Microbiology of oilfield ecosystems Bernard Ollivier IRD Marseille, France Oil well gggeological section Gaz Oil Water Characteristics of oil reservoirs • « Closed » ecosystems • Depth (up to 4000 m; up to several hundreth bars) • Temperature in situ : 30 to 180 °C • Water Salinities : 0 up to saturation (34% salt) ¾Importance of anaerobic microorganisms in oilfield waters 1 μm The best samplings are ¾Cores, but precautions need to be taken when drillinggpg or sampling ¾Oil waters should be collected from oilwell heads where nothing has been already injected. Kindl y provid e d by M. Mago t Studying the anaerobes Anaerobiosis : Methods -Medium pppreparation using a N2 flux. -Hunggqate’s technique -The anaerobic glove box Physico-chemical limitations to the development of microflora in oil reservoirs in situ oil biodegradation in situ oil biodegradation has never been observed in oil reservoirs where temperature is over 82°C Microbial populations in oil reservoirs 250 200 150 100 50 0 0 20 40 60 80 100 120 Tempp()erature (C) Figure kindly provided by M. Magot in situ oil biodegradation Thermophilic to hyperthermophilic Bacteria or Archaea ??? have participated to oil biodegradation Combination of high temperature and high salinity are drastic for microbial life underneath Electron acceptors available in oil field ecosystems 4 H2 + CO2 ---> CH4 + 2 H2O Methanobacterium Methanococcus CO2 + 4 H2 + 2 CO2 ---> Acetate + H + 2 H2O Acetanaerobium 4H4 H2 +H+ H2SO4 --->H> H2S+4HS + 4 H2O DlfiDesulfacinum Desulfotomaculum 2 SO4 - VFA + x H2SO4 --> y H2S + z H2O Desulfotomaculum Archaeoglobus Electron acceptors available in oil field ecosystems ?? 2- - 4H2 + S2O3 --> 2H2S + H2O + 2OH Thermotoga Desulfotomaculum Archaeoglobus 2- S2O3 Oxydation of organic matter Thermoanaerobacter coupldled to red ucti on of fhi thiosulf ate Thermo toga Electron acceptors available in oil field ecosystems ?? - Shllfhewanella putrefaciens Fe3+ - Deferribacter thermophilus - Thermotoga spp. - Archaeoglobus fulgidus - GillttidGarciella ntratireducens - Petrobacter succinatimandens - NO3 - Denitrivibrio acetophilus - Geobacillus spp; Microbi al di versit y of anaerobes in oilfield environments Corrosion in pppipelines Photographs kindly provided by M. Magot The sulfate-reducing bacteria Desulfovibrio gracilis Desulfovibrio capillatus 10% Desulfovibrio longus Desulfovibrio bastinii Desulfovibrio vietnamensis Desulfovibrio gabonensis DlfblbhbdfiDesulfobulbus rhabdoformis Desulfobacterium cetonicum Desulfobacter vibrioformis Thermodesulforhabdus norvegicus Desulfacinum subterraneum Desulfacinum infernum Desulfotomaculum nigrificans Desulfotomaculum halophilum Desulfotomaculum thermocisternum. Desulfotomaculum kuznetsovii Thermodesulfobacterium commune Thermodesulfobacterium thermophilum Archaeoglobus profundus Archaeoglobus fulgidus Archaeoglobus lithotrophicus Methanohalophilus mahii Methanohalophilus euhalobius 5% Methanomethylovorans hollandica Methanococcoides methylutans Methanolobus tindarius The Methanoarchaea Methanosarcina mazei MthMethanosarc ina b ark eri Methanosarcina siciliae Methanohalobium evestigatum Methanomicrococcus blatticolus Methanosaeta concilii Methanocorpusculum parvum Methanocalculus halotolerans Methanospirillum hungatei Methanofollis tationis MthMethanocu lleus b ourgensi s Methanogenium cariaci Methanoplanus limicola Methanomicrobium mobile Methanolacinia ppyaynteri Methanoplanus petrolearius Methanococcus vannielii Methanothermococcus thermolithotrophicus Methanotorris igneus MthMethanoca ldococcus j annaschii Methanothermus fervidus Methanothermobacter thermautotrophicus Methanosphaera stadtmanae Methanobrevibacter ruminantium Methanobacterium subterraneum Methanobacterium