ELSEVIER
FEMS Microbiology Ecology 3 I (I 996) 47-58
A systematic survey for thermophilic fermentative bacteria and
archaea in high temperature petroleum reservoirs
Gino S. Grassia aT*, Keith M. McLean b,‘, PhilippC G&at
Alan J. Sheehy a
a32,John Bauld ‘,
’ Microhiolog?:Research Unit. Faculty of Applied Science, University of Canberra. Belconnen. ACT 2616. Australia
b CSIRO Dil,ision of Exploration Geoscience. Prkwte Bag PO. Wemblx. WA 6014. Australia
L Enr~ironmentol Geoscience nrzd Groundwater Dkision. Australian Geological Surre.v Organisation. GPO Box 378,
Canberra. ACT 2601, Australia
Received 14 March 1996; revised 22 May 1996: accepted 22 May I996
Abstract
Production waters from 36 high temperature petroleum reservoirs were examined for the presence of thermophilic,
fermentative microorganisms.
The direct supplementation
of production waters with glucose and either yeast extract,
peptone, tryptone or casamino acid resulted in the isolation of thermophilic. fermentative microorganisms from 47% of the
petroleum reservoirs examined. Three distinctive morphological
groups were isolated from the production waters of
petroleum reservoirs with depths ranging from 396-3048 metres, temperatures ranging from 21-13O”C, salinities ranging
from 2% 128 g l- ’ and pHs ranging from 6.0-8.5. Group 1 were pleomorphic rod-shaped bacteria, Group 2 were sheathed
rod-shaped bacteria, and Group 3 were coccoid archaea. Partial characterisation
of strains from one seawater-flooded
petroleum reservoir and three non-watefflooded
petroleum reservoirs tentatively identified some strains in Group 1 as
members of the genera Thermoanuerobacter and Tl~ermoanaerobacteriu~?z, Group 2 as members of the Thermotogales order,
and Group 3 as members of the genus Thermococcus. Production water salinity determined the type of microorganisms that
were isolated. Group 1 organisms were found primarily in petroleum reservoirs with salinities less than 30 g/l, while Group
2 and 3 organisms were found to dominate in more saline reservoirs. The successful isolation of thermophilic, fermentative
microorganisms from petroleum reservoirs decreased significantly with increasing salinity and temperature. These findings
support the existence of a deep biosphere where fermentative microorganisms are widespread.
Keyw~ords: Petroleum
reservoir;
Thermophilic
fermenter;
Thermoanuerobocter:
Thermoanaerobacterimn;
Thermotogales;
Thermococcus
1. Introduction
* Corresponding
author. Tel: +61 (6) 201 2381;
I 6361: E-mail: [email protected]
’ Present address: CSIRO Division of Chemicals
Private Bag 10, Clayton. Victoria, Australia.
’ Present address: Total Exploration Production,
Technical Centre, Domaine De Beauplan Route
78470 Saint-Remy-Les-Chevreuse.
France.
Fax: f61
(6)
25
0168-6496/96/$15.00
Copyright
PII SO168-6496(96)00043-8
and Polymers.
Scientific and
De Versailles,
0 1996 Federation
of European
The first descriptions of thermophilic, fermentative microorganisms
from production waters of high
temperature petroleum reservoirs were of partially
characterised strains isolated from oil-water separators on a North Sea (UK) oil production platform
[ 1,2]. In a later study we reported the isolation of
Microbiological
Societies. Published
by Elsevier Science B.V.
