Ultraschall-Extraktion von Lipid-Biomarkern aus Sediment
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Overview
Quelle: Labor von Jeff Salacup – University of Massachusetts Amherst
Das Material, bestehend aus der lebenden “Bio” Anteil an jedem Ökosystem (Blätter, Pilze, Rinde, Gewebe; Abbildung 1) unterscheidet sich grundlegend von dem Material der unbelebten “anorganischen” Aktie (Felsen und ihre konstituierenden Mineralien, Sauerstoff, Wasser, Metalle). Organisches Material enthält Kohlenstoff verbunden zu einer Reihe anderer Kohlenstoff und Wasserstoff Moleküle (Abbildung 2), die sie von anorganischem Material unterscheidet. Carbon ermöglicht breite Valenz (03:56) es, bis zu vier separate kovalente Bindungen mit benachbarten Atomen, in der Regel C, H, O, N, S und P. bilden Es kann bis zu drei kovalente Bindungen auch teilen, mit einem anderen Atom, wie z. B. die Dreifachbindung in der oft giftige Zyanid oder Nitril, Gruppe. Seit 4,6 Milliarden Jahren führte diese Flexibilität zu einer erstaunlichen Vielfalt an chemischen Strukturen, die sich in Größe, Komplexität, Polarität, Form und Funktion unterscheiden. Der wissenschaftlichen Bereich der organische Geochemie befasst sich mit der Identifizierung und Charakterisierung des Gesamtangebots von nachweisbaren organischen Verbindungen, Biomarker, produziert von Leben auf diesem Planeten, sowie andere, durch geologische Zeit genannt.
Abbildung 1. Organisches Material, wie Bäume, Blätter und Moos, unterscheiden sich chemisch und visuell von anorganischem Material wie Pflaster.
Principles
Procedure
Results
At the end of extraction, a total lipid extract (TLE) for each sample is evident. Each vial contains the extractable organic matter from a sediment, soil, or plant tissue. These TLEs can now be analyzed and their chemical constituents identified and quantified.
Applications and Summary
Different classes of biomarkers impart information on specific aspects of the Earth system. For example, in its infancy, organic geochemistry was primarily concerned with the formation, migration, and alteration of petroleum, and many of the chemical tools organic geochemists use today are based on those initial investigations. It was through the investigation of a class of compounds called isoprenoids, having a repeating five carbon pattern (Figure 2), that scientists discovered petroleum comprised the chemically altered remains of ancient primary producers, such as plankton in the ocean (converting to oil, Figure 3) or peat bogs on land (coal, Figure 4). Chemists at large oil companies used the ratios of a variety of compounds, each with its own known rate of alteration, to estimate how old petroleum was, where it came from, and if it was worth exploiting. Today, new biomarkers are being discovered, identified, and characterized in modern and ancient samples analyzed in organic geochemistry labs around the world. Many of today's applications seek to extract environmental information from biomarkers obtained in modern samples (leaves, soil, microbes, water samples, etc.) in order to extend the biomarker's utility to ancient sediments in an effort to reconstruct the climates, environments, and ecosystems of the past. For example, the distribution of a group of biomarkers called glycerol-dialkyl glycerol-tetraethers (GDGTs for short), produced by a suite of archaea and bacteria, were found in modern sediments to change in a predictable manner in response to air or water temperature. Therefore the distribution of these biomarkers in ancient sediments can be used, or through a series of sediments of known age, to reconstruct air and water temperature back several million years.
Figure 2. Isoprene comprises five carbon atoms and two double bonds. When added together in biosynthesis reaction, they can form complex molecules diagnostic for the presence of life. For example, 2, 6,10,15,19-pentamethyleicosane, commonly found in cyanobacterial mats.
Figure 3. Illumination of plankton at Maldives. Copyright PawelG Photo.
Figure 4. Peat bog at 4,500 m elevation in the Ecuadorian Andes. Copyright Dr. Morley Read
Transcript
The first step in paleoclimatology is to collect, or extract, the biomarkers from the sediment they are found in. Environmental samples are composed of non-organic components, such as minerals, water, and metals, and organic components that are created by living organisms in the area. Before these organic components can be used by scientists to elucidate information about the past, they must be removed from their environment. Sonication, which utilizes ultrasonic waves, is the simplest and least expensive of these techniques.
