Title Microbial Diversity in Natural Asphalts of the ...24 such as those that occur in oil sands,...

Page 1

Title: Microbial Diversity in Natural Asphalts of the Rancho La Brea Tar Pits

Authors: Jong-Shik Kim and David E. Crowley

Affiliations: Dept. Environmental Sciences, University of California, Riverside, CA

Corresponding Author:

David Crowley, Department of Environmental Sciences, University of California,

Riverside. Phone 951-827-3785, Fax 951-827-3993

E-mail [email protected]

Manuscript Information:

Text Pages: 22

Figures: 7

Tables: 2ACCEPTED

Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01372-06 AEM Accepts, published online ahead of print on 6 April 2007

on January 26, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 2

ABSTRACT 1

Bacteria commonly inhabit subsurface oil reservoirs, but almost nothing is 2

known yet about microorganisms that live in naturally occurring terrestrial oil seeps and 3

natural asphalts that are comprised of highly recalcitrant petroleum hydrocarbons. Here, 4

we report the first survey of microbial diversity in ca 14,000 year old samples of natural 5

asphalts from the Rancho La Brea Tar Pits in Los Angeles, California. Microbiological 6

studies included analyses of 16S rRNA gene sequences and DNA encoding aromatic 7

ring hydroxylating dioxygenases from two tar pits differing in chemical composition. 8

Our results revealed a wide range of phylogenetic groups within the Archaea and 9

Bacteria domains, in which individual taxonomic clusters were comprised of sets of 10

closely related species within novel genera and families. Fluorescent staining of asphalt-11

soil particles using phylogenetic probes for Archaea, Bacteria, and Pseudomonas 12

showed coexistence of mixed microbial communities at high cell densities. Genes 13

encoding dioxygenases included three novel clusters of enzymes. The discovery of life 14

in the tar pits provides an avenue for further studies on the evolution of enzymes, and 15

catabolic pathways for bacteria that have been exposed to complex hydrocarbons for 16

millennia. These bacteria also should have application for industrial microbiology and 17

bioremediation. 18

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 3

INTRODUCTION 19

Prior studies of subsurface petroleum reservoirs using culture based methods 20

have revealed diverse microbial communities that are able to live on complex petroleum 21

hydrocarbon mixtures (20, 43). Nonetheless, very little is known yet about the true 22

extent of microbial diversity in natural oil reservoirs and terrestrial petroleum deposits 23

such as those that occur in oil sands, shales, and natural asphalts. Initial surveys of 24

underground reservoirs using molecular approaches so far suggest that the majority of 25

microorganisms inhabiting these environments are new species that represent a rich pool 26

of novel genetic diversity with potential importance for industrial and petroleum 27

microbiology (43). Compared to underground oil reservoirs, even less is known about 28

terrestrial habitats where petroleum degrading soil bacteria have come to inhabit heavy 29

oil seeps, tar sands, and natural asphalts. With the advent of improved DNA extraction 30

and purification methods, such bacteria and their genes may now be accessible for 31

detailed study of their diversity and genes that encode petroleum degrading enzymes. 32

The existence of bacteria in petroleum deposits at great depths suggest that 33

many species have evolved specifically to this environment and may be carried to the 34

surface in oil seeps. In soils that are permeated with the asphalt, bacteria may also 35

include indigenous bacteria that survived after asphalt seeped through the soil. Selection 36

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 4

of bacterial communities to petroleum substances occurs rapidly after even short term 37

exposures of soil to petroleum hydrocarbons following oil spills (41, 42). Over time 38

spans encompassing millennia, bacteria that can tolerate this environment would be 39

expected to undergo genetic adaptations that may lead to evolution of new ecotypes and 40

species, and enzymes for growth on petroleum hydrocarbons. During adaptation of 41

communities, genes for petroleum hydrocarbon degrading enzymes that are encoded on 42

plasmids or transposons may exchanged between species. In turn, new catabolic 43

pathways eventually may be assembled and modified for efficient regulation (27). Other 44

cell adaptations leading to new ecotypes may include modifications of the cell envelope 45

to tolerate solvents (28), and development of community level interactions that facilitate 46

cooperation within consortia. 47

Here we report the survey of microbial diversity in natural asphalts at the 48

Rancho La Brea Tar Pits in California. These natural asphalts are located in Hancock 49

Park in downtown Los Angeles, and consist of asphalt-soil mixtures formed by 50

upwelling of heavy oil and asphaltenes in spatially separated seeps that differ in their 51

chemical composition and age. Although the asphalt at Rancho La Brea is commonly 52

called tar, the petroleum hydrocarbons here are correctly referred to as natural asphalt, 53

and are comprised of some of the most recalcitrant carbon compounds in nature (9). Our 54

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 5

survey examined two excavation sites. The first site, Pit 91, has yielded thousands of 55

plant and animal fossils, and is the richest Pleistocene fossil site in the world (10, 16, 56

45). Carbon dating of fossils from the current depth under excavation in Pit 91 range in 57

age from 10,000 to 38,000 years before present (16). The second site, Pit 101 was 58

excavated early last century and was closed in the 1920s, after which the pit was 59

covered with a permanent building as part of a museum display. Microbiological studies 60

included analysis of 16S rRNA genes from DNA extracted from the tar pits and a 61

traditional approach employing cultivation of bacteria on agar. In conjunction with 62

microbial diversity, we also surveyed DNA sequences for aromatic ring hydroxylating 63

dioxygenases that may have application for industrial microbiology and bioremediation 64

of petroleum wastes. 65

66

MATERIALS AND METHODS 67

68

Characterization of chemical properties in Pits 91 and 101. Ten gram 69

samples of the asphalt impermeated soil were physically broken into small aggregates 70

and placed into beakers containing 10 ml deionized water or 10 mM CaCl2 buffer. The 71

suspensions were mixed using a magnetic stir bar for 1 hr after which pH and salinity 72

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 6

were determined. Salinity was determined in water solutions using a conductivity meter. 73

Other samples for metal analyses were acid digested in nitric perchloric acid using 74

digestion bombs and a microwave oven (USEPA SW-846, Method 3051). Heavy metals 75

and cations were analyzed by ICP-MS using an ELAN500 ICP (Perkin-Elmer-Sciex 76

Instruments, Concord, Ontario, Canada). Carbon, nitrogen and sulfur were analyzed 77

using a Flash EA 1112 NC analyzer (Thermo Electron Corporation, Milan, Italy). 78

Sampling and DNA extraction. Previously unexposed samples were removed 79

from approximately 10 cm under the surfaces of Pit 91 and Pit 101 of the Rancho La 80

Brea tar pits in Los Angeles in October 2004. A total of five samples were taken along a 81

3 meter transect from the center of each pit. Samples were removed from the pits with 82

sterile, autoclaved spatulas and were transferred into sterile 50 ml plastic tubes with 83

screw caps and transported to the laboratory for processing. The samples from each pit 84

were pooled prior to extraction. One of the challenges in conducting this survey was the 85

difficulty in extracting high quality DNA for use in cloning and sequencing. DNA was 86

extracted from the asphalt-soil mixtures by first freezing approximately 5 g aliquots of 87

the asphalt in liquid nitrogen. The frozen samples were transferred to a sterile ceramic 88

mortar and were then ground under liquid nitrogen to a fine powder. Approximately 0.5 89

g subsamples were processed to extract DNA by bead beating using the BIO 101 90

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 7

Fastprep DNA extraction kit for soil following the manufacturers protocols. Extracted 91

DNA was concentrated using a Savant Speed Vac system (GMI Inc., Ramsey, MN, 92

USA) and subsamples were combined to obtain a high concentration DNA. The DNA 93

was purified using a QIAquick gel extraction kit (Qiagen, Chatsworth, CA, USA) 94

according to the manufacturers' instructions. The purified DNA was concentrated again 95

for use in construction of clone libraries. 96

97

Phylogenetic analysis. 16S rRNA genes were amplified by PCR and purified 98

with a QIAquick PCR purification kit (Qiagen). The purified PCR products from five 99

runs were combined and used to construct clone libraries using the pGEM-T easy vector 100

(Promega) with selected primer sets. Bacteria were detected using bacterial specific 101

primers, 27F-1492R (22). Archaea were detected using the domain specific PCR 102

primers, Ar4F-Ar958R (17). Pseudomonas spp. were identified using the Pseudomonas 103

selective PCR primers, Ps289F-Ps1258R (47). Dioxygenases were detected using the 104

PCR primer set adoF-adoR for aromatic ring hydroxylating dioxygenases (36, 37). The 105

sequencing primers were T7 and SP6. After obtaining raw sequences using Chromas 2 106

(Technelysium Pty Ltd, Tewantin, Queensland, Australia), putative chimeric sequences 107

were identified using Bellerophon (12) and identified chimeric sequences (43 of 278 108

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 8

bacterial sequences) were excluded. The 16S rRNA sequences were aligned using the 109

NAST aligner and the aligned sequences were compared to the Lane mask (22) using 110

the Greengenes web site (4, 5). Evolutionary distances were calculated with the Kimura 111

2-parameter and a phylogenetic tree was constructed by the neighbor-joining method 112

(31) with MEGA3 for Windows (19). Bootstrap analyses of the neighbor-joining data 113

were conducted based on 1000 samples to assess the stability of the phylogenetic 114

relationships. 115

116

Statistical analyses. The computer program DOTUR (32) was used to calculate 117

species richness estimates and diversity indices. A second program, LIBSHUFF (34) 118

was used to compare the similarities of bacterial clone libraries in Pits 91 and 101. The 119

distance matrices for both programs were obtained using an algorithm located at the 120

