Microbiology. Zhenmei Lu ( 吕镇梅 ) 2010 Spring-Summer 2017 Spring-Summer

Transcription

1 Microbiology Zhenmei Lu ( 吕镇梅 ) lzhenmei@zju.edu.cn 2010 Spring-Summer 2017 Spring-Summer

2 Chapter 23 Microbial Ecosystems

3 23.1 Ecological Concepts Ecosystem: a dynamic complex of plant, animal, and microbial communities and their nonliving surroundings Microbial species diversity: Richness versus Abundance

4 Sampling from Taihu Lake High species richness Low species richness

5 23.1 Ecological Concepts Symbiosis: a relationship between two or more organisms that share a particular ecosystem. Parasitism: Bdellovibrio Mutualism: both benefit. Commensalism: one benefits, the other neither harmed nor helped. Antogonism: kill or inhibit others. Competition: survive on same resources.

6 23.2 Microbial Ecosystems and Biogeochemical Cycling Community 1 Photic zone: Oxygenic phototrophs Community 2 Oxic zone: Aerobes and facultative aerobes Populations, guilds, and communities Community 3 Anoxic sediments: 1. Guild 1: methanogens (CO 2 CH 4 ) acetogens (CO 2 acetate) 2. Guild 2: sulfate-reducing bacteria (SO 2 4 H 2 S) sulfur-reducing bacteria (S 0 H 2 S) 3. Guild 3: denitrifying bacteria (NO 3 N 2 ) ferric iron-reducing bacteria (Fe 3 Fe 2 ) 4. Guild 4: fermentative bacteria

7 23.3 Environments and Microenvironments Oxygen microenvironments Contour map of O 2 concentration of a soil particle

8 23.4 Surfaces and Biofilms Surfaces are important microbial habitats because Nutrients adsorb to surfaces Microbial cells can attach to surfaces Microcolonies Root

9 23.4 Biofilms Attachment (adhesion of a few cells to a suitable solid surface) Colonization (intercellular communication, growth and polysaccharide formation) Development (more growth and polysaccharide) Cell Water channels Surface Polysaccharide Biofilms: assemblages of bacterial cells enclosed in an adhesive matrix excreted by the cells

10 23.4 Surfaces and Biofilms Advantages: 1. Self-defense; 2. Stay in a favorable niche; 3. live together; 4. The typical way bacterial cells grow in nature. Damages: blah, blah, blah How to control: a real challenge

11 23.5 Microbial Mats Microbial mats are very thick biofilms Built by phototrophic and/or chemolithotrophic bacteria

12 23.6 Terrestrial environments O horizon Layer of undecomposed plant materials A horizon Surface soil (high in organic matter, dark in color, is tilled for agriculture; plants and large numbers of microorganisms grow here; microbial activity high) B horizon Subsoil (minerals, humus, and so on, leached from soil surface accumulate here; little organic matter; microbial activity detectable but lower than at A horizon) C horizon Soil base (develops directly from underlying bedrock; microbial activity generally very low) (a) Profiles of a mature soil. (b) Photo of a soil profile showing O, A and B horizons.

13 Microcolonies Sand Sand Silt Clay particle Silt Organic matter Sand Water Air Clay particle A soil microbial habitat. Very few microorganisms are free in the soil solution; most of them reside in microcolonies attached to the soil particles.

14 Scanning electron microscopy of microorganisms on the A microcolony of coccobacilli surface of soil particles Actinomycete spores Fungal hyphae

15 Microbial Sidebar Energy-yielding process Reaction Methanogenesis: Acetogenesis: Sulfate reduction: Inorganic H 2 production: Microbial life in the deep subsurface (1500m) Stevens TO and McKinley JP. Science, 1995, 270: Anderson RT et al. Science, 1998, 281:

16 23.7 Freshwater environments

17 Cell number/nutrients Input (sewage, other wastewaters) O 2 Bacteria, organic carbon, and BOD Algae and cyanobacteria O 2 Effect of the input of organic-rich wastewaters into aquatic systems. (a) In a river, bacterial numbers increase and O 2 Distance downstream NO 3 NH + 4 and PO 3-4 levels decrease with the spike of organic matter. The rise in numbers of algae and cyanobacteria is primarily a response to inorganic nutrients, especially PO (b) Photo of a eutrophic lake.

18 A large bloom of blue-green algae caused water quality to deteriorate severely. Wuxi Blue algae blooming in Taihu Lake in 2007

19 Sampling the deep subsurface Sampling hot (55 ) fissure water from a depth of 3000 m in the Tau Tona South African gold mine Drilling to 600 m

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21 Chapter 22 Methods in Microbial Ecology Stories about Microbial Communities

22 Culture-Dependent Analysis of Microbial Communities

23 22.1 Enrichment Inocula Medium Conditions Techniques Enrichment Bias

24 22.1 Enrichment The Winogradsky Column Lake or pond water Mud supplemented with organic nutrients and CaSO 4 Gradients Column O 2 H 2 S Foil cap Algae and cyanobacteria Purple nonsulfur bacteria Sulfur chemolithotrophs Patches of purple sulfur or green sulfur bacteria Anoxic decomposition and sulfate reduction

25 22.2 Isolation The streak plate Agar dilution tubes Most-Probable Number (MPN) 1 ml (liquid) or 1 g (solid) Enrichment culture or natural sample Dilution 1 ml 1 ml 1 ml 1 ml 1 ml Colonies Paraffin seal Growth Growth No growth 9-ml broth 1/10 (10 1 )

