In situ real-time pathway to study the polyethylene long-term degradation process by a marine fungus through confocal Raman quantitative imaging.

Since plastic waste has become a worldwide pollution problem, studying the ability of marine microorganisms to degrade plastic waste is important. However, conventional methods are unable to in situ real-time study the ability of microorganisms to biodegrade plastics. In recent years, Raman spectroscopy has been widely used in the characterization of plastics as well as in the study of biological metabolism due to its low cost, rapidity, label-free, non-destructive, and water-independent features, which provides us with new ideas to address the above limitations. Here, we have established a method to study the degradation ability of microorganisms on plastics using confocal Raman imaging. Alternaria alternata FB1, a recently reported polyethylene (PE) degrading marine fungus, is used as a model to perform a long-term (up to 274 days) in situ real-time nondestructive inspection of its degradation process. We can prove the degradation of PE plastics from the following two aspects, visualization and analysis of the degradation process based on depth imaging and quantification of the degradation rate by crystallinity calculations. The findings also reveal unprecedented degradation details. The method is important for realizing high-throughput screening of microorganisms with potential to degrade plastics and studying the degradation process of plastics in the future.

Novel SERS Probe Based on Ag Nanobean/Cu Foam for Deep-Sea Extreme Environment Biomolecule Detection.

Here, we report on progress made in coupling advances in surface-enhanced Raman scattering (SERS) techniques with a deep-ocean deployable Raman spectrometer. Our SERS capability is provided by development of a Cu foam-loaded silver-nanobean (Ag/Cu foam) which we have successfully coupled to the tip of a Raman probe head capable of insertion into deep-sea sediments and associated fluids. Our purpose is to expand the range of molecular species which can be detected in deep-sea biogeochemical environments, and our initial targets are a series of amino acids reportedly found in pore waters of seep locations. Our work has progressed to the point of a full dock-based end-to-end test of the essential ship tether-ROV-deep-sea Raman system. We show here the initial results from this test as the essential requirement before at sea full ocean depth deployment. We describe in detail the procedures for preparing the Ag/Cu foam bean and demonstrate in our end-to-end test that this, when coupled to the spectrometer probe tip, yields a SERS signal enhancement of 1.2 × 106 for test molecules and detection of amino acids at 10-6 M levels consistent with reported levels of natural occurrence. Each nanobean unit is for single-use sensing since invasion of the sample fluid into the Ag/Cu foam matrix is not reversible. We describe techniques for bean rotation/replacement at depth to allow for multiple analyses at several locations during each ROV dive.

In situ Raman quantitative monitoring of methanogenesis: Culture experiments of a deep-sea cold seep methanogenic archaeon.

Gas production from several metabolic pathways is a necessary process that accompanies the growth and central metabolism of some microorganisms. However, accurate and rapid nondestructive detection of gas production is still challenging. To this end, gas chromatography (GC) is primarily used, which requires sampling and sample preparation. Furthermore, GC is expensive and difficult to operate. Several researchers working on microbial gases are looking forward to a new method to accurately capture the gas trends within a closed system in real-time. In this study, we developed a precise quantitative analysis for headspace gas in Hungate tubes using Raman spectroscopy. This method requires only a controlled focus on the gas portion inside Hungate tubes, enabling nondestructive, real-time, continuous monitoring without the need for sampling. The peak area ratio was selected to establish a calibration curve with nine different CH4-N2 gaseous mixtures and a linear relationship was observed between the peak area ratio of methane to nitrogen and their molar ratios (A(CH4)/A(N2) = 6.0739 × n(CH4)/n(N2)). The results of in situ quantitative analysis using Raman spectroscopy showed good agreement with those of GC in the continuous monitoring of culture experiments of a deep-sea cold seep methanogenic archaeon. This method significantly improves the detection efficiency and shows great potential for in situ quantitative gas detection in microbiology. It can be a powerful complementary tool to GC.

Raman spectral characteristics of 12CO2/13CO2 and quantitative measurements of carbon isotopic compositions from 50 to 450 °C and 50 to 400 bar.

