Niche differentiation of microbial community shapes vertical distribution of recalcitrant dissolved organic matter in deep-sea sediments.

Sedimentary organic matter provides carbon substrates and energy sources for microorganisms, which drive benthic biogeochemical processes and in turn modify the quantity and quality of dissolved organic matter (DOM). However, the molecular composition and distribution of DOM and its interactions with microbes in deep-sea sediments remain poorly understood. Here, molecular composition of DOM and its relationship with microbes were analyzed in samples collected from two sediment cores (∼40 cm below the sea floor), at depths of 1157 and 2253 m from the South China Sea. Results show that niche differentiation was observed on a fine scale in different sediment layers, with Proteobacteria and Nitrososphaeria dominating the shallow sediments (0-6 cm) and Chloroflexi and Bathyarchaeia prevailing in deeper sediments (6-40 cm), indicating correspondence of microbial community composition with both geographical isolation and the availability of organic matter. An intimate link between the DOM composition and microbial community further indicates that, microbial mineralization of fresh organic matter in the shallow layer potentially resulted in the accumulation of recalcitrant DOM (RDOM), while relatively low abundance of RDOM was linked to anaerobic microbial utilization in deeper sediment layers. In addition, higher RDOM abundance in the overlying water, as compared to that in the surface sediment, suggests that sediment might be a source of deep-sea RDOM. These results emphasize the close relation between the distribution of sediment DOM and different microbial community, laying a foundation for understanding the complex dynamics of RDOM in deep-sea sediment and water column.

Direct Production of Bio-Recalcitrant Carboxyl-Rich Alicyclic Molecules Evidenced in a Bacterium-Induced Steroid Degradation Experiment.

Carboxyl-rich alicyclic molecules (CRAM) are highly unsaturated compounds extensively distributed throughout aquatic environments and sediments. This molecular group is widely referred to as a major proxy of recalcitrant organic materials, but its direct biosynthesis remains unclear. Steroids are a typical anthropogenic contaminant and have been previously suggested to be precursors of CRAM; however, experimental evidence to support this hypothesis is lacking. Here, a steroid-degrading bacterium, Comamonas testosteroni ATCC 11996, was incubated in a liquid medium supplemented with testosterone (a typical steroid) as the sole carbon source for 90 days. Testosterone-induced metabolites (TIM) were extracted for molecular characterization and to examine the bioavailability during an additional 90-day incubation after inoculation with a natural coastal microbial assemblage. The results showed that 1,775 molecular formulas (MFs) were assigned to TIM using ultrahigh-resolution mass spectrometry, with 66.99% categorized as CRAM-like constituents. A large fraction of TIM was respired or transformed during the additional 90-day seawater incubation; nevertheless, 638 MFs of the TIM persisted or increased during incubation. Among the 638 MFs, 394 were commonly assigned in natural deep seawater samples (depths of 500 to 2,000 m) from the South China Sea. Compared to the catabolites of the well-established testosterone degradation pathway, we compiled a list of bio-refractory MFs and potential chemical structures, some of which shared structural homology with CRAM. These results demonstrated direct microbial production of bio-refractory CRAM from steroid hormones and indicated that some of the biogenic CRAM resisted microbial decomposition, potentially contributing to the aquatic refractory dissolved organic matter (DOM) pool. IMPORTANCE CRAM are an operationally defined DOM group comprising a complex mixture of carboxylated and fused alicyclic structures. This DOM group is majorly characterized as refractory DOM in the marine environment. However, the origins of the complex CRAM remain unclear. In this study, we demonstrated that testosterone (a typical steroid) could be transformed into bio-refractory CRAM by a single bacterial strain and observed that some of the CRAM highly resisted microbial degradation. Through molecular comparison and screening, potential chemical structures of steroid-induced CRAM were suggested. This study established the biological connection between steroids and bio-refractory CRAM, and it provides a novel perspective explaining the fate of terrestrial contaminants in aquatic environments.

Experimental Insight into the Enigmatic Persistence of Marine Refractory Dissolved Organic Matter.

