Microbial Diversity of the Brine-Seawater Interface of the Kebrit Deep, Red Sea, Studied via 16S rRNA Gene Sequences and Cultivation Methods

Abstract

The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using
phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were
isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobiumand are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular
fatty acid compositions are consistent with those of otherHalanaerobium representatives, showing unusually large amounts of Δ7 and Δ11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater
interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the
bacterial domain, with the majority affiliated with the genusHalanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine.
In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine
pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched
at the brine-seawater interface.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.7.3077–3085.2001
July 2001, p. 3077–3085 Vol. 67, No. 7
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Microbial Diversity of the Brine-Seawater Interface of the
Kebrit Deep, Red Sea, Studied via 16S rRNA
Gene Sequences and Cultivation Methods
WOLFGANG EDER,
1
* LINDA L. JAHNKE,
2
MARK SCHMIDT,
3
AND ROBERT HUBER
1
Lehrstuhl fu¨r Mikrobiologie und Archaeenzentrum, Universita¨t Regensburg, D-93053 Regensburg,
1
and Institut fu¨r
Geowissenschaften, Universita¨t Kiel, D-24098 Kiel,
3
Germany, and Exobiology Branch,
NASA Ames Research Center, Moffett Field, California 94035
2
Received 12 February 2001/Accepted 17 April 2001
The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of
microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic
culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus
Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools.
Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl
concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other
Halanaerobium representatives, showing unusually large amounts of ⌬7 and ⌬11 16:1 fatty acids. Phylogenetic
analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene
sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus
Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to
the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach
demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea.
They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-
seawater interface.
Hypersaline ecosystems are one of the most unusual and
extreme environments on earth (15, 25, 36). Anaerobic, deep-
sea brine pools, which are located along various tectonic rift
systems, represent a special type of hypersaline environment.
During the last 50 years about 25 deep-sea brine pools (Fig. 1)
with highly saline waters were identified in the Red Sea, an
ocean in statu nascendi within the East African Rift Valley
system (1, 6, 11, 13, 14, 18, 46, 53, 60, 65). The brines of the
Red Sea are typical athalassohaline waters which are in the
main a reflection of the geology, geography, and topography of
the areas where they develop (18, 25, 27). The high salinity is
formed when seawater circulates through subbottom Miocene
evaporite deposits, obtaining geothermal heat and dissolved
solids before surfacing in the depression of the deeps (1, 12, 62,
72). Characteristic of the brine pools is the formation of gra-
dients along the brine-seawater interface, e.g., salinity, pH,
temperature, and oxygen gradients. Brine pools of different
origin are also found in the Gulf of Mexico (e.g., the Orca
Basin) and in the Mediterranean Sea (e.g., the Tyro, Bannock,
or Urania Basin) (19, 37, 44, 61, 63).
The Kebrit Deep (in Arabic, kebrit means sulfur) in the
northern Red Sea was first explored during a Valdivia cruise in
1971 and consists of a basin of approximately 1 by 2.5 km in
size (27, 52, 59). The deep is filled with a brine of 84 m in
thickness at a maximum depth of 1,549 m (27). At the brine-
seawater interface of the Kebrit Deep there is a steep increase
in salinity from 4 to 26% (wt/vol) NaCl (within only 3 m), an
increase in temperature from 21.6 to 23.4°C (within about
7 m), an increase of the CH
4
concentration from 50 nl/liter to
22 ml/liter, and a measurable brine pool H
2
S content of up to
12 to 14 mg of S/liter. Over the same interface the pH drops
from 8.1 to 5.5 and the O
2
concentration decreases from 3.2 ml
of O
2
/liter to zero (23, 27, 64). At the same time, the density
gradient created at the brine-seawater interface acts as an in
situ particle trap for organic and inorganic materials from the
Red Sea water (27–29, 40, 57, 60, 66).
During the last 30 years, detailed geological and geochemi-
cal investigations were carried out in the Kebrit Deep. In
contrast, information about the microbial communities of this
deep is very rare. Recently, novel bacterial and archaeal 16S
rRNA gene sequences have been retrieved from brine sedi-
ments (21, 48, 50). These investigations showed that novel
groups of Archaea and Bacteria (KB1 sequence group) thrive in
the extreme environment of the Kebrit Deep (21). The pres-
ence of archaeal methanogenesis is also suggested by the bio-
chemical characterization of C
40
isoprenoids, an archaeal bio-
marker, in sedimentary organic matter (45), and an apparent
biotic methane oxidation at the brine-seawater interface (23).
Biochemical investigations in similar brine pools (Orca and
Bannock Basins) indicated a high microbial potential at the
brine-seawater interface and suggest the presence of halophilic
microorganisms within the brine (17, 22, 39, 40, 68).
A great diversity of microorganisms have been isolated from
high-salinity environments, including aerobic and anaerobic
organisms of the bacterial and archaeal domains (25). These
halophilic Bacteria include sulfate reducers (reference 4 and
* Corresponding author. Mailing address: Lehrstuhl fu¨r Mikrobiolo-
gie, Universita¨t Regensburg, Universita¨tsstr. 31, D-93053 Regensburg,
Germany. Phone: 0941/943-3180. Fax: 0941/943-2403. E-mail: wolfgang
.eder@biologie.uni-regensburg.de.
3077

references therein) and anaerobic phototrophs, gram-positive
heterotrophs, and cyanobacteria (references 8 and 25 and ref-
erences therein). Some isolates, like Flexistipes sinusarabici,
represent separate lineages (24). In addition, the Halanaerobi-
aceae, which represent a monophyletic lineage within the Bac-
teria, are specifically adapted to their high-salt milieu (49, 55,
71). The halophilic Archaea known to date comprise aerobic
halophiles of the family Halobacteriaceae and anaerobic meth-
anogens of the family Methanosarcinaceae (16, 26, 47).
The goal of this research was to assess, for the first time, the
bacterial diversity of the brine-seawater interface of the Kebrit
Deep, Red Sea. In this initial survey, which is preliminary, a
twofold approach was used. This includes (i) phylogenetic
analysis of 16S rRNA gene sequences as indicators of prokary-
otic diversity and (ii) isolation and cultivation of halophilic
representatives to establish physiological and function poten-
tial within the ecosystem community.
