Download Selenate reduction by a Pseudomonas species: a new mode of

FEMS Microbiology Letters 61 (1989) 195-198
Published by Elsevier
195
FEMSLE 03693
Selenate reduction by a Pseudornonas species:
a new mode of anaerobic respiration
J o a n M. Macy, T h o m a s A. Michel a n d D o n a l d G. Kirsch
Department of Animal Science, University of California, Davis, CA, U.S.A.
Received 9 April 1989
Revision received 9 May 1989
Accepted 10 May 1989
Key words: Pseudomonas sp.; Selenate reduction; Anaerobic respiration
1. SUMMARY
The high levels of selenium (selenate, selenite)
in agricultural drainage water in the San Joaquin
Valley of California, which have led to environmental problems, might be lowered if the
selenate/selenite could be reduced to elemental
insoluble selenium [1]. Two organisms have been
newly isolated which do this in anaerobic coculture. One, a strictly anaerobic, Gram-positive rod,
reduces selenite to elemental selenium. The other,
a Pseudomonas species, was shown to respire
selenate to selenite. Cells grown anaerobically in
Minimal Medium on acetate plus selenate oxidized
14C-acetate to 14C02 with concomitant reduction
of selenate to selenite and small amounts of elemental selenium.
2. INTRODUCTION
Many organisms are known to reduce selenium
oxides [for review of literature see 2-4]. No published reports exist, however, suggesting that any
Correspondence to: Joan M. Macy, Department of Animal
Science, University of California, Davis, CA 95616, U.S.A.
selenate- or selenite-reducing organisms are able
to use these compounds for anaerobic respiration
[2-4]. Since the species of selenium in drainage
water are selenate (SeO42-) and selenite (SeO~-),
it was important to determine whether organisms
exist that can use either of these two compounds
to respire when grown under anaerobic conditions. Recently, Oremland and Hollibaugh have
shown that selenate might be respired by anaerobic
bacteria in sediments [5]. This is the first report of
selenate respiration by a pure bacterial culture.
3. MATERIALS AND METHODS
Media were prepared according to the method
of Hungate [6]. Minimal Medium contained
(g/liter; pH 7.2): NaC1, 1.2; KC1, 0.3; NH4C1,
0.3; K H z P O 4 , 0.2; N a z S O 4 , 0.3; MgC12.H20,
0.4; CaC12 • 2H20 , 0.15; NaHCO 3, 0.6; Trace elements 'SL8' [11]; Vitamins (thiamin- HC1,
pyridoxine. HC1, niacinamide, pantothenic acid,
B12 , biotin, p-aminobenzoic acid, riboflavin, folic
acid). Where required medium also contained:
ascorbate (0.03%, reducing agent), sodium selenate,
20 mM; sodium acetate, 5 or 20 mM; methanol 20
mM; yeast extract, 0.4%; agar, 2%; medium containing yeast extract is referred to as YE medium.
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
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The Gram-negative rods isolated were identified as Pseudomonas aeruginosa and a Pseudomonas sp. (designated strain AX), using the api 20
E system and rapid N F T system of api Analytab
Products, Quebec, Canada.
To demonstrate the oxidation of acetate to CO 2
in the presence of selenate, cells were grown 18 h
in anaerobic (ga phase: 100% N2) Minimal
Medium containing 20 mM sodium acetate, 20
m M sodium selenate and 0.03% ascorbate (reducing agent). Cells were harvested (16000 x g, 20
min), washed once in potassium phosphate (KP)
buffer (50 mM, p H 7.4), and suspended in O2-free
buffer (KP, 50 mM, pH 7.4, 0.03% ascorbate, gas
phase: 100% N2). Aliquots (0.9 ml) of suspension
were added (under a stream of N 2 gas) to anaerobic
Warburg flasks. Flasks were closed with stoppers.
The center well contained 200 /xl of 10% K O H
and a 2 × 4 cm filter-paper wick for CO 2 trapping.
Flasks were incubated at 28°C. Reactions were
started by the addition of 0.1 ml of a solution
containing 70 mM selenate and 31.5 mM 14CU-acetate (specific activity: 50 # C i / m m o l ) . Reactions were stopped by placing flasks at 90 o C for 5
min. To achieve maximal trapping of the CO 2 in
KOH, flasks were incubated, with shaking (24 h at
60 o C). After cooling to room temperature, radioactive counts in the filter paper wick and K O H
were determined by liquid scintillation counting.
