Marine Microbial Biodiversity: Lecture 15

Marine Microbial Biodiversity: Lecture 15

• The lecture covers methods for studying marine microbial biodiversity
• Questions to consider when studying microbial biodiversity:
  • Who is present?
  • What are they doing?
  • Who is doing what specific actions?
  • How are they carrying out these actions?
  • To what extent are these activities present?

Approaches to Microbial Diversity

• A typical workflow for studying microbial diversity includes:
  • Defining the study question and its design
  • Collecting the sample
  • Preserving the sample
  • Processing the sample and acquiring data
  • Analyzing the data
  • Interpreting the results

Approaches to Microbial Diversity: The Scientific Method

• Observe natural phenomena.
• Establish hypotheses.
• Test hypotheses via experiments.
• Establish theory by repeatedly validating results.

Approaches to Microbial Diversity: Actual Methods

• Suggest theory based on what funding/managers want.
• Design minimal experiments to prove the theory is true.
• Modify the theory to fit the information acquired from the data.
• Publish results, renaming theory as hypothesis and following the scientific method.

Problems in Studying Microbial Diversity

• Spatial heterogeneity: Many methods require hundreds of milliliters or tens of grams of sediment or water to test a specific group.
• Inability to culture: A significant portion of microbial diversity cannot be cultured, making it hard to understand their actions.
• Taxonomic ambiguities: Defining "species" can be difficult, with high genomic plasticity and horizontal gene transfer among microbes.
• Technical bias: Different techniques have their unique biases, which need to be considered when interpreting the results.

Sampling for Marine Microbial Biodiversity

• Methods involve use of hand-operated sterile bottles or containers for acquiring specific small volumes.
• Seawater pumps are used for collecting and transferring large volumes of water for various applications.
• The use of different equipment is important depending on the purpose and the need for sampling at different locations.

Sampling Seawater

• Niskin bottles (cylindrical plastic tubes) are frequently used for collecting water from deeper ocean areas.
• These bottles can be equipped with spring-loaded or tensioned caps.
• The deployment and retrieval use a messenger weight, which trips both caps.

Sampling Seawater

• Some methods use rosette systems to hold multiple bottles simultaneously.
• Sample volumes can range from 10 to 20 liters per bottle.
• The bottles are frequently outfitted with sensors to monitor conductivity, temperature, depth, turbidity, dissolved oxygen, fluorescence, and photosynthetically active radiation

Sampling Biomass

• Filtration separates biomass and filtered fluid
• Selective filtration differentiates between particle-associated and free-living microbes; it also separates plankton size classes.

Sampling Fluids

• Fluids (pores, cold seeps, hydrothermal vents) can be sampled with different methods.
• Common sampling strategies use centrifugation or suction methods, often including push corers or syringes

Sampling Biofilms

• Biofilms can be sampled using different methods like syringes, or push corers, as well as by swabbing or scraping the surface.

Sampling Sediments

• Corers collect sediment from the ocean floor using pushing or grabbing methods.
• Different types of tubular or box corers are implemented depending on sampling objectives and location.
• Drillships are employed for offshore drilling, having the capability to handle ultra-deepwater sampling and collect sediment.

Sampling Sediments

• Coring and drilling methods can yield intact sediment and rock structures and their associated microorganism stratifications, revealing the environmental adaptations of diverse microbial habitats

Dredging of Sediments

• Dredging collects unconsolidated rocks from the ocean floor, facilitating mapping and surveys.
• Concerns exist related to potential ecological damage or pollution to wildlife resulting from dredging or disturbance of seabed ecosystems.

Sample Preservation

• Techniques are specific to intended downstream analysis.
• Methods often involve temperature modulation and the use of preservation solutions.
• Temperature-based preservation techniques such as refrigeration (4°C), freezing (-20°C, -80°C, liquid nitrogen), include use of solutions like formaldehyde, glutaraldehyde, RNA later, and PBS (depending on the analysis needs).

Sample Analysis

• Techniques vary depending on the scientific question and analytical principles.
• Methods can be categorized as either culture-dependent or culture-independent, reflecting whether culturing is a step in the investigation or not..
• Analytical approaches fall into qualitative, semi-quantitative, and quantitative categories.
• Techniques employ various principles such as chemical, biochemical, molecular, isotopic, or combined methods.