MATERIALS AND METHODS
Sampling. Brine from the brine-seawater interface of the Kebrit Deep, Red
Sea (Fig. 1), was sampled during RV Sonne cruise SO 121 in 1997 using a rosette
sampler equipped with 24 niskin bottles (10 liters) and a conductivity-tempera-
ture-depths (CTD) unit for monitoring salinity, temperature, transmission, and
pressure (Sea-Bird Electronic, Bellevue, Wash.). The salinity of brine samples
was measured with a hand refractometer (Atago, Tokyo, Japan). Microorgan-
isms were concentrated by pumping anaerobic brine across a crossflow tangential
filtration unit (Pellicon Kasettensystem; Millipore, Eschborn, Germany) (Fig. 2)
under a CO
2
protective atmosphere (cell concentration factor, 400-fold). Con-
centrated brine sample KT-2 was reduced with sodium dithionite (about 0.1
␮M), and KT-3 was used without further treatment. In addition, sample KT-8
was taken from surface sediment of the Kebrit Deep by a chain-sack dredge
(station no. 17034-2). The sample located near the brine-seawater interface
consisted of oily ore rocks and brine (salinity, 15.6%). The samples were trans-
ported to the laboratory by air at ambient temperature and were stored at 4°C.
Strains. Halanaerobium praevalens DSM 2228, Halothermothrix orenii DSM
9562, and Halanaerobacter lacunaris DSM 6640 were obtained from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig,
Germany, and were cultivated by using the indicated DSMZ media.
Culture conditions. Enrichment cultures from samples KT-2 and KT-3 (KT-
2/3) and KT-8 were established in 28-ml serum tubes on board the RV Sonne.
Brine used for enrichments was supplemented with various sterile organic and
inorganic compounds (e.g., yeast extract, peptone, NaNO
3
, and Na
2
SO
4
) and
chemically reduced by the addition of 1 ml of 50% (vol/vol) H
2
S per 10 ml of
brine. In the laboratory, positive enrichments were transferred several times in
supplemented brine and then grown in a synthetic medium whose composition
FIG. 1. Outline of the Red Sea, showing representative brine pools.
The topographical map was generated with the Online Map Creation
program (Geomar, Kiel, Germany [http://www.aquarius.geomar.de]).
FIG. 2. Schematic drawing of the anaerobic filtration system. The crossflow filtration unit was equipped with five Durapore membranes (pore
size, 0.2 ␮m; filter surface, 0.46 m
2
) (Millipore). The cells were kept in suspension and were pumped under pressure over the filter surface. Before
filtration, the complete system was flushed for 10 min with CO
2
to remove oxygen. During the filtration procedure, a protective atmosphere of CO
2
was maintained to prevent cellular damage due to oxygen sensitivity, to prevent precipitation of inorganic compounds, and to keep the pH of the
sample constant. The concentrated brine was collected in tank 1; tank 2 was for storage. Brine flowed into the filtration unit at valve 1, concentrated
cells exited at valve 2, and the filtrate outflow was at valve 4. Valve 3 is a closed valve.
3078 EDER ET AL. APPL.ENVIRON.MICROBIOL.

was based on a chemical analysis of a Red Sea brine-seawater interface (brine
interface medium [BI medium]) (D. Garbe-Scho¨nberg [Institut fu¨r Geowissen-
schaften, Universita¨t Kiel, Germany], personal communication). BI medium
contained (per liter) 100 g of NaCl, 5.11 g of MgSO
4
䡠7H
2
O, 2.0 g of CaCl
2
䡠
2H
2
O, 107 mg of MgCl
2
䡠6H
2
O, 19 mg of MnSO
4
䡠H
2
O, 10 mg of SrCl
2
䡠6H
2
O,
2.7 mg of FeCl
2
䡠4H
2
O, 0.075 mg of NaNO
3
, 0.7 mg of ZnSO
4
䡠7H
2
O, 0.02 mg
of Na
2
MoO
4
䡠2H
2
O, 100 mg of KH
2
PO
4
, and 1.0 g of NaHCO
3
. The pH was
adjusted to 6.5 with HCl. The gas phase over the medium in incubation tubes was
replaced with N
2
, and residual O
2
was chemically reduced by the addition of 1 ml
of 50% (vol/vol) H
2
S per 10 ml of medium. Growth was determined by direct cell
counting with a Thoma chamber (depth, 0.02 mm).
Cell masses of Halanaerobium praevalens,Halothermothrix orenii,Halanaero-
bacter lacunaris, and the novel isolates were obtained by growth at 30°C (60°C for
Halothermothrix orenii) with stirring (100 rpm) in an 80-liter enamel-protected
fermentor (Bioengineering, Wald, Switzerland) pressurized with 300 kPa of N
2
(N
2
-CO
2
[80:20, vol/vol] for Halanaerobacter lacunaris and Halothermothrix ore-
nii).
Light and electron microscopy. Light microscopy, electron microscopy, and
photography were carried out as described elsewhere (31).
Lipid analyses. Freeze-dried cells (about 1 g) were extracted twice using
chloroform-methanol (2:1, vol/vol) under reflux for 1 h. Fatty acid methyl esters
(FAME) were prepared from a portion of the total lipid extract by a modification
of the mild alkaline methanolysis procedure of White et al. (67), which involves
heating at 37°C for 1 h and extraction with hexane-chloroform (4:1, vol/vol).
FAME were separated by thin-layer chromatography on Silica Gel G plates
(Merck, Darmstadt, Germany) using methylene chloride as the solvent. Gas
chromatographic analysis was done using a Perkin-Elmer model Sigma 3B
equipped with a flame ionization detector and a DB-5 megabore column (J&W
Scientific, Folsom, Calif.), with methyl tricosanoate as an internal standard.
Double-bond positions, cis-trans configurations, and confirmations of cyclopro-
pane rings were determined with dimethyl disulfide adducts (69) using gas chro-
matography-mass spectrometry (35).
DNA extraction, PCR, and cloning. Nucleic acids were extracted from 120 ml
of the brine-seawater interface sample KT-2 with the IsoQuick Nucleic Acid
Extraction kit (ORCA Research, Bothell, Wash.) according to the manufactur-
er’s instructions, followed by RNase treatment for 30 min and precipitation of
the nucleic acids with 1 volume of isopropanol. The nucleic acids of the halo-
philic isolates were extracted as described elsewhere (7). PCR amplifications of
the rRNA genes between Escherichia coli positions (5) 9 and 1406 or 9 and 1512
(9bF, bacterial primer; 1406uR and 1512uR, universal primers) were carried out
as described previously (21). PCR products of sample KT-2 were purified (Mi-
crocon 100; Amicon, Witten, Germany), and the 16S rRNA gene fragments were
cloned into the pCR2.1 vector (Invitrogen, Leek, The Netherlands) according to
the manufacturer’s instructions. The resulting ligation products were used to
transform E. coli TOP10F⬘cells. The presence of inserts of the appropriate size
in the transformants was identified by direct PCR screening; amplified ribosomal
DNA (rDNA) restriction analysis was performed as described previously (21).
