Unique deep-sea bacterial assemblages thriving on different natural organic matters delivered via in situ incubators

doi:10.21203/rs.3.rs-1700182/v3

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Unique deep-sea bacterial assemblages thriving on different natural organic matters delivered via in situ incubators

https://doi.org/10.21203/rs.3.rs-1700182/v3

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The transport of organic matters to the deep sea constantly occurs in global oceans and in accompany with simultaneous microbial remineralization. However, little is known about the impact of fast sinking organic complex on oceanic deep ecosystem and its interactions with microbes throughout the process of sinking and settling on the sea bottom. In this report, to observe the response of indigenous microorganisms to the newly input of organic matters, we developed a series of deep-sea in situ incubators loaded on the seabed and seamount with various organic substrates of plant and animal origins. Through high-throughput sequencing, we found the bacterial communities, in situ enriched for 4–12 months, were significant different from the control groups. The bacteria of Marinifilaceae were revealed as the key members; in addition, other key decomposers including Spirochaetaceae, Psychromonadaceae, Vibrionaceae, and Moritellaceae were recruited in most assemblages, with varied abundance accordingly and diversified at the operational taxonomic unit (OTU) level. Additionally, sulphate-reducing bacteria (Desulfobulbaceae and Desulfobacteraceae) and sulphur-oxidizing bacteria (Arcobacteraceae and Sulfurovaceae) were the dominant taxa. Interestingly, PICRUSt analysis demonstrated that nitrogen fixation inside the assemblages was enriched in the enrichments with plant detritus or fatty acids. Within the assemblages stimulated by substrates short of nitrogen sources, the microbial network was dominated by cooperative relationships, whereas competition relationships overwhelmed the communities thriving on protein-rich animal tissue. These unique bacterial assemblages driven by newly input organic matters, constitute the microbial carbon pump involved in carbon, sulphur, and nitrogen cycles in oceanic interior.

General Microbiology

Marine and Freshwater Ecology

Deep sea

In situ incubator

Natural organic matter

Bacterial assemblage

Biogeochemical cycles

Annually, a large amount of organic matter (OM) sinks to the deep sea bottom, among which 10-30 % is of terrestrial origin [1]. Both the terrestrially originated OM and those continuously generated from the oceanic euphotic zone will eventually sink to the deep sea. A single storm event can transport up to 1.8-4.0 teragrams of terrestrially derived OM into the ocean, as previously estimated by Bianchi et al. [2]. The zooplankton and phytoplankton remain, as well as a considerable part of the terrigenous OM originating from animal and plant tissues, are chemically characterized by high-weight proteins or polypeptides, fatty acids, polysaccharides (chitin, cellulose, and hemicellulose), and lignin content [2], respectively. The input of large amounts of OMs, such as plant fragments and animal carcasses, to the sea bottom contribute organic impulse to the deep-sea ecosystem, such as the well-known ecosystems of wood-fall and whale-fall [3-6].

Sinking particulate organic matter (POM), which functions as a conduit for exporting organic materials from the euphotic zone to the deep ocean [7], critically contributes to the establishment of habitats for deep-sea benthic microbial communities [8]. Through the water column, the microbial assemblages associated with sinking particulates in the upper ocean layer, such as photoautotrophs of Cyanobacteria, can be quickly delivered to the abyss, while deep-sea bacteria respond rapidly to the elevated nutrient delivery of sinking POM [9, 10]. The sinking particulates that transit to the deep sea may contain energy-rich organic compounds or relatively energy-replete POM with fatty acids selectively preserved [7, 11]. High OM influx is usually accompanied by high sedimentation rates to the deep sea [12, 13]. The large and fast sinking particles may escape disaggregation, dissolution, solubilization, and even microbial decomposition en route to the deep sea [9], which determines the organic carbon and energy flux pattern of the deep sea and impacts the microbial communities in the deep sea [14, 15]. Considering the vast volume of the deep sea, processes of POM transformation and remineralization via microorganisms in benthic ecosystems are crucial to the global carbon cycle [16]. However, we only have scratches of pieces of information about the microbial processes on fast-sinking particles, let along the dynamic process in both microbial assemblage and organic transformation. Knowledge about the microbial decomposition process in the deep ocean is scarce, which hampers our ability to evaluate the fate and impacts of POM in pelagic waters.

With conductivity, temperature, and depth (CTD) sampling and high-throughput sequencing, quite diverse bacterial heterotrophs have been described in both deep-sea water and sediments; for example, in bathypelagic water, the Gammaproteobacteria Alteromonas, Pseudoalteromomas, Alcanivorax, Halomonas, and Shewanella are relatively abundant [7, 15, 17, 18]. Moreover, in the water column of pelagic oceans, the majority of prokaryotes remain uncultivated, such as SAR324, SAR406, and SAR202 [18-21]. We are unsure about their growth substrates in situ, even for those cultivated in the laboratory.

