MICROBIAL REDUCTION OF PHOSPHATE?
Microbial Diversity project, 1995 by Alison George, School of Molecular and Medical Biosciences, Universiiy of Wales Cardff Cardff UK
Introduction Phosphorus is found in all parts of the biosphere and occurs most commonlyin the form of phosphates in which the P-atom has an oxidation state of +5. Though phosphorus can exist in a range of oxidation states from +5 to -3, it is generally observed that it remains in the +5 state in natural transformations (Fenchel and Blackburn 1979). The biological phosphorus cycle thus appears to be relatively simple, lacking both a gaseous phase and the reduced oxidation states found in the nitrogen and sulphur cycles.
There are, however, numerous reports of trace levels of a reduced form of phosphorus, phosphine (PH3), being present in certain environments. The Wil-O’ the Wisp observed at night-time over peat bogs, swamps, cemeteries, recent battle fields and stagnant waters may not in fact be due to manifestations of the supernatural, but to the spontaneous ignition of gas containing traces of phosphine which evolved during the decomposition of animals in damp soils (Mellor 1940). In the 1920s a debate raged about whether the biological reduction of phosphate really occurred (Rudakov 1927, Rudakov 1929, Liebert 1927) and this argument has not yet been resolved.
A convincing demonstration of the prevalence of biological phosphine production was given in 1988 (Devai 1988). Examining the gases released from a sewage treatment works he found that 9-20% of phosphorus entering the system was converted to phosphine. He confirmed these observations using bacterial enrichments. Analyzing surface sediments of Hamburg harbour (Gassman and Schorn 1993) phosphine was found in the range of 1.2-56.6ngfkg. However, in 1972 Burford and Bremner failed to find any. evidence for evolution of phosphine through microbial reduction of phosphate in waterlogged soils. In summary, it seems clear that trace levels of phosphine are undoubtedly resent in the environment but the mechanism/organism responsible has not been found.
Thermodynamic calculations can be made to predict whether phosphate reduction is an energetically feasible reaction but even these seem to lead to no firm conclusion. Rudakov (1929) and Tsubota (1959) both showed that it was thermodynamically realistic, whereas Liebert (1929) disagreed. Devai (1988) stated that the formation of phosphine “cannot be ruled out or explained by the properties of redox systems”.
was because
orthophosphite
Calculations
reduced
removed
favourable
The
(AGfo
The
(kCalImol)
AGfo
thermodynamically
Calculations
Thermodynamic
and
bacterial
The
exotic in
microbial
advantage
added
400C
a is
explain
source
certain
all
insoluble Phosphate
microbial was
Iverson
There
anaerobic
not
also
reaction
standard
biochemical
characterize
aim
values
present
and
-243.5
As(V)
311 2 P0 4
available.
the
then
to
evidence
of environments
from why is
enrichments
(1968,
of
as
diversity
salts
detergents.
during
necessary
production
phosphates
also
if
with
is
the
does
the
were
Gibbs
could
respiration,
the WiIl-O’
it.
were
(Ahmann
the
in
a
or
is
(Ehrlich
reaction
processes.
evidence
limiting
the
a
1984)
a
+
predi
pH
not
hypophosphite
project
able the
system
obtained
of
phosphate
viable:
steel
free
in
2C 3 1[ 6 0 3
made
not
organisms
-123.75
the
increases.
thermodynamic
appear
phosphine
putrification
microbial
from
phosphate
of
to
ctions
in
noted
Ct
energy
In
may
Wisp
1981,
from
phosphine
be
reduce
by the
is
al
that
such
to
from
a
Why
to
1994)
to
flushing
be
that
variety form
made
rich
+
is
the
Fenchel
see
change
responsible.
phosphine
be
attempt
use
energetically
However,
0 evolution
environments
seen
the
will
surplus
3ff
then
(phosphorus
in
the
common
of
a
and
environment,
experiment,
of
if
of
which
many
thermodynamically
CRC
not
proteins
be
of
data
or
emerging
corrosion
phospholipids,
should
inorganic =
the
U(V1)
—*
and
to
in
+93.9
through
phosphate.