More than 90% of marine dissolved organic matter (DOM) is biologically recalcitrant. This recalcitrance has been attributed to intrinsically refractory molecules or to low concentrations of molecules, but their relative contributions are a long-standing debate. Characterizing the molecular composition of marine DOM and its bioavailability is critical for understanding this uncertainty. Here, using different sorbents, DOM was solid-phase extracted from coastal, epipelagic, and deep-sea water samples for molecular characterization and was subjected to a 180-day incubation. 1H nuclear magnetic resonance spectroscopy and ultra-high-resolution mass spectrometry (UHRMS) analyses revealed that all of the DOM extracts contained refractory carboxyl-rich alicyclic molecules, accompanied with minor bio-labile components, for example, carbohydrates. Furthermore, dissolved organic carbon concentration analysis showed that a considerable fraction of the extracted DOM (86-95%) amended in the three seawater samples resisted microbial decomposition throughout the 180-day heterotrophic incubation, even when concentrated threefold. UHRMS analysis revealed that DOM composition remained mostly invariant in the 180-day deep-sea incubations. These results underlined that the dilution and intrinsic recalcitrance hypotheses are not mutually exclusive in explaining the recalcitrance of oceanic DOM, and that the intrinsically refractory DOM likely has a relatively high contribution to the solid-phase extractable DOM in the ocean.

Investigation of the molecular structure complexity of dissolved organic matter by UPLC-orbitrap MS/MS.

The complex natural organic matter of the Suwannee River fulvic acid (SRFA) standard was analyzed by online reversed-phase chromatography with Orbitrap MS/MS using collision-induced dissociation (CID). The number of isobars per nominal mass could be reduced to a single dominantly abundant species in a chromatographic run, sharing some ions with signals having the identical molecular formula in adjacent chromatographic segments and later serving as a precursor ion for fragmentation. A very large proportion of the same fragment ions existed in adjacent chromatographic fractions. The difference in the fragment ions in adjacent chromatographic fractions could be attributed to a gradual change in the formula composition of precursor ions in a chromatographic run. It could be concluded that dissolved organic matter (DOM) molecules with the same elemental composition in different chromatographic fractions may have very similar molecular structures. In addition, we propose a possible DOM model that might greatly deepen our understanding of the behavior of DOM in aquatic matrices.

Microbial transformation of distinct exogenous substrates into analogous composition of recalcitrant dissolved organic matter.

Oceanic dissolved organic matter (DOM) comprises a complex molecular mixture which is typically refractory and homogenous in the deep layers of the ocean. Though the refractory nature of deep-sea DOM is increasingly attributed to microbial metabolism, it remains unexplored whether ubiquitous microbial metabolism of distinct carbon substrates could lead to similar molecular composition of refractory DOM. Here, we conducted microbial incubation experiments using four typically bioavailable substrates (L-alanine, trehalose, sediment DOM extract, and diatom lysate) to investigate how exogenous substrates are transformed by a natural microbial assemblage. The results showed that although each-substrate-amendment induced different changes in the initial microbial assemblage and the amended substrates were almost depleted after 90 days of dark incubation, the bacterial community compositions became similar in all incubations on day 90. Correspondingly, revealed by ultra-high resolution mass spectrometry, molecular composition of DOM in all incubations became compositionally consistent with recalcitrant DOM and similar toward that of DOM from the deep-sea. These results indicate that while the composition of natural microbial communities can shift with substrate exposures, long-term microbial transformation of distinct substrates can ultimately lead to a similar refractory DOM composition. These findings provide an explanation for the homogeneous and refractory features of deep-sea DOM.

Molecular-level transformation characteristics of refractory organics in landfill leachate during ozonation treatment.

Macromolecular refractory organics in landfill leachate are extremely complex compounds. This study examined the molecular-level transformation characteristics of refractory organics in biologically-treated landfill leachate (i.e., membrane bioreactor (MBR) leachate) during ozonation treatment. Results indicated that higher ozone dosage and longer reaction time enhanced organics removal. When ozone dosage was 32.16 mg/min and reaction time was 30 min, the efficiencies of removing color number, chemical oxygen demand (COD), and dissolved organic carbon (DOC) were 95.16%, 51.13%, and 26.40%, respectively. Furthermore COD / DOC decreased from 3.38 to 2.24, and the content of aromatic substances and macromolecular humic substances (e.g., humic- and fulvic-like substances) substantially decreased. The MBR-treated leachate mainly contained phenolic compounds (82%, aromatic index ≤ 0.50 and H/C < 1.5), and the major elements within the dissolved organic matter in the MBR-treated leachate were C, H, O, N, and S. The CHOS and CHONS compounds in the leachate indicated that it would have a much greater biorefractory property than the natural organic matter (i.e., technical grade humic acid). After the MBR-treated leachate was treated by ozonation for 10 min, the CHO, CHON, CHOS and CHONS compounds were greatly degraded and removed, and the oxidation degree of dissolved organic matter was significantly increased owing to the strong oxidation ability of ozone. At 30 min of ozonation, CHON, CHOS and CHONS compounds were further degraded, and CHOS and CHONS compounds (as the biorefractory substances) were almost completely removed. It was noteworthy that some CHO compounds that mainly contained phenolic compounds (m/z = 250-300, carbon number > 20, and double bond equivalent < 6) with a higher bioavailability and higher saturation degree accumulated. This study provides beneficial references for practical application of landfill leachate treatment using the ozonation process.

