bottle at 4°C and used within one week. 284

Medium for isolation of soil bacteria. The medium containing 0.23 g KH2PO4, 0.23 g K2HPO4, 285

0.23g MgSO4∙7H2O, 0.33 g NH4NO3, 0.25 g NaHCO3 as a group of mineral salts, and 15 g agar in 1 286

L water was sterilized at 121°C for 15 min. 15 g agar (#A7049, Sigma-Aldrich) was treated several 287

times with distilled water to discard any trace nutrients or elements before use (making purified agar). 288

Then the following elements were added to the medium: 5 mg various D-amino acids (D-valine, D-289

methionine, D-leucine, D-phenylalanine, D-threonine, and D-tryptophan), 1 mL vitamin B (vitamin 290

stock solution containing 50 mg each thiamine hydrochloride, riboflavin, niacin, pyridoxine HCl, 291

inositol, calcium pantothenate, and β-aminobenzoic acid and 25 mg biotin in 100 mL distilled water, 292

sterilized through a 0.2-µm syringe filter, stored at 4°C in dark Schott Duran and used within one 293

month), 0.2 L of NSE, 2 mL of selenite-tungstate solution (42) (composition in 1 L distilled water: 294

0.5g NaOH, 3 mg Na2SeO3.5H2O, 4 mg Na2WO4.2H2O; the solution was filter-sterilized, stored at 295

4°C and used within 1 month), and 2 mL of trace element SL-10 (43) (ingredient contained 10 mL of 296

HCl (25%, v/v); 1.5 g of FeCl2∙4H2O; 70 mg of ZnCl2; 100 mg of MnCl2∙4H2O; 6 mg of H3BO3; 190 297

mg of CoCl2∙6H2O; 2 mg of CuCl2∙2H2O; 24 mg of NiCl2∙6H2O; 36 mg of Na2MoO4∙2H2O in a final 298

volume of 1 L; then this solution was passed through a 0.2-μm filter, added directly in the medium 299

after autoclaving). The final volume was 1 L and pH was 6.8±0.2. The complex medium was named 300

as intensive soil extract medium (ISEM). The medium should be prepared freshly and used within 301

one week. In this study, we used 150×20 mm petri dishes (SPL Life Science Co., Ltd., Gyeonggi-do, 302

Korea). The larger dish allows for increased separation of colonies at high dilution concentrations 303

during isolation. 304

Preparation of various media to recover previously uncultured soil bacterial isolates. We used 305

multiple combinations to find a best growth medium as described above to identify the most 306


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important elements for supporting the growth of previously uncultured soil bacteria. Several 307

examinations were carried out based on the strains obtained in this study as follows (i): basic salts 308

(BS) only as a negative control, (ii): BS with selenite-tungstate solution and SL-10; (iii): BS plus D-309

amino acids, (iv): BS with vitamin B, (v): BS with NSE, (vi): NSE only; (vii): mixture of all 310

components (ISEM) as a positive control, and then 15 g purified agar was added to each medium. 311

Agar plates were incubated at 25°C for 4 weeks under aerobic conditions. To prevent the plates from 312

drying out, two cups of distilled water was placed in the incubator. 313

Soil sampling sites and preparation of soil samples. Three soil samples were acquired in South 314

Korea in June 2016, including Ansan (sample A) (Il-dong, Sangnok-gu, Ansan, Gyeonggi-do: 315

37°17’58’’N & 126°53’57 E), Suwon (sample B) (Buksu-dong, Paldal-gu, Suwon, Gyeonggi-do: 316

37°16’42’’ N & 127°00’17’’ E), and Seoul (sample S) (Itaewon-ro, Yongsan-gu, Seoul: 37°31’06’’ N, 317

127°01’04’’ E). For each sample, approximately10 g soil from ten different locations within a 150-m 318

diameter were collected and mixed well. The sample was passed through 0.1-mm sieve and 319

isolated/enriched directly using three methods. A 25-g sieved soil sample was mixed with 250 mL 320

sterile saline (0.9% NaCl, w/v), stirred for 15 min, and allowed to separate between suspension and 321

sediment before use. 322

Newly developed method for isolation of previously uncultured soil bacteria. First, 100 µL of 323

each the dilution of soil suspension was spread onto three agar plates of ISEM (to ensure uniformly 324

distributed suspension on the surface of the medium, 100 μL of each the dilution plus 100 μL of 325

