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

Ziyu Yin; Rikuan Zheng; Lianfu Li; Shichuan Xi; Zhendong Luan; Chaomin Sun; Xin Zhang

doi:10.3389/fmicb.2023.1128064

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

Front Microbiol. 2023 Apr 6:14:1128064. doi: 10.3389/fmicb.2023.1128064. eCollection 2023.

Authors

Ziyu Yin^{1

2

3}, Rikuan Zheng^{1

2}, Lianfu Li^{1

2}, Shichuan Xi^{1

2}, Zhendong Luan^{1

2

3}, Chaomin Sun^{1

2

3}, Xin Zhang^{1

2

3}

Affiliations

¹ CAS Key Laboratory of Marine Geology and Environment and CAS Key Laboratory of Experimental Marine Biology and Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
² Laboratory for Marine Geology and Laboratory for Marine Biology and Biotechnology, Pilot Laboratory for Marine Science and Technology, Qingdao, China.
³ University of Chinese Academy of Sciences, Beijing, China.

Abstract

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.

Keywords: CH4–N2 gas system; Raman spectroscopy; culture experiment; deep-sea methanogenic archaea; in situ; quantitative analysis.