Isolation of unculturable soil bacteria and finding of their antibiotic candidates

5 1.3.5 New developed method for isolation of previously unculturable soil bacteria .... By combination with several active components we chose, a new medium, called ISEM Intentive Soil

Dissertation for Degree of Doctor

Isolation Of Unculturable Soil Bacteria And Finding Of Their Antibiotic Candidates

by Nguyen Manh Tuan

Department of Life Science

Graduate School Kyonggi University

(2017)

Isolation Of Unculturable Soil Bacteria

And Finding Of Their Antibiotic Candidates

by Nguyen Manh Tuan

A dissertation submitted to the faculty of the Graduate School in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Department of Life Science

Graduate School Kyonggi University December 2017

Approved the dissertation of Nguyen Manh Tuan

as qualified for the Degree of Doctor

Contents

List of Tables ……… vii

List of Figures ……… viii

Acknowledgement x

Abstract xi

Chapter I Modification Of Soil Extraction Method To Cultuvate Unculturable Soil Bacteria 1

1.1 Introduction 1

1.2 Aims to Study 3

1.3 Material and method 4

1.3.1 Information of soil property used for the method 4

1.3.2 Preparing new soil extraction (NSE) in 1 litre medium 4

1.3.3 Medium for isolation of soil bacteria 5

1.3.4 Soil sampling site and preparation of soil sample 5

1.3.5 New developed method for isolation of previously unculturable soil bacteria 6

1.3.6 Traditional soil extract method 6

1.3.7 Modified transwell culture system method 7

1.3.8 Bacterial genomic extraction and small-subunit rRNA (SSU rRNA) amplification 7

1.3.9 Identify 16S rRNA gene sequences and accession numbers of 16S rRNA gene sequenses 8

1.3.10 Parameter tools/software programs 8

1.3.11 Role of complex isolate medium compositions to grow previously unculturable soil bacteria 9

1.3.12 DNA extract from soil 9

1.3.13 PCR amplification and pyrosequencing 10

1.3.14 Analysis of pyrosequencing data 10

1.3.15 Determining and comparison of soil extract ingredients investigated 11

1.3.16 Sample preparation for simultaneous profiling analysis of amino acids, organic acids and fatty acids in soil extract 12

1.3.17 Gas chromatography-mass spectrometry 12

1.3.18 Inorganic ingredients 13

1.4 Results 14

1.4.1 Shortage of previous methods and new method development 14

1.4.2 Method validation through comparative analysis of organic/inorganic compounds 19

1.4.3 Method validation through cultivability of unculturable bacteria 21

1.4.4 Method validation through isolation rate of unculturable or new taxonomic bacteria 24

1.4.5 Method validation through taxonomic analysis 29

1.5 Discussion 33

Chapter II Taxonomical Characterization Of Novel Taxa 37

2.1 A Rapid And Simple Method For Identifying Bacterial Polar Lipid Components In Wet Biomass 37

2.1.1 Introduction 37

2.1.2 Aim to study 38

2.1.3 Material and method 39

2.1.3.1 Organisms and growth conditions 39

2.1.3.2 Polar lipids extraction using the improved method 40

2.1.3.3 Polar lipids extraction using the the method of Minnikin et al (1984) 42

2.1.3.4 Polar lipids analysis 42

2.1.4 Results and Discussions 43

2.1.4.1 Polar lipid profiles of Microbacterium lacticum compared 43

2.1.4.2 Polar lipid profiles of Rhodococcus koreensis compared 44

2.1.4.3 Polar lipid profiles of Pseudomonas aeruginosa compared 48

2.1.4.4 Polar lipid profiles of Streptomyces longwoodensis compared 48

2.1.4.5 Polar lipid profiles of Novosphingobium capsulatum compared 48

2.2 Description of Flavobacterium fulvus sp nov., Flavobacterium pedocola sp nov., and Flavobacterium humicola sp nov., novel members belong to the family Flaviobacteriaceae, isolated from soil 51

