Bacteria in the aqua-c environment. Takeshi TERAHARA Assist. Prof. Tokyo University of Marine Science and Technology (TUMSAT)

Transcription

1 Bacteria in the aqua-c environment Takeshi TERAHARA Assist. Prof. Tokyo University of Marine Science and Technology (TUMSAT)

2 How many microorganism live in just 1 ml of seawater? Up to a million of microorganisms!! Microorganism is so small!! It can only be seen with the use of a microscope.

3 What kind of living things are there on Earth? Cyanobacteria Planctomyces Cytophaga Thermotoga Aquifex Bacteria Archaea Eucarya Prokaryotes (No nucleus) Spirochetes Proteobacteria Green Filamentous bacteria Gram posilves Methanobacterium Methanococcus T. celer Thermoproteus Pyrodic-cum Prokaryotes Many are extremophiles. Entamoebae Slime Methanosarcina Halophiles Eukaryotes Complex cells with a nucleus molds Animals Fungi Plants Ciliates Flagellates Trichomonads Microsporidia Diplomonads PhylogeneLc tree of life was originally proposed by Carl Woese.

4 How do living things live? What is needed for? 1. Energy source 2. Material source Organic Metabolism Catabolism Anabolism Organic (smaller) CO 2 Energy O 2 H 2 O Biological material

5 Where do bacteria live? Everything is everywhere, but the environment selects Baas- Becking, L. G. M

6 Earth the water planet 71% Water: 1.4 billion (km 3 ) Seawater Glacier River, Lake, Groundwater 97.5% 1.7% 0.8% hcp://legacy.mos.org/oceans/planet/index.html

7 Bathymetric features of the ocean floor are diverse. EuphoLc zone (On average, 200 m) DisphoLc zone AphoLc zone hcp://legacy.mos.org/oceans/planet/features.html

8 Photoautotroph 1. Energy source : light phototroph 2. Material source : CO 2 autotroph Light Photosynthesis Energy CO 2 Reduced product ( H 2 O H 2 S ) O 2 SO 4 2- etc. Phytoplankton, cyanobacteria can produce oxygen!! Biological material

9 Water bloom An immense spread of phytoplankton bursts. Japan North AtlanLc hcp://earthobservatory.nasa.gov/images/imagerecords/14000/14903/japan_amo_ _lrg.jpg hcp:// science/t/swirling- currents- fuel- huge- blooms- north- atlanlc/

10 Organic Chemoheterotroph (Aerobic) 1. Energy source : chemical material chemotroph 2. Material source : organic material RespiraLon heterotroph CO 2 Energy Organic O 2 Organic CO 2 Energy H 2 O Organic Biological material O 2 H 2 O Biological material

11 Chemoheterotroph (Anaerobic) Dissolved oxygen is deficient on the sediments in the bay such as Tokyo Bay. Anaerobic: No molecular oxygen Organic CO 2 Energy Organic SO 4 2- NO 3 - etc. O 2 Reduced product ( H 2 S NO 2 - etc.) Biological material

12 Organic Sulfate reducing bacteria CO 2 Energy Organic O 2 SO 4 2- Reduced product ( H 2 S ) Biological material Hydrogen sulfide (H 2 S) produce "rocen egg" odor. H 2 S will react with metal ions to produce metal sulfides. These metal sulfides, such as ferrous sulfide (FeS), are insoluble and olen black or brown, leading to the dark color of sediment.

13 Chemoheterotroph (Anaerobic) Organic Oxidized product Energy Organic O 2 FermentaLon Biological material Glucose Ethanol O 2

14 Hydrothermal vent No sunlight for photosynthesis in the darkness of the ocean depths Temperature : may reach over 340ºC Pressure : may reach over 300 atmospheres Chemicals : hydrogen sulfide, the most plenlful compound in vent emissions (toxic to most living things) ph : very acidic as low as 2 (unhealthy for most living things) hcp://

15 Chemoautotroph 1. Energy source : chemical material chemotroph 2. Material source : CO 2 autotroph Glucose H 2 S H 2 etc. Oxidized product Energy Organic CO 2 O 2 HCO 3 - etc. Reduced product (H 2 O CH 4 ) Biological material