Moreover, the bacteria observed in the deep water column sampled with CTD cassettes, filter fractionation techniques, and sediment traps [7] may be different from the bacteria thriving on nutrients during the “hot time” of bulky POM sinking, such as the terrestrial debris transported via hurricanes and water currents or the dropping of marine animal corpses, because large pieces rich in OM do not constantly sink through the water column and settle to the seabed at every location. What we “see” via CTD may be just part of the story; a similar scenario is true for deep-sea sediments. Whether prevalent marine bacteria will be recruited to the community and the identity of the key taxon remineralizing the fresh POM in benthic environments are questions to answer as a prerequisite for discovering the fate of POM that sinks to the seabed. However, the big challenge is to catch up with the sporadic and instantaneous processes in the remote deep ocean. Measuring the responses of the benthic microbiome to the sudden input of OM requires in situ observations during the “hot time” of POM sinking.

We hypothesized that the key bacterial taxa that contribute to the decomposition of newly input POM in the deep sea would be different from the regular predominant bacteria mentioned above. To define the key microbial taxa potentially participating in the transformation and degradation of POM sunk to the deep sea in situ, in this study, we applied long-term in situ enrichments supplemented with natural plant and animal materials rich in polysaccharides, proteins, or fatty acids using our newly developed deep-sea in situ microbial incubator (DIMI), landed at different geographic locations (marginal sea and open sea). The POM input-spiked bacterial assemblages were subjected to bacterial diversity to identify the in situ keystone taxa during the OM remineralization. The results will provide knowledge about the microbial interactions with sinking POM and contribute to our understanding of the roles of the microbial carbon pump in elemental biogeochemical cycles in global oceans.

2.1 Experimental design and in situ incubation experiments

From 2016 to 2018, the in situ incubation experiments were separately carried out on the seafloor in the South China Sea (SCS, the largest marginal sea of the West Pacific Ocean; 18.4925000° N, 116.2717833° E), the deep-sea basin beside the Southwest Indian Ridge in the Indian Ocean (IO, open sea; 33.2789333° S, 50.7510167° E), and a flat-topped seamount in the West Pacific Ocean (PO, open sea; 20.4059567° N, 160.7700883° E) at 3758 m, 4434 m, and 1622 m water depths, respectively (Fig. S1 and Fig. S2a).

In this study, natural plant and animal origin materials were selected for in situ incubation (Table S1). The animal tissue substrates included fish muscle, fish scales, and shrimp muscle; the plant detritus included wood chips, wheat bran, wheat straw, and various seaweeds; in addition, fish oil (EPA and DHA) and vegetable oil as fatty acid were used here. Approximately 10 g of solid substrate was directly placed into a 50 ml tube. For the liquid fish oil and vegetable oil, 5 ml of oil was first absorbed into sintered silicate balls approximately two centimeters in diameter, and three balls were put into a 50 ml tube. All of the assembled tubes were wrapped with 75-μm nylon mesh to prevent potential ingestion by benthic fauna. Parallel tubes with the substrates were sterilized at 115 °C for 30 minutes and then placed into sterilized incubation chambers (ICs) that were made of titanium alloy. ICs filled with OM substrates were designated treatment incubation chambers (TICs). Meanwhile, two cleaned and sterilized empty ICs without any substrates (designated CICs, control incubation chambers) were reserved for collecting in situ seawater during each incubation. Finally, all the TICs and CICs were mounted on a DIMI device (Fig. S2b), which is a deep-sea microorganism in situ incubation system that can be landed on the seafloor at abyssal depths (6000 m) to conduct microbe incubation over the sediment for over 12 months. The details on the deployment and recovery of the DIMI is described in the Supplementary Materials. The temperature and salinity of the surrounding seawater and the depths were recorded using a SBE 37-SM microCAT CTD recorder (Sea-Bird Scientific, Bellevue, WA, USA) mounted on the DIMI during the in situ enrichment.

2.2 Sampling and DNA extraction

After recovery from the deep sea, the enriched biomass in the liquid phase was immediately filtered with a 0.22-μm pore size polycarbonate membrane (Millipore, USA) and stored at -80 °C. The remaining solid substrates were resampled in a sterile plastic bag and stored at -80 °C until subsequent processing. For the controls, two 0.2-μm-pore size polycarbonate membranes were used to harvest the in situ microbial cells from the CICs with no substrates. The solid substrate, as well as the silicate ball, was ground to powder in liquid nitrogen immersion for DNA extraction. Total DNA was extracted and purified with a DNeasy PowerWater Kit (Qiagen, Germany) according to the manufacturer’s protocol. DNA concentrations were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).