2.2.1 Introduction 51

2.2.2 Materials and method 52

2.2.2.1 Sample and isolation of strains 52

2.2.2.2 Phenotypic characterisation 53

2.2.2.3 Genomic DNA isolate 54

2.2.2.4 Phylogenetic analyses 55

2.2.2.5 DNA–DNA hybridization (DDH) 56

2.2.2.6 G+C mol content 58

2.2.2.7 Determining fatty acids components 58

2.2.2.8 Components of menaquinone and polar lipids 59

2.2.3 Results and discussion 61

2.2.3.1 Results of comparision between new isolates and their relative species 61

2.2.3.2 Description of novel isolates of genus Flavobacterium 72

Chapter III Isolation And Characterization Of Novel Antibiotic Candidates 78

3.1 General Information 78

3.1.1 Introduction 78

3.1.2 General background 79

3.1.2.1 Term of antibiotic 79

3.1.2.2 Antibiotic history 80

3.1.2.3 How antibiotics work to kill bacteria 82

3.1.2.4 Current problems with known drugs 86

3.1.2.5 Mechanisms of drug resistance 88

3.1.3 Goal of this study 91

3.2 Screening of Antibiotic-Producing Bacteria Isolated From Soils 92

3.2.1 Materials and methods 92

3.2.1.1 Soil sample 92

3.2.1.2 Isolation of microorganisms 92

3.2.1.3 For members of Actinobacteria 92

3.2.1.4 Modified culture method 93

3.2.2 Screening antibiotic-producing bacteria 93

3.2.2.1 Microorganisms used to test 93

3.2.2.2 Assay of antimicrooganisms activities 94

3.2.2.3 Identification of 16S rRNA gene analysis 94

3.2.3 Results and discussion 94

3.2.3.1 Isolation of anti-microoganisms 94

3.2.3.2 Finding of the best strain to extract antibiotics 101

3.3 Characteristic Of Antibiotic Derived A Novel Species of Genus Sphingobium 104

3.3.1 Material and method 104

3.3.1.1 Fermentation and extraction of antibiotic 104

3.3.1.2 P uri fi cati on of ant ibioti c ext raction b y s i li ca gel colum n chromatography 105

3.3.1.3 Collection of individual compound by Prep-HPLC 105

3.3.1.4 Minimum inhibitory concentration (MIC) test 107

3.3.2 Results and discussion 108

3.3.2.1 Major descriptions of strain UCM-25 108

3.3.2.2 Extraction, purification, separation of antibiotics 110

3.3.2.3 Thin-Layer Chromatography (TLC) assay 110

3.3.2.4 Collecting individual compound 112

3.3.2.5 Minimal Inhibitory Concentration (M.I.C) test 116

3.4 An Antibiotic Producing Streptomyces Species Kills Drug Resistant Pathogens 118

3.4.1 Material and methods 118

3.4.2 Results and Discussion 119

3.4.2.1 Major information for strain T1317-0309 119

3.4.2.2 Screening of fractions collected by Prep-HPLC 126

3.4.2.3 Minimal Inhibitory Concentration (M.I.C) test 127

Chapter IV General Conclusion 131

4.1 Modification Of Soil Extraction Method 131

4.2 Taxonomical Characterization Of Novel Taxa 132

4.3 Isolation And Characterization Of Novel Antibiotic Candidates 133

Reference 135

Appendix 167

Abstract in Korean 202

List of Table

Table 1 Brief summary comparision of results by methods determined 15 Table 2 Li st of soil extract component s defined a new way and general

approaches 17 Table 3 List of bacterial isolates used for evaluating new soil extract (NSE) 21 Table 4 Comparision of convenient methods for extracting bacterial polar lipids 49 Table 5 Comparision of distinct phenotypic characteristics between four isolate strains

and t heir t ype st rai ns of t he most rel at ed speci es of t he genus

Flavobacterium 63

Table 6 Cellular fatty acid composition (%) of four isolate strains and the type strains

of related species of the genus Flavobacterium 67

Table 7 Antibiotic pathways to inhibit microoganisms 83 Table 8 List of isolating bacterial soils that showed target microoganisms 95 Table 9 Antibiotic extract activity of inhibition of target microoganisms using paper

disc 102 Table 10 Activities of each fraction that are collected by Prep-HPLC, which inhibit

target pathogens (Inhibition zone in mm) 113 Table 11 Minimal inhibitory concentration (MIC in μg ml-1) values of Fr-1 and Fr-

2 117

Table 12 Cultural characteristics of Streptomyces sp T1317-0309 on various media

tested at 28oC for 21 days 120 Table 13 Result of identification of strain T1317-0309 based on 16S rRNA gene

sequencing using the eztaxon-e 123

Table 14 Antibacterial activities of the Fr-5 collected from Streptomyces sp

T1317-0309 129

List of Figure

Fi g 1 An impact of various factors on cultivation of uncultured soil bacteria 20

Fi g 2 Achievement of phenotypic diversity of species isolated through soil samples 23

Fig 3 Abundance of bacteria in the soil samples defined by pyrosequencing and a new method 28

Fig 4 Identifing phenotypic diversity of isolates through analysis of 16S rRNA gene sequence 32

Fig 5 Brief proceduces for extraction and purification of polar lipids 41

Fig 6 Polar lipid profiles determined using the two methods 46

Fig 7 Neighbour-joiningtree, based on 16S rRNA gene sequencing, showing the position of strains UCM-R15T, UCM-R21, UCM-R36T and UCM-46T , which are closely related species of the genus Flavobacterium 62

Fig 8 Polar lipid profile of strains UCM-R15T, UCM-R36T, and UCM-46T after two-dimensional chromatography and staining with 5 % molybdatophosphoric acid at 180 oC for 30 min 69

Fig 9 Scanning electron micrograph showing the cells morphology of strains UCM-R15T, UCM-R36T, and UCM-46T grown on R2A medium Bars, 0.5 µm 71

Fig 10 Fleming and his penicillin discovery on the original agar plate 81

Fig 11 Antibiotics and its antibiotic resistance reported 87

Fig 12 Machanism of antibiotic resistance through pathway of efflux pump 89

Fig 13 Distribution (number among total 44 strains) of the strains showing activities against the targets 99

Fig 14 Relative distribution of the active strains in the level of bacterial genus, showing antimicrobial and antifugal activities against the target test 101

Fig 15 Main steps for extracting antibiotic and further analysis 106

Fig 16 Neighbor-joining phylogeny of strain UCM-25 based on near full-length 16S

rRNA gene sequences 109

Fig 17 Thin layer chromatography for compound analysis 111

Fig 18 Collecting individual fraction via a Prep-HPLC system 112

Fig 19 Inhibition zone of these fractions collected killing microoganisms test 114

Fig 20 Thin layer chromatography for compound analysis of T1317-0309 121

Fig 21 A neighbour-joining phylogenetic tree of strain T1317-0309 with related species in genus Streptomyces 125

Fig 22 Profile of secondar y metabolism extracted f rom the fermentation supernatant 126

Fig 23 Zone of inhibtion of these fractions collected showing activity to kill microoganisms tested 126

Acknowledgement

First, I’m really pleased to Prof Jaisoo Kim, who gave me an especial opportunity to study and research in South Korea, as well as great supports for researches and my family during living in Republic of Korea

I would like to thank you all of Professors at the Department of Life Science, Kyonggi University: Prof Byung-Sun Yoon, Prof Sang Seob Lee, Prof Dong-Soo Kong, Prof Byung-Sun Yoo, Prof Ok-Min Lee, and Prof Seong-Ho Ghil for these suggestions to improve my researches I would be glad to Dr Seung Jae Won, who suggested solving my problems during study, as well as his interest in my family life in Korea I am also grateful

to the doctoral communitte members: Prof Seung-Woo Jeong (Kunsan National University) and Prof Song-Bae Kim (Seoul National University) for their valuable suggestions to improve my dissertation An especial thank you to Prof Seung-Woon Myung (Department

of Chemistry, Kyonggi University) and Prof Man-Jeong Paik (College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National University) for their support during my study

To complete my doctoral degree, I received special encouragement from my family and friends that helped me through difficult time, without them my life is certainly very difficult I’m thankful to all of lab members, classmates and Korean students always help

me in study and research

December 2017, Republic of Korea

Nguyen Manh Tuan, Ph.D Student

Abstract

Surely, there are so many unknown metabolic potentials of uncultured soil bacteria A lot of methods have been established by various approaches to explore unculturable soil bacteria But all of these methods have a common obstacle which is limitation of the solid medium to obtain large groups of not-yet cultivated species This study is simply designed

to discover more unculturable soil bacteria in aerobic conditions through a new soil extract (NSE) derived from soil, the "bacterial home" NSE devloped in this study is different from the existing methods since this method uses a mixuture of water and methanol instead of water only to extract soil chemical compounds Moreover, this new method makes easier

to perform and reduces working hours than the existing cultivation methods for unculturable soil bacteria, and also this method can be working well without enrichment and even combining with traditional media

