Marine Microbial Biodiversity Lecture 15 PDF

# Marine Microbial Biodiversity ## University of Naples "Federico II" ## Lecture 15 ## Methods in marine microbial biodiversity 1 ### General (First order) questions in studying microbial diversity - Who's there? - What are they doing? - Who's doing what? - How are they doing it? - To what exte...

# Marine Microbial Biodiversity ## University of Naples "Federico II" ## Lecture 15 ## Methods in marine microbial biodiversity 1 ### General (First order) questions in studying microbial diversity - Who's there? - What are they doing? - Who's doing what? - How are they doing it? - To what extent? ### Approaches to Microbial Diversity Each time we approach the study of microbial diversity we are following a similar workflow generally consisting of: - Definition of the study question and study design - Sample collection - Sample preservation - Sample processing and data acquisition - Data analysis - Interpretation of results ### Problems in studying microbial diversity - **Spatial heterogeneity:** Most methods need a few hundreds milliliters of waters or tens of grams of sediments. - **Inability to culture:** Most of the microbial diversity is still uncultured, with big implications for what we can understand. - **Taxonomic ambiguities of microbes:** Difficult definition of "species", high genomic plasticity, horizontal gene transfer. - **Technical bias of chosen methods:** Each technique has its own bias, that need to be considered while making inferences. ### Sampling for marine microbial biodiversity ### Environmental data - Latitude: 65.6984583 - Longitude: -21.5597800 ### Sampling for microbial biodiversity - **Sediments** - **Background** - **Fluids** ### Sampling Seawater The principal techniques used to sample seawater involve the use of hand-operated sterile bottles or containers to collect small known volumes, or seawater pumps can be used to transfer or circulate huge amounts for various applications on board a marine vessel. - One of the most widely used systems to collect seawater deep in the ocean are the Niskin bottles. These are usually plastic cylindrical tubes equipped with a cap at each end, which is either spring-loaded or tensioned by an elastic rope, and with a messenger weight, which trips both caps shut and seals the tube. - Niskin bottles can also be mounted on a circular **rosette system**, holding up to 24 bottles of 10 or 20 L. These are generally equipped with sensors for conductivity, temperature, and depth (CTD) and sometimes other parameters such as turbidity, dissolved oxygen, fluorescence and photosynthetically active radiation. ### Sampling Biomass | Size category | Size range (µm) | Examples of microbial groups | |-----------------------|------------------|----------------------------------------------------------| | Femtoplankton | 0.01-0.2 | Viruses | | Picoplankton | 0.2-2 (3) | Bacteria, archaea, prasinophytes, haptophytes, some flagellates | | Nanoplankton | 2-20 | Coccolithophores, diatoms, dinoflagellates, flagellates | | Microplankton | 20-200 | Ciliates, diatoms, dinoflagellates, foraminifera, yeasts | Filtration is the most commonly used technique to separate **biomass** and filtered fluid. **Selective filtration** is frequently used to denote divisions between particle-associated and free-living microbes or to discriminate plankton size classes. ### Sampling Fluids Fluids, like sediment pore fluids, or fluids seeping from cold seeps or hydrothermal vents can be sampled using different approaches, that are specific to each case. Common approaches include **centrifugation** or suction from a **push corer** through the use of **syringes**. ### Sampling Biofilms Biofilms can be sampled through a syringe, a **push corer**, or directly **swabbing** or **scraping** the surfaces. ### Sampling Sediments **Corers** are the most used methods for collecting sediment from the ocean floor. They work by pushing or grabbing sediment into containers. Several types of corer with different tubular and box varieties can be used depending on the purpose and location of sampling. - **Multi-corer** - **Box corer** - **Gravity corer** ### Sampling Sediments Drillships are merchant vessels designed for use in exploratory