2.3 Illumina high-throughput sequencing and analysis

The V3-V4 hypervariable region of the bacterial 16S rRNA gene (~454 bp) was amplified from the enrichment and control samples using the primer pair 338F (5'- ACTCCTACGGGAGGCAGCAG-3') and 806R (5'- GGACTACHVGGGTWTCTAAT-3'). The 20 μl reaction solution comprised 4 μl of 5× PCR buffer, 2 μl of each 2.5 mM dNTP, 0.8 μl of each 5 μM primer, 2.5 U of DNA polymerase (rTaq; TaKaRa), and 10 ng of total DNA. Preliminary amplification of some randomly selected samples was conducted to determine the optimal PCR conditions, and the results were as follows: 3 min at 95 °C; 29 cycles of 30 s at 95 °C, 30 s at 53 °C and 45 s at 72 °C; 10 min at 72 °C; and infinite hold at 4 °C. No amplification products were observed for the negative PCR controls. All barcoded amplicons were pooled in equimolar amounts and subjected to paired-end (PE) sequencing using a PE300 strategy on an Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw dataset is available in NODE (https://www.biosino.org/node/) at the accession number OEX011322.

Further analysis for raw data was conducted with the QIIME standard pipeline (version 1.9.1) [22]. In brief, PE reads were merged, yielding ~454 bp fragments, using FLASH software version 1.2.11 [23]. The dataset was then quality filtered to remove ambiguous reads and chimeric sequences using UCHIME version 4.2 [24]. Optimized sequences were clustered into operational taxonomic units (OTUs) using UPARSE version 7 with a 97 % sequence similarity threshold [25]. Taxonomic classification of each representative OTU was performed using the Ribosomal Database Project (RDP) classifier against the SILVA 132 database [26]. Random subsampling of sequences was conducted with an equal depth of 25,904 sequences for the subsequent comparative analysis.

The Chao community richness estimator, as well as the Shannon diversity index, was computed using the QIIME pipeline. Bray–Curtis distance- and UniFrac distance-based β-diversity at the OTU level was calculated using the R 'vegan' package and QIIME, and principal coordinate analysis (PCoA) plots were drawn by the 'ggplot2' package in R. Adonis analysis was carried out to show the important determinants for the variation in microbial community structure based on the Bray–Curtis distance algorithm for 999 permutations through the ‘vegan’ package. A heatmap was constructed to show the community composition at the family level using R version 3.3.2 with the 'vegan' package. The relationships between abundant genera and samples were displayed using CIRCOS software version 0.67-7 [27].

2.4 Phylogenetic analysis

Multiple sequence alignment of 16S rRNA genes was performed using the MAFFT program with default parameters, and a phylogenetic tree was reconstructed with the neighbor-joining method [28]. The reference sequences were retrieved from NCBI.

2.5 Function prediction of each enrichment

Prokaryotic function prediction of each enrichment was performed using PICRUSt based on the KEGG database [29] and using FAPROTAX based on the metabolic functional group database of marine microorganisms [30] with the rarefied OTU tables as inputs. Adonis analysis was carried out to show the contribution of each factor to microbial functional diversity based on the Bray–Curtis distance algorithm for 999 permutations through the ‘vegan’ package.

2.6 Network analysis

Network analyses were carried out using the molecular ecological network analyses (MENA) pipeline at the OTU level [31]. Network complexity was assessed by calculating the number of edges, average degree, and average clustering coefficient. Networks were constructed and visualized in Cytoscape version 3.7.1 [32].

3.1 Deep-sea in situ incubation of OM-decomposing bacteria

After incubation at SCS, IO, and PO for 375, 117, and 348 days, respectively, all incubation tubes, even those with wood chips, exhibited obvious microbial growth indicated by turbidity and color variation (Fig. S3); many organic substrates remained in each centrifuge tube, even for fish oil and vegetable oil we could see oily residue on the surface of sintered silicate balls; most samples smelled of hydrogen sulphide, and under a microscope, the field of view appeared with bacterial cells (Table S1 and Fig. S3). The in situ temperature and salinity of seawater at those sites were approximately 2.39-2.40 °C and 34.62-34.65 ‰, respectively (Table S1).

3.2 Bacterial diversity and its environmental determinants of the OM-decomposing microbial assemblages

The α-diversity (Shannon and Chao indexes) analysis showed that the bacterial diversity of the enrichments was significantly lower than that of the in situ seawater in the CICs (Fig. 1a and 1b, P < 0.01), which indicated that bacterial communities were significantly enriched in situ by the POM supplements.

The β-diversity analysis revealed that the enriched bacterial community structures were significantly different from those of the seawater in CICs and were quite diverse at the three sites, separated by both organic substrate and geographic location (Fig. 1c). Additionally, adonis analyses suggested that the enriched bacterial community structures at OTU level might be influenced by multiple factors, including geographic location, the type of OM, the enrichment time, temperature, water depth, and salinity, among which geographic location (R² = 0.20, P = 0.001), the type of OM (R² = 0.13, P = 0.001), were the two most important determinants of the variation in microbial community structures (Table S2).

