Organic matter (OM) in marine sediments is a major reservoir of organic carbon and nitrogen and plays an important role in the global biogeochemical cycles. Marine sediments also contain a vast amount of microbes. Interaction between sedimentary microbes and OM is an important process among various biogeochemical processes in marine sediments, because it has been suggested that sedimentary microbial ecosystems on most continental margins consist mainly of organic-fueled heterotrophs with relatively minor autotrophic components.

Amino acids are the building blocks of proteins and peptides and key compounds in microbial metabolisms. Amino acids represent one of major fractions of sedimentary OM and are important in undergoing OM mineralization in marine sediments. However, our understanding is still limited about the biogeochemical dynamics of amino acids in marine sediments, in part because currently available methods are not sufficient to constrain sources and transformation processes of amino acids in sediments (e.g., composition analysis of amino acids, enantiomer ratio analysis of amino acids, isotope-labeling experiments).

The research objectives of this thesis are (i) to apply compound-specific nitrogen isotope composition of amino acids (δ(15)N(AA)) at natural abundance as a new tool to estimate sources and transformation of amino acids in marine sediments and (ii) to constrain the biogeochemical dynamics (especially preservation and degradation mechanisms) of amino acids in marine sediments using the δ(15)N(AA) method (combination with other chemical approaches). Towards the first objective (i), I investigated factors for controlling the δ(15)N(AA) signatures of microbes on culture experiments, which is essential to interpret δ(15)N(AA) data of environmental samples (Chapter 2). Next, as the second objective (ii), I analyzed the δ(15)N(AA) values of marine sediment core samples collected in two ocean regions (the Japan Sea in Chapter 3 and the Sagami Trough in Chapter 4). Furthermore, I proposed comparison of δ(15)N of total hydrolysable amino acids (THAA) with δ(15)N of other fractions (chlorin pigment in Chapter 3 and dissolved amino acids in Chapter 4) as a new, effective approach to constrain the biogeochemical dynamics of amino acids.

Nitrogen-isotope fractionation during amino-acids biosynthesis and decomposition by chemotrophic microbes (Chapter 2): δ(15)N(AA) has been demonstrated as a promising tool for estimating the food sources of organisms in the grazing food web. Utility of δ(15)N(AA) analysis to microbial processes in biogeochemistry of detritus organic matter remains uncertain, because the method has been constructed based on the analytical results of aquatic photoautotrophs (cyanobacteria and eukaryotic algae), terrestrial higher plants, and animals (aquatic and terrestrial), but not chemotrophic microbes. I analyzed δ(15)N(AA) in five cultured heterotrophic or chemoautotrophic microbes namely a fungus (Saccaromyces cerevisiae), a bacterium (Escherichia coli) and archaea (Sulfolobus tokodaii, Halobacterium salinarum and Methanothermobacter thermautotrophicus) with controlling their nitrogen sources. When the microbes synthesized amino acids de novo, the relative nitrogen-isotopic fractionation patterns of amino acids relative to glutamic acid (εx/Glu) were generally similar to aquatic photoautotrophs and terrestrial plants, with some exceptions such as phenylalanine of terrestrial C3 plants. Especially, the εx/Glu values of the microbes (n=3) were very close to the aquatic photoautotrophs (n=37) for the most of amino acids such as alanine (0.2±2.2‰ and -0.1±2.2‰), isoleucine (-1.1±0.8‰ and -1.3±1.7‰), and phenylalanine (-3.0±1.0‰ and -2.7±2.2‰, respectively). In the case the microbes assimilated amino acids from the culture media, 15N-enrichment factors of microbes relative to the media amino acids (e.g., +8.2±0.8‰ for glutamic acid and +0.1±0.2‰ for phenylalanine, n=4) were close to animals (+8.0±1.1‰ and +0.4±0.4‰, respectively, n=11). These results suggest that similar processes control the nitrogen-isotope fractionation of amino acids in various organisms covering the three domains (Eukarya, Bacteria, and Archaea). Therefore, δ(15)N(AA) can be used as a powerful tool to clarify microbial metabolisms (de novo synthesis and decomposition of amino acids) and their biogeochemical roles in various environments including marine sediments.

