The McBeth Geomicrobiology Research Group lab in Kirk Hall Room 48 includes equipment for conducting molecular biological, bacteriology, and geochemical analyses and includes:

• PCR and qPCR machines
• Spectrophotometric plate reader with plate for DNA analyses
• Coy glovebox
• Degassing station
• Shaking incubator
• Shaker
• Qubit fluorometer
• Electrophoresis equipment
• Lonza electrophoresis setup
• Micro-centrifuge
• Vortex mixers
• Stirring hotplates
• Oven
• Microwave
• -20C freezer and fridge
• Water bath
• Sonicating water bath
• pH meter

Synchotron Approaches

Micro X-ray fluorescence mapping imaging uses X-rays to map the distribution of elements in a sample at high resolution. A facility like the Canadian Light Source is an excellent source of X-rays for this technique. The X-ray beam is focussed to a small spot size; ; for example 3-5 micrometres on the VESPERS beamline at CLS.

When the X-rays reach the sample, they may hit atoms in the sample, and if they do they can excite the atoms they hit, resulting in release of fluorescence X-rays that are unique for each element. This creates a fingerprint pattern of X-rays which are picked up by a detector over a set interval of time. The fingerprint includes information on what elements are present in the sample and their abundance (Figure 1). 

Figure 1: Elemental fingerprint spectrum for a spot on the elemental maps shown in Figure 2. The x-axis represents the energy of the fluorescence X-rays (in eV) and the y-axis represents the number of X-rays that hit the detector. Argon is present in air and thus occurs as a peak in most spectra. Note that in this example calcium, iron, copper, zinc, and lead each have a characteristic peak. The area under each peak is calculated, and this is used to generate a relative concentration value for that element at this spot.  The position of this spot is shown with a star on the elemental maps in Figure 2.

We move the beam of X-rays across the sample at a set interval (e.g. steps of 5 micrometres), collecting elemental fingerprint data at each point. We can use that fingerprint data to create a relative concentration map for each element over the sample (Figure 2).

Figure 2: Maps of (A) calcium, (B) copper, (C) iron, and (D) lead distribution in a sample. These four maps cover the same region of the sample but represent the relative concentrations of each element across that region. The star on each elemental map mark the pixel representing the spot fingerprint spectrum shown in Figure 1.

We can use this data to look at the distribution of elements within a sample (for example, mercury in fish eyes), and to look for hotspots where an element of interest may be more concentrated.

Our group also uses synchrotron techniques such as X-ray absorption spectroscopy (XAS), and related techniques such as XANES, EXAFS, and NEXAFS, to probe the chemistry of elements in the environments we study. For example, in mine wastes arsenic is an element that is very interesting because of its toxicity when released into the environment. We could use XAS to determine the chemistry of arsenic in mine wastes. This can help us understand the form of the arsenic, and whether it is in a form that is more likely to move in the environment, or a form that is insoluble and stays in a solid form. 

We often study iron, sulfur, uranium, and other metals using XAS techniques. Beamlines at the Canadian Light Source that are useful for studying these elements using XAS include HXMA, VESPERS, SGM, IDEAS, and SXRMB.

We use powder x-ray diffraction (PXRD) to get data we can use to determine mineralogy in samples. This is useful data to complement our XAS analyses, and useful information for understanding mine wastes, soils, and sediments. Synchrotron PXRD offers some advantages over conventional lab X-ray diffractometers; for example it is sometimes possible to obtain higher resolution spectra for small peaks.

Molecular Biology Techniques

High-throughput amplicon sequencing: as geomicrobiologists, a critical background question we ask in every environment we study is “who is there”. In combination with geochemical data, it is very useful information to know what kinds of microbes may be present. This data gives us clues about what microbes may be doing biogeochemically, and what they might do if the geochemistry changes in the system. For example, if we inject a carbon source that feeds the microbes, who might be able to use that carbon source, and what might they do? If we inject acetate, we might feed iron-reducing bacteria if the conditions are anaerobic, and that might be important for the biogeochemistry of the environment we are studying since the iron-reducing bacteria will generate iron that is soluble in the groundwater.

To take this general microbial “census” of microbial communities in our samples, we use high-throughput amplicon sequencing of the V4 region of the 16S rRNA gene.

We analyse our data using the mothur software suite. https://www.mothur.org/

We use the Earth Microbiome Project primers for our amplicon sequencing analyses. For more information see the Earth Microbiome Project Website. http://www.earthmicrobiome.org/

Culturing Approaches

We use enrichment culturing and isolations to complement the sequencing data we get for our field samples. Culturing Bacteria and Archaea from the environments we study allows us to obtain information on the metabolic functions of microbes from these environments. In other words, we can get information on how do the microbes make their living. What compounds in the environment do they use to gain energy? Do they ”breathe” oxygen? Or iron? Do they “eat” ethanol? Or CO₂?

This is important, because sequencing data generally only gives us clues about what the microbes may be doing, based on what their nearest relatives can do. We would like to confirm they are capable of a particular metabolic activity, such as sulfate reduction or iron oxidation, to know what biogeochemical influence they can have in the environment.

In the past few years the McBeth Geomicrobiology Research Group has done enrichment culturing work to obtain enrichments (and some isolates!) of microbes including: halophilic archaea, manganese-oxidizing bacteria, iron-oxidizing bacteria, and sulfate-reducing bacteria.