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Ch. R-6 Isolation and Characterization of Anaerobic Halophilic Bacteria From Oil Reservoir Brines
Reprinted from: Microbial Enhancement of Oil Recovery - Recent Advances, edited by E.C. Donaldson 0 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
115
CH. R-6
ISOLATION AND CHARACTERIZATION OF ANAEROBIC HALOPHILIC BACTERIA FROM OIL RESERVOIR BRINES D. GEVERTZ, J.K. PATEREK, M.E. DAVEY and W.A. WOOD Salk Institute Biotechnology/Industrial Associates, Inc. (SIBIA), P.O. Box 85200, San Diego, CA 92186. ABSTRACT Three metabolic types of obligately anaerobic, halophilic bacteria were isolated from oil reservoir brines and characterized to better understand the ecology of an oil reservoir. These included a methanogenic bacterium, a sulfate-reducing bacterium, and a fermentative bacterium. All species were isolated by plating from enrichment cultures containing substrates specific for each metabolic type. The methanogenic bacterium was isolated by enrichment with trimethylamine. Methane production occurred only with trimethylamine compounds or methanol as substrates. Sodium, magnesium, and potassium were all required for growth. This organism appears to be a member of the genus Methanohalophilus, based on substrate utilization and general growth characteristics. The sulfate-reducing bacterium was isolated by enrichment with a lactate-sulfate medium containing 3% NaC1. This isolate utilized lactate as an electron donor for sulfate reduction and, in addition, contained desulfovirdin, typical of the genus Desulfovibrio. In contrast to the methanogen! only sodium was required for growth. Finally, the sheathed bacterium was isolated by enrichment with glucose. Sodium was also required by this isolate for growth. This organism is a fermentative bacterium surrounded by a sheath, and is capable of reducing elemental sulfur to hydrogen sulfide. These are all characteristics of the genus Thermotoga. INTRODUCTION Conventional waterflooding techniques used in secondary oil recovery leave a large percentage of the oil still in place (ca. 60-70%) (Mungan and Johansen, 1978). As waterflooding progresses, the ratio of oil to water in produced water decreases because water preferentially sweeps zones of highest permeability in the reservoir, leaving oil behind in the low permeability zones. Enhanced oil reccvery (EOR) techniques were developed for use after waterflooding in the final, or tertiary phase of oil recovery (Morrow and Heller, 1985). Some of these processes employ steam, polymers, or surfactants to help recover residual oil. Microbial enhanced oil recovery (MEOR) uses bacteria and their metabolic products to achieve this goal. MEOR can be carried out by several means (Brown g g . , 1986; Finnerty and Singer, 1983). Microorganisms can be grown in fermentors and their products, such as emulsifiers, solvents, polymers or surfactants, can be injected into a reservoir to aid in oil recovery. Secondly, indigenous or injected cells can be stimulated by the injection o f nutrients, generally in the form of carbohydrate, to grow
and p r o d u c e p r o d u c t s i n s i t u .
A l t e r n a t e l y , b a c t e r i a c a p a b l e o f u s i n g compo-
n e n t s p r e s e n t i n c r u d e o i l a r e i n j e c t e d , l i m i t i n g t h e need f o r n u t r i e n t s u pp 1einen t a t ion .
The a b i l i t y o f b a c t e r i a t o s u r v i v e and g r o w u n d e r r e s e r v o i r c o n d i t i o n s i s a A m u l t i t u d e o f extreme e n v i r o n -
p r e r e q u i s i t e f o r a s u c c e s s f u l MEOR p r o c e s s .
ments may b e p r e s e n t i n o i l r e s e r v o i r s w i t h c o n d i t i o n s such a s a n a e r o b i o s i s , h i g h p r e s s u r e , h i g h t e m p e r a t u r e and h i g h s a l i n i t y , a b r o a d s p e c t r u i i i o f pH v a l u e s , l o w n u t r i e n t c o n c e n t r a t i o n s , and t h e p r e s e n c e o f p o t e n t i a l l y t o x i c compounds.
A review o f reservoir conditions i n the top ten oil-producing
s t a t e s r e v e a l e d t h a t a p p r o x i i i i a t e l y o n e - f o u r t h o f r e s e r v o i r s have a c c e p t a b l e c o n d i t i o n s f o r p o t e n t i a l MEOR a p p l i c a t i o n s ( C l a r k
g fl.,
1981).
The c r i t e r i a
f o r s e l e c t i o n o f s u i t a b l e c o n d i t i o n s i n c l u d e d t e m p e r a t u r e s b e l o w 75"C, pH v a l u e s n e a r n e u t r a l i t y , and s a l t c o n c e n t r a t i o n s o f l e s s t h a n t e n p e r c e n t . These e n v i r o n m e n t a l c o n d i t i o n s have l e d t o t h e s u g g e s t i o n t h a t h a l o t o l e r a n t , t h e r i n o p h i l i c b a c t e r i a c a p a b l e o f a n a e r o b i c g r o w t h a r e good c a n d i d a t e s f o r an MEOR p r o c e s s ( M c I n e r n e y , 1983). The i n d i g e n o u s b a c t e r i a p r e s e n t i n an o i l r e s e r v o i r a r e an i m p o r t a n t e l e i n e n t i n an MEOR p r o c e s s f o r a t l e a s t t w o r e a s o n s .
F i r s t , t h e y may b e
s t i m u l a t e d d i r e c t l y b y t h e a d d i t i o n o f n u t r i e n t s and, second, t h e y may i n t e r a c t w i t h exogenous c e l l s i n j e c t e d i n t o t h e f o r m a t i o n .
Therefore, i t i s
e s s e n t i a l t o u n d e r s t a n d t h e i n i c r o f l o r a o f t h e r e s e r v o i r s o as t o p r e d i c t how t h i s p o p u l a t i o n w i l l respond t o p e r t u r b a t i o n by n u t r i e n t s and/or o t h e r bacteria.
Some o f t h e o r g a n i s m t h a t have been i d e n t i f i e d f r o i n o i l r e s e r v o i r
b r i n e s i n c l u d e s u l f a t e - r e d u c i n g b a c t e r i a ( Z o b e l l , 1 9 5 8 ) , pseudoinonads ( I l z u k a and Komagata, 1 9 6 4 ) , inethanogens ( B e l y a e v ( D a v i s , 1967; P f i f f n e r
g a-l.,
fl., 1 9 8 3 ) ,
and B a c i 1 l u s s p e c i e s
1985).
I n t h i s s t u d y , we have i s o l a t e d and c h a r a c t e r i z e d t h r e e n o n s p o r u l a t i n g anaerobic, h a l o p h i l i c b a c t e r i a froiii o i l f i e l d b r i n e s .
T h i s i n f o r i n a t i o n adds
t o our understanding o f t h e inicrobiology o f t h e o i l r e s e r v o i r .
These o r g a n -
isins b e l o n g t o t h e g e n e r a M e t h a n o h a l o p h i l u s , D e s u l f o v i b r i o , and, p o s s i b l y , Thermotoqa, and a r e b e l i e v e d t o be p a r t o f t h e n a t u r a l p o p u l a t i o n p r e s e n t a t this site.
O t h e r s p e c i e s b e l o n g i n g t o t h e s e g e n e r a have been i s o l a t e d f r o i n
m a r i n e and h y p e r s a l i n e e n v i r o n m e n t s w o r l d w i d e . MATERIALS AND METHODS E n r i c hinen t cu 1t u r e s F i e l d b r i n e was c o l l e c t e d f r o m t h e N o r t h Burbank u n i t i n n o r t h e a s t e r n Oklahoma i n s t e r i l e e v a c u a t e d b o t t l e s u n d e r a n a e r o b i c c o n d i t i o n s .
Brine
e n r i c h m e n t s were made b y t h e a d d i t i o n o f f i e l d b r i n e t o c o n c e n t r a t e d n u t r i e n t s o l u t i o n s f o r minimal d i l u t i o n o f t h e b r i n e .
A l l e n r i c h m e n t s w e r e s e t up
117
u n d e r a n a e r o b i c c o n d i t i o n s i n s t o p p e r e d 100 m l serum b o t t l e s and i n c u b a t e d a t 35-45°C. Anaerobic techniques M o d i f i e d Hungate t e c h n i q u e (Hungate, 1 9 6 9 ) , o r an a n a e r o b i c chamber (Coy L a b o r a t o r y P r o d u c t s , Ann A r b o r , M i c h i g a n ) was used f o r a l l m e d i a p r e p a r a t i o n as w e l l as f o r i s o l a t i o n and c u l t i v a t i o n o f b a c t e r i a . Media
~
The medium u s e d f o r p r i m a r y i s o l a t i o n o f m e t h a n o g e n i c b a c t e r i a was made i n a f i e l d b r i n e b a s e and c o n t a i n e d t h e f o l l o w i n g ( i n g / l ) : e x t r a c t , 2.0;
NH4C1, 0.5; y e a s t
t r y p t i c a s e p e p t o n e , 2.0; casamino a c i d s , 2.0; KzHP04, 0.2;
c y s t e i n e e H C 1 , 0.25; methylamineeHC1, 3.0.
sodium b i c a r b o n a t e , 2.0; sodium c a r b o n a t e , 2.0; and t r i The medium was a d j u s t e d t o pH 6.8-7.0
s u l f i d e , 0.25 g / 1 , was used as a r e d u c t a n t .
w i t h KOH.
