Present and future of subsurface biosphere studies in lacustrine sediments through scientific drilling
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Recently, the discovery of active microbial life in deep-sea sediments has triggered a rapid development of the field known as the “deep biosphere.” Geomicrobiological investigations in lacustrine basins have also shown a substantial microbial impact on lake sediments similar to that described for the marine record. Although only 30 % of the lake sites drilled by the International Continental Drilling Program (ICDP) have included microbial investigations, these lakes cover a relatively wide range of salinities (from 0.15 to 33.8 %), pH (from 6.0 to 9.8) and environmental conditions (from very arid to humid subtropical conditions). Here, we analyze results of very recent ICDP lake sites including subsurface biosphere research from southern Patagonia (Laguna Potrok Aike) to the Levantine area (Dead Sea) as well as the East Anatolian high plateau (Lake Van) and Macedonia (Lake Ohrid). These various settings allow the examination of the impact of contrasting environments on microbial activity and their subsequent role during early diagenesis. Furthermore, they permit the identification of biosignatures of former microbial activity recorded in the sediments as well as investigating the impact of microbes in biogeochemical cycles. One of the general outcomes of these preliminary investigations is data to support the hypothesis that microbes react to climatically driven environmental changes that have a direct impact on their subsurface distribution and diversity. This is clear at conspicuous levels associated with well-known climatic periods such as the Medieval Climatic Anomaly or the Little Ice Age. Although more research is needed, this relationship between prevailing microbial assemblages and different climatic settings appears to dominate the lacustrine sites studied until to date.
KeywordsLakes Microbes Biosignatures Genomics
In the past few years, geomicrobiological investigations in the lacustrine subsurface have shown that microbial metabolism, as in their marine counterparts, can have a substantial impact on lake sediments. These impacts include dissolution, alteration and precipitation of minerals and changes in redox conditions (Vaughan and Lloyd 2012). While ocean drilling programs (ODP and IODP) pioneered the study of the “deep biosphere” (e.g., D’Hondt et al. 2002, 2004; Hinrichs and Inagaki 2012; Horsfield et al. 2006; Inagaki 2010; Lever et al. 2013; Lomstein et al. 2012; Røy et al. 2012), lake sediments provide a range of characteristics worth investigating. First, they can represent a wider range of different chemical conditions (e.g., from alkaline to acidic, oxic to anoxic), and these differences can occur in short lateral distances. Secondly, lacustrine sediments can also react very quickly to changing conditions with depth as a result of climatic changes or further input, such as volcanic or seismic events that would trigger dramatic transformations in water chemistry. Additionally, the systematically higher sedimentation rate in the lacustrine realm relative to the oceanic realm allows for tracking microbial populations at high resolution through climatic records. Although this can make interpretations more challenging than in the well-investigated marine settings, lake subsurface studies also bear opportunities for new findings.
The recent development of modern genomics and the emergence of high-throughput gene sequencing technology (e.g., Orsi et al. 2013) have led to a much broader view of the deep biosphere. This eventually led to the concept of the microbiome. Originally coined by Lederberg and McCray (2001) for microbial communities in the human body, it now refers to the entire microbial population within a specific environmental niche. Microbiomes in different environments appear to change in population diversity and density as a function of changes in environmental conditions. When referring to lacustrine sediments, the existing datasets are still limited to fully endorse this statement, but seem to point toward such direction.
Overall, the purpose of this contribution is to increase awareness of the importance of implementing microbial research in future ICDP drilling initiatives. Additionally, we propose a close collaboration with the marine research community to draw upon the much wider experience in sampling and sampling handling already gained during a large number of IODP drilling operations.
Case Studies (in chronological order)
Laguna Potrok Aike, Argentina
This subsaline maar lake is located in a volcanic field in the southernmost tip of Patagonia, east of the Andes cordillera at 51°58′S and 70°22′W (Fig. 3). The lake has been the target of multidisciplinary investigations through continental drilling (Project PASADO: Potrok Aike Lake Sediment Archive Drilling Project; Zolitschka et al. 2013 and references therein) and more than 500 m of sedimentary cores were retrieved. One of the novelties in lacustrine deep drilling was that for the first time in the history of ICDP, a retrieved core was fully dedicated to a detailed geomicrobiological study. A one-meter-long gravity core and a 93-meter-long hydraulic piston core—PTA-5022-1D—were recovered at 100 m water depth and sampled following a newly established sampling strategy under aseptic conditions (Vuillemin et al. 2010). Its application has shown that despite the logistical constraints inherent to each project (e.g., absence of appropriate laboratory facilities on board), it is still possible to sample cores aseptically. Using aseptic techniques to sample cores is a key issue for the credibility of the findings for each of the studies reviewed herein.