G.S. Grassia et al. / FEMS Microbiology
48
thermophilic,
fermentative
microorganisms
in production waters of several high temperature petroleum
reservoirs [3]. The partially characterised strains isolated from these reservoirs were assigned to three
morphologically
distinctive groups (1) non-sheathed
rod-shaped bacteria, (2) sheathed rod-shaped bacte-
Table I
Chemical
and physical
Location
Australia
Bahrain
Dubai
Indonesia
New Zealand
United Kingdom
United States
Venezuela
characteristics
Petroleum reservoir
Alton
Challis *
East Mereenie
Fortescue *
Moonie
Skua ’
Awali
Fateh Mishrif *
Bula
Banyuasin
Semanggi
Mckee
Waihapa
Beatrice *
Hutton *
Murchison *
Thistle *
Wytch Farm
Atkinson
Broussard
Carrie Ball
Delcambre
East Texas
Fitch
Lac des Allemands
Lafayette
Lynch Yates
Milne Point
Moresi
Shirley Barbara
State lease
Rangely Webre
Rankin
Redwash
Webre
Lake Maracaibo
of the petroleum
reservoirs
Water- flooding practiced
no
Yes
no
yes
no
no
“0
yes
no
no
no
no
no
yes
yes
yes
yes
yes
no
no
no
no
no
no
tl0
no
no
yes
no
no
no
no
yes
yes
no
no
Ecology 21 (I9961 47-58
ria, and (3) irregular coccoid archaea. These strains
were also assessed as to their suitability for microbially enhanced oil recovery in high temperature
petroleum reservoirs. Subsequent
studies by other
laboratories also have isolated similar fermentative
microorganisms
from produced waters of high tem-
surveyed
for the presence of thermophilic,
Reservoir characteristics
a
Deoth (m) Temnerature
CC)
1800
1410
1440
2 347
1800
2333
803
2590
396
75
70
60
92
75
96
55
40-l 10
< 50
N/A
500
2100
3 048
2058
3048
3017
2 743
N/A
50
79
88
40-115
37-104
21-109
40- 130
65
113
148
87
111
78
87
73
N/A
3 542
5 527
1851
4182
1700
1859
2913
2 895
1100
2134
1128
3 353
5110
1981
2400
1540
1981
2000
N/A
70
76
41-52
110-112
140
69
88
52
69
80-90
fermentative
DH b Salinitv (me I8.5
6.0
7.3
3.0
2800
43000
100 307
N/A
7.5
5.5
1.7
6.8
6.8
N/A
N/A
7.8
7.5
N/A
110000
16410
49 700
14260
N/A
N/A
13480
28 500
7.1
7.8
7.5
N/A
6.4
5.7
5.6
6.3
6.4
7.5
6.8
6.6
6.1
7.9
7.0
7.5
7.4
6.0
7.1
6.9
7.9
6.3
7.1
N/A
25 755
23810
13000
200 000
192000
181500
100 269
78000
60 160
97 605
110000
110000
14 362
23 200
137000
92 358
102250
90421
63 250
25 280
107000
4000
microorganisms
’ ) NaCl (rns l- ’ j
1081
42 927
60 740
N/A
N/A
103 163
10500
42112
10600
N/A
N/A
13480
23 900
32000
23 647
21000
N/A
195 270
160239
129717
94 108
69310
55 956
91565
99 196
101000
11 168
22 230
80000
86 224
91565
89810
59517
17 138
90 000
N/A
a Characteristics
of petroleum reservoirs surveyed in this study were supplied by oilfield operators. In many cases the information was
incomplete and where possible has been supplemented with data obtained from the Oil and Gas Journal data book [14]. Where data could
not be obtained an N/A has been recorded.
b pH measured at the surface. A lower in situ pH will occur in many instances due to the increased temperatures and partial pressures of
carbon dioxide within petroleum reservoirs.
* Oilfield site is located offshore.
G.S. Grassia et al./ FEMS Microbiology
perature petroleum
reservoirs.
Non-sheathed
rodshaped bacteria identified in production waters include Thermoanaerobacter
[4-61 and Acetoanaerobium romashkovii [7]. Sheathed rod-shaped bacteria
identified in production waters include Thermotoga
elfi [8], Thermotoga subterranea [9] and strains of
Thermotoga [ 10,l I], Petrotoga miothemza, Geotoga
petraea and Geotoga subterranea [12]. Coccoid archaea identified in production waters include strains
of Pyrococcus and Thermococcus [5,13].
In this study we carried out a systematic survey
for the presence of thermophilic,
fermentative
microorganisms
in high temperature petroleum reservoirs having varying temperature, salinity and pH
conditions.
We compare their occurrence
within
non-waterflooded
and watefflooded petroleum reservoirs, and partially characterise a number of strains
isolated from petroleum reservoirs at widely separated geographic locations.
2. Materials
and methods
2.1. Petroleum
reservoirs
49
Table 2
Media used for characterization
different petroleum reservoirs
Component
(g
I_ ’ )
NaCl
NH&I
Na,SO,
MgSO,,.7H,O
H,PO4
K 2HPO,
MgC1,.6H,O
a
CaC1,.2H,O
a
Yeast extract a
Glucose a
Trace elements b (ml>
Vitamins ’ (ml)
Resazurin (ml)
of thermophilic
strains
Medium for specified petroleum
from
reservoir
Alton
Awali
Beatrice
Lake Maracaibo
1.0
0.9
_
10.0
0.9
0.8
0.75
1.5
0.9
0.075
4.0
3.6
1.0
5.0
1.0
30.0
0.9
3.0
0.75
1.5
4.5
0.38
4.0
3.6
1.0
5.0
1.0
4.0
0.9
0.28
0.75
1.5
0.2
0.075
4.0
3.6
1.0
5.0
1.0
0.28
0.75
1.5
0.2
0.075
2.0
7.2
1.O
5.0
1.0
Final pH of basal media is adjusted to between 6.5-6.8. All media
reduced with 4 ml 1-l of neutralized 12% (w/v) Na,S.9H,O.
a These components prepared and sterilized separately from basal
salts media.
b SL-10 solution [17].