The counts in 200 ~1 of the reaction mixture were
determined in the same way. A further aliquot of
the reaction mixture was centrifuged (13000 × g,
40 min), and the amount of radioactivity in 200/~1
of the supernatant was determined. 14C-U-acetate
label in poly-fl-hydroxybutyrate (PHB) was determined in a separate experiment. Cells were
incubated with 14C-U-acetate for 2 h as described
and harvested by centrifugation (20000 x g, 30
min). PHB was extracted according to Smibert [8],
dissolved in chloroform and radioactive counts
were determined by liquid scintillation counting.
To demonstrate the reduction of selenate to
selenite, during the oxidation of acetate to CO2,
the suspension prepared for the labeling experiment described above was incubated with 3.15
mM acetate and 7.0 mM selenate. Ascorbate was
not included in the anaerobic incubation buffer
because it interfered with the selenite analyses (see
below). The suspension was incubated at 28°C.
Portions of the suspension were removed at various times, and the reaction stopped by placing the
material at 90 o C, for 5 min. Samples were centrifuged (20000 × g, 30 min). The supernatant was
removed and frozen. The supernatant was analyzed
for concentrations of selenate, selenite and acetate.
Selenate ion and selenite ion were analyzed by
single-column ion chromatography (HPLC), using
a 4 mM p-hydroxybenzoic acid/sodium borate
mobile phase [9]. A preconcentration column (Ani o n / R guard cartridge, Wescan) was used in place
of a sample loop. A mobile phase of pH 8.0 was
used for the analysis of selenate, while a mobile
phase of pH 7.2 was used for the analysis of
selenite. Elemental selenium concentration was
calculated as the difference between selenate used
and selenite formed. Acetate was determined using
the acetic acid UV test kit of Boehringer Mannheim.
4. RESULTS A N D DISCUSSION
The first step toward demonstration of selenate
respiration was the isolation, under anaerobic conditions, of selenate-reducing bacteria. Material
from biological reactors for selenium removal [10],
was inoculated into anaerobic methanol-acetate
enrichment cultures (Minimal medium plus sodium
selenate, 20 mM; sodium acetate, 5 mM; methanol,
20 mM). Within a week a red color, indicating the
presence of elemental selenium, was visible in the
enrichment cultures. The culture was transferred
(every two days: 10% inoculum) at least thirty
times before organisms from the stable anaerobic
enrichment were isolated.
Organisms from the enrichment were isolated
in YE Medium (plus 2% agar) [8], and three pure
cultures were obtained: (1) Pseudomonas sp AX
92) Pseudomonas aeruginosa and (3) a Gram-positive strictly anaerobic rod-shaped bacterium designated strain 'E'.
In YE Medium (plus 20 mM sodium selenate),
a co-culture of the Pseudomonas sp. plus the strict
anaerobe rapidly selenate to elemental selenium.
Co-cultures of the P. aeruginosa and the strict
anaerobic did not reduce selenate to elemental
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selenium, thus this p s e u d o m o n a d was not further
studied.
The anaerobe (strain ' E ' ) grew in anaerobic Y E
M e d i u m in the absence of selenate or selenite.
Selenate when added was not reduced, selenite
when added was reduced to elemental selenium.
Characteristics of the anaerobe will be described
elsewhere. The Pseudomonas sp. A X only grew in
the anaerobic Y E M e d i u m when selenate was
present, only small a m o u n t s of elemental selenium
were formed. G r o w t h with selenite did not occur.
Anaerobic growth with nitrate (20 m M ) also
occurred. These results suggest that the Pseudornonas sp. A X respires selenate, forming selenite
and that in coculture, selenite formed by the Pseudomonas sp. A X is reduced by the anaerobe to
elemental selenium.
T o prove rigorously that the Pseudomonas sp.
A X can respire using selenate as the terminal
electron acceptor, it was first necessary to determine whether the organism can grow anaerobically on acetate plus selenate, in Minimal Medium.
Theoretically, this should occur (equation [1]) while
complete oxidation of acetate to C O 2 in the absence of an electron acceptor is not possible
(equation [2])
CH3COO-
+H + +4SeO2- ~ 2 C O 2 +4SeO 2- +2H20
( A G o, = _ 556 kJ/mol acetate)
equation [1]
CH3COO- + H + + 2H 2° ---,2CO2 + 4H 2
( AG °' = - + 112 kJ/mol acetate)
equation [2]
(Free energies of formation for selenate and
selenite f r o m [11]; for the remaining c o m p o u n d s
f r o m [12].)