offshore drilling of new oil and gas wells or for deepwater and ultra-deepwater sediment collection. They are equipped with the latest and advanced dynamic positioning systems. Through coring and drilling it is possible to collect cylindrical samples with **intact stratifications** of sediments and rocks. It's possible to also collect **stratifications** of microorganisms with different lifestyles, which they have developed based on the environmental conditions. ### Sampling Sediments Dredging collects loose rocks on the ocean floor. It's useful for mapping the broad-scale distribution of rock types on the seafloor. However, dredging has significant environmental impacts: - It can disturb marine sediments, leading to **water pollution**. - It can destroy important seabed ecosystems. - It can release toxins captured in the sediment. These environmental impacts can significantly hurt marine wildlife populations, contaminate sources of drinking water, and interrupt economic activities such as fishing. ### Sample Preservation Preservation is highly dependent upon the type of downstream analysis that needs to be carried out. There are some common preservation strategies that include the modulation of temperature or the addition of specific preservatives. These are often used in combinations. - **Refrigeration at +4°C:** Culturing, enrichments, live specimens. - **Freezing at -20°C:** Viral counts, chemistry, DNA. - **Freezing at -80°C or liq-N₂:** DNA, RNA, proteins. - **Preserving solutions:** Formaldehyde, glutaraldehyde, RNA later, PBS (counts, DNA, RNA) ### Sample Analysis The technique used depends on the scientific question and the analytical principles used. We can divide techniques in different ways: - **Culture-dependent and culture-independent techniques:** Refers to a grouping of techniques based on the use of culturing as a basic step in the investigation. Since the majority of the microbes is currently uncultured, the decision of using culture-dependent vs independent techniques is very important. - **Qualitative, semi-quantitative and quantitative:** Refers to the type of information obtained from the analysis. Often semi-quantitative and quantitative techniques are all referred as quantitative, although not correct. Bias in the chose technique usually impair our ability to obtain true quantitative data. - **Chemical, biochemical, molecular, isotopic, or a combination:** Refers to the analytical principles used by each technique. ### Geochemical Analysis - **Ion chromatography (IC)** - **Inductively coupled plasma mass spectrometry (ICP-MS)** ### Geochemical Analysis: Major Ions - **The Piper plot is a graphical method used to represent the major ion composition of water samples, typically focusing on both cations and anions. It's widely used in hydrology and geochemistry to classify different types of water and to visualize water chemistry, making it easier to interpret relationships between various water samples.** **In particular, it is useful for identifying the evolution, mixing, and origins of water sources in environments such as groundwater systems, geothermal areas, and surface water bodies.** ### Geochemical Analysis: Major Ions - **Ca-SO₂ waters:** Typical of gypsum ground waters and mine drainage, or waters that have interacted with volcanic gases rich in sulfur. Sulfate-reducers or sulfur-oxidizing microbes live in this type of water. - **Ca-HCO₃ waters:** Typical of shallow, fresh ground waters that interact with carbonate rocks. Sulfate-reducing bacteria may thrive in bicarbonate-rich waters where anaerobic conditions are common and methanogens live. - **Na-Cl waters:** Typical of marine, or deep ancient ground waters. These environments are often home to halophilic and thermophilic microorganisms, or phototrophs in case of sea water. - **Na-HCO₂ waters:** Typical of deeper ground waters influenced by ion exchange. Alkaliphiles and autotrophic microorganisms like methanogens can thrive in such CO₂-rich environments. ### Geochemical Analysis: Major Ions: Cations - **Sodium (Na⁺) and Potassium (K⁺):** Dominates in high-temperature, deep geothermal systems like deep brines, where significant water-rock interactions have occurred. These conditions favor