Preservation mechanism of amino acids in marine sediments of the Japan Sea (Chapter 3): Degradation of amino acids does not achieve completely in surface sediments, although proteins and amino acids are considered to be biologically labile compounds. Several mechanisms have been discussed to explain the persistence of amino acids in subsurface marine sediments. Minerals or organic macromolecules may protect amino acids from microbial decay processes. It has been alsosuggested that in situ production by sedimentary microbes would be an important mechanism for persistence of amino acids. Here, as a new method to estimate microbial alteration of amino acids in marine sediments, I analyzed compound-specific δ(15)N of THAA in marine sediments of the Japan Sea (a surface sediment and a 7-m-long piston core; ca. 46,500 years). The down-core δ(15)N profiles of THAA (δ(15)N(THAA)) were compared with a down-core δ(15)N profile of chlorin (δ(15)N(Chl)), which reflects the δ(15)N values of organic matter produced by photosynthetic organisms in the past ocean. Significant correlations were observed between the δ(15)N(THAA) and the δ(15)N(Chl) in the piston core samples (r2 = 0.87 for phenylalanine (Phe), 0.78 for glutamic acid, 0.77 for alanine, and 0.62 for glycine; n = 13). This result suggests that the major source of THAA is organic matter produced by the organisms in the past ocean (i.e., necromass) and that contribution of in situ sedimentary microbial production to THAA is less than 15% below 1 m depth in the core. The offset values between δ(15)N(THAA) and δ(15)N(Chl) in the sediments of 1-7 mbsf (e.g., Δδ(15)N(Phe-Chl) =+7.3±1.0‰) suggest that the source organisms of THAA contain not only photosynthetic algae and animals but also heterotrophic or chemoautotrophic microbes in the past ocean (water column and surface sediments). These conclusions support the hypothesis that protection of amino acids from microbial decay processes is the important mechanism to explain persistence of amino acids in subsurface marine sediments. In addition, the significant correlations between δ(15)N(THAA) and δ(15)N(Chl) suggest a potential of δ(15)N(THAA) (especially phenylalanine) as a new paleoceanographic proxy of the nitrogen cycle in the past ocean.

Degradation mechanism of amino acids in deep-subsurface marine sediments of the Sagami Trough (Chapter 4): Although amino acids in sediment pore waters are key compounds in metabolic activities of sedimentary microbes and in mineralization of carbon and nitrogen, to date little is known about their biogeochemical dynamics (e.g., sources and transformation processes) in deep-subsurface sediments. As a new approach to constrain the sources of dissolved amino acids in sediment pore waters, I analyzed and compared compound-specific δ(15)N and enantiomer ratio (%D) of THAA in sediment solid phase and dissolved hydrolysable amino acids (DHAA) in sediment pore waters from the same sediment samples. I also conducted enantiomer-specific δ(15)N analysis of alanine for THAA. Samples were collected from deep-subsurface sediments (up to 172.9 m below seafloor) at the Sagami Trough (NW Pacific). Compared to THAA in the same depth, DHAA show higher %D values in alanine, lower %D values in serine, similar %D values in valine, aspartic acid, glutamic acid, and phenylalanine (ΔDDHAA-THAA = +15.0+2.0%, -7.2+6.7%, +2.8+6.1%, +0.2+2.6%, +3.4+4.0%, and -0.7+3.0%, respectively). In the sediments deeper than 9 mbsf, %D values of DHAA were 25.9+2.8% in alanine, 24.8+2.1% in aspartic acid, 11.3+2.8% in serine, and 16.3±2.7% in glutamic acid, and %D changes from THAA (Δ%DDHAA_THAA) were +15.3±2.1% in alanine, -0.4+2.4% in aspartic acid, -8.1±6.2% in serine, and 4.6±3.3% in glutamic acid. Compound-specific δ(15)N(AA) analysis showed that δ(15)N values of alanine are higher in the DHAA than the THAA and that δ(15)N values of glycine and glutamic acid are similar between the two fractions (Δδ(15)NDHAA_THAA = +5.8±2.3‰, +1.9±0.6‰, -0.3±1.1‰, respectively). Differences of δ(15)N between D-alanine and L-alanine in the THAA fractions are significant only at two depths. These results suggest that the DHAA fractions have different δ(15)N(AA) and %D signatures from the THAA fractions, and that hydrolysis of the THAA is not the sole source of the DHAA. Alternatively, the δ(15)N(AA) and %D signatures of DHAA are consistent with the idea that in situ release of proteinaceous materials from sedimentary microbial biomass (such as peptidoglycan of Gram-positive bacteria) is an important source of DHAA. This suggests that recycle of dissolved amino acids by microbes would be an important process during amino-acids degradation in the deep-subsurface sediments.