Greengenes website (4, 5). 121

122

Fluorescent in situ hybridization. FISH was carried out as described 123

previously (49) with minor modifications. Oligonucleotides were 5’end labeled with 124

fluorescent dyes and included Eub338 labeled with Cy3, ARCH915 labeled with Cy5, 125

and Pae997 with Bodipy FL. Details on these probes are available at probeBase (24). 126

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 9

Cells were photographed with a Leica TCS/SP2 UV confocal microscope. 127

128

Cultivation. Culturable bacteria were isolated by serial dilutions of water 129

suspensions of asphalt-soil mixtures on agar plates containing DSMZ medium 371 130

amended with 20% NaCl and 10% TSA and M9 minimum medium. The plates were 131

incubated at 28℃ for 2 to 3 wks after which individual isolates were transferred and 132

processed for sequencing of 16S rRNA gene sequences. Isolates were placed in glycerol 133

medium and transferred to a -80℃ freezer for long term preservation. Bacterial isolates 134

were tested on agar media with 1% asphalt as a sole carbon source. 135

136

137

RESULTS 138

Detailed analyses of the two tar pits revealed differences both in the chemical 139

composition and microbial community composition of the asphalt samples. The asphalt 140

permeated soil from Pit 91 contained a greater concentration of petroleum hydrocarbons, 141

was slightly acidic, and had a relatively low salinity and metal content (Table 1). In 142

contrast, water suspensions of asphalt permeated soil from Pit 101 were alkaline (pH 143

8.4) and contained a high concentration of salts and metals. The salinity of 1:1 asphalt 144

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 10

water suspensions of Pit 101 was 4,610 µS cm-1

, which was 100 times higher than that 145

of Pit 91. Materials from both of the tar pits are impermeable to surface water from 146

rainfall and bacteria in this matrix are subject to water deficits and high salinity. Water 147

contained in the asphalt is present in stratified water pockets and pore spaces that occur 148

throughout the pit and are likely the main sites for microbial growth. The occurrence of 149

microbial activity in the tar pits is visually evident from the continual evolution of 150

methane bubbles at various locations in the pits where there is still viscous liquefied 151

asphalt. 152

Here a total of 235 bacterial clones were sequenced to identify the predominant 153

phylogenetic groups (Fig. 2). The most striking difference in the microbial community 154

composition of the two pits was the presence of halophilic Archaea in the highly saline 155

Pit 101, which were not detected in Pit 91 (Fig. 1). Among these were 27 clones 156

representing clusters of closely related species from two unclassified genera that were 157

most similar to Natronococcus and Natronobacter (Fig. 1). Of the 29 Archaea 158

sequences that were obtained only 2 were outside of the clusters representing the new 159

genera. 160

In addition to Archaea, there were also differences in the distribution of 161

bacterial phyla between the two pits. The predominant bacteria in both pits were 162

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 11

Gammaproteobacteria (purple sulfur bacteria). Pit 91 contained 3 unclassified families 163

(60 clones) in the order Chromatiales, none of which were found in Pit 101 (Fig. 2). 164

Other families of the Gammaproteobacteria in Pit 91 included Xanthomonadaceae (5 165

clones) and Pseudomonadaceae (5 clones). (Fig. 2A and C). 166

Pit 101 contained an greater breadth of diversity of Gammaproteobacteria and 167

Alphaproteobacteria than Pit 91. This included two unclassified families and one new 168

order with two more unclassified families (Fig. 2B). In addition to 16 clones 169

representing the family Rubrobacteraceae, other unique taxa in Pit 101 included 5 170

additional phyla that were represented by 9 clones from the Planctomycetes, 6 clones of 171

Gemmatimonadetes, 1 of BRC1, and 2 clones each of Nitrospira and Verrucomicrobia 172

(Fig. 2D). Taxa that were common to both Pit 91 and Pit 101 included species from 173

Alpha-, Beta-, and Gammaproteobacteria and various representatives from the 174

Acidobacteria, Actinobacteria, Bacteroidetes, and Clostridia (Fig. 2). 175

Sequences obtained using the Pseudomonas selective primers included 14 176

clones of P. stutzeri, which represented 8 new genomovars of this species (Fig. 3). All 177

but 1 of the 14 clones was obtained from Pit 91 suggesting a differential distribution of 178

P. stutzeri in the two tar pits (Fig. 3). 179

Culturable bacteria from both tar pits were represented by relatively few species. 180

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 12

The 10% TSA medium yielded 5 distinct isolates of Pseudomonas spp., 8 isolates of 181

Bacillus spp., and 5 of Citrobacter spp. The 20% salt medium yielded 3 isolates of 182

haloalkalophilic Bacillus sp. from Pit 101 (Fig. 4). All of the isolates obtained on the 183

above media were further shown to grow on M9 medium with asphalt as the sole carbon 184

source. 185

To study the physical distribution of Archaea and Bacteria in the asphalt from 186

Pit 101, samples from the tar pit were microscopically examined using fluorescent in-187

situ hybridization with DNA probes targeted against Bacteria, Archaea, and 188

Pseudomonas sp. (Fig. 5). Cells from each group were observed to occur in dense 189

clusters of colonies along with randomly dispersed individual cells. The very close 190

association of cells from different phylogenetic groups suggests that there may be 191

consortia that cooperatively solubilize, degrade and detoxify the complex substances 192

contained in the asphalt. Such consortia also may be conducive for horizontal gene 193

transfer of plasmids and DNA sequences that encode catabolic genes for use of 194

petroleum hydrocarbons. 195

196

Diversity analyses. Rarefaction curves of Pit 91 and Pit 101 libraries showed 197

significantly different numbers of OTUs (operational taxonomic units) at distance 198

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 13

values of 0.03 corresponding to the species level, 0.05 at the genus level, and 0.1 at the 199

family/class level (Fig. 6A). Only 37 OTUs were obtained from the Pit 91 bacterial 200

library, while 80 OTUs were obtained from the Pit 101 bacterial library. Indices of 201

diversity were higher for the Pit 101 library than the Pit 91 library. The Shannon and 202

Simpson index values were 2.8 and 0.139 for the Pit 91 library compared with 4.2 and 203

0.014 for the Pit 101 library. Richness ACE (abundance-based coverage estimator) and 204

Chao1 (13) were 64 and 58 in Pit 91 and 268 and 242 in Pit 101 (Table 2). There was a 205

significant difference between the rarefaction curves for the Pit 91 and Pit 101 libraries 206

based on 0, 3%, 5% and 10 % differences (Fig. 6A). LIBSHUFF comparisons of each of 207

the libraries indicated that two Pit 91 and Pit 101 community was significantly different 208

(P < 0.001). At an evolutionary distance of 0.03, coverages of the libraries were 80% 209

and 46% in Pit 91 and Pit 101 respectively (Fig. 6B). 210

211

Bacterial dioxygenase sequences. Taxonomic relationships for the 212

dioxygenase sequences that were identified are shown in Fig. 7. The phylogenetic trees 213

for these sequences included reference dioxygenases that were not found in the tar pits, 214

but that provide an indication of the similarities of the new sequences to those of 215

previously described enzymes. Sequences from Pit 91 were predominantly associated 216

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 14

with those of known proteobacterial biphenyl dioxygenases. This cluster of biphenyl 217

dioxygenases was comprised of 12 sequences from Pit 91 and 2 sequences from Pit 101. 218

Overall similarities to known biphenyl dioxygenases ranged from 79 to 95%. Detailed 219

analysis revealed at least 4 subclusters within the biphenyl dioxygenase group. As with 220

the microbial 16S rRNA genes, there were striking differences in the clusters of 221

dioxygenases represented by the two sites. Ring hydroxylating dioxygenases from Pit 222

101 were predominantly comprised of a new group of 18 closely related sequences that 223

represent a novel, phylogenetically deep group of enzymes. This cluster was most 224

closely associated with sequences encoding benzene and toluene dioxygenases, but was 225

sufficiently distant that it is not possible to infer whether these dioxygenases utilize 226

these substances or instead transform other substrates. Two other new clusters that were 227

discovered from sequences from Pit 91 were represented by 1 and 2 clones, respectively, 228

and also appeared to represent deep branches of genes encoding unknown types of 229

dioxygenases. 230

231

DISCUSSION 232

Previous research has suggested that bacteria in deep subsurface oil reservoirs 233

have inhabited those environments since the oil was formed (25). The origin of the 234

