Introduction
Deep-sea sediment is among the least explored biospheres on Earth. Indigenous
microbes differ vastly in community composition and metabolic spectra at
different depths and sites (Orcutt et al., 2011; Teske and Sorensen, 2007).
The distribution of microbes in subsuperficial sediments is determined by the
porosity, nutrient availability and geochemical conditions of the sediment
(Parkes et al., 2000; Webster et al., 2006). In return, genomic features and
the community composition of the indigenous microbial inhabitants may reflect
the in situ conditions and serve as biomarkers containing the geochemical
indicators. However, most of the biomarkers cannot be well preserved and will
be degraded by biological and abiological activities. Although lipids and
other organic carbons present in some minerals allow the interpretation of
microbial activities to some extent (Brocks et al., 2005), the original
metabolic activities are difficult to retrieve in a comprehensive and precise
manner.
Most of the dead microbes are damaged during the sedimentation process, but
some can be maintained in almost their original shape (Taher, 2014; Benison
et al., 2008). Evaporites, which mostly consist of halite and anhydrite
(CaSO4) or gypsum (CaSO4 × 2H2O, temperature
< 38∘; Hill, 1937), are common microbialites with
accretionary organosedimentary structures (Dupraz et al., 2011). Numerous
dead bacteria, algae and metazoans have been detected in gypsum granules
(Petrash et al., 2012; Trichet et al., 2001); bacterial mats growing on
evaporites may become trapped and constitute much larger microbialites
(Babel, 2004). Consequently, microbial inhabitants on the benthic surface may
get trapped in the evaporites (Benison et al., 2008). Anhydrite facies are
not found throughout deep-sea sediments. They usually form around
hydrothermal vents in deep-sea environments (Jannasch and Mottl, 1985). A
strong deep-sea volcanic eruption may break the crustal basalts, resulting in
a drastic emission of hydrothermal gases followed by the crystallization of
anhydrites and the deposition of metal sulfides (Kristall et al., 2006). An
alternative model is that mild hydrothermal activities lead to a slow influx
of solutions into the overlying sediment at temperatures in the sub-seafloor
ranging from 20 to 100 ∘C. This process also results in the formation of
crystalline anhydrites in veins and around warm vents (Jannasch and Mottl,
1985). The latter process may trap microbial inhabitants on the seafloor and
within surface layers in anhydrites. Due to the mild temperature, the trapped
bodies are better preserved as excellent biological evidence for past
geochemical conditions.
A similar mild hydrothermal field is present in the Red Sea. Initially found
in a deep-sea rift in the 1960s (Swallow and Crease, 1965; Girdler, 1970),
the temperature of the Atlantis II brine pool has recently increased to
68 ∘C (Anschutz and Blanc, 1996). In 1972, several sediment cores were
obtained from the southwestern region of the pool (DSDP Site226), and metal
sulfides and evaporites were recognized as major mineral facies in this
brine-filled basin (Shipboard Scientific Party, 1974). In particular, thick
and well-crystalized anhydrite layers were found within the hematite and at
the bottom of the cores. Two major anhydrite units were later defined by
analysis of the adjacent core samples. The lower unit comprised anhydrite
ranging from 12 to 70 wt % (Anschutz et al., 2000). The anhydrite in
the sediments likely resulted from a geyser-type eruption of hydrothermal
solutions into the Atlantis II brine pool followed by the mixing of
calcium-rich solutions with dissolved sulfate-bearing brine and the
precipitation of anhydrites during the cooling process (Ramboz et al., 1988).
The discovery of veins containing sulfides and anhydrite in the sediment
suggests that a mild hydrothermal eruption created the anhydrite facies in
the Atlantis II sediment (Zierenberg and Shanks, 1983; Oudin et al., 1984;
Missack et al., 1989). The formation of anhydrite facies in this manner would
trap microbial cells and organic debris in the bottom water and surface
sediment. These anhydrite layers probably contained important indigenous
microbial inhabitants during the occurrence of the hydrothermal events on the
deep-sea benthic floor of the Red Sea. Coupled with 14C markers to
estimate age, the anhydrite facies contained a large quantity of information
regarding past geochemical changes. The formation process of the Atlantis
II brine pool is still controversial, largely because the source of the brine
is uncertain (Schardt, 2016). The brine water had converted the bottom of the
deep into a hot anoxic hypersaline environment. The microbes in the
anhydrite facies may provide hints about the original benthic conditions and
age of the pool. It is also an interesting question whether oil was generated
in the sediments under the mild hydrothermal activities in the past. If
yes, seeping hydrothermal solutions may bring oil into the seafloor of the
deep, which might be documented by the microbes in the anhydrites.
