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Keywords:

  • Mariana Trench;
  • Extremophile;
  • 16S ribosomal RNA;
  • Phylogenetic tree

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

In an attempt to characterize the microbial flora on the deepest sea floor, we isolated thousands of microbes from a mud sample collected from the Mariana Trench. The microbial flora found at a depth of 10 897 m was composed of actinomycetes, fungi, non-extremophilic bacteria, and various extremophilic bacteria such as alkaliphiles, thermophiles, and psychrophiles. Phylogenetic analysis of Mariana isolates based on 16S rDNA sequences revealed that a wide range of taxa were represented.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The Challenger expedition (1873–1876) is commonly credited as the historical beginning of deep-sea biology[1]. Deep-sea expeditions have been afforded facilities for the collection and microbial analysis of sediment samples from the deep parts of the oceans [2–5]. Some bacteria have been isolated from deep-sea mud and from benthic organisms such as amphipods and sea cucumbers in the bathypelagic zone. Little information is available on the bacterial diversity of the deepest sea floor because most marine microbiologists have focused on barophilic and psychrophilic inhabitants of the deep-sea environment[6].

On 2 March, 1996, the 3 m long submersible Kaiko (meaning ‘trench’ in Japanese), suspended from a parent submersible, touched the bottom of the Challenger Deep (11°21.111′N, 142°25.949′E) in the Mariana Trench at a depth of 10 897 m. Kaiko successfully scooped out a mud sample, the first obtained at such a great depth. We isolated microbes from the sea mud to investigate the microbial flora of the deepest sea floor and characterized the isolates by means of 16S rDNA sequence analysis.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Collection of deep-sea mud

A mud sample was obtained from the bottom of the Challenger Deep at a depth of 10 897 m by means of the unmanned submersible Kaiko as shown in Fig. 1B–D. The grayish brown mud consisting of very fine particles was collected in a sterile 50 ml Falcon tube which was then tightly inserted in a tube holder and brought to the sea surface without being contaminated by ocean bacteria (Fig. 1A, D).

image

Figure 1. Collection of a mud sample at a depth of 10 897 m from the Challenger Deep. The white arrow shows the sampling tube holder (A). This holder is usually set in the box shown at the right side of the white arrow (A). The white arrowhead and open arrowhead show a 50 ml Falcon tube and sample inlet, respectively (A, C, D). The deep-sea mud was scooped out by a manipulator as shown in alphabetical order (B, C, D). The sampling tube filled with the mud was tightly inserted in a sampling tube holder (A).

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2.2Isolation of microorganisms from deep-sea mud

The mud sample was 2-fold diluted with sterile marine broth 2216[7] (Difco Lab.), and 100–200 μl (5–10 mg as dry weight) of the suspension was spread on the marine agar or half-strength nutrient agar plates used as a basal medium. In addition, modified marine agar plates supplemented with 1% skim milk or 1% potato starch with different pH (3, 7, or 10) and NaCl concentrations (0, 2, or 15%) were used for isolation. The alkaline and acidic media contained 1% sodium carbonate and 50 mM citric acid, respectively. The alkaline or acidic source was autoclaved separately as a 10-fold concentration and then added to the plain or the modified Marine agar medium. The agar plates were incubated at 4–75°C at atmospheric pressure (0.1 MPa) or at 100 MPa for 1–4 weeks. Cultivation techniques at high pressure were as described earlier[8].