26 22.2 Isolation The Laser Tweezers Flow cytometry b a Cell Beam focus a b Objective lens of microscope Laser beam Optically trapped cell Severing point Capillary tube Laser Mixture of cells

27 Culture-InDependent Analysis of Microbial Communities

28 22.3 Staining Nonspecific fluorescent stains Nucleic acid staining: DAPI (4,6 -diamido-2- phenylindole) Acridine orange SYBR Green Viable & Dead

29 22.3 Staining Viability staining: Dye unable to penetrate membrane Using with Propidium iodide (Red) nucleic acid staining (penetrating membrane)

30 22.3 Staining Fluorescent Antibodies: Fluorescent antibodies as a cell tag

31 22.3 Staining Green Fluorescent Protein (GFP) as a cell tag

32 22.3 Staining

33 22.4 FISH-Fluorescence in situ hybridization Phylogenetic staining using FISH

34 22.4 FISH Phylogenetic Staining using FISH Sewage sludge

35 22.4 FISH ISRT (in situ reverse transcription) FISH

36 Catalyzed reporter deposition FISH (CARD-FISH) labeling of Archaea

37 22.5 PCR Methods of Microbial Community Analysis Steps in single gene biodiversity analysis

38 Genes commonly used for evaluating specific microbial processes in the environment using PCR

39 22.5 PCR Methods of Microbial Community Analysis DGGE: Denaturing Gradient Gel Electrophoresis

40 Different microbial populations in a community DNA extraction and PCR amplification Mixed 16S rrna gene copies Separate by cloning in E. coli or DGGE Sequence Phylogenetic Identification DGGE operating process

41 Influence of GC Base Content on Temperature Melting Point of DNA GC TA TA GC CG AT CG

42 Culture-Independent Analysis of Microbial Community Structure in the Soil Eubacterial 16S rdna Universal Primers Denaturant 20 % 70 % DNA Extraction PCR Denaturing Gradient Gel Electrophoresis

43 Microbial Species Identification from 16S rdna Bands 16S rdna Band Clone Library PCR pgem-t Sequencing Sequence Analysis

44 Terminal Restriction Fragment Length Polymorphism (T-RFLP) of 16S rrna genes Promoter trna gene Terminator 16S 23S 5S bp V1 V2 V3 V4 V5 V7 V8 V9 Forward Primer PCR & Digestion Reverse Primer A A B B Electrophoresis MspI RsaI A B A,B

45 The microbial community analysis for PCB polluted soil using T-RFLP method

46 Fluorescence Position S rrna gene Forward PCR primer containing fluorescent tag ( ) ITS region bp Position 1513 Position 23 23S rrna gene Reverse PCR primer Community DNA PCR Gel analysis Fragment size (base pairs) Automated ribosomal intergenic spacer analysis (ARISA)

47 22.6 Microarrays and Microbial Diversity: Phylochips Positive Weak positive Negative Phylochips

48 22.7 Environmental Genomics

49 At least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes were identified for the microbial populations (Venter et al., Science,2004)

50 Strategy of finding ideal catalyst for toxic compounds --Metagenomics DNA extraction + Enrichment for bacteria that metabolize substrate (PCB et al.) Genomic DNA extraction Screen Ligation + Ligation Expression + Transformation E. coli + Metagenomic library E. coli

51 22.8 Microbial Activity in Nature Microbial Activity Measurements Sulfate reduction Formalin-killed control H 2 S Lactate 14 CO2 incorporation Photosynthesis Killed Light Dark Time Time Sulfate reduction 14 C-Glucose respiration H 2 present Killed H 2 absent Killed Time Time

52 22.8 Microbial Activity in Nature Microelectrodes

53 Depth in sediment (mm) Oxygen (O 2 ) concentration ( M) Seawater 0 5 O 2 Oxic sediment NO 3 10 Anoxic sediment Nitrate (NO 3 ) concentration ( M)

54 22.8 Microbial Activity in Nature Isotopic fractionation Enzyme substrates Enzyme that fixes CO 2 Fixed carbon

55 22.8 Microbial Activity in Nature Marine carbonate Atmospheric CO 2 Calvin cycle plants Methane Petroleum Isotopic Geochemistry of 13 C and 12 C Cyanobacteria Purple sulfur bacteria Green sulfur bacteria Recent marine sediments 3.5 billion-year-old rocks C ( 0 / 00 )

56 13 C ( 0 / 00 ) Ion source Mass spectrometer Magnet Primary ions Sample ABCDE Multiple detectors Secondary ions Outer shell of sulfate-reducing bacteria cells (green) burned away by primary ion beam Inner core of methanotrophic Archaea cells (red) exposed Cycles of sputtering and data collection (increasing depth into aggregate) Secondary ion mass spectrophotometry (SIMS)

57 22.8 Microbial Activity in Nature FISH-MAR: Microautoradiography

58 22.8 Microbial Activity in Nature Stable isotope probing

59 22.8 Microbial Activity in Nature Stable isotope probing

60 Label cells by FISH Isolate fluorescent cells by flow cytometry Genetic analysis of sorted cells Extract DNA PCR DNA Multiple displacement amplification (MDA) and sequencing Primers Phage DNA polymerase Assay for specific genes (16S rrna genes, metabolic genes, etc.) A B C Sequence genome

61 Thanks for your attention!