FEMS Microbiology Ecology 101 (1992) 113-119
0 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00
Published by Elsevier
113
FEMSEC 00395
Thermophilic denitrifying bacteria: A survey of hot springs
in Southwestern Iceland
Thomas C. Hollocher
* Department
of Biotechnology,
Technological
Institute
av1and Jakob K. Kristj&sson
of Iceland,
Reykjauik,
Keldt~aholt,
and
’ Institute
ayb
of Biology,
University
of Iceland,
Iceland
Received 3 December 1991
Revision received 1 April 1992
Accepted 2 April 1992
Key words: Thermophilic
bacteria; Denitrification;
1. SUMMARY
Samples of water, sediment and bacterial mat
from hot springs in Grandalur and Hveragerdi
areas in southwestern Iceland were screened at
70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing
nitrate or N,O as the terminal oxidant. The
springs ranged in temperature from 65-100°C
and included both neutral (pH 7-S.5) and acidic
(pH 2.5-4) types. Nitrate reducing bacteria
(nitrate + nitrite) and denitrifiers (nitrate + N2)
were found that grew at 70°C but not at 80°C in
nutrient media at pH 8. Samples from neutral
springs that were cultured at pH 8 failed to yield
a chemolithotrophic,
sulfur-oxidizing
and
nitrate-reducing
bacterium, and samples from
acidic springs that were cultured at pH 3.5 seemed
Correspondence
to: J.K. Kristjlnsson, Department of Biotechnology, Technological Institute of Iceland, IS 112 Keldnaholt,
Iceland.
’ Permanent
address:
Department of Biochemistry, Brandeis
University, Waltham, MA 02254, USA.
Nitrate reduction; Thermophilic
Bacillus
entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of
growth solely by N,O respiration. The denitrifiers
appeared to be Bacillus. Two such Bacillus strains
were examined in pure culture and found to
exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosn, and one other strain of thermophilic &cillus. The phenotype is characterized by the ability to grow by reduction of nitrate
to N, with N,O as an intermediate but a virtual
inability to reduce N,O when N,O was the sole
oxidant.
2. INTRODUCTION
To our knowledge the only thermophilic denitrifying bacteria reported are Bacillus species [l81 and possibly Thermothrk thioparus [9]. T.
thioparus was originally reported to grow as a
facultative chemolithotroph
by the reduction of
nitrate or nitrite and oxidation of sulfide or thiosulfate, but gas production was not detected [9].
114
Later it was reported [lo] to denitrify only heterotrophically with production of N, as the primary product and nitrite and N,O as transient
products. Growth by denitrification was reported
to occur both aerobically and anaerobically and
to be supported by amino acids and simple sugars, providing a reduced sulfur source (methionine, glutathione or thiosulfate) was supplied.
However, the rate of N, production reported, 13
of cells, is some 10’ times
nmolxh-‘Xg-’
smaller than that of common mesophilic denitrifiers [ll-131 and quite insufficient to support cell
growth at the rate reported of about 0.5 generations per h. Reduction of nitrate to nitrite is
common among Thermus spp. and can support
anaerobic growth in a few cases. However, no
properly denitrifying Thermus sp. is known [14161. Given the great taxonomic variety among
mesophilic denitrifiers [6,17], it is remarkable that
only two genera are represented among the thermophilic group and one of these by a single
equivocal species. T. thioparus is no longer available from the American Type Culture Collection
nor, as far as we can determine, from any other
source.
The area of Grandalur
and Hveragerdi in
southwestern Iceland is geothermally very active
with hundreds of diverse alkaline and acidic hot
springs. The alkaline springs are characterized by
Cyanobacteria and the green gliding photosynthetic bacterium, Chlorofkxus. They can grow up
to 62°C and 72”C, respectively. Hydrogen- and
sulfide-oxidizers are also known to be present in
high numbers [18]. All of these are primary producers, providing carbon substrates for abundant
aerobic heterotrophs like Thermus and Bacillus
[19]. Anaerobic, fermentative heterotrophs are
also present in high numbers [20].
The acidic springs are almost like thick cultures of sulfur-oxidizing and reducing archaebacteria receiving carbon substrates from hydrogen
and sulfide/sulfur
utilizing autotrophs as well as
from the photosynthetic green algae, Cyanidium
which grows up to 55°C in the walls of the solfataric springs [21].
