Microbial viabilityMicrobial activityMicrobial Community StructureMMG 301 Dr. Frank DazzoMicrobial Ecology: Methodology & Soil MicrobiologySome methods used to study microbes in natural habitats:Microbial abundance:• microscopy, computer-assisted image analysis• measurement of cell constituents, e.g., ATP, muramic acid• filtration / dry weight (aquatic habitats)• viable enumeration techniques: plating, MPN, membrane filtration• quantitative PCR of DNA/RNA using phylogenetic probesMicrobial viability• Combination of fluorescence microscopy using vital stains (e.g.,BacLite Live/Dead) and viable plating techniquesMicrobial activity• Microscopy, redox-sensitive dyes (Cpdox + H+ + e- →→→→ Cpd red)• Microelectrodes• Enzyme activity assays• Gas exchange (e.g., uptake / production of O2, CO2, N2, CH4)• Assess bacterial vs. fungal contributions by use of selectiveantibiotic inhibitors (e.g., chloramphenicol vs. cyclohexamide)• Stable and radioactive isotope studies• In situ rates of substrate utilization and product formationMicrobial Community Structure• Computer-assisted microscopy and image analysis, e.g., CMEIAS• Polyphasic taxonomy & physiological diversity of isolates• Various types of 16S rRNA analysis, FISH, RDP-II bioinformatics• DNA amplification, genomic fingerprinting, functional genomics1. algal autofluorescence2. direct count, acridine orange vital stain3. direct count, DAPI stain for DNA4 & 5: FITC-immunofluorescence using strain-specific anti-LPSantibody; 4- bacteria on slide; 5- bacteria on plant root, LSCM.6. FISH: Fluorescence in situ hybridization (ritc-16S rRNA oligoprobe)7. Live (green) / dead (red) viability stain (Molecular Probes BacLight)8.Green Fluorescent Protein reporter strains for in situ sensing of N-acylhomoserine lactone quorum signal: red = source; green = sensorMicroelectrodes areused to measure thein situ distribution ofO2, pH, and H2S inhot spring microbialmats at sub-mmresolution.Microbial Ecology of Soil• 3-phase (solids, liquids, gases) ecosystem dominated by the solidphase.• Solids consist of soil separates (sand, silt, clay) & soil organic matter.• Clays are the major inorganic soil separate affecting microbialactivity in soil: their size ≤≤≤≤ bacteria, are the greatest surface areacomponent, affect ion /nutrient mobility, buffer pH, water retention,porosity and gas exchange, they form bacterial clay envelopesFormation and movementof soil materials lead todiscrete layers (horizons),producing a maturevertical profile asillustrated here. Microbialbiomass and activity inthis profile varies inproportion to the organicnutrient content: highestnear the surface anddecreases with depth.A soil aggregate of solids (mineral andorganic components), liquids & gases.Most microbes are in microcolonies onsoil particles. They may escape predatoractivities by refuge in small pores.Major groups of soil microorganisms and their significant activities:Bacteria:• numerically abundant (109 cells / g soil) but most non-culturable• along with fungi, most important decomposers of organic matter• specialized groups participate in all biogeochemical cycles• their extracellular polymers help bind soil particles into aggregates• some form beneficial or pathogenic interactions with plantsActinomycetes:• specialized filamentous prokaryotes• participate in decomposition of complex organic compounds• produce many 2°°°° metabolites, e.g., antibiotics,geosmins (earth odor) that give soil itscharacteristic distinctive aromaFungi:• the major component of microbial biomass in soils• major participants in decomposition of organic matter• hyphal growth helps bind soil particles into stable aggregates• some associate with plant roots: major plant pathogens, beneficialsymbionts increase nutrient uptake and decrease diseaseincidenceProtozoa:• major predators of soil bacteria, grazing activities acceleratedecomposition of organic matter in soilCyanobacteria and algae (green algae, diatoms):• photoautotrophs, form surface algal crusts important in H2Oretention• some cyanobacteria carry out free-living and symbiotic N2-fixationViruses:• Numerically abundant, ecology not well defined• Both lytic and lysogenic bacteriophage (latter very common)• Persistance and migration of human enteroviruses pose serioushealth issues with land disposal of sewage and fecal wastesGeosminSpecial considerations for gas/liquid relationships affectingmicrobial activity in the soil environment:• Soil atmosphere/water occupy the pore spaces in the soil matrix• Oxygen flux controls the type of metabolism (aerobic vs.anaerobic) accomplished by the microbes• Oxygen diffusion is 104 – fold faster in gas than through water,hence “water-logged” soils quickly become anaerobicMicrobes in discontinuouswater films on the surface ofsoil particles have good accessto O2. In contrast, microbes incontinuous water-filled poreshave limited O2 fluxes, creatinganoxic microenvironments.Bacterial movement throughsoil can occur when water-filledpore spaces are continuous butceases when they arediscontinuous.(Q?): How can one explain thedetection of fermentativeendproducts of microbialanaerobic metabolism even insandy, well-drained soils?(A): Sandy soils still containsoil aggregates where radialO2 diffusion is restricted, soanoxic microenvironmentsdevelop within their interiorwhere microbes are stillactively conducting anaerobicfermentative metabolism.In highly productive regions, waterlogged soils (bogs, swamps)accumulate high levels of organic matter since microbialdecomposition processes are slow under anaerobic conditions.When drained, the anaerobic →→→→ aerobic conversion accelerates themicrobial mineralization of the accumulated soil organic matter(S.O.M. →→→→ H2O + CO2↑↑↑↑), resulting in soil subsidence. E.g., Someareas of Florida everglades drained for intensive sugarcaneagriculture; ∼∼∼∼1” soil loss/yr. Some areas already reached bedrock.• Soil subsidence in Belle Glade, FL, mediated by aerobicmicrobial mineralization of bog soil high in organic matter.• Profound example of how soil microorganisms can effectphysical properties of
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