ISEM liquid is recommended). These agar plates were incubated at 25°C for 6 weeks. A few colonies 326

appeared after one week of incubation. The number of directly observable colonies was increased 327

after 2 weeks, and tiny colonies were picked up and streaked onto fresh ISEM until morphologically 328

pure colonies were obtained. Cells on fresh ISEM typically require at least one week incubation. 329

Uncultured bacteria generally showed weak growth, and thus in some cases pure colonies were 330


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activated in ISEM broth in a shaking incubator at 25°C, 150 rpm for 1–2 weeks before transferring 331

onto the agar plate. 332

Traditional soil extract culture medium. Approximately 1000 g air-dried soil in 1.3 L of deionized 333

water was autoclaved at 121°C for 1 h and allowed to cool. The supernatant was filtered through 334

WhatmanTM

paper before centrifugation in a 500-mL bottle at 5009 ×g for 30 min at room 335

temperature. One litre of the supernatant (TSE) was obtained. Soil extract agar was enhanced by 336

supplementation (17) with 0.04% K2HPO4, 0.005% MgSO4.7H2O, 0.01% NaCl, 0.001% FeCl3, 0.05% 337

tryptone, 0.05% yeast extract, and 1.5% agar in 1 L of soil extract liquid with a final pH of 6.8. Next, 338

100 µL of each dilution of three soil samples was dispersed onto three soil extract agar plates and 339

cultivated at 25°C for 6 weeks. Colonies were re-streaked until pure colonies were obtained. 340

Modified transwell culture method. Transwell plate system (#35006, SPLInsert™ Hanging, SPL 341

Life Sciences) was used to enrich bacterial community, especially for uncultured soil bacteria, from 342

soil samples, which contains 6-inserts with 6-wells in a plate. An insert has two different sized frames 343

(upper: 28 mm outer, and 26.65 mm inner; lower: 26.6 mm outer, and 23.3 mm inner); 28 mm height, 344

and 4.52 cm2 of area of growth for each insert. The lower frame is covered with a 0.4-µm 345

polycarbonate membrane. Its membrane specification is 25 mm diameter, and 7~10 μm thickness. 346

Approximately 3 g of soil sample was added to a transwell plate, and then 3 mL R2A medium (#MB-347

R2230, MB Cell, Los Angeles, CA, USA; 3.15 g of the powder in 1 L distilled water) was 348

supplemented into the soil-containing wells and then put the insert on the wet soil. Next, 100 µL of 349

the suspension and 1 mL R2A medium was inoculated into the insert. The transwell culture system 350

was covered with parafilm to prevent evaporation. The system was shaken at 120 rpm and 25°C for 4 351

weeks. Seven-fold dilutions of the culture enriched were established in R2A broth medium; 100 µL 352

of each dilution was spread onto three R2A agar plates and incubated at 25°C for 6 weeks. Colonies 353

were subcultured on R2A medium to obtain individual colonies (Table S5). 354


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Identification of 16S rRNA gene sequences and accession numbers of 16S rRNA gene 355

sequences. Near-full-length 16S rRNA sequences were identified and similarity to valid 356

species was calculated using the EzTaxon Database Update 357

(http://www.ezbiocloud.net/eztaxon) for comparison with published uncultured gene 358

sequences via the nucleotide BLAST in NCBI: http://www.ncbi.nlm.nih.gov/. In this study, 359

based on full 16S rRNA similarity to valid published species, with four temporary divisions, 360

candidates of novel species were defined by comparison of 16S rRNA similarity at the 361

threshold of 98.7% (44), 95.3–90.0% novel genus level (45), and novel family level at off 362

limit lower than 90.0%. All sequence data of the isolates were submitted to GenBank 363

database and are listed in the Supplementary Tables. 364

DNA extraction from soil. Using FastDNA® SPIN Kit for Soil (#116560-200, MP Biomedicals), 365

soil DNA from 0.5 g of fresh soil was extracted and purified by following the instruction's guide. 366

DNA quality was checked by 1.2% agarose gel electrophoresis in 0.5 TAE buffer and DNA 367

concentration was determined via MaestroNano spectrophotometer (Mastrogen). Then DNA samples 368

were held at -20°C until use. 369

PCR amplification and pyrosequencing. Pure isolated DNA soil samples were subjected to 370

amplification of the target V1 to V3 regions located in the 16S rRNA gene by PCR using the 371

barcoding primers 27F 5′-CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-AC-372

