Microbial Ecology, Metabolic Diversity , Microbio Exam 3

what are the 2 principle habitats where the majority of microorganisms are found?

soil and water

most microorganisms found in soil and sediments are found where?

in thin layer within a few feet of the surface

where have living microorganisms (bacteria) been found?

in porous rock formations thousands of feet below the surface of the earth

where are microorganisms in bodies of water such as lakes or oceans most concentrated?

at the surface and at the very bottom, in the few inches just above the mud

lowest concentrations of microorganisms in water

in open sea-water and never drop below about 10^5 cells per ml

microorganisms exist in the air, but are considered to be what?

accidental wanderers blown into the atmosphere by strong winds

powerful updrafts can carry microorganisms to heights exceeding 20,000 ft and more

what defines the niche for each particular organism?

the type and quantity or resources and the physiochemical conditions (pH, temp, pressure, light, water availability, oxygen)

the world of a microorganism is often measured in what?

microns

since the world of a microorganism is measured in microns, what is the habitat in which a microorganism lives referred to as?

a microenvironment

microenvironments are characterized by what 3 things?

1. microscopic
2. subject to extreme changes in physiochemical parameters
3. having steep gradients

surfaces

often of considerable importance as microbial habitats

5 properties of surfaces

1. can concentrate nutrients
2. can act as a nutrient
3. can immobilize microorganisms
4. can neutralize certain toxic substances (eg: chlorine)
5. can concentrate microorganisms of diverse species, allowing cross-feeding, etc

surfaces can immobilize microorganisms. what does this allow for?

allows bulk liquid flow past the microorganism, transporting nutrients to the microorganisms, and waste products away from the microorganisms

biofilms

assemblages of microorganisms embedded in a gelatinous matrix associated with an interface (air-water, solid-air, solid-water, water-liquid)

interfaces are in effect, surfaces as far as the microorganism is concerned

5 benefits to living within a biofilm

1. trap nutrients for growth and help prevent detachment of cells in flowing systems
2. cells are protected from attack by reactive chemicals (such as oxidizing biocides)
3. cells are protected from attack by grazing protists and by cells of the immune system
4. cells are protects from the activity of antibiotics
5. cells are buffered from changed in pH, temperature, water activity, salinity, etc

do most microorganisms in the environment exist as biofilms?

yes

what has NIH claimed in regard to biofilms?

that biofilms are responsible for over 80% of all bacterial infections in humans. this is referred to as "The Biofilm Problem"

how do nutrients enter an ecosytem?

in an intermittent fashion

what situation do microorganisms in nature often face with nutrients?

a "feast-or-famine" situation. rarely do microorganisms experience exponential growth for extended periods of time

what are bacteria (and other microbes) able to do because of the intermittent nature of nutrients in the microbial environment?

they are able to store compounds as reserve materials, such as poly-b-hydroxybutyrate, polysaccharides, polyphosphates, etc

what do some bacteria also do to deal with the intermittent fashion of nutrients?

bacteria are also able to "tune down" metabolically, some even increase their surface-to-volume ratio by reduction cell division

what do bacterial growth rates (measured often in minutes or hours) reflect?

the changing environment in which they live

growth rates of microorganisms in the environment

typically only 1%-10% of those achieved in the laboratory

microbial competition

microorganisms form the same species and form different species often compete for the same niche. competition under these circumstances is intense

what are many bacteria able to do in instances of competition?

many bacteria are able to produce substances which are inhibitory to the growth of other microorganisms, such as the production of toxic products like acids, or production of antibiotics or bacteriocins

example of microbial cooperation

microorganisms can be involved in cross feeding, stimulating the growth of a neighbor which can metabolize one or more of its waste products

what is cross-feeding in bacterial communities known as?

syntrophic consortia (considered to be a common situation)

examples of syntrophic consortia

the oxidation of ammonia to nitrate by one organism, and the reduction of nitrate to ammonia by another

it is often said that the number of microorganisms we are able to culture in the laboratory is only _________to________ of the microorganisms found in the environment

1% to 10%

because microorganisms are difficult to grow in pure culture, what is the first step in isolating a microorganism from the environment?

first step is to grow it in enrichment culture

what can happen once an organism is grown in enrichment culture?

it can be isolated in pure culture by dilution to extinction - Most Probable Number Method (MPN), streak plate, etc

what do enrichment cultures provide?

a selective advantage to particular organisms in a mixed environmental sample

how are enrichment cultures made?

by duplicating the environmental conditions where an organism is naturally found and then "spiking" the medium with one or more nutrients

example of an enrichment culture

if we wish to select for cellulose degrading microorganisms, we can take a sample of mud, put it in a tube and add paper to the tube

who were the first enrichment cultures devised by?

