Report for the Joint Use/Research of the Institute for Planetary Materials,
Okayama University
2022 fiscal year first term / second term / others
Day/Month/YearCategory:International Joint Research/ General Joint Research/ Joint Use of Facility/ Workshop
Name of the research project: Lipidomics of archaeal growth in serpentine environments
Principal applicant: Anna Neubeck
Affiliated institution and department: Department of Earth Sciences, Uppsala University,
SwedenCollaborator
Name: Christian Potiszil
Affiliated institution and department: Institute for Planetary Materials, Okayama University atMisasa
Research report:
Research purpose
In order for the origin of life (such as methanogenic, hydrogenotrophic archaea) to have emerged from carbon
dioxide, water, and rocks on the primitive Earth, a consistent, chemically transmutable source of energy wasindispensable. Currently, the process of serpentinization is being recognized as a progressively plausible
candidate for this energy source. Serpentinization of ultramafic crusts would have facilitated a constant supplyof bioessential components, including hydrogen, methane, formate, and ammonia and metals in off-ridge
alkaline hydrothermal springs. Life in serpentinizing systems is dominantly microbial, though it should be
noted that this does not preclude eukaryotes. Nonetheless, bacterial and archaeal biomass in these systems
massively outweighs other classifications. While surficial serpentinizing systems host a significantly higher
biomass and cell abundance than subsurface systems, it is estimated that, in aggregate, subsurface microbialcommunities account for nearly 90% of all bacterial and archaeal biomass on Earth, as well as nearly 15% of
our planet’s total biomass. Prokaryotes in serpentinizing systems are dominantly hydrogen- andmethane-cycling bacteria and archaea, alongside subordinate acetate-, iron-, and sulfate-cycling groups.
Relevant eukaryotes include fungi, nematodes, and rotifers, though they are orders of magnitude less abundant
than the prokaryotes (Bar-On, Phillips, and Milo 2018).

The overall goal of the project is thus to gain better understanding of mineral-microbe interactions inenvironments with active or inactive serpentinization.
The primary objective of this project is to discern lipids derived from methanogenic archaea withinserpentine rocks, with the aim of enhancing the precision of microbial community structure analysis on amacroscopic level in such environments.
The methodology employed in this study involves a comparative analysis of viable microorganisms, utilizing
established DNA extraction and analytical techniques, alongside: (1) isolated lipids obtained from serpentine
rocks and (2) purified lipids obtained from cultured methanogenic archaea that were isolated from thedesignated site of interest.

The model species used in this study is the type strain of Methanobacterium oryzae, first described byJoulian et al. (2000) and an isolated version of a similar (98% similarity) species. The methanogen was
isolated from the target area, in the Chimaera serpentine rocks north of Antalya, Turkey. It is described as a
non-motile, rod-shaped, obligate anaerobic, methanogenic archaea isolated from serpentinizing rice soil nearPila, Luzon, Philippines. Cells occur individually or in chains and range from 0.3-0.4 μm width and 3-10 μm
length. M. oryzae lacks cytochromes—proteins involved with redox catalysis among anaerobes (Fauque andBarton 2012)—which enables their growth at extremely low H2 pressure. Methanogens with cytochromes
grow significantly faster under optimal conditions than those without, having as much as double growthyield, although they have higher H2 pressure requirements (Neubeck et al. 2016). Thus, the M. oryzae is an
ideal methanogen for studying survival in low-temperature serpentine environments.

Preliminary and previous results
Earlier findings have demonstrated the proficient extraction of lipids from serpentine material obtained froman active serpentinization locality located north of Antalya, Turkey, as documented by Rattray et al. (2022).
Furthermore, Neubeck et al. (2017) have extensively investigated the community structure ofmicroorganisms in the same region. Nevertheless, archaea typically occur in significantly lower abundance
and are consequently challenging to detect and classify. In light of this, the lipid data acquired through theaforementioned investigation furnish a superior resolution and accuracy in the assessment of the microbialcolonization of rocks.
Preliminary findings, from experiments using olivine alteration as the primary source of H2 through low
temperature serpentinization, have shown that methanogens may alter the olivine phase of the ultramafic
rocks and grow on the alteration products, see plots below.

