New Cultivation-Based Approaches for Mining the Metabolic Potential of Microorganisms

New cultivation-based approaches for mining the metabolic potential of microorganisms

Karsten Zengler, Diversa Corporation The Introduction of Solid, Defined Media and Pure-Culture Methods Marked a True Revolution*

* from: Biology of the Procaryotes; Lengeler, Drews, Schlegel The Theory…

* from: What's so funny about microbiology? / Joachim Czichos The Reality…

Rich mediumR i Minimal c medium

* from: What's so funny about microbiology? / Joachim Czichos Laboratory conditions (media) are far off from the environment

Concentration of nutrients Media for Organics Seawater Heterotrophs

Peptone – 0.5 – 10 g/L Dextrose – 0.5 – 2 g/L Yeast extract – 0.3 – 5 g/L Meat extract – 0.5 – 10 g/L Pyruvate ~ng/L x 1010 0.3 – 1 g/L Succinate ~ng/L 0.5 – 1 g/L Glucose ~ng/L 0.5 – 20 g/L Amino acids ng/L – µg/L 0.5 – 10 g/L Some bacteria don’t like to live alone

* from: What's so funny about microbiology? / Joachim Czichos Verrucomicrobia 7 vadinBE97 Chlamydiae 13 OP3 Pl anctom ycet es 9 The bacterial world as of today… WS 3 BRC1 NKB19 Firmicutes 1205 OP9 Phyla with cultivated representatives: 27 WS 2 Cyanobacteria 4 Phyla without cultivated representatives: 26 Fusobacteria 25 OP10 SC4 Actinobacteria 1367 NC10 Bacteroidetes 220 Chlorobi 8 Marine Group A Caldithrix 1 Gemmatimonadetes 1 Fibrobacteres 2 Proteobacteria 1808 Deferribacteres 7 Chrysiogenes arsenatis 1 SB R1093 Acidobacteria 3 OP8 OS-K Nitrospirae 6 Termite Group 1 TM6 Synergistes 1 OP5 Spir och aet es 91 ABY1 BD1-5 group OP11 WS 6 TM7 Guaymas1 WS 5 SC3 Chloroflexi 11 Deinococcus-Thermus 24 Thermodesulfobacteria 2 OP1 Thermotogae 25 Coprothermobacter 2 Dictyoglomi 2 Aquificae 13 Desulfurobacterium 1

0. 05 Verrucomicrobia 7 vadinBE97 Chlamydiae 13 OP3 Planctomycetes 9 The bacterial world as of today… WS 3 BRC1 NKB19 Firmicutes 1205 OP9 Phyla with cultivated representatives: 27 WS 2 Cyanobacteria 4 Phyla without cultivated representatives: 26 Fusobacteria 25 OP10 SC4 Actinobacteria 1367 NC10 Bacteroidetes 220 Chlorobi 8 Marine Group A Caldithrix 1 Gemmatimonadetes 1 Fibrobacteres 2 Proteobacteria 1808 others Deferribacteres 7 Chrysiogenes arsenatis 1 5% SB R1093 Firmicutes Acidobacteria 3 25% OP8 OS-K Proteobacteria Nitrospirae 6 Termite Group 1 37% TM6 Synergistes 1 OP5 Spir och aet es 91 Actinobacteria ABY1 Bacteroidetes BD1-5 group 5% 28% OP11 WS 6 TM7 Guaymas1 WS 5 SC3 Chloroflexi 11 Deinococcus-Thermus 24 Thermodesulfobacteria 2 OP1 Thermotogae 25 Coprothermobacter 2 Dictyoglomi 2 Aquificae 13 Desulfurobacterium 1

0. 05 Development of a Novel Cultivation Approach

How can we grow more than the microbial weeds?