Sergei Winogradsky to study soil microorganisms

in order to determine the number of organisms in an unknown sample (environmental or medical origin) it is often not possible to do what?

culture the organisms
*problem of viable non culturable organisms

one solution to viable non culturable organisms

to use staining techniques to reveal the presence of individual cells and to indicate whether the cells are viable

acridine orange (AO)

a nucleic acid stain that can discriminate between actively growing and non-growing bacteria

how does AO work?

AO intercalates into the major groove in DNA and RNA and fluoresces orange when binding single stranded nucleic acid (mostly RNA) and green when binding double stranded nucleic acid (mostly DNA)

actively growing cells contain large amounts of RNA and dead cells contain essentially no RNA

LIVE/DEAD Bac light

another viability stain

uses the presence (live) or absence (dead) on an intact cytoplasmic membrane to distinguish between viable and non-viable bacteria

direct cell count

used to determine the number of bacteria in a sample

this method uses a stain, either a viability stain such as AODC (acridine orange direct count) or a general fluorescent stain such as DAPI (which binds to DNA and causes cells to fluoresce blue)

5 steps of direct cell count

1. cells in sample are stained with fluorescent stain
2. cells are filtered onto a membrane
3. membrane is viewed using a microscope
4. individual cells in each microscope field are counted
5. the total # of cells in the sample is determined by calculating the # of cells in the sample on the filter (number of fields/filter) and multiplying this by the number of samples in 1 ml or 1 g of the sample

3 methods for determining the types of cells in a sample without culturing the sample

1. fluorescence labeled antibodies
2. FISH probing
3. optical tweezers

fluorescence labeled antibodies

used to detect the presence of specific cells in a particular sample. this technique is used
1. to determine the number of a known organism in its natural env
2. to evaluate all the cells carrying a particular antigen in an environmental sample
3. to detect the presence on certain groups of microorganisms which all carry the same antigen

FISH probing (fluorescent in situ hybridization probing)

used to detect members of a particular taxonomic group in an environmental sample

FISH probes

oligonucleotides (usually DNA) which are complementary to a particular 16S rRNA sequence. these probes are complexed to a fluorescent molecule for detection

hybridization of the FISH probe

done in the whole cell in an "undisturbed" sample, so that the probe binds to its target and "lights up" only those cells carrying the specific sequence

FISH can be used to distinguish what?

distinguish between species, genera, families, and domains (eg: bacteria vs archaea) etc

can multiple FISH probes be used on the same sample?

yes, multiple probes can be used on the same sample to evaluate hte presence of many different taxonomic groups in a single sample

one variation on the FISH probe technique

in situ RT-PCR (reverse transcriptase polymerase chain reaction)

in situ RT-PCR

using this technique it is possible to run a PCR reaction in an individual bacterium
it is used in order to amplify specific mRNA in a sample to determine whether a particular gene is being transcribed in a bacterium (without having to culture the bacteria) in its natural env

what happens following PCR amplification in in situ RT-PCR?

a fluorescent oligonucleotide probe is added to the sample to "light up" those cells which are transcribing the particular gene

optical tweezers

Technique in combination with FISH probing to separate individual bacteria from a sample to produce a pure culture

laser light can be used to exert a pressure on individual cells, to either trap or move them in a microscope field

what have optical tweezers also been used to carry out?

in vitro fertilization

microbial activity measurements

radioisotopes and microelectrodes

techniques to investigate the activity of microorganisms in their natural habitats

radioisotopes

radioactive-labeled compounds that can be added to a metabolic pathway in order to assay for the presence of radioactive products/intermediates

^14CO2 (radioisotope)

can be added to phototrophic microorganisms to investigate the generation of ^14C6H12O6 or other reduced ^14C compounds

^35SO4^2- (radioisotope) conversion to H2^35S

can be measured to assess sulfate reduction

^14CO2 (radioisotope) conversion to ^14CH4

can be measured to assess methanogenesis

^35S-methionine (radioisotope)

can be used to measure protein synthesis

^3H-thymidine (radioisotope)

can be used to measure DNA replication

microelectrodes

Miniaturized probes that can measure pH, oxygen, N2O, H2, H2S, etc. can be used to directly detect presence and concentration

often made from 100 microns down to about 10 microns

oxygen electrode

platinum rod functions as cathode when voltage is applied. oxygen is reduced to H2O at gold surface generating a current which can be measured

biological microsensor

used for the detection of nitrate. denitrifying bacteria convert NO3- to N2O which is further reduced to N2 at the cathode where it is detected

isotopic fractionation

metabolic pathways typically discriminate between stable isotopes of certain elements, concentrating the light isotope. therefore, ^12C is used preferentially over the stable carbon isotope, ^13C in most biological reactions

isotopic fractionation in determining whether a substance is of biological origin or geological origin

by comparing the ration of ^13C to ^12C, it is possible to determine whether the substance (ex: rock, fossil, etc) is of biological origin or geological origin

by comparing the ratio of 34S to 32S, it is possible to determine whether the substance is of biological or geological origin

terrestrial environments

in terrestrial environments, microorganisms can be found on almost any surface. the surfaces of plants and animals, rocks and soils are all habitats for microorganisms

many plants and animals produce substances that are toxic to certain microorganisms

soil environments (terrestrial) are composed of what 3 things?

