Discriminative detection and enumeration of microbial life in marine subsurface sediments

Detection and enumeration of microbial life in natural environments provide fundamental information about the extent of the biosphere on Earth. However, it has long been difficult to evaluate the abundance of microbial cells in sedimentary habitats because non-specific binding of fluorescent dye and/or auto-fluorescence from sediment particles strongly hampers the recognition of cell-derived signals. Here, we show a highly efficient and discriminative detection and enumeration technique for microbial cells in sediments using hydrofluoric acid (HF) treatment and automated fluorescent image analysis. Washing of sediment slurries with HF significantly reduced non-biological fluorescent signals such as amorphous silica and enhanced the efficiency of cell detachment from the particles. We found that cell-derived SYBR Green I signals can be distinguished from non-biological backgrounds by dividing green fluorescence (band-pass filter: 528/38 nm (center-wavelength/bandwidth)) by red (617/73 nm) per image. A newly developed automated microscope system could take a wide range of high-resolution image in a short time, and subsequently enumerate the accurate number of cell-derived signals by the calculation of green to red fluorescence signals per image. Using our technique, we evaluated the microbial population in deep marine sediments offshore Peru and Japan down to 365 m below the seafloor, which provided objective digital images as evidence for the quantification of the prevailing microbial life. Our method is hence useful to explore the extent of sub-seafloor life in the future scientific drilling, and moreover widely applicable in the study of microbial ecology.

[1]  K. Porter,et al.  The use of DAPI for identifying and counting aquatic microflora1 , 1980 .

[2]  W. Whitman,et al.  Prokaryotes: the unseen majority. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Maruyama,et al.  Spectral imaging detection and counting of microbial cells in marine sediment. , 2003, Journal of microbiological methods.

[4]  K. Nealson,et al.  Microbial Communities Associated with Geological Horizons in Coastal Subseafloor Sediments from the Sea of Okhotsk , 2003, Applied and Environmental Microbiology.

[5]  David C. Smith,et al.  New cell extraction procedure applied to deep subsurface sediments , 2008 .

[6]  K. Goodman,et al.  Deep bacterial biosphere in Pacific Ocean sediments , 1994, Nature.

[7]  I. Hewson,et al.  Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I , 2007, Nature Protocols.

[8]  T. Hoehler.  Biological energy requirements as quantitative boundary conditions for life in the subsurface , 2004 .

[9]  Tohru Kobayashi,et al.  Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula , 2008, Extremophiles.

[10]  J. Jansson,et al.  Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dualgfp-luxAB Marker System , 1999, Applied and Environmental Microbiology.

[11]  Gerald R. Dickens,et al.  Distributions of Microbial Activities in Deep Subseafloor Sediments , 2004, Science.

[12]  H. Furnes,et al.  Links Between Geological Processes, Microbial Activities&Evolution of Life , 2008 .

[13]  J. Hobbie,et al.  Direct counts of aquatic bacteria by a modified epifluorescence technique1 , 1975 .

[14]  R. Parkes,et al.  Recent studies on bacterial populations and processes in subseafloor sediments: A review , 2000 .

[15]  M. Hovland,et al.  Bacterial Profiles in Deep Sediments of the Santa Barbara Basin, Site 893 , 1995 .

[16]  T. Treude,et al.  Fluids from the Oceanic Crust Support Microbial Activities within the Deep Biosphere , 2008 .

[17]  Scott Rutherford,et al.  Metabolic Activity of Subsurface Life in Deep-Sea Sediments , 2002, Science.

[18]  J. Fuhrman,et al.  Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria , 1998 .

[19]  K. Hinrichs,et al.  Significant contribution of Archaea to extant biomass in marine subsurface sediments , 2008, Nature.

[20]  M. Weinbauer,et al.  Utility of Green Fluorescent Nucleic Acid Dyes and Aluminum Oxide Membrane Filters for Rapid Epifluorescence Enumeration of Soil and Sediment Bacteria , 1998, Applied and Environmental Microbiology.

[21]  F. Inagaki,et al.  Spatial Distribution of the Subseafloor Life: Diversity and Biogeography , 2008 .

[22]  J. Tiedje,et al.  DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community , 1988, Applied and environmental microbiology.

[23]  A. Teske.  Microbial Communities of Deep Marine Subsurface Sediments: Molecular and Cultivation Surveys , 2006 .

[24]  B. Jørgensen,et al.  Exploring subseafloor life with the Integrated Ocean Drilling Program , 2007 .

[25]  D. Prieur,et al.  Extending the Sub-Sea-Floor Biosphere , 2008, Science.

[26]  W. Martens-Habbena,et al.  An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy. , 2005, Environmental microbiology.

[27]  B. Jørgensen,et al.  Biogeographical distribution and diversity of microbes in methane hydrate-bearing deep marine sediments on the Pacific Ocean Margin. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[28]  N. Masui,et al.  Microbiological Assessment of Circulation Mud Fluids During the First Operation of Riser Drilling by the Deep-Earth Research Vessel Chikyu , 2008 .