This video is part of a series on lipid extraction, purification, and analysis from sediments. It will illustrate lipid extraction by ultrasound and present a few applications of the method.
Because of the wide range of biomarkers, there is no single solvent optimized to extract all of them. This is summarized by the so-called ‘like dissolves like’ rule, whereby relatively apolar molecules dissolve in apolar solvents such as dichloromethane, and more polar molecules dissolve in more polar solvents such as methanol. Solvent mixtures for the extraction of specific lipids or groups of lipids are generally optimized empirically.
To accelerate extraction and increase yield, a sonication system is used to apply ultrasound – waves with frequencies greater than 20 kHz, in conjunction with the solvent mixture. When these waves contact the liquid organic phase, they cause the formation of short-lived microbubbles of solvent vapors that rapidly grow and collapse. On collapsing, these bubbles release a tremendous amount of energy as mechanical shear, facilitating lipid solubilization and dramatically increasing the efficiency of extraction.
After the ultrasound assisted solvent-extraction process, the result is a crude extract preparation, called a total lipid extract, that is subjected to further purification to allow qualitative and quantitative examination of lipid signatures. Now that you understand some of the main principles behind lipid extraction by sonication, lets take a look at a protocol for how the procedure is performed.
Collect the necessary sample materials from a chosen location. Some examples are lacustrine and marine sediments, terrestrial soils, microbial cultures, or plant leaves. Collected material is frozen overnight. Following this it is freeze-dried in a freeze dryer for 2 to 3 days. Crush and homogenize the freeze-dried samples prior to extraction with a solvent-rinsed mortar and pestle. To remove organic contaminants, combust the required borosilicate glass pipettes, vials, and weighing tins in an oven. After allowing the glassware to cool in the oven, rinse the metal tools with a mixture of dichloromethane and methanol. Once the sample and glassware are prepared, the sonication procedure can begin.
From this step on, all containers and glassware should be combusted before use. Place the weighing tin on a scale and tare. Rinse the lab spatula with the solvent mixture, then, use it to transfer an appropriate mass of freeze-dried, homogenized sample into the weighing tin and record the mass. Carefully transfer the weighed sample into a labeled vial. Using the squirt bottle of DCM:MeOH, add enough that the sample is covered by 1 to 2 cm of solvent, and cap the vial. Place the vial on a waterproof rack, now ready for sonication. Place the rack directly into the sonication bath. Check that the water level in the sonication bath is just deep enough to submerge the sample vials up to the top of the extraction solvent. Sonicate for 30 minutes at room temperature. After sonication, remove the rack from the sonicator. Let the vials sit to allow sediment settling to occur.
Remove the dichloromethane-methanol upper phase from the extraction vial using a pipette and bulb, and transfer into another pre-weighed and labeled vial. Repeat the sonication process a total of three times for each sample. Collect the extracts into one vial. Allow extracted samples to dry in their vials, caps off, and in the hood, covered loosely with a piece of foil. Label as ‘extracted residue’ and store in the extraction solvent. Now that the biomarkers have been extracted, they must be purified before analysis can take place.
Sonication accelerates several solvent extraction processes and is widely used in geochemical studies. Many archeologists work with geochemists in order to reconstruct the environmental and cultural circumstances under which early human civilizations lived. Pottery, one of the oldest human inventions, when unearthed, can be found to contain residual molecular fossils from wine, rice, or other contents that were once stored within.
To unearth chemical evidence of substances absorbed onto the surfaces, small samples of pottery are sonicated in the presence of organic solvents and extracted compounds can be subsequently identified downstream by spectroscopic methods. This kind of analysis helps archeologists detect the kinds of resources that were available to ancient populations and reconstruct the conditions of their habitat.
Photosynthetic microalgae are found in marine and freshwater ecosystems. Because they grow in seawater-based media and their culture occupies significantly smaller areas, they are now being widely studied as a promising alternative to terrestrial plants for the production of biofuels.
To extract lipids from a microalgal biomass, these researchers describe a sonication-assisted solvent extraction. Acoustic cavitation during sonication effectively disrupts rigid microalgal cell walls in order to liberate lipids. Such techniques aid the characterization of new microalgae from the environment for the production of non-petroleum sources of energy.
You’ve just watched JoVE’s introduction to Sonication-Assisted Extraction of Biomarkers from Sediments. The following videos will explain how the extract is further purified for analysis.
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