Fungal organisms are commonly exploited by the human activity: the production of antibiotics, vitamins, enzymes and other metabolites deserve pharmaceutical, food and biotechnological industries. Fungi have a major place on the ecological dynamics of ecosystems by using different roles, such as symbionts (such as lichens, mycchorhize…), parasites, pathogenic agents and saprophytes, with a degradation activity that permit the transfer of nutrients from the degraded organisms to soils and water. In this study, we will focus on the fungal decomposer community. Here, we will focus on filamentous species, that, contrary to yeasts, are multicellular; however, this distinction is relative, considering dimorphic fungi that may take both forms. The fungus-like organism distinction concern mainly Oomycota, a rein of organisms which has similar feature with fungi but present differences, such as a cellulose cell wall instead of chitin, that made them similar to plants. Fungi, and fungus-like organisms are eukaryotic organisms. Filaments of fungi are also name hyphae (singular: hypha)(1). The filamentous fungi growth is apical: the extreme part of filaments, is the place where cellular division occurs. The hyphae network is called mycelium. A cytoplasmic flow occurs to the hyphal tip, wearing secreted vesicle to the apex. At this position, the wall is thinner . Some fungi are septate, meaning that septa, or cross-walls, are present at regular intervals. Woronin bodies appears close to the cross-walls pores. These are membranous organelles that might close pores when a compartment is damaged, became too old or undergo differentiation. Septa present pores, where cytoplasm and even the nuclei can pass. Hyphae are not made with cells but by interconnected compartments. At the oldest part of the mycelium, present vacuole that may take most of the space on the hypha, restricting the cytoplasm, the nucleus and other organism to a small fraction. When hyphae are too old, compartments may be empty, closed from the rest of the organism. Cells walls are digested by enzymes and the rest is used by the mycelium to produce a thick-wall resting spore, named chlamydospores. The mycelium growth is multidirectional, with several order of branches, and interconnections between branches. Fungi has no predetermined age, size, or shape : they are spatially and temporary undetermined. The cell-wall is composed of Beta-glucan polysaccharide for rigidity, chitin or cellulose (oomycota), with mannoprotein that permit external exchange of molecule; and ergosterol, a steroid that take place of cholesterol of animals, and make the layer more fluid. The nutrition of fungi is chemoheterotrophic. Its solubilize nutrients with external enzymatic catalysis and then absorb nutrient that cannot enter into the organism without depolymerization. . The growing mycelium is normally always attached to the food source, and the shape of the culture is influenced by the need to obtain nutrients. Enzymes are released in the zone of erosion of the hypha that permit the secretion of large molecules (from 20 to 60.10^3 Da). On this located area, fungi is weaker and may separate from the mycelium if its threaten by an external factor. Polymer-degrading fungi may also locally synthesize antibiotics in the closest area of nutrient uptake, probably to compete with other organisms. This has be reported in the Rhizoctonia spp. By Burton and Coley-Smith (1993). Usually, the carbon source differ with fungal strains, from simple methane to larger complex macromolecules, such as lignin, keratin, cellulose... For common laboratory fungal culture, the use of potato-dextrose, malt or cornmeal agar is widespread. These culture media are more acidic than those for bacteria; and contain relatively more nitrogen. Complementary nutrients may by added for appropriate culture such as vitamins, asparagine (as a source of nitrogen for strains that cannot use nitrate, or ammonium), or other more specific nutrients. Spores production is part of the reproduction of fungi organisms: this aspect matter concerning laboratory security rules. Spores are formed in the sporangium, a large multinuclear cell that secrete spores by cytoplasm separation. Some fungi lack a sporangium, and produce spores by several phenomenon, budding, fragmentation… Major taxonomic groups of fungi and fungus like organisms include Chrytridiomycota, Zygomycota, Ascomycota, Deuteromycota and Oomycota. They differs in the sexual reproduction. For example, the life cycle and sexual reproduction of Mucor, a specie employed in bioremediation process that belong to the Zygomycota group, start with an asexual reproduction, with a