Sodium
The gas phase was n i t r o g e n / c a r b o n
d i o x i d e (80:ZO). "BBM" medium f o r i s o l a t i o n o f f e r m e n t a t i v e b a c t e r i a contained the following (in g/l): KNO3, 1.0;
y e a s t e x t r a c t , 0.5;
NaCl, 66.6; NHqS04, 0.13;
CaC12, 15.3; s u c r o s e , 4.0;
MgC12.2H20, 5.1; PIPES [ p i p e r a z i n e -
N, N ' - b i s - ( 2 - e t h a n e s u l f o n i c a c i d ) ] s o d i um s a l t monohydrate, 8.5; p h o s p h a t e , 0.4;
The medium was a d j u s t e d t o pH 6.8 w i t h NaOH. as a r e d u c t a n t .
a-glycerol
and t r a c e e l e m e n t s o l u t i o n , 1.0 m l (Widdel and P f e n n i g , 1 9 8 4 ) . Cysteine.HC1, 0.2 g / 1 , was used
Medium d e s i g n a t e d "MMS" was used t o t e s t s u b s t r a t e u t i l i z a -
t i o n and a n t i b i o t i c s e n s i t i v i t y o f f e r m e n t a t i v e i s o l a t e s (Huber
g
c . ,1 9 8 6 ) .
P o s t g a t e ' s Medium C , w i t h m i n o r m o d i f i c a t i o n s and c o n t a i n i n g 3 5 NaCl, was used f o r t h e i s o l a t i o n o f s u l f a t e - r e d u c i n g b a c t e r i a ; s u l f a t e - f r e e g r o w t h was t e s t e d u s i n g P o s t g a t e ' s Medium D ( P o s t g a t e , 1 9 8 4 a ) .
Oxidation o f short-chain f a t t y
a c i d s , p a l m i t a t e , and b e n z o a t e c o u p l e d t o t h e r e d u c t i o n o f s u l f a t e was t e s t e d u s i n g a m o d i f i c a t i o n o f t h e media b y F o w l e r
d.(1986).
Hydrogen u t i l i z a -
t i o n was t e s t e d w i t h t h e medium o f B a d z i o n g and Thauer ( 1 9 7 8 ) .
A l l media
c o n t a i n e d 1 m g / l i t e r o f r e s a z u r i n as a r e d o x i n d i c a t o r . Growth s t u d i e s G r o w t h s t u d i e s were p e r f o r m e d i n B a l c h t u b e s o r serum v i a l s c o n t a i n i n g 5-10 m l o f medium.
Each t u b e was i n o c u l a t e d f r o m a l i q u i d c u l t u r e o f t h e
i s o l a t e d organism.
Growth was m o n i t o r e d b y i n c r e a s e i n a b s o r b a n c e a t
550-600 nm.
In t h e c a s e o f t h e methanogen, g r o w t h was a l s o m o n i t o r e d
m i c r o s c o p i c a l l y b y f l u o r e s c e n c e a t 420 nm and by p r o d u c t i o n o f methane.
118
Microscopy Routine observations on isolates were made with an Olympus BH-2 phase microscope equipped with a fluorescence attachment. Electron micrographs were taken with a Philips 300 or CM-10 transmission electron microscope. Thin sections were prepared by fixing cells in 3% glutaraldehyde and 1% paraformaldehyde, followed by osmication and embedding. Negatively stained samples were prepared by allowing the cells to attach to a freshly ionized carbon formbar grid, followed by a brief wash in double distilled water, and staining with 2% uranyl acetate. Chemicals Vancomycin, streptomycin, chloramphenicol, rifampicin, Coenzyme M , and bromoethanesulfonic acid (BES) were purchased from Sigma Chemical Company (St. Louis, MO). Clindamycin and cephalothin were purchased from Fluka Chemical Co. (Ron kon koma, NY) . Gas chromatoqraphy
Methane production was monitored using a Hewlett Packard gas chromatograph model 5890 equipped with a thermo-conductivity detector. A carbosieve Sll column was used for separation of the gases. Helium was used as a carrier gas. The oven temperature was held at 35°C for seven minutes and then increased at a rate o f 32°C per minute to a final temperature of 225"C, after 12 minutes. The injector temperature was llO"C, and the detector 230°C. RESULTS Description of site and composition of reservoir brine Reservoir brine was collected from the tank battery o f Tract 5 in the North Burbank unit operated by Phillips Petroleum Company (Bartlesville, OK). This field is a sandstone formation located in northeastern Oklahoma with an average temperature of 45°C and a depth of 3,000 feet. Brine was collected after it passed through the skimmer tank, just prior to reinjection. The brine had a total dissolved solids content of approximately 10% and a pH of 6.2. Chemical analysis showed that the major cations in the brine were: sodium, 3.06; calcium, 0.6%; magnesium, 0.12%; and barium, 0.07%. The major anion was chloride, at a concentration of 6.5%. Preparation of brine enrichments Enrichments were prepared by adding 100 ml of reservoir brine to seruin bottles containing nutrients either as dry solids or concentrated solutions. Various types and combinations of nutrients were utilized in order to examine
119
the microbial population under differing conditions. The addition of nutrient generally resulted in an increase in cell number from 103 cells per ml of brine to 108 cells per ml. Dominant members of the enrichment cultures were isolated by plating on media of the same general composition as that used for enrichment. Pure cultures of organisms were obtained by successive transfers on solid medium or in roll tubes. A methanogenic bacterium, a sulfate-reducing bacterium, and a fermentative isolate were subsequently identified by microbiological and biochemical characterization. Isolation and characterization of microorganisms Methanohalophilus species. The methanogenic isolate (designated BT-5), is a nonmotile, irregular coccus 0.8-1.2pm in diameter, as measured by phase microscopy. An electron micrograph of this organism, shown in Figure 1 , revealed cells of irregular size and shape. The cells stain gram negative and have a tendency to form clumps. Colonies in roll tubes were raised, circular and smooth, with a light brown to brownish-green color.
Fig. 1. Thin section of the methanogenic isolate BT-5. Bar, 1 pm. Some of the distinguishing characteristics of isolate BT-5 are shown in Table I , along with those of other type species. Methanol and methylamines were used by this organism as substrates for methanogenesis. The traditional substrates for methane formation, hydrogen and carbon dioxide, acetate, and formate, were not utilized by this isolate. Bromoethanesulfonic acid (BES), a
120 metabolic inhibitor (Gunsalus g aJ., 1978), and tetracycline both inhibited growth. This organism was insensitive to penicillin, 0-cycloserine, erythromycin, clindamycin, and cephalothin. These observations are consistent with the archeobacterial pattern of sensitivity to antibiotics. Lysis of cells occurred in distilled water, sodium dodecyl sulfate, or Triton X-100. Sodium, potassium and calcium ions were all needed for growth. The sodium chloride range for this organism was 1.5-23%, with the optimum occurring at 11.6-14.5%. The temperature range for growth was 2O-6O0C, with an optimum at 40°C. The optimum pH for this isolate was 6.8. Isolate BT-5 has characteristics similar to F. mahii obtained from the Great Salt Lake (Paterek and Smith, 1988) as shown in Table I. TABLE
I
Comparison of BT-5 Isolate to Members of the Genus Methanohalophilus Characteristic
BT-5
Gram reaction
negative
negative
negative
coccus
coccus
coccus
+
+
+
6.8
7.5
9.2
20-60°C
20-50°C
ND
40°C
35°C
45°C
Salt range (NaC1)
1.5-23%
1.5-23%
1 . 2 - 12.2'6
Optimum salt conc.
11.6-14.5%
11.6%
4.1%
+ +
+ +
ND
Morphology Substrates: methylamines, methanol
M. mahiia -~
M. _~
zhilinaeb
H2/C02, forinate acetate
pH optimum Temperature range Optimum temp.
Inhibitors BES
tetracycline others
BES, bromoethanesulfonic acid ND, not determined +, positive response or reaction - , negative response or reaction a Paterek and Smith, 1985; 1988. Mathrani g aJ., 1988.
cephal othi n
i
chl orinaphenicol
121
Thermotoga-like specie?. The fermentative isolate, designated T5, was morphologically distinct because the cell was surrounded by a sheath or "toga" structure (Huber aJ., 1986). Isolate T5 was variable in length (5-20 pm) and 1.0 pm in diameter, as measured by phase microscopy. The electron micrograph in Figure 2 shows the sheath-like structure surrounding the cell. Isolate T5 exhibited diverse morphologies with regard to cell size and amount of sheath when grown at varying salt concentrations. Cells appear rofi shaped, either singly or in short chains, and stain gram negative. This isolat formed smooth, raised, white colonies on agar plates.
a
Fig. 2. Negative stain of the fermentative isolate T-5. Bar 1 pm. Table I 1 compares the physiological characteristics of is0 ate T5 with members of the genus Thermotoqa. Different carbohydrates were utilized as an energy source by this organism. Approximately 0.1% yeast extract was required for growth. Elemental sulfur was facultatively reduced to hydrogen sulfide. Growth was inhibited by rifampicin at a concentration of 100 pg/ml. Sodium chloride was needed for growth, in the range of 0.25-lo%, with an optimum of 2.5%. The temperature range for growth was 3 0 - 5 5 O C , with an optimum of 5 O O C . This isolate possesses most of the characteristics of the genus Thermotoqa listed in Table 11.