2.316S rDNA sequencing and analysis

Twenty-eight strains were selected on the basis of cell or colony morphology among the Mariana isolates for 16S rDNA analysis. Each strain was cultivated on an agar plate under appropriate growth conditions as shown in Table 2. One loop of cell pellet was suspended in 50 μl of 25 mM Tris-HCl, 50 mM glucose, 10 mM EDTA and 1 mg ml−1 of lysozyme. The cell suspension was incubated at 37°C for 20 min and 5 ml of 10% SDS solution was added to the cell suspension to extract chromosomal DNA. The DNA was purified by phenol/chloroform treatment and then used as template DNA for PCR amplification of 16S rDNA. The following prokaryote-specific primers were used for the gene amplification and sequencing: EU10F (5′-AGAGTTTGATCCTGGCTCAG-3′), EU1500R (5′-GGTTACCTTGTTACGACTT-3′), EU500F (5′-GTGCCAGCAGCCGCGG-3′), EU500R (5′-GTATTACCGCGGCTGCTG-3′), EU1100F (5′-AAGTCCCGCAACGAGCGCA-3′), and EU1100R (5′-TTGCGCTCGTTGCGGGACT-3′). The amplified fragments were purified with Suprec™-02 (Takara Shuzo Co., Ltd., Japan) and sequenced using LI-COR DNA sequencer Model 4000L (LI-COR, Inc., Nebraska, USA).

Table 2.  Mariana isolates used in 16S rDNA analysis
Strain no.Growth conditionsRemarks
 MediumTemp. (°C)pH 
  1. aSabourauD's agar containing glucose (4%) and NaCl (2%)[15].

  2. bHalf strength of nutrient broth.

Mesophile
Neutrophile
HTA064marine agar257.6protease producer
HTA208marine agar257.6 
HTA437marine agar257.6protease producer
HTA456marine agar257.6amylase producer
HTA473marine agar257.6amylase producer
HTA484nutrient agar257.6 
HTA506nutrient agar257.6 
HTA527nutrient agar257.6 
Alkaliphile
HTA212marine agar257.6–10.5amylase producer
HTA218marine agar257.6–9.5amylase producer
HTA275marine agar257.6–9.5amylase producer
HTA333marine agar257.6–10.5 
HTA459marine agar257.6–10.5 
HTA474marine agar257.6–10.5 
Facultative psychrophile
HTA554nutrient agar 4–257.6 
HTA557nutrient agar 4–257.6 
HTA563nutrient agar 4–257.6 
HTA580nutrient agar 4–257.6 
HTA608SabourauD's agara 4–257.6 
Thermophile
HTA426marine agar55–757.6 
HTA454marine agar45–657.6 
HTA462marine agar55–757.6 
HTA14151/2 nutrient agarb45–707.6 
HTA14161/2 nutrient agar45–707.6 
HTA14171/2 nutrient agar45–707.6 
HTA14181/2 nutrient agar55–707.6 
HTA14201/2 nutrient agar45–707.6 
HTA14221/2 nutrient agar45–707.6 

16S rDNA sequences were aligned using the Clustal multiple-alignment program (Clustal W)[9]. Sites involving gaps were excluded from all analysis. A phylogenetic tree was inferred by the neighbor-joining method[10]. The sequences determined in this study were aligned with previously deposited sequences in the GenBank and Ribosomal Database of the University of Illinois at Urbana-Champaign. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL and GenBank nucleotide sequence databases under the accession numbers AB002633 for HTA333, AB002634 for HTA474, AB002635 for HTA275, AB002636 for HTA218, AB002637 for HTA064, AB002638 for HTA459, AB002639 for HTA212, AB002640 for HTA484, AB002641 for HTA563, AB002642 for HTA437, AB002643 for HTA506, AB002644 for HTA454, AB002645 for HTA426, AB002646 for HTA462, AB002647 for HTA1418, AB002648 for HTA1422, AB002649 for HTA1417, AB002650 for HTA1420, AB002651 for HTA1416, AB002652 for HTA1415, AB002653 for HTA473, AB002654 for HTA456, AB002655 for HTA557, AB002656 for HTA554, AB002657 for HTA527, AB002658 for HTA608, AB002659 for HTA580, AB002660 for HTA208, and AB002661 for alkaliphilic Bacillus sp. C-125.

