Takeshi Naganuma (Hiroshima University, Japan)
Microbial communities affect geochemical and hydrogeological processes in deep aquifers, and have been characterized mainly by genetic approaches as well as activity measurements. The commonest gene for this purpose is the 16S ribosomal RNA gene, or 16S rDNA. We have analyzed 16S rDNA assemblages from hydrologically isolated aquifers of porous-but-less-permeable strata in the northernmost part of Japan, and revealed distinctive microbial communities including methanogens and so-far-uncultured species. These microbial communities may affect groundwater chemistry and vice versa. Aquifer microorganisms are usually captured by 0.2-µm-pore-size filters; however, there are microbes passing 0.2-µm pores. The 0.2-µm-passing microbes are likely to occur more abundantly in rock matrices and fractures than limnological and oceanographic habitats, and thus to be focused in aquifer microbiology. We have collected 16S rDNA of the 0.2-µm-passing-but-0.1-µm-captured microorganisms from sedimentary and granite rock aquifers in a uranium deposit area of central Japan. The 16S rDNA analysis also revealed distinctive communities including so-far-unknown lineages. The 16S rDNA-based approach answers who-they-are, but tells no or little about what-they-do. For example, detection of so-far-uncultured or so-far-unknown microorganisms tells nothing about their geochemical functions in situ. In situ activities are characterized and measured by culture-based physiological approaches, which have many limitations and biases though. The two split approaches should be combined to complement each other. An example is quantification of messenger RNA, or mRNA, which is the
“bridge” between DNA (= potential) and protein (= reality, activity). We have developed a quantification of mRNA for redox-responsive proteins of Fe(III)-reducing bacteria collected from a deep aquifer. This approach is applicable to other aquifer processes such as reduction of nitrate, sulfate, CO2, etc. This kind of combined molecular, i.e., DNA/RNA, approaches will enable new tactics to understand biogeochemical processes occurring in deep, less readily accessible aquifers.
International Symposium on GRAPHIC International Symposium on GRAPHIC
4-4-6 April 2006, Kyoto, Japan6 April 2006, Kyoto, Japan
Molecular microbiological approaches Molecular microbiological approaches
to understand biogeochemical to understand biogeochemical
processes in deep aquifers processes in deep aquifers
Takeshi Naganuma1,2, Teruki Iwatsuki2, Satoru Shiimizu3, Masaru Akiyama3& Yoji Ishijima3
1School of Biosphere Science, Hiroshima University
2Japan Atomic Energy Agency
3Horonobe Res. Inst. for Subsurface Environment
Please give your visit to Hiroshima for Please give your visit to Hiroshima for
the
the shrineshrine--onon--waterwater, next time., next time.
HDB-6
Are microbiological features such as community structures affected by hydrogeologic features occurring on the opposite sides of a major fault?
SW of fault
NE of fault Mostly
Archaea as well as Bacteria are prokaryotic microorganisms.
SW of fault SO4-reducers SW of fault NE of fault Group of diversified functional bacteria Proteobacteria
H2S-oxidizers CH4-oxidizers NH4-oxidizers NO3-reducers …
Non-Proteobacteria
SW of fault Mostly occurring on the
SW-side of the fault Biogeochemical functions are unknown; maybe fermentation and SO4-reduction?
Aerobic-anaerobic processes should be affected by vadose zone development.
Every available Every available oxidant is used for oxidant is used for respiration in the respiration in the anaerobic oxidizes hydrogen oxidizes hydrogen sulfide (H sulfide (H22S) .S) . Sulfate (SO Sulfate (SO442-2-) ) oxidizes methane oxidizes methane (CH
(CH44).).
COCO2 2 oxidizes oxidizes hydrogen (H hydrogen (H22) .) . Hydrogen sulfide
(H2S) forms from sulfate.
Methane (CH4) forms from CO2. Heatsplits water (H2O) splits into hydrogen (H2) and oxygen (O).
Chain reaction Chain reaction of anaerobic of anaerobic respiration
Organics (CH2O) O2 Water
Sulfate reduction
Food chain reactions Food chain reactions
COCO22
Tonouranium mineuranium mine
Near-Nagoya
“Tono” area
Kyoto
Tokyo
Tono
Tonomine lab planmine lab plan
Laboratory
TonoTonomine 125 m below ground level (bglmine 125 m below ground level (bgl))
Anaerobic glove Anaerobic glove boxeboxe
pH-pH-redox monitorredox monitor Sampling boreholeSampling borehole
Redox potential lowered by microbial SO4-reduction with natural organics, e.g., lignite, in Tono aquifers
±0 mV +100 mV
-300 mV
Redox potential
30 20
10 0
Days of incubation SO42-+ organics
ÆS2-+ CO2
SO SO44- -reducers
reducers SOSO44- -reducers reducers
HH22--utilizersutilizers HH22--utilizersutilizers
Versatile Versatile Pseudomonas Pseudomonas
Day 3 Day 6 Day 9
Day 13 Day 28
Colony of the bacterium, Pseudomonas stutzeri as an Fe(II)-oxidizer and Fe(III)-accumulater
Magnification by SEM
Feaccumulated on the cell surface of Pseudomonas stutzeri Smaller world
and smaller inhabitants are important, too.
Nano
Nano--microorganismsmicroorganisms
Tentative definition:
Microorganisms that pass through the filters having a cut-off pore size of 0.2 µm.
0.2 µm-filter-pores
Why is 0.2
Why is 0.2 µm important?µm important?
Most food, beverage, medical, clinical, and aquaculture industries rely on filtration-removal of pathogenic microbes using 0.2 µm-filters.
Microbial ecologists have also rely (too) much on the use of 0.2 µm-filters for retrieving environmental
species/genomes.
Microbes smaller than 0.2 µm have
been ignored. 0.2 µm-filter-pores
Tono uranium mine aquifer (150-200 m bgl) Genomes from microorganisms smaller than 0.2 µm
Previously unknown microorganisms were retrieved from the Tono mine deep aquifer.
(Appl. Environ. Microbiol. 71: 1084-1088, 2005)
Genetic info and biogeochemical activities Genetic info and biogeochemical activities
CENTRAL DOGMA of molecular biology DNA: gene = genetic potential
detected/amplified by PCR
mRNA:
mRNA: transcriptiontranscriptionof genetic potentialof genetic potential detected/amplified by RT detected/amplified by RT--PCRPCR
(Potential to Reality)
Protein: expression of genetic potential subject to activity measurement
Now available:
Now available:
quantitative RT quantitative RT--PCRPCR
(Q(Q--RTRT--PCR)PCR)
Messenger RNA (mRNA)
An example of Q
An example of Q--RTRT--PCR of redox stress responsive PCR of redox stress responsive genes associated with
genes associated withFeFe3+3+reduction reduction byby Pseudomonas
Pseudomonas stutzeristutzerifrom the Tonofrom the Tonomine aquifermine aquifer
Fe Fe
3+
3+reduction reduction mRNA transcription mRNA transcription
Genetic info (mRNA transcription) represents biogeochemical activities.
Summary
- Hydrogeology may affect microbial communities that may affect groundwater geochemistry.
Relevant to vadose zone / climate change studies?
- Redox shifts are associated with the changes in microbial communities.
- Nano-sized microorganisms do exist, but their geochemical functions are unknown.
-Genomeinformation provides potentialfunctions, while the advanced technique, Q-RT-PCR, will determine microbial functions in action.