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 15

bacteria in the natural asphalts at Rancho La Brea is unknown, but could reflect recent 235

entry from dust deposition on the surface, bacteria originating from the subsurface oil 236

reservoir that seeped to the surface, or the progeny of soil bacteria that were embedded 237

in the asphalt matrix as heavy oil seeped to the surface. Regardless of their origin, life in 238

asphalt poses extreme conditions in which microbial growth is limited by the lack of air 239

and water, the presence of highly recalcitrant carbon sources, and high concentrations of 240

potentially toxic metals and chemicals. The selectivity of this environment would be 241

expected to require specialized adaptations. Here, 235 clones were described, many of 242

which appear to comprise new genera and families of Proteobacteria (Fig. 2). An 243

analysis of two different pits differing in their chemical properties revealed very little 244

overlap in diversity (Fig. 6A), indicating that site specific differences in salinity and pH 245

strongly influence selection within the asphalt communities. 246

The relatively simple community structures and low complexity of Pits 91 and 247

101 as compared to soil suggest that this environment is highly selective. The 248

phylogenetic trees within the Proteobacteria revealed considerable breadth in taxa at the 249

level of family and order, but also manifested branches containing discrete clusters of 250

related sequences. The occurrence of many closely related species within the 251

Gammaproteobacteria in both pits and Archaea in Pit 101 suggested either an 252

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 16

evolutionary radiation of species or an initial selection of closely related bacteria that 253

share specific traits that enable them to survive in this environment. 254

Important questions arising from this research are the functional properties and 255

adaptations of the taxa that inhabit the tar pits. It is very difficult to infer functionality at 256

the level of phylum and there is sparse information on many of the bacterial species that 257

were found here. Among the predominant bacteria were 60 clones representing three 258

unknown families from the Gammaproteobacteria in the order Chromatiales. This order 259

has previously been characterized by two families, the Ectothiorhodospiraceae and 260

Chromatiaceae, both of which phototrophic anaerobic bacteria that produce sulfur from 261

hydrogen sulfide gas (14, 15). The former produce granules of sulfur on the outside of 262

their cells, while the latter produce internal sulfur granules. The three new families 263

discovered here are not likely to derive energy from photosynthesis given that they live 264

in complete darkness within the asphalt-soil matrix. Nonetheless, an ability to utilize 265

electrons from hydrogen sulfide is consistent with life in the tar pits where hydrogen 266

sulfide and methane are produced during anaerobic metabolism of hydrocarbons 267

contained in the asphalt. 268

Another important cluster in Pit 101 was classified within the 269

Rubrobacteraceae. There were 16 closely related sequences from this family. The 270

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 17

Rubrobacteraceae are in the phylum Actinobacteria and have been previously reported 271

to occur in high ionizing radiation environments and in Australian desert soils (11). 272

Rubrobacter have received considerable attention as being among the most resistant 273

bacteria to ionizing radiation (7). Whether the bacteria from Pit 101 are radiation 274

resistant is unknown, but it can be speculated that this trait could be of importance as an 275

adaptation to protection from DNA damage in mixtures of heterocyclic aromatic 276

hydrocarbons which are potent mutagens (48). 277

The occurrence of Pseudomonas stutzeri sequences, which included 8 new 278

genomovars, is consistent with prior reports on the distribution of this species. P. 279

stutzeri is a well known petroleum hydrocarbon degrader (8) and appears to be a 280

cosmopolitan species that is readily isolated from various petroleum contaminated 281

environments (21, 29). Previously 17 genomovars have been described (21); this 282

research added another 8 candidate genomovars. As noted for several taxa in the 283

communities from the tar pits, the existence of closely related sequences of P. stutzeri 284

could be explained either by selection for bacteria that shared essential characteristics 285

that allow them to survive in the asphalt or as an evolutionary radiation of ecotypes. 286

With the exception of Pseudomonas sp, all of the identified taxa were dissimilar to those 287

reported earlier for two studies investigating high temperature oil reservoir, which are 288

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 18

the only other studies in the literature reporting a bacterial survey of a natural petroleum 289

habitat using culture independent methods (3, 26). 290

The discovery of many halophilic Archaea sequences in Pit 101 is particularly 291

intriguing and provides an opportunity for future studies on the role of Archaea in 292

petroleum hydrocarbon degradation. Based on studies conducted with enrichment 293

cultures of bacterial petroleum degraders, biodegradation typically involves consortia in 294

which the species composition of the degrader community is strongly influenced by 295

salinity (18, 44). Whether this selection also occurs with Archaea is not known. Archaea 296

have been reported to occur in crude oil sludge, but not in crude oil samples in oil 297

stockpiles in Japan (40) or in petroleum reservoirs in California (26). Various 298

Halobacteria isolated from soil and sediments that are capable of degrading 299

hydrocarbons under saline conditions have been reported in the literature. These include 300

species tentatively identified as Halobacterium (2), Haloferax (51), and Haloarcula (40). 301

A prior report of Archaea that can degrade aromatic hydrocarbons under anaerobic 302

conditions using Fe(III) as an electron acceptor was published in 2001 (39). Although 303

not yet cultivable, genes from these bacteria potentially could be used for improving 304

culturable hydrocarbon degraders used for bioaugmentation, or could serve as a source 305

of catabolic genes that could be seeded into the environment on plasmids to facilitate 306

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 19

adaptation of indigenous strains for degradation of recalcitrant hydrocarbons (38). It 307

will also be important to determine whether the large unknown cluster of ring 308

hydroxylating dioxygenases identified in Pit 101 are carried by the Archaea, which is 309

suggested by their co-occurrence in this particular site. 310

As expected from prior experience with environmental samples, relatively few 311

isolates were obtained using culture based methods. The culturable bacteria included 312

strains of Pseudomonas spp., Citrobacter spp. and Bacillus spp. that were similar to 313

known strains from oil contaminated environmental samples (Table 2). The ability to 314

culture these strains, especially isolates of Pseudomonas provides opportunities for full 315

genome analysis and examination of genetic exchange that may have occurred within 316

this group of bacteria, as well as determination of plasmid borne genes or catabolic 317

pathways. 318

From an applied perspective, the most practical aspect of this research may be 319

the confirmation of heavy oil seeps and natural asphalts as sources of novel genes for 320

biodegradation of petroleum hydrocarbons. Here, we focused on genes encoding 321

aromatic ring hydroxylating dioxygenases that are important for degradation of BTEX 322

and aromatic chemicals such as polychlorinated biphenyls (PCBs) and polycyclic 323

aromatic hydrocarbons (PAHs) that are common environmental pollutants. The 324

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 20

maintenance of genes encoding dioxygenases by bacteria that have undergone selection 325

for life in an anaerobic system is somewhat of a paradox, but is common in known oil 326

degrading bacteria. Previously described toluene degrading bacteria placed under 327

anaerobic conditions have been shown to first use dioxygenases to degrade toluene until 328

all of the oxygen is consumed, after which the cells switch to a benzylsuccinate pathway

329

that is coupled to denitrification (33). Among the sequences that were obtained, a large 330

number that were similar to those encoding known biphenyl dioxygenases that function 331

for degradation of PCBs. Subtle variations in key regions of these genes can lead to 332

large differences in substrate range and specificity for different PCB congeners. In the 333

future, enrichment culture methods may be used to identify still other enzymes that can 334

target specific substrates. 335

Relatively little is known yet about anaerobic petroleum hydrocarbon 336

degradation, although there has been steady progress in this field (1, 23, 30, 50). 337

Tentative mechanisms that function for anaerobic degradation of alkylbenzenes and 338

nonaromatic hydrocarbons are proposed to involve hydrolases and carboxylases, and are 339

coupled to sulfate or nitrate reduction (35, 46). In our survey, anaerobic bacteria that 340

were identified included members of Gammaproteobacteria (60 clones of purple sulfur 341

bacteria), Bacteroidetes, Clostridia, and Acidobacteria, none of which have been 342

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 21

studied with respect to their possible contributions to anaerobic degradation of 343

petroleum hydrocarbons. Homologous genes for the benzylsuccinate synthase

(bss), 344

which is the key enzyme for anaerobic toluene degradation have been cloned (33), and 345

may provide an entry point for future studies on the relevance of this pathway in the tar 346

pit bacteria. In addition to direct catabolism of hydrocarbons, anaerobic bacteria may 347

also contribute to hydrocarbon degradation by syntrophy, in which metabolically linked 348

consortia function to consume fatty acids and degradation products of hydrocarbons to 349

generate methane (46). 350

Looking toward future research on the Rancho La Brea microorganisms, the 351

discovery of closely related bacterial clusters and genes encoding new dioxygenases is 352

of particular interest for understanding the evolutionary biology of bacteria during 353

adaptation to the extreme environment posed by life in asphalt. Detailed studies on 354

efficient regulator-promoter pairs are now being conducted to understand and design 355

improved operons for xenobiotic degrading bacteria (6). New approaches using high-356

throughput DNA sequencing will be the next step for obtaining insight into the function 357

and diversity of oil inhabiting bacteria and the catabolic pathways for degradation of 358

petroleum hydrocarbons. 359

360

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 22

Nucleotide sequence accession numbers. All sequences were deposited in the 361

GenBank under accession numbers DQ001614-DQ001723, DQ001626-DQ001638, 362

DQ001641- DQ001642, DQ001644, DQ001646, DQ001647 (for Bacteria), DQ192039-363

DQ192061 (for isolates), DQ062817-DQ062856 (for dioxygenase), AY860441-364

AY860440 and AY939988-AY940011 (for Archaea), AY940013, AY940019-22, 365

AY940024-26, AY940028-29, AY940032, and AY860446-48 (for Pseudomonas 366

stutzeri). 367

368

ACKNOWLEDGMENTS 369

The authors gratefully acknowledge the assistance and review comments of Dr. 370

John Harris and Mr. Christopher Shaw, and the cooperation of the George C. Page 371

Museum at Hancock Park, Rancho La Brea Tar Pits. This project was supported in part 372

by National Research Initiative Competitive Grant from the USDA Cooperative State 373