NSE contains essential ingredients required for soil microorganisms including chemoorganotrophic, chemolithotrophic, and heterotrophic bacteria based on our anlytical results By combination with several active components we chose, a new medium, called ISEM (Intentive Soil Extraction Medium for unculturable soil bacteria), was created and showed successful cultivation of bacterial strains involved in 7 phyla: approximately 49% and 55% of the total isolates (n=258) was belonging to the previously uncultured bacteria and new taxonomic candidates including species, genus and even family level, repectively Therefore, we expect that this discovery could open up a new avenue to explore unknown mechanisms and new knowledge through sussessful cultivation of a number of bacteria hidden in various soil environments

Although the use of freeze-dried cells to determine polar lipid components have successfully been used, several methods have been developed based on wet cells However, wet cell methods showed limitation with narrow scope in applications due to non-polarity

of extracted phospholipids In this study, wet biomasses of five species of Gram-positive

and Gam-negative bacteria including Microbacterium lacticum, Rhodococcus koreensis,

Pseudomonas aeruginosa, Streptomyces longwoodensis and Novosphingobium capsulatum

were used to analyze lipid components, revealing equal quality compared to the most applied procedure with wet biomasses except a few minor spots Moreover, the improved method also ensures simplicity of lipid extraction

Four Gram-staining negative, non-endospore-forming, non- non-motile strains were found soil in Yuseong-gu, Daejeon, South Korea Through analysis of these 16S rRNA gene

sequences suggested they should be belonged the genus Flavobacterium; strain UCM-R15T

and UCM-R21 most closely related to F enshiense DK69T (97.38-97.46 %), F

saliperosum S13T (96.28-96.35 %), F suncheonense GH29-5T (96.28-96.35 %), F

limnosediminis JC2902T (96.14-96.22 %), F cauense R2A-7T (95.17-95.67 %), and F

columnare IFO 15943T (94-88-95.25 %); for strain UCM-R36T showed 97.53 % to F

suncheonense GH29-5T, 96.57 % to F enshiense DK69T, 96.43 % to F limnosediminis

JC2902T, 96.29 % to F saliperosum S13T, 95.74 % to F cauense R2A-7T; UCM-46T shared

closely related to F suncheonense GH29-5T (98.27 %), F cauense R2A-7T (96.06 %), F

enshiense DK69T (95.5 %), F saliperosum S13T (95.44 %), and F limnosediminis JC2902T

(95.44 %) All four strain cannot reduce/digest nitrate or urea Major menaquinone MK-6 was detected in all isolate strains Fatty acid as C15:0 iso, C17:0 iso 3-OH, C15:1 iso G, summed feature 9 (C17:1 iso ω7c and/or C16:0 10-methyl, C15:0 iso 3-OH, C15:0 anteiso, and C16:0 iso were found in all the strains Phosphatidylethanolamine was found in three strains as major polar lipid; phosphatidylserine in strain UCM-R15T, UCM-R36T, not UCM-46T; and phosphatidylmonomethylethanolamine was only occurred in strain UCM-R15T; moreover unidentified polars (L2 or L3) were appeared at 160-180 °C after 15 min, while these remaining spots were fast appeared Genomic DNA G+C content of strains UCM-R15T, UCM-R21, UCM-R36T, UCM-46T was 35.3, 36.2, 39.0, and 38.4 mol%, respectively The results of DNA-DNA hybridizations between our strains and comparison strains were lower than 70 % limit off; combining with their physiological and biochemical characteristics,

we suggest that three strains represent novel members within the genus Flavobacterium,

for which the name Flavobacterium fulvus sp nov is proposed, the type strain is

UCM-R15T (=KACC 18666T =NBRC 111764T); Flavobacterium pedocola sp nov is proposed,

the type strain is UCM-R36T (=KACC 18668T =NBRC 111765T); Flavobacterium

humicola sp nov is proposed, the type strain is UCM-46T (=KACC 18575T =NBRC

111657T)

More than 100 soil samples were collected from many forests in Korea during 2013

We found 43 strains from more than 3,000 isolates, which showed antimicrobial activity

against Paenibacillus larvae as an agent of American Foulbrood (AFB) as well as common microbial human pathogens such as Bacillus subtilis, Staphylococcus aureus, Escherichia

coli, Candida albicans and Aspergillus niger On the basis of 16S rRNA gene sequence

analysis, they belong to 12 genera (mostly actinobacteria): Streptomyces (28), Kitasospora (4), Bacillus (2), Streptacidiphilus, Amycolatopsis, Paenibacillus, Promicromonospora,

Rhodococcus, Pseudomonas, Burkholderia, Undibacterium and Actinomadura Among

them, Streptomyces species are dominant (28/43 strains, 65.12%) Burkholderia and

Undibacterium spp were reported first as antimicrobial agents, especially against

Gram-positive bacteria

Among them, our work focused on strain UCM-25 that is belong to genus

Sphingobium, which is no antibiotic-producing report in this genus so far Strain UCM-25T

was classified as a novel species of genus Sphingobium, for which name Sphingobium

aromaticivorans Fr-1 and Fr-2 derived from this new species as prep-HPLC fractions of

culture extractant can inhibit Bacillus subtilis KEMB 51201-001 (16; 64 μg ml-1, MIC

respectively), Staphylococcus aureus KEMB 4659 (16; 64 μg ml-1), Pseudomonas

aeruginosa KACC 10185 (16; 128 μg ml-1), Escherichia coli KEMB 212-234 (32; 128 μg

ml-1), Paenibacillus larvae KACC 14031 (16; 32 μg ml-1), Streptococcus agalactiae

CCARM 4504 (64; 128 μg ml-1), Streptococcus pyogenes CCARM 4520 (32; 256 μg ml-1),

Enterococcus faecalis CCARM 5168 (128; 128 μg ml-1), and Enterococcus faecalis

CCARM 5171 (64; 128 μg ml-1)

Strain T1317-0309 was isolated from a soil sample collected in South Korea, showing strong activity against some Gram-positive bacteria, but not Gram-negative bacteria, yeast and fungus Based on the analysis of 16S rRNA gene sequence, the strain was involved in

genus Streptomyces Fr-5 was collected and purified among five fractions through a

prep-HPLC and detected at 280 nm Fr-5 was not able to be dissolved in water, but well in methanol, ethanol, ethyl acetate Based on TLC analysis and order of prep-HPLC peaks, Fr-5 seemed to be non-polar property in its molecule The compound from Fr-5 inhibited Gram-positive bacteria including antibiotic resistant-bacteria with its MIC values (range of