environments,
Handbook
the
environments
ie
may
then
+3.2 great
from
3P11 3
if observe
reduction
Fe(ffl)
Blackburn
free
bacteria
viable.
the
(Mellor
(Lovley
oxidation
kJ/mole
it
energetics
reacts
from
play
and
product
excess.
compounds
not
energy
phosphine
a
may
+
-92.26
(Lovley
syntrophic
and
he
6C0 2
graveyards
a
for
There
ATP
only
employ
with
1940).
et
of
give
1979).
and
role
suggested
favourable
and
Fe 2 P
due
of states
Chemistry al
For
quantify
phosphate
from
and
ferrous
of
÷
1991).
formation/electrode
gastric
and
is
if
certain
or
as
to
-56.69
example,
it
in
61120
this was
an
Sewage
It
association
phosphate
+111
terminal
carbon as
the
Phillips
feces
and
anaerobic
is
extraordinary
an
that
is
iron
phosphate
evolved
one,
juices
and
fact
also
bacteria
and
battlegrounds).
successful,
electron
to
but
living
sludge
to
dioxide this
Physics).
and
1988) electron
that
(Maybe
a
+1
stored
phosphine from
vital
from
and
reduction when
steel
was
becomes
respectively)
tissue
it
an
reduction
also
acceptor?
to
the
forms
Fe 2 P.
diversity
gases
nutrient
potential
phosphate
ecological
to
due
this
acceptors
at
the
phosphate
corrosion.
presents
is
pH
isolate
around
There
more
to
less could
a
was
are
many
to
rich
is
for
in
the
in
In
I) —
Vitamin
IMKH 2 PO 4 1MK 2 HPO 4
Six
SL-12
following
This
1MNa 2 S
MgCl 2 .6H 2 0
1M
NaC1
KC1
NI{ 4 Cl
CaC1 2 .2H 2 0
dissolved
Enrichment
adapted
In
electron
F:
E:
D:
C:
B: A:
Innocula
Six
terminal
carried reduction
general
desulfuricans
as
anaerobic
The
established.
Materials
vitamin
this
NaHCO 3
that
The
Thiodendren
A
A
The
Raw
solution different
six
exact
mixture
mixture
B
marsh
enrichment
for
black
out
from
sandy
strategy
donor
vitamin
solutions
sewage electron
in
solution
solution
of
the
at
on
1
environments
biological
was
innocula
that However,
phosphate
litre
and
outer
(under
(pH
pH
of
of
black
may
enrichments
inorganic
and
veiled
the
from
the
autoclaved
employed
solution
acceptor.
used
7
of
added:
Methods
be
surface
only
and
the
sulphate
6.8)
lab
lab distilled
V
100%
20.Og
0.62g
0.06g
0.5g
0.25g
were
the
able
to
mud
mechanism
the
4.
pH to
SRB
SRB
phosphate
phosphate
enrich
inlet
to
phosphine
of
for
used
C02)
and
scant
to
from
Preliminary
and
of
reducing
enrichments
generate
enrichments
a
try
pipe sulphate
the
rusty
cooled in
for
that
literature
the
to
the
(if
enrichment
of
lOmi
lOmi
was
lml
3Omls
imi
imi
imi
sulphate
enrich
iron
Sippiwissit
was
the
layer
initial
phosphine
the
one
reducing
under
thermodynamic
used
nail
(acetate
Falmouth
(lactate
more
reports
exists)
of
for
MARINE
sulphate
enrichments.
reducing
washed
N 2 /C0 2
a
as
was
phosphate
microbial
likely
bacteria
(Iverson
as
the
as insinuate
of
sewage
varied.
marsh
the
the
up
(80/20)
reducing
at
MEDIUM
bacteria. sole
phosphate
calculations
electron
election
on
low
but
mat
andOlson,
reducing
(pH
electron
that
works
Sippiwissit
with
The
pH
and
from
=
phosphine
The
bacteria
donor).
donor).
so
6.8).
no
reduction
enrichment
aliquots
(pH
the
bacteria
demonstrated
the
acceptor,
following
1984).
sulphate
=
beach.