thermophilic and halophilic microorganisms. - **Calcium (Ca²⁺):** More abundant in lower-temperature, less-evolved geothermal waters or in waters that have interacted with carbonate rocks. - Sulfate-reducing bacteria play a key role in the formation of minerals like gypsum or calcite. - **Magnesium (Mg²⁺):** Often present in cooler, shallower geothermal systems, where the water has had less interaction with rocks or has mixed with meteoric (surface) waters. Alkaliphiles, halophiles and phototrophs are more abundant in these environments. ### Geochemical Analysis: Major Ions: Anions - **Chloride (Cl⁻):** Represents deep geothermal waters, typically associated with high-temperature fluids that have circulated through deep crustal rocks. These waters host thermophilic methanogens and sulfur-reducing microbes. - **Sulfate (SO₄²⁻):** Indicates interaction with shallow, oxidized waters, often associated with surface processes such as steam-heated waters, or volcanic gases. Microbial communities that participate in sulfur oxidation and sulfate reduction are common in these environments. - **Bicarbonate (HCO₃⁻):** Reflects the influence of CO₂-rich waters, often representing shallow or intermediate-depth geothermal fluids influenced by subsurface degassing. Autotrophic CO₂-fixing microorganisms and methanogens are common in these environments. ### Geochemical Analysis: Trace Metals - **(A)** - **(B)** - **(C)** - **(D)** ### Microbiological Analysis Given our current inability to grow a large portion of marine microbes, the choice between culture dependent and independent technique is very important. - **Culture-independent techniques:** Allow you to probe the natural diversity of microbial communities **avoiding the bottleneck of culturing**. However, the inferences about functional diversity are entirely dependent upon information obtained from pure microbial cultures. - **Culture-dependent approaches:** While highly selective for a small subset of microorganisms, they have the advantage of resulting in a new model system that can be used for probing functional diversity using physiology, genetics and biochemistry. - Some approaches are a hybrid between the two techniques, for example combining enrichments with **molecular tools**. **A successful approach to the study of microbial diversity requires both approaches combined**. ### Culture-dependent approaches ### Microbial culturing Microbial culturing is the process of growing microbes in a controlled environment. - **Culture Medium:** A substance (solid or liquid) that provides food and nutrients for microbes to grow. - **Sterile:** An environment, object, or substance were no microbes are present. It's crucial to prevent unwanted microbial growth ( **contamination**). - **Petri Dish:** A shallow, round dish used to hold the agar or other culture medium. - **Inoculation:** The process of placing microbes onto the culture medium to grow them. - **Incubation:** Keeping the microbes in a controlled environment to help them grow. - **Colony:** A group of microbes that have multiplied and become visible as a small, round spot on the solid culture medium. ### Enrichment culture For an enrichment culture, a medium and a set of incubation conditions are established that are selective for the desired organism and counterselective for undesired organisms. Effective enrichment cultures duplicate as closely as possible the resources and conditions of a particular ecological niche. ### TABLE 19.1 Some enrichment culture methods for phototrophic and chemolithotrophic bacteria | Light-phototrophic bacteria: main C source, CO₂ | | |---------------------------------------------|-------------------------------------------------------------------------------------------------------------------| | Incubation condition | Organisms enriched | Inoculum | | Incubation in air | Cyanobacteria | Pond or lake water; sulfide-rich muds; stagnant water; raw sewage, moist, decomposing leaf litter, moist soil exposed to light | | NO₂ as nitrogen source, 55°C | Thermophilic cyanobacteria | Hot spring microbial mat | | Anoxic incubation | Purple nonsulfur bacteria, heliobacteria | Same as above plus hypolimnetic lake water (dp Section 20.8), pasteurized soil (heliobacteria), microbial mats for thermophilic species | | H₂S as electron donor | Purple and green sulfur bacteria | | | Fe², NO₂ as electron donor | Purple bacteria | | | | | | | Dark-chemolithotrophic bacteria main C source, CO₂ (medium must lack organic C) | | | Electron donor | Electron acceptor | Organisms enriched | Inoculum | | Incubation in air: aerobic respiration | | | | | NH4⁺ | O₂ | Ammonia-oxidizing Bacteria (Nitrosomonas) or Archaea (Nitrosopumilus) | Soil, mud, sewage effluent, seawater | | NO2⁻ | O₂ | Nitrite-oxidizing bacteria (Nitrobacter, Nitrospira) | | | H₂ | O₂ | Hydrogen bacteria (various genera) | | | H₂S, S, S₂O₂ | O₂ | Thiobacillus spp. | | | Fe², low pH | O₂ | Acidithiobaollus ferrooxidans | | | | | | | | Anoxic incubation | | | | | S², SO3² | NO3⁻ | Thiobacillus denitrificans | Mud, lake sediments, soil | | H₂ | NO3⁻ | Paracoccus denitrificans | | | Fe², neutral pH | NO3⁻ | Acidovorax and various other gram-negative autotrophic bacteria | | ### TABLE 19.2 Some enrichment culture methods for chemoorganotrophic and strictly anaerobic bacteria | Electron donor (and nitrogen source) | Electron acceptor | Typical organisms enriched | Inoculum | |:-------------------------------------|:------------------|:---------------------------------------------------------------------------------------------|:----------------------------------------------------------------------------------------------------------------------| | **Incubation in air: aerobic respiration** | | | | | Lactate + NH4⁺ | O₂ | Pseudomonas fluorescens | Soil, mud; lake sediments; decaying vegetation, pasteurize inoculum (80°C for 15 min) for all Bacillus enrichments | | Benzoate + NH4⁺ | O₂ | Pseudomonas fluorescens | | | Starch + NH4⁺ | O₂ | Bacillus polymyxa, other Bacillus spp. | | | Ethanol (4%) +1% yeast extract, pH 6.0 | O₂ | Acetobacter, Gluconobacter | | | Urea (5%)+1% yeast extract | O₂ | Sporosarcina ureae | | | Hydrocarbons (e.g., mineral oil, gasoline, toluene) + NH4⁺ | O₂ | Mycobacterium, Nocardia, Pseudomonas | | | Cellulose + NH4⁺ | O₂ | Cytophaga, Sporocytophaga | | | Mannitol or benzoate, N₂ as N source | O₂ | Azotobacter | | | CH4+NO3⁻ | O₂ | Methylobacter, Methylomicrobium | Lake sediments, thermocline (dp Section 20.8) of stratified lake | | | | | | | **Anoxic incubation: anaerobic respiration** | | | | | Organic acids | NO3⁻ | Pseudomonas (denitrifying species) | Soil, mud, lake sediments. | | Yeast extract | NO3⁻ | Bacillus (denitrifying species) | | | Organic acids | SO4²⁻ | Desulfovibrio, Desulfotomaculum | | | Acetate, propionate, butyrate | SO4²⁻ | Fatty acid-oxidizing sulfate reducers | As above; or sewage digester sludge; rumen contents: marine sediments | | Acetate, ethanol | S⁰ | Desulfuromonas | | | Acetate | Fe³⁺ | Geobacter, Geospirillum | | | Acetate | ClO3⁻ | Various chlorate-reducing bacteria | | | | | | | | **Anoxic incubation: fermentation** | | | | | Glutamate or histidine | None | Clostridium tetanomorphum or other proteolytic Clostridium species | Mud, lake sediments, rotting plant or animal material; dairy products (lactic and propionic acid bacteria); rumen or intestinal contents (enteric bacteria); sewage sludge; soil; pasteurize inoculum for Clostridium enrichments | | | | | | | Starch + NH4⁺ | None | Clostridium spp. | | | Starch + N, as N source | None | Clostridium pasteurianum | | | Lactate + yeast extract | None | Veillonelia spp. | | | Glucose or lactose + NH4⁺ | None | Escherichia, Enterobacter, other fermentative organisms | | | Glucose + yeast extract (pH 5) | None | Lactic acid bacteria (Lactobacillus). | | | Lactate + yeast extract | None | Propionic acid bacteria | | | Succinate + NaCl | None | Propionigenium | | | Oxalate | None | Oxalobacter | | | Acetylene | None | Pelobacter and other acetylene fermenters | | ### Enrichment culture ### Enrichment culture - **Phormidium** - **Salileptolyngbya** - **Anabaena** - **Limnothrix** ### Microcosms Microcosms are artificial, simplified ecosystems that are used to simulate and predict the behaviour of natural ecosystems under controlled conditions. Open or closed microcosms provide an experimental area to study natural ecological processes. They're generally used to study the whole microbial