GAGTTTGATCMTGGCTCAG-3′ and 518R 5′-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-373

X-ACWTTACCGCGGCTGCTGG-3′; (‘X’ directs the unique barcode for each subject) 374

(http://oklbb.ezbiocloud.net/content/1001). The reaction was conducted as follows: initial 375

denaturation at 95°C for 5 min, followed by 30 cycles of (denaturation at 95°C for 30 sec, annealing 376

at 55°C for 30 sec, and extension at 72°C for 30 sec), and final elongation at 72°C for 5 min. Next, 377


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the amplicons were evaluated by 2% agarose gel electrophoresis and observed with a Gel Doc system 378

(Bio-Rad, Hercules, CA, USA). A QIA quick PCR purification kit (#28106, Qiagen, Hilden, 379

Germany) was used to purify the PCR products. An Ampure beads kit (Agencourt Bioscience, 380

Beverly, MA, USA) was used to enhance the quality of the sample and remove non-target products 381

following the manufacturer’s instructions. Quality and target size were estimated with a Bioanalyzer 382

2100 (Agilent, Santa Clara, CA, USA) using a DNA 7500 chip. Next, PCR products were mixed by 383

emulsion PCR and deposited on Picotiter plates. Target sequencing was conducted with a GS Junior 384

Sequencing system (Roche, Basel, Switzerland) according to the manufacturer’s instructions. 385

Analysis of pyrosequencing data. Pyrosequencing results were analysed as follows. Unique 386

barcodes for each amplicon as a standard were sorted from distinctive samples and readings were 387

obtained. Removal of either these non-target sequences including the barcode, linker, and primers or 388

more than two ambiguous nucleotides, low-quality score of less than 25 through Trimmomatic 389

version 0.321 (46), and reads shorter than 300 bp from the original sequencing reads. Additionally, 390

the Bellerophone method was used to discard chimeric sequences, and then a full sequence was 391

compared both in the forward and reverse directions via BLASTN (47). The similarity of each full 392

sequence to valid published type strains was determined using the EzTaxon-e database 393

(http://eztaxon-e.ezbiocloud.net) or uncultured bacterium clone in GenBank database 394

(https://blast.ncbi.nlm.nih.gov). Chao1 estimation at a 3% distance (48) and Shannon diversity index 395

(49) were used to confirm the level of richness and diversity of each sample. Phylogenetic analysis of 396

microbial communities was estimated via the Fast UniFrac (50) combined with principle coordinate 397

analysis. Finally, XOR analysis of CLcommunity program (Chunlab Inc., Seoul, Korea) was used to 398

compare the number of operational taxonomic units among samples. 399

Determining and comparison of soil extract ingredients. To determine the impacts of different 400

ingredients, the rhizosphere soil (Robinia pseudoacacia L.) at Kyonggi University was collected and 401


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prepared via three methods: the first as NSE; the second as TSE1 was autoclaved at 121°C for an 402

hour, allowed cool and settling, next the supernatant was passed through a 0.22-µm filter 403

nitrocellulose membrane using a vacuum pump, followed by a rotary evaporator bath at 40°C to 404

reduce water to 200 mL; the third method as TSE2 was similar to TSE1 except sterilization (at 121°C) 405

was replaced with shaking overnight at room temperature to compare variations in soil components at 406

high temperature. Three samples obtained from the supernatant after soil extraction methods were 407

lyophilized at -54°C to produce 6.36 g powder (NSE), 4.65 g for TSE1, and 4.73 g for TSE2. These 408

powders were stored at 4°C until analysis. For inorganic composition and carbohydrates, soils were 409

prepared and analysed directly from a final volume of 1 L of deionized water without adding any 410

substrates. Experiments were three repeated to ensure accuracy, and chemical data were prepared and 411

analysed individually in triplicate. 412

Sample preparation for simultaneous profiling analysis of amino acids, organic acids, and fatty 413

acids in soil extract. Amino acids (AAs), organic acids (OAs), and fatty acids (FAs) were 414

simultaneously profiled in soil samples as their ethoxycarbonylation (EOC), methoximation (MO), 415

and tert-butyldimethylsilyl (TBDMS) derivatives as described previously (51, 52). Briefly, 2.5 mg 416

soil extract was dissolved in distilled water containing 0.1 μg of norvaline, 3,4-dimethoxybenzoic 417