Do we have to move away from the traditional understanding of a pure culture? Development of a Novel Cultivation Approach

• novel

• easy to automate

• universal

• detection of small amount of cells – 100 cells

• high-throughput

• low organic media

• cultivation together and apart FACS Technology

• Premier Diversa screening platform

• 3-Dimensional, single cell format

• Screening rates of 50,000 cells/second

• Multiple Screening systems

• expression

• hybridization

• bioassays

• Encapsulation program Sorting of Single and Multiple Occupied Microcapsules

Multiple occupied Microcapsules

Single occupied Microcapsules

Free Bacteria

Traditional:

1. Grow 2. Isolate Traditional:

1. Grow 2. Isolate

HTC:

1. Isolate 2. Grow Cultivation of Sargasso Sea Microbes

Collect Environmental Sample Output Constant Media Flow

4 Different Media

Microcapsules with a Variety of Bacterial Purify Cells Colonies (Mixed Population) Input

Encapsulate Individual Pack Column Cells in with Microcapsules Microcapsules

4 different types of media: • Marine rich media 1/100 • Sea water + amino acids • Sea water + inorganic nutrients • Sea water Microcolonie in Marine Rich Medium after 5 Weeks Microcolonies in Pure Seawater after 5 Weeks

50 µm High Throughput Cultivation - Sargasso Sea

GMD21C08 a GMD14H10 GMD14H07

uncultured clone CD4H6, coral reef (AY038494) uncultured clone CD4D8, coral reef (AY038561) uncultured clone CL120-56, freshwater Crater Lake (AF316766) uncultured clone CL500-3, freshwater Crater Lake (AF316767)

GMD16E07 GMD15D02 uncultured clone #0319-7F4, arid soil (AF234144) uncultured clone CLEAR-30, marine salinity lake sediments (AF146252) uncultured clone MERTZ_21CM_160, Antarctic shelf sediments (Af424489) uncultured clone MERTZ_21CM_213, Antarctic shelf sediments (AF424493) uncultured clone BD2-16, deep sea sediments (AB015544) uncultured clone ACE-29, marine salinity lake sediments (AF142804) uncultured clone Sva0503, marine sediments (AJ241009) Planctomyces/Pirellula

Isosphaera/Gemmata

uncultured clone CL500-15, freshwater Crater Lake (AF316773) uncultured clone OM190, Atlantic coastal seawater (U70712) Candidatus 'Brocadia anammoxidans' (AF375994) Candidatus 'Kuenenia stuttgartiensis' (AF375995)

0.10

Planctomyces and Relatives Verrucomicrobia and Chlamydiae sequences were used as outgroups High Throughput Cultivation - Sargasso Sea

GMD37A6 GMD38C5 GMD37A2 GMD37B10 uncultured clone pS115, seawater over coral reef (U87512) uncultured clone pS12F, seawater over coral reef (U87483 ) GMD37B4 GMD37C8 Rhodospirillum rubrum (D30778) Aquaspirillum itersonii (Z29620) alpha proteobacterium MBIC1474 (AB024595) Maricaulis maris (AB008849) Hyphomonas hirschiana (AF082794) Paracoccus denitrificans (X69159) Roseobacter litoralis (X78312) Erythrobacter longus (M59062) Azospirillum lipoferum (Z29619) Phaeospirillum molischianum (M59067) Caulobacter vibrioides (M83799)

0.10

Alpha Proteobacteria Gamma and Beta Proteobacteria sequences were used as outgroups High Throughput Cultivation - Sargasso Sea

Cytophaga-Flavobacterium-Bacteroides

uncultured clone kpc26, Atlantic coastal seawater (AF195429) uncultured clone kpc29, Atlantic coastal seawater (AF195430) GMDJE10E6 GMDsbC7 uncultured clone Keppib30, Atlantic coastal seawater (AF188171) uncultured clone kpj254f, Atlantic coastal seawater (AF195426) uncultured clone kpa13rc, Atlantic coastal seawater (AF195433) uncultured clone kpj56rc, Atlantic coastal seawater (AF195434) uncultured clone MT70, hypersaline black mud (AF211301) uncultured clone MPD-32, hypersaline evaporation pond (AF348716) uncultured clone MPD-21, hypersaline evaporation pond (AF348715) uncultured clone MPD-14, hypersaline evaporation pond (AF348711) uncultured clone CE61, hypersaline black mud (AF211300) uncultured clone MPD-11, hypersaline evaporation pond (AF348708) uncultured clone BD2-15, deep sea sediments (AB015543) uncultured clone BD7-14, deep sea sediments (AB015587) Salinibacter ruber (AF323500) Rhodothermus marinus (X80994)

