UNIVERSITY of ALASKA
COLLEGE, ALASKA 99701
INSTITUTE OF MARINE SCIENCE
Biological Effects of Copper
and Arsenic Pollution
Cont. 18050 DLW
R71-8
-------
INSTITUTE OF MARINE SCIENCE
UNIVERSITY OF ALASKA
COLLEGE, ALASKA
BIOLOGICAL EFFECTS OF COPPER AND ARSENIC POLLUTION
Prepared by
D. K. Button and S. S. Dunker
Principal Investigator: D. K. Button
Final Report to the
Environmental Protection Agency
Contract No. 18050 DLW
Report No. R71-8 D. W. Hood
April 1971 Director
-------
ABSTRACT
Inhibitory effects of copper toward marine microorganisms were studied.
Phosphate was found to prevent copper inhibition. An organism capable of
metabolism at low phosphate concentrations was selected. Growth and phosphate
flux kinetics were described. These phosphate limited systems were found to
be copper sensitive only under conditions of manganese deficiency. Toxicity
occurred in this normally rather resistant yeast and in hydrocarbon oxidizing
microorganisms at about 10 M. The protective influence in reagent grade
phosphate was traced to its usual contamination with trace amounts of manganese.
Among a number of metabolic inhibitors found to inhibit phosphate transport
was arsenate. Phosphate also competitively reduced arsenate uptake. Reduced
phosphate uptake rates occurred at 10 M arsenate and high death rates in
cultures otherwise sustaining normal growth rates were Induced with 10"
—8
to 10 M arsenate in phosphate deficient systems. These levels are exceeded
in environmental and potable water systems.
Evidence is presented consistent with the view that both arsenate and
phosphate are accumulated by the same active transport system. The system
is peculiar in that velocities increase as the hydrogen ion is increased,
saturation of the system does not occur, and the transport temperature
coefficient is very large.
Evidence suggests that both arsenate and copper are important at existing
concentrations in the environment. When nutrients are dilute these anti-
metabolites prevent microbial metabolism thus affecting the steady state
chemistry which they control.
It is abundantly clear that toxicity levels of antimetabolites depend
not only on populations and chelate concentrations but in a major way on free
nutrients and trace metal levels.
-------
INTRODUCTION
It is well known that phosphate is actively accumulated by many
organisms (Blum, 1966; Goodman and Rothstein, 1968) and that the accumulation
is directly effected by arsenate (Mitchell, 1964; Jung & Rothstein, 1965).
However little data exists describing phosphate incorporation kinetics in
the concentration range found in natural water systems. One reason for
this is that organisms with the capacity to live in low phosphate natural
systems have sufficient affinity and capacity to satisfy their growth require-
ments at less than the usual background phosphate content of chemically defined
laboratory media. This paper describes a method to handle nutrient uptake
kinetics and suggests that environmental levels of arsenate are such that
they make this phosphate analogue an important antimetabolite in population
dynamics.
That heavy metals such as copper are inhibitory is also well known (Breslow
and Girotti, 1966; McBrien & Hassall, 1965; Steemann-Nielsen & Uiura-Anderson,
1969; Steeman-Nielsen & Wium-Anderson, 1970; Nielsen, 1969; Cabadaj & Gdoven,
1970; and Grande, 1966). This paper further describes the strong influence the
availability of divalent metabolites such as manganese has on the level at which
copper becomes inhibitory. Implications of the combined influence of these
antimetabolites are discussed.
METHODS
A marine yeast was selected from the collection of Dr. K. Natarajan,
Institute of Marine Science, University of Alaska, on the basis of ease of growth
on low phosphate medium. This pink yeast was isolated from Amukta Pass near
the Aleutian Islands. It was found to be nitrate positive, lactose negative,
asporogenous and otherwise similar to Rhodotorula rubra.
-------
-2-
As with most yeasts the sodium chloride concentration did not affect
maximum growth rates and was maintained low for chemical simplicity. Other
constituents were maintained at near limiting values for the rather low
populations employed. The constituents of the medium were (per liter):
glucose, 5 mg; NaCl, 40 rag; KC1, A mg; NH^SO^ 1 mg; MgSO^, 2.5 mg; CaCl2 •
2 H20, 5 mg; ZnSO^ • 7 H20, 70 yg; Fe(NH4>2S04 • 6 H20, 50 yg; MnS04 • 7 H20,
15 pg; Co(N03)2 • 6 H20, 0.15 ug; vitamins B^ B12 and biotin, 10~10 M in
distilled water followed by filtration. Continuous culture was in a single
phase 250 cc stirred glass reactor at 25°C as previously described (Button
and Carver, 1956).
Limiting substrate concentration and growth rates in continuous culture
were calculated from the usual relationships (Button, 1969) modified to
analyze conditions where limiting substrate concentration S is low and ap-
proaches the quantity supplied as impurities in the feed S.. Thus at steady
state:
(Sa - Sb - S) (1)
where X is the cell population and Y the yield constant. The yield constant
can be determined graphically from cell population at a range of added substrate
values according to equation 1 where S and S, are constant. The value of S,
D b
is then given by the steady state population X, when S is zero.
D cl
Xb = Y (Sb - S) (2)
A portion of added substrate was isotopically labeled so that the radioactivity
ratio, R, of extracellular steady state reaction medium to total isotope
concentrations gives S from the concentrations S and S, :
a b
S = R (Sa + Sb) (3)
-------
-3-
Substituting S, from equation 2 into equation 3 and solving for S:
R (S -
S = rS—-
The background substrate concentration S, was evaluated at the beginning
of each experiment according to equation 2. The portion of total substrate
remaining outside the organism at steady state R was determined by a rapid
separation procedure. Reactor contents were withdrawn with a syringe fitted
with a two way valve which allowed immediate filtration separation of the
dilute yeast population from surrounding medium. The single phase reactor was
fitted with a temporary filtered air source to replace removed liquid during
sampling so that the sample was not diluted with unreacted feed. The medium
supply system was shut down for a sufficient period to prevent rapid replacement
of the removed sample volume from appreciably disturbing the steady state.