2.4Cultivation of Mariana isolates under high pressure

Seventeen isolates were selected at random as representatives of extremophilic or non-extremophilic bacteria. A marine agar plate was inoculated with each isolate and overlaid with low melting agarose (1% w/v) dissolved in the same medium. Half-strength nutrient agar was used for culturing the last three strains shown in Table 3. Each agar plate was slipped into a plastic bag and sealed with a heat sealer. The bacteria were cultivated under temperature and pH conditions appropriate for each isolate (Table 2) at 30 MPa or 60 MPa for 2 weeks. All isolates were cultivated at 4°C under 100 MPa for 2 weeks.

Table 3.  Growth pattern of Mariana isolates under high hydrostatic pressure
Strain no.Temp. (°C)pHHydrostatic pressure (MPa)
   3060100a
  1. aAll isolates were cultivated at 4°C under 100 MPa. Symbols: −, no growth; +, slight growth; ++, growth; +++, good growth.

Mesophile
Neutrophile
HTA064257.6++++++
HTA208257.6+++++
Alkaliphile
HTA212259.5++
HTA275259.5++++
HTA459259.5++++
HTA474259.5+++
Facultative psychrophile
HTA55487.6++++
HTA55787.6+++++
HTA56387.6+++++
HTA58087.6++++
HTA60887.6+++++
Thermophile
HTA426557.6
HTA454557.6
HTA462557.6+
HTA1416557.6
HTA1417557.6
HTA1418557.6

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Isolation of microorganisms from deep-sea mud

Various non-extremophilic bacteria were isolated from the mud sample with a frequency of 2.2×104–2.3×105 colonies per g dry sea mud under the conditions described in Table 1. This is the first report of the isolation of viable bacteria from the bottom of the Challenger Deep. Previously, by means of microscopic observations, ZoBell[2] confirmed the existence of viable bacterial cells in mud collected at a depth of 10 400 m from the Philippine Trench, but none of these bacteria were cultured. We have obtained thousands of isolates from the Challenger Deep. Some were found to be producers of enzymes such as proteases and amylases.

Table 1.  Recovery of colony-forming extremophilic bacteria at a depth of 10 897 m of the Challenger Deep
CategoryIsolation conditionsBacteria recovered (colonies g−1 dry sea mud)
  1. –: no growth obtained.

AlkaliphilepH 9.5–10.04.1×102–1.2×103
 25°C, 0.1 MPa 
Barophile100 MPa
 25°C, pH 7.6 
Thermophile55–75°C5.8×102–3.5×103
 pH 7.6, 0.1 MPa 
Psychrophile4°C2.0×102
 pH 7.6, 0.1 MPa 
Halophile15% NaCl, 25°C
 pH 7.6, 0.1, 100 MPa 
AcidophilepH 3
 25°C, 0.1, 100 MPa 
Non-extremophile25°C, 0.1 MPa2.2×104–2.3×105
 pH 7.2±0.4 

Although barophilic, halophilic, and acidophilic bacteria have not yet been detected in mud samples from the Challenger Deep, various other types of extremophilic bacteria were isolated at 0.1 MPa (Table 1). Among the extremophiles found were many alkaliphiles and thermophiles which would be expected to thrive in an extreme environment different from the in situ conditions of high hydrostatic pressure and low temperature in the Challenger Deep. The number of facultative psychrophilic isolates recovered showed a tendency to be smaller than those of alkaliphiles and thermophiles. In addition, we successfully isolated filamentous fungi and actinomycetes at 0.1 MPa with almost the same frequency (2.0×102 per g dry sea mud) as that of facultative psychrophilic bacteria. All isolates were stored in the gas phase area of a nitrogen freezer (−165 to −171°C) for further investigation.