0.10

Cytophaga-Flavobacterium-Bacteroides and Relatives Chlorobiaceae sequences were used as outgroups High Throughput Cultivation Targeted Biotopes

• Marine samples (sediment and water samples from surface and deep sea)

• alkaline Lake sediment (Kenya)

• Invertebrates (e.g., sponges)

67% of all cultures (960) • Soils (e.g., Ghana) investigated reached OD600 >0.1 Red: environmental clone library GHCex.B3 8 new groups GH1.2.A10 Ghana GH2.2.D2 Yellow: microcolonies Pseudomonas syzygii Ralstonia solanacearum GH1-1-A7 Pelobacter acetylenicus GHCex.D11 Ralstonia pickettii Desulfuromonas acetoxidans GHCex.B8 Ralstonia eutropha GHCex.H3 Azospirillum lipoferum GHCex.H11 GHCex.H1 GH3.2.F8 GH1-1-A10 GH1.2.B4 GHCex.C5 GH2.2.D5 GHCex.A8 Burkholderia cepacia Agrobacterium tumefaciens GH1-1-A12 GHCex.B7 GH2.2.C6 GH1-1-B11 Acidimicrobium ferrooxidans Agrobacterium radiobacter GH3.2.F2 GHCex.C6 GHCex.E8 Burkholderia norimbergensis GHCex.D4 GH2.2.C2 Comamonas acidovorans Clone env.MC4 GH2.2.D3 GH1-1-A5 Nocardioides simplex Aquaspirillum psychrophilum GH2.2.C5 Acidovorax delafieldii GH2.2.C11 Rhizobium giardinii GH2.2.C1 GHCex.B12 GH2.2.C8 GH1-1-B12 Rhodococcus opacus GH2.2.D1 Variovorax paradoxus Mycobacterium chelonae GH2.2.D11 GH1-1-A6 GH1-1-B5 Rhizobium galegae Xylophilus ampelinus Mycobacterium leprae GHCex.F2 GHCex.E4 GHCex.A4 GH3.2.F10 GHCex.C7 GH2.2.D8 GHCex.E6 GH3.2.E12 Alcaligenes latus GHCex.A10 GHCex.F4 GHCex.G8 GH1-1-B3 Rhizobium leguminosarum Bordetella sp GHCex.A2 GHcex.A9 Bordetella avium GHCex.E11 GHCex.E7 Pelistega europaea GHCex.E1 Devosia riboflavina GH1.2.B10 Bacillus simplex GHCex.F6 GH4.2.H4 GHCex.F12 Pseudaminobacter salicylatoxid GH1.2.B5 Achromobacter xylosoxidans Bacillus methanolicus GH2.2.D12 GHCex.G9 GH1.2.A7 Ensifer adhaerens GH1-1-B10 GHCex.F11 Sinorhizobium terangae GHCex.C9 clone pM10 GH1.2.B12 GH1.2.A9 GHCex.F5 GH2.2.C4 GHCex.B2 Bacillus cereus Mycoplana ramosa GHCex.D9 Bacillus sphaericus GH1.2.B2 GHCex.C10 GHCex.E5 Brucella A4/I GHCex.G7 GHCex.B5 Ochrobactrum grignonense GHCex.H8 GHCex.E10 GHCex.F10 GH3.2.E8 GHCex.A6 GHCex.A7 GH1.2.A2 GHCex.G2 env.RB_40, clone RB40, 1546 GH1.2.B6 Oxalobacter formigenes