Initial rates of uptake were measured from the slopes of organism radio-
activity with respect to time, usually over a six minute period. When the
-4
pH was altered, 10 M Tris buffer was added to the medium, otherwise similar
to that described. Organisms for these experiments were provided by a phosphate
limited continuous culture at half maximum growth rate operated solely for this
purpose. These systems were cleaned where necessary by exposing to a fresh
phosphate limited culture for phosphate scavenging. No phosphate precipitates
or radiochemical exchange with the glassware could be detected below pH 7.0
32
according to microfiltration and long term storage experiments with PO,
labeled medium.
Cell mass and volumes were calculated from an integration of electronic
counter and plate count population, size distribution and total volume from
electronic counter data, microbalance determination of dry weight in evacuated
heated chambers and checked by comparison vrlth glucose limited continuous
-------
-4-
cultures according to the equation X = y (S - S) (Button, 1969) where cell
yields, steady state populations and the glucose distribution is known.
A gamma spectrometer was used to count arsenate, scintillation or planchett
counting was used to count P depending on the radioactivity available. Usual
scintillants and precautions were used throughout.
RESULTS
During steady state growth phosphate controls growth velocity between
_a
zero and 2 x 10 M at pH 4.0 as shown in Fig. 1. The flux of phosphate
through the cell membrane can be calculated from initial uptake experiments
at concentrations greater than sufficient to achieve the maximum growth rate
of the organism, and thus measure the capacity of the phosphate transport
system. This flux is shown in Fig. 2. Arsenate is incorporated in a similar
manner and arsenate flux is also shown as calculated from initial uptake data.
Line slopes are approximately one throughout, indicating absence of a
saturation mechanism. This lack of saturation indicates diffusion limitation
of phosphate and arsenate transport so the system was examined for an active
transport mechanism. Recent data show that phosphate and arsenate show the
same affinity for the cell.
As shown in Fig. 3 plunging cells exposed to 10 M phosphate into higher
concentrations loads the cells to such an extent that previously formed
phosphate pools leak out. Preliminary experiments with high concentrations of
phosphate suggested this from the fact that initial uptake rates were very
high for the first minute, and then total radioactivity incorporated into the
cell decreased.
Exchanged phosphate amounts to a minimum concentration of 5 x 10~ M
inside the cell concentrated from the 10 M external solution. The actual
pool size is probably much larger, since all free phosphate would not be expected
-------
-5-
be the case. In fact, toluene completely stopped phosphate incorporation
as shown in Table I.
Table 1
Phosphate Uptake Inhibition
Inhibitor
_
Toluene
NaN3
1AA
DCCD
AsO."3
A
AsO,"3*
Q
Concentration
_
_5
10 M
10"5 M
10"5 M
10~7 M
10~7 M
10"8 M
Uptake rate
M/g cells/rain
12.6
1.6
8.7
11.3
4.3
0.4
5.1
Inhibition
0
100
56
31
12
36
94
20
*10 minutes pretreatment with inhibitor
Metabolic Inhibitors such as sodium azide were found to be inhibitory,
however, N, N-dicyclohexyldicarbodiimide (DCCD), a bound adenosine tripho-
sphotase inhibitor, was found to be relatively Ineffective. Since this system
does appear to be an active transport system of sorts, ATP does not appear
to be involved as suggested above and phosphoenol pyruvate is thought to
drive some sugar transport systems, iodacetate was tested. It is a well known
inhibitor of glyceraldehyde-3-phosphate dehydrogenase, a step preceding
phosphoenol pyruvate formation. The degree of inhibition was some three times
that of DCCD at 10 M and it is conceivable that phosphate transport is
driven by this glycolytic intermediate.
Most striking was the inhibition of phosphate transport by arsenate, a
phosphate analogue. Arsenate treated cells yielded only 6% of control activity
-------
-6-
Fig. 1. Steady state phosphate concentration as a function of
growth rate. Growth rates were calculated from continuous
culture dilution rate and viability data. The maximum
growth rate w is 0.175 hr~ . Phosphate concentrations
are calculated from extracellular radioactivity according
to the relationship
R
s
Data spread is shown as standard deviation from the results
of approximately 10,000 hours of continuous culture operation
data.
-------
1.0
.75
o .50
.25
STEADY STATE
PHOSPHATE
I 2
PHOSPHATE, M x I08
10,000
-------
-8-
Fig. 2. Arsenate and phosphate uptake as a function of
concentration. Solid symbols from initial uptake
data, open symbols from steady state continuous
culture data. Velocity units, moles per mg cells
dry wt per minute; concentration, molar.
-------
8
CD
O
10
12
10
PHOSPHATE
8
LOG
ARSENATE
-------
-10-
Fig. 3. Phosphate removed from cells by plunging cells
equilibrated with 10~ M phosphate into higher
concentrations to allow free or pool phosphate to
exchange back out. The first column shows radio-
activity incorporated from labeled phosphate* The
second column shows the concentration of cold
phosphate and the third column shows the phosphate
radioactivity remaining in the filtrate as an
indication of phosphate removed from the original
concentration supplied.
-------
PHOSPHATE POOLS
OOOO
_Q_Q_QP..
• '
PO,
UPTAKE
/ o
COLD
P04
COUNT
COUNT
EXCHANGE
PQ4
PC, REMOVED
MOLES XIO12
1723
1469
1270
333 *
288 *
IO'7M
lo-4
JO"2
lO'7
ID"2
2.7
5.5
5.6
2.5*
2.3*
* HEAT KILLED CONTROL
-------
-12-
to exchange back out. Hot ethanol as well as trichloracetic acid extrac-
_2
tion by conventional techniques shows an internal concentration of 10 M
32 —6
extractable P material from 10 M phosphate.
