Extraction & Isolation Of Marine Dissolved Organic Matter And Its Molecular Characterization

Study of marine dissolved organic matter is not only the basis but also the key point of research of marine carbon sink process and mechanism.Currently,the study of DOM molecular composition,organic structural aspects characteristics of recalcitrant molecular structures has advanced rapidly,but it is still in the "darkness before dawn".The lack of adequate solid mDOM samples often impedes icts the progress of its biogeochemical studies.In this thesis,DOMs in different aquatic ecosystems were extracted,concentrated,and purified by a SPE method.Gram-scale solid DOM samples were successfully achieved,characterized by state-ofthe art analytical instruments;this progress will set the stage for preparation of reference standards of mDOM in the future.The major contents and conclusions are as follows:1.The extraction method of DOM from large volume seawaters by Solid Phase Extraction（SPE）using PPL resin was developed.The automatic extraction device was set up and developed to extract gram-scale DOM,and the procedure was optimized.A series batches of coastal seawater were processed with this device and the DOC recovery was in the range of 45%～55%,and about 230 mg/day of DOM could be absorbed using the developed technique.This PPL-SPE method is reliable,efficient and stable.A variety of advanced analytical instruments were used to characterize the samples,the results showed that the elemental compositions,spectral characteristics of PPL-DOM samples prepared in different batches were reliable and stable.Using FT-ICR MS,a largenumber of the same molecular formulas in DOM samples in different batches were identified and assigned,the results indicate the similarity of samples at the molecular level.2.DOM composition is obviously affected by the aquatic ecosystems and sources of DOM.DOM samples were extracted,isolated from waters collected from coastal aquaculture water,tidal creek of mangrove wetland and intermediate seawater of northern South China Sea（SCS）by the PPL-SPE method.The mass spectra and solid-state ¹³C NMR spectra results indicate that the sub-fractions and molecular compositions of DOM were obviously affected by their sources.The analytical results of Orbitrap MS showed that the main classes of compounds in DOM of several aquatic ecosystems were CHO,CHON and CHOS,respectively,but there were significant differences in the averaged numbers and abundances（intensity-weighted）of different classes of compounds.In comparisons of these DOMs,the abundance of S-containing compounds in DOM of the mangrove wetland was relatively greater（ca.25%）,it may be affected by those functional microorganisms in mangrove sediments,sulfur-oxidation and sulfate reduction processes.The DOM molecules of northernSCS were more assembled in its van Krevelen diagram,and about 95%of its molecular formulae were identified among all molecules of rest of DOMs.In addition,solid-state ¹³C NMR spectra demonstrate characteristics of alkyl C,aromatic,carbohydrate and carboxyl,but sources of DOM samples greatly affected the spectral shapes and integral values.There were significant more contribution from carbohydrate-like signals from Aoshan Bay and Sanggou Bay.However,distinct methoxyl C signals from lignin were detected in tidal DOM of the mangrove wetland.3.The molecular composition of DOM in the marine sediments of Bohai Sea was significantly correlated with the abundance of microbial community.The sediment DOM（sDOM）collected from different depths of 0-60 cm sediments of M8 station,Bohai Sea,was extracted using PPLcartridges.FT-ICR MS results showed that the major classes of compounds in the sediments were CHO,CHON and CHOS,respectively,and there are little CHONS class at certain depths.We observed that apparent differences were observed between CHON and CHO moleculas in terms of number and relative abundance（intensity-weighted）,the maximum and minimum relative abundances of CHON and CHO classes were observed at 40 cm,respectively.The ratios of CHOS class was stable,and the ratio of relative abundance was greater than ratios of molecular numbers.By comparison,sDOM molecular composition vary with depths,it is found that CHON classes may be the main organic matter components changed in sediments.In addition,through the correlation analysis,it was found that there was an obvious correlation between the abundance of molecular classes and the abundances of bacteria and archaea communities.The abundance of anaerobic bacteria was negatively correlated with the abundance of CHO class,while the abundance of aerobic bacteria was positively correlated with the relative abundance of CHO class.4.In comparison of spectral and molecular characteristics of different sub-fractions of surface water DOM（Laoshan Reservoir）and marine DOM（Aoshan Bay）,we observed that there might be presences of similar molecular structures between marine DOM and surface water DOM.DAX-8/XAD-4 resin-in-tandem was used to fractionate and to concentrate fresh water from Laoshan reservoir and surface water from Aoshan Bay;two sub-fractions,hydrophobic acid（HPOA）and Amphiphilic acid（AMPA）,were obtained and analyzed by organic elemental composition,fluorescence spectral characteristics and ultra-high resolution mass spectrometry.The results showed significant differences in two distinct categories of DOMs.The C/N ratio and δ¹³C values indicated that there are more N-containing components and a large terrigenous organic contribution for both DOMs.However,bulk analysese of FTIR and solid-state ¹³C NMR illustrated that there were still a large number of similar functional groups in these DOM organic molecules except for differences in the relative abundance of functional groups.The EEMS results revealed that the chromophoric DOM（cDOM）of HPOA was characterized by strong humic-like signals,while the cDOM of AMPA was characterized by more protein-like signals(microbia derivatives or autochthonous origins.FT-ICR mass spectra showed that the molecular weights of fractions ranged from 250-500 Da,which contained a large number ofsame molecular formulae;over 50%of same molecular formulae belonged to the class of so-called carboxylic-rich refractory alicyclic molecules（CRAM）.The mass ions detected in four DOM samples were selected for analysis of tandem mass spectrometry.Losses of m/z 44 were often occured,and fragments from methyl,hydroxyl and carbonyl were also dominant.Overall,the results implied existences of structual similarities among moleculae of fraction of marine DOM and surface water DOM.