’ Wolin solution [ 181.
surveyed
The location and characteristics of the 28 onshore
and 8 offshore petroleum reservoirs surveyed between 1988- 1995 are shown in Table 1. Of the
petroleum
reservoirs
investigated,
7 were waterflooded with seawater (Beatrice,
Challis,
Fateh
Mishrif, Hutton, Murchison, Fortescue and Thistle),
one with river water (Redwash), one with groundwater (Milne Point) and two by unspecified
sources
(Rankin and Wytch Farm). Samples of production
water were collected from operating oil wells through
sampling valves located on the well head or as close
to the well head as permitted. Samples were collected, after flushing the lines for at least 10 min,
into sterile 200 ml glass or plastic screw cap bottles
filled completely to expel air and then tightly sealed.
All samples were transported at ambient temperatures and stored at 4°C until processed.
2.2. Enrichment
Ecology 21 (1996) 47-58
and cultivation
Enrichment cultures were established by adding
sterile nutrients directly to production water and by
inoculating (2.5% v/v) sterile anaerobic culture me-
dia. Nutrient-supplemented
production waters were
prepared inside an anaerobic cabinet by transferring
50 ml of sample water to 125 ml serum vials containing combinations of nutrients added from sterile
anaerobic stock solutions. Also added to all enrichment cultures were 10 gl-’
elemental sulfur as a
hydrogen sink and 4 ml l- ’ of sodium sulfide solution (12% w v- ’ stock solution) as a reducing agent.
The nutrient-supplemented
production waters were
then overpressured with CO, to the calculated Pco,
required to achieve the pH values found in the
reservoir under study [15]. Enrichment cultures were
also established
in anaerobic
phosphate-buffered
(Table 2) and CO,-bicarbonate
buffered medium
[16]. The concentrations
of Na+, Mg2+, Ca2+ and
Cl- were varied to mimic the conditions prevailing
in the reservoir under study. Studies on subsequent
isolated strains employed only the phosphate-buffered
medium. All media preparation and manipulations
were carried out using strictly anaerobic techniques
[ 19,201. Media were dispensed under oxygen-free
nitrogen, carbon dioxide or nitrogen and carbon
dioxide (80%:20%) mixture using a modified Hungate technique [21]. Carbon sources, vitamins and
C.S. Grussia et (11./ FEMS Micro&dog!
50
mined on a gas chromatograph
equipped with a
thermal conductivity detector after separation on a 5
ft X l/8 inch stainless steel column packed with
Carbosieve B 120/140 mesh (Supelco Inc.). Carrier
gas was nitrogen or helium at 40 ml min- ‘, injector
and detector temperature set at 90°C and column set
at 80°C. Antibiotic sensitivity was tested by adding
antibiotics to give final concentrations
of 100 pg
ml-‘. Sodium azide was tested at a final concentration of 500 pg ml-l.
A Leitz Diaplan microscope equipped with phase
contrast and photomicroscopy
system was used for
direct examination and cell size determination.
For
electron
microscopy
negative
staining
used the
method of Cole and Popkin [231 and grids were
examined in a Phillips 2000 electron microscope at
an operating voltage of 60 kV.
DNA was isolated by the method of Marmur [24]
and the G + C content determined by thermal denaturation analysis [25].
reducing agents were added from sterile, anaerobic
stock solutions. Enrichment cultures were incubated
at a range of temperatures from 40 to 85°C and at the
reservoir temperature
provided it did not exceed
100°C. Pure cultures were isolated from positive
enrichments by repeated application of the agar shake
method [16] or by plating on media (Table 2) containing 10 g I-’ Gelrite (Kellco Div. of Merck and
Co. Inc.. San Diego, CA) in a glovebox under an
oxygen-free atmosphere of 10% hydrogen. 10% carbon dioxide and 80% nitrogen. Plates were incubated
at temperatures ranging from 55 to 75°C in anaerobic
jars flushed periodically with pure nitrogen to prevent growth inhibition due to the production and
build up of hydrogen. Single colonies were picked
and the step repeated at least two further times.
Purity was also checked by microscopy.