0.125~4 μg per ml), such as Bacillus subtilis KEMB 51201-001 (0.5 μg per ml), Bacillus

anthracis KEMB 211-146 (0.5), Staphylococcus aureus KEMB 4659 (1), Paenibacillus larvae KACC 14031 (0.125), Streptococcus agalactiae CCARM 4504 (0.5), Streptococcus pyogenes CCARM 4520 (0.5), Enterococcus faecalis CCARM 5168 (0.5), Enterococcus faecalis CCARM 5171 (0.5), Enterococci CCARM 5025 (0.5), Enterococci CCARM 5024

(0.5), Staphylococcus epidermidis KACC 13234 (1), methicillin-resistant Staphylococcus

aureus CCARM 3095 (0.5), methicillin-resistant Staphylococcus aureus CCARM 3192 (4),

methicillin-resistant Staphylococcus aureus CCARM 3155 (1.25), methicillin-resistant

Staphylococcus aureus CCARM 3710 (1.25), methicillin-resistant Staphylococcus aureus

CCARM 3A041 (1.25), and Propionibacterium acnes subsp acnes KCTC 3314 (0.25)

These results indicates that the antibiotic candidate isolated and purified in this study can

be useful as an antibiotic against multi-antibiotic resistant pathogens because of its lower MIC values against many antibiotic-resistant pathogens than ones of commercial antibiotics

Chapter I Modification Of Soil Extraction Method

To Cultuvate Unculturable Soil Bacteria

1.1 Introduction

Since the method of solid medium culture have been established, currently we have

benefited greatly from secondary metabolites of microorganism, which were cultured in

the laboratories For example: the first antibiotic as named Penicillin derived from

Penicillium in 1928 (Fleming, 1929), after that, many new antibiotics were found, which

support to prevent the disease effectively

Through molecular tools revealed that prokaryotic species are much more diverse and

abundant in soil, they contain a lot of unexplored potential metabolites (Doroghazi et al.,

2014; Lewis et al., 2010; Ling et al., 2015; Milshteyn et al., 2014) Lack of complex

factors/conditions in laboratory conditions were given for these bacteria that have not been

isolated yet (Stewart, 2012; Vartoukian et al., 2010) Since, the term of "unculturable

bacteria" published, passing nearly a decade, several methods have been developed They

were things to be become more simply seeming to "bring bacteria" from nature to

laboratory by creating artificial media/conditions similar to natural environment through

modification of growth media's components (Vartoukian et al., 2010), modification of

growth conditions (Stevenson et al., 2004), using inorganic compounds or metals as

electrodes (Hobbie & Hobbie, 2013; Pierra et al., 2015), various factors (Davis et al., 2005)

and helper bacteria (Burmølle et al., 2009; D'Onofrio et al., 2010)

Furthermore, sophisticated techniques were also developed as iChip for an in situ

cultivation (Nichols et al., 2010), micro-bioreactor (Amanullah et al., 2010), optical

tweezers (Zhang and Liu, 2008), or micro-manipulator (Fröhlich and König, 2000), etc.,

allowing to pick up individual cells in soil samples Hence a new artificial advanced

medium for maintaining these cells is requested Although the most power tool developed recently allows to know any metabolic diversity of microorganisms without isolated species by the single-cell sequencing, however, it still has many unresolved challenges in the near future due to limitation of cultivation (Gawad et al., 2016; Heath et al., 2016; Yuan

et al., 2017; Liu & Trapnell, 2016; Vallejos et al., 2017; Mocali & Benedetti, 2010; Daniel, 2005; Medema & Fischbach, 2015)

Although scientists can be successful to enrich slow-growing microorganisms through using diffusion chamber (Bollmann et al., 2007; Kaeberlein et al., 2002) or soil substrate membrane (Ferrari et al., 2005), however, the number of bacteria have been isolated on agar plates stopped at restriction level So far, we have only known less than 1% of the total soil bacteria Without cultivation, there is no way to detect and identify novel organisms,

to gain any phenotypic and functional information, and to determine the functions of unknown genes (Pham and Kim, 2012)

A big question is given, what is the most important factor to cultivate uncultured bacteria? Which is still lack of artificial media in laboratory? In this study, we try to answer that question, with a simple culture method developed without specific supplies through successful new bacterial isolates

Definition of uncultured bacteria and major reasons for that:

There are too many bacteria can’t grow under laboratories, especially on agar plates using current commercial media for example nutrient, R2A, trypticase soy agar, Luria-Bertani, etc These bacteria are called uncultured bacteria or not-yet-cultivated bacteria

By the above mentioned reasons, factors such as temperature, pH, nutrient specificity and oxygen levels have been shown to affect isolation (Kopke et al., 2005)

Relation between bateriocins, antibacterial agents and species living together, they are present in natural habitats, but may be absent in artificial media (Tamaki et al., 2005)

Lack of knowledge about bacterial biofilms, as well as their interactions among species (Hall-Stoodley et al., 2004; Stoodley et al., 2002; Marsh, 2005; Ten Cate, 2006; Belenguer et al., 2006; Mikx and Van der Hoeven, 1975; Davey, 2008)

Structure of the bacterial community and survival, which maybe relative to sensing mechanism (De Kievit et al., 2001; Konaklieva and Plotkin, 2006; Ten Cate, 2006) Shortage of molecular signals, which are only present in natural environments, have resisted culture in laboratories (Lewis, 2007, Nichols et al., 2008; Barcina et al., 1990; Colwell, 2000)

quorum-Together with other reasons leading to scientists have been unable to reach the group

1.3 Material and method

1.3.1 Information of soil property used for the method

Rhizosphere soil was collected at Kyonggi University (154-42 Gwanggyosan-ro, dong, Yeongtong-gu, Suwon, Gyeonggi-do, South Korea; 37o30'04''75'''N, 127o03'58''26'''E) during June 2016, where has diverse vegetation Fresh soil was dried at room temperature for 24 h, then discarded any plant debris, gravel and rock by a sieve with ahole of 0.2 mm Next, the soil was dried at 110oC for 24 h, then cool it at room temperature to determine physical properties of soil Soil contain approximately 78% sand, 17% silt and 5% clay pH