Because these organisms are found at low temperatures and because our
studies of maltose transport showed similar linear uptake kinetics and
operated very poorly at low temperature, we studied the effect of temperature
on the phosphate transport system. Fig. 4 shows an Arrhenius plot of these
data which yields an activation energy of 52,000 cal per mole. This value
also is high and suggests a mechanism more complex than simple diffusion and
one that requires reserve capacity for operation at low temperature.
Mitchell (1954) has suggested that only one of the four possible phosphate
species might be transported. Accordingly we studied the velocity of phosphate
transport with respect to hydrogen ion concentration as shown in Fig. 5. Both
the initial uptake rate and the concentration at half maximum growth rate show
that the resistance to phosphate incorporation is greater when the pH is raised.
Most experiments were performed at pH 4 where the flux is maximum. Another
reason for this choice of pH is the fact that NH_ is the only ligand present
with an appreciable binding constant, and this species virtually disappears
at pH 4. This simplifies consideration of extracellular chemical complexation.
Recent observations show a similar pH dependence for arsenate uptake, that
is significantly higher at pH 4 than pH 7.
A number of metabolic inhibitors were tested to see what sorts of anti-
metabolites effected transport from a medium 10 M in phosphate. Organic
solvents such as ethanol, dimethyl sulfoxide and toluene were tested because
in some of our other systems showing similar kinetics, membrane disruptants
have been found to increase substrate flux . This however, was not found to
D. K. Button, unpublished data
-------
-13-
if the cells were presented with 10 M arsenate 10 minutes before phosphate
uptake was measured as shown.
Since phosphate uptake is inhibited by both arsenate and metabolic
inhibitors, part of the inhibition was probably due to reduced respiration of
this strict aerobe. To see if arsenate inhibition was common with a phosphate
transport step the degree of arsenate transport inhibition by phosphate was
measured as shown in Fig. 6. Since arsenate transport kinetics are linear,
concentrations and velocities are presented rather than their reciprocals as
normally used when describing saturatable systems. The data show that arsenate
transport is reduced 50% by about 10 M phosphate.
The effect of arsenate on the viability of low phosphate systems is
shown in Fig. 7. Line slopes show that growth rates are slightly reduced by
—8 —7
10 M arsenate and that 10 M arsenate is sufficient to produce a death
rate of 0.15 hr~ from a control growth rate of 0.12 hr~ .
Many marine isolates are inhibited by low levels of heavy metals. Table
II shows some of the characteristics of hydrocarbon oxidizing organisms
selected mostly from Cook Inlet, Alaska. Table III shows their sensitivity
to copper. All showed reduced growth rates and most were killed by the
addition of 10 M copper as shown including £. rubra used in all of the
preceding experiments. However early experiments showed that high phosphate
concentrations prevent copper inhibition of growth rates or viability at any
level and generated the need to understand phosphate incorporation kinetics
described in the foregoing. This understanding allowed experimentation with
steady state continuous culture systems at low but not limiting levels of
phosphate. Fig. 8 shows a pair of glucose limited continuous cultures popu-
lation data. One set has a moderate (10 M) level of phosphate but trace
metals are at only the level provided by reagent grade chemical contamination.
The second set are with a full complement of trace metals but with added
-------
-14-
Fig. 4. Initial rate of phosphate uptake measured over a six
-6
minute period with respect to temperature from a 10
M medium according to the Arrhenius equation. Velocity
is expressed as moles phosphate per gram cells dry weight
per minute. Temperature shown is degrees centigrade.
Temperature plotted is the reciprocal of the absolute
temperature.
-------
40°
(I/T)
-------
-16-
Fig. 5. Effect of hydrogen ion concentration on phosphate
flux. Open symbols, steady state concentration of
phosphate in a continuous culture system diluted at
0.09 hr"1. Closed symbols, initial uptake rate from
a 10 M phosphate solution. Concentrations are
expressed as total extracellular phosphate.
-------
CONCENTRATION
AT STEADY STATE
in
^
X x
LU
O-CJ
CO
8
pH
-------
-18-
Fig. 6. Initial rate of arsenate transport with respect
to arsenate concentration with three concentrations
of phosphate.
-------
UJ
o
<
a:
e>
UJ
UJ
O)
UJ
o
0 PHOSPHATE
10
8
ARSENATE X 10° MOLAR
-------
-20-
Fig. 7. Growth rates in low phosphate solution at various
levels of arsenate. Inoculation was from a phosphate
limited continuous culture and populations measured
by plate counting.
-------
o
o
X
o
TIME, HOURS
-------
-22-
Flg. 8. Effect of heavy metals on the growth rate of IU rubra.
Data are steady or transient state populations in
continuous culture of two runs. Circles are electronic
counter counts, triangles are plate counts. Both runs
are glucose limited at 5 x 10~ M. Medium used in the
upper run contains 10** M phosphate but no added zinc,
manganese, cobalt or molybdenum. The lower run medium
contains these added trace elements but phosphate is
reduced to 3 x 10 M.
-------
TIME. DAYS
-------
Table II. Hydrocarbon oxidizing organism characteristics.
Culture
54
72
80
114
179
181
197
198
Dimensions
Gram uM
+ 0.6 x 1.0
+ 0.5 x 2.0
+ 1.0 x 1.2
+ 0.6 x 3.0
+ 0.6 x 3.0
- 0.5
+ 0.5 x 20
+ 0.6 x 3
Shape
rod
rod
coccoid
rod
rod
cocci
branched
filaments
rod
Upper
temp.
limit
30
30
37
30
25
25
-
25
u u
max max
broth kerosene Ea
hr-1 hr'1 K/cal/mole
0.23
0.35 0.20 16.0
0.35 0.087
0.35 0.020 16.0
0.35 - 15.2
0.15
_
0.35 - 14.5
Emulsifi-
cation*
2
1
1
2
2
0.5
-
0.5
Type**
Mycobacterium
Nocardia
Arthrobacter
Nocardia
Nocardia
Micrococcus
Streptomyces
Mycobacterium
I
NJ
**
Genus most closely resembling isolate.
Grams crude oil emulsified per gram dry weight of organism.