community present in a certain habitat, or how the effects of disturbance can change the relative abundance of each species. Another use for microcosms is to determine the ecological role of key species. ### Winogradsky column - The Winogradsky Column consists of a glass container into which mud, water, sources of carbon, sulphur, minerals and vitamins are placed. The column is then exposed to sunlight for a few months until two gradients (gradual changes by composition of the created environment)are formed: one based on the amount of oxygen present and another based on the different concentration of sulfur. - Algae and cyanobacteria develop quickly in the upper portions of the water column; by producing O₂ allow the growth of heterotrophs. - Fermentative processes in the mud lead to the production of organic acids, alcohols, and H₂, suitable substrates for sulfate-reducing bacteria. - Hydrogen sulfide triggers the development of purple and green sulfur bacteria that use sulfide as a photosynthetic electron donor. ### Winogradsky Column - **Carbon inorganic:** CO₂ (atm), CaCo₃, Egg shells - **Carbon organic:** Organic matter, Cellulose, Sucrose - **Nitrogen:** NO₃², NH₄⁺, Urea (Fertilizer) - **Sulfur:** SO₂⁴, SO², Organic sulfur (Egg yolk) - **Phosphorus:** P₂O₅ (Fertilizer), PO₄²-, PS₄³- (matches) - **Hydrogen:** H₂O - **Oxygen:** O₂ (atm), H₂O - **Light:** Direct ambient exposure - **Trace elements:** Fe (Iron citrate); Ca, Mg, Fe, Cu, Mn, Se, Mo, I, Zn (Multielement); Cu, Bo, Mn, Zn (Fertilizer) - **Vitamins:** A, D, E, K, C, B1, B2, niacin, B6, folic acid, B12, biotin, pantothenic acid (Multielement) ### Isolation: Pure Culture technique - The environmental sample is diluted and spread on the solid medium. - The plate is incubated until single colonies are visible. - A single, well-isolated colony is selected using a sterile inoculating loop and streaked across the solid medium. ### Isolation: Most Probable Numbers - **Most Probable Numbers (MPN) technique** relies on our ability to selectively culture specific trophic groups. - A serial dilution of a sample is made on a selective medium (e.g. thiosulfate oxidation), and the viable colonies for each dilution are counted on a plate. - When a 10-fold serial dilution is used, the last tube showing growth should have originated from ten or fewer cells. - Therefore, it represents a method for both obtaining pure cultures and to estimate viable cell numbers. ### Isolation: High-Throughput Culture - High-throughput methods require dilution (or cell sorting) of a sample to yield a single cell in each well of a microtiter plate. - From there, each well is robotically monitored over time for cell growth or a specific target gene. - High-throughput methods allow to test many alternative growth conditions simultaneously in an attempt to replicate the realized niche, or to allow the organism to occupy its fundamental niche by relieving it from competition. - High-throughput methods were used for the isolation of Pelagibacter ubique. ### Cultured VS Uncultured - While the initial enrichment and isolation can be based on different techniques (different types of incubators, cell sorting, co-cultures), pure cultures are the ONLY officially approved approach that can lead to the description of a new microbial species. - New species names are officially valid only if they appear on the journal of the International Committee on the Systematic of Prokaryotes (ICSP) in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). - Approved name appear as Genus species sp. nov. in their first appearance, and Genus species from then on. Not officially approved species appear instead as "Candidatus Genus species" or "Ca. Genus species". - Officially described species are deposited in at least two culture collections, from where they are available to the community for analysis. ### Cultured VS Uncultured - Pure cultures give a model organism for manipulative experiments, physiological, biochemical, and genetic studies, allowing for new microbial functions to be characterized. - Even a single isolate from a previously uncultured group can dramatically change our view of the ecological role of the group. - Being "Uncultured" is an operational definition, not an