In contrast to IODP operations, the size and configuration of lake drilling platforms prevent the establishment of a sampling laboratory with minimal conditions of asepsis right at the drilling site. As a result, an ad hoc laboratory was installed in a container on the campsite by the lakeshore and the retrieved cores were transported every 90 min from the platform to the laboratory. A complete in situ sampling procedure allowed recovering aseptic samples as well as determining the presence of active microbes. Once in the laboratory, the exterior surfaces of the core liners were first disinfected with isopropylic alcohol and sprayed with fungicide. Sampling windows were cut in the liner every 1 or 1 m and at higher resolution for the upper 15 m using a portable circular saw. Conversely, windows were cut every 5 cm in the empty liner of a gravity core and sealed with strong adhesive tape prior to coring. The latter allowed a faster sampling at a higher resolution at the uppermost part of the sedimentary column that encompasses the largest concentration of microbes. The interior surfaces of the core liners were not disinfected prior to coring. However, it is assumed that a contamination by preexisting microbes can be discarded because any potential microbes would have been washed out while descending the drilling tools throughout the water column.
A quick activity test was used to test microbial activity in the sediments immediately after coring. In situ adenosine 5′-triphosphate (ATP) measurements were taken as indication of living organisms within the sediments. The presence of ATP is a marker molecule for living cells since it is not known to form abiotically. ATP can be easily detected with high sensitivity and high specificity using an enzymatic assay because light is emitted as a result of the reaction. The further application of this test to water samples can aid in the evaluation of the degree of contamination of the drilling water, which percolated along the inside of the core liner.
Three and five milliliters of sediment were extracted from freshly opened windows using previously sterilized syringes that had an open-end cutoff in order to use them as minicores. Once the window was cut, the first extracted sample was for methane analyses to prevent it to escape due to volume expansion when exposed to ambient pressure. Hence, 3 ml of sediment was chemically stabilized and sealed in vials for headspace analysis. The sediments were further sampled for different techniques using 5-ml syringes and portioned out as follows: the first 1-ml portion of sample was placed in an Eppendorf tube and kept frozen for further DNA extraction; a second 1-ml portion was chemically fixed for DAPI (4′,6-diamidino-2-phenylindole) cell count; a third 1-ml portion of the sediment was mixed with 1 ml of deionized water in an Eppendorf tube and centrifuged for 5 min. Commercially available water testers (Biotrace International) were carefully submerged in the supernatant of the Eppendorf tube, and its ATP content measured with the Uni-Lite® NG luminometer from Biotrace International as an index of in situ microbial activity. The remnant sediment in the syringe was coated with plastic foil, sealed into hermetic aluminum foil bags and flushed with nitrogen prior to be sealed with a heating device in order to prevent oxidation. These samples can be further used for microbial culture experiments when back at the home laboratory. Once the sampling was accomplished, the windows were sealed with strong adhesive tape. This sampling procedure was carried out nonstop by both day and night shift teams. A more detailed description of the procedures as well as the different methods used can be found in Vuillemin et al. (2010).
One of the general outcomes of these pioneer investigations in Laguna Potrok Aike is data to support the hypothesis that microbes react to climatically driven environmental changes that have a direct impact on their subsurface distribution and diversity. This is clear at conspicuous levels associated with well-known climatic periods such as the Medieval Climatic Anomaly (MCA) or the Little Ice Age (LIA). The discrepancy between the in situ microbial activity (ATP) measurements and the total cell counting by DAPI (4′,6-diamidino-2-phenylindole) at these levels is reflecting the accumulation of dead microbial cells. This is interpreted as the result of the initial colonization of the substrate by microbes (Vuillemin et al. 2013a, b). It also shows that microbes resolve the scarcity of both nutrients and energy, becoming dominantly lithotrophs or organotrophs in the last glacial and Holocene sediments, respectively. These results are critical to understanding the limits of life in the subsurface of lakes under contrasting physicochemical conditions. On a practical side, this study shows that in the search of biosignatures in the sediments such as diagenetic concretions, it is crucial to use a wide range of high-resolution geochemical and imagery techniques that can be further blended with microbiological results. Only through a careful examination of these two—apparently independent—datasets, it is possible to disentangle the actual role of different microbial metabolisms during early diagenesis.