-------
-25-
Table III
Culture
54
72
80
114
179
181
197
198
11. ruba
Added Cu"""
0 10"6 M 10"3 M
5 x 104* 0 0
3 x 10* 0 0
1 x 106 0 0
1 x 106 3 x 105 0
5 x 10* 1 x 103 1 x 103
70 0 0
70 - 5 x 103
70 0 0
370 20 0
10~4 M
0
0
0
1 x 104
1 x 102
0
0
0
0
*Cultures were grown in nutrient broth, harvested and suspended
in mineral mannose medium pH 7.2 except R. rubra which was
suspended in mineral salts glucose medium pH 4.0. Columns above
report the population in broth culture 24 hr after inoculation
with various quantities of added copper shown.
-------
-26-
phosphate at only 3 x 10~ M so that added copper at 10 M is in a greater
than 1:1 ratio with remaining phosphate after population growth. It is clear
that the trace metal deficient steady state is repeatedly perturbed by added
copper showing no accumulated resistance and that manganese and to a lesser
extent zinc reduce the Inhibitory effects of copper. The low phosphate high
trace metal system remains unperturbed under the same conditions of perturbation
as shown in the upper data set. Atomic absorption analysis of our stock
phosphate revealed a manganese peak of sufficient size to provide an alternate
manganese source and thus provide protection from copper inhibition in high
phosphate systems.
DISCUSSION
The isotope dilution bioassay technique presents a useful method to
handle nutrient incorporation kinetics of a common metabolite incorporated
by a high affinity system. The data spread for phosphate uptake appears
large but the absolute concentrations involved are extremely small, less than
1% of that found in most marine systems. Although it appears that phosphate
uptake rates extrapolated to a finite concentration at zero concentration,
extensive data collection (10,000 hours of continuous culture operation) did
not substantiate this point or reduce the data spread. Initial uptake data
were obtained to evaluate the capacity of the phosphate transport system
and are the basis for describing the phosphate incorporation system in growing
systems as nonsaturatable. The mechanism of phosphate uptake remains obscure.
However the linear concentration velocity relationship has appeared before
and it may be a rather general type of incorporation kinetics. Active
transport is clearly implicated in that internal free phosphate concentrations
are greater than external concentrations. Metabolic inhibitors such as
iodoacetate and sodium azide also support the view that active transport is
-------
-27-
involved, possibly driven by phosphoenol pyruvate hydrolysis since DCCD
appears ineffective.
The fact that arsenate inhibits phosphate transport, phosphate inhibits
arsenate transport, and that the uptake kinetics are similar suggest that
this is another example of a transport system that cannot distinguish between
the two ions.
If the mechanism involves diffusion limitation of a transfer process
across a membrane one might expect a low temperature coefficient for transport
and that membrane solubilizing agents might enhance the transport rate. We
found neither to be the case and none of the usual transport models appear
attractive to explain the results presented.
The environmental implications of these findings are interesting. Table
IV shows some typical arsenate levels occurring in potable water and marine
Table IV
Q
Environmental Arsenic
Soil 10,000 ppbb
Hot springs 8,500
Lakes 2-50
Rivers 0.2 - 25
Desert ground water 8-27
Sea water 1-5
Rain water 0.02 - 14
^feoephl, K. H., Handbook of Geo-
chemistry, New York, 1969, p. 33-
1-1.
v» —8
1 ppb is equivalent to 1.4 x 10 M
-------
-28-
systems. Dr. Michael Pilson reports marine arsenic concentrations of between
—8 7
10 and 10 M with the concentration in some areas exceeding that of phosphate.
Since arsenic is particularly effective in low phosphate systems it seems clear
that existing arsenic levels are sufficient to effect the metabolism rate of
at least part of a diversified population in low phosphate natural systems.
It seems likely that low levels of arsenate have had a role in contributing
to some of our experimental irreproducibility. For example, sodium chloride
tolerance experiments using this organism probably more accurately reflected
the arsenate level in the reagent grade sodium chloride used than the salt
tolerance of the marine yeast.
The survey of various hydrocarbon oxidizing marine organisms at hand
showed major copper toxicity at 10 M. However most of the foregoing was an
effort to track down the rather variable sensitivity a rather resistant marine
yeast exhibited toward copper. The final continuous culture shows that
copper inhibition is related in a major way to trace metal nutrition. This is
not particularly surprising when one realizes that the prosthetic groups of
many enzymes can be replaced by heavy metals under certain conditions. It
does however mean that many organisms sensitive to heavy metals are probably
even more sensitive than reported when in natural systems which are trace metal
and particularly manganese deficient. Effects of this copper - manganese
interaction await careful quantitation.
It seems likely that growth rates of organisms in the natural environment
are reduced by antimetabolites such as arsenate and heavy metals and at
existing concentrations so that blooms only occur when nutrient build-up has
been sufficiently great to provide a positive driving force to autocatalytic
microbial reproduction sufficient to overcome the negative influence of anti-
personal communication, Or. Michael Pilson, Univ. of R. I.
-------
-29-
metabolites discussed. It is interesting that this "bloom or bust" type
behavior of aquatic populations is probably one that maximizes the standing
crop from available nutrients. The alternative of a stable slow growing
steady state population in the ocean for example would distribute available
nutrients so widely that major amounts of energy would be diverted to endogenous
metabolism in the smaller primary forms and to collection mechanisms in the
upper trophic levels. The expected result would be a lower yield of fish for
example. However some environmental arsenate levels may greatly exceed
threshold toxicity levels and seriously disturb the normal metabolic processes.
Reinforcement of these effects would be expected by additional antimetabolites
such as heavy metals.
-------
-30-
REFERENCES
Blum, J. J. 1966. Phosphate uptake by phosphate - starved Euglena. J.
Gen. Physiol., 49: 1125.
Breslow, E., and A. W. Girotti. 1966. The interaction of cupric and zinc
ions and the effect of cytidylic acid. J. Biol. Chem. 241; 5651.
Button, D. K. 1969. Thiamine limited steady state growth of the yeast,
Cryptococcus albidus. J. Gen. Microbiol., 58; 15-21.