Fig. 1. Average (of triplicates) CH4 gain in “unfed” samples (i.e., without added H2/CO2) over theexperimental period. For treatment abbreviations, see Table 1.

Actually Conducted Research
So far, a method has been developed to extract standard lipid compounds using a modified bligh dyer
extraction (Bligh & Dyer 1959; Boumann et al., 2006; Sturt et al., 2004). The standards have also been used
to confirm the validity of an orbitrap based method to detect them (Rattray et al., 2022). In the future, theisolated culture of M. oryzae will be extracted and run to obtain the required data to complete the project.
The data are expected to be obtained in the coming financial year and after data analysis will result in apublication.

Expected research outcome
The expected research outcome is an extensive understanding of the lipid structure and composition of the M.
oryzae wild type and type strain, by using the methods and techniques described previously in Rattray et al.
2022. Archaea often have what is called an S-layer which is made of proteins that form a paracrystalline
structure that is hydrophobic and is often referred to as a 'molecular sieve' (Schultze-Lam et al., 1996)(Figure 3). Most archaea seemingly have an S-layer, but a few are missing one. Several anionic groups on
this layer render the surface charge negative and allows for interaction with cations. A few archaea, like some
types of methanogens, may have additional cell wall structures that are similar to the peptidoglycan found inbacteria like, for example, pseudomurein (Meyer and Albers, 2020). It is often these cell wall structures that
determine the extent, if any, of mineral precipitation onto the surface of the microorganism which directlyaffects their preservation potential in the rock record. Thus, we hope to identify the lipid composition ofthese both methanogens, which would aid in future identification of these species in the serpentine
geological rock record.

Figure 1. Cell wall structural differences between archaea (a-c), Gram-positive (d),and Gram-negative bacteria (e). The proteinaceous S-layer is often but not always
present in archaea and bacteria, whereas a peptidoglycan layer is always found in bacteria, either thick andexternal (Gram-positive) to the membrane or thin and sandwiched between two membranes (Gram-negative).

Some archaea also have a pseudopeptidoglycan layer between the membrane and the external S-layer (Sleytret al. 2014).

References
Bar-On, Yinon M., Rob Phillips, and Ron Milo. 2018. “The Biomass Distribution on Earth.” Proceedings of
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Fauque, Guy D., and Larry L. Barton. 2012. “Chapter 1 - Hemoproteins in Dissimilatory Sulfate- andSulfur-Reducing Prokaryotes.” In Advances in Microbial Physiology, edited by Robert K. Poole, 60:1–90.
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Joulian, C, B K Patel, B Ollivier, J L Garcia, and P A Roger. 2000. “Methanobacterium Oryzae Sp. Nov., a
Novel Methanogenic Rod Isolated from a Philippines Ricefield.” International Journal of Systematic andEvolutionary Microbiology 50 (2): 525–28. https://doi.org/10.1099/00207713-50-2-525.
Neubeck, Anna, Susanne Sjöberg, Alex Price, Nolwenn Callac, and Anna Schnürer. 2016. “Effect of Nickel
Levels on Hydrogen Partial Pressure and Methane Production in Methanogens.” PLOS ONE 11 (12):e0168357. https://doi.org/10.1371/journal.pone.0168357.
Jayne E. Rattray, Alexandra Zetterlind, Rienk H. Smittenberg, Christian Potiszil, Anna Neubeck,"Complexity of the serpentinization lipidome", Organic Geochemistry, Volume 174, 2022
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Anna Neubeck, Li Sun, Bettina Müller, Magnus Ivarsson, Hakan Hosgörmez, Dogacan Özcan, Curt Broman, Anna Schnürer, 2017, Microbial Community Structure in a Serpentine-Hosted Abiotic Gas Seepage
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