In the present study, we sampled a sediment core near Site226 and detected
an anhydrite layer. The dominant species were alkane- and oil-degrading
bacteria indicating an oxic, oil-spilling benthic condition when the layer
was formed. The present study sheds light on the importance of anhydrites in
deep-sea sediment as an archive of microbial inhabitants that can serve as
biomarkers of past geochemical events.
Materials and methods
Physicochemical measurements of sediment layers
In 2008, a 2.25 m gravity sediment core was obtained from the southwestern
basin (approximately 2180 m below sea level) of the Atlantis II Deep
(21∘20.76′ N, 38∘04.68′ E) in the Red Sea (Fig. S1 in
the Supplement) (Bower, 2009). The core was frozen at -80 ∘C and then
sliced aseptically into 75 sections of 3 cm each. Microbes from sediment
slices of 12–15, 63–66, 105–108, 183–186 and 222–225 cm were first
suspended in phosphate-buffered saline and shaken by a vortexer for 30 s.
After 30 min, the supernatant was filtered through a 0.22 µm black
polycarbonate filter. After 6-diamidino-2-phenylindole (DAPI) staining, the
microbes from each layer were counted under an epifluorescence microscope
(n = 3) (Gough and Stahl, 2003). The pore water from the top five layers
was collected by centrifugation. The concentration of dissolved organic
carbon (DOC) in the pore water was determined using the combustion method
(Trichet et al., 2001). The concentrations of ammonium, nitrite and nitrate
were measured using a TNM-I analyzer (Shimadzu Corp., Kyoto, Japan). To separate
large particles (> 63 µm) from small particles
(< 63 µm), the sediment samples were passed through a
63 µm stainless steel sieve. The percentage of small particles (dry
weight) was calculated for all slices.
The age of the sections was estimated with a radiometric dating method that
utilizes the naturally occurring radioisotope 14C. The
monospecific Globigerinoides sacculifer specimens, ranging in size
from 250 to 350 µm, were manually selected with caution and then
subjected to 14C measurement in the National Ocean Sciences Accelerator
Mass Spectrometry (AMS) Facility at the Woods Hole Oceanographic Institute,
USA. The raw AMS 14C ages were converted to calendar ages using the
CALIB 6.0 program (http://calib.qub.ac.uk/calib/) with the dataset
Marine 09 (Reimer et al., 2009). A reservoir correction has been considered
for the 14C difference between atmospheric and surface waters (Bard,
1988).
DNA extraction and amplification
The boundary of the anhydrite layer was determined by naked eye observation
and particle size measurement. Crystals were manually collected from the
layers, followed by ultrasonic cleaning. The homogenized crystals were then
analyzed by X-ray diffraction (XRD) (Rigaku, Tokyo, Japan) using Cu K-alpha
radiation of 40 kV and 30 mA. The following procedure was conducted for DNA
extraction from the crystals with caution to avoid contamination. Surface
contamination was removed by rinsing with 70 % alcohol in autoclaved
distilled deionized water, followed by pulsed ultrasonic cleaning for 2 h.