Iui-of soil ~5.7 that was defined directly from fresh soil

1.3.2 Preparing new soil extraction (NSE) in 1 litre medium

Approximately, 1000 g of the dry soil sieve at room temperature was divided into two equal parts (500 g soil for each) in a 2L flask, then was mixed with 1.3 litre of 80% methanol (#494291, grade methanol, Sigma Aldrich) that is created in deionized water, incubated overnight at room temperature (should be below than 25oC), tranfferred supernatant to a new flask Secondly, 1.3 liter of 80% methanol was added in the soil, mixed well for an hour Combining the two supernatant, filtered through WhatmanTM paper (#1001-150, 

150mm, GE healthcare UK) Methanol was evaporated with N2 (~ 40°C) The NSE was adjusted the final volume of 200ml deionized water, was sterilized through a 0.22 µm filter membranes nitrocellulose (#GSWP04700, Merck Millipore Ltd.) using a vacuum pump The pure supernatant was passed through via a 0.2 µm PVDF membrane (#WHA67791302, Sigma Aldrich) if necessary to make sure none of bacteria in the soil extraction, the pure NSE was storedin dark schott duran bottle at 4oC using within one week

1.3.3 Medium for isolation of soil bacteria

Medium component including 0.23 g KH2PO4, 0.23 g K2HPO4, 0.23 g MgSO4•7H2O, 0.33 g NH4NO3, 0.25 g NaHCO3 as a group of mineral salts, 15 g agar in 1 litre water, sterilized at 121oC for 15 min Then, adding final volume of 5 mg of each various D-amino acids (D-valine, D-methionine, D-leucine, D-phenylalanine, D-threonine and D-tryptophan), 1 ml vitamin B (vitamin stock solution containing 50 mg each thiamine

hydrochloride, riboflavin, niacin, pyridoxine HCl, inositol, calcium pantothenate and

β-aminobenzoic acid and 25 mg biotin in 100 ml distilled water, sterilized through a 0.2 µm syringe filter, which was kept at 4oC in dark schott duran using within one month), 0.2 litre

of NSE, 2 mL of selenite-tungstate solution (Tschech & Pfennig, 1984), and 2 mL of trace element SL-10 (Widdel et al., 1983) The final volume of various mixed components is 1L,

pH is ranged in 6.8±0.2 Complex medium component was called Intentive Soil Extraction Medium (ISEM) The medium should be prepared freshly and use within a week In this study, we used petri dish with Φ 150 x 20 mm (SPL Life Science Co., Ltd, South Korea) The bigger dish allows for increased separation of colonies at the high dilution concentrations during isolation

1.3.4 Soil sampling site and preparation of soil sample

Three soil samples were taken in South Korea during June 2016, included in Ansan as named sample A (Sangnok-gu, Ansan-si, Suwon, Gyeonggi-do; 37o31'56''N, 126o86'65''E);

in Buksu-dong/sample B (Buksu-dong, Paldal-gu, Suwon, Gyeonggi-do; 37o28'59''N,

127o01'70''E); and in Seoul/sample S (Itaewon-ro, Yongsan-gu, Seoul; 37o53'51''N,

126o97'44''E) For each sample, approximately 10 g soil atten different locations in 150 meters in diameterwere collected and mixed well together The sample was passed through 0.1 mm sieve, isolated/enriched directly for the three methods Taking the 25 g sieved soil sample was mixed well in 250 mL sterile saline (0.9% NaCl, w/v) stirring within 15 min, allowed to sediment Then, 100 µl of suspension was transferred into the transwell insert

for the modified method that was shown in detail the next part Both of remaining the methods, six folds were preprared in the 0.9% NaCl, were ready for next step

1.3.5 New developed method for isolation of previously unculturable soil bacteria

About 100 µL of each the dilution (10-2 to 10-6) was spread onto three agar plates of ISEM (to ensure uniformly distributed suspension on the surface of the medium, 100 μL of each the dilution plus 100 μL of ISEM liquid is recommended) These agar plates were incubated at 25oC for 6 weeks A few colonies were appeared after one week incubation The number of directly visual colonies was increased after 2 weeks, tiny colonies were picked up, streaking onto fresh ISEM until getting pure morphological colonies Cells on the fresh ISEM usually require at least a week to grow Actually, the unculturable bacteria are generally weak growth, thus in some cases pure colony is activated in ISEM broth with shaking from one to two weeks before transferring to the agar plate Unusually, unculturable bacteria need much time to grow therefore, in some case, tiny colonies were cultivated on ISEM liquid until the culture will becomce opaque, then it was transferred onto the agar plates

1.3.6 Traditional soil extract method

About 1000 g air-dried soil in 1.3 L of deionized water was be autoclaving at 121oC for an hour, allowed to cool The supernatant was filtered by using the WhatmanTM paper

before centrifuge using 500 ml of bottle at 5009 ×g, 30 min, at room temperature Finally,

obtaining 1L of the supernatant (TSE) A soil extract agar was enhanced by supplement34

of 0.04% K2HPO4, 0.005% MgSO4.7H2O, 0.01% NaCl, 0.001% FeCl3, 0.05% tryptone, 0.05% yeast extract, 1.5% agar in 1 L the soil extract liquid, the final pH of 6.8 Then, 100

µL of the each fold was dispersed onto three soil extract agar plates, cultivated at 25oC for

6 weeks Appearing colonies were re-streak until getting pure colony on the medium

1.3.7 Modified transwell culture system method

Approximately, addition of 3 g of the soil sample in the well of a transwell plate, then

3 mL R2A medium (#MB-R2230, MB Cell, South Korea; 3.15 g of the powder in 1L distilled water) was supplemented into the soil-wells, placing 0.4 µm polycarbonate membrane (#35006, SPLInsert™ Hanging, SPL Life Sciences) as the insert on the wet soil Then 100 µL of the suspension and 1ml R2A medium were inoculated into the insert The transwell culture system was covered by parafilm tape to prevent evaporation The system was shaked at 120 r.p.m, 25oC for 4 weeks After that, seven folds in R2A medium were established, 100 µL of each dilution was spread on three R2A agar plates that were incubated at 25oC for 6 weeks Colonies were subcultured on R2A medium to get individual colony