The present Dead Sea is located at ~425 m below sea level (b.s.l.) in the Levantine Basin, at the border between Israel, Jordan and the Palestinian Authority. At 31.4°N, 35.4°E, the lake lies along the Dead Sea Transform fault system (DST), turning it into a prime target for paleoseismic studies. The site is also ideal to study the impact of climate in the hydrology of the region, to extend the already long record of earthquakes for the area, and to investigate the presence of active microbes that could survive under extreme conditions of salinity (Dead Sea present salinity of 348 g L−1). ICDP drilling operations started in winter 2010, having retrieved 720 m of sediment from two different coring sites. Multidisciplinary investigations are currently ongoing, with a strong focus on paleoclimate and paleoseismicity reconstructions (Project DSDDP: Dead Sea Deep Drilling Project; Stein et al. 2011a, b). Based on the correlation of the main lithological units observed in the cores with numerous outcrop studies, down-hole logging and first 14C and U-Th dating, the composite core from the deepest area of the basin is interpreted to encompass the last two glacial–interglacial cycles (Neugebauer et al. 2014).
Within this framework, core catchers from the longest core were prioritized for the geomicrobiology study since, in contrast to Laguna Potrok Aike, no special hole was drilled for subsurface biosphere investigations. In the modern Dead Sea, the microbial content in the water column is very limited, preventing possible contamination of the sampled sediments with water column populations. Additionally, as in the previously described sites, we sampled the center of the individual core catchers (Fig. 6), disregarding their outermost part to minimize the possibility of contamination with drilling water. After each 12-h drilling shift, core catchers were brought onshore to a specially tailored facility for sampling under sterile conditions. Due to its unique salinity characteristics among all other aquatic environments (ten times higher than sea water), the Dead Sea drilling offers the opportunity to address life limits in a unique way. It also allows the assessment of the impact of microbial communities on the sedimentary record of extreme hypersaline environments.
These preliminary results indicate that the Dead Sea subsurface microbial community does not depend only on salinity. Although halophilic species are the dominant form obtained from sequencing techniques, Archaea of the Halobacteria class predominate within halite–gypsum sediments, while sequences related to hypersaline methanogens and fermenters are found in the shallowest aad facies. Thus, as in Laguna Potrok Aike, the sedimentary microbial communities in the Dead Sea seem to reflect the conditions in the water column at the time of sedimentation, linking their distribution to the prevailing climate. Additional investigations are necessary to better understand the microbial origin and evolution through time. Analogously, the traces of activity left by these microbes on the sediment are complex and not fully yet understood. However, Fe–S mineralization has been observed associated with EPS in some aragonitic-rich sediments, showing that even under extremely saline conditions, microbes can affect their environment (Thomas et al. 2014).
This lake is located between Albania and Macedonia on the Balkan Peninsula (41.1° N and 20.8° E) at an altitude of 639 m above sea level (a.s.l.; Fig. 3). It is considered the oldest lake in continuous existence in Europe containing more than 200 endemic species (Wagner et al. 2014 and references herein; Wilke et al. 2010), which makes it a particularly interesting place to investigate the subsurface biosphere. It was successfully drilled in spring 2013. Although there were neither a planned geomicrobiological study nor cores specially retrieved for this purpose, a core-catcher sampling was implemented in the deepest site following the precautions mentioned for previous drilling projects. Samples were immediately frozen for subsequent studies. While this sampling is not ideal for in situ microbial activity investigation or incubation studies, immediately frozen samples are perfect for certain biogeochemistry and geomicrobiological investigations, such as biomarker analyses, DNA extraction and subsequent microbial ecology techniques (PCR and sequencing) studies. Ongoing analyses have allowed the extraction of DNA down to 134 m sediment depth. Hence, they may deliver a first view of the subsurface microbial activity and its genetic continuity in this unique site characterized by a large number of endemic eukaryote species.
