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.

A deep-sea sulfate-reducing bacterium generates zero-valent sulfur via metabolizing thiosulfate.

Zero-valent sulfur (ZVS) is a crucial intermediate in the sulfur geobiochemical circulation and is widespread in deep-sea cold seeps. Sulfur-oxidizing bacteria are thought to be the major contributors to the formation of ZVS. However, ZVS production mediated by sulfate-reducing bacteria (SRB) has rarely been reported. In this study, we isolated and cultured a typical SRB designated Oceanidesulfovibrio marinus CS1 from deep-sea cold seep sediment in the South China Sea. We show that O. marinus CS1 forms ZVS in the medium supplemented with thiosulfate. Proteomic and protein activity assays revealed that thiosulfate reductase (PhsA) and the sulfide:quinone oxidoreductase (SQR) played key roles in driving ZVS formation in O. marinus CS1. During this process, thiosulfate firstly was reduced by PhsA to form sulfide, then sulfide was oxidized by SQR to produce ZVS. The expressions of PhsA and SQR were significantly upregulated when O. marinus CS1 was cultured in a deep-sea cold seep, strongly indicating that strain CS1 might form ZVS in the deep-sea environment. Notably, homologs of phsA and sqr were widely identified from microbes living in sediments of deep-sea cold seep in the South China Sea by the metagenomic analysis. We thus propose that SRB containing phsA and sqr genes potentially contribute to the formation of ZVS in deep-sea cold seep environments.

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.

A novel bacterial thiosulfate oxidation pathway provides a new clue about the formation of zero-valent sulfur in deep sea.

Zero-valent sulfur (ZVS) has been shown to be a major sulfur intermediate in the deep-sea cold seep of the South China Sea based on our previous work, however, the microbial contribution to the formation of ZVS in cold seep has remained unclear. Here, we describe a novel thiosulfate oxidation pathway discovered in the deep-sea cold seep bacterium Erythrobacter flavus 21-3, which provides a new clue about the formation of ZVS. Electronic microscopy, energy-dispersive, and Raman spectra were used to confirm that E. flavus 21-3 effectively converts thiosulfate to ZVS. We next used a combined proteomic and genetic method to identify thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) playing key roles in the conversion of thiosulfate to ZVS. Stoichiometric results of different sulfur intermediates further clarify the function of TsdA in converting thiosulfate to tetrathionate (-O3S-S-S-SO3-), SoxB in liberating sulfone from tetrathionate to form ZVS and sulfur dioxygenases (SdoA/SdoB) in oxidizing ZVS to sulfite under some conditions. Notably, homologs of TsdA, SoxB, and SdoA/SdoB widely exist across the bacteria including in Erythrobacter species derived from different environments. This strongly indicates that this novel thiosulfate oxidation pathway might be frequently used by microbes and plays an important role in the biogeochemical sulfur cycle in nature.

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.

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.

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.