3.3 Bacterial composition of deep-sea in situ enrichments with natural organic materials

A total of 2457 OTUs were retrieved from the incubation samples (Table S3) and 1461, 961, and 651 OTUs were obtained from IO, SCS, and PO enrichment sites, respectively (Fig. 2a), however, only 144 OTUs were shared among the three sites (Fig. 2b). The dominant bacteria were affiliated with the classes Bacteroidia (average abundance of 28.4 %), Gammaproteobacteria (20.4 %), Deltaproteobacteria (13.5 %), Spirochaetia (12.0 %), Campylobacteria (8.4 %), Clostridia (7.2 %) and Fusobacteriia (6.1 %) across the deep-sea in situ enrichments (Fig. 2c). Although Gammaproteobacteria (25.4 %) and Deltaproteobacteria (8.8 %) also thrived in the communities of the in situ seawater in the CICs, however, unlike the enriched communities, Alphaprotebacteria (14.1 %), Dehalococcoidia (11.1 %), Actinobacteria (3.7 %), Gemmatimonadetes (4.1 %), and Phycisphaerae (3.1 %) were additionally detected in large numbers. These results indicated that the newly input POMs had a significant impact on the indigenous microorganisms in the deep sea.

At the family level, the dominant bacterial groups were shared across these deep-sea enrichments even though the sites were far away on the oceanic scale (Fig. 3 and Fig. S4). The bacteria were affiliated with the families Marinifilaceae, Spirochaetaceae, Psychromonadaceae, Vibrionaceae, Moritellaceae, Desulfobulbaceae, Desulfobacteraceae, Family_XII_Clostridiales, Fusobacteriaceae, Arcobacteraceae, Sulfurospirillaceae, and Sulfurovaceae (Fig. 3 and Fig. S4). Most of these bacterial members were anaerobes or facultative anaerobes, like Marinifilaceae [33], Spirochaetaceae [34], Psychromonadaceae [35], Fusobacteriaceae [36], sulphur-oxidizing bacteria (SOBs) of Arcobacteraceae and Sulfurovaceae [37-39], sulphur-reducing bacteria of Sulfurospirillaceae [40], and sulphate-reducing bacteria (SRBs) of Desulfobulbaceae and Desulfobacteraceae [41, 42]. In the in situ seawater collected by CICs, the putative anaerobic bacteria Woeseiaceae/JTB255 and SAR202 were recovered, which have been recognized as cosmopolitan and abundant core members of deep-sea surface sediments [43] and benthic water [44-46], respectively. However, they were not detected in the enrichments with newly input OM (Fig. 3). Thus, unique deep-sea bacterial assemblages were stimulated by the newly input POM, which were fast-growing and more competitive in OM-rich habitats than in oligotrophic ones.

3.4 Keystone bacteria showing a preference for organic substrates and geographic locations at the OTU level

To discriminate the keystone species enriched by different types of POM, we analyzed the distribution of OTUs of each dominant family. Bacteria of the family Marinifilaceae were the most predominant, thriving in all POM enrichments and showing the highest abundance among all bacterial communities (Fig. S4). Interestingly, OTU6861 was mainly dominant in the consortia enriched with plant detritus, such as wood detritus, seaweeds, and wheat bran in the SCS and PO sites, while both OTU4252 and OTU5909 dominated the consortia of substrates-rich in animal tissue (Fig. 4). Moreover, OTU5914 not only occurred as the most predominant member in animal tissue enrichments at the SCS site but also among all the enrichments of different POM types at the PO site (Fig. 4). Therefore, these OTUs exhibited discrepant preferences for plant detritus or animal tissue.

Spirochaetaceae was the second predominant group in the enrichments (Fig. S4). OTU5914 occurred in most plant detritus and animal tissue enrichments at both PO and SCS sites (Fig. 4), particularly with relative abundances of 90.0 % and 52.0 % in the wheat bran enrichment at the PO site and shrimp muscle enrichment at the SCS site, respectively. However, little was detected in the enrichments at the IO site. These results indicated that Spirochaetaceae perhaps showed a preference for geographic locations.

In addition, the heterotrophic bacteria Moritellaceae, Psychromonadaceae [35], and Vibrionaceae [3, 47, 48] of Gammaproteobacteria are also the predominant member and varied with substrate and location at the OTU level (Fig. 4), which are described in detail in the text of the Supplementary Information.

Collectively, the keystone species for each predominant group were divergent at the OTU level in response to different POMs and geographic locations, which were also confirmed by adonis analyses (Table S2). Meanwhile, this result implied that these predominant groups were characterized by functional diversity, that is, some species may mainly degrade polysaccharides and/or lignin, and some may mainly degrade proteins and/or fatty acids, or both.