Representative transformants were selected based on the fingerprinting pattern
of the rRNA gene clones, and the corresponding plasmid DNAs were obtained
using a QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany).
Sequencing of rRNA genes. 16S rRNA gene sequences of the halophilic iso-
lates and 16S rDNA clone sequences of sample KT-2 were sequenced with an
ABI Prism 310 capillary DNA sequencer (PE Applied Biosystems, Foster City,
Calif.), using the ABI Prism BigDye Terminator Cycle Sequencing Ready Re-
action kit (Perkin-Elmer), at the Institute for Genetics, University of Regens-
burg, Regensburg, Germany. The bacterial sequences were determined using a
set of specific and universal primers (21).
Phylogenetic analyses. For the analyses, an alignment of about 11,000 homol-
ogous full and partial primary sequences available in public databases (ARB
project [41; W. Ludwig and O. Strunk, http://www.mikro.biologie.tu-muenchen
.de/pub/ARB/documentation/arb.ps]) was used. The new bacterial 16S rRNA
gene sequences (1,365 to 1,473 nucleotides) were fitted in the 16S rRNA tree by
using the automated tools of the ARB software package (http://www.mikro.biologie
.tu-muenchen.de/pub/ARB/documentation/arb.ps). Distance matrix (Jukes-Cantor
correction), maximum-parsimony, and maximum-likelihood (fastDNAml) meth-
ods were applied as implemented in the ARB software package (42, 51). Insig-
nificant branching points were shown by multifurication. Phylogenetic distances
were determined using distance matrix analysis without applying a correction
factor. Each sequence alignment was checked manually, and the sequences were
analyzed with the CHECK_CHIMERA program of the Ribosomal Database
Project (43) to detect the presence of possible chimeric artifacts.
Nucleotide sequence accession numbers. The 16S rRNA gene sequences were
submitted to EMBL and have been assigned accession numbers AJ309519 to
AJ309527.
RESULTS
Transmission-temperature-depth profile of the brine-sea-
water interface. The data were collected during RV Sonne
cruise SO 121. Figure 3 shows the transmission-versus-depth
profile of the Kebrit Deep brine-seawater interface, including
data for temperature. At a depth of 1,468 ⫾3 m, 240 liters
of brine was obtained (station no. 17029-8; 24°43.16⬘N,
36°16.42⬘E). The original sample temperature was 22°C, with a
pH of 6.5. O
2
was not detected, and the water smelled strongly
of H
2
S. Over the 1-m length of the niskin bottles the salinity
varied from 4.6% at the top to 9.6% at the bottom. Samples
KT-2/3 were retrieved from the upper part of this brine-sea-
water interface, indicated by the beginning of an increase in
temperature and salinity (4.6 to 9.6%) and an decrease in
transmission (Fig. 3).
Enrichment and isolation. In order to isolate representative
microorganisms from the Kebrit brine, enrichment cultures
were established in 28-ml serum tubes on board ship by adding
different sterile organic and inorganic nutrients directly to the
brine. Two successful enrichments designated KT-2/3-3 (from
the brine-seawater interface, 1,468 ⫾3 m) and KT-8-13 (from
a brine surface sediment near the brine-seawater interface)
were grown anaerobically in original brine supplemented with
0.1% (wt/vol) thiosulfate and 0.01% (wt/vol) of a mixture of
equal parts of yeast extract, meat extract, peptone, and brain
heart infusion (C-Org). In the laboratory, the enrichment cul-
FIG. 3. Transmission-temperature-depth profile of the brine-sea-
water interface of Kebrit Deep, Red Sea. The salinities at water depths
of 1,465, 1,467, 1,469, 1,470, and 1,471 m (corrected depths) are 4.0,
4.2, 17.2, 24.5, and 26.0% (wt/vol) NaCl, respectively.
VOL. 67, 2001 MICROBIAL DIVERSITY OF DEEP BRINE-SEAWATER INTERFACE 3079

tures were transferred several times in the original brine and
then into synthetic BI medium. From each enrichment culture,
a single cell, which was optically trapped by using a strongly
focused infrared laser beam (“optical tweezers”), was sepa-
rated under visual control from the mixed culture and was
grown as a pure culture (selected cell cultivation [2, 30, 32]).
The pure cultures KT-2/3-3 and KT-8-13 (for simplicity, des-
ignated the enrichment cultures) were chosen as representa-
tives for the experiments described below. Unless indicated
otherwise, the novel isolates were cultivated in BI medium
supplemented with 0.01% C-Org. Several further enrichment
cultures resulted in single clones after selected cell cultivation,
but they showed 16S rRNA gene sequences identical to that of
either KT-2/3-3 or KT-8-13 and therefore were not further
characterized (data not shown).
Morphology. Cells of isolates KT-2/3-3 and KT-8-13 were
gram-negative, motile rods with rounded ends; the average cell
length was 1 to 2 ␮m, and the average cell width was 0.3 to 0.4
␮m. Addition of glucose (0.1%, wt/vol) resulted in an increase
of the cell length up to 6.5 ␮m (for KT-8-13). During growth,
cells of the two isolates appeared singly or in pairs. No evi-
dence of spore formation was observed. In transmission elec-
tron micrographs, the new isolates exhibited monopolar, poly-
trichous flagellation with up to three flagella per cell (Fig. 4).
Growth and physiological characterization. Isolates KT-2/
3-3 and KT-8-13 grew chemoorganoheterotrophically under
strictly anaerobic culture conditions. Both isolates were obli-
gate halophiles, with an optimal NaCl requirement of between
10 and 20% (wt/vol). They grew at NaCl concentrations of
between 5 and 34% at pH 6.5. In the presence of H
2
(5%,
vol/vol), thiosulfate or S
0
(but not sulfate) was reduced to H
2
S
without stimulating the growth rate.
Isolate KT-8-13 grew at temperatures of between 18 and
48°C, with an optimum of between 30 and 45°C. KT-8-13 was
able to grow heterotrophically on C-Org (0.001 and 0.1%) and
brain heart infusion (0.01%). No growth was observed on glu-
cose (0.1%), fructose (0.1%), saccharose (0.1%), maltose
(0.1%), lactose (0.1%), xylose (0.1%), betaine (0.1%), choles-
terol (0.1%), acetate (0.01%), yeast extract (0.01%), peptone
(0.01%) (Merck), Trypticase-peptone (0.01%) (BBL), meat
extract (0.01%), or Casamino Acids (0.01%). H
2
S concentra-
tions of 0.5 to 50% (vol/vol) were tolerated by isolate KT-8-13.
