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A combined protocol for the simultaneous identification of fungi and bacteria in water samples: Implication in chitin determination
BIANCALANA, F.; KOPPRIO, G. A.; LARA, R. J.; ALONSO, C.
Conferencia; ECSA 56 Coastal systems in transition: From a 'natural' to an 'anthropogenically-modified' state; 2016
LEIBNIZ CENTER FOR TROPICAL MARINE ECOLOGY, ELSEVIER, ECSA
Chitin is a highly abundant natural polymer in aquatic environments where it derives from multiple sources, playing a crucial role in the biogeochemical cycles. It is known that processes of biodegradation and/or production of chitin occur in the seston, principally in the fraction ≤ 20 µm. This fraction corresponds to the nano- and picoplancton, in which not only some components possess chitin in their walls, but also some of the main known chitin-processing organisms thrive (i.e. fungi, bacteria). The objective of this study was the development of a technique for the identification of microorganisms associated with the production and/or degradation of chitin in aquatic environments. A new protocol was set up combining the methodology of chitin determination -modified Wheat germ agglutinin method (WGA - FITC)- and the protocol for bacterial identification using in situ hybridization linked to catalyzed reporter deposition (CARD - FISH).The optimized protocol allowed the identification and quantification of particles and organisms that possessed chitin in their walls, and also served to evidence associations among them, performing well in marine, fresh and brackish water samples. Not only fungal hyphae and reproductive structures were stained with WGA but also diatoms and cyanobacteria which thus showed containing chitin in their walls. This protocol was also established to develop methodological approaches to analyze specific groups of bacteria in relation to particles containing chitin (Betaproteobacteria, Gammaproteobacteria and Bacteroidetes). This novel technique appears as a promising approach to identify chitin sources and organisms responsible for its processing in a wide range of aquatic systems.