122
TABLE I 1
Comparison o f T5 I s o l a t e with Members of t h e Genus Thermotoga T5
T. maritimaa
negative
negative
negative
negative
present
present
present
Characteristic Gram reaction
Sheath-like s t r u c t u r e present
neapolitanablc
thermarumC
Moti 1 i t y
+
+
+/ -
+
Carbohydrates fermented
+
+
+
+
Obligate anaerobe
+
+
+
+
Elemental s u l f u r reduced
+
+
I
30-55"C
55-90°C
55-90°C
55-84°C
80°C
80" C
Temperature range Optimum temperature S a l t range (NaC1) Optimum s a l t conc.
5OoC
80°C
0.25- 10% 0.24-3.75% 2.5%
Sensitivity t o rifampicin (100 pg/ml)
+
2.7%
NO
0.2-0.55%
ND
0.35% 4
N D , not determined +, p o s i t i v e response o r reaction
-, negative response o r reaction
g aJ., 1986. Jannasch e t fl., 1988. WindbergeFg aJ., 1989.
a Huber
Desulfovibrio species. The sulfate-reducing bacterium, designated SSR, i s a m c t i l e , curved rod, 2-5 pm in length and 1 pm i n diameter, as measured by phase microscopy. An electron micrograph o f t h i s organism, shown in Figure 3 , revealed comma-shaped c e l l s with polar f l a g e l l a . These organisms occur e i t h e r singly o r in p a i r s and s t a i n gram negative. Raised, black colonies were formed on a g a r p l a t e s containing ferrous iron.
123
Fig. 3.
N e g a t i v e s t a i n o f t h e s u l f a t e - r e d u c i n g i s o l a t e SSR.
Bar, 1 pm.
Table 111 l i s t s t h e c h a r a c t e r i s t i c s o f i s o l a t e SSR, a l o n g w i t h t h o s e o f o t h e r h a l o p h i l i c species o f Desulfovibrio.
Hydrogen, f o r m a t e , and l a c t a t e
were u t i l i z e d as e l e c t r o n donors f o r s u l f a t e r e d u c t i o n .
Acetate, ethanol,
p r o p i o n a t e , b u t y r a t e , p a l m i t a t e , and benzoate were n o t used as e l e c t r o n donors, and no growth was observed w i t h t h e s e s u b s t r a t e s .
Growth i n s u l f a t e -
An i n c r e a s e i n t h e v i s c o s i t y o f t h e c u l t u r e o v e r t i m e was apparent i n media c o n t a i n i n g y e a s t e x t r a c t , which i s t y p i c a l o f s a l t - r e q u i r i n g s t r a i n s o f D e s u l f o v i b r i o (Ochynski and Postgate,
f r e e media o c c u r r e d w i t h p y r u v a t e and fumarate.
1963).
The pigment d e s u l f o v i r d i n was p r e s e n t i n t h i s organism, as evidenced
b y f l u o r e s c e n c e o f c o n c e n t r a t e d c e l l s under u l t r a v i o l e t l i g h t a f t e r t h e a d d i t i o n o f 1 N sodium h y d r o x i d e (Postgate, 1984a). Sodium c h l o r i d e , i n t h e range o f D.5-15%, was r e q u i r e d f o r t h e growth o f t h i s organism.
The optimum sodium c h l o r i d e c o n c e n t r a t i o n ranged f r o m 2-6%.
Potassium c h l o r i d e c o u l d n o t s u b s t i t u t e f o r sodium c h l o r i d e , i n d i c a t i n g t h a t i s o l a t e SSR i s n o t a s t r a i n o f
g.
s a l e x i g e n s (Postgate, 1984b).
t u r e range f o r growth was 25-45"C, w i t h an optimum o f 35-40°C.
The tempera-
TABLE 111 Comparison o f SSR I s o l a t e t o O t h e r H a l o p h i l i c S t r a i n s o f D e s u l f o v i b r i o
Characteristic Grain r e a c t i o n
negative
negative
negative
vibrio
vibrio
vibrio
Morphology Desul f o v i r d i n
+
t
+
*
t
t
t
t
E l e c t r o n donors f o r sulfate reduction: hydrogen, formate, l a c t a t e ethanol acetate, butyrate, palmitate benzoate
NO
Tempera t u r e r a n g e
25-45°C
25-45"C
25-459c
T e m p e r a t u r e optimum
40°C
30-36OC
30-36OC
Optiriium s a l t c o n c . (NaC1)
2-6%
2.5%
N a+
ND
I o n requirement
2.5-5'0 c1-
ND, n o t d e t e r m i n e d +, p o s i t i v e response or r e a c t i o n - , n e g a t i v e response o r r e a c t i o n
a P o s t g a t e , 1984b. b l i d d e l , 1988.
DISCUSSION T h r e e p h y s i o l o g i c a l t y p e s o f b a c t e r i a were i s o l a t e d from r e s e r v o i r b r i n e s . The m e t h a n o g e n i c i s o l a t e , BT-5, was o b t a i n e d by e n r i c h m e n t w i t h t r i m e t h y l a m i n e , a i y p i c a l s u b s t r a t e f o r h a l o p h i l i c methanogens.
H a l o p h i l i c methanogens
c o m p r i s e a u n i q u e g r o u p because t h e y do n o t u s e t h e t r a d i t i o n a l s u b s t r a t e s f o r m e t h a n o g e n e s i s , &,
h y d r o y e n i c a r b o n d i o x i d e , a c e t a t e , and f o r m a t e .
Instead,
t h e s e methanogens g e n e r a t e methane f r o m m e t h a n o l o r N-methyl compounds (Mathrari
d., 1988;
P a t e r e k and S m i t h , 1 9 8 8 ) .
The o n l y r e c o g n i z e d genus
o f h a l o p h i l i c methanogens, M e t h a n o h a l o p h i l u s , c o n t a i n s t w o members. compares t h e c h a r a c t e r i s t i c s o f
y.
z h i l i n a e (Mathrani
( P a t e r e k and S m i t h , 1 9 8 8 ) , and i s o l a t e BT-5.
M.
ct aJ.,
1988),
Table I
F. mahii
z h i l i n a e was i s o l a t e d froin an
a l k a l i n e h y p e r s a l i n e l a k e , and i s d i s t i n g u i s h e d f r o i n t h e o t h e r t w o o r g a n i s m s
125
w,
isolated by its pH optimum of 9.2. Isolate BT-5 closely resembles y. from the Great Salt Lake. Methanogenesis has been studied in a number of hypersaline lakes (Giani gJ d., 1984; Oremland g fl., 1982; Paterek and Smith, 1985) and resulted in the isolation of several halophilic species. The fermentative sheathed isolate from brine closely resembles members o f the genus Thermotoga. 1. maritima, the first described member of this genus, was isolated from geothermally heated areas of marine sediments in Italy and the Azores (Huber g fl., 1986). Cells are characterized by the presence o f a sheath-like structure and an extreme temperature requirement, the highest reported for eubacteria. Two additional species, 1. neapolitana (Jannasch al., 1988) and 1. thermarum (Windberger d., 1989) differ from I. maritima in physiological properties, but still retain the sheath-like structure and a high temperature requirement. Isolate T5 resembles these species both morphologically and physiologicslly, although it is capable of growth at mesophilic temperatures. Another possibility is that this isolate belongs to the genus Thermosipho, a new genus within the Thermotoqales (Huber 9 fl., 1989). One distinguishing characteristic of Thermosipho is growth at lower temperatures than Thermotoga. Further characterization o f the T5 isolate by genetic techniques, such as DNA homology to other species and 16s ribosomal RNA sequencing, will establish its relationship to these genera. Reservoir brines would constitute a new environment for isolation of Thermotoqa or Thermosipho. Sulfate-reducing bacteria have long been associated with oil deposits (Davis, 1967; Zobell, 1958). Their presence can have a serious economic impact on the oil industry, causing problems such as corrosion of pipes, plugging o f injection wells and souring of fuel gas and oil (Cord-Ruwisch g aJ., 1987; Iverson and Olsen, 1984). The sulfate-reducing bacterium described here is a member of the genus Desulfovibrio and, based on its general characteristics, is probably a salt-requiring strain o f !. desulfuricans (Postgate, 1984b). Sulfate reduction has been reported to occur in several hypersaline environments such as the Dead Sea (Truper, 1969), Big Soda Lake (Smith and Oremland, 1987) and solar salt ponds (Klug g d., 1985). Carbohydrates, such as molasses, have traditionally been used as substrates for MEOR processes; initiation of growth depends on metabolism o f carbohydrate by fermentative bacteria. The Thermotoga-like isolate, T5, is capable of using carbohydrates directly, and is undoubtedly one of a number o f fermentative bacteria present in reservoir brines. Metabolism o f carbohydrate will result in the production of fermentation end products and other types o f metabolic by-products. One class of by-products produced in response to hypersaline conditions are osmoregulants, compounds used to maintain internal osmotic balance (Le Rudulier and Bouillard, 1983). Osmoregulants such a s
126
betaine and choline can, in turn, be broken down to methylamines, wh ch serve as substrates for methanogenesis (King, 1984a). The end products of carbohydrate metabolism, for example, hydrogen and acetate, can be utilized as electron donors for sulfate reduction, giving rise to the growth of ulfatereducing bacteria (Widdel , 1988). In many ecosystems, methanogenesis and sulfate reduction are competing processes for the same substrates; namely, hydrogen and acetate (Nedwell and Banat, 1981; Oremland and Taylor, 1978; Schonheit g, a-l., 1982). Hypersaline environments present a situation in which different substrates give rise to methane and hydrogen sulfide and, consequently, these metabolic types are not competitive (King, 1984b; Oremland -et_al., 1982). Surprisingly, sulfide is generally not produced in our carbohydrate enrichments, perhaps because of limited sulfate in the brine, exhaustion of other nutrients by fermentative bacteria, competition for substrates with other organisms, or the buildup of toxic end products. These interactions illustrate the type of community metabolism which will likely take place in an MEOR process upon the addition of nutrients, and underscores the need for understanding the microbiology of the oil reservoir before designing a MEOR process. Patterns of how the indigenous population responds to nutrient stimulation can then be established, s o as to predict and control their behavior. The reservoir conditicns at this site dictate that bacteria must be able to tolerate, survive, and grow anaerobically at salinities of up to 10% and at temperatures of approximately 45°C. The three organisms discussed here are capable o f proliferating under these conditions. These organisms are readily isolated from reservoir brine at this site and have been identified in brines from other locations in the Burbank field. We have isolated another Thermotoga-like organism from reservoir brine collected from a site in Texas as well. This suggests that these organisms were selected by, or adapted to, this extreme environment. Since these organisms are easily cultured on synthetic media in the laboratory, it raises questions about the source and concentration of nutrients present in the reservoir environment. Based on the composition of this brine, nutrients are most likely present at low concentrations. One form of adaptation to such nutrient deprivation is the formation of ultramicrobacteria, which are metabolically quiescent (Novitsky and Morita, 1978; Roszak and Coldwell, 1987). The ability to survive under these conditions would indicate that the organisms best suited for MEOR are those already present ir the reservoir. Screening programs have been devised based on this concept, e . ~ .examining , bacteria obtained from brine and evaluating their ability to respond to nutrients and to produce exopolymers, surfactants and other products beneficial to MEOR (Grula g, d., 1982; Lazar, 1983; Pfiffner
127
e t fl.,
1986).
