A simple separation method for downstream biochemical analysis of aquatic microbes

Area of Science:

• Microbiology
• Biochemistry
• Environmental Science

Background:

• Accurate chemical analysis of aquatic microbes requires pure subpopulations.
• Traditional filtration methods fail to separate microbes due to smaller cells contaminating larger fractions.

Purpose of the Study:

• To develop a reliable method for separating aquatic microbial subpopulations based on subtle size differences.
• To enable the determination of bulk biochemical composition (proteins, polysaccharides+nucleic acids, lipids) of separated microbial groups.

Main Methods:

• Utilized a sucrose density separation technique to isolate microorganisms.
• Employed (14)C-bicarbonate labeling for biochemical fractionation of autotrophs and heterotrophs.
• Investigated separation mechanisms, fixation, and pre-concentration using tangential flow filtration for oligotrophic waters.

Main Results:

• Demonstrated successful separation of eukaryotic microbes from prokaryotes in cultures and field samples.
• Showcased the ability to detect differences in the biochemical makeup of separated microbial populations.
• Showed that simultaneous isopycnal and rate-zonal separation yields finely-separated density and size fractions.

Conclusions:

• Sucrose density separation is a viable method for isolating aquatic microbial subpopulations with distinct biochemical profiles.
• This technique overcomes limitations of filtration for microbial separation and analysis.
• Future applications include exploring stoichiometric, biochemical, and genetic variations in diverse aquatic environments.