Nitrate is normally absent from geothermal
fluids when they emerge from the ground and
when detected it is usually much lower than nor-
mally found in surface waters in the same area.
Nitrate in hot springs is therefore expected to be
produced by biological mineralization of nitrogen-containing organic matter. Nitrate-reducing
and denitrifying bacteria would still be expected
to have similar roles in thermal ecosystems as in
non-thermal systems.
In an attempt to discover novel thermophilic
denitrifiers, particularly any that might grow solely
by N,O respiration, a survey was made of hot
springs in the above area during March-June
1990. What follows is a summary of findings. The
survey failed in its original goal to find new
thermophilic denitrifiers and likewise failed to
discover an Icelandic thermophile
capable of
chemolithotrophic
nitrate respiration with thiosulfate as the electron donor. It did, however,
serve to reinforce the view that Bacillus represents a widely dispersed genera of thermophilic
denitrifiers.
3. METHODS
3.1. Samples and culturing
Samples of water, sediment, and bacterial mat
were obtained from ten springs (65-100°C) along
the river valley, Grzndalur, which is located about
5 km north of Hveragerdi, Iceland. Seven were
neutral springs (pH 7-8.5) and three were acidic
(pH 2.5-4). Particular attention was given to
springs showing growth of filamentous, sulfurproducing bacteria. Inocula consisted of lo-200
~1 of water or slurry of bacterial mat, sand or
plant detritus. Samples were used routinely to
inoculate 25 ml of anaerobic media contained in
50 ml serum bottles. The bottles were sealed with
black butyl rubber stoppers (DC96,35, Rubber
BF, Vaartweg, The Netherlands) that were secured with aluminum crimp caps. These extremely impermeable seals allowed retention of a
particular gas during autoclaving and subsequent
incubation at 70°C or 80°C. The rubber was also
sufficiently heat resistant to allow repeated sterilization of the outer surface of the stopper by
alcohol combustion.
At one neutral spring (75”C, pH 7.51, an in situ
enrichment experiment [22-241 was undertaken
115
for the purpose of finding N,O-respiring bacteria.
N,O was allowed to bubble into the spring at
lo-20 ml per min through a wad, composed of
cotton and wool cloth, that was partly immersed
in the sediment. The wad was recovered after 5
weeks and pieces were used to inoculate media.
Primary enrichment cultures were incubated
for 2-6 days, depending on whether cell growth
was obvious, and, in any case, were then used to
inoculate fresh bottles. This procedure allowed
the visual detection of cell growth in the face of
initially turbid samples. The isolation of organisms was attempted by means of limiting dilution
for obligate anaerobes
or facultative
microaerophiles or by streaking on agar plates for
facultative aerobes. Agar plates were incubated
at 65-70°C. In order to minimize evaporation,
plates were stacked and contained in heavy plastic bags. Stab and slant-tubes were incubated
inside of plastic jars with screw lids. The N-oxide
added for anaerobic respiratory growth was N,O
or nitrate. To check if the isolates could grow
fermentatively they were inoculated into culture
bottles initally sparged with N,. Relative bacterial
growth was estimated in stationary phase by absorbance at 600 nm.
3.2. Media
The media used are listed in Table 1. The
compositions of media 1, 2, 8 and 9 are based on
medium 162 of Degryse et al. [25] but having only
l/10-1/5
of the mineral base. The ingredients
for growth media, l-10, were added in each case
to 1 liter of water of geothermal origin from the
hot tap. This water had a pH of about 8.5 and
was buffered chiefly by bicarbonate/carbonate
(total concentration about 0.35 mM) and silica
(SiO, is typically 71-146 ppm [261X In consequence, the final pH of the media was about 8.
The tap water provided the main source of CO,
for growth of possible autotrophic denitrifying
bacteria.