2.3. Growth, metabolic
and cellular charucterisation
Growth was determined
by measuring the increase in absorbance at 600 nm. Temperature characterisation studies were carried out in thermostatically
controlled circulating water baths capable of maintaining temperature to within f 0.1°C. Fermentation
end products were quantified by GC using the method
of Teunissen et al. [22]. H, and CO2 were deter-
Table 3
Petroleum
reservoirs
that yielded thermophilic.
fermentative
3. Results
Thermophilic,
fermentative microorganisms
were
isolated from the production waters of 19 petroleum
reservoirs located in Australia, Bahrain, Dubai, In-
microorganisms
Petroleum reservoir (Location)
Culture conditions
Alton and Moonie (Australia)
Challis (Timor Sea. Australia)
Awali (Bahrain)
70
40-60
50-70
2.8
43
16
Fateh Mishrif (Dubai)
Bula (Indonesia)
Banyuasin and Semanggi (Indonesia)
McKee and Waihapa (New Zealand)
Carrie Ball (USA)
Mime Point (Alaska. USA)
Redwash (USA)
Webre (USA)
Beatrice, Hutton. Thistle, and Murchison
(North Sea, UK)
Lake Maracaibo (Venezuela)
40-60
40-50
50-70
65-79
60
75
50-70
50
30-85
49
I4
N.D.
13-28
100
23
25
107
25-30
55-79
3
Temperature
N.D. = not determined.
(“C)
Ecolo,q~ 21 (IYY61 47-58
Description
Salinity (g I-
of major morphological
groups
’)
Rods in pairs, chains, filaments and sheathed rods
Sheathed rods and single rods
Rods in pairs, chains, filaments and sheathed rods
in pairs and chains, motile
Sheathed rods in pairs and chains, motile
Sheathed rods
Rods. and pleomorphic rods and coccoid cells
Pleomorphic rods, many in pairs and chains
Single rods
Sheathed rods
Motile rods and sheathed rods
Sheathed rods. large, uniform, motile rods
Pleomorphic rods in pairs and chains, sheathed rods,
and irregular-coccoid
cells
Pleomorphic rods in pairs, chains and filaments.
and rods with terminal spheroids
G.S. Grassia et al. / FEMS Microbiology Ecology 21 (1996) 47-58
donesia, New Zealand, United Kingdom,
United
States and Venezuela (Table 3). Pleomorphic rodshaped bacteria which occurred singly, in pairs,
chains and filaments, and sheathed rod-shaped bacteria which occurred singly, in pairs or chains were
51
isolated
from production
waters of both nonwatefflooded and watefflooded petroleum reservoirs.
The isolation of these bacteria did not differ significantly between seawater-flooded
(Beatrice, Challis,
Fateh Mishrif, Hutton, Murchison and Thistle), river
Fig. 1. Electron (a) and phase contrast (b, c, d. e) micrographs of thermophilic, fermentative strains isolated from production waters of high
temperature petroleum reservoirs. a: Negatively stained cells of strain A5G9 showing the presence of a coccoid cell attached to rod-shaped
cells; b: strain A3YE showing cells with terminal spheroids; c: sheathed strain BlYE; d: strain BS2 shown growing as several cells in a
single sheath; e: irregular coccoid-shaped
cells of strain a Al. Bar = I cm (a) and 10 pm (b. c. d. e).
52
G.S. Grassia et al. / FEMS Microbiology Ecolog)l21 11996147-58
waterflooded
(Redwash)
or groundwater-flooded
(Milne Point) reservoirs.
In contrast,
extremely
thermophilic
(TmaX 9 1“C) irregular coccoid-shaped
archaea were isolated only from production waters of
the seawater-flooded
North Sea petroleum reservoirs
(Beatrice,
Hutton, Murchison
and Thistle).
Extremely thermophilic archaea could not be isolated
from any of the other petroleum reservoirs examined.
Thermophilic
fermentative
microorganisms
were
successfully isolated from all production waters examined with salinity values of 40 g l- ’ or less,
except those from Lynch Yates. Those reservoirs
with waters of higher salinity either failed all attempts to isolate thermophilic
fermenters or supported growth only to 60°C. Moderately thermophilic
(T,,, 60°C) and halophilic strains were isolated from
the hypersaline waters of Fateh Mishrif, Webre and
Carrie Ball reservoirs. A sheathed strain from Fateh
Mishrif grew at salinities of up to 200 gl-’
with
optimal growth between 50 and 100 gl-’ at 60°C.
The moderate thermophiles isolated from these reservoirs often grew slowly and were difficult to maintain in culture.