GHCex.C12 GH1.2.A4 GH3.2.E5 clone mb1228 GH1.2.B7 Strain My14 GHCex.B11 uncultured alpha proteobacterium Herbaspirillum G8A1 GHCex.D2 GH4.2.H3 GH3.2.E9 GHCex.F8 GH1.2.B3 GH3.2.F1 clone S027 clone A40t GH4.2.G9 GH3.2.E11 GHCex.G11 GHCex.G3 GHCex.H9 GH3.2.E10 GHCex.G4 GH4.2.H11 GHCex.A1 Rhodomicrobium vannielii GH4.2.G3 GHCex.D6 GHCex.E9 GH4.2.H7 GHCex.H10 Rhodoplanes elegans GH3.2.F3 clone iii1-8 GHCex.C3 Herbaspirillum frisingense clone 32-20 Bradyrhizobium elkanii GH4.2.G1 clone Rb25 GH4.2.H6 GH1-1-B1 clone Rb12 GHCex.F9 Rhodopseudomonas acidophila clone Tm7 GH3.2.F7 GH1-1-B6 Strain SBR2076 GHCex.H6 Asticcacaulis biprosthecium Clone env.PAD58 GHCex.B4 Sphingomonas JSS-28 GH4.2.H9 GHCex.G6 GHCex.C8 GH4.2.G12 clone OPB92 GHCex.D5 Stenotrophomonas maltophilia clone WCHB1-56 GHCex.A11T3 GH4.2.G4 clone WCHB1-07 Brevundimonas vesicularis GH4.2.G5 GHCex.G5 Stenotrophomonas africae GH2.2.D6 GH4.2.H2 clone WCHB1-58 GH2.2.C10 GHCex.H7 GH4.2.G8 Rhodobium marinum GH1-1-A2 Pirellula staleyi GHCex.A3 Frateuria aurantia GHCex.C1 GHCex.G10 Rhodanobacter lindaniclasticus Pirellula AJL5 GH1-1-B2 GHCex.C11 Gemmata obscuriglobus Methylobacterium extorquens GHCex.E3 Aquifex pyrophilus Beggiatoa alba Pseudomonas fluorescens GH4.2.H8 0.10 GH2.2.C12 GHCex.B3 GH1.2.A10 Ghana GH2.2.D2 Pseudomonas syzygii Ralstonia solanacearum GH1-1-A7 Pelobacter acetylenicus GHCex.D11 Ralstonia pickettii Desulfuromonas acetoxidans GHCex.B8 Ralstonia eutropha GHCex.H3 Azospirillum lipoferum GHCex.H11 GH3.2.F8 GHCex.H1 GH1-1-A10 GHCex.C5 GH1.2.B4 GH2.2.D5 Burkholderia cepacia GHCex.A8 Agr obacterium tumefaciens GH1-1-A12 GHCex.B7 anaerobic, GH2.2.C6 GH1-1-B11 Acidimicrobium ferrooxidans Agrobacterium radiobacter GH3.2.F2 GHCex.C6 GHCex.E8 Burkholderia norimbergensis GHCex.D4 chemotrophic, GH2.2.C2 Comamonas acidovorans GH1-1-A5 Clone env.MC4 GH2.2.D3 Nocardioides simplex Aquaspirillum psychrophilum GH2.2.C5 Acidovorax delafieldii GH2.2.C11 thermophilic Rhizobium giardinii GH2.2.C1 GHCex.B12 GH2.2.C8 GH1-1-B12 Rhodococcus opacus GH2.2.D1 Variovorax paradoxus Mycobacterium chelonae GH2.2.D11 GH1-1-A6 GH1-1-B5 Rhizobium galegae Xylophilus ampelinus Mycobacterium leprae