Sippiwissit
enrichments
6.7)
Desulphovibrio
of
is
was
Therefore
has
evolved
each
media
whilst
salts
the
not
to
that
of
salt
were
act
were
was
same
the
from
been
the
the
the as
detector
0.22p.m
ultrapure
acid,
HPLC
as
exchange Phosphate
l2Oml
innoculum
that
conditions growth,
Bacterial
(see reducing
Agar
b)
The
a)
to
differences In In
(enrichments
sterile case
Secondary
carbon
suiphite
added added
bottles
sterile
This
1:100.
follows:
situ
y attempt
situ
SRB,
are
25g
method
Marine
(enteric),
controls
shake
acetonitrile solution
analysis
innoculum
hybridization
filter
HCI
was
as
in
capable hybridization
source.
and
under
For
sodium
water.
bacteria
(IC
as
Flavo,
Isolations
was
gaseous
possible
and
A
of
between
to
the
enrichments
and
a
from
series followed
medium
and
each
PAK
stock
B)
430
for
pHJwith
Archea identii’ N 2 /C0 2
has
were
derived.
phosphite
electron
of
CL,
was
tetraborate The
at
degassed
The
but
both
in
(autoclaved
innoculum
Waters
phosphate
growing
a
A
f3,
were
solution
electron
form
pH
bacteria
pH
a
made
dispensed
HC)
enrichments
above).
6
again
was
was
eluens
and
total
pH7
which
and
7
(8
donors value
The
the
were
made
as
and
conductivity
0/20).
column
were
that
before
universal
carried
for
with
universal
at
of
and
X
decahydrate
donors
a
tubes
same
observed
was
at
and
4
made
types
these
of
sewage).
was
possible
of
Isolations
ilitre
each
as
that
the
according
each
pH4
measured
no
7
Bauer The
were
phosphite
use.
made
above.
eluted
out
were
±
made
electron
gas
from
had
pH to
probes
sulphate of
pH
ultrapure
0.2.
to
probes
pH,
pH
at
on
meter
The
incubated give
organism
phase
electron
enable
conditions.
incubated
by
(lab
lactate
with
(Borax)
with
pH7
by
the
were
lactate,
Innoculum
of
Half
enrichments
to
by
for
mixing
flow
dissolving
a
for
exercise).
the
donor
enrichments
present
running
the
of
and
gluconate/borate.
a)
final
water.
HPLC
a
37°C
of
made
as
45°C
the
remaining
comparison
donor.
rate
and
no
had at
acetate,
this
method
pH4.
the
in
20m1
concentration
300C.
media
as
Isolations
hybridization
the
electron
was
and
at of
hybridization
from
been
electron media
(Waters)
The
from
Probes
the
3
the
dark
16g
5°C.
stock
In
the
added
with
sodium
of
contained
250m1
solution
eluens
enrichments
enriched,
HPLC
enrichment
the
was
sodium
to
black
phosphate
at
Jorg
donor
were
the
donor.
used
X,
be
30°C.
to
equipped
The
enrichments
then
temperature.
of
suiphite
was
greatest
made
glycerol
the
2Oml
was
temperature.
sulphate
Overman
was
were:
made
gluconate,
eluens
10mM.
and
b)
10%
dispensed
The
medium
filtered
2mllminute.
with
present
reduced
between
from
no
iN
to
under
with
or
growth.
aim CO 2
was
reducing
phosphate
in
butanol
compare
for
the
formate
with
H 2
which
through
at
of
18g
an
ilitre
at
to
into
made
the
to
sulphate
bacteria
greatest
a
was
this
20mM
H 2
act
ratio
anion
4
boric
In
same
The
serum.
and
using
mud
were
the was
up
any
of
also
as
this
and
a
c)
of
a IL,
may
serum
given
available,
with
Phosphine
The
alcohol)
chloride to Enrichment
raw
The
E
experiment
enrichments
source
Samples
enrichment
the
ultimate
have
sewage
same
a
bottles
that
luminous
of
enrichments.
concentration
were
the
selected
been
method
lactate
is
the
were
was on
“spontaneously
experiment
these
and
ultimate
with
added
evolved
ideal
organic
flame.
the
or
the
waving
was
for
formate
demonstrated
acetate
only
A
analytical
to
HPLC
Phytic
experiment
in
employed
phosphate
during
enrichment
It
the
the
the
innoculum
with
combines
and
medium
neck
flammable
medium
phosphate
acid
the
tool, E
sustained
as
the
enrichment
across
in
enrichment
with
or
for
used
this
was
violently
a
greatest
to
phosphatidyl
enrichment
in
GC
lactate
analysis
a
project
reduced
and
a
no
air
flame.
final
method
growth
with
if
only
procedure.
with
growth.
