Abstract
Nitrogen flows through bacteria and algae in aquatic microbial communities are an important part of the nitrogen cycle, which plays a central role in aquatic ecosystems. However, work on uptake and retention of nitrogen in bacteria versus algae in natural microbial communities has long been hampered by a lack of
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adequate methodology, especially in turbid waters and sediments. This thesis deals with the development, validation and application of a new method for analysis of nitrogen and carbon flows through bacteria and algae in aquatic microbial communities. The method concerns analysis of incorporation of the stable isotopes 15N and 13C in hydrolysable amino acids (HAAs), including the bacterial biomarker D-alanine (D-Ala) in combination with stable isotope labeling. After detailed presentation and assessment of the analytical aspects of the method, the method was applied in three different stable isotope labeling studies: In the first study, we investigated the short term (24 h) incorporation of nitrogen and carbon from urea and amino acids by incubating surface sediment samples from the Scheldt Estuary with dual labeled (15N and 13C) urea and amino acids. Analysis of 15N and 13C incorporation into THAAs yielded some interesting results concerning the uptake dynamics of the two substrates and the underlying mechanisms (including uncoupling of 15N and 13C uptake and incorporation) while analysis of 15N and 13C incorporation into D-Ala revealed a small to negligible bacterial contribution to total microbial 15N incorporation. The second study concerns an in situ 15N pulse chase experiment investigating the incorporation and retention of nitrogen in the sediment of the subtropical Brunswick Estuary (Australia). Analysis of 15N in HAAs over a 30 day period revealed very efficient recycling of 15N by the microbial community within the sediment with a major role for bacteria. In the third study, the method was used to investigate the fate of peptidoglycan, a bacterial cell wall component, in sediment. The bacterial community of an intertidal mudflat in the Scheldt Estuary was labeled with 13C and we subsequent traced the fate of the 13C in various bacterial cell components (D-Ala (specific for peptidoglycan), THAAs (representing bulk proteinaceous material) and bacteria-specific PLFAs (representing cell membranes of living bacteria)) over a 4.5-month period in situ. Results revealed a relatively strong retention of 13C in D-Ala providing direct in situ evidence for relative accumulation of peptidoglycan during reworking and degradation of bacterial biomass in sediments. Altogether, the applications of the method presented in this thesis as well as those in other 15N- and/or 13C-labeling studies served as a thorough validation of the method which proved to be very suitable for its originally intended application (analysis of 15N incorporation by bacteria). Together with the additional features like analysis of 13C in bacteria and 15N and 13C in total (microbial) biomass, this makes the method a very useful, versatile new tool for studies on flows of nitrogen and carbon through bacteria and algae in aquatic microbial communities.
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