Anhydrite crystals (20 g) (Fig. 1a) of different sizes were treated with
1 µL (2U) Turbo DNase I (Ambion, Austin, Texas, USA) for 30 m in a
37 ∘C incubation before being ground for DNA extraction in a sterile
hood. The anhydrite powder was used for DNA extraction with the PowerSoil DNA
Isolation Kit (MO BIO, Carlsbad, USA), followed by a purification step
according to the manufacturer's instructions. The DNA concentration was
quantified with a Quant-iT PicoGreen Kit (Invitrogen, USA). Of the raw DNA extract, 20 pg was used for DNA amplification using a MALBAC kit
(Yikon Genomics, Jiangsu, China) according to the manufacturer's manual (Zong et al.,
2012). The MALBAC amplification method has been evaluated recently in
metagenomic studies (Wang et al., 2016). Two MALBAC amplification assays were
conducted using 21 PCR cycles to acquire a sufficient amount of DNA
for subsequent sequencing. A negative control was also incorporated in the
assay. The DNA concentration of the MALBAC-amplified sample and the negative
control was measured with a Bioanalyzer (Agilent Technologies, CA, US). The products of
the MALBAC amplification and negative control were examined by gel
electrophoresis to confirm the size ranges of the amplicons. Three replicates
of MALBAC amplifications for each sample were mixed and used for Illumina
sequencing on a Hiseq2000 platform (Illumina, San Diego, US). As a control,
10 g of sediment from a position at 168 cm from the top of the core was
used for DNA extraction. There were no recognizable anhydrite crystals in
this layer. DNA sequencing was conducted as described above.
Anhydrite crystals and genome binning.
Anhydrite crystals in a Petri dish (90 mm in diameter) (a) were used for DNA
extraction. The amplified genomic DNA was sequenced and then reassembled.
Based on the G + C content and read coverage, the binned contigs with high
coverage levels (b) were selected for examination of the tetranucleotide
frequency consistency in the PCA analysis (c).
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Binning of metagenomes
The initial Illumina 2 × 110 bp paired-end reads were subjected to
quality assessments using the NGS QC Toolkit with default parameters (Patel
and Jain, 2012). The Illumina sequencing data were deposited in the NCBI SRA
database (accession number SRA356974). The 35 bp MALBAC adapters at the two
ends of the sequencing reads were removed. Assembly of the trimmed Illumina
2 × 75 bp paired-end reads was performed using SPAdes 3.5 (Nurk et
al., 2013). The read coverage for the assembled contigs was calculated using
SAMtools (Li et al., 2009). The 16S/18S rRNA genes in the contigs were
identified using rRNA_HMM (Huang et al., 2009). Using classify.seqs command
in mothur package (Schloss et al., 2009), taxonomic sorting of the 16S rRNA
genes was conducted against the SILVA database with a confidence threshold of
80 %. The relative abundance of the species in the metagenomes was
roughly estimated based on the coverage of the 16S/18S rRNA genes. Binning of
the draft genomes was performed based on the read coverage and G + C
content of the contigs (Fig. 1b), followed by principal component analysis
(PCA) of the tetranucleotide frequencies (TNF) of their respective contigs
using a previously described pipeline (Fig. 1c) (Albertsen et al., 2013). The
R scripts (R Core Team, 2013) for the binning process were obtained from
https://github.com/MadsAlbertsen/multi-metagenome. To evaluate the
completeness of the draft genome, conserved single-copy genes (CSCGs) were
counted in the genome. The CSCGs were identified by searching the CDSs (coding DNA sequences)
against a database of essential bacterial genes (107 essential genes)
(Albertsen et al., 2013) using hmmsearch (3.0) with default cutoffs for each
protein family (Krogh et al., 1994).
Genomic analyses
The CDSs of the draft genome were predicted using
Prodigal (version 2.60) (Hyatt et al., 2010). KEGG annotation of the CDSs was
performed using BLASTp against the KEGG database (Kanehisa et al., 2012) with
a maximum e-value cutoff of 1e-05. The KEGG pathways were reconstructed using
the KEGG website (http://www.kegg.jp). CDSs were also annotated against
the NCBI NR database, and MEGAN (MEtaGenome ANalyzer) was used for taxonomic affiliation and
SEED/subsystem annotation of the CDSs (Overbeek et al., 2005). The draft
genome was submitted to NCBI (accession number LKAP00000000). The average
nucleotide identity (ANI) was calculated using the algorithm integrated in
the web service of EzGenome (Goris et al., 2007). The DNA–DNA hybridization
(DDH) estimate value was calculated using the genome-to-genome distance
calculator (GGDC2.0) (Meier-Kolthoff et al., 2013; Auch et al., 2010a, b).
Detection and phylogeny of 16S ribosomal RNA (rRNA) genes
The 16S rRNA gene sequence was identified from the draft genome sequence.