3.5 Function prediction of the deep-sea bacterial communities

Adonis analyses based on the PICRUSt function prediction suggested that the type of POM contributed more to microbial functional diversity (R² = 0.185, P = 0.01) than other factors (Table S4). Moreover, enrichments with plant detritus or animal tissue tended to cluster together, while fatty acid enrichments were scattered among all the enrichments (Fig. S5). Furthermore, the enrichment patterns of key metabolic pathways of the bacterial communities varied among OM substrates. For example, several key modules for carbohydrate metabolism were obviously enriched in plant detritus enrichment, such as glycolysis (KEGG modules: M00001 and M00002), pentose phosphate pathway (M00007), pectin degradation (M00081), D-galacturonate degradation (M00631), and galactose degradation (M00632) (Table S5). Intriguingly, the module for nitrogen fixation was particularly enriched in the nitrogen-deficient enrichments of plant detritus and fatty acid enrichments. The relative abundance of the nitrogen fixation module (M00175) in plant detritus enrichments was 2.7 times higher than that in protein enrichments (Table S5). In fatty acid enrichments, the modules thought to be responsible for fatty acid oxidation were significantly enriched (Table S5), such as beta-oxidation (M00087), acyl-CoA synthesis (M00086), the ethylmalonyl-CoA pathway (M00373 and M00774), and acylglycerol degradation (M00098).

In addition, the FAPROTAX predictions revealed that the top ten functional groups were chemoheterotrophy, fermentation, respiration of sulphur compounds, sulphate respiration, hydrogen oxidation, sulphur respiration, nitrate reduction, thiosulfate respiration, oxidation of sulphur compounds, and iron respiration. These results indicated that the bacterial decomposition of newly input POM during the “hot time” in the deep sea was an anaerobic process achieved via fermentation and diverse forms of anaerobic respiration, and these processes were perhaps tightly coupled with the cycles of nitrogen, sulphur, and hydrogen (Fig. S6).

3.6 Microbial co-occurrence networks within the bacterial assemblages of different POMs

To further understand the co-occurrence relationships among bacteria during the process of OM degradation, we constructed three co-occurrence networks for plant detritus-, animal tissue-, and fatty acid-enriched communities, respectively (Fig. 5). Each network diagram consisted of 58 to 75 OTUs, mainly belonging to the dominant families mentioned above (Fig. 5 and Table S6).

The co-occurrence relationships of the bacterial OTUs were closely related to the enrichment substrates. The relationships among bacteria within the communities of animal tissue were negative in 90.7 % of cases (Fig. 5a and Table S7). However, cooperative relationships among different bacteria taxa were observed in the plant detritus and fatty acid incubations (Fig. 5b, Fig. 5c, and Table S7). These two kinds of distinct relationships could be explained as follows: nutrients in animal tissue are replete and full of not only nitrogen, carbon, and phosphate sources but also other nutrients, such as vitamins. In contrast, many kinds of nutrients are quite limited in fatty acids and plant detritus. Therefore, bacteria in animal detritus enrichments showed competitive relationships and were more independent from each other. However, the fatty acid- and plant detritus-enriched consortia displayed more cooperative relationships to maintain nutrient balance and sustain the whole community. The details of the co-occurrence of the keystone species are described in the Supplementary Materials.

Organic matter decomposition and mineralization in global oceans is a key process in global carbon cycling [16]. Characterizing the microorganisms involved in these processes in the deep sea is important for understanding how element cycling works in this vast ecosystem. Previous studies have used serial filtration and sediment trapping techniques to characterize suspended and sinking particle-associated microbes [7, 18]. Filtration methods can be biased by the volume of water filtered [49] and undersample fast-sinking particles. In this report, in situ incubations were conducted to observe bacteria involved in the transformation and mineralization of OM within the sinking “compact” biomasses in the deep sea of open areas (the Pacific Ocean and the Indian Ocean) and a marginal sea (the South China Sea). Our results showed that the organic pulse with different POMs enriched unique bacterial communities different from those in deep-sea water, or deep-sea sediment [50]. The in situ degradation and further mineralization of POM were mainly carried out by anaerobic processes via a special bacterial assemblage, though our incubation chambers were open to deep seawater. High-molecular-weight OM, usually in the form of insoluble particles, tends to go through anaerobic processes even in oxic seawater, as reported previously [51].

4.1 The bacterial assemblages in enriched communities are unique

Most enriched microbial assemblages were composed of bacteria belonging to the classes of Bacteroidia, Gammaproteobacteria, Deltaproteobacteria, Spirochaetia, Campylobacteria, Clostridia, and Fusobacteriia, including families of Marinifilaceae, Spirochaetaceae, Psychromonadaceae, Vibrionaceae, Moritellaceae, Desulfobulbaceae, Desulfobacteraceae, Family_XII_Clostridiales, Fusobacteriaceae, Arcobacteraceae, Sulfurospirillaceae, and Sulfurovaceae (Fig. 1c and Table S2). However, they were only present as a minority in the surrounding seawater of empty chambers (Fig. 3). Routinely, these bacteria should exist as a rare species of famine waiting for the feast of sinking bulky POM on the cold barren seabed, as suggested previously by Jorgensen [8].