The way ahead
At present, there is a limited number of subsurface microbial studies in lacustrine settings. Despite this scarcity, the results of the first geomicrobiological investigations in ICDP scientific drilling sites are quite promising. It already provides critical information to answer some open questions dealing with the impact of active microbes in the sediments. Laguna Potrok Aike, so far the best-studied lacustrine site for this purpose, has delivered some interesting conclusions. As for the Dead Sea and Lake Van sediments, it appears that the initial environmental conditions prevailing in the water column of these lakes affected both the composition and distribution of the microbial mass, which in some cases, have remained active within the sediments. A comparison between contrasting climatic zones derived from paleoclimatic proxies and microbial units derived from cluster analyses of geomicrobiological proxies in Patagonian lake Potrok Aike (Vuillemin et al. 2013b) provides an excellent approach to investigate the influence of different paleoenvironments on the distribution of microbial communities. It also appears that salinity and alkalinity are not the limiting factors controlling microbial development and survival because active microbes have been detected as deep as 200 m below the lake floor in the Dead Sea sediments, one of the saltiest water bodies in the world. In their fight for survival, microbes can be fueled using sedimentary organic matter and/or mineral matter depending on that resource’s availability in the environment. As a result, the development and distribution of conspicuous microbial communities can reasonably be related to former climatically driven environmental changes. A rigorous statistical treatment of coupled geomicrobiological and paleoenvironmental proxies is critical to confirm or dismiss this correlation.
Several diagenetic processes directly associated with microbes have been identified, in particular the formation of authigenic minerals that can be used as biosignatures of past microbial activity in the geological record. The study of these minerals, coupled with modern phylogenetic techniques and pore water analyses, is critical in determining the exact role of microbial metabolism in the complex reactions leading to their development. With the emergence of high-throughput gene sequencing technology, microbes can be grouped in microbiomes that in the sedimentary realm are best defined as microbial facies. As with lithological types, these microbial facies are representative of a given depositional environment. With regard to lacustrine sediments, existing data necessary to fully establish a relationship between climate and microbial facies remain limited and require further study.
The significance and validity of the results of these types of investigations are largely dependent on the quality, rapidity and prevailing conditions during the initial sampling. Closer collaboration with the marine research community will enable standardization of sampling methods. Through this collaboration, the lake community could benefit from the various experiences in sampling and sample handling already gained during a large number of IODP operations. Such methodological coordination will allow (1) reduction in the impact of contamination, (2) determination of the best method(s) to accomplish onsite cell counting, in situ microbial activity and substrate turnover rate measurements, (3) selection of the appropriate sampling protocol for further molecular characterization and (4) standardization of sample archival. Due to the different nature of each drilling project, the need for standardized biological sampling, processing and analysis is quite significant. New ICDP sites such as Lake Towuti in Indonesia (Russell et al. 2014) incorporate and implement an appropriate sampling strategy for subsurface studies from the initial phase of the project.
As in the marine realm, the main challenge for the development of subsurface biosphere studies in lacustrine settings is to obtain intellectual reciprocity from other scientific disciplines participating in ICDP drilling initiatives. It is important for future ICDP projects in the terrestrial realm to involve geomicrobiologists in discussions about the effects of an active biosphere on the subsurface environment. Paleoenvironmental reconstructions largely depend on the use of proxies that need to be fully understood because this is crucial in any attempt to reconstruct past environmental changes. Many of these proxies are based on organic compounds (biomarkers) and inorganic constituents, including stable isotope compositions. Thus, a clear understanding of the impact of different microbial communities on proxies is vital.
The authors are thankful to all participants of the PASADO and DSDDP projects for their help and patience with field and laboratory sampling. AV and DA are particularly thankful to the PIs and research team of the PASADO project for devoting an entire core for this pioneer study. We kindly acknowledge the J. Pawlowski team (Department of Genetics and Evolution, University of Geneva) for their assistance and advice on different microbiology methods. CT and DA thank D. Ionescu from MPI Bremen, Germany, for his help and for sharing his knowledge of the Dead Sea microbiology. DA kindly acknowledges discussions and suggestions from H. Mills, J. de Leeuw and the team of the Advancing Subsurface Biosphere and Paleoclimate Research Workshop (ECORD/ICDP Magellan Plus Workshop Series Program). C. Glombitza and an anonymous reviewer along with associated editor L. Soreghan are thanked for constructive comments that improved the original manuscript. This research has been possible through the combination of multiple funding agencies including ICDP and all partners from the different drilling projects; generous funding from the Swiss National Science Foundation (Grants 200020-119931/2—PASADO—and Projects 200021-132529 and 200020-149221/1—DSDDP) to DA; and the University of Geneva, Switzerland.
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