acid, and pentadecanoic acid as internal standards. The solution pH was adjusted to ≥12 with 5.0 M 418

sodium hydroxide and mixed with dichloromethane (2.0 mL) containing 40 μL ethyl chloroformate, 419

which was converted to the EOC derivative. This was converted to the MO derivative via a reaction 420

with methoxyamine hydrochloride at 60°C for 60 min. The aqueous phase as sequential EOC/MO 421

derivatives was acidified (pH ≤ 2.0 with 10% sulphuric acid), saturated with sodium chloride, and 422

extracted with diethyl ether (3 mL×2). The extracts were evaporated to dryness using a gentle 423

nitrogen stream. Dry residues containing AAs, OAs, and FAs were reacted at 60°C for 30 min with 424

TEA (5 μL), toluene (15 μL), and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (20 μL) to 425


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form the TBDMS derivative. All samples were prepared individually in triplicate and examined 426

directly by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. 427

Inorganic ingredients. The cations Li+, Na

+, Mg

2+, K

+, Ca

2+, and NH4

+ were detected using a 428

Dionex ICS3000 (Sunnyvale, CA, USA) with an Ionpac CS12A column (4×250 mm/Dionex) and 429

detector (suppressed conductivity, CSRS URTRA (4 mm), recycle mode). Oven temperature was 430

30°C, injection volume was 25 μL, samples were eluted with 20 mM methanesulfonic acid at flow 431

rate 1 mL/min, and run time was 20 min according to the IonPac®

CS12Amanual (Thermo Fisher 432

Scientific). A Dionex ICS3000 was also used to detect the anions F-, Cl

-, Br

-, NO2

-, NO3

-, SO4

2-, and 433

PO42-

with standards, and the column was an Ionpac AS20 (4×250 mm, Dionex);the detector was a 434

suppressed conductivity ASRS URTRA II (4mm), recycle mode. Gradient elution was conducted for 435

0–8 min (12 mM KOH), 8–12 min (30 mM KOH), 12–17 min (30 mM KOH), 17–18 min (12 mM 436

KOH), and 18–20 min (12 mM KOH) at a flow rate 1 mL/min.The oven temperature was 30°C and 437

injection volume was 25 μL. All processes were conducted as described in the IonPac®AS20 Anion-438

Exchange Column product manual (Thermo Fisher Scientific). 439

440

ACKNOWLEDGEMENTS 441

This research was supported by the Basic Science Research Program through the National Research 442

Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1A09916982). 443

444

REFERENCES 445

1. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schäberle TF, 446

Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman 447


.asm.org/

Dow

nloaded from

http://aem.asm.org/

KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. 2015. A new antibiotic kills pathogens 448

without detectable resistance. Nature 517:455-459. https://doi.org/10.1038/nature14098. 449

2. Lewis K, Epstein S, D’Onofrio A, Ling LL. 2010. Uncultured microorganisms as a source of 450

secondary metabolites. J Antibiot (Tokyo) 63:468–476. https://doi.org/10.1038/ja.2010.87. 451

3. Doroghazi JR, Albright JC, Goering AW, Ju KS, Haines RR, Tchalukov KA, Labeda DP, Kelleher 452

NL, Metcalf WW. 2014. A roadmap for natural product discovery based on large-scale genomics 453

and metabolomics. Nat Chem Biol 10:963–968. https://doi.org/10.1038/nchembio.1659. 454

4. Milshteyn A, Schneider JS, Brady SF. 2014. Mining the metabiome: Identifying novel natural 455

products from microbial communities. Chem Biol 21:1211–1223. 456

https://doi.org/10.1016/j.chembiol.2014.08.006. 457

5. Gawad C, Koh W, Quake SR. 2016. Single-cell genome sequencing: current state of the science. 458

Nat Rev Genet 17:175–188. https://doi.org/10.1038/nrg.2015.16. 459

6. Heath JR, Ribas A, Mischel PS. 2016. Single-cell analysis tools for drug discovery and 460

development. Nat Rev Drug Discov 15:204–216. https://doi.org/10.1038/nrd.2015.16. 461

7. Vartoukian SR, Palmer RM, Wade WG. 2010. Strategies for culture of ‘unculturable’ bacteria. 462

FEMS Microbiol Lett 309:1–7. https://doi.org/10.1111/j.1574-6968.2010.02000.x. 463