In-House Standard Method for Molecular Characterization of Dissolved Organic Matter by FT-ICR Mass Spectrometry.

Electrospray ionization (ESI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been widely used for molecular characterization of dissolved organic matter (DOM). However, ESI FT-ICR MS generally has poor repeatability and reproducibility because of its inherent ionization mechanism and structural characteristics, which severely hindered its application in quantitative analysis of complex mixtures. In this article, we developed an in-house standard method for molecular characterization of DOM by ESI FT-ICR MS. Instead of obtaining reproducible results by determining the instrument parameters, we adopted an approach of object control on the mass spectrum to solve the problem of poor reproducibility. The mass peak shape, resolution, and relative intensity distribution of a natural organic matter standard were adjusted by optimizing the operating conditions to obtain a repeatable result. The quality control sample was run 26 times by the different operators in a 6-month-long period to evaluate the reproducibility. Results showed that the relative standard deviation (%) of repeatability and reproducibility are 1.02 and 2.35 for average H/C, respectively. The in-house standard method has been validated and successfully used for the characterization of more than 4000 DOM samples, which is transferable to other laboratories.

Molecular investigation into the transformation of dissolved organic matter in mature landfill leachate during treatment in a combined membrane bioreactor-reverse osmosis process.

This study investigated the effectiveness of a combined membrane bioreactor (MBR) and reverse osmosis (RO) process for treating leachate produced by a large-scale anaerobic landfill. The MBR process had limited treatment efficiency for removing organic pollutants, but when combined with RO, the integrated system completely removed macromolecular compounds (i.e., humic- and fulvic-like substances) and produced effluent that satisfied the applicable discharge standard. The landfill leachate contained many types of DOM that had high molecular weight and were highly unsaturated. Although the MBR process removed some DOM that had a relatively low saturated degree (mainly aliphatic compounds (2.0 ≥ H/C ≥ 1.5) with relatively high bioavailability), many bio-refractory compounds were not removed. The RO system greatly reduced the content of residual DOM in MBR effluent and was effective for removing heteroatom DOM, especially polycyclic aromatics (AI > 0.66) and polyphenols (0.66 ≥ AI > 0.50). The effluent from the combined process of MBR and RO treatment mainly contained a small number of aliphatic compounds and phenolic compounds (AI ≤ 0.50 and H/C < 1.5) that had higher bioavailability than DOM in the raw leachate and posed little environmental risk.

Validation and Evaluation of High-Resolution Orbitrap Mass Spectrometry on Molecular Characterization of Dissolved Organic Matter.

Molecular composition of dissolved organic matter (DOM) is a hot topic in subjects such as environmental science and geochemistry. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been applied to molecular composition characterization of DOM successfully. However, high instrument and maintenance costs have constrained its wider application. A high-resolution Orbitrap mass spectrometer (Orbitrap MS) can provide approximately 500,000 resolving power (at m/z 200), which is potentially capable of characterizing the molecular composition of DOM. In this paper, the application of high-resolution Orbitrap MS was evaluated by comparing with FT-ICR MS in the aspect of resolution, mass distribution, detection dynamic range, and isotopic peak intensity ratio. The impact of instrument parameters of Orbitrap MS was further investigated, which includes ionization, ion transfer, and mass detection. The result shows that the high-resolution Orbitrap MS is capable or even preferable for molecular characterization of DOM. However, the peak intensity distributions are dependent on the instrument parameters, which could affect the environmental impact assessment caused by the sample itself. The result indicates that development of a universal and comparable method is of great demand.