1.3.8 Bacterial genomic extraction and small-subunit rRNA (SSU rRNA) amplification

Pure individual colonies were extracted genomic DNA using GenElute™ Bacterial Genomic DNA Kit (#NA2110, Sigma aldrich) Two universal primers 27F-AGAGTTTGATCMTGGCTCAG and 1492R-TACGGYTACCTTGTTACGACTT were used to amplify SSU rRNA PCR compositions and running conditions were carried out following previously study (Klindworth et al., 2013) Briefly, 80ng DNA template, 0.3 mg/mL Bovine Serum Albumin (#B8667, Sigma Aldrich), 250 µM dTNP (#R1121, Thermo Fisher Scientific), 0.02 U Phusion DNA Polymerases (#F530S, Thermo Fisher Scientific), 5× Phusion HF Buffer presence of 1.5 mM MgCl2 (#M8787, Sigma Aldrich), 0.5 mM of each primer in the total 50 µL The PCR reaction was performed at 95oC for 5min: initial denaturation, 25 cycles repeated of: denaturation 95oC for 40s), 2 min for annealing at 55oC and extension at 72oC for 1 min Final extension step at 72oC for 7 min The PCR product was examined in 1.3% gel agarose, stored at -20 oC within one month for further analysis Sequencing was performed with an Applied Biosystems 3730XL DNA

analyser using a Big Dye terminator cycle sequencing kit v.3.1 (Applied Biosystems) A nearly complete sequence was compiled with SeqMan software (DNASTAR Inc.)

1.3.9 Identify 16S rRNA gene sequences and accession numbers of 16S rRNA gene sequenses

Near-full-length 16S rRNA sequences were identified, calculating similarity to valid species through the EzTaxon Database Update (http://www.ezbiocloud.net/eztaxon) to compare with published unculturable gene sequences via the nucleotide BLAST in NCBI: http://www.ncbi.nlm.nih.gov/ In this study, based on full 16S rRNA similarity to valid published species, we were temporary division four, among candidates of novel species are defined through comparison of 16S rRNA similarity at the threshold of 98.7% (Browne et al., 2016), the 95.3~90.0% novel genus level (Yarza et al., 2008), and novel family level at off limit lower than 90.0% These sequences for candidates of uncultured or novel species

or new genera or family were released in the GenBank database that were listed in the Supplementary Tables; for isolates without accession numbers are ready if requested

1.3.10 Parameter tools/software programs

CLUSTAL X2.1, BioEdit, MEGA7.0.21 and the Kimura’s two-parameter model with bootstrap values based on 1000 replications to build topology phylogenetic tree based on full 16S rRNA gene sequences

1.3.11 Role of complex isolate medium compositions to grow previously

unculturable soil bacteria

We used multiple compositions to make isolate medium that mentioned above To evaluate which kind of them is a primary element to support growing prevously unculturable soil bacteria Several examinations were carried out based on the isolate strains in this study, following (i): basic salts (BS) only as a negative control, (ii): BS with the selenite-tungstate solution and the SL-10; (iii): BS plus D-amino acids, (iv): BS with vitamin B, (v): BS with NSE, (vi): NSE only; (vii): mixture of all the componentsas a positive control, 15g agar (#A7049, Sigma Aldrich) was treated several times with distilled water to discard any trace nutrients or elements, then added in each medium Agar plates were incubated at 25oC for 4 weeks in aerobic condition The 126 previously unclturable isolates, and 5 levels of new generaobtained using our method, were performed The experiments were confirmed independently biological repeats

1.3.12 DNA extract from soil

Using FastDNA® SPIN Kit for Soil (#116560-200, MP Biomedicals) to extract and purify soil DNA Fresh soil (0.5 g) was followed the instruction's guide DNA was checked quality through 1.2% agarose gel electrophoresis in 0.5× TAE buffer Preparing DNA concentration for futher experiments was made via nano Maestrogen, held at -20oC untill use The experiment was extracted three times independently before mixing the soil DNA together for each soil sample

1.3.13 PCR amplification and pyrosequencing

Pure isolation of DNA soil samples was applied to amplify target V1 to V3 regions located in the 16S rRNA gene through PCR amplification using barcoding primers of 27F

5’-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-ACWTTACCGCGGCTGCTGG-3’;

(http://oklbb.ezbiocloud.net/content/1001) The reaction was investigated as following initial denaturation at 95°C for 5 min, allowed by 30 cycles of (denaturation at 95°C for 30

s, annealing at 55°C for 30 s, and extension at 72°C for 30 s), andat final 72 °C an elongation for 5 min Then, the amplicon were checked under 2% agarose gel electrophoresis and observe via a Gel Doc system (BioRad, Hercules, CA, USA) QIAquick PCR purification kit (#28106, Qiagen, Valencia, CA, USA) was used to purified PCR products Ampure beads kit (Agencourt Bioscience, MA, USA) was used to enhance quality of sample and removal of non-target products following the manufacturer’s instructions, estimated quality and target sizeon a Bioanalyzer 2100 (Agilent, Palo Alto,

CA, USA) using a DNA 7500 chip Next, mixture of PCR products was performed through emulsion PCR, which was deposited on Picotiter plates The target sequencing was done at Chunlab, Inc (Seoul, Korea), supported with GS Junior Sequencing system (Roche, Branford, CT, USA) according to the manufacturer’s instructions

1.3.14 Analysis of pyrosequencing data

Analysis of pyrosequencing result was carried out following the principle described previously (Chun et al., 2010; Hur et al., 2011), unique barcodes of each amplicon as a standard, were sorted from distinctive samples obtained reading Removal of either these non-target sequences contaminated during the processing including the barcode, linker, and primersor more than two ambiguous nucleotides, low quality score that has average score

less than 25 through trimmomatic 0.321 (Bolger et al., 2014), and reads shorter than 300

bp from the original sequencing reads Using the bellerophone method to discard chimera sequences also (Huber et al., 2004) A perfect sequence was compared both of forward half and reverse half sequences via BLASTN(Huber et al., 2004) Each full sequence was estimated similarity with either valid published type strains using the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net) or uncultured bacterium clone in Genbank database (https://blast.ncbi.nlm.nih.gov) Using Chao1 estimation at the 3% distance (Chao, 1984) and Shannon diversity index (Ludwig & Reynolds, 1988) to confirm level of richness and diversity of each sample Phylogentic analysis of microbial communities was estimated via the Fast UniFrac (Hamady et al., 2010), combining principle of coordinate analysis (PCoA) Finally, the XOR analysis of CLcommunity program (Chunlab Inc., Seoul, Korea) was used to determine comparing number of operational taxonomic unit (OTUs) among samples