8. Stewart EJ. 2012. Growing unculturable bacteria. J Bacteriol 194:4151–4160. 464

https://doi.org/10.1128/JB.00345-12. 465

9. Ward DM, Weller R, Bateson MM. 1990. 16S rRNA sequences reveal numerous uncultured 466

microorganisms in a natural community. Nature 345:63–65. https://doi.org/10.1038/345063a0 467


.asm.org/

Dow

nloaded from

http://aem.asm.org/

10. Stott MB, Crowe MA, Mountain BW, Smirnova AV, Hou S, Alam M, Dunfield PF. 2008. 468

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand. 469

Environ Microbiol 10:2030-2041. https://doi.org/10.1111/j.1462-2920.2008.01621.x 470

11. Hobbie JE, Hobbie EA. 2013. Microbes in nature are limited by carbon and energy: The 471

starving-survival lifestyle in soil and consequences for estimating microbial rates. Front 472

Microbiol 4:1–11. https://doi.org/10.3389/fmicb.2013.00324. 473

12. Pierra M, Carmona-Martínez AA, Trably E, Godon JJ, Bernet N. 2015. Microbial 474

characterization of anode-respiring bacteria within biofilms developed from cultures previously 475

enriched in dissimilatory metal-reducing bacteria. Bioresour Technol. 195:283–287. 476

https://doi.org/10.1016/j.biortech.2015.07.010. 477

13. Davis KER, Joseph SJ, Peter H, Janssen PH. 2005. Effects of growth medium, inoculum size, 478

and incubation time on culturability and isolation of soil bacteria. Appl Environ Microbiol 479

71:826–834. https://doi.org/10.1128/AEM.71.2.826-834.2005. 480

14. Burmølle M, Johnsen K, Al-Soud WA, Hansen LH, Sørensen SJ. 2009. The presence of 481

embedded bacterial pure cultures in agar plates stimulate the culturability of soil bacteria. J 482

Microbiol Methods 79:166–173. https://doi.org/10.1016/j.mimet.2009.08.006. 483

15. D'Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E, Epstein S, Clardy J, Lewis K. 2010. 484

Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chem Biol 485

17:254–264. https://doi.org/10.1016/j.chembiol.2010.02.010. 486

16. Taylor CB. 1951a. Nature of the factor in soil-extract responsible for bacterial growth-stimulation. 487

Nature 168:115-116. https://doi.org/10.1038/168115a0. 488

17. Taylor CB. 1951b. The nutritinal requirements of the predominant bacterial flora of the soil. J 489

Appl Microbiol 14:101-111. https://doi.org/10.1111/j.1365-2672.1951.tb01999.x. 490


.asm.org/

Dow

nloaded from

http://aem.asm.org/

18. Hamaki T, Suzuki M, Fudou R, Jojima Y, Kajiura T, Tabuchi A, Sen K, Shibai H. 2005. Isolation 491

of novel bacteria and actinomycetes using soil-extract agar medium. J Biosci Bioeng 99:485–492. 492

https://doi.org/10.1263/jbb.99.485. 493

19. Vilain S, Luo Y, Hildreth MB, Brozel VS. 2006. Analysis of the life cycle of the soil saprophyte 494

Bacillus cereus in liquid soil extract and in soil. Appl Environ Microbiol 72:4970–4977. 495

https://doi.org/10.1128/AEM.03076-05. 496

20. Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M. 2002. Improved culturability of soil 497

bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, 498

Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ Microbiol 68:2391-2396. 499

https://doi.org/10.1128/AEM.68.5.2391-2396.2002. 500

21. Schoenborn L, Yates PS, Grinton BE, Hugenholtz P, Janssen PH. 2004. Liquid serial dilution is 501

inferior to solid media for isolation of cultures representative of the phylum-level diversity of 502

soil bacteria. Appl Environ Microbiol 70:4363–4366. https://doi.org/10.1128/AEM.70.7.4363-503

4366.2004. 504

22. Janssen PH. 2008. Accessing uncultured microorganisms: From the environment to organisms 505

and genomes and back. ASM Press: Washington, DC 506

23. Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A, Belanger A, Kanigan T, Lewis K, 507

Epstein SS. 2010. Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial 508

species. Appl Environ Microbiol 76:2445–2450. https://doi.org/10.1128/AEM.01754-09. 509