A s u c c e s s f u l MEOR process w i l l u l t i m a t e l y depend on a sound
u n d e r s t a n d i n g o f m i c r o b i a l p h y s i o l o g y and ecology. ACKNOWLEDGEMENTS We acknowledge Michael McCaffery and Lance Washington a t t h e U n i v e r s i t y o f C a l i f o r n i a a t San Diego f o r p r e p a r i n g e l e c t r o n micrographs o f o u r i s o l a t e s . T h i s work was funded by P h i l l i p s Petroleum Company, B a r t l e s v i l l e , Oklahoma. REFERENCES
w-
Badziong, W. and Thauer, R.K., 1978. Growth y i e l d s and growth r a t e s o f f o v i b r i o v u l a r i s (Marburg) growing on hydrogen p l u s s u l f a t e and hydrogen m o s u l ? a t e as t h e s o l e energy sources. Arch. M i c r o b i o l . , 117: 209-214. Belyaev, S.S., Wolkin, R . , Zenealy, W.R., DeNiro, M.J., E p s t e i n , 5 . and 1983. Methanogenic b a c t e r i a f r o m t h e Bondyuzhskoe o i l f i e l d : Z e i k u s , J.G., g e n e r a l c h a r a c t e r i z a t i o n and a n a l y s i s o f s t a b l e - c a r b o n i s o t o p i c f r a c t i o n a t i o n . Appl. E n v i r o n . M i c r o b i o l . , 45: 691-697. Moses, V., Robinson, J.P. and Springham, D.G., 1986. M i c r o b i a l Brown, M.J., enhanced o i l r e c o v e r y : p r o g r e s s and p r o s p e c t s . C r i t . Rev. B i o t e c h . , 3: 159-197. C l a r k , J.B., Munnecke, D.M. and Jenneman, G.E., 1981. I n s i t u m i c r o b i a l enhancement o f o i l p r o d u c t i o n . Devel. I n d u s t . M i c r o b i o l . 22: 695-701. Cord-Ruwisch, R., K l e i n i t z , W. and Widdel, F., 1987. S u l f a t e - R e d u c i n g b a c t e r i a and t h e i r a c t i v i t i e s i n o i l p r o d u c t i o n . J. Pet. Tech., January: 97-106, D a v i s , J.B., 1987. Microbes i n i n j e c t i o n waters. Petroleum M i c r o b i o l o g y , E l s e y i e r P u b l i s h i n g Co., New York, New York, pp. 449-470. and S i n g e r , M.E., 1983. M i c r o b i a l enhancement o f o i l F i n n e r t y , W.R., r e c o v e r y . B i o t e c h n o l o g y , 1: 47-54. Fowler, V.J., Widdel, F., Pfenning, N., Woese, C.R. and S t a c k e b r a n d t , E., 1986. P h y l o g e n e t i c r e l a t i o n s h i p s o f s u l f a t e - and s u l f u r - r e d u c i n g eubact e r i a . Sys. Appl. M i c r o b i o l . , 8: 32-41. G i a n i , D . , G i a n i , L., Cohen, Y. and Krumbein, W.E., 1984. Methanogenesis i n t h e h y p e r s a l i n e S o l a r Lake ( S i n a i ) . FEMS M i c r o b i o l . L e t t . , 25: 219-224. G r u l a , E.A., R u s s e l l , H.H., B r y a n t , D., Kenaga, M. and H a r t , M., 1983. I s o l a t i o n and s c r e e n i n g of C l o s t r i d i a f o r p o s s i b l e use i n m i c r o b i a l enhanced o i l r e c o v e r y . I n t e r n a t i o n a l Conference on t h e M i c r o b i a l Enhancement o f O i 1 Recovery. I n : E.C. Donaldson and J.B. C l a r k ( E d i t o r s ) , Proceedings, U.S. Department o f Energy, B a r t l e s v i l l e Energy Technology Center, B a r t l e s v i l l e , Oklahoma, pp. 43-47. 1978. P r e p a r a t i o n o f Coenzyme Gunsalus, R . P . , Romesser, J.A. and Wolfe, R.S., M analogues and t h e i r a c t i v i t y i n t h e methyl Coenzyme M r e d u c t a s e system o f Methanobacterium thermoautotrophicum. B i o c h e m i s t r y , 17: 2374-2377. KSnig, H., Thomm, M., Woese, C.R., S l e y t r , U.B. Huber, R., Langworthy, T.A., and S t e t t e r , K.0, 1986. Thermoto a m a r i t i m a sp. nov. r e p r e s e n t s a new genus o f u n i q u e e x t r e m e l y t h e r m o p ~ i l F E d G Z t e r i a growing up t o 90°C. Arch. M i c r o b i o l . , 144: 324-333. Huber, R., Woese, C.R., Langworthy, T.A., F r i c k e , H. and S t e t t e r , K.O., 1989. Thermosi ho A f r i c a n i s new-genus new-species r e p r e s e n t s a new genus o f t h e r d b a c t e r i a w i t h i n t h e Thermotogales. Sys. Appl. M i c r o b i o l . , 12: 32-37. Hungate, R.E., 1969. A r o l l t u b e method f o r t h e c u l t i v a t i o n o f s t r i c t anaerobes. Methods M i c r o b i o l . , 3: 117-132.