1.3.15 Determining and comparison of soil extract ingredients

investigated

To define the ingredients, the rhizosphere soil was collected, designed for three modes that were including #1 as a the SF; mode #2 was the same all of the procedures for preparing the TSE, then the supernatantwas passed via a 0.22 µm filter membranes nitrocellulose using a vacuum pump; vaporizing at 40oC to reduce water to 200 mL #3 was also carried out almost similar to the #2 except the sterilizing at 121°C that was replaced by shaking overnight at room temperature to compare variation of soil components by high temperature Three unlike supernatant soil extract methods were lyophilized at -54°C, then receiving 6.36 g powder (#1), 4.65 g for #2, and 4.73 g for #3 These powder were stored

at 4°C for further analysis For inorganic composition and carbohydrates, soils were prepared, analyzed directly from a final volume of 1L of deionized water without adding any substrates The experiments were comfirmed biological repeats for each analysis

1.3.16 Sample preparation for simultaneous profiling analysis of amino

acids, organic acids and fatty acids in soil extract

Simultaneous profiling analysis of amino acids (AAs), organic acids (OAs) and fatty acids (FAs) was attempted in soil samples as their ethoxycarbonylation (EOC), methoximation (MO) and tert-butyldimethylsilyl (TBDMS) derivatives according to our previously described method (Paik & Kim, 2004) Briefly, soil extract of 2.5 mg was dissolved in distilled water containing 0.1 μg of norvaline, 3,4-dimethoxybenzoic acid and pentadecanoic acid as internal standards (ISs) And this solution adjusted to pH ≥12 with 5.0M sodium hydroxide was added to dichloromethane (2.0mL) containing 40μL of ethyl chloroformate (ECF), which was converted to the EOC derivative Then this was converted

to the MO derivative by a reaction with methoxyamine hydrochloride at 60°C for 60 min The aqueous phase as sequential EOC/MO derivatives was acidified (pH ≤ 2.0 with 10% sulfuric acid), saturated with sodium chloride, and extracted with diethyl ether (3 mL×2) The extracts were evaporated to dryness using a gentle nitrogen stream Dry residues containing AAs, OAs, and FAs were reacted at 60°C for 30 min with TEA (5 μL), toluene

(15 μL), and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (20 μL) to form

TBDMS derivative All samples were prepared individually in triplicate and examined directly by GC-MS in SIM mode

1.3.17 Gas chromatography-mass spectrometry

GC-MS profiling analysis in selected ion monitoring (SIM) mode was performed using an Agilent 7890N gas chromatograph interfaced with an 5975C mass-selective

detector (70 eV, electron impact mode) and installed with an Ultra-2 (5% phenyl-95%

methylpolysiloxane bonded phase; 25 × 0.20 mm i.d., 0.11m film thickness) cross-linked capillary column (Agilent Technologies, Atlanta, GA, USA) The temperatures of the injector, interface, and ion source were 260, 300, and 230°C, respectively Helium was used

as the carrier gas at a flow rate of 0.5 mL/min in constant flow mode The samples were

introduced in split-injection mode (10:1), and the oven temperature was initially at 60°C for 2 min and programmed to 255°C at a rate of 25°C/min and finally to 300oC at a rate of

7°C/min with a holding time of 2.5 min In scanning mode, the mass range was 50-750 m/z

at a rate of 0.43 scans/s

1.3.18 Inorganic ingredients

Cations such as Li+, Na+, Mg2+, K+, Ca2+ and NH4+ were determined by Dionex ICS3000 (Dionex, USA) with Ionpac CS12A column (4 × 250 mm/Dionex, USA) and detector (suppressed conductivity, CSRS URTRA (4 mm), recycle mode) Oven temperature was 30°C, injection volume was 25 μL, elution was done by 20 mM methanesulfonic acid at flow rate 1 mL/min, and run time was 20 min This work was assisted by the IonPac® CS12A manual (Thermo Fisher Scientific) Dionex ICS3000 (Dionex, USA) was also used to determine anions such as F-, Cl-, Br-, NO2-, NO3-, SO42-

and PO42- with standards, and the column was Ionpac AS20 (4 × 250mm, Dionex, USA) and the detector was suppressed conductivity, ASRS URTRA II (4mm), recycle mode Gradient elution was fixed by 0-8min (12mM KOH), 8-12min (30mM KOH), 12-17min (30mM KOH), 17-18min (12mM KOH), 18-20min (12mM KOH) at flow rate 1mL/min Oven temperature was 30°C and injection volumn was 25 μL All the processes were referred by IonPac®AS20 Anion-Exchange Column product manual (Thermo Fisher Scientific)

1.4 Results

1.4.1 Shortage of previous methods and new method development

So far, many methods have been applied to cultivate uncharacterized species in the laboratories by using soil extractants using water (Taylor, 1951a,b) or aqueous buffers (Hamaki et al., 2005; Vilain et al., 2006), diluted medium or serial dilution culture (Janssen

et al., 2002; Schoenborn et al., 2004), coculture with helper bacteria (Burmølle et al., 2009; D'Onofrio et al., 2010) that may provide essential trace nutrients (amino acids, vitamins, carbon sources, siderophores and other yet-unidentified components) for growth of its adjacent bacteria, and electron donors/acceptors (Hobbie & Hobbie, 2013; Pierra et al., 2015) Even though, all of them had clear achievements than previous methods, but they still revealed some limitation regardless of expected results because of low isolation efficiencies of unculturable bacteria or no idea on subculture To overcome these problems, this study devloped a new method using a mixture of methanol/water (4:1; 80%) to extract bacterial nutrients from soil instead of water or aquous buffers This is the first time to use other than water or aqueous buffers We called this extract as new soil extract (NSE) and the brief procudure is as follows 500 g dry soil was prepared and incubate with 1.3 L 80% methanol overnight at room temperature Then supernatant was transferred to a new flask and new 1.3 L 80% methanol was added into the remained soil, and mixed well for an hour Two supernatants were combined, filtered and evaporated The pure NSE was stored at 4°C before use Here, we compared effective covering species using different methods, which showed in Table 1

Table 1 Brief summary comparision of results by methods determined

Our method General soil extract method Modified method

o Uncultured bacteria: 2/82 (2.4%)

o Uncultured bacteria: 7/80 (8.8%)

 New species: 0%

 Know species: 7 (100%)

 New species: 7/15

 Know species: 8/15

o Cultured: 67/82=81.7%

o New genus: 1/84 (1.2%)

o New family: 0

o Known species: 60/84 (71.4%)

o Uncultured bacteria: 13/84 (15.5%)