Geochemical analysis

• Ion chromatography (IC) and Inductively coupled plasma mass spectrometry (ICP-MS) are used to determine the major ions and trace metals in the environment.
• The Piper plot is a useful graphical tool for representing and visualizing major ion data from water samples, making it helpful in the classification of water types and the interpretation of relationships among them.

Geochemical analysis: Trace Metals

• Diagrams illustrate that different conditions influence behaviors related to the chemical forms that trace metals adopt within the environment.

Microbiological Analysis

• Culture-independent methods are important for probing the natural diversity of microbial communities without the constraint of culturing.
• Culture-dependent methods are valuable for creating a new model system enabling the exploration of functional diversity through various biological methods.

Microbial Culturing

• It involves cultivating microbes in controlled environments.
• Crucial factors include sterile environment, culture media containing nutrients, inoculation process, and incubation within controlled conditions.
• A colony represents a group of multiplied microbes.

Enrichment Culture

• It facilitates cultivation by selecting for particular organisms, mimicking the selective pressures in a specific ecological niche.
• Different media are used to support microbial growth, often under conditions not favorable to the majority of organisms.

Microcosms

• Mimicking natural ecosystems, microcosms provide controlled environments for studying ecological processes within a particular habitat to understand how disturbances influence the populations of microbes.

Winogradsky Column

• A layered system mimicking the environment on the ocean floor is used to study different microbial strategies.
• The system consists of strata including those with high oxygen levels or sulfur gradients, supporting microbial growth and interactions in a simulated natural environment.

Isolation: Pure Culture

• The process starts with diluting and spreading samples on solid growth media to produce individual colonies.
• Well-isolated colonies are chosen via sterile tools.

Isolation: Most Probable Numbers

• A serial dilution and plating method helps in determining numbers of microbes within samples.
• This method works under conditions where direct counts are difficult or unreliable.

Isolation: High-Throughput Culture

• It involves diluting or sorting samples to obtain a single cell per well of a microtiter plate.
• Culture-based methods are robotically monitored over time.

Cultured VS Uncultured

• Pure cultures create model organisms for physiological, biochemical, and genetic studies that enable researchers to characterize new microbial functions.
• Isolating organisms from previously uncultured groups can provide valuable insights into microbial ecology and the roles microbes play within ecosystems.

Cultured VS Uncultured

• "Uncultured" is an operational term, not a definitive characteristic relating to the inherent attribute of an organism.
• Scientists often refer to uncultured microbes as "microbial dark matter" within their studies.

Culture-Independent Approaches

• Early methods of study leveraged microscopy, later evolving through molecular biology.
• The development of DNA-based analysis provided significant advances for investigating microbial diversity.
• DNA sequencing revolutionized the study of microbial diversity, particularly in the establishment of the archaea domain and the discovery of numerous new microbial species.

Fluorescent Microscopy

• DNA chemicals that fluoresce under UV light are used to count and quantify microbes in varied samples.
• This approach allows for population abundance measurement, while viability staining distinguishes living and dead cells.
• FISH (fluorescence in situ hybridization) uses specific fluorescent probes localized at specific targets, and this is crucial for studying microbial spatial organization within ecosystems.

Confocal Laser Scanning Microscopy

• Improves image formation contrast and resolution related to micrographs.
• Optical sectioning yields 3D images of an object's structure and arrangement.

Flow Cytometry

• Measures physical and chemical traits of cells and particles, utilizing fluorescent markers, light scattering, and sorting.
• Allows for analyzing population abundance, viability, and separating cell subpopulations.

Activity Measurements

• Microbial metabolic activity can be quantified by employing selected substrates and monitoring the resultant rates, however, sometimes this may not provide a completely accurate account of in-situ rates.
• In some instances, direct chemical analysis can be a sufficient method for evaluating microbial activity in environments, such as looking at the fate of lactate in sediment samples with sulfate-reducing bacteria, using specific assays to measure sulfide levels.

This Week's Reading

• Studies on cultivating marine bacteria illustrate significant developments and focus on overcoming hurdles with cultured vs. uncultured organisms.