16 SymbioLc relalonship with tubeworm and bacteria Hydrothermal vent H 2 SO 4 H 2 S Tubeworms do not feed due to no mouth or stomach! Tubeworms depend for their nutrilon upon symbiolc bacteria that live inside of them!! Sulfur oxidizing bacteria Inside tubeworm Organic Tubeworm hcp:// j/gallery/yusui/yusui_6.html O 2

17 Methane hydrate Topic : aclvity of methanogens in methane hydrate- bearing sediments H 2 Methanogen (Archaea) H 2 O Energy CO 2 HCO 3 - CH 4 Biological material hcp://

18 InvesLgaLng the Ocean 1. How to collect samples (For example: seawater, sediment) 2. How to study marine bacteria 3. Unique characterislcs of marine bacteria (For example: NaCl tolerance)

19 How to collect seawater Niskin bocle: cylindrical container open at each end

20 Picked up with a crane

21 Suspended on steel wire and sunk into sea Collected at depth as you want to measure!

22 Cylindrical container close at each end

23 How to measure salinity and temperature in the Ocean ConducLvity - Temperature - Depth Sensor (CTD) To measure the conduclvity (salinity), temperature and underwater pressure (depth) of the ocean, electrically.

24 Pressure increases by 1 atm (1 atm 1 kg/cm 2 ) with each 10 m of depth. Cup noodle vs. Water pressure At a depth of 1000 m in ocean, 100 kg/cm 2!

25 How to collect sediment Sediment sampler (Ekman-Birge type bottom sampler)

26 Messenger weight triggers to grab sediment. When the sampler reaches the bocom, a messenger is sent down.

27 Sediment sample collected from Tokyo Bay

28 How to study marine bacteria Culturability = Colony counts Cell counts CulLvaLon Microscope Amann et al. (1995) Most of bacteria are unculturable!

29 Molecular techniques for studying bacteria CulLvaLon 1% Culturable Microscope Molecular techniques DNA PCR Electrophoresis T- RFLP FISH Cloning & sequencing DGGE

30 Fluorescent In Situ Hybridiza-on (FISH) Cell 1. Probe with fluorescent dye : matches the DNA of target type of bacteria 2. Add the probe to a sample, the probe binds to any bacteria that have similar DNA. 3. Under a microscope, only target- bacteria can be observed. h#p:// educa6on_modules/marine_bacteria/explore_trends/

31 Terminal Restric-on Fragment Length Polymorphism (T- RFLP) 1. PCR Fluorescent primer 3. Electrophoresis 4. DetecLon of fragments 2. RestricLon enzyme digeslon Fragment height Fragment length One fragment length : One species Different fragments : Bacterial species Fragment height : Bacterial richness

32 Denaturing Gel Gradient Electrophoresis (DGGE) GCGCGCGC CGCGCGCG GCGCGCGC CGCGCGCG GCGCGCGC CGCGCGCG Low PCR products GC content A GC (guanine plus cytosine) rich sequence can be incorporated into one of the primers. PCR products of different sequence can be separated in an acrylamide gel. High Low Gradient High Poly- acrylamide gel containing a linearly increasing gradient of DNA denaturants (usually urea and formamide) One band : one bacterial species Band strength : Bacterial richness

33 Direct visualizalon of marine bacterial diversity DNA extraclon T- RFLP DGGE FISH

34 Unique characterislcs of marine bacteria Sediment samples (0.1 g each) were suspended in 1 ml of saline solulon. 0.1 ml of each suspension was spread over the medium IncubaLon at 27 C for 3 weeks.

35 Examples of bacterial colonies AcLnomycetes (Streptomyces) Bacteria

36 AcLnomycetes isolated from marine sediment is more tolerant of NaCl than bacteria isolated from soil. NaCl 0% NaCl 4% NaCl 6% NaCl 8% Soil Marine Species is same, but characterislcs is different!

37 The color of some colonies changed to light purple, then to dark purple with the increasing NaCl concentralon. NaCl 0% NaCl 2% NaCl 4% NaCl 6%

38 Bacteria in the aqualc environment (ocean) H 2 O O 2 FISH Organic CO 2 Organic T- RFLP H 2 S H 2 SO 4 DGGE HCO 3 - CH 4