The carbon isotopic composition of CO2 is traced to its different origins and widely used in the fields of geology, biology, and chemistry. Raman spectroscopy can be performed in situ, is nondestructive, and requires no sample preparation; these characteristics enable Raman spectroscopy to be considered a new alternative method to measure the carbon isotopic composition of CO2. In this work, Raman spectra of high-purity 13CO2, 12CO2, and six 12CO2-13CO2 binary mixtures with known mixing ratios were collected using a High Pressure Optical Cell (HPOC) at 50-450 °C and 50-400 bar. The results showed that the characteristic peak positions of both 13CO2 and 12CO2 shift to lower wavenumbers with increasing temperature and decreasing pressure, but the peak positions of 13CO2 show a larger shift. The peak position difference of the corresponding characteristic peaks between 13CO2 and 12CO2 is greater than 15 cm-1 under the above temperatures and pressures, and the peaks can be distinguished. However, ν-13 overlays νH.B.12 near 1265 cm-1, ν+12 overlaps νH.B.13 near 1288 cm-1. The existence of 13CO2 can cause a change in the Fermi diad splitting of 12CO2 and affect the establishment of CO2 Raman densimeters. The positive correlation obtained between the peak intensity ratio and the content ratio is affected by temperature, pressure, and 13CO2 content. I+13/I+12 and I-13/I-12 were selected as the quantitative indices to establish Raman quantitative analysis models for the determination of the carbon isotopic composition of CO2, which can be applied to in-situ measurements of high-temperature and high-pressure systems.

Study of Microbial Sulfur Metabolism in a Near Real-Time Pathway through Confocal Raman Quantitative 3D Imaging.

As microbial sulfur metabolism significantly contributes to the formation and cycling of deep-sea sulfur, studying their sulfur metabolism is important for understanding the deep-sea sulfur cycle. However, conventional methods are limited in near real-time studies of bacterial metabolism. Recently, Raman spectroscopy has been widely used in studies on biological metabolism due to its low-cost, rapid, label-free, and nondestructive features, providing us with new approaches to solve the above limitation. Here, we used the confocal Raman quantitative 3D imaging method to nondestructively detect the growth and metabolism of Erythrobacter flavus 21-3 in the long term and near real time, which possessed a pathway mediating the formation of elemental sulfur in the deep sea, but the dynamic process was unknown. In this study, its dynamic sulfur metabolism was visualized and quantitatively assessed in near real time using 3D imaging and related calculations. Based on 3D imaging, the growth and metabolism of microbial colonies growing under both hyperoxic and hypoxic conditions were quantified by volume calculation and ratio analysis. Additionally, unprecedented details of growth and metabolism were uncovered by this method. Due to this successful application, this method is potentially significant for analyzing the in situ biological processes of microorganisms in the future. IMPORTANCE Microorganisms contribute significantly to the formation of deep-sea elemental sulfur, so studies on their growth and dynamic sulfur metabolism are important to understand the deep-sea sulfur cycle. However, near real-time in situ nondestructive metabolic studies of microorganisms remain a great challenge due to the limitations of existing methods. We thus used an imaging-related workflow by confocal Raman microscopy. More detailed descriptions of the sulfur metabolism of E. flavus 21-3 were disclosed, which perfectly complemented previous research results. Therefore, this method is potentially significant for analyzing the in-situ biological processes of microorganisms in the future. To our knowledge, this is the first label-free and nondestructive in situ technique that can provide temporally persistent 3D visualization and quantitative information about bacteria.

The diel vertical distribution and carbon biomass of the zooplankton community in the Caroline Seamount area of the western tropical Pacific Ocean.

Zooplankton can affect and regulate the biological carbon pump in the biogeochemical cycles of marine ecosystems through diel vertical migration (DVM) behaviour. The diel vertical distribution and migration of a zooplankton community were studied at a continuous survey station in the Caroline Seamount area of the western tropical Pacific Ocean. Using a MultiNet sampling system, 346 zooplankton species/taxa were collected and identified. The vertical distribution patterns of abundance and composition of the zooplankton community differed between daytime and nighttime. The highest biodiversity index occurred in the 100-200-m ocean depth layer, but some zooplankton species remained in the deep-water layer below 300 m. The DVM patterns of the various dominant species differed, even when the species belonged to the same order or family. Dissolved oxygen and seawater temperature were the main environmental factors affecting the diel vertical distribution of the zooplankton community. The oxygen minimum zone was identified as performing the dual role of "ecological barrier" and "refuge" for zooplankton. The active carbon flux mediated by the zooplankton DVM in the Caroline Seamount area was 14.5 mg C/(m2·d). Our findings suggest that zooplankton DVM can affect and mediate the biological carbon pump in the Caroline Seamount area.

The spatial-temporal evolution of heavy metal accumulation in the offshore sediments along the Shandong Peninsula over the last 100 years: Anthropogenic and natural impacts.