Cellular fatty acid composition. The cellular fatty acid com-
positions of isolates KT-2/3-3 and KT-8-13 in comparison with
those of representatives of three different halophilic genera are
given in Table 1. The novel bacterial halophiles Halanaerobium
praevalens,Halothermothrix orenii, and Halanaerobacter lacu-
naris were cultured under optimal growth conditions (see Ma-
terials and Methods) and were harvested in the exponential
growth phase. The FAME analysis of the Kebrit Halanaero-
bium isolates, KT-2/3-3 and KT-8-13, showed large amounts of
16:0 and 16:1 fatty acids and minor amounts of 18:0 and 18:1
fatty acids, consistent with the data for Halanaerobium praeva-
lens (Table 1) (71). The two Kebrit isolates contained a rela-
tively large proportion of monounsaturated 16:1 with unusual
bond positions at ⌬7 and ⌬11, closely matching the 16:1 isomer
composition of Halanaerobium praevalens. Halothermothrix
orenii contained little unsaturated FAME, while Halanaer-
obacter lacunaris had 16:1 with the commonly encountered ⌬9
position but an unusual 18:1 for a bacterium, with the bond at
⌬9. Hopanoids were not detected in the organisms analyzed
(data not shown).
16S rRNA phylogeny. (i) Halophilic isolates. A comparative
analysis of the 16S rRNA gene sequences revealed that isolates
KT-2/3-3 and KT-8-13 belong to the Halanaerobiaceae within
the monophyletic lineage of the Halanaerobiales (49, 55). Fur-
ther sequence alignments and phylogenetic analyses showed
the taxonomic and phylogenetic positions of the new isolates to
be among those of the members of the genus Halanaerobium
(Fig. 5). The sequence similarity of isolates KT-2/3-3 and KT-
8-13 is 97.1%. The sequence similarities of KT-2/3-3 and KT-
8-13 to other Halanaerobium species ranged from 95.7 to 99%
and from 95.5 to 99.4%, respectively.
(ii) Brine-seawater interface. Specific (9bF) and universal
(1406uR) 16S rRNA primers were used to amplify bacterial
sequences from bulk DNA derived from the brine-seawater
interface sample KT-2 (salinity, 4.6 to 9.6% NaCl). About 40
clones were obtained from the extracted nucleic acids. After
FIG. 4. Transmission electron micrographs of a dividing cell of Halanaerobium sp. strain KT-2/3-3 showing flagella (a) and of a single flagellated
cell of Halanaerobium sp. strain KT-8-13 (b). The cells were air dried and platinum shadowed. Bars, 1 ␮m.
3080 EDER ET AL. APPL.ENVIRON.MICROBIOL.

cloning, the 16S rRNA gene fragments were further charac-
terized by restriction endonuclease digestion. Based on a com-
parison of the restriction patterns on agarose gels, eight dif-
ferent bacterial groups were identified. From a representative
of each restriction pattern group, the 16S rRNA gene sequence
was determined and aligned with 16S rRNA sequences derived
from the ARB database. Clone sequences KT-2K20 and KT-
2K29 (representing 26% of the clones) were chimeras: KT-
2K20 of KT-2K1 and KT-2K23/KT-2K28, and KT-2K29 of
KT-2K1 and KT-2K38/KT-2K12.
The analysis of the clone sequences KT-2K1, KT-2K12, KT-
2K23, KT-2K28, KT-2K34, and KT-2K38 showed a high phy-
logenetic diversity within the bacterial domain. The phyloge-
netic positions of the derived clone sequences were supported
by the different tree reconstruction methods (see Materials
and Methods). KT-2K23 and KT-2K28 (together, 41% of the
derived clones) were affiliated with cultivated members of the
genus Halanaerobium (Fig. 5). They showed highest sequence
similarity to Halanaerobium sp. strain KT-8-13 (99.9%),
Halanaerobium fermentans (99.3%), and Halanaerobium sp.
strain KT-2/3-3 (97%). Sequence clones KT-2K1, KT-2K12,
and KT-2K38 (representing 26% of the clones) showed the
closest relationship to Clostridium subterminale or Propionibac-
terium acnes, whereas KT-2K34 (7% of the clones) is related to
sequence clone PVB OTU 4 (U15116) within the ␥-proteobac-
teria. The G⫹C contents of the rRNA gene sequences range
from 51 to 57%.
DISCUSSION
In brine-filled Red Sea deeps, the brine-seawater interface
represents a unique microbial ecosystem mainly determined by
a steep salt gradient (4 to 26% NaCl) within only a few meters.
The combination of salt, oxygen, and pH gradients may be
responsible for organisms specifically adapted to this environ-
ment. To date, nothing is known about the morphological and
physiological features of these organisms. The presence of
microorganisms in hypersaline brines (e.g., the Gulf of Mexico
and the Mediterranean Sea) was suggested by biochemical
investigations which included measurements of ATP or CH
4
,
lipid analysis, and epifluorescence microscopy (17, 20, 40, 54,
68). The novel halophilic isolates from the Kebrit Deep (KT-
2/3-3 and KT-8-13) are the first organisms to have been culti-
vated and isolated from a brine-seawater interface or the
deeper anaerobic, brine pool.