The above bacteria propagating on newly input POM are different from bathypelagic bacteria in the global ocean water column and deep-sea sediments [18, 50]. In the oceans, Gammaproteobacteria are the most prevalent among deep-sea pelagic prokaryotes, including the genera Alteromonas, Halomonas, Pseudoalteromonas, Psychrobacter, and so on [7, 15, 17, 18], and in deep-sea sediments, the phyla Chloroflexi, Planctomycetes, and Actinobacteria are usually predominant [50, 52]. However, they are not the components thriving on the newly input POM, suggesting the remolding of bacterial assemblages. Although uncultivated SAR324, SAR406, and SAR202 are prevalent in deep-sea pelagic oceans [18], they are probably not involved in the remineralization of the macromolecular OM, e.g., polysaccharides and proteins, as only SAR202 occurred in a few enrichments with very low abundance. Similarly, Woeseiaceae/JTB255, the prevalent uncultivated bacteria in surface sediment as well as those present in the bottom water, were also missing in all enrichments (Fig. 3 and Table S3).

Intriguingly, in contrast to the bacterial diversity sampled via the CTD water sampler mentioned above [18], our observation was partially consistent with those associated with sinking particles sampled by sediment traps in the oceanic interior described below. In abyssal sinking POM, DeLong and colleagues found that Arcobacteraceae (Campylobacterales) was the most predominant bacterial group [7], which also was the predominant member in all our enrichments (Fig. 3); additionally, the genera Colwellia, Moritella, and Shewanella of Gammaproteobacteria constituted the second predominant group in their report [7], which are frequently reported as psychrophilic and piezophilic deep-sea bacteria. Congruently, those Gammaproteobacteria taxa were present among the top 50 predominant members in almost all our enrichments. In particular, Moritella was even among the top 5 ones in some enrichments (Table S3).

Even in eutrophic coastal sediment, our parallel in situ incubation also revealed some predominant taxa shared with deep-sea enriched assemblages, e.g., Desulfobulbaceae, Desulfobacteraceae, Arcobacteraceae, Vibrionaceae, Spirochaetaceae, and Fusobacteriaeae [53]. However, several special groups, including Marinilifaceae, Moritellacea, and Psychromonadaceae, that dominated the deep-sea enriched consortia were barely detected in our coastal enrichments [53]. Moreover, in deep-sea chemoorganotrophic ecosystems of wood and whale falls, the taxa of Spirochaetaceae, Marinilifaceae, Vibrionaceae, and Fusobacteriaeae also occur in bacterial assemblages with high abundance [3]. In contrast, the bacteria Desulfobulbaceae, Psychromonadaceae, Moritellaceae, Arcobacteraceae, Sulfurospirillaceae, and Sulfurovaceae were rarely enriched in the wood-falls sunk in the Mediterranean Sea compared to our deep-sea incubations [47].

4.2 Bacterial assemblages may form a unique ecological niche

Some of the keystone taxa enriched in situ in the deep sea were quite diverse in phylogeny from those in other conventional habitats but were closely related to those from wood- and/or whale-fall ecosystems. For example, Spirochaetaceae are typical intestinal anaerobic bacteria [34], most OTUs of which in this study were obviously separated from those known to be from the animal intestine in the phylogenetic tree (Fig. S7). However, they formed several taxonomic branches with those from wood-fall ecosystems, and our coastal enrichments (Fig. S7). Therefore, members of Spirochaetaceae displayed obvious ecotypic differentiation.

A total of 29 OTUs of Desulfobulbaceae were found in the in situ enrichments. In the phylogenetic tree, they neighboured those from the chemosynthetic wood-fall or whale-fall ecosystems but separated from those from hydrothermal vents (Fig. S8). Arcobacteraceae have been identified as emerging pathogens potentially cause health risks to humans and animals [54] and also occur in hydrothermal vents like Sulfurovaceae [37, 55]. However, phylogenetic analysis showed that the dominant species of Arcobacteraceae (represented by OTU9977, OTU4225, OTU6340, and OTU1752) were obviously separated from those associated with humans and animals or with hydrothermal origins, but clustered onto a branch with those from the deep-sea wood-falls (Fig. S9). The same phenomenon was observed for the most abundant bacterium of Sulfurovaceae, OTU4956 (Fig. S10). This indicates that SOBs in chemoorganoheterotrophic ecosystems display obvious ecotypic differentiation from those in chemolithoautotrophic ecosystems. Thus, bacterial assemblages via the deep-sea in situ enrichment may form a unique ecological niche, like whale-fall and wood-fall ecosystems, described as one of the four chemosynthetic deep-sea ecosystems [48].

4.3 The key bacterial taxa play important and different roles in POM mineralization

Functional prediction results showed that the hydrolysis of proteins and polysaccharides, oxidation of fatty acids, fermentation, sulphate reduction, and sulphur oxidation were distinctly enriched in the enrichment assemblages (Table S5 and Fig. S6), suggesting that multiple functional groups were involved in the in situ mineralization of POMs in the deep sea.