GHCex.F2 GHCex.E4 GHCex.A4 GH3.2.F10 GHCex.C7 GHCex.E6 GH2.2.D8 GH3.2.E12 GHCex.A10 Alcaligenes latus GHCex.F4 GH1-1-B3 GHCex.G8 Rhizobium leguminosarum Bordetella sp GHCex.A2 ? GHcex.A9 Bordetella avium GHCex.E11 GHCex.E7 Pelistega europaea GHCex.E1 Devosia riboflavina GH1.2.B10 Bacillus simplex GHCex.F6 GH4.2.H4 GHCex.F12 Pseudaminobacter salicylatoxid GH1.2.B5 Bacillus methanolicus GH2.2.D12 Achromobacter xylosoxidans GHCex.G9 GH1.2.A7 Ensifer adhaerens GH1-1-B10 GHCex.F11 Sinorhizobium terangae GHCex.C9 clone pM10 GH1.2.B12 GH1.2.A9 GHCex.F5 GH2.2.C4 GHCex.B2 Bacillus cereus Mycoplana ramosa GHCex.D9 Bacillus sphaericus GH1.2.B2 GHCex.C10 GHCex.E5 Brucella A4/I GHCex.G7 GHCex.B5 Ochrobactrum grignonense GHCex.H8 ? GHCex.F10 GH3.2.E8 GHCex.E10 GHCex.A6 GH1.2.A2 GHCex.A7 GHCex.G2 env.RB_40, clone RB40, 1546 GH1.2.B6 Oxalobacter formigenes GHCex.C12 GH1.2.A4 GH3.2.E5 clone mb1228 GH1.2.B7 Strain My14 GHCex.B11 uncultured alpha proteobacterium Herbaspirillum G8A1 GH3.2.E9 GHCex.D2 GH4.2.H3 GHCex.F8 ? GH3.2.F1 GH1.2.B3 clone A40t clone S027 GH4.2.G9 GH3.2.E11 GHCex.G11 GHCex.G3 GH3.2.E10 GHCex.H9 GHCex.G4 GH4.2.H11 GHCex.A1 Rhodomicrobium vannielii GH4.2.G3 GHCex.D6 GHCex.E9 phototrophic GH4.2.H7 GHCex.H10 Rhodoplanes elegans GH3.2.F3 GHCex.C3 Herbaspirillum frisingense clone iii1-8 GH4.2.G1 clone 32-20 Bradyrhizobium elkanii GH1-1-B1 GH4.2.H6 clone Rb25 clone Rb12 GHCex.F9 Rhodopseudomonas acidophila GH3.2.F7 clone Tm7 GH1-1-B6 Strain SBR2076 Asticcacaulis biprosthecium GHCex.H6 Clone env.PAD58 Sphingomonas JSS-28 GHCex.B4 GH4.2.H9 GHCex.C8 GHCex.G6 GH4.2.G12 GHCex.D5 Stenotrophomonas maltophilia clone OPB92 GHCex.A11T3 GH4.2.G4 clone WCHB1-56 ? Brevundimonas vesicularis GH4.2.G5 clone WCHB1-07 Stenotrophomonas africae GH2.2.D6 GH4.2.H2 GHCex.G5 GH2.2.C10 clone WCHB1-58 GH4.2.G8 Rhodobium marinum GH1-1-A2 Pirellula GHCex.H7 staleyi GHCex.A3 Frateuria aurantia GHCex.C1 GHCex.G10 Rhodanobacter lindaniclasticus Pirellula AJL5 GH1-1-B2 GHCex.C11 Gemmata obscuriglobus Methylobacterium extorquens GHCex.E3 Aquifex pyrophilus Beggiatoa alba Pseudomonas fluorescens phototrophic GH4.2.H8 0.10 GH2.2.C12 Encapsulation and Growth of Anaerobes