ivanovii Methanobacterium bryantii The fermentative thermophilic bacteria 5% Fervidobacterium islandicum DSM 5733T 100 Fervidobacterium gondwanense ACM 5017T 100 Fervidobacterium nodosum ATCC 35602T Thermopppallium natronophilum DSM 9460T 95 100 Marinitoga piezophila DSM 14283T Marinitoga camini DSM 13578T 100 100 Geotoga subterranea ATCC 51225T T 100 Geotoggpa petraea ATCC 51226 Petrotoga mobilis DSM 10674T 100 Petrotoga mexicanus DSM 14811T 97 Petrotoga sibirica DSM 13575T Petrotoga olearia DSM 13574T 100 Petrotoga miotherma DSM 10691T 100 Thermotoga lettingae DSM 14385T 100 Thermotoga elfii DSM 9442T Thermophilic Thermotoga subterranea DSM 9912T Thermotoga thermarum DSM 5069T Thermotoga hypogea DSM 11164T Thermotoga naphthophila DSM 13996T 100 Thermotoga petrophila DSM 13995T Hyperthermophilic 96 Thermot oga neapolit ana DSM 4359T Thermotoga maritima DSM 3109T Thermosipho geolei DSM 13256T 100 Thermosipho melanesiensis CIP 104789T Thermosipho japonicus DSM 13481T 100 Thermosipho africanus DSM 5309T Aquifex pyrophilus DSM 6858T Petrotoga mexicana • Isolated from a Mexican oil field. • Motile rod with toga, Gram negative, Thermophilic and halophilic. • Anaerobe fermenting sugars into acetate, lactate, L-alanine, H2 et CO2. • Thiosu lfate-reducer. opt. Temp.: 55°C (30-65°C), opt. pH : 666,6 (5(58,8-65)6,5) opt. NaCl : 3 % (1-20 %). Thermotoga hypogea • Isolated from an African oil field, (Cameroon) •The most thermophilic microorganism, domain Bacteria • Optimum Temperature : 70 - 75°C • Range Temperature: 56 - 92°C • Anaerobe reducing thiosulfate Thermotoga elfii O. D. 580 nm 0.6 + Thiosu lfat e 040.4 - Thiosu lfat e 0.2 1 μm 0 20 40 60 Hours Isolation of Thermotoga elfii from oil reservoirs Figure kindly provided by M. Magot Isolation of Archaeoglobus fulgidus. from oil reservoirs The fermentative halophilic bacteria Halanaerobium praevalens Halanaerobium alcaliphilum Halanaerobium sacch. subsp. senegalense Halanaerobium sacch. subsp. saccharolyticum Halanaerobium kushneri Halanaerobium acetethylicum Halanaerobiaceae Halanaerobium congolense 10% Halanaerobium lacurosei Halanaerobium fermentans Halanaerobium salsuginis Halothermothrix orenii Halocella cellulosilytica Sporohalobacter lortetii Acetohalobium arabaticum Se len ihal anaerob act er sh riftii Orenia salinaria Orenia marismortui Orenia sivashensis Halobacteroidaceae Natroniella acetigena Halobacteroides halobius Halobacteroides elegans Halanaerobacter lacunarum Halanaerobacter chitinivorans Halanaerobacter salinarius Natronincola histidinovorans HlHalanaero bium congo lense • Isolated from an oilfield water in Congo • Heterotrophic thiosulfate-reducing microbe •Opppt. temp. : 42°C (>20-<45°C), • Opt. pH : 7,0 (6,3-8,5), • Op t. Na Cl : 10% (4-24%) A question of major concern in petroleum microbiology ? Conclusion and questions ? ¾ Reports in scientific literature indicate that there is a wide range of microorganisms (mainly anaerobes) inhabiting oil reservoirs. Temperature and salinity are limiting physico-chemical factors for their development ¾ Methanoarchaea have been significant intervenors in oil degradation in shallow deep subsurface petroleum reservoirs, what about the deep hot ones ? ¾ Which anaerobes (mesophilic to hyperthermophilic) might have been possibly involved in hydrocarbon oxidation (synthrophs or sulfate-reducing bacteria, etc..)? ¾ What about the biogeochemical role to be played by common microbial populations inhabiting oil reservoirs (Unknown metabolism? Utilization of hydrogen or volatile fatty acids by members of the order Thermotogales or by sulfate-reducing bacteria using minerals (sulfate, iron ?) or methanoarchaea as terminal electron acceptors)? Thanks for your attention.