The N-oxide added to screen samples (from all
10 hot springs) for anaerobic respiratory growth
was N,O at 0.1 or 1 atm (media l-4) or 15 mM
IWO, (media 1, 2, 5, 6, 8). For experiments
Table 1
Composition of liquid media used for enrichment (media l-61, for growth on single carbon sources (medium 7) and for agar media
Media
Tryptone
2g
Yeast
extract
5g
2g
50 mg
8
9
10
2g
2g’
3.1 g
50 mg
0.1 g
2g
2g
3.1 g
Phosphate
buffer a
(ml)
10
10
15
15.1
15.1
15.1
15.2
10
10
62
Basal
Trace
elements b salts ’
(ml)
(ml)
0.35
0.35
mo3
1.5 M
(ml)
NH&I
0.3 M
(ml)
%%%
Other additions
0.75 M
(ml)
25
25
25
25
0.005
0.005
0.005
0.35
0.35
0.35
125
Single organic compounds ’
10 g agar for stab tubes
28 g agar for plates/slants
35 g agar for plates
” Phosphate buffer. Prepared by mixing 200 mM KI-I,PO, with 200 mM Na2HP0,.2H20
to achieve a pH of 7.2.
b Trace element mixture. 12.8 g of trisodium nitrilotriacetate (Titriplex I), 1 g of FeCI,.4H,O, 0.8 g of NaSeO,.SH,O, 0.7 g of
NaMo0,.2H,O,
0.5 g of MnC12.4H20, 0.3 g of CoCl,.6HsO, 0.2 g of ZnCI,, 50 mg of CuC12.2H20, 20 mg each of HsBO,,
and NiC12.6H20, and 1 liter of tap water.
c Salts mixture. 1.32 g of Titriplex I, 5 ml of 10 mM ferric citrate, 0.4 g of CaSO,*2H,O, 2 g of.MgC12*6H20, 5 ml of trace
element mixture, and 1 liter of tap water.
d The medium was supplemented with 0.4% w/v of a particular compound, except for L-valine and sodium formate for which 0.2
and 0.8% were used. The gas phase was N2 or NsO. For nitrate respiring or denitrifying bacteria, medium 7 also contained 30
mM IWO,. Nitrate was omitted when testing the isolates for fermentative ability. The following compounds were tested: Xylose,
glucose, galactose, myo-inositol, maltose, sucrose, lactose, formate, acetate, propionate, pyruvate, tartrate, succinate, citrate,
urea, L-serine, L-proline, L-valine, and L-arginine.
116
involving growth yields, medium 2 was supplemented with 2-50 mM KNO,. For samples from
acidic hot springs, media l-6 were titrated with
HzSO, to a pH of about 3.5. Initial enrichment
was done both at 70°C and 80°C.
3.3. Assays
N,O, CO, and N, in the headspace of culture
bottles were detected simultaneously by gas chromatography with use of a thermistor detector and
a dual annular column containing Porapak Q and
molecular sieve. Culture bottles were sparged with
He before being inoculated. Nitrite formed by the
reduction of nitrate was assayed by the sulfanilamide diazotization method [27].
4. RESULTS AND DISCUSSION
4.1. Overview of survey
Samples from acidic springs failed to show
growth in any of the media tested under anaerobic conditions at pH 3.5. Thus neither acidophilic
fermenting nor N-oxide respiring bacteria were
found.
No organism that could respire solely on N,O
with production of N, was observed with any
sample.
No autotrophic bacterium that could grow by
oxidation of thiosulfate and reduction of nitrate
was detected.
Out of nineteen samples from ten neutral
springs, two scored positive at 70°C in nutrient
media for nitrate reducing bacteria (nitrite production), two for denitrifying bacteria (Nz production) and one for fermenting bacteria. The
bacteria responsible for denitrification appeared
in both cases to be Bacillus and one was isolated.
The bacteria responsible for nitrate reduction
appeared to be ‘Therinus in one case and a microaerophile in the other. The latter was isolated,
as was also one anaerobic fermenting organism.
Only one sample of the nineteen scored positive for growth at 8O”C, and one novel organism
was isolated from the cultures.
No evidence was .obtained for novel thermophilic denitrifiers.
4.2. Pure cultures
Four partially characterized bacteria from this
survey are described below.
IT1 379-H-90. A non-fermenting,
motile microaerophile able to grow by the anaerobic reduction of nitrate to nitrite. This Gram- and sporenegative bacterium grew well at 70°C in anaerobic nitrate medium 2 as ‘a narrow rod 2-4 pm
long linked end-to-end in chains of 2-20 cells.