24. Amanullah A, Otero JM, Mikola M, Hsu A, Zhang J, Aunins J, Schreyer HB, Hope JA, Russo 510

AP. 2010. Novel micro-bioreactor high throughput technology for cell culture process 511

development: Reproducibility and scalability assessment of fed-batch CHO cultures. Biotechnol 512

Bioeng 106:57–67. https://doi.org/10.1002/bit.22664. 513


.asm.org/

Dow

nloaded from

http://aem.asm.org/

25. Zhang H, Liu KK. 2008. Optical tweezers for single cells. J R Soc Interface 5:671–690. 514

https://doi.org/10.1098/rsif.2008.0052. 515

26. Fröhlich J, König H. 2000. New techniques for isolation of single prokaryotic cells. FEMS 516

Microbiol Rev 24:567–572. https://doi.org/10.1111/j.1574-6976.2000.tb00558.x. 517

27. Kaeberlein T, Lewis K, Epstein SS. 2002. Isolating ‘uncultivable’ microorganisms in pure 518

culture in a simulated natural environment. Science 296:1127–1129. 519

https://doi.org/10.1126/science.1070633. 520

28. Bollmann A, Lewis K, Epstein SS. 2007. Incubation of environmental samples in a diffusion 521

chamber increases the diversity of recovered isolates. Appl Environ Microbiol 73:6386–6390. 522

https://doi.org/10.1128/AEM.01309-07. 523

29. Ferrari BC, Binnerup SJ, Gillings M. 2005. Microcolony cultivation on a soil substrate 524

membrane system selects for previously uncultured soil bacteria. Appl Environ Microbiol 525

71:8714–8720. https://doi.org/10.1128/AEM.71.12.8714-8720.2005. 526

30. Pham VHT, Kim J. 2012. Cultivation of unculturable soil bacteria. Trends Biotechnol 30:475–527

484. https://doi.org/10.1016/j.tibtech.2012.05.007. 528

31. Liebeke M, Brözel VS, Hecker M, Lalk M. 2009. Chemical characterization of soil extract as 529

growth media for the ecophysiological study of bacteria. Appl Microbiol Biotechnol 83:161–173. 530

https://doi.org/10.1007/s00253-009-1965-0 531

32. Harris D.C. 2010. Quantitative chemical analysis, 8th

edition. WH Freeman and Company 41 532

Madison Avenue, New York, NY 10010. 533


.asm.org/

Dow

nloaded from

http://aem.asm.org/

33. Friedel JK, Scheller E. 2002. Composition of hydrolysable amino acids in soil organic matter 534

and soil microbial biomass. Soil Biol Biochem 34:315–325. https://doi.org/10.1016/S0038-535

0717(01)00185-7. 536

34. Stenger VA. 1996. Solubilities of various alkali metal and alkaline earth metal compounds in 537

methanol. J Chem Eng Data 41:1111–1113. https://doi.org/10.1021/je960124k. 538

35. Reasoner DJ, Geldreich EE. 1985. A new medium for the enumeration and subculture of 539

bacteria from potable water. Appl Environ Microbiol 49:1–7. 540

36. Davis KE, Joseph SJ, Janssen PH. 2005. Effects of growth medium, inoculum size, and 541

incubation time on culturability and isolation of soil bacteria. Appl Environ Microbiol 71:826-542

834. https://dx.doi.org/10.1128%2FAEM.71.2.826-834.2005. 543

37. Kakumanu ML, Williams MA. 2012. Soil diffusion system enriches the growth of diverse and 544

previously uncultivated bacterial taxa. Soil Sci Soc Am J 76:463–474. 545

https://doi.org/10.2136/sssaj2011.0227. 546

38. Joseph SJ, Hugenholtz P, Sangwan P, Osborne CA, Janssen PH. 2003. Laboratory cultivation of 547

widespread and previously uncultured soil bacteria. Appl Environ Microbiol 69:7210–7215. 548

https://doi.org/10.1128/AEM.69.12.7210-7215.2003. 549

39. Sait M, Davis KE, Janssen PH. 2006. Effect of pH on isolation and distribution of members of 550

subdivision 1 of the phylum Acidobacteria occurring in soil. Appl Environ Microbiol 72:1852-7. 551