I2X
Ilzuka, H. and Komagata, K, 1964. Microbiological studies on petroleum and natural gas. 11. Determination of pseudomonads isolated from oil-brines and related materials. J. Gen. Appl. Microbiol., 10: 223-231. Iverson, W.F., and Olsen, G.J., 1984. Problems related to the sulfate-reducing bacteria in the petroleum industry. In: R.M. Atlas (Editor), Petroleum Microbiology, MacMillan Publishing Co., New York, New York, pp. 619-641.. Janna:ch, H.W., Huber, R., Belkin, S and Stetter, K.O., 1988. Thermotoqa neapolitana sp. nov. of the extremely therinophilic, eubacterial genus motoga. Arch. Microbiol., 150: 103-104. King, G.M., 1984a. Metabolism of trimethylamine, choline and glycine betaine by sulfate-reducing and methanogenic bacteria in marine sediments. Appl. Environ. Microbiol., 48: 719-725. King, G.M., 1984b. Utilization of hydrogen, acetate and "non-competitive" substrates by methanogenic bacteria in marine sediments. Geoinicrobiol. J., 3: 275-306. Klug, M., Boston, P . , Francois, R., Gyure, R., Javor, B . , Tribble, G. and Vairavamurthy, A., 1985. Sulfur reduction in sediments of marine and evaporite environments. In: D. Sagan (Editor), The Global Sulfur Cycle, NASA Technical Memorandum 87570, July (1985), pp. 128-157. Lazar, I., 1983. Some characteristics of the bacterial inoculuin used for oil release from reservoirs. In: J.E. Zajic, D.C. Cooper, T.R. Jack and N. Kosaric (Editors), Microbial Enhanced Oil Recovery, PennWell Publishing Co., Tulsa, Oklahoma, pp. 73-82. Le Ruaulier, D. and Bouillard, L . , 1983. Glycine betaine, an osmotic effector in Klebsiella pneumonia and other members of the Enterobacteriaceae. Appl. Environ. Microbiol., 46: 152-159. Mathrani, I.M., Boone, D.R., Mah, R.A., Fox, G.E. and Lau, P.P., 1988. Methanohalophilus zhilinae sp. nov., alkaliphilic, halophilic, methylotrophic, methanogen; Int. J. Sys. Bacteriol., 38: 139-142. Mclnerney, M.J., 1983. Fhysiological types of microorganisms useful for enhanced oil recovery. In: E.C. Donaldson and J.B. Clark (Editors), Proceedings of the 1982 International Symposium on Microbial Enhanced Oil Recovery. Department of Energy, Bartlesville, Oklahoma, p p . 38-42. Morrow, N.R. and Heller, J.P., 1985. Fundamentals of enhanced oil recovery. In: E.C. Donaldson, G.V. Chilingarian and T.F. Yen (Editors), Enhanced Oil Recovery, 1. Fundamentals and Analysis, Elsevier, Amsterdam, The Netherlands, pp. 47-74. Mungan, N., and Johansen, R.T., 1978. Miscible Fluid Displacement. In: D.C. Bond (Editor), Secondary and Tertiary Oil Recovery Processes, The Interstate Oil Compact Commission, Oklahoma City, Oklahoma, p p . 40-66. Nedwell, D.R. and Banat, I.M., 1981. Hydrogen as an electron donor for sulfate-reducing bacteria in slurries of salt marsh sediment. Microb. c - c r l . , 7: 305-313. Nov;<-%ky, J.A. and Morita, R.Y., 1978. Possible strategy for the survival of m a .-I? bacteria under starvation conditions. Mar. Biol., 48: 289-295. Ochynsl:;, F.W. and Postgate, J.R., 1963. Some biological differences between :rFih water and salt water strains of sulphate-reducing bacteria. In: C.H. .. Oppanheimer (Editor), Marine Microbiology, C.C. Thomas, Springfield, i:l'?ois, p p . 426-441. Oremlarr'. R.S., Marsh, L.M. and DesMarais, D.J., 1982. Methanogenesis in Big :-oda Lake: an alkaline, moderately hypersaline desert lake. Appl. F?v?rcm. Microbiol., 43: 462-468. Oremland. R.S., Marsh, L.M. and Polcin, S . , 1982. Methane production and >imul-csr,eoussulphate reduction in anoxic, salt marsh sediments. Nature, 296: 143-145. and T a y l o r , B.F., 1978. Sulfate -eduction and methanogenesis Oremland, I!.':. in marire s-dime-its. Geochim. Cosmochim. Acta, 42: 209-214. Paterek, J.R. a n d Smith, P.H., 1985. Isolation and characterization of a halophilic methanogen from Great Salt Lake. Appl. Environ. Microbiol., 50: 877-881.
=-
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Paterek, J.R. and Smith, P.H., 1988. Methanohalophilus mahii [gen. nov., sp. nov.] a methylotrophic halophilic methanogen. Int. J. Sys. Bacteriol., 38: 122-123 Pfiffner, S.M., Jenneman, G.E., Walker, Jr., G.B., Javaheri, M., McInerney, M.J. and Knapp, R.M., 1985. Isolation and characterization of halotolerant, thermophilic, anaerobic, biopolymer-producing and biosurfactantprcducing bacteria. In: E.J. Zajic and E.C. Donaldson (Editors), Microbes and Oil Recovery (Vol . 1), International Bioresources Journal, pp. 285-294. Pfiffner, S.M., McInerney, M.J., Jenneman, G.E. and Knapp, R.M., 1986. Isolation of halotolerant, thermotolerant, facultative polymer-producing bacteria and characterization of exopolymer. Appl. Environ. Microbiol., 51: . .. 1224-1229. Postgate, J.R., 1984a. The Sulfate-Reducing Bacteria, Second Edition Cambridge, University Press, Great Britain, pp. 1-50. Postgate, J.R., 1984b. Genus Desulfovibrio. In: N.R. Kreig and J.G. Holt (Editors), Bergey's manual of systematic bacteriology, Vol. 1 , Wil iams and Wilkins, Baltimore, Maryland, pp. 666-672. Roszak, D.B. and Coldwell, R.R., 1987. Survival strateqies of bacter a in the natural environment. Microbiol. Revs., 51: 365-379.Schonheit, P., Kristjansson, J.K., and Thauer, R.K., 1982. Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate. Arch. Microbiol., 132: 285-288. Smith, R.L., and Oremland, R.S., 1987. Big Soda Lake (Nevada). 2. Pelagic Sulfate Reduction. Limnol. Oceanogr., 32: 794-803 Truper, H.G., 1969. Bacterial sulfate reduction in the Red Sea hot brines. In: E.T. Degens and D.A. Ross (Editors), Hot Brines and Recent Heavy Metal Deposits in the Red Sea, Springer-Verlag, New York, NY, pp. 263-271. Widdel, F., and Pfennig, N., 1984. Dissimilatory sulfate- or sulfur-reducing bacteria. In: N.R. Krieg and J.G. Holt (Editors), Bergey's Manual of Systematic Bacteriology, Volume 1, Williams and Wilkins, Baltimore Maryland, pp. 663-679. Widdel, F., 1988. Microbiology and ecology of sulfate- and sulfur-reducing bacteria. In: A.B.J. Zehnder (Editor), Biology of Anaerobic Microorganisms, John Wiley and Sons, Inc., New York, New York, pp. 469-585. Windberger, E., Huber, R., Tricone, A.. Fricke, H., and Stetter, K.O.. 1989. Thermotoga thermarum sp. nov. and Thermotoqa neapol itana occurring in African continental solfataric sprinas. Arch. Microbiol.. 151: 506-512 Zobell, C.E., 1958. Ecology of sulfatelreducing bacteria. Producers Monthly, 22: 12-29.
115
CH. R-6
ISOLATION AND CHARACTERIZATION OF ANAEROBIC HALOPHILIC BACTERIA FROM OIL RESERVOIR BRINES D. GEVERTZ, J.K. PATEREK, M.E. DAVEY and W.A. WOOD Salk Institute Biotechnology/Industrial Associates, Inc. (SIBIA), P.O. Box 85200, San Diego, CA 92186. ABSTRACT Three metabolic types of obligately anaerobic, halophilic bacteria were isolated from oil reservoir brines and characterized to better understand the ecology of an oil reservoir. These included a methanogenic bacterium, a sulfate-reducing bacterium, and a fermentative bacterium. All species were isolated by plating from enrichment cultures containing substrates specific for each metabolic type. The methanogenic bacterium was isolated by enrichment with trimethylamine. Methane production occurred only with trimethylamine compounds or methanol as substrates. Sodium, magnesium, and potassium were all required for growth. This organism appears to be a member of the genus Methanohalophilus, based on substrate utilization and general growth characteristics. The sulfate-reducing bacterium was isolated by enrichment with a lactate-sulfate medium containing 3% NaC1. This isolate utilized lactate as an electron donor for sulfate reduction and, in addition, contained desulfovirdin, typical of the genus Desulfovibrio. In contrast to the methanogen! only sodium was required for growth. Finally, the sheathed bacterium was isolated by enrichment with glucose. Sodium was also required by this isolate for growth. This organism is a fermentative bacterium surrounded by a sheath, and is capable of reducing elemental sulfur to hydrogen sulfide. These are all characteristics of the genus Thermotoga. INTRODUCTION Conventional waterflooding techniques used in secondary oil recovery leave a large percentage of the oil still in place (ca. 60-70%) (Mungan and Johansen, 1978). As waterflooding progresses, the ratio of oil to water in produced water decreases because water preferentially sweeps zones of highest permeability in the reservoir, leaving oil behind in the low permeability zones. Enhanced oil reccvery (EOR) techniques were developed for use after waterflooding in the final, or tertiary phase of oil recovery (Morrow and Heller, 1985). Some of these processes employ steam, polymers, or surfactants to help recover residual oil. Microbial enhanced oil recovery (MEOR) uses bacteria and their metabolic products to achieve this goal. MEOR can be carried out by several means (Brown g g . , 1986; Finnerty and Singer, 1983). Microorganisms can be grown in fermentors and their products, such as emulsifiers, solvents, polymers or surfactants, can be injected into a reservoir to aid in oil recovery. Secondly, indigenous or injected cells can be stimulated by the injection o f nutrients, generally in the form of carbohydrate, to grow
and p r o d u c e p r o d u c t s i n s i t u .
A l t e r n a t e l y , b a c t e r i a c a p a b l e o f u s i n g compo-
n e n t s p r e s e n t i n c r u d e o i l a r e i n j e c t e d , l i m i t i n g t h e need f o r n u t r i e n t s u pp 1einen t a t ion .