Particularly, Marinifilaceae, Spirochaetaceae, Psychromonadaceae, and Vibrionaceae may be the direct decomposers and fermenters of macromolecular OM. Marinifilaceae family contains some psychrotolerant and facultatively anaerobe bacteria that are capable of degrading a large variety of macromolecules polysaccharides, e.g., starch, cellulose, and alginate, and protein, e.g., casein and gelatin, and generate various small molecular organic acids via fermentation [33, 56-58]. In addition, through omics data, we found that Marinifilaceae OTU6861, which mainly occurred in plant detritus enrichments, possessed the metabolic potential in hydrolyzing cellulose and xylan, while OTU4252 and OTU5909 within this family have no corresponding potential, but hydrolyze proteins (unpublished data in another separate paper attached for reference). Previous studies showed that some members of Spirochaetaceae played an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut [34]. Moreover, Pleomorphochaeta multiformis MO-SPC2 [59] and Spirochaeta perfilievii [60] within Spirochaetaceae, could utilize mono-, di- and poly-saccharides (xylan, trehalose, and pectin) and protein, and can generate formate, acetate, ethanol, pyruvate, and hydrogenvia fermentation. Therefore, bacteria OTU5914 and OTU4953, sharing high similarity with P. multiformis MO-SPC2 (99 %) and S. perfilievii (97 %), respectively, may play key roles in the degradation of polysaccharide and/or protein in plant detritus and animal tissue enrichments in this study. Additionally, previous studies also found that some members within Vibrionaceae and Psychromonadaceae, could degrade various macromolecular substrates, e.g., starch, xylan, alginate, mannan, gelatin, casein, chitin, and lecithin [35, 61], suggesting that those enriched taxa may play an important role in the mineralization of plant detritus and animal tissues in the deep sea in situ as well.

Although the above taxa have not been found or proved to have fatty acid oxidation capacity in previous reports, the functional prediction results showed that fatty acid oxidation-related modules were significantly enriched in fatty acid enrichment (Table S5), suggesting that bacteria with potential in fatty acid oxidation occurred in the fatty acid enrichments.

Common SRBs and SOBs were found in most of the enrichments (Fig. 3). As well, respirations of sulphur compounds, e.g., sulphate, sulphur, thiosulfate, and oxidation of sulphur compounds were detected by function prediction (Fig. S6). Both Arcobacteraceae and Sulfurovaceae are typical chemoautotrophic marine SOBs [37-39]. In our enrichments, Arcobacteraceae was more abundant, prevalent, and diverse than Sulfurovaceae (Fig. 3 and Fig. 4); in contrast, the bacterial Sulfurovaceae is the predominant SOB in the hydrothermal plume close to a vent [62]. Evidently, they are not directly involved in macromolecular OM decomposition but oxidizing sulphides generated by SRBs, such as Desulfobulbaceae and Desulfobacteraceae, which might use the most common microbial fermentation products (e.g., acetate, propionate, butyrate, lactate, and hydrogen) as an energy source, coupled with sulphate, sulphite, and/or thiosulfate reduction, like cultured strain KaireiS1 and members of Desulfofrigus, Desulfofaba, and Desulfotalea [41, 42]. Moreover, the chemoautotrophic lifestyle of Arcobacteraceae and Sulfurovaceae in the enrichments will suppress the accumulation of hydrogen sulphide; otherwise, sulphide may have detrimental effects on other microorganisms. These results indicated POM anaerobic degradation coupled with sulphate reduction inside the organic particles. The inorganic chemical energy retained in sulphide was conserved by SOBs via sulphur oxidation and preserved in the form of new organic carbon to avoid energy escape from the particle ecosystem to the extremely oligotrophic surroundings.

At the community level based on functional prediction, the metabolic modules for nitrogen fixation were selectively enriched in the enrichments of plant detritus and fatty acids (Table S5). This result indicated nitrogen fixation was accompanied by POM remineralization in nitrogen-nutrient-depleted enrichments. A recent study showed that some members within Acidobacteria, Firmicutes, Nitrospirae, Gammaproteobacteria, and Deltaproteobacteria are active diazotrophs in the deep-sea sediments, among which Desulfobacterales and Desulfuromonadales of Deltaproteobacteria are the most abundant diazotrophs [63]. In this study, the dominant Desulfobacteraceae belonging to Desulfobacterales, might play the role of nitrogen fixation, in addition to sulphur metabolism. Moreover, the Marinifilaceae OTU6861 also possesses the ability of nitrogen fixation (unpublished data, in another separate paper attached for reference). Same as our observation in the enrichments, more and more deep-sea nitrogen fixation has been found to occur at regional sites of enhanced productivity, such as methane seeps, mud volcanoes, and hydrothermal vents [64, 65]. Cross the ocean, nitrogen fixation accompanies chemoheterotrophic processes, as we observed, which are stimulated by OMs inside organic aggregates short of nitrogen sources in the deep water column. Nitrogen deficiency may be one of the key factors restricting the degradation and transformation of nonnitrogenous OM; therefore, diazotrophs play an important role in the biogeochemical process of POMs in the deep sea, which agrees with one of the predictions on nitrogen fixation made by Chakraborty: “nitrogen fixation can occur on large particles with high concentrations of polysaccharides and polypeptides in fully oxygenated marine waters” [66].