https://doi.org/10.1128/AEM.72.3.1852-1857.2006. 552

40. Jones RT, Robeson MS, Lauber CL, Hamady M, Knight R, Fierer N.A. 2009. Comprehensive 553

survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. ISME J 554

3:442-53. https://doi.org/10.1038/ismej.2008.127. 555

41. Kielak AM, Barreto CC, Kowalchuk GA, van Veen JA, Kuramae EE. 2016. The ecology of 556

Acidobacteria: moving beyond genes and genomes. Front Microbiol 7:744. 557


.asm.org/

Dow

nloaded from

http://aem.asm.org/

https://doi.org/10.3389/fmicb.2016.00744.eCollection 2016. 558

42. Tschech A, Pfennig N. 1984. Growth yield increase linked to caffeate reduction in 559

Acetobacterium woodii. Arch Microbiol 137:163–167. 560

43. Widdel F, Kohring GW, Mayer F. 1983. Studies on dissimilatory sulfate-reducing bacteria that 561

decompose fatty acids. Arch Microbiol 134:286–294. 562

44. Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, Goulding D, Lawley 563

TD. 2016. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive 564

sporulation. Nature 533:543–546. https://doi.org/10.1038/nature17645. 565

45.Yarza P, Richter M, Peplies J, Euzeby J, Amann R, Schleifer KH, Ludwig W, Glöckner FO, 566

Rosselló-Móra R. 2008. The all-species living tree project: A 16S rRNA-based phylogenetic tree 567

of all sequenced type strains. Syst Appl Microbiol 31:241–250. 568

https://doi.org/10.1016/j.syapm.2008.07.001. 569

46. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence 570

data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170. 571

47. Huber T, Faulkner G, Hugenholtz P. 2004. Bellerophon: A program to detect chimeric sequences 572

in multiple sequence alignments. Bioinformatics 20:2317–2319. 573

https://doi.org/10.1093/bioinformatics/bth226 574

48. Chao A. 1984. Non-parametric estimation of the number of classes in a population. Scand J Stat 575

11:265–270. 576

49. Ludwig JA, Reynolds JF. 1988. Statistical ecology: A primer on methods and computing. Wiley, 577

New York. 578


.asm.org/

Dow

nloaded from

http://aem.asm.org/

50. Hamady M, Lozupone C, Knight R. 2010. Fast UniFrac: facilitating high-throughput 579

phylogenetic analyses of microbial communities including analysis of pyrosequencing and 580

PhyloChip data. ISME J 4:17–27. https://doi.org/10.1038/ismej.2009.97. 581

51. Paik MJ, Kim KR. 2004. Sequential ethoxycarbonylation, methoximation and tert-582

butyldimethylsilylation for simultaneous determination of amino acids and carboxylic acids by 583

dual-column gas chromatography. J Chromatogr A 1034:13–23. 584

https://doi.org/10.1016/j.chroma.2004.02.032. 585

52. Paik MJ, Lee HJ, Kim KR. 2005. Simultaneous retention index analysis of urinary amino acids 586

and carboxylic acids for graphic recognition of abnormal state. J Chromatogr B Anal Technol 587

Biomed Life Sci 821:94–104. https://doi.org/10.1016/j.jchromb.2005.04.011. 588

589

590

591

592

593

594

595

596

597

598

599

600

601

602


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Figure legends 603

FIG 1 Proportion of uncultured/cultured bacteria and new taxonomic candidates isolated from 604

soil samples by using ISEM (newly developed method), traditional soil extract culture medium 605

and modified transwell culture method. 606

(a) Percentage of uncultured species (126 individual species among the total 258; 11 of 243; 39 of 607

252, respectively). Points of significance compared to two paired samples for means: t-test as 608

indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). (b) Percentage of novel genera 609

candidates (14/258; 0/243; 1/252, respectively). 610

(c) Proportion of novel bacterial species and known species among 126, 11, and 39 previously 611

uncultured species via the investigated methods, respectively. Error bars indicate standard deviation. 612

613

FIG 2 Abundance of bacteria in soil samples determined by pyrosequencing. 614

(a) The abundance determined at a 1% ETC cut-off. (b) Distribution and relative abundance of 615

identified species: approximately 8143, 8314 and 6836 individual species detected in A, B and S soil 616

samples, respectively; scale of x-axis: log2. 617

618

FIG 3 Network topology tree for microbial cultivation based on full-length 16S rRNA gene 619

sequencing. Only bootstrap support values ≥50% are shown in the tree. Accession numbers for 16S 620

rRNA gene sequences revealed close relationships with previously uncultured bacteria, and those 621

cultured as new species and novel genera are shown in the tree. 622

623

624

625

626


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Table 1. Components of soil extracts prepared in three different ways 627