(which
there
were
was
concentration
to
lactate
organic
on
choline
at
lg/l
to is
to
inorganic
showed B
all.
a
detect
NaC1.
attempt
The
trace
as
enrichment
This
phosphate
the
(dissolved
(Merck
2
of very
was
phosphate
electron
phosphine
of
negative
to
P 2 11 4
10mM.
poor
done
ignite
with
sources
index).
present
in
donor.
growth)
by
controls
but
the
electron
reagent
gas,
The
opening
were
in
Therefore,
gases
and
was
Of
this
sodium
and
in
added
burns
grade
all
donor
that
case
not
the
this
the the
in
amount
an
sterile
at
low
Secondary
procedure
(indicating
spirillum
precipitation
nutrient
It
black
Table
enrichments.
showed innoculum
only were
Enrichment
salt
After
RESULTS
the
the
should
electron
pH
marsh.
control
time
innoculum)
layer
a
1.
those
8
of
and
black
broth,
were
days
be
a
Growth
growth
is
of
enrichment
that
donor.
as’
little
acetate
F,
noted
derived
selective
pH
with
of
No
writing 4
4
4
4
4
on
7
7
7
7
7
precipitate
the
the
AND
observed
the
they
inorganic
the
inorganic
growth.
growth
did
no in
with
inoculum.
that enrichments
or
rusty
The
primaiy
from
may
culture
the
electron
not
ie
lactate
from
DISCUSSION
lactate
this
growth
only
was
controls
in
sustain
(with
material.
not
nail. innoculum
.
phosphate
Table
is
B
the
was
seems
enrichments
observed
enrichment
donor
be
a
‘+
primaiy
and
no
were
relative
is
enrichment
The
growth.
as
‘=
1
(with
very
not
bacterial
sulphite
The
to below
Electron
fragile
growth,
added.
B,
enrichments
analyzed
Hydrogen
enrichments be
observed
Hydrogen
Formate
in
Formate
Suiphite
Acetate
Sulphite
Lactate
scale
Acetate
Lactate
thin,
either
the
bacteria
that
the
for the
Secondary
shows
as
as
growth) black
at
‘-‘
preferred
and
donor
had
phosphate
enrichments
with
they
electron
no
for
pH
in
=
present
compared
grown
the
sulphate
electron
using
no
turbidity.
every
did
often
4
most
was
enrichments
growth
with
levels
electron
not
donors,
bottle.
sulphate-reducing
were
at
reducing
appear
observed
of
with
have
reducing
donor,
lactate
pH
to
of
the
The
all
a
but
7
donors.
the
growth
time
There
culture
to
was
at
small
turbidity
++
most
no
bacteria
as
growth
sewage
in
be).
pH
layer
Growth
to
(spirillum)
that
the
the
phosphate
is
rods
4
observed
+
+
fully
+
turbid
of +
- + - -
-
more
The
showed from
enrichment
with
electron
was
say
innoculum,
mud
with
derived
or
develop
enrichment
-
E.
enrichments
acetate
growth
Sippiwissitt
cocci,
observed
SRB
sample
or
in
a
Coil
donor
small
with
from
the
but
mat
from
but
as
on
and
at
B E ______
After incubating for 13 days the other enrichments were studied under the microscope to establish whether the turbidity that was often present was due to precipitation of some the inorganic components of the medium or due to bacterial growth. The following observations were made:
Enrichments A (sewage inoculum) Small amounts of growth were observed at both pH 4 and 7.
Enrichments C and D (lab collection of SRB enrichments as innoculum) Trace amounts of growth were observed with lactate as the electron donor, but there was no growth with acetate as the electron donor.
