| Marine dissolved organic matter(DOM)constitutes the main carbon pool for biogeochemical cycles and plays an important role in global carbon dynamics.The microbial carbon pump(MCP)provides a mechanistic illustration of transformation of recalcitrant dissolved organic matter(DOM)in the ocean.Here,we demonstrate the critical roles of algae-associated microorganisms(mainly heterotrophic bacteria)in the transformation of recalcitrant dissolved organic matter through laboratory cultures of a model diatom,Skeletonema dohrnii.Our experimental results showed that in addition to affecting the growth and the physiology of S.dohrnii,algae-associated bacteria are important in processing and converting algal DOM into CRAM-like DOM.Facilitated by the associated bacteria,the amount and the chemodiversity of DOM derived from algae varied during the growth and decomposition of algal cells,and enriched recalcitrant DOM formed in the later growth stage.The properties and diversity of DOM increased with the growth and decay of algal cells,indicating the transformation from active DOM to inert organic matter.Our results confirmed that the direct involvement of algae-associated microbes in the production of CRAM-like DOM.Detailed community structure analysis of the algae-associated bacterial community and its predicted functions confirmed the involvement of certain bacterial groups(e.g.,Flavobacteriia)in biosynthesis,metabolism,and degradation.S.dohrnii is a common red tide microalgae occurring in the coastal waters and throughout the world.The associated heterotrophic or autotrophic bacteria play vital roles in regulating algal growth,production,and physiology.In this study,we investigated the detailed bacterial community structure associated with the growth of S.dohrnii’s using high-throughput sequencing-based on 16S rDNA.Our results demonstrated that Bacteroidetes(48.04%)and Proteobacteria(40.66%)in all samples accounted for the majority of bacterial populations.There was a significant linear regression relationship between the abundance of bacterial phyla and culture time.Notable shifts in bacterial community composition were observed during algal growth:Flavobacteriales accounted for the vast majority of sequences at the order level.Furthermore,the relative abundance of Rhodobacterales was gradually reduced during the whole growth process of S.dohrnii(0-12 days).However,beyond that,the relative abundance of Marinobacter was slowly increasing.It is noteworthy that five fluorophores(Peaks T1,T2,I,M,and A)were detected during the growth stage of S.dohrnii.The characteristic indexes(fluorescence index,humification index,and biological index)of chromophoric dissolved organic matter(CDOM)also varied with the culture time.In addition,the taxa of bacteria had certain effects on CDOM and they were inextricably linked to each other.Bacterial transformation and processing of phytoplankton-derived organic matter are extremely important for the formation of ubiquitous organic matter(OM)in aquatic ecosystems.Heterotrophic bacteria convert OM into biomass and recycle inorganic components,contributing to the production of microbial food webs.While phytoplankton-derived organic matter is commonly studied,the transformation and processing of dissolved OM(DOM)and lysate OM(LOM)by culturable epiphytic bacteria remains poorly understood.In this study,cultivable epiphytic bacteria from the marine diatom,Skeletonema dohmii,were isolated,purified,and identified.Three bacteria,Roseobacteria sp.,Marinobacter sp.,and Bacillus sp.,were selected to study the transformation and processing of S.dohrnii-derived DOM and LOM using excitation-emission matrix(EEM)fluorescence methods,and bacterial abundance,dissolved organic carbon(DOC)concentration,and transparent exopolymer particle(TEP)content were measured.Meanwhile,the bacterial transformation of DOM and LOM was further evaluated by the fluorescence index,biological index,β/α,and humification index.The primary fluorophores,peak A(humic-like),peak C(humic-like),peak M(humic-like),peak B(protein-like),and peak T(tryptophan-like),were present in the sample.The fluorescence of DOM and LOM was dominated by protein-like signal that became increasingly humic-like over time,suggesting that more complex molecules(e.g.,recalcitrant OM)are being produced.The fluorescence of DOM and LOM was dominated by a protein-like signal that became increasingly humic-like over time,suggesting that epiphytic bacteria produced more complex molecules.Results showed that the bacteria utilized LOM more rapidly than DOM.While the three bacteria transformed OM to different degrees,all were able to facilitate microbial reprocessing of OM into refractory OM.Although the algae-derived organic matter is commonly studied,the transformation and processing of DOM by epiphytic bacteria for phytoplankton have rarely been investigated,especially under warming and acidification.Heterotrophic bacteria are assumed to play an important role in processing of phytoplankton-derived dissolved organic matter(DOM).Although the algae-derived organic matter is commonly studied,the transformation and processing of DOM by epiphytic bacteria for phytoplankton have rarely been investigated,especially under warming and acidification.In this study,Bacillus pumilus is used to explore the ecologically important marine diatom S.dohrnii-derived DOM under different conditions,utilizing fluorescence excitation-emission matrix(EEM)combined with parallel factor analysis(EEM-PARAFAC).Fluorescence regional integration and the peak selecting method are used to generate B,T,N,A,M,and C peaks in the EEM fluorescence spectroscopy.The main known fluorophores including that protein-like components(peaks B and T),unknown components(peak N),and humic-like component(peaks A,M,and C).Our experimental