The closest relatives based on 16S sequence similarity were determined using
the web service of EzTaxon (Kim et al., 2012). The neighbor-joining
phylogenetic tree was constructed using MEGA version 5.0 (Tamura
et al., 2011) with the Kimura 2-parameter model. The phylogenetic tree was
supported with bootstrap values based on 1000 replications.
Fluorescence in situ hybridization (FISH) of
<italic>Alcanivorax</italic> bacteria
FISH probes for 16S rRNA gene of Alcanivorax bacteria were designed
based on the 16S rRNA gene sequence extracted from the Alcanivorax
draft genome. Two 16S rRNA fragments, 5′-CCTCTAATGGGCAGATTC-3′ and
5′-CCCCCTCTAATGGGCAGA-3′, were selected as candidate probes with
Probe_Design in the ARB package (Ludwig et al., 2004). The coverage
efficiency of the probes was then examined in the Silva database (Quast et
al., 2013). The 6-FAM-labeled probe used to target the alkB gene was
5′-ATGGAGCCTAGATAATGAAGT-3′ (Wang et al., 2010). A pure culture of
Alcanivorax borkumensis Sk2 (Yakimov et al., 1998) was
first used to examine the probes before performing the assay, and a culture
of Escherichia coli was used as a negative control. Two grams of
anhydrite crystals were sonicated for 30 min in 1 U DNase I solution
(Takara, Dalian, China). The crystals were washed with deionized water and
then ground into a powder with a BeadBeater in a germ-free environment. The
supernatant was mixed with 37 % formaldehyde (final concentration,
1–4 %). To fix the cells in phosphate buffer saline solution, the sample
was maintained at 4 ∘C for 3–4 h. After centrifugation at
13 000 r min-1 for 3 min, the supernatant was discarded. The
remaining microbes were soaked in 200 µL of PBS buffer, followed by
the addition of 200 µL of ethanol (Pernthaler et al., 2002). The sample
was filtered through 3 µm and 0.22 µm membranes
sequentially (diameter, 25 mm; Millipore, Massachusetts, USA). After
dehydration of the membrane using alcohol, 2 µL of dying solution
containing oligonucleotide probes and 20 µL of buffer
(360 µL of 5 M NaCl, 40 µL of 1 M Tris / HCl,
700 µL of 100 % formamide, 2 µL of 10 % SD and
water to a total volume of 2 mL) was added. The hybridization of the probes to the
microbes was performed for 2 h at 46 ∘C. Rinsing buffer
(700 µL of 5 M NaCl, 1 mL of 1 M Tris / HCl, 500 µL of
0.5 M EDTA, 50 µL of 10 % SDS and water to a total volume of
50 mL) was used to remove free probes. For counterstaining, 50 µL
of 4′,6′-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Massachusetts, USA)
solution (1 µg mL-1) was added to the sample. After
incubation for 3 min, the sample was washed in Milli-Q water (Millipore, Massachusetts, USA) and 96 % ethanol for 1 min (Pernthaler et
al., 2002). The microscopic observation was conducted using an Olympus BX51
(Olympus, Tokyo, Japan).
Results
Physicochemical profile and cell counts
A thick anhydrite layer was present at the bottom of the sediment core based
on naked-eye observation of the color and grain size. The anhydrite layer at
depths ranging from 177 to 198 cm consisted of coarse, agglutinated crystals,
which corresponded to the high percentage of large grains (78 wt %
larger than 63 µm) (Fig. 2). The XRD analysis further confirmed
that the crystals in this layer were anhydrite. In contrast, halite comprised
the evaporites at depths of 12, 63, 105 and 222 cm. For the samples at
different depths, the DOC concentration was measured, and the highest value
was recorded at 183 cm (80.9 mg L-1), which was even higher than the
surface layer at 12 cm (Fig. 3). In the 12 cm layer, the cell density was
3.2 × 105 cells per cm3, whereas in the layers at 63 cm,
105 and 222 cm, it was reduced by 88, 92 and 96 %, respectively
(Fig. 3). The cell density was also calculated as the number of cells per
gram of sediment. The results revealed a value of 7.1 × 105
cells per gram at a depth of 12 cm, which declined more than 70 % in the
deeper layers. Although the cell density in the 183 cm layer
(6.7 × 104 cells cm-3) was markedly lower than that in
the 12 cm layer, it was higher than those in the 105 and 222 cm layers.