A number of thermophilic
fermentative
strains
were partially characterised after being purified from
Alton, Awali, Lake Maracaibo and Beatrice production water enrichments (Table 4). All strains were
strictly anaerobic heterotrophs and none, except (YA 1
(TmaX91°C>, were capable of growth above 81°C. All
but one of these strains grew on carbohydrates in the
presence of added yeast extract. The exception was
CLAl. which grew only on yeast extract or other
complex nutrients such as peptone. Partial characterisation of reservoir strains allowed them to be categorised into one of three distinct groups (Table 4).
Group 1 strains were pleomorphic
rods which
occurred singly, in pairs, short chains and longer
filaments.
Chains occasionally
contained
coccoid
cells (Fig. la), a feature characteristic of many glycolytic, thermophilic anaerobes [26]. They were isolated from reservoirs in Australia, the Middle East
and Venezuela on media containing
yeast extract
(*glucose).
Similar morphologies
were also observed in production
waters from New Zealand
reservoirs. All strains grew over the 45-80°C temperature range, with an optimum of around 7O”C,
and all grew optimally at NaCl concentrations
of
0- 10 g l- ’ at 70°C. Growth was supported individu-
ally by yeast extract, tryptone, peptone, casamino
acids or by a range of carbohydrates, but not cellulose, in the presence of yeast extract. The addition of
carbohydrates generally enhanced the growth of these
strains. Group 1 strains fermented glucose to a mixture of end products including
lactate, acetate,
ethanol, hydrogen and carbon dioxide. The combination of end products varied between strains (Table
4). All strains in Group 1 with the exception of
A3G9 were capable of reducing thiosulfate and several also reduced elemental sulfur. Only A3G9 from
this group grew in the presence of 100 p,gml-’
of
rifampicin. G + C ratios ranged from 33.5-48.3%.
Group 2 strains were sheathed bacteria which
occurred singly, in pairs and in chains. Strains A3YE
and V7 always possess a spheroid at one cell end
(Fig. lb). These membrane bound structures often
contained several cells during the late exponential
growth phase. The remaining strains in this group
possess a visible extension of the sheath at both cell
ends (Fig. lc) or were enclosed in a sheath that
contained
several individual
cells (Fig. Id). The
strains grew in the temperature range 50-80°C and
growth was supported individually by yeast extract,
tryptone and peptone or by a range of carbohydrates
in the presence of yeast extract. Group 2 strains also
fermented glucose to a mixture of lactate, acetate,
ethanol, hydrogen and carbon dioxide. Elemental
sulfur was reduced by all strains except V7 while
thiosulfate reduction was variable. All Group 2 strains
grew in the presence of 100 pgml-’
of rifampicin.
G + C ratios ranged from 31-45% and strains A3YE
and BlYE contained long chain fatty acids in the
with
range C,, to C,, which co-chromatographed
long chain dicarboxylic acids of Thermotoga maritima and Fervidobacterium
nodosum (Leeming,
personal communication).
Group 3 is represented by a single strain isolated
from the Beatrice reservoir which is an extremely
thermophilic
(T,,, 91°C) member of the archaea.
Similar cells were isolated from other North Sea
reservoirs. Coccoid cells grew singly and in clusters
(Fig. le) on yeast extract and other complex nutrients that were fermented to hydrogen and carbon
dioxide. Hydrogen sulfide was formed in the presence of elemental sulfur and cells contained refractile intracellular sulfur globules. Thiosulfate was not
reduced. The strain was resistant to 100 kgml-’
of
4
A3Gl
A3G6.
Ah0n
Awali
Lake
ASG9
BIGI
VI5
microorgamsms
and width
Maracaibo
Maracaho
(brn)
coccr
chains.
with
wth
cocci
tilaments
wth
cocci
Rods in paws. chains.
cocci
chams
with
and filaments
cocci
Rods ,n par<.
with
pairs. chains.
filaments
Rods.
filaments
Rods rn paxrs. chains.
with cow
par*,
pars
tilamentr
Rod,.
Rods.
Lake Maracalbo
V7
Sheathed
termmal
spherords
with
rods, pairs
spheroid
and chains
Sheathed
terminal
with
rods smgly.
pairs. chains
2-5
2-4
3-10
3-10x
3-10
3-7
L-10
2-6
X 0.5-0.6
X 0.5-l
X 0 5-0.8
I
x 0 4-0.5
x 0.4-0.5
X 0.4-0.6
X 0.5-0.75
AWall
Beatrice
BIYE
Bal.Bor?
strains
= not derermmed.