3.216S rDNA sequencing and analysis

The total 16S rDNA sequences of Mariana isolates shown in Table 2 were determined. These sequences were aligned and compared with the 16S rDNA sequences of 27 reference strains categorized as actinomycetes, low G+C Gram-positive bacteria, and proteobacteria belonging to the α and γ subdivisions[11]. Evolutionary distances between Mariana isolates and reference strains were computed, and a phylogenetic tree was constructed using the NJ algorithm and the pairwise evolutionary distances (Fig. 2).

image

Figure 2. Unrooted phylogenetic tree showing the relationship of Mariana isolates to reference organisms. The numbers indicate the percentages of bootstrap samples, derived from 10 000 samples, that supported the internal branches[16]. Bootstrap probability values less than 50% were omitted from this figure. Bar = 0.01 Knuc unit. Black box, alkaliphile; gray box, neutrophile; white box, thermophile; ABC, facultative psychrophile.

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Five of the six alkaliphiles were categorized as actinomycetes. The 16S rDNA sequences of alkaliphilic strain HTA474 and HTA459 were 99% similar to that of Diezia maris and 98% similar to that of Aureobacterium testaceum. On the other hand, the 16S rDNA sequence of HTA333 categorized as a member of the proteobacteria γ subdivision was almost identical to that of Brevundimonas diminuta.

Thermophiles categorized as low G+C Gram-positive bacteria were distant from reference strains used in this study, whereas the 16S rDNA sequences of HTA426 were comparatively similar to that of Bacillus kaustophilus or Bacillus thermoleovorans. Four of the five facultative psychrophiles were categorized as members of the proteobacteria γ subdivision. These four psychrophiles which constitute two subclasses may be members of the genus Acinetobacter. Another facultative psychrophile, HTA563, categorized as a member of the low G+C Gram-positive bacteria had a 16S rDNA sequence 99% identical to Staphylococcus epidermidis. We consider this isolate to be indigenous to the site, and exclude the possibility that it grew as a contaminant. Neutrophiles were widely distributed as shown in Fig. 2. Two strains, HTA437 and HTA506, which constitute a subclass, may be members of the genus Bacillus. The 16S rDNA sequences of HTA484 and HTA208 were 98% identical to that of Bacillus cereus and 99% identical to that of Pseudomonas stutzeri.

3.3Growth pattern of Mariana isolates under high pressure

Seventeen isolates described in Table 3 were cultivated under high hydrostatic pressure. Thermophilic bacteria did not grow at pressures higher than 30 MPa during 2 weeks of cultivation, whereas strain HTA462 grew slightly at 30 MPa. The neutrophilic strain HTA064 showed better growth at 600 MPa than at 300 MPa and also grew slightly even at 100 MPa. The same was observed with strain HTA208. Alkaliphiles were more sensitive to inhibition at 60 MPa than the neutrophiles, although three of them (HTA212, HTA275, and HTA459) grew slightly at 100 MPa. Facultative psychrophilic bacteria showed growth patterns similar to mesophilic bacteria, neutrophiles and alkaliphiles under high hydrostatic pressure. It was found that some of the Mariana isolates grew slowly at 100 MPa, which is similar to the in situ conditions in the Mariana Trench, although no barophilic or barotolerant bacteria have been isolated yet following 2 week cultivation under 100 MPa.

We have obtained many isolates of varying taxonomic affiliations, and many additional types of microbes may exist in the mud of the deepest sea floor. The microbes present in the aquatic environment are free-living or attached. The attached microbes are usually found with the flocculate aggregates of particulate matter constituting ‘marine snow’[12–14]. Theoretical settling rates of the particulate matter, including phytoplankton, range from 1.0 to 0.1 m per day or 5000 m in 1–50 years[1]. Accordingly, almost all isolates obtained in this study would have been brought down with the particulate matter to the bottom of the Challenger Deep in the course of a long period of time.

We conclude that the deepest sea mud of the Challenger Deep is a depository of active or dormant microbes under high hydrostatic pressure and low temperature.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We thank Drs. C. Kato, F. Abe, H. Kobayashi, and M. Nagahama for useful suggestions. Thanks are also due to Mr. N. Masui and colleagues for technical assistance. We thank the Kaiko operation team and the crew of M.S. YOKOSUKA for collecting the mud sample.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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