Grain size and age of selected layers
The percentages of the small particles (< 63 µm) in dry weight
are shown for 75 slices of the sediment core (small squares on the line).
The age estimates (black circles) of the selected layers were performed
using radioisotope 14C of the monospecific Globigerinoides sacculifer specimens. Age errors ranged between 25 and 40 years. Anhydrite
and control layers for metagenomic study were indicated by arrows.
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Nutrient measurements and cell counts in the different sediment
layers.
The pore water samples were analyzed for five layers of a sediment core
obtained from the Atlantis II Deep (21∘20.76′ N, 38∘04.68′ E) in 2008. DOC: dissolved organic carbon.
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The sediment as a whole is a highly reductive environment, as indicated by
the low nitrate, low nitrite and extremely high ammonium concentrations
(Fig. 3). To determine the time of the anhydrite layers at 177–198 cm, an
age estimate was performed for several layers. The sediment ages were
estimated based on the radioisotope 14C of G. sacculifer,
assuming a linear increment from the top (Fig. 2). The results obtained for
the layers above and below the anhydrite layer indicated a narrow range of
750–770 years between 153 and 198 cm (Table 1).
Age estimates of the sediment layers.
Layer
Age
Age error
(cm)
(year)
(year)
3–6
320
25
21–24
475
35
45–48
490
30
90–93
500
25
129–132
560
35
153–156
750
30
198–201
770
30
222–225
880
30
Eight sediment layers were selected for the age estimates using radioisotope
14C of G. sacculifer collected from the respective layers. The
age was corrected by the 400-year reservoir age with an error range.
Microbial communities in anhydrite crystals and neighboring
control sediment.
Phyla and genera in the anhydrite crystals and control layer were predicted
using 16S/18S rRNA gene fragments extracted from the corresponding
metagenomes (D-T: Deinococcus-Thermus). The relative abundance of the genera
can be estimated by the coverage level (> 5) of the 16S/18S rRNA
fragments by reads.
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Draft genome of the dominant bacterial species in anhydrites
About 1.8 Gbp Illumina raw sequencing data were obtained for the anhydrite
sample and 3.1 Gbp data were obtained for the adjacent control layer. The
size of the anhydrite and control metagenomes was 59 and 84 Mbp,
respectively, after assembly (Table S1 in the Supplement). The microbial
communities differed remarkably according to the taxonomic assignment of the
16S/18S rRNA gene fragments in the two metagenomes (Fig. 4). At the genus
level, only Ochrobactrum and Alkanindiges were common
inhabitants in both samples. Alcanivorax and Bacillus were
also dominant genera in the anhydrite and the control, respectively. At the
phylum level, excluding the Proteobacteria, the two metagenomes had
distinctive phyla. The anhydrite contained archaea that were represented by
the methanogenic Methanoculleus (Barret et al., 2012) and fungi
that consisted of the Ascomycota. In contrast, the control sediment contained
mainly Firmicutes, Bacteroides, Actinobacteria, and Deinococcus-Thermus. At
last, an invertebrate species, Prototritia sp. belonging to
Arthropoda, was identified in the anhydrite.
Phylogenetic tree of 16S rRNA genes.
Bootstrap values (expressed as percentages of 1000 replications) are shown
at the branches of the neighbor-joining tree.
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Genome binning of an <italic>Alcanivorax borkumensis</italic> genome
The binned draft genome from the anhydrite metagenome was 3 069 971 bp and
comprised 77 contigs. A partial 16S rRNA gene sequence (805 bp) was
extracted from the draft genome. Because the sequence was almost identical to
that of A. borkumensis Sk2 (99.9 %) (see also genomic alignment
in Fig. S2), we considered the binned draft genome to be from a strain of
A. borkumensis. As shown in Fig. 5, a phylogenetic tree based on the
16S rRNA gene sequences of the genus Alcanivorax indicated that the
strain clustered with A. borkumensis Sk2, an exclusive and
ubiquitous hydrocarbon-degrading bacterium (Schneiker et al., 2006; Sabirova
et al., 2011). The strain name of the A. borkumensis in the sediment
core was ABS183. It was the only microbial species that could be reliably
separated from the metagenome.