Interestingly, cooperative relationships dominate the bacterial relationships within the assemblages enriched with polysaccharides or fatty acids. In contrast, the relationships of the dominant taxa in protein enrichments were almost competitive (Fig. 5). Presumably, the co-occurrence of the key taxa within each assemblage is determined by the availability of nutrients and the supply of energy, both influenced by the OM provided. We hypothesized that the relative scarcity of nitrogen sources may be one of the main factors leading to the cooperative relationship among microorganisms, in addition to the type of organic substrates themselves. Therefore, in communities supported by polysaccharides or fatty acids, different members may cooperate to deal with the shortage of nitrogen sources.

Based on the results of this study, we aimed to delineate the roles of microbial key taxa in the degradation of POM and the relationships among them. As shown in Figure 6, the key taxa belonging to Marinifilaceae, Spirochaetaceae, Psychromonadaceae, and Vibrionaceae were inferred to be the direct depolymerizers and degraders of polysaccharides and/or protein polymers in the plant detritus or animal tissue. They depolymerize the natural polymers into monomers and simultaneously produce low-molecular weight compounds by fermentation (Fig. 6). These fermentation products can be supplied to SRBs of Desulfobulbaceae and Desulfobacteraceae or sulphur-reducing bacteria of Sulfurospirillaceae to generate reduced hydrogen sulphide. The reduced sulphur compounds further provide energy for chemoautotrophic bacteria of SOBs belonging to Arcobacteraceae and Sulfurovaceae, these primary producers that may feed the community in return by newly synthesized carbon sources and reduce the potential negative effects of high concentrations of sulphide. Therefore, bacterial assemblages developed over the new organic inputs and formed a unique ecological niche in the deep sea.

In conclusion, this report revealed unique deep-sea benthic bacterial communities responsive to diverse natural POM input, by which macromolecules in the natural plant detritus and animal tissue sinking to the seabed is decomposed and remineralized in situ in the deep sea. This suggested these communities which are different from those dominating the water column and sediment, play an unneglectable role in carbon biogeochemical cycling in global oceans.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors give thanks to all the scientists, engineers and technicians and all crew members of several oceanic cruises of COMRA. We thank Prof. Marcus Elvert (Organic Geochemistry Group, MARUM—Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany) for comments on an earlier version of the manuscript. This work was financially supported by the National Natural Science Foundation of China (No.42030412), the China Ocean Mineral Resources R&D Association (COMRA) program (No. DY135-B2-01), High-Tech Research and Development Program of China (No. 2012AA092102), and the Scientific Research Foundation of Third Institute of Oceanography, MNR (2019021).

Author contributions

Zongze Shao conceived the idea, applied the financial supports, organized and performed in situ incubation and processed samples on board during the cruise of COMRA DY40 in the Indian Ocean. Chunming Dong organized and conducted in situ incubation and processed samples on board during the cruise of COMRA DY45 in the Pacific Ocean and the South China Sea. Zongze Shao, Jianyang Li, and Zhaobin Huang participated in equipment deployment during the cruise of COMRA DY45 in the South China Sea and participated in the next year's cruise for equipment recovery with Qiliang Lai. Jianyang Li and Chunming Dong conducted further sample processing. Jianyang Li analyzed the 16S rRNA sequence data. Zongze Shao, Jianyang Li, and Chunming Dong interpreted the results and wrote the manuscript. Donghui Zhou and Zongze Shao were involved in equipment development and maintenance. Linfeng Gong was responsible for building the analysis platform. Guangyi Wang reviewed the paper.

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Unique deep-sea bacterial assemblages thriving on different natural organic matters delivered via in situ incubators

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Experimental design and in situ incubation experiments

2.2 Sampling and DNA extraction

2.3 Illumina high-throughput sequencing and analysis

2.4 Phylogenetic analysis

2.5 Function prediction of each enrichment

2.6 Network analysis

3 Results

3.1 Deep-sea in situ incubation of OM-decomposing bacteria

3.2 Bacterial diversity and its environmental determinants of the OM-decomposing microbial assemblages

3.3 Bacterial composition of deep-sea in situ enrichments with natural organic materials

3.4 Keystone bacteria showing a preference for organic substrates and geographic locations at the OTU level

3.5 Function prediction of the deep-sea bacterial communities

3.6 Microbial co-occurrence networks within the bacterial assemblages of different POMs

4 Discussion

4.1 The bacterial assemblages in enriched communities are unique

4.2 Bacterial assemblages may form a unique ecological niche

4.3 The key bacterial taxa play important and different roles in POM mineralization

5 Summary And Conclusion

Declarations

References

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