Etoh
in media
AC. = acetate:
= lactate;
Lat.
production.
were grown
Beatrice
N.D.
of greatest
* All
aAl
Group 3. Archaea
and Bu65
BearrICe
AWall
Bo3
and BS4
BS?
rods in
and filaments
in Table
= ethanol.
shown
rod rn pairs
rodr in pairs
0.5-I
I-6
Z-4
and Lake
.O dramerer
x 0.4-0.5
X 0.5-0.8
4-10X03-0.4
?-10x0.5-I
2 for 24 h. Alton
m pairs and
aggregares
Cocci
and chains
Sheathed
and chains
Sheathed
spheroids
Rods wrth termmal
chains
Sheathed
Group 2b. Shealhed cells with terminal spheroids at both cell ends
Ahon
A3YE
BSI.
from
length
isolated
Average
(optrma)
(70)
I (70-75)
(70)
(60-65)
(55s79)
(70)
(65-70)
(70-75)
(65-70)
strains
(70-75)
Maracaibo
60-91
< 75 (60-70)
50-76
60-<65
< 65 (55-60)
55-79
45-8
55-79
45-70
37-79
55-79
4S&RI
45-79
(5-9)
(6)
(7)
(6)
(6-7)
(7)
(7)
(7-8)
(7)
(6-7)
(7)
(6)
17)
ts-
(5)
(IO)
Awali
Q-30)
(20-35)
at 70°C.
5-50
S-60
l-30(10)
(30)
IO)
(O-10)
(5)
(O-15)
5-70(10-15)
15-55
(g/l)
range
Awali
(5m IO)
O-15(0-15)
I-20
O-25
l-35
O-15
O-20
O-25
l-25
(optima)
Salinity
(Australia).
were incubated
4-9
5-8
6-8.5
5-8
6-8
6-9
6-8.5
5-9
5.5-8
6-8
6.2-8
4.9-8.5
5 S-8.5
(optmla)
pH range
of the Allon
range
waters
(“C)
Temperature
productron
rod-shared cells that demonstrate mixed acid fermentation
Morphology
fermenratwe
Group 2% Sheathed cells with single terminal spheroids
Lake
v5
and A3G8
A3G5.
AhOrl
A3G4,
AhOIl
A3G I, A3G?.
1. Pleomorrhir
of thermophrlrc.
A3Cd
Grout
Characrrrirtic~
Table
Lx.,
strains,
CO,.
Etoh.
Etoh.
CO>.
Hz
H,
Hz
Hz
H 2
H2
H,
Hz
H,
and Beatrice
Cyclorerine
Penrcillm.
Rlfamprcm
Rifamplcin
Rrfampicrn
Rifampicin
Rlfampicm
Rifampicm
Rifamprcin
Rifamprcm
rEZSlStZI”Ce
petroleum
strains
-
+
-
-
+
-
+
+
+
at 60-7O’C
+
+
+
+
+
+
_
_
+
End product\
55
3 I .5-37.
39.7
N.D.
36.6-39.7
44.7
40.5
42 2
35.4
33.5
42.5
39.0-4
+
+
48.3
+
+
given
I
I .o
rn order
G + C (Tm)
reservoirs
MolW
-
So reduction
Wenezuela)
reduction
s+-
Maracabo
Antibrotrc
and Lake
at erther 60 or 65°C
Etoh.
Hz
HL
CO?.
CO,.
CO,,
AC.. CO,.
AC., CO>,
AC., CO,.
CO?. Hz
AC,
Lat.,
AC.. Etoh.
AC., Lx..
CO,.
CO,.
H,
(UK)
AC.. CO,,
Etoh.
AC., Etoh.
Eroh.
Et&,
Lat.,
AC.. Lx.
Lat.,
Lat..
Lx..
Etoh.
AC., Lx.,
Etoh,
Ac.,C02.
AC., Lat.,
Lat..
fermentation
of glucose
a
Beatrice
End products
(Bahrain),
54
penicillin
was 55%.
G.S. Grassia et al. /FEMS
and cycloserine.
Microbiology
The G + C of this strain
4. Discussion
A number of thermophilic, fermentative bacteria
and archaea were isolated from high temperature
petroleum reservoirs throughout the world. The isolated strains were placed into one of three groupings
based on morphological and physiological characteristics. Group 1 strains were recovered from low
salinity
reservoirs
in Australia,
Venezuela
and
Bahrain and were also observed in enrichment cultures from a reservoir in New Zealand. These strains
were glycolytic thermophiles which fermented glucose to one or more of lactate, acetate, ethanol,
hydrogen and carbon dioxide. None of the strains
grew on cellulose and spores were not observed. The
morphology and physiology of strains in Group 1
suggest that they are similar to members of the
genera Thermoanaerobacter
and Thermoanaerobacterium [27]. Members of the genus Thermoanaerobacter are capable of reducing thiosulfate to hydrogen sulfide, a trait shown by isolated strains A3GlA3G8 and A5G9, and a species of Thermoanaerobacter described from a low salinity petroleum
reservoir [4]. Strains V5, V15 and BlGl are capable
of reducing thiosulfate only to elemental sulfur which
is a differentiating
characteristic of the genus Thermoanaerobacterium.