The anthropogenic and natural impacts on the temporal and spatial variations of heavy metals in sediments under the Shandong Peninsula coastal current are still unclear. Here, the concentrations\burial fluxes of Cr, Cu, Zn, As, and Pb in three sediment cores retrieved from the Bohai Sea and the Yellow Sea along the Shandong Peninsula were analyzed to study the spatial-temporal variability of heavy metal accumulation over the last century. The results showed that the buried heavy metal fluxes were relatively low at the end of the Shandong Peninsula coastal current. The enrichment factor (EF) and geoaccumulation index (Igeo) indicated that those metals did not severely pollute the sediments except As that reached a moderate enrichment. Principal component analysis (PCA) revealed that Cr, Cu, Zn, and Pb were mainly derived from natural weathering and As was determined by anthropogenic contamination. The strength of the Shandong Peninsula coastal current, the Yellow River estuary location, and sediment discharge load significantly influenced the concentrations of natural-origin heavy metals by affecting sediment grain size and the source-sink process. The emission of pollutants from agricultural and industrial activities in the Shandong Peninsula region resulted in As enrichment since the 1950s. Moreover, the EF values of heavy metals in sediment cores from China's coastal seas showed apparent spatial variations of heavy metal pollution but had coherent temporal variability with China's economic development process. Heavy metals pollution has weakened in most coastal seas since the 2000s, likely due to the extensive industrial upgrading and the implementation of pollution control. These results have a reference significance for studying the evolution and source-sink process of the heavy metals in offshore sediments and tracing anthropogenic impacts in different periods.

A Piecewise Model for In Situ Raman Measurement of the Chlorinity of Deep-Sea High-Temperature Hydrothermal Fluids.

The chlorinity of deep-sea hydrothermal fluids, representing one of the crucial deep-sea hydrothermal indicators, indicates the degree of deep phase separation of hydrothermal fluids and water/rock reactions. However, accurately measuring the chlorinity of high-temperature hydrothermal fluids is still a significant challenge. In this paper, a piecewise chlorinity model to measure the chlorinity of high-temperature hydrothermal fluids was developed based on the OH stretching band of water, exhibiting an accuracy of 96.20%. The peak position, peak area ratio, and F value were selected to establish the chlorinity piecewise calibration model within the temperature ranges of 0-50 ℃, 50-200 ℃, and 200-300 ℃. Compared with that of the chlorinity calibration model built based on a single parameter, the accuracy of this piecewise model increased by approximately 4.83-12.33%. This chlorinity calibration model was applied to determine the concentrations of Cl for high-temperature hydrothermal fluids in the Okinawa Trough hydrothermal field.

Discovery of supercritical carbon dioxide in a hydrothermal system.

Supercritical CO2 appearing as bubbles in hydrothermal vents was identified in the south part of the Okinawa Trough using in situ Raman spectroscopy. Significantly, the N2 peak in supercritical CO2 is much larger than those in seawater and vent fluids, indicating that supercritical CO2 enriches N2 from the surrounding environment. Considering that the partial pressures of CO2 and N2 in the Earth's proto-atmosphere were ~10-20 MPa, supercritical CO2 with high N2 was likely the dominant CO2 phase near the water-air interface in the early history of the Earth, which promoted the synthesis, pre-enrichment and preservation of amino acids and other organic matters that are essential to the origin of life.

Marinobacter fonticola sp. nov., isolated from deep sea cold seep sediment.

In this study, we report a novel Gram-negative bacterium, designated as strain CS412T, isolated from deep-sea sediment collected in a cold seep area of the South China Sea. Growth of strain CS412T occurred at 4-40 °C (optimum, 28 °C), pH 5.0-11.0 (optimum, pH 6.0) and with 0-19 % (w/v) NaCl (optimum, 1-2 %). Phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain CS412T belonged to the genus Marinobacter. The closest phylogenetic neighbours of strain CS412T were Marinobacter pelagius HS225T (96.9 %), Marinobacter szutsaonensis NTU-104T (96.8%), Marinobacter santoriniensis NKSG1T (96.4%) and Marinobacter koreensisdd-M3T (96.3 %). The genomic DNA G+C content of strain CS412T was 58.0 mol%. The principal respiratory quinone was ubiquinone-9 (Q-9). The polar lipids of CS412T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipidand and four glycolipids. The major fatty acids of CS412T contained cyclo-C19 : 0ω8c, C16 : 0, C18 : 1ω7c and C18 : 1ω7c 11-methyl. The results of phylogenetic, physiological, biochemical and morphological analyses suggested that strain CS412T represents a novel species of the genus Marinobacter, and the name Marinobacter fonticola sp. nov. is proposed with the type species CS412T (=CCTCC AB 2019197T=KCTC 72475T).