The a b i l i t y o f b a c t e r i a t o s u r v i v e and g r o w u n d e r r e s e r v o i r c o n d i t i o n s i s a A m u l t i t u d e o f extreme e n v i r o n -
p r e r e q u i s i t e f o r a s u c c e s s f u l MEOR p r o c e s s .
ments may b e p r e s e n t i n o i l r e s e r v o i r s w i t h c o n d i t i o n s such a s a n a e r o b i o s i s , h i g h p r e s s u r e , h i g h t e m p e r a t u r e and h i g h s a l i n i t y , a b r o a d s p e c t r u i i i o f pH v a l u e s , l o w n u t r i e n t c o n c e n t r a t i o n s , and t h e p r e s e n c e o f p o t e n t i a l l y t o x i c compounds.
A review o f reservoir conditions i n the top ten oil-producing
s t a t e s r e v e a l e d t h a t a p p r o x i i i i a t e l y o n e - f o u r t h o f r e s e r v o i r s have a c c e p t a b l e c o n d i t i o n s f o r p o t e n t i a l MEOR a p p l i c a t i o n s ( C l a r k
g fl.,
1981).
The c r i t e r i a
f o r s e l e c t i o n o f s u i t a b l e c o n d i t i o n s i n c l u d e d t e m p e r a t u r e s b e l o w 75"C, pH v a l u e s n e a r n e u t r a l i t y , and s a l t c o n c e n t r a t i o n s o f l e s s t h a n t e n p e r c e n t . These e n v i r o n m e n t a l c o n d i t i o n s have l e d t o t h e s u g g e s t i o n t h a t h a l o t o l e r a n t , t h e r i n o p h i l i c b a c t e r i a c a p a b l e o f a n a e r o b i c g r o w t h a r e good c a n d i d a t e s f o r an MEOR p r o c e s s ( M c I n e r n e y , 1983). The i n d i g e n o u s b a c t e r i a p r e s e n t i n an o i l r e s e r v o i r a r e an i m p o r t a n t e l e i n e n t i n an MEOR p r o c e s s f o r a t l e a s t t w o r e a s o n s .
F i r s t , t h e y may b e
s t i m u l a t e d d i r e c t l y b y t h e a d d i t i o n o f n u t r i e n t s and, second, t h e y may i n t e r a c t w i t h exogenous c e l l s i n j e c t e d i n t o t h e f o r m a t i o n .
Therefore, i t i s
e s s e n t i a l t o u n d e r s t a n d t h e i n i c r o f l o r a o f t h e r e s e r v o i r s o as t o p r e d i c t how t h i s p o p u l a t i o n w i l l respond t o p e r t u r b a t i o n by n u t r i e n t s and/or o t h e r bacteria.
Some o f t h e o r g a n i s m t h a t have been i d e n t i f i e d f r o i n o i l r e s e r v o i r
b r i n e s i n c l u d e s u l f a t e - r e d u c i n g b a c t e r i a ( Z o b e l l , 1 9 5 8 ) , pseudoinonads ( I l z u k a and Komagata, 1 9 6 4 ) , inethanogens ( B e l y a e v ( D a v i s , 1967; P f i f f n e r
g a-l.,
fl., 1 9 8 3 ) ,
and B a c i 1 l u s s p e c i e s
1985).
I n t h i s s t u d y , we have i s o l a t e d and c h a r a c t e r i z e d t h r e e n o n s p o r u l a t i n g anaerobic, h a l o p h i l i c b a c t e r i a froiii o i l f i e l d b r i n e s .
T h i s i n f o r i n a t i o n adds
t o our understanding o f t h e inicrobiology o f t h e o i l r e s e r v o i r .
These o r g a n -
isins b e l o n g t o t h e g e n e r a M e t h a n o h a l o p h i l u s , D e s u l f o v i b r i o , and, p o s s i b l y , Thermotoqa, and a r e b e l i e v e d t o be p a r t o f t h e n a t u r a l p o p u l a t i o n p r e s e n t a t this site.
O t h e r s p e c i e s b e l o n g i n g t o t h e s e g e n e r a have been i s o l a t e d f r o i n
m a r i n e and h y p e r s a l i n e e n v i r o n m e n t s w o r l d w i d e . MATERIALS AND METHODS E n r i c hinen t cu 1t u r e s F i e l d b r i n e was c o l l e c t e d f r o m t h e N o r t h Burbank u n i t i n n o r t h e a s t e r n Oklahoma i n s t e r i l e e v a c u a t e d b o t t l e s u n d e r a n a e r o b i c c o n d i t i o n s .
Brine
e n r i c h m e n t s were made b y t h e a d d i t i o n o f f i e l d b r i n e t o c o n c e n t r a t e d n u t r i e n t s o l u t i o n s f o r minimal d i l u t i o n o f t h e b r i n e .
A l l e n r i c h m e n t s w e r e s e t up
117
u n d e r a n a e r o b i c c o n d i t i o n s i n s t o p p e r e d 100 m l serum b o t t l e s and i n c u b a t e d a t 35-45°C. Anaerobic techniques M o d i f i e d Hungate t e c h n i q u e (Hungate, 1 9 6 9 ) , o r an a n a e r o b i c chamber (Coy L a b o r a t o r y P r o d u c t s , Ann A r b o r , M i c h i g a n ) was used f o r a l l m e d i a p r e p a r a t i o n as w e l l as f o r i s o l a t i o n and c u l t i v a t i o n o f b a c t e r i a . Media
~
The medium u s e d f o r p r i m a r y i s o l a t i o n o f m e t h a n o g e n i c b a c t e r i a was made i n a f i e l d b r i n e b a s e and c o n t a i n e d t h e f o l l o w i n g ( i n g / l ) : e x t r a c t , 2.0;
NH4C1, 0.5; y e a s t
t r y p t i c a s e p e p t o n e , 2.0; casamino a c i d s , 2.0; KzHP04, 0.2;
c y s t e i n e e H C 1 , 0.25; methylamineeHC1, 3.0.
sodium b i c a r b o n a t e , 2.0; sodium c a r b o n a t e , 2.0; and t r i The medium was a d j u s t e d t o pH 6.8-7.0
s u l f i d e , 0.25 g / 1 , was used as a r e d u c t a n t .
w i t h KOH.
Sodium
The gas phase was n i t r o g e n / c a r b o n
d i o x i d e (80:ZO). "BBM" medium f o r i s o l a t i o n o f f e r m e n t a t i v e b a c t e r i a contained the following (in g/l): KNO3, 1.0;
y e a s t e x t r a c t , 0.5;
NaCl, 66.6; NHqS04, 0.13;
CaC12, 15.3; s u c r o s e , 4.0;
MgC12.2H20, 5.1; PIPES [ p i p e r a z i n e -
N, N ' - b i s - ( 2 - e t h a n e s u l f o n i c a c i d ) ] s o d i um s a l t monohydrate, 8.5; p h o s p h a t e , 0.4;
The medium was a d j u s t e d t o pH 6.8 w i t h NaOH. as a r e d u c t a n t .
a-glycerol
and t r a c e e l e m e n t s o l u t i o n , 1.0 m l (Widdel and P f e n n i g , 1 9 8 4 ) . Cysteine.HC1, 0.2 g / 1 , was used
Medium d e s i g n a t e d "MMS" was used t o t e s t s u b s t r a t e u t i l i z a -
t i o n and a n t i b i o t i c s e n s i t i v i t y o f f e r m e n t a t i v e i s o l a t e s (Huber
g
c . ,1 9 8 6 ) .
P o s t g a t e ' s Medium C , w i t h m i n o r m o d i f i c a t i o n s and c o n t a i n i n g 3 5 NaCl, was used f o r t h e i s o l a t i o n o f s u l f a t e - r e d u c i n g b a c t e r i a ; s u l f a t e - f r e e g r o w t h was t e s t e d u s i n g P o s t g a t e ' s Medium D ( P o s t g a t e , 1 9 8 4 a ) .
Oxidation o f short-chain f a t t y
a c i d s , p a l m i t a t e , and b e n z o a t e c o u p l e d t o t h e r e d u c t i o n o f s u l f a t e was t e s t e d u s i n g a m o d i f i c a t i o n o f t h e media b y F o w l e r
d.(1986).
Hydrogen u t i l i z a -
t i o n was t e s t e d w i t h t h e medium o f B a d z i o n g and Thauer ( 1 9 7 8 ) .
A l l media
c o n t a i n e d 1 m g / l i t e r o f r e s a z u r i n as a r e d o x i n d i c a t o r . Growth s t u d i e s G r o w t h s t u d i e s were p e r f o r m e d i n B a l c h t u b e s o r serum v i a l s c o n t a i n i n g 5-10 m l o f medium.
Each t u b e was i n o c u l a t e d f r o m a l i q u i d c u l t u r e o f t h e
i s o l a t e d organism.
Growth was m o n i t o r e d b y i n c r e a s e i n a b s o r b a n c e a t
550-600 nm.
In t h e c a s e o f t h e methanogen, g r o w t h was a l s o m o n i t o r e d
m i c r o s c o p i c a l l y b y f l u o r e s c e n c e a t 420 nm and by p r o d u c t i o n o f methane.