production of sporangiophores . Spores are released, then fix and germinate to a somatic hyphae. The mating occurs when complementary types of aerial branches of hyphae growth towards each other, mate, produce a progametangia that separate from each other. The final gametangia will fuse, and create a diploid compartmented zygospore. Then, the meiosis occurs and produce a new hypha. The fungal kinetic growth present thew wide-known model: after the inoculation in laboratory, first occurs a lag phase, followed by an exponential growth, that then decelerate until becoming stationnary, and finally and autolysis phase. On laboratory condition, when a culture is saturated, the organism start to sporulate; spore may spread and contaminate other non-sterile experiment, but can also threaten the human health while breathing. Source : Introduction to Modern Mycology, Blackwell Sciences - J.W. Deacon (1997)
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A short explanation of the biodegradation of pollutants by fungi Let’s talk about mycoremediation. Different pollutants are present in the environment, soils, water stream, air and oceans: We can consider different pollutants : polycyclic aromatic hydrocarbons, or PAH, a families of stable chemical products composed from many aromatic rings that make them really stable; pesticides, that make huge damages in the environment ; synthetic dyes, from the textiles industries, are often found in wastewaters and came from the water from your washing machine; TNT and RDX, explosives compounds used in bombs and spread spread over environment after war conflicts ; oils, fragrances and diesels form petroleum hydrocarbons; heavy metals and metalloïds; endocrine disruptors, chemicals compounds that can interact with endocrine organs and lead to a disrupt in human and animal metabolisms – theses compounds, including phtalates, phenols, organochlorides metallo-organic compounds and medicines has a wide range of origins, including pesticides, medicine and plastic micro products… In order to counteract these different compounds and their negative effect in the environment, fungi present several properties that make them suitable from remediation : They can be tolerant to extreme conditions, they developement permit them to reach chemicals pollutants, that are sometimes adsorbed to surfaces and over biomass, blocked in tiny pores, precipitated or dissolved in the water. They also don’t need these pollutants for they development. A plus for they application is that they sometimes bring waters and nutrient to the contaminated environment, permitting the revival of the ecosystem. In the family tree of fungi, most pollutant degraders belong to the phyla Ascomycota and Basidomycota, and Mucoromycotina, a subdivison of Zygomycota. The studied fungi are filamentous fungi : unlike yeasts, they spatially growth forming a hyphae network. Because of they major role in the rhizosphere, they are able to cop with plants by absorbing some pollutants; they can also host endomycotic bacteria. Fungi generally solubilize external nutrient with enzymes before absorption. Fungal degradation is also advantageous in remediation because of the enzyme are not highly specific and can degrade different and related compounds. Its is know that some Cladophialora ans Exophiala dedrade toluene. Aspergillus and Penicillium can degrade some pesticides, TNT, PAHs and hydrocarbons. PAHs can be degraded by several (sub)phylums, such as Kickwellomycotina, Chytridiomycota, Glomeromycota… The subphylum of Mucoromycotina has been less studied concerning the degradation properties, but, the genera Cunninghamella, Mucor and Rhizopus belonging to this subphylum can degrade PAH, textile dyes and TNT. How does fungi degrade pollutants. We can divide degradation into two types : external degradation, and internal degradation. The external oxidation involve enzymes such as laccases and peroxydases, resulting in cleavages, aromatic-ring fission, oxidative-coupling products… Internal degradation happened inside the fungal organism. First, an initial attack in the cytochrome P450 enzyme permit a further treatment by pollutants, such as conjugate formation using transferase, or further catabolism. The biodegradation lead product from pollutants that act as reactants. The mineralization of pollutants can ultimaltely lead to the production of H20/CO2, due to oxidations/reductions. But generally, remediation lead to products that are less toxic, such as bound residues, metabolites excretion… Source : Untapped potential : exploiting fungi in bioremediation of hazardous chemical, Harms, Schlosser ans Wick, 2011, Applied and Industrial Microbiology.
