This study aimed at investigating the carbon flow within communities of benthic diatoms, which are major primary producers in estuarine environments and important for the food web in marine intertidal sediments. Although intertidal sediments and their benthic diatom inhabitants have been extensively studied, our knowledge on the carbon flow in these ecosystems is limited and for a large part still unexplored. One important reason for this limited research was the incapability of the available methods and techniques for tracking the production and fate of the individual components in specific pools of biochemical macromolecules. The majority of in-situ studies that has been published dealt with various aspects of the diatom lipid composition using stable isotope techniques. However, in order to study the carbon flow in greater detail it is crucial to include other carbon pools such as carbohydrates, amino acids, nucleic acids, and extracellular polymeric substances (EPS) in addition to lipids. For a long time compound specific stable isotope analysis (CSIA) of carbon was restricted to the established technique of gas chromatography/isotope ratio mass spectrometry (GC/IRMS). Because most biological compounds are polar and non-volatile, the vast majority of compounds can only be analysed by GC/IRMS after derivatisation (i.e.a chemical modification to increase volatility, but has the disadvantage that it also alters the 13C/12C ratio of the compounds and hence needs substantial correction, which may affect the accuracy and reproducibility of the measurement). Another drawback of GC/IRMS analysis is the requirement of time consuming stringent testing of the analytical procedures in order to determine the proper correction factors. Moreover, not all biological compounds can be analysed by GC/IRMS. The introduction of liquid chromatography / isotope ratio mass spectrometry (LC/IRMS) has opened new avenues for the analysis of 13C in non-volatile, aqueous soluble organic compounds and enables the study of metabolic pathways for a much broader range of biological compounds. Especially the analysis of carbohydrates, amino acids and nucleic acids benefit importantly from the use of LC/IRMS because the synthesis of derivatives is not required. However, the challenge of LC/IRMS method development is to meet the analytical constraints of the LC/IRMS interface, which prohibits the use of carbon-containing eluents and columns displaying carbon bleeding. Hence, this prevents the use of most existing traditional LC methods. Summary 239 This thesis is divided in two parts: (1) Stable isotope methodology (Chapter 2-4) and (2) Carbon cycling in benthic diatom mats (Chapter 5-8). Part 1 highlights the development of novel analytical procedures to perform LC/IRMS stable 13C analysis of individual components in specific biochemical pools. Chapter 2 discusses the development of an LC/IRMS method to analyze stable carbon isotope ratios in the most important carbohydrates in microorganisms. Subsequently, Chapter 3 presents an LC/IRMS method for analyzing stable carbon isotope ratios in DNA and RNA nucleotides. In addition, a method for DNA and RNA extraction was developed and validated and an enzymatic hydrolysis protocol was designed to the study DNA and RNA biosynthesis in benthic diatom mats. An LC/IRMS method was already available for the separation of all amino acids as are encountered in benthic diatoms, as well as a method to separate short chain organic acids (SCOA) including volatile fatty acids. For lipid analysis, GC/IRMS remains the technique of choice. Finally, the performance of GC/IRMS and LC/IRMS CSIA analysis was tested by the measurement of δ13C values in carbohydrates (Chapter 4). In general, LC/IRMS achieved the highest precision and a broader range carbohydrates could be analyzed using this technique. LC/IRMS analysis is much easier to carry out and is less time consuming, because laborious sample preparation and challenging system validation are not required. In conclusion, LC/IRMS can be used to quantify the biosynthesis of metabolites and can be applied both for natural 13C-abundance as well as for the analysis of 13C-labeled material. The developed LC/IRMS methods are not restricted to study carbon flow in communities of benthic diatom, but can also be used to study metabolic processed in many other research areas. Part 2 reports of the study of the fate of carbon fixed by communities of benthic diatoms. The established methods of GC/IRMS and the newly developed LC/IRMS methods were combined to trace 13C into carbohydrates, amino acids, fatty acids, EPS (i.e. water-extractable and EDTA-extractable EPS) and in SCOA. With a preliminary in-situ13C bicarbonate labeling experiment, the successful application of LC/IRMS was demonstrated in the study of carbon flow in communities of benthic diatoms. It was also concluded that this technique could be a valuable tool for other biological studies (Chapter 5). Water-extractable EPS was the major component produced and consisted mainly of glucose with minor contributions from other carbohydrates and amino acids. Diatoms were the predominantly primary producers in this study. Gammaproteobacteria, Bacteroides, and Deltaproteobacteria were the major heterotrophic bacterial groups (Chapter 6). The exudation of low-molecular weight Summary240organic compounds (such as SCOA) by diatoms was most probably responsible for the initial fast transfer of organic carbon to the heterotrophic bacteria. The transfer of organic carbon from the diatoms to the heterotrophic bacteria through water-extractable EPS was slower but the turnover of this EPS explained 75% of the total carbohydrate processing in the sediment. The different groups of heterotrophic bacteria benefited equally from the organic matter that was released by the diatoms suggesting a limited specialization in this microbial food web. In Chapter 7, the carbon flow within the diatom mats was investigated during one year in order to cover seasonal variations. The inorganic carbon fixed by the diatoms was recovered in carbohydrates, amino acids, fatty acids and nucleic acids. Independent on the season, fixed carbon was initially stored as carbohydrate (glucose), while nitrogen- and or phosphorus-rich compounds (e.g. amino acids and RNA/DNA) were synthesized more slowly. It seemed that the dense diatom mat was unable to acquire sufficient nutrients for the synthesis of nitrogen- or phosphorus- containing structural cell material during the period of photosynthesis and, hence, that the fixed carbon was mainly stored as reserve material or exuded as EPS. In summer, a change in the fate of carbon fixed by benthic diatoms was observed and more neutral storage lipids were synthesized, which could hint to stress for the diatom mat. The lipids may serve as storage of carbon and energy. The lipids may also serve as an electron sink that could protect the diatoms from oxidative stress, which is induced by high photosynthetically active radiation (PAR) and high temperature. In addition, due to the presence of bioturbating organisms and grazers, urea produced by this fauna could be an important nitrogen source during the summer months and affecting the diatom’s metabolism. In Chapter 8, the seasonal dynamics of EPS and SCOA exuded by benthic diatoms and the use of these exudates as a carbon source by heterotrophic bacteria were investigated. The production rates of the carbohydrates and amino acids that originated from EPS were remarkably different between seasons. This resulted in a more heterogeneous composition of the EPS in spring and summer when compared to the rest of the year and suggested a different function for these exopolymers. It was conceived that the exudation of carbohydrates served mainly to balancing energy and motility of diatoms.The role of the extracellular amino acids was conceived as to interconnect polysaccharide chains in the EPS and hence forms a structure that is important for adhesion of the diatom cell and for defense against grazing. In order to compare our results with other studies that use other operational defined fractions to Summary 241 extract EPS, we compared two water-extractions (Milli-Q and artificial seawater) and the extraction of EDTA and a cation-exchange (DOWEX) protocol. No difference was found between water-extractions, however although it seemed that the EDTA and DOWEX protocol extracted the same type of EPS in terms of composition, the EDTA extraction was 4-fold more efficient compared to the DOWEX agent.From February until June the biomass and production of diatoms and bacteria were closely coupled and especially sulfate reducing bacteria (SRB) benefited from associating with SCOA-releasing diatoms. From August on, the coupling of biomass and production of diatoms and bacteria became less strong and EPS produced by diatoms promoted the growth of other bacterial taxa rather than SRB. The seasonal variation of exudates produced by diatoms therefore played an important role in shaping the community composition and diversity of the associated bacteria. |