Based on 16S rRNA gene sequence comparisons, these Ke-
TABLE 1. Comparison of the cellular fatty acid compositions of isolates KT-2/3-3 and KT-8-13 with those of Halanaerobium praevalens,
Halothermothrix orenii, and Halanaerobacter lacunaris
Fatty acid
a
% in:
KT-2/3-3 KT-8-13 Halanaerobium
praevalens
Halothermothrix
orenii
Halanaerobacter
lacunaris
14 0.8 1.2 9.6 3.5 3.7
14:1, ⌬7 0.2 0.2 2.9 ND 0.5
14:1, ⌬9 0.1 0.0 0.9 ND ND
i15 0.1 ⬍0.1 0.1 0.1 0.2
a15 0.1 ⬍0.1 ⬍0.1 0.1 0.2
15 0.2 0.4 0.3 0.2 0.6
15:1, ⌬7 0.1 0.1 0.2 ND ND
15:1, ⌬9 0.1 0.1 0.4 ND ND
i16 0.2 0.1 ⬍0.1 0.4 0.2
16 17.3 24.8 17.9 54.9 20.9
16:1, ⌬7cis 7.3 10.1 12.3 ND 1.2
16:1, ⌬9cis 34.2 35.0 28.3 1.4 34.2
16:1, ⌬9trans 8.5 2.2 4.8 0.5 4.9
16:1, ⌬11cis 20.4 13.3 17.5 ND 1.1
i17 0.2 ⬍0.1 0.1 0.2 0.1
a17 0.2 0.2 0.1 0.3 ND
17 0.2 0.2 ⬍0.1 0.6 0.5
cy17 1.7 1.6 0.2 0.2 0.6
17:1 0.2 0.6 0.1 ND 2.6
i18 ND
b
⬍0.1 0.1 0.1 ND
a18 ND ND ⬍0.1 ⬍0.1 ND
18 2.5 1.9 2.7 29.4 5.3
18:1, ⌬9cis 0.5 1.8 0.9 3.3 10.3
18:1, ⌬9trans 0.5 0.3 0.1 2.0 2.2
18:1, ⌬11cis 2.0 5.0 0.4 1.0 1.0
18:1, ⌬11trans 0.6 ND ND 0.1 0.4
18:1, ⌬13cis 1.4 0.4 ND ND ND
i19 ND ND ND 0.3 1.4
a19 0.5 0.4 0.1 1.2 7.5
19 ND ND ⬍0.1 0.2 0.4
a
Fatty acids are designated by total number of carbon atoms:number of double bonds. The number after ⌬indicates the position of the double bond relative to the
carboxylic (⌬) end of the molecule, with cis and trans geometry indicated. cy, cyclopropyl ring; iand a,iso- and anteiso-branched fatty acids, respectively. The
concentrations of total fatty acids of KT-2/3-3, KT-8-13, Halanaerobium praevalens, Halothermothrix orenii, and Halanaerobacter lacunaris were 160.9, 228.2, 1,017.4,
187.3, and 45.6 ␮g g (dry weight)
-1
, respectively.
b
ND, none detected.
VOL. 67, 2001 MICROBIAL DIVERSITY OF DEEP BRINE-SEAWATER INTERFACE 3081

FIG. 5. 16S rRNA gene-based phylogenetic tree of the bacterial domain, including the 16S rDNA sequences from brine-seawater interface
sample KT-2 and the 16S rRNA gene sequences of the new halophilic isolates from Kebrit Deep, Red Sea. The KB1 group marks a cluster of
closely related environmental sequences, which were obtained from a sediment sample (depth, 1,515 m) of the Kebrit Deep (21). The topology
of the tree is based on results of a maximum-parsimony analysis. Reference sequences were chosen to represent the broadest diversity of Bacteria.
Only sequence positions that share 50% or more residues identical to the 16S rRNA sequences of a corresponding group were included for tree
reconstruction. Accession numbers for the sequences are indicated. The scale bar represents 0.10 fixed mutation per nucleotide position.
3082 EDER ET AL. APPL.ENVIRON.MICROBIOL.

brit isolates were identified as members of the Halanaerobiales,
a group of anaerobic, halophilic, fermentative bacteria. The
Halanaerobiales represent a separate lineage in the bacterial
domain (49, 55). Within this order, the isolates KT-2/3-3 and
KT-8-13 showed the closest relationship to cultivated members
of the genus Halanaerobium and most likely comprise new
species in the genus (3, 9, 38, 49, 55, 56, 71). Prior to our
investigations, members of the genus Halanaerobium have
been shown to occur only in sediments from salt lakes and
offshore oil fields; this study thus increases our knowledge of
the ecological distribution of these organisms (3, 9, 55, 56).
The results of the FAME analysis for the new Halanaerobium
species are consistent with the 16S rRNA data in that the new
species exhibit fatty acid patterns similar to those of
Halanaerobium praevalens (71). The FAME are dominated by
16:0 and 16:1 and minor amounts of 18:0 and 18:1. In contrast
to Halothermothrix orenii, the new isolates show the presence
of large amounts of unsaturated fatty acids (Table 1) (10). The
presence of relatively large amounts of ⌬7 and ⌬11 16:1 is very
unusual and may serve as a biomarker for Halanaerobium.
Small amounts of ⌬11 16:1 are also present in type I meth-
anotrophs; however, these bacteria also contain ⌬8, ⌬9, and
⌬10 16:1 (33, 34). Like most other Halanaerobium representa-
tives, Halanaerobium sp. strains KT-2/3-3 and KT-8-13 grew
over a wide NaCl range (5 to 34% NaCl). This physiological
flexibility allows the organisms to grow within the salt gradient
of the brine-seawater interface and in the highly saline lower
brine pool. The brine-seawater interface also exhibits a density
gradient which acts as an in situ particle trap for organic ma-
terial (Fig. 3) (29, 40, 57, 58, 66), providing an appropriate
environmental niche for heterotrophic bacteria. Indeed, these
chemoorganotrophic halophiles may contribute significantly to
the anaerobic degradation of suspended organic material en-
riched at the brine-seawater interface of the Kebrit Deep.
However, the occurrence of Halanaerobium representatives is
not restricted to the Kebrit Deep. Recently, a new member of
the genus Halanaerobium (isolate S5L4, AJ309521) was ob-
tained from the brine-seawater interface (salinity, 24.2%) of
the Shaban Deep, Red Sea (W. Eder and R. Huber, unpub-
lished results).
For phylogenetic analysis, the brine sample was concen-
trated anaerobically on board ship using a crossflow tangential
filtration unit operated under a protective CO
2
atmosphere
(Fig. 2). Two hundred forty liters of water of the brine-seawa-
ter interface was concentrated about 400-fold. Anaerobic sam-
pling of the brine is essential to circumvent the precipitation of
reduced inorganic compounds such as Fe(II) in the presence of
O
2
or the toxic effect of O
2
on strict anaerobes like methano-
gens (60, 70). From the concentrated brine sample KT-2, the
nucleic acids were extracted, and the 16S rRNA gene se-
quences were PCR amplified and cloned. The 16S rRNA gene
fragments were further characterized by amplified rDNA re-
striction analysis, and different restriction pattern groups were
identified. The phylogenetic analysis of a representative of
each restriction group showed that the majority of the se-
quences had high sequence similarity to cultivated microorgan-
isms within the bacterial domain. From the same bulk DNA,
archaeal PCR products were obtained, but these were not
further investigated for this study (Eder and Huber, unpub-
lished).
Using different tree reconstruction methods (see Materials
and Methods), the majority of the bacterial clone sequences
represented by KT-2K23 and KT-2K28 showed the closest
relationship with members of the genus Halanaerobium (55)
and exhibited the highest sequence similarity to the isolated
Halanaerobium sp. strain KT-8-13. Although KT-8-13 was en-
riched from the sedimentary portion of the brine pool, this
sequence was not detected in a previous in situ analysis of the
brine-sediment interface (21). That phylogenetic analysis iden-
tified a deep-branching KB1 sequence group (Fig. 5) (21). This
KB1 group could not be retrieved from the brine-seawater
interface sample, suggesting that representatives of the KB1
group are specifically adapted to the higher salinities within the
lower brine body (Fig. 3) and the sediments (the salinity of the
pore water is up to 26%), while KT-8-13, like KT-2/3-3, is more
naturally adapted to the interface environment.