-Remember, my internship is about fungal strains that can dissolve plastic pollutants, such as Biphenol A, on waste-waters... Hello my friends ! In order to measure the degradation of plastic by fungi in a liquid media, we have to measure the pollutant concentration before and during the fungal enzymatic activity. In order to quantitate the three pollutants, we use a UPLC – Ultra Performant Liquid Chromatography. This machine permit to identify components in a liquid phase with a huge precision. On this article, I will give more explanation about this machine …. A pokemon evolution is a change in the shape of the pokemon, resulting a new pokemon that will be stronger than the previous one, more powerful, faster, more beautiful... As an example, Paras, the mushroom pokemon, evolve to Parasect at the level 24; Parasect is (relatively) better... For our chromatography technology, it's the same! UPLC (Ultra-Performant-Liquid-Chromatography) technology, released in 2004, is much more accurate to separate compounds in a mixture than HPLC (Hight-Performat-L-C), first designed in 1952. UPLC is a patented trademark from the Waters Corporation. Its a technology similar to the HPLC with advanced features. This is how the machine look like : The process cover by this machine is called UHPLC – but the trademark is commonly used to name the process, removing the H. This malapropism happen in other situations, such as “-Margaux, do you have a Kleenex?” or “-Margaux, could you give me your scotch?” instead of “-Margaux, do you have a tissue, and could you give me the [whisky] adhesive tape...” The chromatography separate components in a mixture, permit their identification and can quantitate and even isolate them. UPLC deliver data, visual spectrum and associated values for each peaks that can be analysed quickly by scientists, even with internship students. It is composed by several complementary features: Pumps and Solvent Reservoir, The Automatic Sampling, The Column, the PDA detector and the tubing.
UPLC bonus: a lower volume of solvent is need to inject the mobile phase: Where 30µL were previously needed, only 4.97 µL is used with the new technology. The binary solvent distribution is managed by the PLAST4 method: from the beginning until the fifth minute, the water solvent is injected, and hydrophilic compounds are first passing through the columns. The water percentage decrease hugely and Methanol is add from the fifth minutes during 30 seconds, and hydrophobic compounds start to go through the columns. In the spectrum, a huge number of peak appeared at this period. The Methanol is injected until the seventh minutes.
UPLC malus: lifetime of columns are usually shorter...
Fun fact: A shift in time of released compound happened during my experiment: the same compound was released belatedly than at the beginning. I have to know its an error from the machine or if it’s something else … I first wanted to draw a scheme but now I am too lazy ! Hope you have understand something useful :D Bibliography :
Wiki : https://fr.wikipedia.org/wiki/Chromatographie_en_phase_liquide_%C3%A0_haute_performance https://en.wikipedia.org/wiki/High-performance_liquid_chromatography https://fr.wikipedia.org/wiki/UPLC https://en.wikipedia.org/wiki/Van_Deemter_equation video : https://www.canal-u.tv/video/cerimes/chromatographie_liquide_a_haute_performance.9085 http://www.lachimie.fr/analytique/chromatographie/HPLC/HPLC.php http://www.waters.com/waters/fr_FR/UPLC---Ultra-Performance-Liquid-Chromatography-Beginner%27s-Guide/nav.htm?cid=134803622&locale=fr_FR http://www.waters.com/waters/fr_FR/Original-UPLC-UHPLC-system-with-sub-2-micron-particle-technology-for-separations/nav.htm?cid=514207&locale=fr_FR PWP : https://www.slideshare.net/manubhau/ultra-performance-liquid-chromatography-pptby-manoj-ingale https://www.slideshare.net/ShaimaaAhmadeen/1-14411077 http://www.americanlaboratory.com/913-Technical-Articles/1569-Effective-UPLC-Implementation/ From February until May 2017, I am doing an internship at the environmental microbiology department of the UFZ Helmoltz Center for Environmental research (Leipzig, Germany). The studies of Dr. Dietmar Schlosser, my internship supervisor, are focus on some strains of fungi that are able to degrade micro-pollutants(1) found in in the environment, with a industrial origin, such as pharmaceuticals (ibuprofen…), endocrine disruptor (bisphenol A…), hormones... The impact of these pollutants is various and hazardous: it has been recorded that it might change the gender of aquatic animals!(2) I started to test the degradation ability of 5 different fungal strains: Phoma sp., Coniothyrium sp, Stachybotrys chlorohalonata and two others unidentified (yet) marine strains. After cultivate the strains, they are inoculated in contact of a mixture of three micro-pollutants (Bisphenol A, Dibutylphtalate, Diethylphtalate) with a salt medium to mimic a marine environment. The next step is to collect samples approximately each days over one week, and analyze it by UPLC (Ultra Performant Liquid Chromatography) – I will explain how does this machine works on another blogpost :) Mycoremediation might be one solution to water pollution, let’s see what we can do with this potential ! (-in a next blogpost also haha) don’t forget to follow me on instagram :) Sources:
(1) - "Unttapped potential: exploiting fungi in bioremediation of hazardous chemicals" -Harms & Schlosser, 2011 (2) - "Sex Hormones originating from different lifestock production systems: fate and potential disrupting activity in the environment" - G Lange and all, 2002 |