本論文は、海洋堆積物中におけるアミノ酸の生物地球化学的動態の解明を目的として行われた研究である。全5章から構成される。

第1章はイントロダクションであり、海洋堆積物中におけるアミノ酸の生物地球化学的動態を研究する背景・意義が述べられている。海洋堆積物における有機物埋没プロセスは、地球表層環境の炭素・窒素・酸素などの物質循環に重要な役割を果たしていると考えられている。しかし海洋堆積物中における有機物と微生物の相互作用にはブラックボックスな部分が多く、有機物埋没フラックスの制御要因についても不明点が多い。海洋堆積物中有機物の中でも、アミノ酸は特に重要な画分を占め、有機物分解の際にも重要な中間産物になっていると考えられている。本論文の趣旨は、アミノ酸の化合物レベル窒素同位体組成(δ(15)N)およびD/L比を、アミノ酸の起源(生産者)を推定する手法として新たに提唱もしくは発展させ、海洋堆積物に初めて応用することで、海洋堆積物中アミノ酸の生物地球化学的動態(特に残存メカニズムと分解メカニズム)を制約することである。

第2章では、アミノ酸δ(15)Nを生物地球化学的手法として発展させるための微生物培養実験の研究について述べられている。3ドメイン(真核生物、バクテリア、アーキア)にまたがる5種類の微生物について、窒素源をコントロールした培養実験が行われ、アミノ酸δ(15)Nの変動パターンが調べられた。さらに、先行研究により報告されている藻類・陸上植物・動物のアミノ酸δ(15)Nデリタとの比較が行われた。その結果、(1)微生物がアミノ酸を新たに生合成した時は、グルタミン酸に対するアミノ酸δ(15)N相対パターンは特に藻類と一致する、(2)微生物が培地からアミノ酸を取り込んで無機化した時には、餌のアミノ酸δ(15)Nの上昇値は動物と一致する、ということが明らかになった。これらの結果は、アミノ酸の窒素同位体分別は、3ドメインにまたがる様々な生物について同様のプロセスが制御していることを示唆し、海洋堆積物など環境中のアミノ酸δ(15)Nデータを解釈する際に有用な情報である。

第3章では、アミノ酸δ(15)Nと光合成色素δ(15)Nの比較による、アミノ酸残存メカニズム制約の研究について述べられている。「全加水分解性アミノ酸δ(15)Nと光合成色素δ(15)Nの比較」から微生物によるアミノ酸の生産・二次的不可を推定するという新たなアプローチが提唱され、日本海秋田県沖で採取された海洋堆積物コア試料に初めて応用が行われた。その結果、コアの1m以深では、アミノ酸と光合成色素のδ(15)N深度プロファイルは高い相関を示し、堆積物中では過去の海洋の生物の死骸がアミノ酸として残存しており、堆積物中微生物による生産の寄与は15%未満であることが示唆された。特に、アミノ酸と光合成色素のδ(15)Nの差からは、過去の海洋の従属栄養もしくは化学合成独立栄養微生物の死骸が重要であることが示唆された。また、アミノ酸δ(15)Nを古海洋窒素循環の新たなプロキシとして使える可能性が示唆された。

第4章では、固相アミノ酸と溶存態アミノ酸のδ(15)N・D/L比の比較による、アミノ酸分解メカニズム制約の研究について述べられている。「固相アミノ酸と間隙水溶存態アミノ酸についてδ(15)NおよびD/L比の比較」から溶存態アミノ酸に対する堆積物中微生物からの放出の寄与を推定するという新たなアプローチが提唱され、北西太平洋相模トラフで採取された海洋堆積物コア試料に初めて応用が行われた。固相アミノ酸と溶存態アミノ酸との間で、δ(15)NおよびD/L比の両方で有意な差が見られ、溶存態アミノ酸の起源は固相からの加水分解だけでなく、堆積物中微生物からのタンパク性分子(特にグラム陽性バクテリアのペプチドグリカン)の放出が重要であることが示唆された。このことから、アミノ酸分解に際して、堆積物中微生物によるリサイクルが重要なプロセスであることが示唆された。

第5章では、本論文のまとめと今後の展望が記述されている。本研究では、海洋堆積物中アミノ酸の残存メカニズムについては過去の海洋の微生物の死骸の残存が重要なプロセスで、分解メカニズムについては堆積物中微生物によるリサイクルが重要なプロセスであることが示唆された。本研究で新たに提唱された研究アプローチは、他の環境の海洋堆積物や海水などの試料にも応用可能であり、海洋堆積物中の有機物動態について今後より詳細な理解が進んでいくことが期待できる。

なお研究に関しては、横山祐典准教授(東京大学大気海洋研究所)、大河内直彦博士、力石嘉人博士、高野淑識博士、小川(大河内)奈々子博士、菅寿美博士(海洋研究開発機構)との共同研究の成果である。しかし、論文提出者が主に分析、解析及び解釈を行なったもので、論文提出者の論文への貢献は本質的な部分で特に高く、寄与は十分であると審査委員全員が判断した。

以上の理由より。審査委員会は本論文を提出した山口保彦氏に博士(理学)の学位を授与できると認めた。