Methanococcus thermolithotrophicus

Phase contrast Autofluorescence (F420) The Phototrophs:red algae: Catenella repens

Red algae SSC FL3 (chlorophyll) FL3

SSC FSC

Cyanobacteria Diatoms FL3 (chlorophyll) FL3 FL3 (chlorophyll) FL3

SSC SSC

Cyanobacteria: 91% Planktothrix The Fungi: High Throughput Cultivation for Filamentous Fungi

Many morphological different fungi from a single environmental sample (average of 40 fungi/sample) 11830870 Hypocrea rufa 11877246 Hypocrea koningii 1730334 Hypocrea lutea 21 Trichoderma aureoviride/harzianum (-1) 20 Trichoderma aureoviride/harzianum (-2) 530266 Hypocrea sp. 34 Trichoderma parceramosum/ghanense (-1) 35 fungal isolates from 2 Trichoderma virens (-0) 174964 Hypomyces chrysospermus 30 unidentified mycorrhizal fungus (-1) 10 unidentified mycorrhizal fungus (-2) 29 unidentified mycorrhizal fungus (-1) a single soil sample (Ghana) 9 unidentified mycorrhizal fungus (-2) 6 unidentified mycorrhizal fungus (-0) 37 Epichloe sp. endophytic fungus (-5 for ITS2) 13345003 Illosporium carneum 11830874 Myrothecium verrucaria 11830876 Myrothecium verrucaria 11877344 Myrothecium atroviridae 11830873 Myrothecium roridum 18S rDNA tree, ITS identification mapped on 11830875 Myrothecium roridum 11877343 Myrothecium leucotrichum (with number of nucleotide changes from closest match) 11877346 Myrothecium inundatum 11830871 Myrothecium roridum 11830872 Myrothecium roridum 11877342 Myrothecium sp. BBA69174 4850335 Geosmithia putterillii 6172364 Nectria cinnabarina 19 Fusarium sp. NRRL22412 (-4 for ITS2) 33 Fusarium sp. NRRL22412 (-0) Hypocreales 4 Fusarium sp. NRRL22412 (-0) 3415124 Gibberella pulicaris (16) 26 Nectria vilior/Glionectria tenua (-3 for ITS1) 28 Nectria cinnabarina (-32) 3 1 S e pe donium bole tiphagum (- 2 3) 11877345 Myrothecium cinctum 975348 Verticillium dahliae 6537145 Verticillium dahliae 6606309 Plectosphaerella cucumerina 'Verticilleales' (1) 7 Verticillium nigrescens (-18) 2979414 Kionochaeta ramifera 2979415 Kionochaeta spissa 2979416 Kionochaeta spissa 2979413 Kionochaeta ivoriensis Sordariomycetes 38 Chaetosphaeria chloroconia (-28) Sordariales (1) 174060 Chaetomium elatum (20) 175571 Ophiostoma stenoceras Ophiostomales (1) Sporothrix schenckii 11527210 Endomyces scopularum 8919684 Ophiostoma piliferum 175570 Ophiostoma ulmi 27 Ophiostomales (NO I TS MATCH) 6440649 Pseudogymnoascus roseus 12 Pseudogymnoascus roseus (-8) 2735847 Ascozonus woolhopensis 3426001 Geomyces pannorum 3426000 Geomyces asperulatus Leotiomycetes 3688198 Bulgaria inquinans 17 Penicillium raperi (-1) (1) 11 Eupenicillium javanicum 709801 Eupenicillium javanicum 2582977 Chromocleista malachitea 3 Penicillium westlingii/citrinum (-2) 2582973 Penicillium namyslowskii 5030945 Penicillium namyslowskii 169432 Penicillium chrysogenum 6172360 Penicilliopsis clavariaeformis 2588675 Hemicarpenteles ornatus 2588668 Chaetosart orya cremea 13 Aspergillus flavipes (-12) 12862439 Aspergillus flavipes 2588669 Aspergillus flavipes 12862440 Aspergillus flavipes 2588678 Aspergillus terreus 23 Penicillium aculeatum/pinophilum (-11) 176095 Talaromyces flavus 6172367 Chromocleista cinnabarina Eurotiales 6172369 Chromocleista cinnabarina 6063406 Talaromyces emersonii (6) 2582976Talaromyces eburneus 58322 Blastomyces dermatitidis 173895 Blastomyces dermatitidis 1046282 Chrysosporium parvum Eurotiomcyetes (7) 8164018 Paracoccidioides brasiliensis Ascomycota 8469174 Paracoccidioides brasiliensis Onygenales (1) Pezizomycotina (32) 14 Auxarthron sp. (-19 for ITS2) 24 Exophiala spinifera ( -0) 1040746 Phaeococcomyces exophialae 1040726 Exophiala jeanselmei 1040738 Exophiala nigra 4138277 Exophiala nigra 1040745 Phaeoannellomyces elegans 14422285 Epicoccum nigrum 1040725 Exophiala dermatitidis 1040760 Exophiala dermatitidis 18 Capronia coronata (-11 for ITS2) 35 Capronia coronata (-12 for ITS2) 36 Capronia coronata (-26) 16 Phialophora lignicola (-42) Chaetothyriomycetes 3779216 Ceramothyrium linnaeae 32 Graphium calicioides (NO CLOSE ITS MATCH) (6) 4586331 Graphium calicioides 1209731 Capronia pilosella 22 Gongronella sp. (NO I TS MATCH) 8 Gongronella sp. (NO ITS MATCH) 5 Gongronella sp. (NO ITS MATCH) 11078022 Gongronella butleri Zygomycota 11078025 Hesseltinella vesiculosa 6707223 Cunninghamella elegans Mucorales (3) 6561318 Cunninghamella polymorpha 11078023 Halteromyces radiatus