118
Microscopy Routine observations on isolates were made with an Olympus BH-2 phase microscope equipped with a fluorescence attachment. Electron micrographs were taken with a Philips 300 or CM-10 transmission electron microscope. Thin sections were prepared by fixing cells in 3% glutaraldehyde and 1% paraformaldehyde, followed by osmication and embedding. Negatively stained samples were prepared by allowing the cells to attach to a freshly ionized carbon formbar grid, followed by a brief wash in double distilled water, and staining with 2% uranyl acetate. Chemicals Vancomycin, streptomycin, chloramphenicol, rifampicin, Coenzyme M , and bromoethanesulfonic acid (BES) were purchased from Sigma Chemical Company (St. Louis, MO). Clindamycin and cephalothin were purchased from Fluka Chemical Co. (Ron kon koma, NY) . Gas chromatoqraphy
Methane production was monitored using a Hewlett Packard gas chromatograph model 5890 equipped with a thermo-conductivity detector. A carbosieve Sll column was used for separation of the gases. Helium was used as a carrier gas. The oven temperature was held at 35°C for seven minutes and then increased at a rate o f 32°C per minute to a final temperature of 225"C, after 12 minutes. The injector temperature was llO"C, and the detector 230°C. RESULTS Description of site and composition of reservoir brine Reservoir brine was collected from the tank battery o f Tract 5 in the North Burbank unit operated by Phillips Petroleum Company (Bartlesville, OK). This field is a sandstone formation located in northeastern Oklahoma with an average temperature of 45°C and a depth of 3,000 feet. Brine was collected after it passed through the skimmer tank, just prior to reinjection. The brine had a total dissolved solids content of approximately 10% and a pH of 6.2. Chemical analysis showed that the major cations in the brine were: sodium, 3.06; calcium, 0.6%; magnesium, 0.12%; and barium, 0.07%. The major anion was chloride, at a concentration of 6.5%. Preparation of brine enrichments Enrichments were prepared by adding 100 ml of reservoir brine to seruin bottles containing nutrients either as dry solids or concentrated solutions. Various types and combinations of nutrients were utilized in order to examine
119
the microbial population under differing conditions. The addition of nutrient generally resulted in an increase in cell number from 103 cells per ml of brine to 108 cells per ml. Dominant members of the enrichment cultures were isolated by plating on media of the same general composition as that used for enrichment. Pure cultures of organisms were obtained by successive transfers on solid medium or in roll tubes. A methanogenic bacterium, a sulfate-reducing bacterium, and a fermentative isolate were subsequently identified by microbiological and biochemical characterization. Isolation and characterization of microorganisms Methanohalophilus species. The methanogenic isolate (designated BT-5), is a nonmotile, irregular coccus 0.8-1.2pm in diameter, as measured by phase microscopy. An electron micrograph of this organism, shown in Figure 1 , revealed cells of irregular size and shape. The cells stain gram negative and have a tendency to form clumps. Colonies in roll tubes were raised, circular and smooth, with a light brown to brownish-green color.
Fig. 1. Thin section of the methanogenic isolate BT-5. Bar, 1 pm. Some of the distinguishing characteristics of isolate BT-5 are shown in Table I , along with those of other type species. Methanol and methylamines were used by this organism as substrates for methanogenesis. The traditional substrates for methane formation, hydrogen and carbon dioxide, acetate, and formate, were not utilized by this isolate. Bromoethanesulfonic acid (BES), a
120 metabolic inhibitor (Gunsalus g aJ., 1978), and tetracycline both inhibited growth. This organism was insensitive to penicillin, 0-cycloserine, erythromycin, clindamycin, and cephalothin. These observations are consistent with the archeobacterial pattern of sensitivity to antibiotics. Lysis of cells occurred in distilled water, sodium dodecyl sulfate, or Triton X-100. Sodium, potassium and calcium ions were all needed for growth. The sodium chloride range for this organism was 1.5-23%, with the optimum occurring at 11.6-14.5%. The temperature range for growth was 2O-6O0C, with an optimum at 40°C. The optimum pH for this isolate was 6.8. Isolate BT-5 has characteristics similar to F. mahii obtained from the Great Salt Lake (Paterek and Smith, 1988) as shown in Table I. TABLE
I
Comparison of BT-5 Isolate to Members of the Genus Methanohalophilus Characteristic
BT-5
Gram reaction
negative
negative
negative
coccus
coccus
coccus
+
+
+
6.8
7.5
9.2
20-60°C
20-50°C
ND
40°C
35°C
45°C
Salt range (NaC1)
1.5-23%
1.5-23%
1 . 2 - 12.2'6
Optimum salt conc.
11.6-14.5%
11.6%
4.1%
+ +
+ +
ND
Morphology Substrates: methylamines, methanol
M. mahiia -~
M. _~
zhilinaeb
H2/C02, forinate acetate
pH optimum Temperature range Optimum temp.
Inhibitors BES
tetracycline others
BES, bromoethanesulfonic acid ND, not determined +, positive response or reaction - , negative response or reaction a Paterek and Smith, 1985; 1988. Mathrani g aJ., 1988.
cephal othi n
i
chl orinaphenicol
121
Thermotoga-like specie?. The fermentative isolate, designated T5, was morphologically distinct because the cell was surrounded by a sheath or "toga" structure (Huber aJ., 1986). Isolate T5 was variable in length (5-20 pm) and 1.0 pm in diameter, as measured by phase microscopy. The electron micrograph in Figure 2 shows the sheath-like structure surrounding the cell. Isolate T5 exhibited diverse morphologies with regard to cell size and amount of sheath when grown at varying salt concentrations. Cells appear rofi shaped, either singly or in short chains, and stain gram negative. This isolat formed smooth, raised, white colonies on agar plates.
a
Fig. 2. Negative stain of the fermentative isolate T-5. Bar 1 pm. Table I 1 compares the physiological characteristics of is0 ate T5 with members of the genus Thermotoqa. Different carbohydrates were utilized as an energy source by this organism. Approximately 0.1% yeast extract was required for growth. Elemental sulfur was facultatively reduced to hydrogen sulfide. Growth was inhibited by rifampicin at a concentration of 100 pg/ml. Sodium chloride was needed for growth, in the range of 0.25-lo%, with an optimum of 2.5%. The temperature range for growth was 3 0 - 5 5 O C , with an optimum of 5 O O C . This isolate possesses most of the characteristics of the genus Thermotoqa listed in Table 11.
122
TABLE I 1
Comparison o f T5 I s o l a t e with Members of t h e Genus Thermotoga T5
T. maritimaa
negative
negative
negative
negative
present
present
present
Characteristic Gram reaction
Sheath-like s t r u c t u r e present
neapolitanablc
thermarumC
Moti 1 i t y
+
+
+/ -
+
Carbohydrates fermented
+
+
+
+
Obligate anaerobe
+
+
+
+
Elemental s u l f u r reduced
+
+
I
30-55"C
55-90°C
55-90°C
55-84°C
80°C
80" C
Temperature range Optimum temperature S a l t range (NaC1) Optimum s a l t conc.
5OoC
80°C
0.25- 10% 0.24-3.75% 2.5%
Sensitivity t o rifampicin (100 pg/ml)
+
2.7%
NO
0.2-0.55%
ND
0.35% 4
N D , not determined +, p o s i t i v e response o r reaction
-, negative response o r reaction
g aJ., 1986. Jannasch e t fl., 1988. WindbergeFg aJ., 1989.
a Huber
Desulfovibrio species. The sulfate-reducing bacterium, designated SSR, i s a m c t i l e , curved rod, 2-5 pm in length and 1 pm i n diameter, as measured by phase microscopy. An electron micrograph o f t h i s organism, shown in Figure 3 , revealed comma-shaped c e l l s with polar f l a g e l l a . These organisms occur e i t h e r singly o r in p a i r s and s t a i n gram negative. Raised, black colonies were formed on a g a r p l a t e s containing ferrous iron.
123
Fig. 3.
N e g a t i v e s t a i n o f t h e s u l f a t e - r e d u c i n g i s o l a t e SSR.
Bar, 1 pm.
Table 111 l i s t s t h e c h a r a c t e r i s t i c s o f i s o l a t e SSR, a l o n g w i t h t h o s e o f o t h e r h a l o p h i l i c species o f Desulfovibrio.
Hydrogen, f o r m a t e , and l a c t a t e
were u t i l i z e d as e l e c t r o n donors f o r s u l f a t e r e d u c t i o n .
Acetate, ethanol,
p r o p i o n a t e , b u t y r a t e , p a l m i t a t e , and benzoate were n o t used as e l e c t r o n donors, and no growth was observed w i t h t h e s e s u b s t r a t e s .
Growth i n s u l f a t e -
An i n c r e a s e i n t h e v i s c o s i t y o f t h e c u l t u r e o v e r t i m e was apparent i n media c o n t a i n i n g y e a s t e x t r a c t , which i s t y p i c a l o f s a l t - r e q u i r i n g s t r a i n s o f D e s u l f o v i b r i o (Ochynski and Postgate,
f r e e media o c c u r r e d w i t h p y r u v a t e and fumarate.
1963).