ACKNOWLEDGMENTS
We are grateful to Karl O. Stetter for stimulating and critical dis-
cussions. For helpful discussions we thank Reiner Botz, Roger Sum-
mons, and Gerald Maroske. Furthermore, we thank Reinhard Rachel
and Peter Hummel for electron microscopy and Thomas Hader, Kon-
rad Eichinger, and Marcus Koch for technical assistance. We are
grateful for the valuable help of the crew on board the RV Sonne (SO
121 cruise) and for the valuable support of cruise leader Peter Stoffers.
This work was financially supported by the BMBF (grant no.
03G0121B to Karl O. Stetter and grant no. 03G0121A to Peter Stof-
fers) and by the Fonds der Chemischen Industrie (to Karl O. Stetter).
The work of L.L.J. was supported by a grant from NASA’s Exobiology
program.
REFERENCES
1. Ba¨cker, H., and M. Schoell. 1972. New deeps with brines and metalliferous
sediments in the Red Sea. Nat. Phys. Sci. 240:153–158.
2. Beck, P., and R. Huber. 1997. Detection of cell viability in cultures of
hyperthermophiles. FEMS Microbiol. Lett. 147:11–14.
3. Bhupathiraju, V. K., M. J. McInerney, C. R. Woese, and R. S. Tanner. 1999.
Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacte-
rium from an oil brine. Int. J. Syst. Bacteriol. 49:953–960.
4. Brandt, K. K., B. K. C. Patel, and K. Ingvorsen. 1999. Desulfocella halophila
gen. nov., sp. nov., a halophilic, fatty-acid-oxidizing, sulfate-reducing bacte-
rium isolated from sediments of the Great Salt Lake. Int. J. Syst. Bacteriol.
49:193–200.
5. Brosius, J., T. J. Dull, D. D. Sleeter, and H. F. Noller. 1981. Gene organi-
zation and primary structure of a ribosomal RNA operon from Escherichia
coli. J. Mol. Biol. 148:107–127.
6. Bruneau, L., N. G. Jerlov, and F. F. Koczy. 1953. Physical and chemical
methods (⫹Appendix), p. 99–112. Reports of the Swedish deep-sea expe-
dition, vol. III. Physics and chemistry, no. 4.
7. Burggraf, S., H. Huber, and K. O. Stetter. 1997. Reclassification of the
crenarchaeal orders and families in accordance with 16S ribosomal RNA
sequence data. Int. J. Syst. Bacteriol. 47:657–660.
8. Caumette, P. 1993. Ecology and physiology of phototrophic bacteria and
sulfate-reducing bacteria in marine salterns. Experientia 49:473–481.
9. Cayol, J.-L., B. Ollivier, B. K. C. Patel, E. Ageron, P. A. D. Grimont, G.
Prensier, and J. L. Garcia. 1995. Haloanaerobium lacusroseus sp. nov., an
extremely halophilic fermentative bacterium from the sediments of a hyper-
saline lake. Int. J. Syst. Bacteriol. 45:790–797.
10. Cayol, J.-L., B. Ollivier, B. K. C. Patel, G. Prensier, J. Guezennec, and J. L.
Garcia. 1994. Isolation and characterization of Halothermothrix orenii gen.
nov., sp. nov., a halophilic, thermophilic, fermentative, strictly anaerobic
bacterium. Int. J. Syst. Bacteriol. 44:534–540.
11. Charnock, H. 1964. Anomalous bottom water in the Red Sea. Nature 203:
591.
12. Craig, H. 1969. Geochemistry and origin of the Red Sea brines, p. 208–242.
In E. T. Degens and D. A. Ross (ed.), Hot brines and recent heavy metal
deposits in the Red Sea. Springer-Verlag, New York, N.Y.
13. Cruise report Menor 1. 1983. Preussag Meerestechnik, confidential report
PREE-CR-03-4 (1403 AH). Ministry of Petroleum and Mineral Resources,
Deputy Ministry for Mineral Resources, Jeddah, Saudi-Arabia.
14. Cruise report Menor 2. 1984. Preussag Meerestechnik, confidential report
PREE-CR-04-10 and PREE-CR-05-4 (1404AH und 1405AH). Ministry of
Petroleum and Mineral Resources, Deputy Ministry for Mineral Resources,
Jeddah, Saudi-Arabia.
VOL. 67, 2001 MICROBIAL DIVERSITY OF DEEP BRINE-SEAWATER INTERFACE 3083

15. DasSarma, S., and P. Arora. 1999. Halophiles. In Embryonic encyclopedia of
life sciences. [Online.] Nature Publishing Group, London, England. http://
www.els.net.
16. Davidova, I. A., H. J. M. Harmsen, A. J. M. Stams, S. S. Belyaev, and A. J. B.
Zehnder. 1997. Taxonomic description of Methanococcoides euhalobius and
its transfer to the Methanohalophilus genus. Antonie Leeuwenhoek 71:313–
318.
17. De Domenico, M., and E. De Domenico. 1989. Bannock Basin: first approach
to water microbiology, p. 100–102. In M. B. Cita, A. Camerlenghi, and C.
Corselli (ed.), Anoxic basins of the eastern Mediterranean: results of the
third conference, Bergamo (Italy), December 14–16, 1988. Ricerca Scienti-
fica Educazione Permanente, Mailand, Italy.
18. Degens, E., and D. A. Ross. 1969. Hot brines and recent heavy metal deposits
in the Red Sea. Springer-Verlag, New York, N.Y.
19. de Lange, G. J., and H. L. ten Haven. 1983. Recent sapropel formation in the
eastern Mediterranean. Nature 305:797–798.
20. Dickins, H. D., and E. S. van Vleet. 1992. Archaebacterial activity in the Orca
Basin determined by the isolation of characteristic isopranyl ether-linked
lipids. Deep-Sea Res. 39:521–536.
21. Eder, W., W. Ludwig, and R. Huber. 1999. Novel 16S rRNA gene sequences
retrieved from highly saline brine sediments of Kebrit Deep, Red Sea. Arch.
Microbiol. 172:213–218.
22. Erba, E. 1991. Deep mid-water bacterial mats from anoxic basins of the
Eastern Mediterranean. Mar. Geol. 100:83–101.