1. mineral soils
2. organic soil
3. Rhyzosphere

mineral soils

derived initially from the weathering of rock and other inorganic material - most soils

organic soil

derived from sedimentation in bogs and marshes

Rhyzosphere

the habitat (usually high in organic material) that surrounds the roots of plants

soil as a microbial habitat

-most extensive microbial activity occurs on the surfaces of soil particles (biofilms), usually within the rhyzosphere
-soil aggregates
-numbers of microorganisms in organic rich mineral soils often approach and exceed 10^12 bacteria per gram

soil aggregates

combination of organic material, mineral material, water and microorganisms

aquatic habitats

oceans, estuaries, salt marshes, lakes, ponds, rivers and springs

dominant phototrophic organisms in most aquatic habitats are what?

bacteria

what 2 zones exist in aquatic habitats?

aerobic and anaerobic zones

aerobic and anaerobic zones of aquatic habitats

aerobic zones- cyanobacteria and algae
anaerobic zones- anoxygenic phototrophic bacteria

the open ocean contains low concentrations of what?

essential nutrients(particularly iron)

low concentrations of essential nutrients in the open ocean results in what?

a relative photosynthetic activity that is less than found at inshore environments

Prochlorophytes

considered the dominant oxygenic phototroph in the open ocean. this organism is related to (but distinct form) the cyanobacteria

microbial communities found in lacustrine (lake) environements

variable
lakes often stratify and different bacteria are found at different levels depending on their metabolic activity

rivers

rivers in addition to lake are heavily influenced by the activity of man (agricultural pollutants, sewage, erosional run-off)
this influences both the oxygen concentration and nutrient content of the water

low nutrient waters

generally have the bulk of bacterial numbers and activity in the photic zones and at the surface and the bottom

hydrothermal vents

deep ocean hot springs that occur at spreading centers where hot basalt and magma near the sea floor cause the floor to slowly drift apart. sea water seeping into these cracked regions mixed with hot mineral and is emitted

2 types of hydrothermal vents

warm vents and hot vents (black smokers)

warm vents

emit water at temperatures at 6-23 degrees Celsius, characterized by low flow rates

hot vents (black smokers)

emit water at temperatures at 270-380 degrees Celsius, characterized by high flow rates

fluids form hydrothermal vents have been shown to be rich on what?

H2S, Mn2+, H2, NH3, and CO

seawater in hydrothermal vents

rich in CO3^2- and HCO3- (from CO2), and O2, but contains very low amounts of organic material

what is the basis of the food chain in and around hydrothermal vent communities?

the chemolithotrophic activity of bacteria living in or adjacent to the vents

animals in hydrothermal vent communities

many animals are present in these communities, the food chain based upon symbiotic relationships with or grazing of the chemolithotrophs

tube worms

-giant (up to 2 meters long)
-macrophytic members of the phylum Pogonophora
-found in hydrothermal vent communities
-contain symbiotic sulfur oxidizing autotrophs in their gastrointestinal tracts

tube worm symbiotic relationship with sulfur oxidizing autotrophs in their gastrointestinal tracts

tube worm blood transports O2, CO2 and H2S which are transported to the symbiotic bacteria, which provide the tube worms with reduced carbon for energy production and biosynthesis

Global Carbon Cycle

characterized by the flow of carbon compounds through multiple reservoirs: the atmosphere, the land, the oceans, and other aquatic environments, sediments and rocks and biomass (microbes, plants and animals)

largest carbon reservoir

present in the sediments and rocks of the Earth's crust

turnover of carbon in this reservoir is measured on a geologic time scale

the most rapid means of carbon turnover involves what?

CO2 from the atmosphere

how is CO2 removed and returned to the atmosphere (and from aquatic habitats)?

removed by photosynthetic organisms and is returned to the atmosphere by respiration of animals nad chemoorganotrophic microbes

the majority of carbon fixed from CO2 is through what process?

photosynthesis (with lesser amounts from chemolithotrophy)

photosynthesis occurs only in what environments?

environments containing light

the predominant photosynthetic organisms in the terrestrial environment are what?

the higher plants