The pigment d e s u l f o v i r d i n was p r e s e n t i n t h i s organism, as evidenced
b y f l u o r e s c e n c e o f c o n c e n t r a t e d c e l l s under u l t r a v i o l e t l i g h t a f t e r t h e a d d i t i o n o f 1 N sodium h y d r o x i d e (Postgate, 1984a). Sodium c h l o r i d e , i n t h e range o f D.5-15%, was r e q u i r e d f o r t h e growth o f t h i s organism.
The optimum sodium c h l o r i d e c o n c e n t r a t i o n ranged f r o m 2-6%.
Potassium c h l o r i d e c o u l d n o t s u b s t i t u t e f o r sodium c h l o r i d e , i n d i c a t i n g t h a t i s o l a t e SSR i s n o t a s t r a i n o f
g.
s a l e x i g e n s (Postgate, 1984b).
t u r e range f o r growth was 25-45"C, w i t h an optimum o f 35-40°C.
The tempera-
TABLE 111 Comparison o f SSR I s o l a t e t o O t h e r H a l o p h i l i c S t r a i n s o f D e s u l f o v i b r i o
Characteristic Grain r e a c t i o n
negative
negative
negative
vibrio
vibrio
vibrio
Morphology Desul f o v i r d i n
+
t
+
*
t
t
t
t
E l e c t r o n donors f o r sulfate reduction: hydrogen, formate, l a c t a t e ethanol acetate, butyrate, palmitate benzoate
NO
Tempera t u r e r a n g e
25-45°C
25-45"C
25-459c
T e m p e r a t u r e optimum
40°C
30-36OC
30-36OC
Optiriium s a l t c o n c . (NaC1)
2-6%
2.5%
N a+
ND
I o n requirement
2.5-5'0 c1-
ND, n o t d e t e r m i n e d +, p o s i t i v e response or r e a c t i o n - , n e g a t i v e response o r r e a c t i o n
a P o s t g a t e , 1984b. b l i d d e l , 1988.
DISCUSSION T h r e e p h y s i o l o g i c a l t y p e s o f b a c t e r i a were i s o l a t e d from r e s e r v o i r b r i n e s . The m e t h a n o g e n i c i s o l a t e , BT-5, was o b t a i n e d by e n r i c h m e n t w i t h t r i m e t h y l a m i n e , a i y p i c a l s u b s t r a t e f o r h a l o p h i l i c methanogens.
H a l o p h i l i c methanogens
c o m p r i s e a u n i q u e g r o u p because t h e y do n o t u s e t h e t r a d i t i o n a l s u b s t r a t e s f o r m e t h a n o g e n e s i s , &,
h y d r o y e n i c a r b o n d i o x i d e , a c e t a t e , and f o r m a t e .
Instead,
t h e s e methanogens g e n e r a t e methane f r o m m e t h a n o l o r N-methyl compounds (Mathrari
d., 1988;
P a t e r e k and S m i t h , 1 9 8 8 ) .
The o n l y r e c o g n i z e d genus
o f h a l o p h i l i c methanogens, M e t h a n o h a l o p h i l u s , c o n t a i n s t w o members. compares t h e c h a r a c t e r i s t i c s o f
y.
z h i l i n a e (Mathrani
( P a t e r e k and S m i t h , 1 9 8 8 ) , and i s o l a t e BT-5.
M.
ct aJ.,
1988),
Table I
F. mahii
z h i l i n a e was i s o l a t e d froin an
a l k a l i n e h y p e r s a l i n e l a k e , and i s d i s t i n g u i s h e d f r o i n t h e o t h e r t w o o r g a n i s m s
125
w,
isolated by its pH optimum of 9.2. Isolate BT-5 closely resembles y. from the Great Salt Lake. Methanogenesis has been studied in a number of hypersaline lakes (Giani gJ d., 1984; Oremland g fl., 1982; Paterek and Smith, 1985) and resulted in the isolation of several halophilic species. The fermentative sheathed isolate from brine closely resembles members o f the genus Thermotoga. 1. maritima, the first described member of this genus, was isolated from geothermally heated areas of marine sediments in Italy and the Azores (Huber g fl., 1986). Cells are characterized by the presence o f a sheath-like structure and an extreme temperature requirement, the highest reported for eubacteria. Two additional species, 1. neapolitana (Jannasch al., 1988) and 1. thermarum (Windberger d., 1989) differ from I. maritima in physiological properties, but still retain the sheath-like structure and a high temperature requirement. Isolate T5 resembles these species both morphologically and physiologicslly, although it is capable of growth at mesophilic temperatures. Another possibility is that this isolate belongs to the genus Thermosipho, a new genus within the Thermotoqales (Huber 9 fl., 1989). One distinguishing characteristic of Thermosipho is growth at lower temperatures than Thermotoga. Further characterization o f the T5 isolate by genetic techniques, such as DNA homology to other species and 16s ribosomal RNA sequencing, will establish its relationship to these genera. Reservoir brines would constitute a new environment for isolation of Thermotoqa or Thermosipho. Sulfate-reducing bacteria have long been associated with oil deposits (Davis, 1967; Zobell, 1958). Their presence can have a serious economic impact on the oil industry, causing problems such as corrosion of pipes, plugging o f injection wells and souring of fuel gas and oil (Cord-Ruwisch g aJ., 1987; Iverson and Olsen, 1984). The sulfate-reducing bacterium described here is a member of the genus Desulfovibrio and, based on its general characteristics, is probably a salt-requiring strain o f !. desulfuricans (Postgate, 1984b). Sulfate reduction has been reported to occur in several hypersaline environments such as the Dead Sea (Truper, 1969), Big Soda Lake (Smith and Oremland, 1987) and solar salt ponds (Klug g d., 1985). Carbohydrates, such as molasses, have traditionally been used as substrates for MEOR processes; initiation of growth depends on metabolism o f carbohydrate by fermentative bacteria. The Thermotoga-like isolate, T5, is capable of using carbohydrates directly, and is undoubtedly one of a number o f fermentative bacteria present in reservoir brines. Metabolism o f carbohydrate will result in the production of fermentation end products and other types o f metabolic by-products. One class of by-products produced in response to hypersaline conditions are osmoregulants, compounds used to maintain internal osmotic balance (Le Rudulier and Bouillard, 1983). Osmoregulants such a s
126
betaine and choline can, in turn, be broken down to methylamines, wh ch serve as substrates for methanogenesis (King, 1984a). The end products of carbohydrate metabolism, for example, hydrogen and acetate, can be utilized as electron donors for sulfate reduction, giving rise to the growth of ulfatereducing bacteria (Widdel , 1988). In many ecosystems, methanogenesis and sulfate reduction are competing processes for the same substrates; namely, hydrogen and acetate (Nedwell and Banat, 1981; Oremland and Taylor, 1978; Schonheit g, a-l., 1982). Hypersaline environments present a situation in which different substrates give rise to methane and hydrogen sulfide and, consequently, these metabolic types are not competitive (King, 1984b; Oremland -et_al., 1982). Surprisingly, sulfide is generally not produced in our carbohydrate enrichments, perhaps because of limited sulfate in the brine, exhaustion of other nutrients by fermentative bacteria, competition for substrates with other organisms, or the buildup of toxic end products. These interactions illustrate the type of community metabolism which will likely take place in an MEOR process upon the addition of nutrients, and underscores the need for understanding the microbiology of the oil reservoir before designing a MEOR process. Patterns of how the indigenous population responds to nutrient stimulation can then be established, s o as to predict and control their behavior. The reservoir conditicns at this site dictate that bacteria must be able to tolerate, survive, and grow anaerobically at salinities of up to 10% and at temperatures of approximately 45°C. The three organisms discussed here are capable o f proliferating under these conditions. These organisms are readily isolated from reservoir brine at this site and have been identified in brines from other locations in the Burbank field. We have isolated another Thermotoga-like organism from reservoir brine collected from a site in Texas as well. This suggests that these organisms were selected by, or adapted to, this extreme environment. Since these organisms are easily cultured on synthetic media in the laboratory, it raises questions about the source and concentration of nutrients present in the reservoir environment. Based on the composition of this brine, nutrients are most likely present at low concentrations. One form of adaptation to such nutrient deprivation is the formation of ultramicrobacteria, which are metabolically quiescent (Novitsky and Morita, 1978; Roszak and Coldwell, 1987). The ability to survive under these conditions would indicate that the organisms best suited for MEOR are those already present ir the reservoir. Screening programs have been devised based on this concept, e . ~ .examining , bacteria obtained from brine and evaluating their ability to respond to nutrients and to produce exopolymers, surfactants and other products beneficial to MEOR (Grula g, d., 1982; Lazar, 1983; Pfiffner
127
e t fl.,
1986).
A s u c c e s s f u l MEOR process w i l l u l t i m a t e l y depend on a sound
u n d e r s t a n d i n g o f m i c r o b i a l p h y s i o l o g y and ecology. ACKNOWLEDGEMENTS We acknowledge Michael McCaffery and Lance Washington a t t h e U n i v e r s i t y o f C a l i f o r n i a a t San Diego f o r p r e p a r i n g e l e c t r o n micrographs o f o u r i s o l a t e s . T h i s work was funded by P h i l l i p s Petroleum Company, B a r t l e s v i l l e , Oklahoma. REFERENCES
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