Ingredients (mgL-1

) Soil extraction methods

NSE TSE1 TSE2 NSE TSE1 TSE2

1. Amino acid (Continued)

Alanine 0.47 0.10 0.18 Erucic acid nd 0.071 0.06

Glycine 0.32 0.16 0.40 Behenic acid 1.80 nd nd

Valine 0.09 nd nd Nervonic acid 0.07 nd nd

Leucine 0.47 0.05 0.06 Lignoceric acid 2.60 0.093 0.08

Isoleucine 0.20 0.03 0.03 Cerotic acid 0.09 0.182 0.14

Proline 0.21 0.63 0.69 Total 13.10 0.79 0.79

γ-Aminobutric acid 0.76 0.62 0.69 3. Organic acid

Pipecolic acid 0.09 nd nd 3-Hydroxybutyric acid 0.96 2.07 1.88

Pyroglutamic acid 0.85 0.23 0.32 Pyruvic acid 0.40 4.31 5.75

Serine 0.38 nd nd Acetoacetic acid 1.47 28.75 27.25

Threonine 1.38 nd nd Lactic acid 9.42 nd nd

Phenylalanine 0.22 0.03 0.04 Glycolic acid 8.03 nd nd

Cysteine 0.22 0.12 0.29 2-Hydroxybutyric acid 0.09 nd nd

Aspartic acid 0.27 0.08 0.31 Malonic acid 0.85 1.21 1.75

Glutamic acid 0.55 0.40 0.39 Succinic acid 1.43 2.18 1.78

Asparagine 0.57 0.41 0.52 Fumaric acid 0.05 nd nd

Ornithine 0.77 0.46 0.45 Oxaloacetic acid 0.17 18.35 44.84

Glutamine 0.55 0.67 0.66 α-Ketoglutaric acid 0.11 nd nd

Lysine 0.12 0.25 0.24 Malic acid 0.73 3.32 3.33

Tyrosine 9.29 0.10 0.09 2-Hydroxyglutaric acid 0.50 0.93 0.72

Tryptophane 0.72 0.50 0.51 Cis-Aconitic acid 0.27 0.28 0.33

Total 18.50 4.84 5.87 Citric acid 0.46 0.81 0.83

2. Fatty acid

Isocitric acid 0.20 0.48 0.54

Decanoic acid 0.47 nd nd Total 25.14 62.69 89.00

Lauric acid 0.48 nd nd 4. Inorganic compounds

Myristoleic acid 0.15 nd nd Na+ 58.87 83.65 85.05

Myristic acid 1.18 0.07 0.08 NH4+ 6.93 17.76 19.45

Isopentadecylic acid 0.09 nd nd Mg2- 29.24 27.52 27.95

Isopalmitic acid 0.11 nd nd K+ 15.98 58.95 61.32

Palmitoleic acid 0.35 0.09 0.08 Ca2+ 87.93 114.59 119.49

Palmitic acid 1.29 0.12 0.11 Cl- 43.34 120.21 116.32

Linoleic acid 0.40 nd nd NO3- 489.67 398.43 384.51

Oleic acid 0.40 nd nd SO42- 5.41 130.15 128.64

Stearic acid 2.54 0.09 0.17 PO42- 10.66 4.74 4.18

Arachidic acid 1.08 0.07 0.07 Total 748.03 956.00 946.91

Method NSE was developed in this study; method TSE1 involves autoclaving at 121°C for 1 h; 628

method TSE2 does not involve autoclaving; nd: not detected. 629


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Table 2. Number of bacterial isolates used to evaluate new soil extract (NSE) and other media 630

Soil

samples

Species level Genus level Family level

Total Previously

uncultured

novel species

Previously

uncultured

known species

Previously

uncultured

novel genus

Previously

cultured novel

genus

Previously

uncultured

novel family

A 24 12 2 3 - 41

B 28 9 3 1 - 41

S 34 10 3 1 1 49

Total 86 31 8 5 1 131

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653


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654

655

656

(Fig. 1) 657

658


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659

660

661

662

(Fig. 2) 663

664

665

666

667

668


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669

(Fig. 3) 670

671

672


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effective soil extraction method for cultivating ...oct 05, 2018 · 62 diffusion chamber s (27,...

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