Cabadaj, R., and T. Gdovin. 1970. Tolerance of sheep to cupric sulfate
(CuSO. • 5 H.O). Vet. Med. (Prague), 15: 21-29.
H L ~~~
Goodman, J. and A. Rothstein. 1957. The active transport of phosphate into
the yeast cell. J. Gen. Physiol., 40; 915.
Grande, M. 1966. Effect of copper and zinc on salmpnid fishes. Third Int.
Conf. on Water Pollution Res., Munich, Germany.
Jung, C. and Rothstein. 1965. Arsenate uptake and release in relation to
the inhibition of transport and glycolysis in yeast., 14; 1093-1112.
Mitchell, P. 1954. Transport of phosphate across the osmotic barrier of
Micrococcus pyogenes; specificity and kinetics. J. Gen. Microbiol.,
11: 73.
Nielsen, L. K. 1969. Influence of copper on the photosynthesis and growth
of Chlorella' pyrenoidosa. Dan. Tidsskr. Farm., 43; 249-254.
Steeman-Nlelson, E., and S. Wium-Anderson. 1969. The effect of deleterious
concentrations of copper on the photosynthesis of Chlorella pyrenoidosa.
Physiol. Plant., 22i 1121-1133.
Steemann-Nielson, E., and S. Wium-Anderson. 1970. Copper ions as poison in
the sea and fresh water. Marine Biol., £: 93-97.
-------
-31-
APPENDIX I
DATES AND TITLES OF CONTINUOUS CULTURE
BUNS AND EXPERIMENTS
-------
-32-
CONTINUOUS CULTURE DATA
Run
Dates
Experiments (Rh-rubra)
76
77
79
80
81
86
87
88
90
91
92
94
95
5/15/68 - 5/26/68
6/6/68 - 7/8/68
8/1/68 - 8/23/68
9/4/68 - 10/7/68
10/15/68 - 12/18/68
11/10/69 - 1/17/69
3/13/69 - 2/24/69
3/13/69 - 4/4/69
4/9/69 - 5/12/69
5/19/69 - 6/19/69
6/24/69 - 7/8/69
7/8/69 - 7/23/69
8/6/69 - 9/9/69
Cu inhibition @ low constant
phosphate concentration.
Added Cu4*, P6C12, HgCl2, ZnSo4
to observe effect on phosphate
limited culture.
Effect of copper on glucose
limited steady state continuous
culture.
Glucose limited steady state
culture w/o copper.
Added MnSo4, varying amounts of
substrate - glucose and copper to
reactor @ 50% maximum growth rate.
Phosphate limited steady state
continuous culture.
Phosphate limited steady state
continuous culture with P-^.
Phosphate limited steady state
continuous culture with p32.
Phosphate limited steady state
continuous culture with respect to
pH and determination of S.
Determination of S @ phosphate
limited steady state continuous
culture @ rO.3, rO.5 + rO.8.
Phosphate limited steady state
culture determination of S @ differ-
ent pH values.
Increasing amounts of phosphate
added to phosphate free steady state
continuous culture.
Phosphate limited steady state
continuous culture samples taken for
S @ rO.3, rO.5 + rO.8.
-------
-33-
Run
Dates
Experiments (Rh-rubra)
96
96R
96Rx2
100
101
105
106
107
110
113
114
120
127
9/23/69 - 10/6/69
10/6/69 - 10/23/69
10/23/69 - 12/8/69
12/4/69 - 1/8/70
1/16/70 - 2/12/70
2/16/70 - 2/26/70
3/2/70 - 3/14/70
3/2/70 - 4/11/70
4/15/70 - 5/8/70
6/8/70 - 6/13/70
6/22/70 - 7/9/70
8/18/70 - 9/14/70
10/7/70 - 11/11/70
Low phosphate steady state
continuous culture•
Phosphate limited steady state
continuous culture with respect to
pH.
Various sampling method for S in
steady state continuous culture
phosphate limited @ rO.3.
Phosphate limited steady state
continuous culture and determination
of S @ rO.3, rO.5 + rO.8 using new
quick sampling method.
Phosphate limited steady state
continuous culture c determination
for S @ rO.8.
Low phosghate steady state continuous
culture c determination for S @
rO.8.
Phosphate limited steady state
continuous culture c determination
for S @ rO.8.
^_l_
Effect of Ni on K for phosphate.
S
1^1
Determination of S with Ni ,
@ rO.5.
Determination of S with copper, @
rO.3.
Phosphate limited continuous culture
@ steady state c sample taken for S
@ rO.5.
14
Determination of_S with_C and copper
samples taken @ rO.5 + rO.8.
With a phosphate limited continuous
culture contamination of effect of
CU++ MnSO. + ZnSO..
4 4
-------
-34-
D. K. Button - FROM NOTEBOOKS
Ref.
Date
Expt.
DKB B4
P13
DKB B4
P14
DKB B4
P31
DKB B6
P4
DKB B6
P6
DKB B6
P9
DKB B6
P10
DKB B6
DKB B6
P14
DKB B6
P15
DKB B6
P16
DKB B6
PI 7
DKB B6
P18
DKB B6
P19
DKB B5
P32
DKB B5
P56
1/5/68
1/11/68
1/20/68
6/9/70
6/10/70
7/2/70
7/2/70
7/17/70
7/20/70
7/22/70
7/24/70
7/28/70
11/11/69
1/20/70
Inoculation + isolation of a low
phosphate requiring organism.
Set up culture in yeast extract, isolated
3 colonies and transferred to low glucose
media and allowed to grow through one log
phase.
A check for amounts of phosphate in
media salts.
Copper inhibition test for hydrocarbon
microbacterium isolate.
Copper inhibition of continuous culture @
steady state @ pH7.
Copper/ inhibition of continuous culture
using 64cu.
The use of low pH copper to prevent clumping
copper @ 3 x 10~6 H.
N.N'-Dicyclohexylcarbodiimide inhibition of
phosphate transport.
