Constraints in the colonization of natural and engineered subterranean igneous rock aquifers by aerobic methane‐oxidizing bacteria inferred by culture analysis

Abstract

The aerobic methane-oxidizing bacteria (MOB) are suggested to be important for the removal of oxygen from subterranean aquifers that become oxygenated by natural and engineering processes. This is primarily because MOB are ubiquitous in the environment and in addition reduce oxygen efficiently. The biogeochemical factors that will control the success of the aerobic MOB in these kinds of underground aquifers remain unknown. In this study, viable and cultivable MOB occurring at natural and engineered deep granitic aquifers targeted for the disposal of spent nuclear fuel (SNF) in the Fennoscandian Shield (approximately 3-1000 m) were enumerated. The numbers were correlated with in situ salinity, methane concentrations, conductivity, pH, and depth. A mixed population habiting freshwater aquifers (approximately 3-20 m), a potential source for the inoculation of MOB into the deeper aquifers was tested for tolerance to NaCl, temperature, pH, and an ability to produce cysts and exospores. Extrapolations show that due to changing in situ parameters (salinity, conductivity, and pH), the numbers of MOB in the aquifers dropped quickly with depth. A positive correlation between the most probable numbers of MOB and methane concentrations was observed. Furthermore, the tolerance-based tests of cultured strains indicated that the MOB in the shallow aquifers thrived best in mesophilic and neutrophilic conditions as opposed to the hyperthermophilic and alkaliphilic conditions expected to develop in an engineered subterranean SNF repository. Overall, the survival of the MOB both quantitatively and physiologically in the granitic aquifers was under the strong influence of biogeochemical factors that are strongly depth-dependent.

The origin, source, and cycling of methane in deep crystalline rock biosphere

“…As most of the deep crystalline bedrock habitats are mainly anaerobic, other electron acceptors than oxygen could be more relevant in these environments. Nevertheless, the detection of both aerobic and anaerobic methanotrophs from deep crystalline bedrock prove that both of these microbial groups have a niche in the depths (Table 1 ) (Kalyuzhnaya et al, 1999 ; Chi Fru, 2008 ; Hirayama et al, 2011 ; Nyyssönen et al, 2012 ; Bomberg et al, 2015 ; Purkamo et al, 2015 ; Rajala et al, 2015 ). In addition, aerobic methylotrophs appear to frequently occupy geological lignite and coal formations that are usually considered anaerobic, at depths of over 1 km (Mills et al, 2010 ; Stępniewska et al, 2013 , 2014 ).…”

“…In addition to methanogens, methanotrophs have also been detected from deep bedrock of Äspö and Forsmark. Clone libraries of methanotrophy marker gene pmo A were dominated by Methylomonas and Methylocystis (Chi Fru, 2008 ). Methylomonas and Methylobacter dominated enrichment cultures from Äspö groundwater from below 400 m depth (Kalyuzhnaya et al, 1999 ).…”

“… 6 ND, not detected; MG, methanogens; MT, aerobic methanotrophs; ANME, anaerobic methanotrophs . 7 [1] Ahonen et al, 2004 , [2] Anttila et al, 1999 , [3] Bomberg et al, 2015 , [4] Borgonie et al, 2011 , [5] Chi Fru, 2008 , [6] Gihring et al, 2006 , [7] Hallbeck and Pedersen, 2008a , [8] Hallbeck and Pedersen, 2008b , [9] Hallbeck and Pedersen, 2012 , [10] Haveman et al, 1999 , [11] Itävaara et al, 2011a , [12] Kaija et al, 1998 , [13] Kalyuzhnaya et al, 1999 , [14] Kieft et al, 2005 , [15] Kietäväinen et al, 2013 , [16] Kotelnikova and Pedersen, 1997 , [17] Kotelnikova and Pedersen, 1998 , [18] Lin et al, 2005a , [19] Lin et al, 2006b , [20] Luukkonen et al, 1999 , [21] Morelli et al, 2010 and references therein, [22] Moser et al, 2003 , [23] Moser et al, 2005 , [24] Nyyssönen et al, 2012 , [25] Nyyssönen et al, 2014 , [26] Onstott et al, 2009 , [27] Osburn et al, 2014 , [28] Pedersen and Ekendahl, 1990 , [29] Pedersen and Haveman, 1999 , [30] Pitkänen and Partamies, 2007 , [31] Pitkänen et al, 1996 , [32] Pitkänen et al, 1998 , [33] Purkamo et al, 2013 , [34] Purkamo et al, 2015 , [35] Rajala et al, 2015 , [36] Sherwood Lollar et al, 2006 , [37] Stotler et al, 2009 , [38] Stotler et al, 2010 , [39] Ward et al, 2004 . …”

Hydrogeological controls on microbial activity and habitability in the Precambrian continental crust

“…(2023) furthered supported this interpretation by reporting that even in old and supposedly anoxic groundwaters, oxygen can be produced in situ through microbial dismutation of chlorite and nitric oxide, thus supporting a series of hitherto underappreciated aerobic microbial activities via what they called “dark oxygen”. Other studies have also reported aerobic metabolic potential in deep terrestrial subsurface settings, including several deep aquifers in the Fennoscandian Shield where viable methanotrophic bacteria were detected (Chi Fru, 2008), and artesian water samples from a 2.8‐km‐deep borehole in Western Siberia, Russia, where substantial amounts of heterotrophic bacteria capable of aerobic respiration were reported (Kadnikov et al., 2020). While oxygen and aerobic organisms could be delivered to the subsurface by penetration of (paleo)meteoric waters (Borgonie et al., 2011, 2015) and likely are in some cases, the recent work also suggests for other hitherto underappreciated oxidant sources.…”

“…This revealed a redox potential much higher on the redox (Eh) ladder than previously expected in a subsurface setting. Ruff et al (2023) furthered supported this interpretation by reporting that even in old and supposedly anoxic groundwaters, oxygen can be produced in situ through microbial dismutation of chlorite and nitric oxide, Fru, 2008), and artesian water samples from a 2.8-km-deep borehole in Western Siberia, Russia, where substantial amounts of heterotrophic bacteria capable of aerobic respiration were reported (Kadnikov et al, 2020). While oxygen and aerobic organisms could be delivered to the subsurface by penetration of (paleo)meteoric waters (Borgonie et al, 2011(Borgonie et al, , 2015 and likely are in some cases, the recent work also suggests for other hitherto underappreciated oxidant sources.…”