Growth was proportional to nitrate concentration
up to about 50 mM and stationary phase* was
reached in about 24 h with maximum density of
lo’-10’
CFU per ml. Growth on nitrate.was
partially inhibited by N,O (1 atm). Coloniescould
grow on glass surfaces; In defined medium, the
organism grew well by’,nitrate respiration on acetate, propionate,:tartrate
or amino acids, poorly
on pyruvate or myo-inositol, and not at all on
succinate, citrate, formate, urea or any sugar
tested. It was viable for at least 1 mo at 4°C if
kept anaerobic;
IT1 380-H-90. A weakly motile, Gram- and
spore-negative, fermenting anaerobe, able to grow
at 70°C in medium 1 or 2 as a thin, tapered,
generally C-shaped rod which was linked end-toend in chains of 2-5 cells. Cell length was about
2-3 pm. It grew well in defined medium on
sucrose, maltose and glucose, less well on lactose,
and not at all on galactose, myo-inositol, xylose,
urea or any organic acid or amino acid tested.
Stationary phase densities of 107-10’ CFU per ml
were reached within 30 h typically. Growth was
inhibited by small amounts of 0, or nitrite, and
cells were killed by air within a few hours at room
temperature. A small amount of gas (probably
Hz) was produced during fermentation of sugars.
Cells remained viable for at least 2 weeks at 4°C
if kept anaerobic;
IT1 381-H-90. A non-motile organism of uncertain taxonomic affiliation that grew probably
as a fermentative anaerobe in medium 1 or 2
under N, or N,O at 80°C but seemingly not at
70°C. Growth was also not observed at 87°C in
one experiment. Both wet and stained preparations revealed extremely long, thin (5 0.3 pm),
unbranched filaments in loose coils and spools.
The filaments were Gram-negative, stained well
with safranine but less well with basic fuchsin, or
117
crystal violet. Growth was sparse, typically requiring 5-10 days, and appeared on the bottom of
bottles as thin mats which, when disturbed, broke
into fibers resembling dust motes. Attempts to
grow the organism as an anaerobe on single carbon sources or as an aerobe were unsuccessful. It
was viable for 2-3 weeks at 4°C under anaerobic
conditions. Its microscopic appearance was grossly
reminiscent of the mycelial growth habit found
within the euactinomycetes division of Actinomycetes [28], notwithstanding its staining properties, lack of branching and likely anaerobic growth
processes. No structures were observed that took
spore stain or were obviously highly refractile in
wet preparations. This apparent lack of spores
also does not fit Actinomycetes. Two genera of
thermophilic Actinomycetes are recognized, Thermomonospora [29] and Thermoactinomycetes [30],
but neither contains species able to grow at 80°C.
In its microscopic appearance, the organism
closely resembled the organism described by
Bauld and Brock [311 as a small flexibacterium
(unidentified) which was found to co-exist with
Synechococcus and Cl~lo~ojlaxus in the surface
layers of algal-bacterial mats in neutral and alkaline hot springs.
Growth at 80°C but not 70°C was unexpected
and raises the possibility that growth was connected to the thermal destruction of a growth
inhibitor in the nutrient media.
ITI 382-H-90. A motile, heterotrophic, Nz-producing denitrifier and facultative aerobe, probably Bacillus, that grew at 70°C in anaerobic nitrate medium 1 or 2 as sporulating rods with
transient production of nitrite and N,O. It grew
vigorously on aerobic plates at 65°C as white,
opaque colonies. No growth was observed in nutrient media under N,O. Denitrification with N,
production occurred extensively only in nitrate
stab tubes or in liquid media containing cloth or a
cotton ball. Otherwise, growth in liquid media
chiefly was by nitrate respiration with nitrite production. Immature spores, which formed terminally with distension, stained Gram-positive, but
vegetative cells (even those from young cultures)
and mature spores did not. Vegetative cells were
linked end-to-end in chains of 6-8 cells, but
spore-forming cells were individual or in pairs.
Mature spores were slightly ellipsoidal. Cultures
reached stationary phase in about 24 h and contained about lo9 CFU per ml. The organism grew
well in defined medium on acetate and nitrate;
other carbon sources have not yet been screened.