Schematic model of metabolism and cross-membrane transporters.
The model was predicted based on the genes in the draft genome of
Alcanivorax borkumensis ABS183.
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The genome of A. borkumensis ABS183, despite containing gaps, was
slightly smaller than that of A. borkumensis Sk2 (accession number
NC_008260; 3,120,143 bp), suggesting that the draft genome of A. borkumensis ABS183 was nearly complete. Also, there were not detectable
alignment gaps between the two genomes (Fig. S2). The identification of a
complete list of single-copy genes also supported the completeness of the
genome. The DDH estimation between A. borkumensis ABS183 and Sk2 was
97.1 % ± 1.3 %, which was higher than the standard cut-off
value of 70 % for genome relatedness between pairs of species (Wayne et
al., 1987). The ANI value between ABS183 and Sk2 was 99.9 %, which was
also higher than the standard ANI criterion for species identity
(95–96 %) (Richter and Rossello-Mora, 2009). These results further
confirmed that ABS183 was a strain of A. borkumensis.
Fluorescence in situ hybridization (FISH) of
Alcanivorax borkumensis ABS183 embedded in anhydrite crystals.
DAPI staining and FISH using 16S rRNA probes are shown in (a) and (b). The
merged image of (a) and (b) is shown in Fig. 7c. DAPI staining and FISH were
also performed using two samples that were filtered with 3 µm (d–g) and
0.22 µm (h–k) membranes, respectively. Alcanivorax bacteria
were released from the large crystals filtered through the 3 µm
membranes (d–g). The bacteria were stained with DAPI (d), Alcanivorax borkumensis ABS183 probes
(f) and the alkB probe (f), respectively, and overlaid (g). Using a
sample filtered through a 0.22 µm membrane, a dividing
Alcanivorax borkumensis ABS183 cell was labeled using the same method and
probes (h–j). The microscopic fields shown in (h)–(j) are merged in (k).
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The genome of A. borkumensis ABS183 contains two copies of the
alkane-1 monooxygenase gene (alkA; 10502_28 and 2890_35), which is
an essential functional gene for alkane utilization by Alcanivorax
bacteria (Fig. 6) (Schneiker et al., 2006). Neighboring the alkA
genes, alkBGHJ genes, a GntR family transcriptional regulator gene,
and a rubredoxin gene were identified. The gene order of the related genes
was consistent with that of the homologs in the genome of strain Sk2
(Schneiker et al., 2006). The alk genes were completely absent from
the control metagenome. Moreover, the genome of A. borkumensis
ABS183 contains genes responsible for the reduction of nitrogen oxides (KEGG
genes: K00370-K00374 and K00362-K00363; nitrate reductase I genes and nitrite
reductase genes). The reduction process was believed to generate ammonia for
the efficient synthesis of amino acids (Schneiker et al., 2006). Ammonia
might be generated through nitrate reduction as indicated by the presence of
the related genes encoding nitrate and nitrite reduction enzymes (Fig. 6).
Ammonia might be imported by transmembrane transporters and assimilated into
glutamate. A high demand for fatty acids was a characteristic of A. borkumensis to perform rapid energy and organic carbon storage. A. borkumensis ABS183 was probably able to synthesize long fatty acids because
the fas and fabBFGIKZ genes responsible for the elongation
of fatty acids were all present in its draft genome. In contrast, the
essential fas gene (K11533) and other relevant genes were not found
in the control metagenome. Crude oil generally contains aromatic compounds,
and the current sediment at the sampling site also contained oil (Wang et
al., 2011). As expected, the two metagenomes possessed a complete set of
genes responsible for the degradation of aromatic compounds. Based on the
homology of the genes, the Ochrobactrum and Alkanindiges
species probably played a role in this degradation.
