Measurement of Microbial Activities

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Chapter 11

• Measurement of Microbial Activities

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Why measure activity?

• Microbial contribution to nutrient cycling
• Transfer of energy between trophic levels
• Impact of a disturbance to the system

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Measuring oxygen concentration in aqueous
solutions
O2 electrode method
Colorimetric method
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Methods used to measure microbial activities

• Microelectrodes
• pH
• H2S
• O2

O2 4e- 2H2O ? 4OH-
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Ex situ Sealed LaboratoryMicrocosms

• Remove gas sample periodically and measure O2
  disappearance or CO2 appearance AdvantageAllows
  one to design complex experiments that can be
  operated under controlled conditions
• standardization of some parameters
• soil moisture
• temperature
• DisadvantageDestroys soil structure that may be
  important in controlling microbial activity

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Dissolved oxygen consumption by microbes
degrading organic matter in wastewater
Add water sample, remove sample for DO
measurement, seal container, incubate in dark

• Carbon respiration (heterotrophic activity)
• consumption of oxygen or other terminal electron
  acceptor
• Biochemical Oxygen Demand

DO0 - DO5 amount of organic C oxidized to CO2

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In Situ Field Studies
Basal respiration

• Place a chamber over a plot of surface soil
• Measure respiration over time
• advantage
• minimal physical disturbance to system
• disadvantage
• cannot control soil moisture and temperature
• measurements are more variable

Used as an indicator of soil health or
condition.
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Apparatus to determine oxygen demand of sediment
in presence of overlying aqueous phase
Sealed (closed) flow-through system
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Basal In-situ respiration in an uncontaminated
soil
Inject air through probe into subsurface. Halt
air injection and allow microbes to
consume O2. Withdraw gas samples over 2-8h
intervals (O2 used/hr)
To measure O2 CO2
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Determination of basal rate of oxygen consumption
in absence of contaminant
Determine basal respiration rate of microbial
community by measuring amount of O2 consumed/h
at contaminated site over 2-10 h period
O2 consumed
1 2 3 4 5 6 7 8 9 10
hours
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In-situ respiration in a hydrocarbon-contaminated
soil
Inject air through probe into subsurface. Halt
air injection and allow microbes to
consume O2. Withdraw gas samples over 2-8h
intervals (O2 used/hr)
To measure O2 CO2
oil
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Determination of rate of oil biodegradation
Determine rate of oil biodegradation by measuring
amount of O2 consumed/h at contaminated
site over 2-10 h period
O2 consumed
1 2 3 4 5 6 7 8 9 10
hours
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Carbon dioxide measurement

• Respiratory gases
• Radiolabeled carbon sources (14C-acetate)

Sealed top
alkaline solution
CO2 trap
Saturation kinetics
CO2
14C-acetate
Water sample
bacterium
time
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Liquid Scintillation Counting
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Lineweaver-Burke Plot
t incubation time f fraction of added
substrate taken up v rate of substrate uptake A
amount of substrate added Sn natural
substrate concentration K substrate
concentration at 1/2 Vmax
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Methods used to measure microbial activities
3H-thymidine
Incubate under in-situ conditions

• Incorporation of radiolabeled thymidine into
  cellular DNA
• Measure of secondary production in aquatic
  environments

Cell replication
Scintillation counting
3H
3H
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Methods used to measure microbial activities

• Adenylate energy charge (ATP, AMP, ADP)

ATP 1/2 ADP
AEC
ATP ADP AMP
High AEC gt0.8 (active microbial community Low
AEC lt0.4 (dead or moribund community
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Methods used to measure microbial activities

• Enzyme assays
• Dehydrogenase assay
• assesses oxidation-reduction reactions inside cell

Active cells
Starved cells
DAPI-DNA stains all cells
Tetrazolium salt- 5-cyano-2,3-ditoly tetrazolium
chloride stains only cells that are respiring
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Methods used to measure microbial activities

• Enzyme assays
• Hydrolysis of fluorogenic substrates
• phosphatase, lipase, esterase enzyme activity

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Localization of enzyme activity in flocs

• Phosphatase activity detected via yellow-green
  fluorescence of ELF
• Activity is
• localized within floc matrix
• not associated with protozoans

50 µm
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Modern Molecular Methods

• Stable isotope probing
• Which microbial populations are active
• Dont need to cultivate populations
• Expression microarrays
• Which genes are expressed and at what level?
• 2D-polyacrylamide gel electrophoresis
• Separates proteins in 2 dimensions
• Charge and size
• Matrix assisted laser desorption time-of-flight
  mass spectrometry

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Typical experiment
Question How does a microorganism whose genome
has been sequenced respond to a
perturbation in its environment?
Inoculum of Geobacter sulfurreducens
Soil contaminated with cadmium
5-min exposure
10-min exposure
Extract mRNA
15-min exposure
25-min exposure
Spot on chip containing
Reverse-transcription PCR with fluorescently
labeled random primers
TGGAC
CGGAC
Gene probe
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Gene chip
Each spot contains a probe sequence of a
different gene
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Functional Genomics
Microarray Investigation

• How do environmental perturbations influence gene
  expression in microbes whose genomes have been
  sequenced?
• 674 genes evaluated
• Each row represents 1 gene
• 4 different time points
• Red up-expression
• Green down-expression
• Black no change

Effect of cadmium on gene expression
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Typical experiment
Question How does a microorganism whose genome
has been sequenced respond to a
perturbation in its environment?
Inoculum of Geobacter sulfurreducens
Soil contaminated with cadmium
5-min exposure
10-min exposure
Extract proteins
15-min exposure
25-min exposure
2-D gel electrophoresis
proteomics
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Proteomics
2-D gel electrophoresis
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Proteomics
Mass spectrometry
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How do we scale observations and measurements
made at the laboratory bench scale to the field
scale?

• Physical model experimental systems scaled to
  adapt knowledge acquired at the bench for
  accurate interpretation of observations in the
  field

Size
Sandbox
Geoblock
Control
Complexity
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Computational modeling
Small scale, high control minimum complexity
Simple algorithms, Simple predictions
Experimental system
Computer program
Results
Results
Iteration
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CCl4-saturated grease plug
CCl4
Mesoscale Experimental System
DNAPL sensors
2m
clay layer
Evaluate microbial degradation of
carbon tetrachloride in unsaturated heterogeneous
porous medium
bacteria
capillary fringe
sand
2m
3m
macropores
saturated zone
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Benefits of mesoscale experimental systems
Large enough size and time scales to allow
coupling of hydrological, geophysical,
geochemical and biological processes.
Accuracy of Biodegradation Rate Constant
Influences Prediction Accuracy
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Computational modeling
Medium scale, moderate control increasing
complexity
Complex algorithms, predictions on a practical
scale
Experimental system
Computer program
Results
Results
Iteration
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Summary

• Many different techniques to measure microbial
  activities
• Different experimental systems can be used to
  control environmental variables
• Scalability of measurements is important
• Ultimate goal is to incorporate activity
  measurements into predictive computational models
  that are accurate in predicting phenomenon at a
  relevant scale