Fermentative growth was not observed in medium
2. Colonies on plates remained viable for at least
4 weeks at 4°C. The organism resembled B.
stearothermophilus
in size, gross appearance,
spore shape, colony characteristics, thermophilic
habit and apparent inability to grow fermentatively in nutrient media [2,5].
4.3. Comments
On previous occasions in this laboratory, thermophilic denitrifying heterotrophs have been isolated- incidentally from Icelandic hot springs.
These bacteria are also apparently Bacillus. One
of these, designated FV-A and now well characterized as an aerobe, was observed to be similar
to IT1 382-H-90 in many ways, but did not require soft agar or a fibrous matrix in order to
denitrify. FV-A, like 382-H-90, produced N, from
nitrate with the transient appearance of some
nitrite and N,O but could not grow on or even
reduce N,O when it was the sole oxidant. When
FV-A was grown on limiting (2.5 mM) nitrate,
N,O afforded little or no enhancement of cell
yield. At the same time, N,O seemed not to
prevent its growth on nitrate.
It would appear that the first two Icelandic
denitrifying Bacillus to be partially characterized
exhibit the denitrification
phenotype first observed with P. aeruginosa [32-361 and subsequently with the thermophilic marine Bacillus
strain FE-l [7]. That phenotype is one in which
growth is supported by reduction of nitrate to N,
via N,O as an intermediate, but growth on N,O
alone is poor or absent because of the inability to
reduce N,O.
With the possible exception of T. thioparus,
the known thermophilic Nz-producing denitrifiers
are Bacillus or Bacillus;like. Of the five examined
so far for growth on N,O (the two discussed
herein, strain FE-l and B. stearothermophilus [7],
and the sporulating bacterium of Garcia [S]), the
first three of these were unable to reduce N,O in
absence of nitrate or nitrite. The fourth, B.
118
was curiously different [71 in
that, although it was active in reducing N,O in
absence or presence of nitrite, it was nevertheless
unable to grow solely by means of N,O respiration. The bacterium studied by Garcia [8] would
appear to be the only thermophilic Bacillus sp. so
far described that is capable of normal growth on
N,O. No bacterium of that kind was discovered
to grow at 70°C or 80°C in the present survey.
The denitrifying thermophilic Bacillus strains
described above cannot grow as denitrifiers much
above 70°C and T. thioparus has been -reported
to denitrify up to 75°C [lo]. Given these limited
examples, it is possible that the global temperature limit for denitrification may be around 75”C,
just as the limit for photosynthesis is about 73°C
from the examples of Chloroj7exus aurantiacus
[31] and Synechococcus lividus [37,38]. If this were
the case, it is unlikely that nitrate reductase would
be the enzyme to limit the pathway. Some Thermus strains can grow by nitrate respiration at
least up to 80°C [14-16; unpublished results of
the Technological Institute) and an extremely
heat-stable nitrate reductase has been discovered
in Thermus [14]. In addition, because numerous
mesophilic denitrifiers (e.g., Pseudomonas fluerescens) can dispense with the N,O reduction
step [6] and still derive enough free energy from
the other steps of the pathway for growth, it is
unlikely that the nitrous oxide reductase system
would set the temperature limit. We suggest
therefore that the nitrite reductase or nitric oxide
reductase system will prove to set the upper limit
for the pathway.
Since T. thioparus seems no longer to be available and we were not successful in this work to
reisolate it, it’s denitrifying phenotype cannot be
confirmed. It should, however, be stressed that
we could observe it visibly and microscopically as
very long threads with sulfur granules attached to
it, often growing massively in high-sulfide springs.
It is therefore an important organism in many
geothermal ecosystems and it is a worthy challenge to reisolate it.
The results of this study indicate that nitrate
reduction is common in hot spring ecosystems
although denitrification does not seem so widely
distributed. It is, however, still an open question
steurothermophilus,
if more thermophilic denitrifiers cannot be found,
and we intend to pursue that further.
ACKNOWLEDGEMENT
This study was supported by a Fulbright-Hayes
Research Fellowship in Iceland, March-June
1990, to T.C. Hollocher and by grants from the
Icelandic National Research Council and the
Nordic Fund for Industrial. Research and Development to J.K. Kristjansson.
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