Detection of bacteria in anhydrite crystals by DAPI and FISH
To determine whether complete microbial cells could be maintained in the
anhydrite crystals, DAPI and FISH assays were conducted to visualize the
microbes. The DAPI results revealed the presence of complete cells that were
released or embedded in the crystals (Fig. 7a, d and h). However, the FISH
assay, which was used to detect A. borkumensis ABS183 with two
probes specific to the 16S rRNA gene, showed some fluorescence-labeled
microbes (Fig. 7b, e and i). These microbes could also be envisioned with the
FISH assay using the alkB gene probe (Fig. 7f and j). The
alkB is one of the functional genes that participate in alkane
degradation (Schneiker et al., 2006). The rod shape of the fluorescent
microbes is consistent with the microscopic features reported previously
(Sabirova et al., 2011). These results indicated that some microbes in the
microscopic fields were A. borkumensis ABS183, as revealed in the
anhydrite metagenome.
Discussion
In the present study, we detected complete microbial cells and analyzed their
metagenome in the anhydrite crystals from a deep-sea anoxic basin. The
dominant bacterial species was A. borkumensis ABS183, an aerobic
bacterium that is capable of degrading alkanes in crude oil.
Alcanivorax is one of the bacterial indicators for the spilling of
oil in waters and surface sediment (Yakimov et al., 2007). However, the
Atlantis II brine pool is anaerobic and increasingly hydrothermal (Bougouffa
et al., 2013b). The brine sediment in the basin was also found to be anoxic.
Thus, A. borkumensis ABS183 could not be current inhabitants of the
hydrothermal anoxic basin. This difference did not explain the stratification
of microbial communities in the different sediment layers of the brine-filled
basin. A recent study showed that Alcanivorax was not present in all
sediment layers of a sediment core from the Atlantis II basin (Wang et al.,
2015). A reasonable explanation for this finding is that the anhydrite layer
at 177–198 cm in the sediment core was formed at a previous benthic site
when hydrothermal solution was injected into the seafloor. The organisms
living in the benthic water and subsurface sediment were subsequently sealed
and protected in the anhydrite crystals. Because the metabolism of A. borkumensis bacteria was specifically used for the degradation of alkanes
and other hydrocarbons in crude oil (Yakimov et al., 2007), the benthic site
in which the anhydrite layer formed was probably an oil-spilling or
oil-forming environment in the Atlantis II basin. The current hot sediments
in the basin are biogenic and abiogenic sources of crude oil (Simoneit,
1988). Seeping of the oil has resulted in proliferation of A. borkumensis bacteria in the bottom water. Similarly, oil-utilizing bacteria
were nourished after the oil-spilling disaster in the Gulf of Mexico
(Gutierrez et al., 2013). The A. borkumensis bacteria were important
producers of organic carbons as they could convert alkanes and nitrate into
organic matter. Fatty acids and lipopolysaccharides that were yielded by
A. borkumensis bacteria were nutrients for the whole ecosystem.
Based on the results in the present study, we propose that mild eruptions of
hydrothermal solutions injected calcium-rich solutions into the seafloor and
produced anhydrite veins by mixing with sulfate in the bottom water of the
Atlantis II rift basin. The anhydrite layer was then covered by sulfide
minerals and biological debris such as the planktonic foraminifera G. sacculifer. In this study, we narrowed the age of the thick anhydrite layer
to 750–770 years using the 14C isotope of the G. sacculifer
specimens. This result also indicates a relatively young sediment age and a
high accumulation rate of precipitated metals in the Atlantis II basin.
Because the upward movement of hydrothermal solutions might transfer some
foraminifera specimens from lower layers to the anhydrite layer, we did not
use the foraminifera between anhydrite crystals. In our previous study, we
showed evidence of oil formation in the Atlantis II brine pool (Wang et
al., 2011). The organic carbon content can be converted to aromatic compounds
under the hydrothermal conditions in the pool based on chemical and
metagenomic evidence (Wang et al., 2011). However, the bottom of the anoxic
brine pool was not a habitat of Alcanivorax species (Bougouffa et
al., 2013a; Blanc and Anschutz, 1995), suggesting that Alcanivorax
flourished in the basin before the formation of brine water layers over the
sediment (Blanc and Anschutz, 1995).