Research, Education, and Extension Service. 374

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 23

REFERENCES 375

376

1. Aitken, C. M., D. M. Jones, and S. R. Larter. 2004. Anaerobic hydrocarbon 377

biodegradation in deep subsurface oil reservoirs. Nature 431:291-294. 378

2. Bertrand, J. C., M. Almallah, M. Acquaviva, and G. Mille. 1990. 379

Biodegradation of hydrocarbons by an extremely halophilic archaebacterium. 380

Lett. Appl. Microbiol. 11:260-263. 381

3. Bonch-Osmolovskaya, E. A., M. L. Miroshnichenko, A. V. Lebedinsky, N. A. 382

Chernyh, T. N. Nazina, V. S. Ivoilov, S. S. Belyaev, E. S. Boulygina, Y. P. 383

Lysov, A. N. Perov, A. D. Mirzabekov, H. Hippe, E. Stackebrandt, S. 384

L'Haridon, and C. Jeanthon. 2003. Radioisotopic, culture-based, and 385

oligonucleotide microchip analyses of thermophilic microbial communities in a 386

continental high-temperature petroleum reservoir. Appl. Environ. Microbiol. 387

69:6143-6151. 388

4. DeSantis, T. Z., P. Hugenholtz, N. Larsen, M. Rojas, E. L. Brodie, K. Keller, 389

T. Huber, D. Dalevi, P. Hu, and G. L. Andersen. 2006. Greengenes, a chimera-390

checked 16S rRNA gene database and workbench compatible with ARB. Appl. 391

Environ. Microbiol. 72:5069-5072. 392

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 24

5. DeSantis, T. Z., Jr., P. Hugenholtz, K. Keller, E. L. Brodie, N. Larsen, Y. M. 393

Piceno, R. Phan, and G. L. Andersen. 2006. NAST: a multiple sequence 394

alignment server for comparative analysis of 16S rRNA genes. Nucl. Acids Res. 395

34:W394-399. 396

6. Diaz, E., and M. A. Prieto. 2000. Bacterial promoters triggering biodegradation 397

of aromatic pollutants. Curr. Opin. Biotech. 11:467-475. 398

7. Ferreira, A. C., M. F. Nobre, E. Moore, F. A. Rainey, J. R. Battista, and M. S. 399

da Costa. 1999. Characterization and radiation resistance of new isolates of 400

Rubrobacter radiotolerans and Rubrobacter xylanophilus. Extremophiles 3:235-401

238. 402

8. Ferrero, M., E. Llobet-Brossa, J. Lalucat, E. Garcia-Valdes, R. Rossello-403

Mora, and R. Bosch. 2002. Coexistence of two distinct copies of naphthalene 404

degradation genes in Pseudomonas strains isolated from the western 405

Mediterranean region. Appl. Environ. Microbiol. 68:957-962. 406

9. Freemantle, M. 1999. What's that staff? Asphalt. Chem. Engineer. News 77:81. 407

10. Harris, J. M. 2001. Rancho La Brea : Death trap and treasure trove. (Natural 408

History Museum of Los Angeles County, Los Angeles). Terra 38:1-56. 409

11. Holmes, A. J., J. Bowyer, M. P. Holley, M. O'Donoghue, M. Montgomery, 410

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 25

and M. R. Gillings. 2000. Diverse, yet-to-be-cultured members of the 411

Rubrobacter subdivision of the Actinobacteria are widespread in Australian arid 412

soils. FEMS Microbio. Ecol. 33:111-120. 413

12. Huber, T., G. Faulkner, and P. Hugenholtz. 2004. Bellerophon; a program to 414

detect chimeric sequences in multiple sequence alignments. Bioinformatics 415

20:2317-2319. 416

13. Hughes, J. B., J. J. Hellmann, T. H. Ricketts, and B. J. M. Bohannan. 2001. 417

Counting the uncountable: Statistical approaches to estimating microbial 418

diversity. Appl. Environ. Microbiol. 67:4399-4406. 419

14. Imhoff, J. F. 2006. The Chromatiaceae, p. 846-873. In M. Dworkin, S. Falkow, 420

E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (ed.), The Prokaryotes, 3rd 421

ed, vol. 6. Springer, New York, N.Y. 422

15. Imhoff, J. F. 2006. The family Ectothiorhodospiraceae, p. 874-886. In M. 423

Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (ed.), 424

The Prokaryotes, 3rd ed, vol. 6. Springer, New York, N.Y. 425

16. Janczewski, D. N., N. Yuhki, D. A. Gilbert, G. T. Jerfferson, and S. J. 426

O'Brien. 1992. Molecular phylogenetic inference from saber toothed cat fossils 427

of Rancho La Brea. Proc. Nat. Acad. Sci. USA 89:9769-9773. 428

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 26

17. Jurgens, G., F. O. Glockner, R. Amann, A. Saano, L. Montonen, M. 429

Likolammi, and U. Munster. 2000. Identification of novel Archaea in 430

bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent 431

in situ hybridization. FEMS Microbiol. Ecol. 34:45-56. 432

18. Kleinsteuber, S., V. Riis, I. Fetzer, H. Harms, and S. Muller. 2006. Population 433

dynamics within a microbial consortium during growth on diesel fuel in saline 434

environments. Appl. Environ. Microbiol. 72:3531-3542. 435

19. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for 436

molecular evolutionary genetics analysis and sequence alignment. Brief. 437

Bioinform. 5:150-163. 438

20. L'Haridon, S., A. L. Reysenbacht, P. Glénat, D. Prieur, and C. Jeanthon. 439

1995. Hot subterranean biosphere in a continental oil reservoir. Nature 377:223-440

224. 441

21. Lalucat, J., A. Bennasar, R. Bosch, E. Garcia-Valdes, and N. J. Palleroni. 442

2006. Biology of Pseudomonas stutzeri. Microbiol. Mol. Biol. Rev. 70:510-547. 443

22. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt 444

and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John 445

Wiley & Sons, Ltd., Chicester, United Kingdom. 446

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 27

23. Lovley, D. R. 2000. Anaerobic benzene degradation. Biodegradation 11:107-116. 447

24. Loy, A., M. Horn, and M. Wagner. 2003. probeBase - an online resource for 448

rRNA-targeted oligonucleotide probes. Nucl. Acids Res. 31:514-516. 449

25. Magot, M., B. Ollivier, and B. K. C. Patel. 2000. Microbiology of petroleum 450

reservoirs. Antonie van Leeuwenhoek 77:103-116. 451

26. Orphan, V. J., L. T. Taylor, D. Hafenbradl, and E. F. Delong. 2000. Culture-452

dependent and culture-independent characterization of microbial assemblages 453

associated with high-temperature petroleum reservoirs. Appl. Environ. Microbiol. 454

66:700-711. 455

27. Rabus, R., M. Kube, J. Heider, A. Beck, K. Heitmann, F. Widdel, and R. 456

Reinhardt. 2005. The genome sequence of an anaerobic aromatic-degrading 457

denitrifying bacterium, strain EbN1. Arch. Microbiol. 183:27-36. 458

28. Ramos, J. L., E. Duque, M. T. Gallegos, P. Godyoy, M. I. Ramos-Gonzalez, 459

A. Rojas, W. Teran, and A. Segura. 2002. Mechanisms of solvent tolerance in 460

gram-negative bacteria. Ann. Rev. Microbiol. 56:743-768. 461

29. Roling, W. F. M., M. G. Milner, D. M. Jones, F. Fratepietro, R. P. J. 462

Swannell, F. Daniel, and I. M. Head. 2004. Bacterial community dynamics and 463

hydrocarbon degradation during a field-scale evaluation of bioremediation on a 464

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 28

mudflat beach contaminated with buried oil. Appl. Environ. Microbiol. 70:2603-465

2613. 466

30. Rueter, P., R. Rabus, H. Wilkes, F. Aeckersberg, F. A. Rainey, H. W. 467

Jannasch, and F. Widdel. 1994. Anaerobic oxidation of hydrocarbons in crude-468

oil by new types of sulfate-reducing bacteria. Nature 372:455-458. 469

31. Saitou, N., and M. Nei. 1987. The neighbor-joining method a new method for 470

reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425. 471

32. Schloss, P. D., and J. Handelsman. 2005. Introducing DOTUR, a computer 472

program for defining operational taxonomic units and estimating species 473

richness. Appl. Environ. Microbiol. 71:1501-1506. 474

33. Shinoda, Y., Y. Sakai, H. Uenishi, Y. Uchihashi, A. Hiraishi, H. Yukawa, H. 475

Yurimoto, and N. Kato. 2004. Aerobic and anaerobic toluene degradation by a 476

newly isolated denitrifying bacterium, Thauera sp strain DNT-1. Appl. Environ. 477

Microbiol. 70:1385-1392. 478

34. Singleton, D. R., M. A. Furlong, S. L. Rathbun, and W. B. Whitman. 2001. 479

Quantitative comparisons of 16S rRNA gene sequence libraries from 480

environmental samples. Appl. Environ. Microbiol. 67:4374-4376. 481

35. Spormann, A. M., and F. Widdel. 2000. Metabolism of alkylbenzenes, alkanes, 482

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 29

and other hydrocarbons in anaerobic bacteria. Biodegradation 11:85-105. 483

36. Taylor, P. M., and P. H. Janssen. 2005. Variations in the abundance and 484

identity of class II aromatic ring-hydroxylating dioxygenase genes in 485

groundwater at an aromatic hydrocarbon-contaminated site. Environ. Microbiol. 486