Initial uptake of phosphate c glucose
5 mg/1.
A viability check with phosphate + copper.
Initial uptake from continuous run, using
phosphate @ concentrations of 1 x 10"^ M •*
1 x 10~8 M.
Effect of pH on phosphate uptake.
Phosphate uptake of C. utilis + C. Albidus.
Phosphate concentration gradient in Rh
rubra.
Phosphate uptake in synthetic media.
Expt . for Rh rubra mutant .
-------
-35-
Ref. Date Expt.
0KB B5
P66
DKB B5
P67
DKB B5
P73
2/25/70
2/27/70
5/4/70
Low phosphate transport of mutants.
Continuation of phosphate mutant expt.
Preparation of phosphate transportless
mutants .
-------
-36-
INITIAL UPTAKES (HEAVY METALS)
Ref.
DaCes
Expts.
SSD BK2
P9
SSD BK2
P10
SSD BK2
P14/15
SSD BK2
P69-73
SSD BK3
P32
SSD BK3
P33
SSD BK3
P35
SSD BK3
P38
SSD BK3
P45
SSD BK4
P33
8/12/70
8/13/70
8/17/70
12/2/70 ->
12/7/70
12/21/70
12/29/70
12/30/70
11/7/71
1/14/71
3/22/71
Copper inhibition of phosphate uptake
pilot run.
Copper inhibition of phosphate uptake
pilot run 11.
Copper inhibition of phosphate uptake
run III.
As-04 @ 10"6 M, 10"5 M, 10~7 M, 10~8 M +
10-9 M pilot expts.
As-73 c AsO, initial uptake.
Phosphate inhibition of AsO, uptake.
Comparison of As-0, + phosphate flux
under identical conditions.
AS-73 pool concentration expt.
AS-0, toxicity to Rh rubra.
Effect of pH on AS-0, uptake.
Notes: * » day, * = expts. were repeated
-------
-37-
INITIAL UPTAKES (OTHER)
Ref.
SSD BK2
P12
SSD BK2
P13
SSD BK2
P16
SSD BK2
P17
SSD BK2
P18
SSD BK2
P19
SSD BK2
P27
SSD BK2
P35
SSD BK2
P37
SSD BK2
P39
SSD BK2
P44
SSD BK2
P47
SSD BK2
P53
SSD BK2
Pll
Dates
8/14/70
8/17/70
8/18/70
8/20/70
8/21/70
8/21/70
8/31/70
9/14/70
9/21/70
9/29/70
10/2/70
10/7/70
10/22/70
8/13/70
Expts.
Phosphate concentration gradient a range
of dilutions from 1 x 10~6 M phosphate •*
10-7 M phosphate.
Effect of temperature on phosphate uptake,
5°C. 15°C, 25°C.
Effect of temperature on phosphate uptake,
5°C, 15°C, 25°C.
Phosphate concentration gradient a range
of dilutions from 1 x 10"^ M phosphate •*•
5 x 10~6 M phosphate.
Effect of temperature on phosphate uptake,
10°C, 20°C + 25°C.
Phosphate inside Rh rubra cells (pools) .
Glucose requirement of phosphate transport.
Effect of temperature on phosphate uptake,
1°C.
Initial uptake regarding pH c phosphate
limited cells.
Effect of sodium + potassium.
Effect of pH on phosphate limited cells
(final expt.).
Repeat of salt concentration effect using
sodium and potassium c phosphate limited
cells.
Effect of pH on phosphate limited cells c
tris buffer.
INHIBITORS
N.N'-dicyclohexyldicarbodiimide (DCCD) effe
on phosphate uptake.
-------
-38-
Ref.
Dates
Expts.
SSD BK2
P56
SSD BK2
P58
SSD BK2
P74
SSD BK2
P75
SSD BK3
P21
SSD BK3
P36
SSD BK4
P7
SSD BK4
P8
SSD BK4
P12
SSD BK4
P14
SSD BK4
P25
SSD BK4
P26
11/11/70
11/13/70
12/7/70
12/11/70
12/16/70
1/4/71
2/16/71
2/17/71
2/22/71
2/24/71
3/8/71
3/9/71
Icdoacetic acids, effect on phosphate
uptake.
Sodium azide's effect on phosphate
uptake.
Sodium dichromate effect on phosphate
uptake.
Effect of high temperature on phosphate
uptake, 25°C, 30°C + 35°C.
Effect of high temperature on phosphate
uptake, 25°C, 408C + 50°C.
Effect of low temperature on phosphate
uptake, 25°C, 40°C, 50°C, 1°C.
Phosphate inhibition of AS-0, uptake,
25°C, 40°C, 50°C, °C. *
Phosphate uptake c sodium chloride.
Saline inhibition check for viability.
Phosphate inhibition @ low temperature,
5°C, 158C + 25°C.
Effect of EtOH on phosphate transport.
Repeat of EtOH on phosphate transport
and including effect of dimethylsulfoxide
(DMSO) 20%.
-------
-39-
APPENDIX II
EFFECT OF CLAY ON THE AVAILABILITY OF DILUTE ORGANIC
NUTRIENTS TO STEADY-STATE HETEROTROPHIC POPULATION
SOME FACTORS INFLUENCING KINETIC CONSTANTS FOR
MICROBIAL GROWTH IN DILUTE SOLUTION
-------
-41-
Made in the United States of America
Reprinted from LIMNOLOGY AND OCEANOGRAPHY
Vol. 14, No. 1, January 1969
pp. 95-100
EFFECT OF CLAY ON THE AVAILABILITY OF
DILUTE ORGANIC NUTRIENTS TO STEADY-STATE
HETEROTROPHIC POPULATIONS
BY D. K. BUTTON
Reprinted from Limnology and Oceanography. _14_(1): 95-100
(1969), by permission of the Editor.