The strain A3G9 differs from
other strains in Group 1 because it is unable to
reduce thiosulfate and produces lactate and acetate as
the principal fermentation
end products from glucose. In addition its G + C ratio was higher than the
other isolated strains and it was capable of growth in
the presence of 100 p_g ml-l rifampicin. Lactate and
acetate are the principal glucose fermentation
end
products of Caldicellusiruptor
saccharolyticus
[28]
and Caldicellusiruptor
lactoaceticus [29], a number
of unidentified
strains from a New Zealand hot
spring [30] and spore-forming
strains from a
petroleum reservoir [ 111. However, unlike Caldicellusiruptor species this strain does not use cellulose
and spore-formation
has not been observed.
Group 2 strains are glycolytic thermophiles with
distinctive morphologies. These strains were isolated
from petroleum reservoirs in Australia, Venezuela,
Ecology 21 (1996) 47-58
Bahrain and the United Kingdom and were also
observed in enrichment cultures from reservoirs in
Indonesia,
Alaska, USA and Dubai. The strains
A3YE and V7 have a morphology similar to that of
members of the genus Fervidobacterium
[31-331.
These strains resemble members of this genus by
their temperature range for growth, their fermentative metabolism and end products, their growth at
low salinity and the possession of dibasic fatty acids
in the range C,, to C,, (only A3YE tested) which
are characteristic of the Thermotogales [32,34]. The
G + C ratio of A3YE is similar to F. islandicum
while the G + C ratio of V7 is somewhat higher than
other species in the genus. The remaining strains in
the group are morphologically
and physiologically
close to other members of the Thermotogales namely
Thermotoga, Thermosipho, Geotoga and Petrotoga
[12,34-371. These strains resemble members of the
above genera because they grew over a similar temperature range, fermented glucose to similar end
products, all grew in the presence of 100 p,gml-’
rifampicin and contained dibasic fatty acids in the
range Cz6 to C,, (only B 1Gl tested). The strains had
G + C ratios in the range 31-40% typical of these
genera. Growth was possible in the presence of NaCl
concentrations
of up to 70 g/l. Species from the
genera Geotoga, Petrotoga, and Thermotoga have
also been described from petroleum reservoirs [8- 131.
Group 3 contains a single strain of an extremely
thermophilic archaea that resembles members of the
genus Thermococcus [38]. This strain is a coccoidshaped organism able to grow up to 91°C and is able
to grow on yeast extract, peptone and other complex
nutrients. Carbohydrates do not support growth. This
organism was isolated from the Beatrice reservoir
and morphologically
similar organisms were isolated
from other North Sea reservoirs investigated in this
study. A range of hyperthetmophilic
coccoid archaea, including Thermococcus
species, have also
been isolated from offshore reservoirs in the North
Sea, UK, and the North Slope of Alaska, USA [ 131,
and from an onshore reservoir in the Paris Basin,
France [5].
All the thermophilic strains characterised in this
study were isolated from reservoirs with salinities
less than 40 g l- ’ and reservoir salinity appears to
influence the type of organism present. Strains resembling
Thermoanaerobacter
and Thermoanaer-
G.S. Grassia et al. / FEMS Microbiology
obacterium species were isolated from those reservoirs with salinities less than 30 g l- ’ and, with the
exception of B lGl, none of these strains were able
to grow above 2.5 gl-’ NaCI. Strains V7 and A3YE
resembling
Fervidobacterium
species were recovered only from the Alton, Moonie and Lake Maracaibo reservoirs that have very low salinity (less than
4 g 1-l > and neither strain grew above 20 g 1-l
NaCl. Ferridobacterium
nodosum, F. islandicum and
F. gondwanense
are all similarly
restricted
to
thermophilic
habitats with low salinity. Sheathed
organisms similar to other members of the Thermotogales were isolated from reservoirs with salinities
higher than 10 g 1-l and these strains were capable
of growth up to 70 g l- ‘. These salinity ranges are
typical of members of the genera Thermotoga (T.
maritima, T. neapolitana and T. eljii; O-60 gl-’
NaCl), Thermosipho (T. africanus; l-36 g 1-l NaCl),
Geotoga (G. subterrunea 5-100 gl-’
NaCl) and
Petrotoga (P. miotherma; 5- 100 g 1-l NaCl). Extremely thermophilic
archaea resembling
Thermococcus species were recovered only from North Sea
reservoirs having salinity less than 30 g 1- ’ and grew
optimally at this salinity.