G r o w t h of the Pseudomonas sp. A X in
anaerobic Minimal M e d i u m with acetate (20 m M )
and selenate (20 m M ) did indeed occur. The
organism was subcultured (10% inoculum) at least
thirty times. The culture reached an absorbance
(600 nm) of roughly 0.4 and the generation time
was approximately four hours. After about 24 h,
the culture had a slightly red color (i.e., elemental
selenium), which was associated with the cells.
While growth in Minimal M e d i u m on the nonfermentable c a r b o n source, acetate, implied that
respiration with selenate occurred, it was necessary to demonstrate that acetate is oxidized to
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Fig. 1. Oxidation of 14C-U-acetate (3.15 mM) to 14CO2 in the
presence of selenate (7 mM) by the Pseudomonas sp. AX
grown in Minimal Medium plus 20 mM acetate and 20 mM
selenate and suspended (0.17 mg protein per ml) in O2-free
phosphate buffer (50 mM, pH 7.4). dpm: acetate (A), CO2 (m),
cells (e).
C O 2 with c o n c o m i t a n t stoichiometric reduction of
selenate to selenite (Figs. 1 and 2). As shown in
Fig. 1, a large percentage of the acetate was f o u n d
associated with the cells; 31.6% of this acetate was
recovered in PHB. Acetate not incorporated into
ceils was oxidized to C O 2. During the first 50 rain
of the experiment, roughly 60% of the added
acetate was recovered as CO2; during this time
recovery of label was also approximately 90% or
higher. After 50 min a m o u n t of label recovered as
C O 2 decreased significantly, as did total a m o u n t
of label recovered. This is p r o b a b l y due to the
difficulty of trapping increasing a m o u n t s of C O 2,
resulting in decreased C O 2 trapping efficiency and
decreased recovery of label. In Fig. 2 can be seen
the reduction of selenate during the oxidation of
acetate to C O 2. Small a m o u n t s of elemental
selenium were formed during the first 25 min of
incubation. Thereafter, selenate was reduced only
to selenite, in accordance with the stoichiometry
of the reaction as written in equation [1].
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ACKNOWLEDGEMENTS
12
We t h a n k J.L. I n g r a h a m , B.W. H o l l o w a y a n d
D.R. Lovely for critical review of this m a n u s c r i p t ,
a n d W. Pfeiffer for helpful discussions.
10
REFERENCES
P.
~
4-
2
0
0
25
50
75
100
125
Time (minutes)
Fig. 2. Reduction of selenate (7 raM) to selenite, during the
oxidation of acetate (3.15 mM) to CO2 by a suspension of the
Pseudornonas sp. AX (see legend Fig. 1). Selenate (o), selenite
(A), selenium (n), acetate (/,).
T h u s the Pseudomonas sp. A X is able to carry
out a n a e r o b i c respiration of selenate, f o r m i n g
primarily selenite. This is the first report describing this m o d e of respiration b y a pure bacterial
culture. T h e selenate-respiring Pseudomonas sp.
A X together with the selenite-reducing a n a e r o b e
(strain ' E ' ) m a y prove to be the ideal m e a n s of
r e m o v i n g selenium oxides from c o n t a m i n a t e d
agricultural drainage water, e n a b l i n g reutilization
of the water.
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[2] Maiers, D.T., Wichlacz, P.L., Thompson, D.L., Bruhn,
D.F. (1988) Appl. Environ. Microbiol. 54, 2591-2593.
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[4] Doran, J.W. (1982) Adv. Microb. Ecol. 6, 1-32.
[5] Oremland, R.S., Hollibaugh, J.T. (1988) Abstract AS51A,
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[7] Pfennig, N., Widdel, F., Triiper, H.G. (1981) in The
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[8] Smibert, R.M., Krieg, N.R. (1981) in Manual of Methods
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[9] Mehra, H,C. and Frankenberger, W.T. (1988) Chromatographia 25, 585-588.
[10] Groves, R., Greenaway, P., Smith, B., Kovac, K., Taylor,
E. (1988) Performance Evaluation of Research Pilot Plant
for Selenium Removal; California Department of Water
Resources (April).
[111 Mc Keown, B. and Marinas B. (1986) in Symposium on
Selenium in the Environment, pp. 7-15, CSU, Fresno.
[12] Thauer, R.K., Jungermann, K., Decker, K. (1977)
Bacteriol. Rev. 41, 100-180.