Although there were differences in microbial communities between the
anhydrite crystals and the control sediment, Ochrobactrum sp. was
one of the common inhabitants. Previous studies have shown that
Ochrobactrum species could metabolize aromatic compounds aerobically
and anaerobically (Zu et al., 2014; Mahmood et al., 2009), which explains
their presence in both metagenomes assessed in the current study. Moreover,
we determined the concentrations of nitrogen oxides in the different sulfide
layers, although only low concentrations were detected. Ochrobactrum
species were potentially able to anaerobically degrade polycyclic aromatic
compounds using nitrate as an oxygen donor (Mahmood et al., 2009; Wu et al.,
2009). Such a chemolithoheterotrophic lifestyle is in accordance with the
current in situ environment of the sediment in the Atlantis II. Regardless of
the environmental changes indicated by the findings in the present study, the
spreading of Ochrobactrum sp. was seemingly not affected. Although
the metagenomes in the present study contained an abundance of essential genes
for degrading a variety of aromatic compounds, the microbial degradation of
these compounds might have been attenuated by a lack of oxygen and a high
level of salinity (Klinkhammer and Lambert, 1989). Anaerobic degradation of
compounds is more difficult than aerobic degradation, often requiring oxygen
donors such as nitrate and sulfate (Mahmood et al., 2009; Wu et al., 2009).
Based on its ability to survive under anoxic conditions,
Ochrobactrum sp. is probably able to maintain a higher level of
fitness in the control sediment compared with Alcanivorax. In the
present study, the Alkanindiges identified in both metagenomes was
also a well-known alkane degrader (Klein et al., 2007; Bogan et al., 2003).
Because of its presence in both anhydrites and the adjacent sulfide layer, we
assumed that the Alkanindiges bacterium was also capable of
surviving aerobically and anaerobically in the oil-producing sediment. Hence,
the change from an oxic to an anoxic benthic environment caused a dramatic
shift in the microbial communities, resulting in the extinction of the
obligate aerobic alkane-utilizer Alcanivorax and continuous
residency of anaerobic oil-degraders. The availability of nitrogen oxides and
the dissolution of sulfate from anhydrite crystals were possibly critical to
the metabolic activities of the anaerobes. In addition, the Bacillus
and fungi present in the control sediment were probably present in the form
of dormant spores. In a recent report, Ochrobactrum and
Bacillus were confirmed to be dominant species in some upper sulfide
layers in the Atlantis II (Wang et al., 2015). Altogether, in the present
study, the current microbial inhabitants in the sulfide layers were largely
different from those in the anhydrite crystals.
The geochemical data collected herein suggested that the sub-superficial
anhydrite layer could release organic carbon contents into the sediment, as
reported previously. Our measurement of DOC at 80.9 mg L-1
in the anhydrite layer was higher than the generally accepted maximum value
of 50 mg L-1 for marine sediments (Cameron et al., 2006). The
abnormally high DOC was considered a notable alteration of the local
environments and probably resulted from the breakdown of anhydrite crystals.
Anhydrites in the Atlantis II brine sediment were likely maintained by the
high salinity and temperature and then slowly dissolved. This phenomenon may
be explained by the slight undersaturation of the anhydrite in the Atlantis II
sediment (Anschutz et al., 2000). Such anhydrite layers are widely
distributed in Middle Eastern sediments (Alsharhan and Nairn, 1997). Hence,
our findings shed light on the formation of micro-environments by anhydrite
evaporites in the deep sediments. In this study, there was an inconsistency
between the cell density and the DOC at the 12 cm depth layer, in which the
DOC could not support a 10-fold higher biomass. This phenomenon probably
resulted from the formation of petroleum compounds under the hydrothermal
effects (Wang et al., 2011). In the petroleum, hydrophobic organic compounds
(HOCs) consisting of polycyclic aromatic hydrocarbons (PAHs) could not be
counted in our DOC measurements (J. Pearsons, personal
communication, 2014). The nutrient supply is
critical for microbes to survive in deep-sea sediment. Apart from the
chemolithoautotrophic microbes, numerous other inhabitants take advantage of
the buried organic matter. Importantly, the trapped organic matter serves as
a nutrient supply following the dissolution of organic-rich anhydrite
crystals. Therefore, our findings highlighted the importance of the nutrients
released from the anhydrite facies for microbes in deep-sea subsuperficial
sediment.