results showed that under higher temperature and pressure of CO2(pCO2)conditions,S.dohrnii-derived DOM fluorescence was dominated by a protein-like signal that slower waning throughout the experiment,becoming an increasingly humic-like substance,implying that processing by the epiphytic bacteria(B.pumilus)produced more complex molecules.This study reveals and confirms the direct participation of heterotrophic bacteria in the transformation and generation of algae-derived DOM in the laboratory,underlining the influence of global warming and ocean acidification on this process.Marine organic matter(OM)is one of the major carbon pools on Earth and plays a crucial role in global carbon cycle.The bacterial transformation and processing of diatom-derived OM is extremely important for the cycling of production and energy in marine ecosystems.Marine bacteria convert OM into biomass and recycle inorganic components;this process contributes to the production of microbial food webs.In this study,a cultivable bacterium(Roseobacter sp.SD-R1)from the marine diatom Skeletonema dohrnii were isolated and identified.A combined Fourier-transform ion cyclotron resonance mass spectrometry(FT-ICR MS)/untargeted metabolomics approach was used to synthesize the results of bacterial transformation with dissolved OM(DOM)and lysate OM(LOM)under warming and acidification conditions through laboratory experiments.Our experimental results showed a significant negative correlation(p<0.05)between bacterial abundance and dissolved organic carbon concentration.Roseobacteria sp.SD-R1 had different preferences for the conversion of molecules in S.dohrnii-derived DOM and LOM treatments.The effects of warming and acidification contribute to the increased number and complexity of molecules of carbon,hydrogen,oxygen,nitrogen,and sulfur after the bacterial transformation of OM.After 30 days of laboratory experiments,OM treatments resulted in a number of differentially regulated metabolites compared with OM-initial.In addition,metabolites were differentially expressed after warming and acidification-treated OM(i.e.,DOM and LOM),but the expression patterns of DOM and LOM treatments were similar within groups(ⅰ,26℃+pCO2400 ppm;ⅱ,26℃+pCO2 1000 ppm;ⅲ,30℃+pCO2 400 ppm;and iv,30℃+pCO2 1000 ppm).The reasons for these differences are that different substrates promote cellular differentiation and metabolite exchange in microorganisms.Ultimately,the chemical complexity generated by bacterial metabolism provides new insights into the mechanisms that shape OM complexity and diversity.Moreover,changes in ocean climate(warming and acidification)exacerbate this process and further elaborate the important role of microbes in microbial carbon pump.To better understand the underlying mechanisms,seawater samples were collected in October and November of 2020 from sampling stations in three subregions:the mouth of the Bay of Bengal,Southern Sri Lanka,and Western Sumatra.We calculated and evaluated different hydrological parameters and organic carbon concentrations.In addition,we used excitation emission matrix(EEM)spectroscopy combined with parallel factor analysis(PARAFAC)to analyze the natural water samples directly.Parameters associated with chromophoric DOM did not behave conservatively in the study areas as a result of biogeochemical processes.We further evaluated the sources and processing of DOM in the eastern Indian Ocean by determining four fluorescence indices(the fluorescence index,the biological index,the humification index,and the freshness index β/α).Based on EEM-PARAFAC,we identified six components(five fluorophores)using the peak picking technique.Commonly occurring fluorophores were present within the sample set:peak A(humic-like),peak B(protein-like),peak C(humic-like),and peak T(tryptophan-like).The fluorescence intensity levels of the protein-like components(peaks B and T)were highest in the surface ocean and decreased with depth.In contrast,the ratio of the two humic-like components(peaks A and C)remained in a relatively narrow range in the bathypelagic layer compared to the surface layer,which indicates a relatively constant composition of humic-like fluorophores in the deep layer.Based on East Indian Ocean DOM samples,the molecular composition and component characteristics of surface(5 m),deep chlorophyll maximum(DCM),and deep(2000 m)layer DOM in the eastern Indian Ocean(EIO)were investigated using Fourier transform ion cyclotron resonance mass spectrometry(FT-ICR MS)and three-dimensional fluorescence spectroscopy.Thousands of individual DOM formulas(approximately 3716-6986 formulas)were detected at 100-700 Da,showing a Gaussian distribution.The elements carbon(C),hydrogen(H),oxygen(O),nitrogen(N)and sulfur(S)were detected and constituted four formula classes in solid-phase extracted marine DOM samples.Furthermore,the order of the percent intensity of the formulas was CHO>CHNO>CHOS>CHNOS.Carboxylic-rich alicyclic molecule(CRAM)compounds,as part of recalcitrant DOM(RDOM),were detected at 61.32%-78.77%(by intensity).In addition,the concept of islands of stability(IOS,approximately 3.99%-11.22%)has been proposed in this study,representing the most stable components in the marine environment.Such molecular formulas as described above probably contribute to increased RDOM content in the EIO and potentially reflect enhanced accumulation or sequestration of RDOM in the deep layer.The variation in the spectroscopic indices(FI,β/α,BIX,and HIX)and fluorescent components(C1 to C4)with depth indicates a shift from protein-like to humic-like components,leading to gradual aging of the water column.In brief,this study relies on data from marine DOM in the EIO to provide a molecular and chemical background for global models of marine DOM production,transformation and sequestration. |