5 changes

= novel culture = hit in Pharma screen High Throughput Cultivation Novel Tool to Access Natures Biodiversity

• easy to multiplex • cultivating novel microorganisms • hundreds of different bacteria and fungi per sample High Throughput Cultivation How do we determine different cultures and reduce redundancy ?

Options: 16S rRNA gene sequencing: time consuming, expensive Mass Spectroscopy: no database, sample preparation FT-IR (Fourier transform infrared spectroscopy): no database fast (30 min/384 samples), inexpensive Bacterial Deconvolution by FT-IR*

* FT-IR: Fourier-transform Infrared Spectroscopy Hanford Site

SX-108 Zone 1

SX-108 Zone 2

SX-108 Zone 3

SX-108 Zone 4

Figure 2. Location of core samples from slant borehole beneath tank SX108. Jim Fredrickson, Fred Brockman/PPNL Pooled sample Total cells

SX-108 zone 1 2.9 x 104 SX-108 zone 2 2.8 x 104 SX-108 zone 3 9.0 x 105 SX-108 zone 4 9.3 x 105

10 10 137Cs Na 99 Nitrate Tc

20 20

30 30

40 40 Depth below ground surface (m) Depthground below surface(m)

50 50 0 5 10 15 20 123456789 mol/L 99Tc in log (pCi/L) or 137Cs in log (pCi/g) Jim Fredrickson, Fred Brockman/PPNL SX-108 Zone 1 SX-108 Zone 2 Aquatic bacterium RUB_NH4_10 96% ALPHA Aquatic bacterium RUB_NH4_10 98% ALPHA aquatic bacterium RUB_NH4_10 99% ALPHA Bradyrhizobium elkanii 100% ALPHA Arthrobacter chlorophenolicus 99% HIGH GC G+ Burkholderia cepacia 100% BETA Arthrobacter chlorophenolicus 97% HIGH GC G+ Gordonia polyisoprenivorans 100% HIGH GC G+ Bradyrhizobium elkanii 100% ALPHA Mesorhizobium amorphae 98% ALPHA Ralstonia solanacearum 96% BETA Methylobacterium sp. 99% ALPHA Ralstonia solanacearum 98% BETA Pseudomonas fluorescens 99% GAMMA Burkholderia cepacia 99% BETA Pseudomonas sp. BUVI 100% GAMMA Methylobacterium radiotolerans 99% ALPHA Ralstonia pickettii 100% BETA Methylobacterium fujisawaense 99% ALPHA Rhizobium sp. RM1-2001 100% ALPHA Pseudomonas fluorescens 99% GAMMA Ralstonia pickettii 100% BETA SX-108 Zone 4 Arthrobacter chlorophenolicus 99% HIGH GC G+ uncultured eubacterium WD208 98% ALPHA Arthrobacter globiformis 99% HIGH GC G+ unidentified Actinomycete BD4-12 100% HIGH GC G+ Arthrobacter ramosus 98% HIGH GC G+ Bacillus megaterium 100% HIGH GC G+ SX-108 Zone 3 Gordonia polyisoprenivorans 100% HIGH GC G+ Arthrobacter chlorophenolicus 99% HIGH GC G+ Mesorhizobium amorphae 98% ALPHA Blastobacter denitrificans 99% ALPHA Methylobacterium sp. 99% ALPHA Ralstonia solanacearum 98% BETA Ralstonia sp. APF11 100% BETA Gordonia polyisoprenivorans 100% HIGH GC G+ Rhizobium sp. RM1-2001 100% ALPHA Pseudomonas fluorescens 100% GAMMA Streptococcus mitis 99% LOW GC G+ Ralstonia pickettii 100% BETA Staphylococcus epidermidis 97% LOW GC G+ Targeted Isolation: Acidobacteria