Enrichments E (SRB mud as innoculum)
pH Electron donor Observation 4 Lactate ++ Rods and cocci. Long ifiamentous organisms wrapped around sediment (figure_1) 4 Acetate - 4 Hydrogen ++ Short rods and cocc. 4 Sulphite - 4 Formate - 7 Lactate ++ Rods and cocci 7 Acetate - 7 Hydrogen - 7 Sulphite ++ Rods & cocci & the occasional long spirochete 7 Formate - Table 2. Growth in enrichmentsE (withSRBmud as the innoculum).
ii
p
Microscopic analysis of these enrichments did not detect any signs of life. The turbidity present in the flaskswas due solelyto precipation.
Figure
Time
Compact
‘Pure’
Figure
morphotypes
Some
Bacterial
only
(from
After
‘I
constraints
0
present
culture
colonies
4.
an
enrichments
3.
white
incubation
Composition
isolations
Colony
at
were
of
colonies:
were
the
did
long
formation
a..
•
analysed
top
of
not
period
noticibly
rods
pH
of
centimetre
allow
the
7)
under
of
in
colonies
(tjcc1z
at
further
compact
7
the
dilutions
days
the
of
agar
the
analysis
microscope
U&
derivedfrom
growth
whereas
shake
agar
of
Long
Diffuse
10’
of
(Fig.3).
,.
could tubes
— these
0
others
to
(Fig.
rods
the
colonies:
atpH
i0 3 .
only
cultures.
and
4).
agar
were
be
The
highly 7.
0
shake
observed
more
round
motile
series.
diffuse.
white
in
cocci
medium
The
colonies
2
of
colony
pH
were 7
not
enrichments. was
Phospholipid,
donor.
and
of
Enrichment
With
appeared including,
Figure
Rhodospirillum
was
The
fluoresced
for
In
predominently
probe
The
substrates
to
7,
situ
therefore
the
no
bacteria
phytic
bacterial
cells
and
2.
hybridization
and
observable
enrichment
that
unfortunately,
Hybridization
spirochetes
were
on
brighter
acid
other
Microscopic
the
organisms
when
present
difficult
growth.
organic
sulphate
centenum
mostly
as
universal
than
difference
added
than
was
a
in
were
to
phosphate
phosphate
those
the
within
proble
the
reducing
small
others
assess
sulphate
analysis
to
used
observed
enrichment
controls
probes.
intended.
enrichment
between
rods
these
results
as
but
by
bacteria
source a
revealed
reducing
eye
and
positive
this
enrichments
in
(except
the
the
at
The
whether
Again,
cocci
was
medium,
pH
(this
growth
bacteria
enrichment
that
mud).
control
for
bacteria
4
also
which
is
and
significant
any
the
were
bacterial
hardly
was
true
formed
from
pH
organism.
one
As
turbidity
were
at
present
capable
7
of
observed
suprising
can
with the
pH7
tested
the
growth
growth
a
very
enrichment
be
mificy
with
was sterile
universal
of
in
positive
seen
growing
in
skinny
given
was
had
due
these
lactate
white
all
innoculum).
in
observed
to
occured
that
probe
the with
at
t fig.
compared
enrichments
immulsion.
as
at
pH
4.
the
s.-4
enrichments,
the
J?G
the
only
2
too.
4
emulsion
some
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are 4 HPLC analysis for phosphate and phosphire Phosphate (in the form of PONafl pH7) was easily and accurately determined by this method. However no hypophosph,ite24 could be detected (I used standards of low concentration up tolOOI.tMbecause I predicted that if any phosphate had been reducted, at this early stage in the enrichment only low levels of the reduced form would be found). Levels of phosphate measured in certain enrichments are shown below in Table 3:
Sample Growth? IPhosphate] mM B lactate pH 7 enrichment + 19.46 B acetate pH 7 enrichment + 22.68 E lactate pH 7 enrichment + 22.26 A lactate pH 7 enrichment trace 22.33 E formate pH 7 enrichment - 15.54
Table 3. Phosphate concentrations of selected enrichments after Xdays on incubation.