High temperature-induced proteomic and metabolomic profiles of a thermophilic Bacillus manusensis isolated from the deep-sea hydrothermal field of Manus Basin.

Thermophiles are organisms that grow optimally at 50 °C-80 °C and studies on the survival mechanisms of thermophiles have drawn great attention. Bacillus manusensis S50-6 is the type strain of a new thermophilic species isolated from hydrothermal vent in Manus Basin. In this study, we examined the growth and global responses of S50-6 to high temperature on molecular level using multi-omics method (genomics, proteomics, and metabolomics). S50-6 grew optimally at 50 °C (Favorable, F) and poorly at 65 °C (Non-Favorable, NF); it formed spores at F but not at NF condition. At NF condition, S50-6 formed long filaments containing undivided cells. A total of 1621 proteins were identified at F and NF conditions, and 613 proteins were differentially expressed between F and NF. At NF condition, proteins of glycolysis, rRNA mature and modification, and DNA/protein repair were up-regulated, whereas proteins of sporulation and amino acid/nucleotide metabolism were down-regulated. Consistently, many metabolites associated with amino acid and nucleotide metabolic processes were down-regulated at NF condition. Our results revealed molecular strategies of deep-sea B. manusensis to survive at unfavorable high temperature and provided new insights into the thermotolerant mechanisms of thermophiles. SIGNIFICANCE: In this study, we systematically characterized the genomic, proteomic and metabolomic profiles of a thermophilic deep-sea Bacillus manusensis under different temperatures. Based on these analysis, we propose a model delineating the global responses of B. manusensis to unfavorable high temperature. Under unfavorable high temperature, glycolysis is a more important energy supply pathway; protein synthesis is subjected to more stringent regulation by increased tRNA modification; protein and DNA repair associated proteins are enhanced in production to promote heat survival. In contrast, energy-costing pathways, such as sporulation, are repressed, and basic metabolic pathways, such as amino acid and nucleotide metabolisms, are slowed down. Our results provide new insights into the thermotolerant mechanisms of thermophilic Bacillus.

A New Approach to Measuring the Temperature of Fluids Reaching 300 ℃ and 2 mol/kg NaCl Based on the Raman Shift of Water.

The OH stretching band of water is very sensitive to temperature and salinity for the existence of hydrogen bonds between H2O molecules. In this study, the OH stretching band was deconvoluted into two Gaussian peaks, with peak 1 at approximately 3450 cm-1 and peak 2 at approximately 3200 cm-1. The positions of peaks 1 and 2 both shifted to higher wavenumbers with increasing temperature from 50 ℃ to 300 ℃. The effects of salinity in the range of 0-2 mol/kg NaCl on the OH stretching band were also studied. Linearity for the relationship between Raman shift of peak 1 and temperature increased as the salt concentration increased from 0 to 2 mol/kg, while peak 2 displayed an opposing trend. Two temperature calibration models were developed based on the temperature-dependent changes in the Raman frequency shifts of peaks 1 and 2 (precision of 0.9 ℃ and 1.0 ℃, respectively). The calibration models for temperature were successfully applied to determining the temperatures of deep-sea hydrothermal fluids in the Okinawa Trough hydrothermal field. The degree of mixing of hydrothermal fluids and ambient seawater during in situ Raman measurements was estimated by the difference in temperatures determined through these calibration models and those measured through thermocouple sensors.

A Direct Quantitative Raman Method for the Measurement of Dissolved Bisulfate in Acid-Sulfate Fluids.

Raman spectroscopy has been applied to the quantitative analysis of the concentration of bisulfate in acid-sulfate fluids at different temperatures. The quantitative analysis method is based on the peak area ratios of [Formula: see text](ν1) and H2O (ν2), where PA([Formula: see text]/H2O) = [[Formula: see text]] × (0.0066 × T + 1.3070) at a temperature range of 0-100 ℃. We found that the molal scattering coefficient of bisulfate increases slightly at the elevated temperature may be due to the changes of fraction of water molecules that are hydrogen-bonded. The method can also be applied to analyze physicochemical parameters of other acid fluids, such as hydrogen phosphate, bicarbonate, etc., and especially to the in situ detection of deep sea acid-sulfate hydrothermal fluids in the future.

Description of Bacillus kexueae sp. nov. and Bacillus manusensis sp. nov., isolated from hydrothermal sediments.