intrinsic attribute of the organism. The ultimate goal is to culture the uncultured. - Recently the community has started referring to uncultured microbes as "microbial dark matter", a term that while great for science communication purposes raises much debate. ### Culture-independent approaches ### Culture-independent approaches There are a number of culture independent approaches to study microbial diversity. They can be distinguished based on either the approach (microscopy, molecular, biochemical) or based on the type of information they provide. Earlier culture-independent approaches were based on different **microscopy techniques**, from **optic** and **phase contrast** to **transmission** and **scanning electron microscopy**. The big leap in culture-independent techniques started in the seventies with the evolution of **molecular biology techniques**, which allowed scientists to probe the microbial world in new ways (mainly DNA at the time). This process brought about a big revolution. The establishment of a third domain of life (the **Archaea**) is a direct result of the application of molecular biology techniques to the study of microbial diversity. ### Fluorescent Microscopy Counting microbes in natural samples can be achieved by using **DNA staining chemicals** that **fluoresce** under UV light. The staining is nonspecific to any DNA (double or single strands depending on the dye). These approaches are used to obtain quantitative data on population abundance. **Viability staining**, instead, differentiates **live cells** from **dead ones**, therefore yielding both abundance and viability data at the same time. **FISH (fluorescence in situ hybridization)** uses specific fluorescent nucleic acid probes that, detecting and localizing specific targets, give important information about the micron-scale spatial organisation of microbes in ecosystems. ### Confocal Laser Scanning Microscopy - Confocal laser scanning microscopy (CLSM) is an **optical imaging technique** for **increasing optical resolution** and **contrast of a micrograph** by means of using a **spatial pinhole** to block out-of-focus light in image formation. - Capturing **multiple two-dimensional images** at **different depths** in a sample enables the reconstruction of **three-dimensional structures** (a process known as **optical sectioning**). ### Flow Cytometry - Flow cytometry (FC) is a technique used to detect and measure **physical and chemical characteristics** of a population of cells or particles. - In this process, a sample containing cells, **generally labeled with fluorescent markers**, is suspended in a fluid and injected into the flow cytometer instrument. - The sample is focused to ideally flow **one cell at a time** through a **laser beam**, where the light scattered is characteristic to the cells and their components. - Certain flow cytometers have the additional capability for allowing **sophisticated fluorescence-activated cell-sorting technology (FACS)**, which allows scientists to separate physically a user-defined sub-population of cells of interest away from a complicated mixture for further analysis such as downstream genomics or proteomics. - The technology is increasingly used in the isolation of both **culturable** and **hitherto non-culturable bacteria** present in environmental samples. ### Activity measurements - Specific metabolic activities can be measured by incubating natural samples with selected substrates. However, the resulting rates are often not representative of in situ rates since the community is stimulated by substrate addition. - In some cases, **direct chemical measurements** of microbial reactions are sufficient for assessing **microbial activity** in an environment. - For example, the fate of **lactate oxidation** by **sulfate-reducing bacteria** in a sediment sample can be easily tracked through monitoring of SO4²⁻ reduction to H₂S using simple chemical assays, generally measuring total sulfide (S²) with the **Methylene Blue Method**. ### This week read Rodrigues CJC, de Carvalho CCCR. Cultivating marine bacteria under laboratory conditions: Overcoming the "unculturable" dogma. *Front Bioeng Biotechnol*. 2022 Aug 17:10:964589. doi: 10.3389/fbioe.2022.964589.