7:140-146. 487

37. Taylor, P. M., J. M. Medd, L. Schoenborn, B. Hodgson, and P. H. Janssen. 488

2002. Detection of known and novel genes encoding aromatic ring-489

hydroxylating dioxygenases in soils and in aromatic hydrocarbon-degrading 490

bacteria. FEMS Microbiol. Lett. 216:61-66. 491

38. Top, E. M., D. Springael, and N. Boon. 2002. Catabolic mobile genetic 492

elements and their potential use in bioaugmentation of polluted soils and waters. 493

FEMS Microbiol. Ecol. 42:199-208. 494

39. Tor, J. M., and D. R. Lovley. 2001. Anaerobic degradation of aromatic 495

compounds coupled to Fe(III) reduction by Ferroglobus placidus. Environ. 496

Microbiol. 3:281-287. 497

40. Usami, R., T. Fukushima, T. Mizuki, Y. Yoshida, A. Inoue, and K. Horikoshi. 498

2005. Organic solvent tolerance of halophilic archaea, Haloarcula strains: 499

Effects of NaCl concentration on the tolerance and polar lipid composition. J. 500

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 30

Biosci. Bioengin. 99:169-174. 501

41. Van der Meer, J. R. 1994. Genetic adaptation of bacteria to chlorinated 502

aromatic-compounds. FEMS Microbiol. Rev. 15:239-249. 503

42. Van der Meer, J. R., W. M. Devos, S. Harayama, and A. J. B. Zehnder. 1992. 504

Molecular mechanisms of genetic adaptation to xenobiotic compounds. 505

Microbiol. Rev. 56:677-694. 506

43. Van Hamme, J. D., A. Singh, and O. P. Ward. 2003. Recent advances in 507

petroleum microbiology. Microbiol. Mol. Biol. Rev. 67:503-549. 508

44. Ward, D. M., and T. D. Brock. 1978. Hydrocarbon biodegradation in 509

hypersaline environments. Appl. Environ. Microbiol. 35:353-359. 510

45. Ward, J. K., J. M. Harris, T. E. Cerling, A. Wiedenhoeft, M. J. Lott, M. D. 511

Dearing, J. B. Coltrain, and J. R. Ehleringer. 2005. Carbon starvation in 512

glacial trees recovered from the La Brea tar pits, southern California. Proc. Nat. 513

Acad. Sci. USA 102:690-694. 514

46. Widdel, F., and R. Rabus. 2001. Anaerobic biodegradation of saturated and 515

aromatic hydrocarbons. Curr. Opin. Biotechnol. 12:259-276. 516

47. Widmer, F., R. J. Seidler, P. M. Gillevet, L. S. Watrud, and G. D. Di 517

Giovanni. 1998. A highly selective PCR protocol for detecting 16S rRNA genes 518

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 31

of the genus Pseudomonas (sensu stricto) in environmental samples. Appl. 519

Environ. Microbiol. 64:2545-2553. 520

48. Xue, W. L., and D. Warshawsky. 2005. Metabolic activation of polycyclic and 521

heterocyclic aromatic hydrocarbons and DNA damage: A review. Toxicol. Appl. 522

Pharmacol. 206:73-93. 523

49. Zarda, B., D. Hahn, A. Chatzinotas, W. Schonhuber, A. Neef, R. I. Amann, 524

and J. Zeyer. 1997. Analysis of bacterial community structure in bulk soil by in 525

situ hybridization. Arch. Microbiol. 168:185-192. 526

50. Zhang, C. L., and G. N. Bennett. 2005. Biodegradation of xenobiotics by 527

anaerobic bacteria. Appl. Microbiol. Biotechnol. 67:600-618. 528

51. Zvyagintseva, I. S., S. S. Belyaev, I. A. Borzenkov, N. A. Kostrikina, E. I. 529

Milekhina, and M. V. Ivanov. 1995. Halophilic archaebacteria from the 530

Kalamkass oil field. Microbiology 64:67-71. 531

532

533

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 32

Figure Legends

FIG. 1. Phylogeny of halophilic Archaea identified in asphalt soil mixture from Pit 101

of the Rancho La Brea Tar Pits. Genera are indicated by vertical bars on right of the tree

and values in parenthesis are clone numbers. The robustness of the topology was

estimated by bootstrap resampling of the neighbor joining method. Bootstrap values

greater than 75 are shown at branch points. Scale bar denotes 0.02 changes per

nucleotide.

FIG. 2. Phylogeny of the Bacteria from near full length 16S rRNA gene sequences

identified in heavy oil from in Pits 91 and 101 of the Rancho La Brea Tar Pits. Genera

are indicated by vertical bars on right of the tree and values in parenthesis are clone

numbers. The robustness of the topology was estimated by bootstrap resampling.

Bootstrap values greater than 75 are shown at branch points. Scale bar denotes change

per nucleotide. Proteobacteria in Pit 91(A) and Pit 101 (B), and other bacteria in Pit 91

(C) and Pit 101 (D). Accession numbers are indicated in parentheses on right of the tree.

Scale bar denotes 0.02 changes per nucleotide.

FIG. 3. Phylogeny of Pseudomonas stutzeri. 16S rRNA gene sequences identified in

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 33

asphalt from Pits 91 and 101 of the Rancho La Brea Tar Pits using PCR primers for

Pseudomonas sp. Candidate genomovars (gv) are indicated by vertical bars on right of

the tree. The robustness of the topology was estimated by bootstrap resampling of the

neighbor joining method. Bootstrap values greater than 50 are shown at branch points.

Scale bar denotes 0.005 changes per nucleotide.

FIG. 4. Phylogeny of cultured bacteria isolated from the Rancho La Brea Tar Pits.

Bootstrap values greater than 75 are shown. Bold letters indicate isolates were obtained

from Pit 91 or Pit 101. Scale bar denotes 0.02 changes per nucleotide.

FIG. 5. Microorganisms associated with heavy oil -soil as revealed by fluorescent in situ

hybridization (FISH) using a combination of dyes that stain different organisms. Cells

of the Archaea are stained red, Bacteria are yellow, and Pseudomonas (a genus of

Bacteria) is blue. The microbial colonies can be seen as clumps of different color cells

that are growing together, which suggests coexistence in diverse consortia.

FIG. 6. Rarefaction curves of observed OUT richness using the DOTUR (A) and

coverage calculated using the LIBSHUFF (B) from the two bacterial 16S rRNA gene

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 34

libraries in Pit 91 and Pit 101 of Rancho La Brea.

FIG. 7. Phylogeny of aromatic ring hydroxylating dioxygenases identified from

Rancho La Brea Tar Pits. (A) Pit 91; (B) Pit 101. Bootstrap values greater than 75 are

shown. Bold letters denote clones sampled from the asphalt-soil mixtures. Sequences

shown in regular typeface are known dioxygenases from GenBank used here for

classification. Enzyme classes are indicated by vertical bars at right side of the trees.

Scale bar indicates 0.05 changes per nucleotide.

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 35

Fig. 1.

A101-4

Arc12

Arc1

Arc14

A101-3

Arc6

Arc8

Arc16

Arc23

Arc3

Arc22

A101-2

Arc10

Arc4

Arc2

Arc28

Arc11

Arc26

Arc15

Arc18

Arc24

A101-1

Arc9

A101-5

Arc21

Arc7

Arc17

Natronorubrum sp. Wadi Natrun-19 (AB046926)

Arc29

Natronococcus amylolyticus Ah-36 (D43628)

N. occultus NCMB2192 (Z28378)

N. zabuyensis DS7 (AY433789)

Natrinema versiforme XF10(AB023426)

N. altunense AB3 (AY277583)

Natrialba aegyptiaca 40 (AF251941)

N. asiatica 172P1 (D14123)

Natronobacterium wudunaoensis Y21 (AJ001376)

N. chahannaoensis C112 (AJ004806)

N. innermongolia HAM-2 (AF009601)

Arc27

N. chagannuoerensis X21 (AJ003193)

Natronolimnobius innermongolicus N-1311 (AB125108)

Halobacterium salinarum DSM3754 (AJ496185)

Halorubrum saccharovorum NCIMB2081 (X82167)

Haloferax gibbonsii ATCC33959 (D13378)

Halogeometricum borinquense PR3 (AF002984)

Halobaculum gomorrense DSM9297 (L37444)

Haloarcula hispanica ATCC33960 (U68541)

Halosimplex carlsbadense ATCC BAA-75 (AF320478)

Halorhabdus utahensis AX-2 (AF071880)

Methanofollis formosanus ML15 (AY186542)

Thermoplasma volcanium GSS1 (AJ299215)

Methanobacterium formicicum DSMZ1535 (AF169245)

Methanococcus vannielii SB (AY196675)

Archaeoglobus profundus 234 (AF322392)

Thermococcus celer DSM2476 (AY099174)

Pyrococcus woesei DSM3773 (AY519654)100

100

82

100

100

99

99

91

85

99

99

88

91

76

99

100

0.02

Unclassified genus 1 (16)

Natronococcus (1)


Natronobacterium (1)

Halobacteriaceae (family)