Phosphate levels do not decrease in proportion to the ammount of growth observed. The large discrepancies in the concentration of phosphate measured can be explained in terms of the amount of phosphate-containing precipite forming in a given enrichment. Phosphate readily forms insoluble precipitates with calcium and magnesium even when these ions are present in very low levels. This precipitation renders the phosphate undetectable in the aqueous phase of the enrichment from which the sample for HPLC analysis is derived. Given the small amount of bacterial growth in the enrichhients, it is likely that the dominant factor governing the amount of phosphate measured is precipitation of the phosphate rather than biologicalprocessing.
The value of a measuring the amounts of phosphate used up in the enrichments as an indication of phosphate reduction is limitedbecause it is difficultto measure of how much phosphate the cells would be using in ‘normal daily life’, not as an electron acceptor. Ideally the method would be applied to the secondary enrichmentswhich would have had less carry-over of nutrients and for which suitable controls were availablefor comparison. However, not enough growth was observed in these enrichments to be able to observe significantsdecreases in the concentration of phosphate.
The ultimate erperiment Unfortunately (or fortunately for some) this experiment did not yield explosive results, or even one little luminous flame! 3 cultures seemed to make the flame go “phut” a tiny bit, but that could have just been due to air currents as the gas phase emerged from the serum flask or wishfulthinkingon my part.
4(9: Conclusions The results shown above neither confirm or rule out the possibility of biological phosphate reduction. They do however nicely demonstrate that bacteria were surviving/growing under very stringent conditions in terms of their choice of electron donor/acceptor and in terms of the pH in which they were placed. Given that estuarine bacteria require up to a week to switch on the necessary enzymic mechanisms and to grow suffiently to cause a significant decrease in levels of the highly biodegradable surfactant SDS, it seems very unlikely that I would have observed a significant reduction in the levels of phosphate in the enrichments given the limited time scale of the project.
It is quite reassuring to read that Devai (1988) had to wait 56 days before he could observe significant reductions in the phosphate levels in his in vitro experiments to detect phosphate reduction. However, the lack of flither publications from him on this topic would seem to indicate that maybe his enrichments for phosphate-reducing bacteria were not so sucessfiil after all.
Though phosphine is undoubtedly present in trace quantities in certain environments, the mechanism for its production remains a mystery. The secret of Will-O’the Wisp lives on.
References
Burford and Bremner (1972) “Isphosphate reduced to phosphine in waterlogged soils?” Soil Biology and Biochemistry 4, 489-495
Devai, Feffoldy, Wittner and Plosz (1988) “Detection of phosphine: new aspects of the phosphorus cycle in the hydrosphere” Nature 333, 343-345
Ehrlich (1981) “Geomicrobial transformations. phosphorus” in Bacterial and Mineral Cycling ppi37-i46, Dekker, NY.
Ahmam et al (1994) “Microbegrows by reducing arsenic” Nature 371, p750
Lovley and Phillips (1988) “Novelmode of microbial energy metabolism: organic carbon oxidation coupled to dissimilatroy reduction of iron of manganese” Applied and Environmental MIcrobiology 54, 1472-1480
Lovley et al (1991) ‘Microbial reduction of uranium” Nature 350, 413-416
Fenchel and Blackman (1979) Section 7.2 “Phosphorus” in “Bacterial and Mineral Cycling”, Academic Press, London
4G Gassman and Schorn (1993) “Phosphine from Harbour Surface Sediments” Naturwissenschaften 80, 78-80
Iverson (1968) “Corrosion of iron and formation of iron phosphide by Desulphovibrio desulfuricans” Nature 217, 1265-1267
Iverson and Olson (1984) “Anaerobic corrosion of iron and steel: a novel mechanism” p.623-627 in Kiug and Reddy (ed) Current Perspectives in microbial ecology, American Society for Microbiology, WashingtonDC
Liebert (1927) “Reduzieren mikroben phosphate” Zentbl. bakt. parasit. kole, Abt. II, 72, 369-374
Mellor (1949) “Phosphorus” ch.4 in “A comprehensive treatise on inorganic and theoretical chemistry”vol 8, pp802822 Rudakov (1929) “Die reduktion- der mineralischen phosphate auf biologischem wege. II Mittehung” Zenbi. bakt. paristkde, Abt II, 79, 229-245
Tsubota (1959) “Phosphate reduction in the paddy field” Soil Plant Fd Tokyo 5, 10-15