23. Faber, E., R. Botz, J. Poggenburg, M. Schmidt, P. Stoffers, and M. Hart-
mann. 1998. Methane in Red Sea brines. Org. Geochem. 29:363–379.
24. Fiala, G., C. R. Woese, T. A. Langworthy, and K. O. Stetter. 1990. Flexistipes
sinusarabici, a novel genus and species of eubacteria occurring in the Atlantis
II Deep brines of the Red Sea. Arch. Microbiol. 154:120–126.
25. Grant, W. D., R. T. Gemmell, and T. J. McGenity. 1998. Halophiles, p.
93–132. In K. Horikoshi and W. D. Grant (ed.), Extremophiles: microbial life
in extreme environments. Wiley-Liss, New York, N.Y.
26. Grant, W. D., M. Kamekura, T. J. McGenity, and A. Ventosa. Family
Halobacteriaceae. In G. Garrity (ed.), Bergey’s manual of systematic bacte-
riology, vol. 1. The Archaea, cyanobacteria, phototrophs and deeply branch-
ing genera, in press. Springer Verlag, New York, N.Y.
27. Hartmann, M., J. C. Scholten, P. Stoffers, and F. Wehner. 1998. Hydro-
graphic structure of brine-filled deeps in the Red Sea—new results from the
Shaban, Kebrit, Atlantis II, and Discovery Deep. Mar. Geol. 144:311–330.
28. Hemleben, C., W. Roether, and P. Stoffers. 1996. Meteor-Berichte 96–4;
O
¨stliches Mittelmeer, Rotes Meer, Arabisches Meer, Cruise No. 31. Meteor-
Berichte, Institut fu¨r Meereskunde der Universita¨t Hamburg, Hamburg,
Germany.
29. Henneke, E., and G. J. de Lange. 1990. The distribution of DOC and POC
in the water column and brines of the Tyro and Bannock Basins. Mar. Chem.
31:113–122.
30. Huber, R., S. Burggraf, T. Mayer, S. M. Barns, P. Rossnagel, and K. O.
Stetter. 1995. Isolation of a hyperthermophilic archaeum predicted by in situ
RNA analysis. Nature 376:57–58.
31. Huber, R., W. Eder, S. Heldwein, G. Wanner, H. Huber, R. Rachel, and K. O.
Stetter. 1998. Thermocrinis ruber gen. nov., sp. nov., a pink-filament-forming
hyperthermophilic bacterium isolated from Yellowstone National Park.
Appl. Environ. Microbiol. 64:3576–3583.
32. Huber, R., H. Huber, and K. O. Stetter. 2000. Towards the ecology of
hyperthermophiles: biotopes, new isolation strategies and novel metabolic
properties. FEMS Microbiol. Rev. 24:615–623.
33. Jahnke, L. L., and P. D. Nichols. 1986. Methyl sterol and cyclopropane fatty
acid composition of Methylococcus capsulatus grown at low oxygen tensions.
J. Bacteriol. 167:238–242.
34. Jahnke, L. L. 1992. The effects of growth temperature on the methyl sterol
and phospholipid fatty acid composition of Methylococcus capsulatus (Bath).
FEMS Microbiol. Lett. 93:209–212.
35. Jahnke, L. L., R. E. Summons, L. M. Dowling, and K. D. Zahiralis. 1995.
Identification of methanotrophic lipid biomarkers in cold-seep mussel gills:
chemical and isotopic analysis. Appl. Environ. Microbiol. 61:576–582.
36. Javor, B. 1989. Hypersaline environments: microbiology and biochemistry.
Springer-Verlag, Berlin, Germany.
37. Jongsma, D., A. R. Fortuin, W. Huson, S. R. Troelstra, G. T. Klaver, J. M.
Peters, D. van Harten, G. J. de Lange, and L. ten Haven. 1983. Discovery of
an anoxic basin within the Strabo Trench, eastern Mediterranean. Nature
305:795–797.
38. Kobayashi, T., B. Kimura, and T. Fujii. 2000. Haloanaerobium fermentans sp.
nov., a strictly anaerobic, fermentative halophile isolated from fermented
puffer fish ovaries. Int. J. Syst. Evol. Bacteriol. 50:1621–1627.
39. La Ferla, R., and E. Crisafi. 1991. Preliminary study on vertical distribution
of microorganisms in the Bannock Basin waters (Eastern Mediterranean
Sea). Mar. Ecol. Prog. Ser. 75:309–311.
40. LaRock, P. A., R. D. Lauer, J. R. Schwarz, K. K. Watanabe, and D. A.
Wiesenburg. 1979. Microbial biomass and activity distribution in an anoxic,
hypersaline basin. Appl. Environ. Microbiol. 37:466–470.
41. Ludwig, W. 1995. Sequence databases (3.3.5), p. 1–22. In A. D. L. Akker-
mans, J. D. Van Elsas, and F. J. De Bruijn (ed.), Molecular microbial ecology
manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
42. Ludwig, W., O. Strunk, S. Klugbauer, N. Klugbauer, M. Weizenegger,
J. Neumaier, M. Bachleitner, and K.-H. Schleifer. 1998. Bacterial phylogeny
based on comparative sequence analysis. Electrophoresis 19:554–568.
43. Maidak, B. L., J. R. Cole, T. G. Lilburn, C. T. J. Parker, P. R. Saxman, J. M.
Stredwick, G. M. Garrity, B. Li, G. J. Olsen, S. Pramanik, T. M. Schmidt,
and J. M. Tiedje. 2000. The RDP (Ribosomal Database Project) continues.
Nucleic Acids Res. 28:173–174.
44. MEDRIFF Consortium. 1995. Three brine lakes discovered in the seafloor of
the eastern Mediterranean. EOS 76:313–318.
45. Michaelis, W., A. Jenisch, and H. H. Richnow. 1990. Hydrothermal petro-
leum generation in Red Sea sediments from the Kebrit and Shaban Deeps.
Appl. Geochem. 5:103–114.
46. Miller, A. R. 1964. Highest salinity in the world ocean? Nature 203:590–591.
47. Ollivier, B., P. Caumette, J.-L. Garcia, and R. A. Mah. 1994. Anaerobic
bacteria from hypersaline environments. Microbiol. Rev. 58:27–38.
48. Olsen, G. J., D. J. Lane, S. J. Giovannoni, N. R. Pace, and D. A. Stahl. 1986.
Microbial ecology and evolution: a ribosomal RNA approach. Annu. Rev.
Microbiol. 40:337–365.