A101-4

Arc12

Arc1

Arc14

A101-3

Arc6

Arc8

Arc16

Arc23

Arc3

Arc22

A101-2

Arc10

Arc4

Arc2

Arc28

Arc11

Arc26

Arc15

Arc18

Arc24

A101-1

Arc9

A101-5

Arc21

Arc7

Arc17


Arc29











Arc27


















100

82

100

100

99

99

91

85

99

99

88

91

76

99

100

0.02

A101-4

Arc12

Arc1

Arc14

A101-3

Arc6

Arc8

Arc16

Arc23

Arc3

Arc22

A101-2

Arc10

Arc4

Arc2

Arc28

Arc11

Arc26

Arc15

Arc18

Arc24

A101-1

Arc9

A101-5

Arc21

Arc7

Arc17


Arc29











Arc27


















100

82

100

100

99

99

91

85

99

99

88

91

76

99

100

0.02


Natronococcus (1)


Natronobacterium (1)

Halobacteriaceae (family)ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 36

Fig. 2(A)

Alkalilimnicola halodurans (AJ404972)Thialkalivibrio denitrificans (AF126545)

Ectothiorhodosinus mongolicum (AY299804)Ectothiorhodospira shaposhnikovii (M59151)

Thiobaca trueperi (AJ404006)Thiocystis violacea (Y11315)

Chromatium okenii (AJ223234)

Salinisphaera shabanensis (AJ421425)91-44

91-7091-48

Xanthomonas vasicola (Y10755)X. sacchari (Y10766)X. melonis (Y10756)

E91-46Lysobacter enzymogenes (AJ298291)

91-80Nevskia ramosa (AJ001010)

Halomonas pantelleriensis (X93493)H. muralis (AJ320530)

H. sulfidaeris (AF212204)Marinobacter flavimaris (AY517632)

Cellvibrio japonicus (AF452103)Pseudomonas veronii (AF064460)

P. anguilliseptica (X99540)P. balearica (U26418)

91-143P. stutzeri (U26262)

91-2091-105

P. chloritidismutans (AY017341)91-2591-101

91-14191-112

91-6391-49

Hydrogenophaga palleronii (AF019073)91-50

Caenibacterium thermophilum (AJ512945)Pandoraea pulmonicola (AF139175)

Massilia timonae (U54470)Pigmentiphaga kullae (AF282916)

91-41Sterolibacterium denitrificans (AJ306683)

Thermodesulforhabdus norvegica (U25627)91-45

Rhodovibrio sodomensis (M59072)91-58

Candidatus Devosia euplotis (AJ548825)E91-34

Azospirillum doebereinerae (AJ238567)Skermanella parooensis (X90760)

Inquilinus limosus (AY043374)E91-35

91-82Phaeospirillum fulvum (D14433)

91-7891-102

E91-4391-118

Porphyrobacter donghaensis (AY559428)Sphingomonas asaccharolytica (Y09639)

S. oligophenolica (AB018439)91-23

Sandaracinobacter sibiricus (Y10678)91-4

91-1891-54

91-12591-9

91-151Roseovarius tolerans (Y11551)

Parvibaculum lavamentivorans (AY387398)Brevundimonas subvibrioides (AJ227784)

B. vesicularis (AJ007801 )Methylosinus sporium (Y18946)

Mesorhizobium tianshanense (AF041447)Rhizobiales bacterium VKM-B1336 (AJ542534)

91-124Rhizobium galegae (X67226)

R. huautlense (AF025852)

100

100100

98

100

100

78

100

88

100

100

100

100

100

98

100

81

100

79

97

98

100

99

98

84

73

98

95

86

94

74

99

100

98

100

100

100

100

100

82

100

77

96

97

99

94

8794

98

100

0.02

γ (70)

Chromatiales (60)

Chromatiaceae

Ectothiorhodospiraceae

Unclassified family 1 (42)



Xanthomonadaceae (5)

Pseudomonadaceae (5)

β (6)

θ (1)

α (16)

Rhizobiales (1)

Sphingomonadales (7)

Rhodobacteraceae (4)

Rhodospirillaceae (3)


Burkholderiales (3)

Rhodocyclales (1)






91-7091-48








91-143P. stutzeri (U26262)

91-2091-105


91-14191-112

91-6391-49











91-7891-102

E91-4391-118




91-1891-54

91-12591-9







100

100100

98

100

100

78

100

88

100

100

100

100

100

98

100

81

100

79

97

98

100

99

98

84

73

98

95

86

94

74

99

100

98

100

100

100

100

100

82

100

77

96

97

99

94

8794

98

100

0.02






91-7091-48








91-143P. stutzeri (U26262)

91-2091-105


91-14191-112

91-6391-49











91-7891-102

E91-4391-118


S. oligophenolica (AB018439)






91-7091-48








91-143P. stutzeri (U26262)

91-2091-105


91-14191-112

91-6391-49











91-7891-102

E91-4391-118




91-1891-54

91-12591-9







100

100100

98

100

100

78

100

88

100

100

100

100

100

98

100

81

100

79

97

98

100

99

98

84

73

98

95

86

94

74

99

100

98

100

100

100

100

100

82

100

77

96

97

99

94

8794

98

100

0.02

γ (70)

Chromatiales (60)

Chromatiaceae

Ectothiorhodospiraceae






β (6)

θ (1)

α (16)

Rhizobiales (1)





Burkholderiales (3)

Rhodocyclales (1)

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 37

Fig. 2(B)


Halomonadaceae (5)

Chromatiales (3)


β (8)

Caulobacteraceae (4)

Rhizobiales (5)




Unclassified family 3 (5)Unclassified order (6)


101-108101-210

E101-47101-71

101-203Pseudomonas chloritidismutans (AY017341)

101-194P. stutzeri (U26262)101-7

P. balearica (U26418)P. veronii (AF064460)P. anguilliseptica (X99540)

101-139101-13

101-209E101-9

Marinobacter flavimaris (AY517632)101-107Halomonas sulfidaeris (AF212204)101-159H. pantelleriensis (X93493)

H. muralis (AJ320530)101-11101-120

101-138101-155

Cellvibrio japonicus (AF452103)101-80Thiobaca trueperi (AJ404006)

Chromatium okenii (AJ223234)Thiocystis violacea (Y11315)


Thialkalivibrio denitrificans (AF126545)101-185

101-178Alkalilimnicola halodurans (AJ404972)

101-208Salinisphaera shabanensis (AJ421425)


101-95101-5

Lysobacter enzymogenes (AJ298291)Xanthomonas vasicola (Y10755)X. sacchari (Y10766)X. melonis (Y10756)

101-112101-134Massilia timonae (U54470)

E101-4Sterolibacterium denitrificans (AJ306683)

101-39Hydrogenophaga palleronii (AF019073)

Caenibacterium thermophilum (AJ512945)101-201

Pandoraea pulmonicola (AF139175)101-19

101-68Pigmentiphaga kullae (AF282916)

101-92101-142



Inquilinus limosus (AY043374)101-114Rhodovibrio sodomensis (M59072)

101-193101-213

101-99101-31

101-73Sandaracinobacter sibiricus (Y10678)

101-127Porphyrobacter donghaensis (AY559428)E101-6Sphingomonas asaccharolytica (Y09639)

S. oligophenolica (AB018439)E101-25

101-43Parvibaculum lavamentivorans (AY387398)

Roseovarius tolerans (Y11551)101-189

Methylosinus sporium (Y18946)101-16

Candidatus Devosia euplotis (AJ548825)Rhizobium galegae (X67226)R. huautlense (AF025852)

101-116101-196

E101-27Mesorhizobium tianshanense (AF041447)Rhizobiales bacteriumVKM-B1336 (AJ542534)

101-153101-65

Brevundimonas vesicularis (AJ227784 )B. subvibrioides (AJ007801)

101-140101-126

99

99

99

99

99

98

98

99

99

99

87

89

84

99

91

8090

87

99

99

99

99

99

98

99

8499

99

99

93

99

99

99

99

94

99

99

99

99

99

94

99

92

99

99

90

99

90

92

97

87

89

98

86

75

0.02

Burkholderiales (6)


Ectothiorhodo

-spiraceae (2)

Chromatiaceae (1)

α (26)

γ (24)


Halomonadaceae (5)

Chromatiales (3)


β (8)

Caulobacteraceae (4)

Rhizobiales (5)




Unclassified family 3 (5)Unclassified order (6)


101-108101-210

E101-47101-71

101-203Pseudomonas chloritidismutans (AY017341)

101-194P. stutzeri (U26262)101-7

P. balearica (U26418)P. veronii (AF064460)P. anguilliseptica (X99540)

101-139101-13

101-209E101-9

Marinobacter flavimaris (AY517632)101-107Halomonas sulfidaeris (AF212204)101-159H. pantelleriensis (X93493)

H. muralis (AJ320530)101-11101-120

101-138101-155

Cellvibrio japonicus (AF452103)101-80Thiobaca trueperi (AJ404006)

Chromatium okenii (AJ223234)Thiocystis violacea (Y11315)


Thialkalivibrio denitrificans (AF126545)101-185

101-178Alkalilimnicola halodurans (AJ404972)

101-208Salinisphaera shabanensis (AJ421425)


101-95101-5

Lysobacter enzymogenes (AJ298291)Xanthomonas vasicola (Y10755)X. sacchari (Y10766)X. melonis (Y10756)

101-112101-134Massilia timonae (U54470)

E101-4Sterolibacterium denitrificans (AJ306683)

101-39Hydrogenophaga palleronii (AF019073)

Caenibacterium thermophilum (AJ512945)101-201

Pandoraea pulmonicola (AF139175)101-19

101-68Pigmentiphaga kullae (AF282916)

101-92101-142



Inquilinus limosus (AY043374)101-114Rhodovibrio sodomensis (M59072)