Two Gram-staining-positive, strictly aerobic bacilli, designated as strains Ma50-5T and Ma50-6T, were isolated from the hydrothermal sediments of Manus Basin in the western Pacific Ocean. Based on 16S rRNA gene sequence, strains Ma50-5T and Ma50-6T were most closely related to Bacillus alveayuensis (97.0 and 97.2 % identity, respectively). The 16S rRNA gene sequence identity between strains Ma50-5T and Ma50-6T was 97.4 %. The identities between strains Ma50-5T and Ma50-6T and other closely related organisms were below 97.0 %. The G+C contents of the genomic DNA of strains Ma50-5T and Ma50-6T were 43.4 and 47.6 mol%, respectively. The major fatty acids (>10 %) of both strains were iso-C15 : 0 and iso-C17 : 0. The predominant isoprenoid quinone detected in both strains was menaquinone-7. Phylogenetic, physiological, biochemical and morphological analyses suggested that strains Ma50-5T and Ma50-6T represent two novel species of the genus Bacillus, for which the names Bacillus kexueae sp. nov. (type strain Ma50-5T=KCTC 33881T=CCTCC AB 2017020T) and Bacillus manusensis sp. nov. (type strain Ma50-6T=KCTC 33882T=CCTCC AB 2017019T), respectively, are proposed.

In Situ Raman Spectral Characteristics of Carbon Dioxide in a Deep-Sea Simulator of Extreme Environments Reaching 300 ℃ and 30 MPa.

Deep-sea carbon dioxide (CO2) plays a significant role in the global carbon cycle and directly affects the living environment of marine organisms. In situ Raman detection technology is an effective approach to study the behavior of deep-sea CO2. However, the Raman spectral characteristics of CO2 can be affected by the environment, thus restricting the phase identification and quantitative analysis of CO2. In order to study the Raman spectral characteristics of CO2 in extreme environments (up to 300 ℃ and 30 MPa), which cover most regions of hydrothermal vents and cold seeps around the world, a deep-sea extreme environment simulator was developed. The Raman spectra of CO2 in different phases were obtained with Raman insertion probe (RiP) system, which was also used in in situ Raman detection in the deep sea carried by remotely operated vehicle (ROV) "Faxian". The Raman frequency shifts and bandwidths of gaseous, liquid, solid, and supercritical CO2 and the CO2-H2O system were determined with the simulator. In our experiments (0-300 ℃ and 0-30 MPa), the peak positions of the symmetric stretching modes of gaseous CO2, liquid CO2, and supercritical CO2 shift approximately 0.6 cm-1 (1387.8-1388.4 cm-1), 0.7 cm-1 (1385.5-1386.2 cm-1), and 2.5 cm-1 (1385.7-1388.2 cm-1), and those of the bending modes shift about 1.0 cm-1 (1284.7-1285.7 cm-1), 1.9 cm-1 (1280.1-1282.0 cm-1), and 4.4 cm-1 (1281.0-1285.4 cm-1), respectively. The Raman spectral characteristics of the CO2-H2O system were also studied under the same conditions. The peak positions of dissolved CO2 varied approximately 4.5 cm-1 (1282.5-1287.0 cm-1) and 2.4 cm-1 (1274.4-1276.8 cm-1) for each peak. In comparison with our experiment results, the phases of CO2 in extreme conditions (0-3000 m and 0-300 ℃) can be identified with the Raman spectra collected in situ. This qualitative research on CO2 can also support the further quantitative analysis of dissolved CO2 in extreme conditions.

Comparative transcriptome analysis of Rimicaris sp. reveals novel molecular features associated with survival in deep-sea hydrothermal vent.

Shrimp of the family Alvinocarididae are the predominant megafauna of deep-sea hydrothermal vents. However, genome information on this family is currently unavailable. In the present study, by employing Illumina sequencing, we performed the first de novo transcriptome analysis of the gills of the shrimp Rimicaris sp. from the hydrothermal vent in Desmos, Manus Basin. The analysis was conducted in a comparative manner with the shrimp taken directly from the vent (GR samples) and the shrimp that had been maintained for ten days under normal laboratory condition (mGR samples). Among the 128,938 unigenes identified, a large number of differentially expressed genes (DEGs) between the GR and mGR samples were detected, including 2365 and 1607 genes significantly upregulated and downregulated, respectively, in GR. The DEGs covered diverse functional categories. Most of the DEGs associated with immunity were downregulated in GR, while most of the DEGs associated with sulfur metabolism and detoxification were upregulated in GR. These results provide the first comprehensive transcriptomic resource for hydrothermal vent Rimicaris and revealed varied categories of genes likely involved in deep-sea survival.