Fred Brockman (PNNL), Cheryl Kuske and Sue Barns (LANL) Limited Biomass…

Total environmental sequencing (metagenomics):

+ Tremendous amount of information - For complex communities no individual genome coverage

Single genome sequencing:

+ Link between diversity and function - Access to individual species Genomes to Life

- Diversa - Los Alamos National Laboratory (LANL) - Pacific Northwestern National Laboratory (PNNL)

“Access genomes and function of so-far uncultivated microorganism” GTL scheme

Sample High-throughput cultivation FISH

Rolling circle whole Genome Microarray amplification Sequencing

Gene expression experiments Proteomics GTL scheme In Situ CULTIVATION

GENOMICS PROTEOMICS METABOLOMICS Acknowledgment

Diversa:Diversa PNNL: Martin Keller Fred Brockman Gary Woodnutt Jay Short LANL: Greg Clark Cheryl Kuske Imke Haller Susan Barns Trevin Holland Gerardo Toledo Marion Walcher 35

30 Novel strains (%), <97% similarity Diversity: 25

Costa Rican soil 20 wk 1 wk 2 15 wk 3 wk 4 10 wk 5

0 nd nd nd 10e-6SE OHHL BHL ATP cAMP 90% 80% 70% Different strains (%) 60% 50% wk 1 wk 2 40% wk 3 30% wk 4 wk 5 20% 10% 0% 10e-6SE OHHL BHL ATP cAMP

OHHL: N- oxohexanoyl-DL-homoserine lactone BHL: N-butyryl homoserine lactone FT-IR reference strains (13):

Variable cut-off Phylogeny of NCE-producing fungus

Fungi Ascomycota Euascomcycotina 6715471 Trichoderma viride Sordariomycetes 6715473 Hypocrea/Trichoderma koningii Hypocreomycetidae Hypocreales Hypocrea /Trich od erma kon ingii Nectriaceae, Hypocrea rufa Hypocreaceae, 530266 unidentified Clavicipitaceae 97 7619936 Hypocrea lutea 1730334 Hypocrea lutea 7619937 Hypomyces chyrsospermus 100 174964 Hypomyces chr ysospermus 84 E050679 Hypomyces 7619928 Cordyceps tricentri 13345005 Nectria parmeliae 7619917 Cordyceps jezoensis 11125693 Hirsutella citriformis 7619919 Cordyceps ophioglossoides 9887229 Claviceps purpurea AD00146MIXI4 Clavicipitaceae? 11125692 Paecilomyces fumosoreseus 58 AD00134DI2 Cordyceps 66 7619934 Beauvaria bassiana 64 1332409 Paecilomyces tenuipes 85 6172366 Tritiarchium sp. 11877339 Myrothecium leucotrichum 9798925 Metarhizium anisopliae 69 12642504 Metarhizium anisopliae 100 7619935 Metarhizium anisopliae 57 13241783 Cordyceps khaoyaiensis 5 changes