From reservoirs with salinities higher than 40
g1-’ only a few attempts to isolate thermophilic
fermentative
bacteria were successful. Moderately
thermophilic
fermenters capable of growth at 200
g1-’ salinity at 60°C were isolated from the Fateh
Mishrif, Webre and Carrie Ball reservoirs, but these
microorganisms proved difficult to maintain and were
not characterised. The absence of halophilic, thermophilic bacteria from petroleum reservoirs have been
reported previously [10,39] and suggests that combinations of high salinity and high temperature may
not be supportive of microbial growth because incompatible physiological
and biochemical solutions
result for each extreme (Fig. 2). However, Hulothermothriw orenii a species of truly thermophilic CT,,,
7O”C), moderately halophilic (40-200 g l- ’ NaCl)
bacterium has been recently isolated from sediments
of a saline lake [45]. While it is possible that high
temperature-high
salinity petroleum reservoir habitats are unable to support microbial growth it is also
possible that delays in returning samples to the laboratory or unsuitable incubation conditions may have
resulted in the failure to isolate organisms.
This study has demonstrated that organisms with
Ecology 21 (1996) 47-58
55
v/
250-
a Successful
200-
0
0
0
isolation in other studies
0
cl
+
150 -
+
3
.3b
0
2
tz
0
+0
lW-
cl0
00
.+o*
Cl
0
ol
0
80
.
oeo
0
+
0
.
0
:
50-
.
+
.+
.
.
00 0
80
o
0
0
0
l
.
0
20
40
60
80
100
I
120
Temperature (OC)
Fig. 2. Temperature and salinity conditions used in attempts to
isolate petroleum reservoirs microorganisms
from production waters. a Data obtained from literature [1,2,5,8-10,12.13,39-441.
similar characteristics
appear to be globally distributed in production waters of petroleum reservoirs,
that the organisms are adapted to the salinity and
temperature
conditions
of the reservoir, and that
these factors determine the types of organisms present. Sheathed organisms similar to members of the
order Thermotogales
were present in all reservoirs
where thermophilic
fermenters were isolated irrespective of whether they were non-waterflooded
or
waterflooded
with seawater, groundwater
or river
water. Similar results were observed for the nonsheathed bacteria that resemble Thermounuerobucter
and Thermoanuerobacterium
species. This suggests
that these organisms are indigenous
to petroleum
reservoirs rather than being contaminants
that have
successfully
inhabited reservoirs after being introduced during drilling or with waterflooding.
This study also demonstrated that thermophilic,
fermentative microorganisms
are widespread within
high temperature petroleum reservoirs and that their
isolation
decreases
significantly
with increasing
petroleum reservoir salinity and temperature combinations. Strains isolated from petroleum reservoir
56
G.S. Grassia et al. / FEMS Microbiology
waters were broadly grouped according to morphology and their temperature and salinity requirements.
The regular isolation
of fermentative
organisms
within non-waterflooded
and waterflooded petroleum
reservoirs around the world is consistent with such
organisms having been successful residents of these
deep subsurface environments and possible others in
the crust of the Earth 133,461. We feel that our
findings support the existence of a deep biosphere
where fermentative microorganisms
are widespread.
Acknowledgements
This work was supported by Live Oil Services
NV. We thank the following operators for kindly
providing production water samples together with
chemical
and physical
reservoir
data:
AGL
Petroleum,
BHP Petroleum Pty. Ltd., and ESSO
Australia Ltd., Bahrain National Oil Co., Dubai
Petroleum Co., AGL Petroleum (Scram) Ltd., Petrocarp Exploration Ltd. with the assistance of Exxon
Chemicals New Zealand Ltd., BP Exploration (UK),
Conoco UK Ltd., BP Exploration (Alaska) Inc., BWN
Pty. Ltd., Petroleos de Venezuela SA and Corpoven
SA. We also thank Joanna Bruce for technical assistance, Rhys Leeming (CSIRO Division of Oceanography, Hobart, Tasmania) for lipid analyses, and the
Electron Microscopy Unit, RSBS, Australian National University
for assistance with electron microscopy. J.B. publishes with permission of the Executive Director, Australian Geological Survey Organisation.
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