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10/29/2013

Organisms do not exist isolated in the environment; instead they exist in


microbial communities analysis methods of microbial communities
Microbial community

CEE_ 361 Lecture 10

Isolation and characterization


of different microorganisms

Viability and Quantification


Using staining techniques

-Enrichment culture technique


-Isolation in pure culture
-Physiological characterization

-Enumeration of total
microorganisms in a sample
using fluorescent stains
-Enumeration of viable cells in a
sample using viable stains
-Identification of microorganisms
using genetic stains

10/28/13
Methods in Microbial Ecology
PART 2

Surveying and identifying


biodiversity in a sample
-Detection of signature genes
-Determination of the gene
sequence variation
-Creation of phylogenetic trees
-Reveal the Entire Gene
complement of a community
(Metagenome) Environmental
genomics sequencing of total
DNA cloned from a microbial
community

Measuring microbial activity in nature


Microbial activity can be measured by using:
1) Radioisotopes unstable atomic nucleus loses energy by emitting radiation
(e.g., 14C 14N)
2) Stable isotopes stable atomic nucleus with no apparent radioactive decay
(but if radioactive have lives to long to be measured) (e.g., 13C (1.1% of
carbon), and 12C (98.89% of all natural carbon on Earth)
3) Microelectrodes
Although some techniques may allow for a more targeted assessment of
individualized activities, for most part activity measurements in natural
communities are collective estimates for the entire microbial activity
(Why?)
Both the types and rates of major metabolic reactions in a habitat can be
determined
Together with biodiversity estimates, these parameters define the microbial
ecology of that habitat
Isotopes different types of atoms (nuclides) of the same chemical element differing in the
number of neutrons

Measuring Microbial Activities in Nature


-Measurement of net activity of microbial
community
-Identification of both types and rates of
major metabolic reactions in a habitat using
-Radioisotopes
-Microelectrodes
-Stable isotopes

Measuring microbial activity in nature use of radioisotopes


Direct chemical measurements of microbial transformations are many times
sufficient to assess microbial activities in an environment
E.g., fate of lactate oxidation by sulfate-reducing bacteria
in a sediment sample can be easily followed by a
chemical assay: Lactate is consumed and SO4- is reduced
to H2S (anaerobic respiration)

However,
When high sensitivity is required (e.g., low levels of particular molecule)
Turnover rates need to be determined
Fate of portions of a molecule need to be followed
Radioisotopes (e.g., 35S, 14C) are generally used
How can microbial activity be distinguished from
abiotic processes?
A killed cell control is required to prove that the
transformations are not due to abiotic processes

10/29/2013

Measuring microbial activity in nature use of microelectrodes


Microelectrodes are very small glass electrodes (tips: 2 to 100m diameter), used
to study microbial activity
-> they measure among others pH, O2, N2O, CO2, H2, H2S
a

Fig (a) schematic of a O2 microelectrode;


Platinum rod functions as a cathode, and when
voltage is applied, O2 is reduced to H2O,
generating current at the gold surface this
current is proportional to the O2 concentration
in the sample

Measuring microbial activity in nature use of stable isotopes


Stable isotopes: nonradioactive isotopes metabolized differentially by microoganisms
Isotope fractionation can reveal the biological origin of various substances.
- Lighter isotope is incorporated preferentially over heavy isotope through
enzymatic activity
Carbon exists primarily as 12C
(~99%), but about 1% exists as 13C.

The natural abundance of these


isotopes in a given compound
changes when C is metabolized by
organisms

Fig (b) study of microbial mats using microelectrodes;


core taken from hot spring
Fig (c) Upper layer (dark green) contains
cyanobacteria, beneath which are several layers of
anoxygenic phototrophic bacteria (orange).
While the whole thickness of the mat is ~ 2cm, note how
O2, pH and H2S vary within the first 3mm of the mat!

Measuring microbial activity in nature use of stable isotopes

Plant material and petroleum will


have higher 12C than carbonate rocks

Biochemical reactions favor lighter isotope

(sulfur found primarily as 32S (95%)


with some 34S (4.2%) and additional
22 isotopes (<1% abundance)

Measuring microbial activity in nature use of stable isotopes

Carbon isotope composition of various substances


values are given in parts per thousand () and calculated using the formula

(13C /12Csample) (13C /12Cstd )


x1000
(13C /12Cstd )
The standard (std) is a belemnite sample,
from the PeeDee rock formation (mostly
rock) due to unusually high 13C:12C ratio
established as 13C value of 0 most
geochem materials show negative ratio
Belemnite = Any of various extinct
cephalopod mollusks of the order
Belemnoidea that lived from the Triassic into
the Tertiary Period (~200 million years old).
Belemnites had a large, cone-shaped internal
shell with a complex structure that served as
a support for muscles and as a hydrostatic
device. Belemnites were closely related to the
present-day squids and cuttlefishes.

The activity of sulfate-reducing bacteria is easy to


recognize from their fractionation of sulfur in sulfides

10/29/2013

Linking Specific Genes and Functions to Specific Organisms

Flow cytometry application

Flow cytometry and multiparametric analysis


Natural communities contain large populations
Flow cytometer examines specific cell parameters very fast
Cell size
Cell shape
Fluorescence
Parameters can be combined
and analyzed (multiparametric analysis)

Apprill 2007 Coral Reefs Visibly healthy corals exhibit variable pigment concentrations
and symbiont phenotypes, 26:387397

Linking Specific Genes and Functions to Specific Organisms


Microautoradiography (MAR)

Microbial community
14C- organic compound for chemoorganotrophs
(or 14C- CO2 for autotrophs) and incubation

Cells affixed on a slide, exposed


to photographic emulsion

Radioactive decay from the incorporated


substrate exposes silver grains in the emulsion

Fluorescent in situ Hybridization (FISH)


Identifies organisms present by e.g., targeting
RNA in ribosomes with specific fluorescent-labeled
probes (see lecture methods part 1)

MAR- FISH
Combines the identification and activity measurements of a microbial community
Fig (a) a Proteobacteria (FISH analysis) and an autotroph (MAR- measured uptake of 14CO2)
Fig (b) uptake 14C-glucose by a mixed culture of E. coli (yellow cells FISH analysis) and Herpetosiphon
aurantiacus (filamentous green cells)
Fig (c) MAR of the same field of cells shown in b 14C-glucose assimilated by E. coli only