101-193101-213

101-99101-31

101-73Sandaracinobacter sibiricus (Y10678)

101-127Porphyrobacter donghaensis (AY559428)E101-6Sphingomonas asaccharolytica (Y09639)

S. oligophenolica (AB018439)E101-25

101-43Parvibaculum lavamentivorans (AY387398)

Roseovarius tolerans (Y11551)101-189

Methylosinus sporium (Y18946)101-16

Candidatus Devosia euplotis (AJ548825)Rhizobium galegae (X67226)R. huautlense (AF025852)

101-116101-196

E101-27Mesorhizobium tianshanense (AF041447)Rhizobiales bacteriumVKM-B1336 (AJ542534)

101-153101-65

Brevundimonas vesicularis (AJ227784 )B. subvibrioides (AJ007801)

101-140101-126

99

99

99

99

99

98

98

99

99

99

87

89

84

99

91

8090

87

99

99

99

99

99

98

99

8499

99

99

93

99

99

99

99

94

99

99

99

99

99

94

99

92

99

99

90

99

90

92

97

87

89

98

86

75

0.02

Burkholderiales (6)


Ectothiorhodo

-spiraceae (2)

Chromatiaceae (1)

α (26)

γ (24)

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 38

Fig. 2(C)

91-13291-72

91-7791-2891-60

91-14Agreia pratensis (AJ310412)E91-36

Leifsonia rubra (AJ438585)91-14691-17Terrabacter tumescens (X53215)Streptosporangium roseum (X70425)

91-6991-98Pseudonocardia alni (X76954)

Crossiella equi (AF245017)Acidimicrobium ferrooxidans (U75647)

Conexibacter woesei (AJ440237)Thermoleiphilum minutum (AJ458464)

Rubrobacter radiotolerans (X87134)Desulfotomaculum luciae (AF069293)

Moorella thermoautotrophica (L09168)91-13091-120

91-133Caloramator fervidus (L09187)

Nitrospira moscoviensis (X82558)Gemmatimonas aurantiaca (AB072735)

91-8891-131

Verrucomicrobium spinosum (X90515)91-144

Planctomyces brasiliensis (AJ231190)Planctomyces maris (AJ231184)

Pirellula staleyi (AJ231183)Pirellula marina (X62912)

Holophaga foetida (X77215)E91-12

E91-3991-113

91-103Gillisia mitskevichiae (AY576655)

Arenibacter troitsensis (AB080771)Robiginitalea biformata (AY424899)

91-95Pedobacter cryoconitis (AJ438170)

E91-1991-3291-10491-139

91-111E91-11

100100

100

100

100

89100

100

100

91

100

100

100

93

99

92

99

78

96

87

9585

79

100 96

99

96

99

100

0.02

Actinobacteria (11)

Clostridia (3)

TM7 (2)

Cyanobacteria (1)

Acidobacteria (1)

Bacteroidetes (9)

TM6 (1)

91-13291-72

91-7791-2891-60










91-8891-131





E91-3991-113




E91-1991-3291-10491-139

91-111E91-11

100100

100

100

100

89100

100

100

91

100

100

100

93

99

92

99

78

96

87

9585

79

100 96

99

96

99

100

0.02

91-13291-72

91-7791-2891-60










91-8891-131





E91-3991-113




E91-1991-3291-10491-139

91-111E91-11

100100

100

100

100

89100

100

100

91

100

100

100

93

99

92

99

78

96

87

9585

79

100 96

99

96

99

100

0.02

Actinobacteria (11)

Clostridia (3)

TM7 (2)

Cyanobacteria (1)

Acidobacteria (1)

Bacteroidetes (9)

TM6 (1)

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 39

Fig. 2(D)

101-135Pseudonocardia alni (X76954)

101-195Crossiella equi (AF245017)

Streptosporangium roseum (X70425)Terrabacter tumescens (X53215)

101-10101-48

Agreia pratensis (AJ310412)Leifsonia rubra (AJ438585)

Acidimicrobium ferrooxidans (U75647)101-169

101-172101-29

101-26101-151

101-180Thermoleiphilum minutum (AJ458464)

101-212Conexibacter woesei (AJ440237)

101-104101-124

101-103101-79

R. radiotolerans (X87134)101-3

101-179101-49101-156101-1101-64101-161101-181101-188

101-12101-168101-70

101-52101-98

Gemmatimonas aurantiaca (AB072735)101-85

101-50101-109

101-89Geothrix fermentans (U41563)

Holophaga foetida (X77215)E101-54101-207


Moorella thermoautotrophica (L09168)Desulfotomaculum luciae (AF069293)

101-192101-191

BRC1 uncultured bacterium za47 (AJ604544)101-106

Nitrospira moscoviensis (X82558)101-147

101-197101-154

101-83V. spinosum (X90515)

101-74E101-2

Pirellula staleyi (AJ231183)101-75

Pirellula marina (X62912)101-18

101-66Planctomyces maris (AJ231184)Planctomyces brasiliensis (AJ231190)

E101-45101-111101-122101-121


Robiginitalea biformata (AY424899)101-198

Gillisia mitskevichiae (AY576655)101-146

101-58Arenibacter troitsensis (AB080771)

99

99

99

99

99

99

99

99

99

90

85

99

99

99

99

99

99

99

99

99

9999

99

99

99

99

99

97

98

99

87

99

99

82

94

86

91

97

92

97

83

97

85

0.02

Actinobacteria

(27)

Rubrobacteraceae

(16)

Gemmatimonadetes

(6)

Acidobacteria (2)

Clostridia (3)

BRC1 (1)

Nitrospira (2)

Verrucomicrobia

(2)

Planctomycetes

(9)

Bacteroidetes

(4)




101-10101-48



101-172101-29

101-26101-151



101-104101-124

101-103101-79


101-179101-49101-156101-1101-64101-161101-181101-188

101-12101-168101-70

101-52101-98


101-50101-109





101-192101-191



101-197101-154

101-83V. spinosum (X90515)

101-74E101-2




E101-45101-111101-122101-121





99

99

99

99

99

99

99

99

99

90

85

99

99

99

99

99

99

99

99

99

9999

99

99

99

99

99

97

98

99

87

99

99

82

94

86

91

97

92

97

83

97

85

0.02




101-10101-48



101-172101-29

101-26101-151



101-104101-124

101-103101-79


101-179101-49101-156101-1101-64101-161101-181101-188

101-12101-168101-70

101-52101-98


101-50101-109





101-192101-191



101-197101-154

101-83V. spinosum (X90515)

101-74E101-2



101-66




101-10101-48



101-172101-29

101-26101-151



101-104101-124

101-103101-79


101-179101-49101-156101-1101-64101-161101-181101-188

101-12101-168101-70

101-52101-98


101-50101-109





101-192101-191



101-197101-154

101-83V. spinosum (X90515)

101-74E101-2




E101-45101-111101-122101-121





99

99

99

99

99

99

99

99

99

90

85

99

99

99

99

99

99

99

99

99

9999

99

99

99

99

99

97

98

99

87

99

99

82

94

86

91

97

92

97

83

97

85

0.02

Actinobacteria

(27)

Rubrobacteraceae

(16)

Gemmatimonadetes

(6)

Acidobacteria (2)

Clostridia (3)

BRC1 (1)

Nitrospira (2)

Verrucomicrobia

(2)

Planctomycetes

(9)

Bacteroidetes

(4)

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 40

Fig. 3.

P.stutzeri AN10 (U22427; gv. 3)

P.stutzeri KC (AF067960; gv. 9)

P.stutzeri MW56 (AJ006108; gv


P91-14

P.stutzeri DSM50227 (U26415; gv. 3)

P.stutzeri 4C29 (AJ270456; gv 15)

P.stutzeri CLN100 (AJ544240; gv. 10)

P.stutzeri 28a22 (AJ312167; gv. 13)

P91-2

P101-20

P91-21

P91-31

P91-32

P91-22




P91-13

P.stutzeri MT-1 (AB004241; gv. 18)


P.stutzeri JM300 (X98607; gv. 8)

P.stutzeri ATCC17641 (AJ006106; gv. 8)

P91-4

P91-35

P91-12

P.stutzeri DNSP21 (U26414; gv. 5)

P91-15

P91-1

P91-18

P.stutzeri ATCC17591 (U26261; gv. 2)



P91-3


P.stutzeri DSM6084 (AJ005167; gv. 4)


P.stutzeri ZoBell (U26420; gv. 2)

P.stutzeri ST27MN3 (U26419; gv. 4)




P.stutzeri CCUG11256 (U26262; gv. 1)


P.chlororaphis IAM12354 (D84011)

P.putida IAM1236 (D84020)

P.fluorescens IAM12022 (D84013)99

97

96

0.005

19

20

21

22

2324

26

25

Candidate genomovars

54

59

50

61

66

60



P.stutzeri MW56 (AJ006108; gv. 3)


P91-14


P.stutzeri 4C29 (AJ270456; gv.15)



P91-2

P101-20

P91-21

P91-31

P91-32

P91-22




P91-13





P91-4

P91-35

P91-12


P91-15

P91-1

P91-18




P91-3













P.fluorescens99

97

96

0.005

19

20

21

22

2324

26

25


54

59

50

61

66

60



P.stutzeri MW56 (AJ006108; gv


P91-14


P.stutzeri 4C29 (AJ270456; gv 15)



P91-2

P101-20

P91-21

P91-31

P91-32

P91-22




P91-13





P91-4

P91-35

P91-12


P91-15

P91-1

P91-18




P91-3













P.fluorescens IAM12022 (D84013)99

97

96

0.005

19

20

21

22

2324

26

25


54

59

50

61

66

60



P.stutzeri MW56 (AJ006108; gv. 3)


P91-14


P.stutzeri 4C29 (AJ270456; gv.15)



P91-2

P101-20

P91-21

P91-31

P91-32

P91-22




P91-13





P91-4

P91-35

P91-12


P91-15

P91-1

P91-18




P91-3













P.fluorescens99

97

96

0.005

19

20

21

22

2324

26

25


54

59

50

61

66

60

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 41

Fig 4.

