Advantages and Disadvantages of Various Methods for Detecting Microbial Activity

The list of available techniques is shown in Table 9.1. The rate of incorporation of labeled DNA and protein precursors (thymidine and leucine, respectively) is the most popular method for testing homogeneous frozen objects, such as sea and glacier ice,

Technique

Habitat

Temp (°C)

References

Advantages of technique

Methodological limitations

Incorporation of labeled precursors

DNA (3H-thymidine) and proteins (14C-Leucine)

Glacial ice bacteria

-15

Christner (2002)

• High sensitivity

• Clear physiological and biochemical interpretation of data

• Can be combined with subsequent analysis of labeled constituents

• Disturbance of natural community by substrate addition and thaw-refreezing

• Technique is destructive (cannot be used repeatedly on the same sample)

South Pole Snow

-17 to-12

Carpenter et al. (2000)

Proteins (various 3H and 14C-amino acids)

Arctic sea ice

-20 to + 1

Ritzrau (1997) and Junge et al. (2006) "

Lipids (14C-acetate)

Siberian permafrost

-20 to 0

Rivkina et al. (2000)

Gas evolution

co2

Barrow, Alaska

-40 to 0

Panikov et al. (2006)

• High precision and sensitivity, availability of respective analytical instruments

• Relevance to high-priority green-house gases research

• Overestimation of activity resulted from release of gases accumulated in sample before measurements

• Underestimation because of time delay between formation and release of gases

Tussock tundra, Alaska

-12 to 0

Mikan et al. (2002)

ch4

Siberian permafrost

-16.5 to 0

Rivkina et al. (2002)

n2o

Alpine tundra, Colorado

-5 to 0

Brooks et al. (1997)

Gradient of gases

Mountain glacier, Bolivia

-40 to 0

Campen et al. (2003)

(continued)

Technique

Habitat

Temp (°C)

References

Advantages of technique

Methodological limitations

Gas uptake

o2

Antarctic peat

-1 to + 1

Wynn-Williams (1982)

• Low sensitivity

Light 14CO, uptake

Alpine, Tibet

-10 to + 20

Kato et al. (2005)

• Highly sensitive and simple technique

• Technique is destructive because of requirement to extract the labeled cell constituents

Endolithic lichen,

Antarctica

-24 to + 5

Kappen and Friedmann (1983) and Kappen (1993)

• Does not require substrate addition and thaw of frozen sample

Dark 14CO, uptake

Permafrost and tundra. North Slope of Alaska

-80 to 0

Panikov and Sizova (2007)

• Results are not affected by gases accumulation prior to measurements

Organic matter decomposition

Net N mineralization and nitrification

Taiga and tundra soils, Alaska

-5 to + 5

Clein and Schimel (1995)

• Assessment of the in situ processes

• Provides data for entire outdoor ecosystem

• Poor temporal resolution

• Low sensitivity

Tundra, Alaska

-30 to + 5

Schimel et al. (2004)

• Difficulties in data interpretation stemming from stochastic and seasonal variations of temperature and other environmental factors

Plant litter weight loss

Tussock tundra, Alaska

-30 to + 5

Hobbie and Chapin (1996)

Loss of K, Mg, P, phenolics and carbohydrates

Subarctic woodland, Canada

ND

Moore (1983)

Oxidation of 14C-labeled compounds added to frozen sample

Barrow tundra, Alaska

-40 to 0

Panikov et al. (2006)

• High sensitivity

• High specificity

• No effect of gases present before analysis

• Disturbance of microbial community by thaw-refreezing and addition of substrate

>> Cu

Low-temperature cells

Arctic sea ice

-20 to -2

Junge et al. (2004)

• High spatial resolution at

• Technique is not quantitative

J*-

staining and micro

micro-scale

• Possible changes in micro-

2 2

scopy in the walk-in

environment by staining and

S

cold room

microscopy

polar snow, supercooled cloud droplets, etc., but sometimes it is also used for soils. This technique is sensitive, and characterizes two basic intracellular processes, DNA and protein synthesis. The major disadvantage is that the procedure is destructive, and requires preliminary ice or soil thaw which could be sources of artifacts. In addition, there are some general uncertainties (Karl 1980), e.g., strong dependence of results on the amount of added nucleoside or amino acid: if it is too small, then endogenous synthesis is not suppressed and the incorporation rate is underestimated; if the amount is too large, then the trophic status of the sample is changed.

Microscopy in combination with oligonucleotide probes and stains visualizing active cells (like 5-cyano-2,3-ditoyl tetrazolium chloride, CTC) is a potentially powerful tool; however, so far it provides only qualitative information on the state of cells in frozen samples, rather than on activity or growth rates.

The microbial activity in heterogeneous habitats (frozen soils, permafrost) is estimated most often either through exchange rates of gases (CO2, O2, CH4, N2O), or by recording decomposition processes, e.g., plant litter weight loss or N net mineralization. The second approach characterizes the in situ process, which is a great advantage but is destructive and not sensitive. The major reason for low sensitivity is that decomposition dynamics provides a time-averaged integral curve rather than an instant rate of a particular microbial activity related to the current temperature or other environmental factors. Besides, recorded data are usually difficult to interpret because the observed dynamics are a sum of several simpler processes having often opposite signs, e.g., production-consumption, decay-synthesis, immobilization-mobilization. Decomposition of labeled individual compounds (e.g., 14C-glucose) is much less complex, but should be classified as potential substrate-induced microbial activity.

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