-------
-43-
EFFECT OF CLAY ON THE AVAILABILITY OF
DILUTE ORGANIC NUTRIENTS TO STEADY-STATE
HETEROTROPHIC POPULATIONS1
D. K. Button"
Institute of Marine Science, University of Alaska, College 99701
ABSTRACT
The effect of clays on the availability of small organic molecules to microorganisms
was determined. Recovered suspended sedimentary material and Bentonite was tested
for its ability to compete with yeast and bacteria for thiamine or glucose in continuous
culture systems at low organism concentrations. The added clays do not render the
organics tested unavailable to microorganisms or remove them from solution to a detectable
or significant extent. The addition of clays can, however, effect a perturbation to the
steady system, possibly by altering the inorganic chemistry of the dissolved phase.
INTRODUCTION
Chemical analyses of certain systems
have been interpreted to show that clays
and sediments are effective in adsorbing
organic compounds from solution. These
clay-organic associations include starch
(Lynch, Wright, and Cotnoir 1956), pro-
teins and amino acids (Sieskind and Wey
1959), and carbohydrates (Bader, Hood,
and Smith 1960). The amount of adsorp-
tion determines the effect of clays on the
dissolved organic chemistry of sediment-
laden water systems. Suspended sediment
loads normally run from 0.5 to 100 mg/
liter in the glacier-fed bays and inlets of
southeast Alaska and even higher in
streams draining areas of intensive mining
or other earth-moving activities.
The extent of the clay-organic associa-
tion was further investigated by measur-
ing the influence of clay particles on the
nutrient adsorption kinetics of some het-
erotrophic cultures. This was done by
subjecting steady-state low population nu-
trient-limited cultures to perturbation by
injected clay.
1 Substantial support for this work was provided
by U.S. Department of Interior, Office of Water
Resources and Federal Water Pollution Control
Administration, Grants A-007-ALAS and WP-
1240-01. Institute of Marine Science Contribution
No. 48.
21 am indebted to Emma Dieter who provided
technical assistance.
THEORY
In a nutrient-controlled continuous cul-
ture system, the limiting nutrient governs
the steady-state cell population (Monod
1950; Herbert, Elsworth, and Telling 1956;
Button and Carver 1966) and is divided
between the growing organisms and extra-
cellular solution according to the equation:
= Y(S0-S),
(1)
where S0 is the initial limiting nutrient con-
centration and Y the yield constant, grams
of organisms produced per gram of lim-
iting substrate used, (S0-S). In continu-
ous culture at 100% viability the average
growth rate is numerically equal to the
dilution rate and is set by the concentra-
tion of S at a particular temperature. This
rate increases as a monotonic increasing
function of substrate concentration fre-
quently approximated by the Michaelis-
Menten equation:
K. + S-
Thus one can determine changes in avail-
able substrate concentration, S, by observ-
ing changes in the steady-state concentra-
tion of organisms, X, at a particular rate.
If one chooses a value of S0 near the
Michaelis constant for growth, K,, and sets
the dilution rate (which is equal to the
growth rate at 100% viability and perfect
population dispersal) at a large fraction
95
-------
-44-
96
D. K. BUTTON
of the maximum growth rate, j^mnv, small
changes in available limiting substrate are
reflected by large changes in the cell popu-
lation. For example, K, for Escherichia coli
is 4 mg glucose/liter (Monod 1950), about
the same as for most heterotrophs near
their upper temperature limit under usual
laboratory conditions. If one operates at a
dilution or growth rate of 0.5 /imnx, where
S = K,, and sets S0 at 5 mg/liter, the
resulting population at Y = 0.5 will be
0.5 mg/liter, according to equation (1).
Under these conditions a 10% reduction
in available substrate would reduce the
steady-state bacterial population to half its
original value.
CLAY PREPARATION
Initial nutrient adsorption experiments
were run with sediments collected from
routine hydrographic stations in southeast
Alaska. Sufficient seawater was filtered to
obtain a few hundred mg of sediment. The
sediment was weighed, resuspended, and
autoclaved 15 min at 121C. This sterile
suspension was cither introduced into the
feed chamber (a 20-liter carboy) or the
culture vessel (a 250-ml boiling flask) of
the continuous culture apparatus, depend-
ing on the experiment. Sterile conditions
were maintained by injecting the suspen-
sions with a syringe through rubber cov-
ered ports. Standard clay was prepared by
grinding Montmorillonitc No. 27 (Benton-
ite. Belle Fourche, S. Dak., Ward's Natu-
ral Science Establishment, Inc., Rochester,
N.Y.) in a porcelain mortar and pestle.
This was fractionated by sedimentation
and a cut of approximately 1.2 X 10-I3-g
particles was obtained with 90% of the
particles between 1.5 and 2.5 /u in diam-
eter. These particles remained indepen-
dent from one another and from the
microorganisms, this was clear in phase
contrast examination of the suspensions.
CULTURE METHODS
Nutrient adsorption by clay was mea-
sured in a single-phase continuous culture
apparatus of the type described by Button
and Carver (1966). Cryptococcus albidus,
o
z
"* c
m O
a
o
-------
-45-
CLAY-NUTH1ENT ASSOCIATION
97
A third organism was selected for use in
experiments involving low buffer capacity
and high pH—the only one of those tested
that would tolerate these conditions. This
organism, a pink yeast tentatively identi-
fied as belonging in the genus Rhodotorula,
was isolated from Amukta Pass near the
Aleutian Islands by K. Natarajan. It pos-
sessed the requisite characteristics of high
growth rate and stable behavior in con-
tinuous culture without adhering to glass
surfaces. Culture conditions were 25C, pH
6.5 in the medium described above except
that Na2HPCvH2O was reduced to 3 mg/
liter. This is referred to as the "low phos-
phate" medium.
COUNTING AND SIZING
Populations were counted on plates pre-
pared from the liquid culture medium plus
1.5% agar. Where possible an electronic
counter (Coulter Counter Model B, Hia-
leah, Fla.) was used for counting and sizing
the organisms and the clay. Agreement
between methods was normally within sta-
tistical error when more than 1,000 colo-
nies were counted. Yeast cells could be
counted in the presence of clay with the
counter owing to their greater average vol-
ume; however, all populations reported in
the presence of clay were determined from
plate counts. Populations for the K, mea-
surement were determined from optical
density in a 5-cm cell at 625 Tap..
