Week 2 - techniques for environmental microbiology

What is the geo-bio interface?

The zone where biological, chemical, and geological processes intersect—especially in soils, sediments, and water

3 components - Cellular physiology, Aqueous environment, Mineralogy

What is understood from the geo-bio interface

Microbial roles in elemental cycles

Interactions between microbes and minerals, gases, and organic matter

Responses to pollutants, climate change, and anthropogenic disturbance

Describe sediment sampling for aquifer profiles

Used to study groundwater flow and contamination

Extract cores maintaining anoxia and hydraulic integrity

Cores help analyse mineral composition, microbial populations, and chemical gradients

Describe marine/lake sampling

Gravity Corers: Drop under their own weight, sealing top and bottom upon impact

Jenkin Surface Mud Sampler: Specially designed for surface mud collection

Describe groundwater sampling

Niskin Bottle → collects deeper water and larger samples, sealing airtight at desired depth triggered by messenger system

Johnson-ZoBell (J-Z) → sucks water up into the bottle via a glass tube, maintaining sterility with a rubber stopper to create a vacuum used for anaerobic and deep-water sampling

CTD diver measures Conductivity, Temperature and Depth in situ

Filtered immediately to trap biomass

Describe processing for water samples

• Filtration or centrifugation to concentrate microbial cells.

• Large volumes needed if microbial populations are low.

• Filters used:

  • 0.1 µm for viruses

  • 0.22 µm for bacteria and archaea

• Filters frozen or used immediately for DNA extraction, culturing, chemical analysis

Describe processing for sediment/soil samples

• Homogenised in sterile containers

• Sample structure is destroyed when processed.

• Physical structure and microbial symbioses are important considerations.

• Aliquoted for:

  • Microbial DNA/RNA

  • Enzyme activity

  • Geochemistry

Storage conditions depend on intended analysis. Describe some of these storage conditons.

Purpose	Storage Conditions
Direct microbial activity	Store at 4°C
Long-term storage	Freeze at -20°C to -70°C (liquid nitrogen or dry ice)
Cellular component analysis	Fix in formaldehyde or alcohol
Cultivation-based studies	Avoid freezing (alters viability)

Describe some analytical techniques.

Method	Used For
pH/Redox Probes	Environmental conditions Measures acidity/alkalinity. Determines oxidation-reduction potential.
Ion Chromatography (IC)	Identifiers anions/cations in aqueous samples
ICP-AES / ICP-MS	Measures metals (major and trace)
Gas Chromatography (GC)	Gas composition analysis (e.g. CH₄, CO₂, VOCs)
TIC/TOC Analysis	Measure carbon content in pools (organic/inorganic)

How can direct chemical measurements be used to measure microbial activity?

• Measure biological transformations of compounds - substrate depletion (e.g., CH₄), product accumulation (e.g., NO₃⁻, H₂S)

• Sensitive to environmental conditions.

How can specific metabolic inhibitors be used to measure microbial activity?

• Used to block microbial pathways and study activity.

• Examples:

  • Sodium molybdate (sulphate reduction inhibitor).

  • Bromoethane sulfonic acid (methanogenesis inhibitor).

How can radiotracers be used to measure microbial activity?

• ¹⁴C-labeled compounds used to track microbial metabolism.

• ³⁵SO₄²⁻ for sulphate reduction studies.

What are unculturable microoragnisms?

Microbes that cannot be grown in standard lab conditions.

Describe key techniques part of the molecular revolution

• 16S rRNA Gene Sequencing – Identifies bacterial/archaeal taxonomy.

• PCR – Amplifies microbial DNA.

• Shotgun Metagenomics – Sequences entire microbial communities.

• Fluorescent In-Situ Hybridization (FISH) – Uses fluorescent probes to detect microbes.

Why take samples

Difficult to measure a whole ecosystem

Heterogeneity makes replication important

Downside of samples

Microenvironment is changed during, so components behave differently

Sampling sediments/soil

Topsoil with trowel

Deeper with boreholes/corers → corers preserve stratification and microbial distribution

In lakes with a Jenkin sampler

Store in sterile bags or tubes at 4°C (short term) or -80°C (long term DNA work)

Geoprobe for sediments

Sampling water

Johnson-ZoBell → sucks water up into the bottle via a glass tube, maintaining sterility with a rubber stopper to create a vacuum

Groundwater pump

Niskin bottle for deeper water and larger samples, sealing airtight at desired depth

CTD diver measures Conductivity, Temperature and Depth in situ

How do you concentrate biomass in a water sample

Filtration and centrifuge

Methods of sample storage

Freeze between -20 → -70C using liquid nitrogen/dry ice for direct measurement of cellular components

Fixative solution (formaldehyde, alcohol, commercial) for cell counts (cant freeze due to ice crystals rupturing)

Cultivation or activity measurements as soon as possible after sampling or after storage at 4^oC

Geochemical analysis of samples

pH probe

Redox probe

Ion Chromatography (IC)

Inductively Coupled Plasma - Atomic Emission Spectrometry

(ICP-AES)

GC

TIC/TOC

Electron microscopy

Xray techniques

3 ways to measure microbial numbers

Direct counts

Biomass estimation

Culturing

Direct counts

Use a microscope

Can be difficult to distinguish between living and dead cells and debris (aided by fluorescent dyes)

Good for larger cells

• Acridine Orange Direct Counts (AODC) – Stains nucleic acids.

• DAPI Staining – Specific for DNA detection.

• Flow Cytometry – Uses fluorescent markers to count cells in liquid samples.

Overestimates by up to 1-2 orders of magnitude

Biomass estimation

Measures biomass and total numbers

Can target ATP cell envelope components

• ATP Assay (Luciferase Enzyme) – Estimates active biomass.

• Phospholipid Fatty Acid (PLFA) Analysis – Determines microbial community composition.

Culturing

Isolation of selected media

Targets specific organisms

Plate counts → slective media for target microbes.

Underestimates diversity

Most Probable Number (MPN) Method – Estimates viable bacterial populations.

Methods of measuring microbial activity

Radiotracers → uptake of radiolabelled compounds, degredation of C14 measured