I91-4Pseudomonas mendocina (Z76664)

I91-9P. mendocina (M59154)

P. pseudoalcaligenes (Z76666)I101-5I91-7P. alcaliphila (AB030583)

I91-8P. putida (D37923)

I91-2P. stutzeri (U26262)P. chloritidismutans (AY017341)

P. balearica (U26418)I91-5

Citrobacter freundii (AJ233408)C. braakii (AF025368)C. murliniae (AF025369)I101-10I91-1I91-3I91-6

I91-10Gracilibacillus dipsosauri (X82436)

Na101-1Na101-2

Na101-4Filobacillus milosensis (AJ238042)

Tenuibacillus multivorans (AY319933)Bacillus halodenitrificans (AY543168)

B. firmus (X60616)B. shackletonii (AJ250318)

B. circulans (X60613)B. vireti (AJ542509)

B. psychrosaccharolyticus (X60635)I101-8B. simplex (X60638)

I101-4I101-1I101-2I101-7I101-6B. muralis (AJ628748)I101-3I101-9

95

100

92

75

92

100

100

99

99

100

100

99

0.02

Pseudomonas

Citrobacter

Bacillus

I91-4Pseudomonas mendocina (Z76664)

I91-9P. mendocina (M59154)

P. pseudoalcaligenes (Z76666)I101-5I91-7P. alcaliphila (AB030583)

I91-8P. putida (D37923)

I91-2P. stutzeri (U26262)P. chloritidismutans (AY017341)

P. balearica (U26418)I91-5

Citrobacter freundii (AJ233408)C. braakii (AF025368)C. murliniae (AF025369)I101-10I91-1I91-3I91-6

I91-10Gracilibacillus dipsosauri (X82436)

Na101-1Na101-2

Na101-4Filobacillus milosensis (AJ238042)

Tenuibacillus multivorans (AY319933)Bacillus halodenitrificans (AY543168)

B. firmus (X60616)B. shackletonii (AJ250318)

B. circulans (X60613)B. vireti (AJ542509)

B. psychrosaccharolyticus (X60635)I101-8B. simplex (X60638)

I101-4I101-1I101-2I101-7I101-6B. muralis (AJ628748)I101-3I101-9

95

100

92

75

92

100

100

99

99

100

100

99

0.02

Pseudomonas

Citrobacter

BacillusACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 42

Fig. 5.

Archaea

Bacteria

Pseudomonas

Archaea

Bacteria

Pseudomonas

10 µM

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 43

Fig. 6(A)

0

20

40

60

80

100

0 20 40 60 80 100 120

Number of Sequences Sampled

Nu

mb

er

of

OT

Us

Ob

se

rved

10% difference

5% difference

3% difference

No difference Pit 91

Pit 101

0

20

40

60

80

100

0 20 40 60 80 100 120

Number of Sequences Sampled

Nu

mb

er

of

OT

Us

Ob

se

rved

10% difference

5% difference

3% difference

No difference Pit 91

Pit 101

Pit 91

Pit 101

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 44

Fig. 6(B)

0

0.2

0.4

0.6

0.8

1

0 0.05 0.1 0.15 0.2 0.25 0.3

Evolutionary Distance (D)

Co

vera

ge (

C)

Pit 91

Pit 101

0

0.2

0.4

0.6

0.8

1

0 0.05 0.1 0.15 0.2 0.25 0.3

Evolutionary Distance (D)

Co

vera

ge (

C)

Pit 91

Pit 101

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 45

Fig. 7(A)

0.05

Proteobacterial

biphenyl dioxygenase

Pseudomonas

monoaromatic dioxygenase

Pseudomonas

polyaromatic dioxygenase

T clsuter

Rhodococcus


Unknown dioxygenase I

Unknown dioxygenase II

Unknown dioxygenase III

S/T clsuter

S clsuter

Tar91-4Tar91-6

Tar91-73Tar91-64

P.pseudoalcaligenes KF707 [M83673]bphA1b-biphenylTar91-58

Tar91-75Tar91-54

Tar91-63Comamonas testosteroni B-356 [U47637]bphA-biphenyl/chlorobiphenyl

Tar91-2Pseudomonas sp. KKS102 [D17319]biphenyl

Tar91-59Tar91-9Tar91-51

Rhodococcus globerulus P6 [X80041]bphA1-biphenylP.putida BE81 [M17904]benzeneP.putida F1 [J04996]todC1-toluene

Pseudomonas sp. [U15298]tcbAa-chlorobenzeneTar91-66Tar91-69Tar91-70Tar91-71Tar91-72mw0-108 [AF400543]

Tar91-1Tar91-8

mw8-121 [AF400540]Tar91-67

R.erythropolis TA421 [D88021]bphC-biphenylmw0-112 [AF400544]

P.putida G7 [M83949]nahAc-naphthaleneP.putida NCBI9816 [M23914]ndoB-naphthalene100

100

97

100

100

88

94

99

99

80

97

9999

100

97

0.05

Proteobacterial


Pseudomonas


Pseudomonas


T clsuter

Rhodococcus



Unknown dioxygenase II

Unknown dioxygenase III

S/T clsuter

S clsuter

Tar91-4Tar91-6

Tar91-73Tar91-64


Tar91-75Tar91-54



Tar91-59Tar91-9Tar91-51



Tar91-1Tar91-8

mw8-121 [AF400540]Tar91-67



100

97

100

100

88

94

99

99

80

97

9999

100

97

Tar91-4Tar91-6

Tar91-73Tar91-64


Tar91-75Tar91-54



Tar91-59Tar91-9Tar91-51



Tar91-1Tar91-8

mw8-121 [AF400540]Tar91-67



100

97

100

100

88

94

99

99

80

97

9999

100

97

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 46

Fig. 7(B)

0.05

Pseudomonas


Pseudomonas


Rhodococcus



S/T clsuter

S clsuter

Proteobacterial


T clsuter

Tar101-83Tar101-69

Tar101-90Tar101-94Tar101-2Tar101-54Tar101-59Tar101-9Tar101-1Tar101-77

Tar101-65Tar101-92Tar101-78

Tar101-63Tar101-60Tar101-61Tar101-58Tar101-55

Pseudomonas sp. [U15298]tcbAa-chlorobenzeneP.putida BE81 [M17904]benzeneP.putida F1 [J04996]todC1-tolueneR.globerulus P6 [X80041] bphA1-biphenyl

mw0-108 [AF400543]

Pseudomonas sp. KKS102 [D17319]biphenylC.testosteroni B-356 [U47637]bphA-biphenyl/chlorobiphenylP.pseudoalcaligenes KF707 [M83673]bphA1b-biphenyl

Tar101-53Tar101-86

mw8-121 [AF400540]R.erythropolis TA421 [D88021]bphC-biphenyl

mw0-112 [AF400544]P.putida G7 [M83949]nahAc-naphthalene

P.putida NCBI9816 [M23914]ndoB-naphthalene99

98

99

99

78

92

91

99

0.05

Pseudomonas


Pseudomonas


Rhodococcus



S/T clsuter

S clsuter

Proteobacterial


T clsuter

Tar101-83Tar101-69


Tar101-65Tar101-92Tar101-78



mw0-108 [AF400543]


Tar101-53Tar101-86




98

99

99

78

92

91

99

Tar101-83Tar101-69


Tar101-65Tar101-92Tar101-78



mw0-108 [AF400543]


Tar101-53Tar101-86




98

99

99

78

92

91

99

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 47

Table 1. Chemical properties of asphalt impermeated soil samples from Pit 91 and Pit 101 of Rancho La Brea Tar Pits. 1

Sample pH EC C N S C/N Na Ca Mg K Al Cd Cr Fe Zn Cu Mn Pb Ni

site 1:1(CaCl2) 1:5(H2O) (µs/cm) (%) ratio (µg/g)

Pit 91 6.3 5.36 46 22.04 0.3 0.97 74.16 13 2902 265 86.1 356 2.2 2.65 484 33.9 23.4 13 ND* 89.9

Pit 101 8.44 7.59 4610 7.04 0.18 0.53 46.74 178 3696 4245 1011 11395 1.52 19.9 14133 126 37.1 178 58.1 39.4

ND: not detected2

ACCEPTED on January 26, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/

Page 48

Table 2. Richness and diversity estimations in 16S rRNA gene libraries from Pit 91 and 3

Pit 101 of La Brea Tar Pit. 4

Richness Diversity

Library OTU ACE Boot Chao1 Jack Shannon Simpson Coverage

Pit 91 37 64 46 58 58 2.8 0.139 0.8

Pit 101 80 268 105 242 265 4.2 0.014 0.455

6

7

ACCEPTED


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Date post:	04-Jan-2020
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Title Microbial Diversity in Natural Asphalts of the ...24 such as those that occur in oil sands,...

Documents