49. Oren, A. 2000. Change of the names Haloanaerobiales,Haloanaerobiaceae
and Haloanaerobium to Halanaerobiales,Halanaerobiaceae and Halanaero-
bium, respectively, and further nomenclatural changes within the order
Halanaerobiales. Int. J. Syst. Evol. Bacteriol. 50:2229–2230.
50. Pace, N. R., D. A. Stahl, D. J. Lane, and G. J. Olsen. 1986. The analysis of
natural microbial populations by ribosomal RNA sequences. Adv. Microb.
Ecol. 9:1–55.
51. Page, R. D. M., and E. C. Holmes. 1998. Molecular evolution: a phylogenetic
approach. Blackwell Science, Oxford, England.
52. Pautot, G. 1983. Les fosses de la Mer Rouge: approche ge´omorphologique
d’un stade initial d’ouverture oce´anique re´alise´e a` l’aide du Seabeam.
Oceanol. Acta 6:235–244.
53. Pautot, G., P. Guennoc, A. Coutelle, and N. Lyberis. 1984. Discovery of a
large brine deep in the northern Red Sea. Nature 310:133–136.
54. Pease, T. K., E. S. van Vleet, and J. S. Barre. 1992. Diphytanyl glycerol ether
distributions in sediments of the Orca Basin. Geochim. Cosmochim. Acta
56:3469–3479.
55. Rainey, F. A., T. N. Zhilina, E. S. Boulygina, E. Stackebrandt, T. P. Tourova,
and G. A. Zavarzin. 1995. The taxonomic status of the fermentative halo-
philic anaerobic bacteria: description of Haloanaerobiales ord. nov., Halobac-
teroidaceae fam. nov., Orenia gen. nov. and further taxonomic rearrange-
ments at the genus and species level. Anaerobe 1:185–199.
56. Ravot, G., M. Magot, B. Ollivier, B. K. C. Patel, E. Ageron, P. A. D. Grimont,
P. Thomas, and J. L. Garcia. 1997. Haloanaerobium congolense sp. nov., an
anaerobic, moderately halophilic, thiosulfate- and sulfur-reducing bacterium
from an African oil field. FEMS Microbiol. Lett. 147:81–88.
57. Ryan, W. B. F., E. M. Thorndike, M. Ewing, and D. A. Ross. 1969. Suspended
matter in the Red Sea brines and its detection by light scattering, p. 153–157.
In E. Degens and D. A. Ross (ed.), Hot brines and recent heavy metal
deposits in the Red Sea. Springer-Verlag, New York, N.Y.
58. Sackett, W. M., J. M. Brooks, B. B. Bernard, C. R. Schwab, H. Chung, and
R. A. Parker. 1979. A carbon inventory for Orca Basin brines and sediments.
Earth Planet. Sci. Lett. 44:73–81.
59. Schoell, M. 1974. Valdivia VA 01/03, Hydrographie II und III. Bundesanstalt
fu¨r Bodenforschung, Hannover, Germany.
60. Scholten, J. C., P. Stoffers, D. Garbe-Scho¨nberg, and M. Moammar. 1999.
Hydrothermal mineralization in the Red Sea, p. 369–395. In D. S. Cronan
(ed.), Handbook of marine mineral deposits. CRC Press, Boca Raton, Fla.
61. Scientific staff of Cruise Bannock 1984–12. 1985. Gypsum precipitation from
cold brines in an anoxic basin in the eastern Mediterranean. Nature 314:
152–154.
62. Shanks, W. C., and J. L. Bischoff. 1977. Ore transport and deposition in the
Red Sea geothermal system: a geochemical model. Geochim. Cosmochim.
Acta 41:1507–1519.
63. Shokes, R. F., P. K. Trabant, B. Presley, and D. F. Reid. 1977. Anoxic,
hypersaline basin in the northern Gulf of Mexico. Nature 196:1443–1446.
64. Stoffers, P., M. Moammar, M. Abu-Ouf, D. Ackermand, O. Alassif, Y. Al-
Hazim, S. Boldt, R. Botz, W. Eder, A. El-Garafi, M. El-Mamoney, D. Fleit-
mann, D. Garbe-Scho¨nberg, S. Geiselhart, D. Goedecke, M. Hartmann, S.
Klauke, K. Moussa, N. Mu¨lhan, T. Mu¨ hlstrasser, J. Poggenburg, W. Rehder,
M. Schmidt, M. Schmitt, D. Schoeps, J. Scholten, M. Shbalaby, A. Wismann,
and E. Yohannes. 1998. Cruise report SONNE 121, Red Sea. Hydrography,
hydrothermalism and paleoceanography in the Red Sea. No. 88. Geologisch-
Pala¨ontologisches Institut der Universita¨t Kiel, Kiel, Germany.
65. Swallow, J. C., and J. Crease. 1965. Hot salty water at the bottom of the Red
Sea. Nature 205:165–166.
66. Trefry, J. H., B. J. Presley, W. L. Keeney-Kennicutt, and R. P. Trocine. 1984.
Distribution and chemistry of manganese, iron and suspended particles in
Orca Basin brine. Geomar. Lett. 3:125–130.
67. White, D. C., W. M. Davis, J. S. Nickels, J. D. King, and R. J. Bobbie. 1979.
3084 EDER ET AL. APPL.ENVIRON.MICROBIOL.

Determination of the sedimentary microbial biomass by extractable lipid
phosphate. Oecologia 40:51–62.
68. Wiesenburg, D. A., J. M. Brooks, and B. B. Bernard. 1985. Biogenic hydro-
carbon gases and sulfate reduction in the Orca basin brine. Geochim. Cos-
mochim. Acta 49:2069–2080.
69. Yamamoto, K., A. Shibahara, T. Nakayama, and G. Kajimoto. 1991. Deter-
mination of double-bond positions in methylene-interrupted dienoic fatty
acids by GC-MS as their dimethyl disulfide adducts. Chem. Phys. Lipids
60:39–50.
70. Zeikus, J. G. 1977. The biology of methanogenic bacteria. Bacteriol. Rev.
41:514–541.
71. Zeikus, J. G., P. W. Hegge, T. E. Thompson, T. J. Phelps, and T. A. Lang-
worthy. 1983. Isolation and description of Haloanaerobium praevalens gen.
nov. and sp. nov., an obligately anaerobic halophile common to Great Salt
Lake sediments. Curr. Microbiol. 9:225–234.
72. Zierenberg, R. A., and W. C. I. Shanks. 1986. Isotopic constraints on the
origin of the Atlantis II, Suakin and Valdivia brines, Red Sea. Geochim.
Cosmochim. Acta 50:2205–2214.
VOL. 67, 2001 MICROBIAL DIVERSITY OF DEEP BRINE-SEAWATER INTERFACE 3085