 New species: 7

 Known species: 6

o Cultured: 71/84 (84.5%)

43 known families; 1 possible

new family level

26 known families, none of new

Bacteroidetes: 2

Total genera: 60

Table 2 List of soil extract components defined a new way and general approaches

#1 was developed by us; #2 defined using autoclaving at 121oC for an hour; #3 without autoclaving Soil components without any supplements were derived the sample but only different methods used Mean ± standard deviation; nd: not detected

Ingredients

(mgL -1 )

Soil extraction methods compared

#1 #2 #3 Continued #1 #2 #3

1 Amino acid 2 Fatty acid

Leucine 0.468 0.052 0.061 Lignoceric acid 2.603 0.093 0.079 Isoleucine 0.201 0.026 0.034 Cerotic acid 0.094 0.182 0.144

γ-Aminobutric acid 0.756 0.621 0.689 3 Organic acid

Pipecolic acid 0.092 nd nd 3-Hydroxybutyric acid 0.962 2.068 1.881 Pyroglutamic acid 0.847 0.234 0.318 Pyruvic acid 0.404 4.310 5.746

Phenylalanine 0.219 0.033 0.036 Glycolic acid 8.031 nd nd Cysteine 0.216 0.117 0.293 2-Hydroxybutyric acid 0.094 nd nd Aspartic acid 0.272 0.076 0.308 Malonic acid 0.845 1.205 1.754 Glutamic acid 0.552 0.398 0.392 Succinic acid 1.425 2.176 1.784

Ornithine 0.766 0.463 0.454 Oxaloacetic acid 0.173 18.353 44.839 Glutamine 0.547 0.668 0.664 α-Ketoglutaric acid 0.114 nd nd

Tyrosine 9.286 0.100 0.085 2-Hydroxyglutaric acid 0.496 0.932 0.721 Tryptophane 0.722 0.504 0.507 Cis-Aconitic acid 0.272 0.279 0.329

Total 18.472 4.834 5.865 Citric acid 0.458 0.813 0.831

2 Fatty acid Isocitric acid 0.196 0.478 0.537

Lauric acid 0.478 nd nd 4 Inorganic molecule

1.4.2 Method validation through comparative analysis of

organic/inorganic compounds

Heterotrophic soil bacteria are dependent for nutrition and bacterial growth on molecular-weight organic substances (LMWOS) and inorganic compounds (Liebeke et al., 2009), in this study, we tried to compare major LMWOS such as amino acids, fatty acids, organic acids and carbohydrates, and inorganic ions in three different extraction methods (#1: new extraction developed in this study; #2: traditional extraction without autoclave;

low-#3 traditional extraction with autoclave) (Table 2)

In case of amino acids, #1 showed the total yield of 18.472 mgL-1 that is much higher than the two comparative methods, 4.834 and 5.865 mgL-1, respectively (P<0.006) Also,

it extracted 21 amino acids including four more amino acids (valine, pipecolic acid, serine and threonine) and very high concentration of tyrosine (3.650 mgL-1) than other methods

So more concentration and diversity of amino acids in NSE may cause better cultivability

of unculturable soil bacteria as mentioned above However, there was significant difference between #2 and #3, their total was changed under using with/without influence of

autoclaving (P#2 vs #3<0.02)

In fatty acid analysis, the result was incomparable because #1 extracted much higher total concentration (13.114 mgL-1 vs 0.789-0.793 mgL-1) and more number (n=16 vs n=8)

of fatty acids than other two (Table 2) As well as amino acids, more amount and number

of fatty acids may make better cultivability of unculturable soil bacteria

In case of organic acids, the result showed different pattern from amino acids and fatty acids in that #1 had lower total yield of organic acids (25.132 mgL-1 vs 62.686-89.000 mgL-1; P < 0.03) but more number (n=16 vs n=11) additionally including lactic acid,

glycolic acid, 2-hydroxybuyric acid, fumaric acid and -ketoglutaric acid (Table 2) Total amount of organic acids for #2 and #3 seemed to be significantly influenced by two major

components such as acetoacetic acid and oxaloacitic acid, making not much meaningful in total amount

The total yields of each approach were 748.03, 965.00 and 946.91 mgL-1, respectively Although #1 showed lower concentration of total inorganic compounds than two others, a mixture of water-methanol seemed to dissolve substances not much different from the amounts extracted by water and recovered all tested inorganic ions as water extraction (#2

& #3) (Table 2) Temperature did not significantly affect dissolution of inorganic

compounds in water between #2 and #3 as well as organic compounds (P > 0.7)

Fig 1 An impact of various factors on cultivation of uncultured soil bacteria Diverse nutrient sources were used, results recorded all of the species growth occur on the ISEM growth, not growth on either the BS only or combination of BS with vitamin B Among components investigated, revealing NSE is the most important factor, it seems containing complete necessary nutrients without adding the others However, the effective components supplemented such as vitamin, D-amino acids and trace nutrient are determined to help these species grow better based on colonies size appearing in the media

1.4.3 Method validation through cultivability of unculturable bacteria

For comparison, we examined basic salts (BS), BS plus micro-nutrients, BS plus vitamin B, BS plus D-amino acids and BS plus NSE 131 bacterial isolates including unculturable bacteria and novel genus candidates, obtained from our developed culture method (Table 3), were tested to know their effectiveness As a result, while NSE and BS plus NSE showed 100% growth efficiency as visible colonies, BS plus D-amino acids revealed 8% and others had 0% (Fig 1) Therefore, NSE is verified as an effective medium for isolation of unculturable soil bacteria than previously used ones

Table 3 List of bacterial isolates used for evaluating new soil extract (NSE)

Previously uncultured novel genus level

Previously cultured novel genus level

Previously uncultured novel family level

Fig 2 Achievement of phenotypic diversity of species isolated through soil samples (a) Comparing percentage of uncultivable species (126 individual species among the total 258;

11 of the 243; 39 of the 252, respectively) Points of significance compared to the paired

two sample for means t-test where indicated by *P < 0.005; **P < 0.01; ***P < 0.009; (b)

Average achievement of number of novel genera level revealed significantly increased using improved method (~ four species for each sample) while either none or only one species defined from three soil samples via two remaining methods; (c) The number of new bacterial species and known species cultured among total of 126, 11 and 39 previously

uncultivable species via the methods investigated, respectively