Contribution of Bicarbonate Assimilation to Carbon Pool Dynamics in the Deep Mediterranean Sea and Cultivation of Actively Nitrifying and CO2-Fixing Bathypelagic Prokaryotic Consortia

Abstract

Covering two-thirds of our planet, the global deep ocean plays a central role in supporting life on Earth. Among other processes, this biggest ecosystem buffers the rise of atmospheric CO₂. Despite carbon sequestration in the deep ocean has been known for a long time, microbial activity in the meso- and bathypelagic realm via the "assimilation of bicarbonate in the dark" (ABD) has only recently been described in more details. Based on recent findings, this process seems primarily the result of chemosynthetic and anaplerotic reactions driven by different groups of deep-sea prokaryoplankton. We quantified bicarbonate assimilation in relation to total prokaryotic abundance, prokaryotic heterotrophic production and respiration in the meso- and bathypelagic Mediterranean Sea. The measured ABD values, ranging from 133 to 370 μg C m^-3 d^-1, were among the highest ones reported worldwide for similar depths, likely due to the elevated temperature of the deep Mediterranean Sea (13-14°C also at abyssal depths). Integrated over the dark water column (≥200 m depth), bicarbonate assimilation in the deep-sea ranged from 396 to 873 mg C m^-2 d^-1. This quantity of produced de novo organic carbon amounts to about 85-424% of the phytoplankton primary production and covers up to 62% of deep-sea prokaryotic total carbon demand. Hence, the ABD process in the meso- and bathypelagic Mediterranean Sea might substantially contribute to the inorganic and organic pool and significantly sustain the deep-sea microbial food web. To elucidate the ABD key-players, we established three actively nitrifying and CO₂-fixing prokaryotic enrichments. Consortia were characterized by the co-occurrence of chemolithoautotrophic Thaumarchaeota and chemoheterotrophic proteobacteria. One of the enrichments, originated from Ionian bathypelagic waters (3,000 m depth) and supplemented with low concentrations of ammonia, was dominated by the Thaumarchaeota "low-ammonia-concentration" deep-sea ecotype, an enigmatic and ecologically important group of organisms, uncultured until this study.

Keywords: ammonium-oxidizing Thaumarchaeota; anaplerotic reactions; dark bicarbonate assimilation; deep-sea microbial community; mediterranean sea.

Figure 1

Location of the ST1-ST7 stations (A), assimilation of bicarbonate in the dark (ABD) rates (B) and relative contribution of ABD to the total carbon demand (C) measured in aphotic water column along the sampling stations in the Atlantic Ocean and Mediterranean Sea. The area of the Mediterranean deep-sea hypersaline anoxic lakes (DHAB) is indicated as red rectangle. Abbreviations used: ABD, assimilation of bicarbonate in the dark; PHP, prokaryotic heterotrophic production; PR, prokaryotic respiration.

Figure 2

Location of all currently known Mediterraneran DHAL and dark primary production (ABD) rates, measured in oxic/anoxic surficial layer of these deep-sea lakes.

Figure 3

Growth curves (triangles) and nitrite production (circles) of ATA (A), KRY (B), SAL5 (C) enrichments and their total and cell-specific bicarbonate assimilation rates, ABD and csABD, respectively (D). Error bars represent standard deviations of measurements from triplicate cultures.

Figure 4

Phylogenetic affiliation of the archaeal 16S rRNA (A) and putative 4-hydroxybutyryl-CoA dehydratases 4-HBD (B) gene sequences, retrieved from three stably nitrifying enrichments ATA, KRY and SAL5. The tree was constructed using MacVector 11.1.2 software by Neighbor-Joining method and Jukes–Cantor distance matrix. Non-parametric bootstrapping was performed upon 1,000 iterations to infer tree topology and the nodes with the percentage of bootstrap re-sampling above 70% are indicated. The 16S phylogenetic tree was rooted with Haloferax lucentense 16S ribosomal RNA gene (AH003665), while the 4-hbd tree was out-grouped with 3-hydroxybutyryl-CoA dehydrogenase of N. maritimus SCM1 (Nmar_1028, ABX12924). The frequencies of all phylotypes of 16S rRNA and 4-hbd genes, present in clone libraries, are evidenced at the end of each sequence as percentage of analyzed clones. The scale bar represents the number of fixed mutations per nucleotide position.

Figure 5

Neighbor-joining phylogenetic nucleotide tree of thaumarchaeotal amoA obtained from the three stably nitrifying enrichments ATA, KRY, and SAL5. The marine clades 1–2 of high ammonia concentration ammonium-oxidizing archaea (HAC-AOT) and the marine clades 3–6 of low ammonia concentration ammonium-oxidizing archaea (LAC-AOT), as defined by Sintes et al. (2016), are color-coded as aquamarine and yellow, respectively. Sequences of enrichments are also color-coded according to types of clone library origins, as blue (ATA), green (KRY), and red (SAL5). Significant bootstrap values are shown as open (>50) and red-filled (>70) circles at branch nodes.

Figure 6

Phylogenetic affiliation of the bacterial 16S rRNA gene sequences, retrieved from the stably nitrifying enrichments ATA, KRY, and SAL5. The tree was constructed by Neighbor-Joining method and Jukes–Cantor distance matrix using MacVector 11.1.2. Non-parametric bootstrapping was performed upon 1,000 iterations to infer tree topology and the nodes with the percentage of bootstrap re-sampling above 70% are indicated. The 16S phylogenetic tree was rooted with Sunxiuqinia elliptica 16S ribosomal RNA gene (GQ200196). The frequencies of all phylotypes of 16S rRNA, present in clone libraries, are evidenced at the end of each sequence as number of corresponding clones, respectively. The scale bar represents the number of fixed mutations per nucleotide position.