RESULTS
A thiamine-limited continuous culture of
C. albidus was maintained at steady state
at half maximum growth rate of 0.132 hr1
corresponding to a doubling time of 5.25 hr
at 25C. The feed contained 2 X 10-12 M
thiamine, and the Michaelis constant (ex-
tracellular concentration at (j. = 0.5uma,) for
this nutrient was 4.7 X 10'13 M (unpub-
lished data). This resulted in a reactor
population of 2.0 X 107 cells/liter. Clay
recovered from Glacier Bay, Alaska, was
then injected into the feed to a level of
10 mg/liter of medium. This remained in
suspension owing to the agitation caused
by air bubbles from the sparger used to
saturate the feed medium with air. The
time allowed for equilibration was 48 hr,
during which 6.3 volumes of feed flushed
through the 500-ml reactor. The final pop-
ulation in the reactor after equilibration
was 2.4 X107 cells/liter, essentially the
same as before adding the clay. A fall in
the cell population had been expected due
to the addition of the clay-thiamine (vita-
min Bj) term in the equilibrium:
Clay - B!
+ Cells ^ Clay + BI
+ Cells^Clay + Cells-B,. (3)
However, since the above evidence indi-
cated a low binding constant for the clay-
thiamine association, the binding ability of
clay was further tested with the carbon
source—glucose. Experimental conditions
to test the affinity of clay for glucose were
the same as above except that thiamine
was added to 10-° M, and glucose was
added to 6 mg/liter. Under these condi-
tions, the Michaelis constant for glucose is
3.0 mg/liter as shown in the plot of equa-
tion (1) in Fig. 1 according to the method
of Button and Carver (1966). The reactor
population before and after adding 140 mg
of Bentonite of 10 liters of feed is shown
in Fig. 2. The upward portion of the
curve indicates that the experiment was
terminated before a true steady state was
reached. However, the glucose removed
by the clay cannot have exceeded 0.7 mg/
liter and appears to approach zero.
Since pH is a factor in the chemistry
and charge of the clay surface (Sieskind
and Wey 1959), the apparent lack of ad-
sorption was tested again at neutrality. For
this purpose E. coli was set up in continu-
ous culture at pH 7, with a feed glucose
concentration of 6 mg/liter and at a dilu-
tion or growth rate of 0.35 hr1. The yeast
used in the experiments above was not
used here because its glucose transport
system apparently fails above pH 5. The
total lack of effect of Bentonite particles
injected directly into the reactor on the
steady-state cell population is shown in
Fig. 3. As the clay particle concentration
changed from zero to 1012 particles/liter
-------
-46-
D. K. BITITON
8 9 10
TIME, DAYS
12
FIG. 2. Steady-state population of Cryptococ-
cits albidus, glucose limited at 6 nig/liter growing
at 0.132 hr'1 before and after adjusting the level
of clay in the feed to 14 mg/liter.
and then exponentially towards zero the
bacterial population remained unchanged.
A monomolecular glucose layer on the clay
would have reduced the dissolved glucose
level 4 mg/liter to 2 mg/liter and caused
immediate and complete washout of the
culture.
Phosphate was the only nutrient in large
excess in these experiments. To rule out
the possibility of phosphate protecting the
clay from associations with the organic
compounds, the low phosphate medium
was used with the pink yeast described
above. This yeast possessed characteristics
allowing low population culture techniques
without buffers or crielates, and it behaved
very well in continuous culture. It has a
thiamine requirement that is satisfied by
something less than the 9 X 104 molecules/
organism required by C. albidus at half
maximum growth rate.
The chemical species having the highest
binding capacity for cations in the low
phosphate medium is NH3 which is present
at only 9 X 10"7 M at pH 6.5. The steady-
state population in the presence of the 2 X
10"5 M phosphate, or low phosphate me-
dium, is shown in Fig. 4. The initial addi-
tion of Bentonite to the reactor at 4.5 days
caused a temporary 80% decrease in the
cell population. The level added was 20
mg of clay/liter. However, subsequent
additions of 10 and 100 mg/liter of clay
PARTICLE CONCENTRATION per liter
O Q. 0 o
_ .0 ^ „= K3
. !
^
I ci
\
\
\ ORGANISMS
-O. ~ \
\
S
s
.AY \
\
. V
) 10 20
TIME, HOURS
30
Fie. 3. Steady state of population of Esch-
erichia coli growing at 0.35 hr1 before and after
the introduction of 10" clay particles/liter into the
reactor.
to the feed had no effect on the population.
Under these conditions, there was some
coalescing of the-clay in the feed vessel
so that the reactor clay concentration was
somewhat lower than the feed concentra-
tion. In Fig. 5, the effect of 100 mg of clay
added to one of duplicate batch cultures
9 hr after inoculation is shown. The cul-
tures were limited to about 2 X 107 orga-
nisms/liter by the 3 mg/liter of glucose
present initially. The added clay had no
effect on the final cell population; the two
cultures behaved as duplicates within the
normal variability of batch cultures.
DISCUSSION
The vitamin thiamine is a cation in solu-
tion with a positive charge residing at the
nitrogen (3-position) of the thiazolium ring
under the conditions reported here. Since
clays have been reported to possess ion
exchange capacity for cations (Hendricks
1941) and to have surface affinity for
neutral molecules (Bader et al. 1960), it is
surprising that no binding of glucose or
thiamine was detected in any of the experi-
ments reported here.
-------
-47-
CLAY-NUTHIENT ASSOCIATION
99
0 I
234 56789 10 II
TIME, DAYS
FIG. 4. Steady-state yeast population, glucose
limited in unbuffered medium, as clay was added
to the reactor and to the feed.
Temporary reduction of a yeast popula-
tion owing to the addition of clay in the
low phosphate experiments was detected.
Since the populations returned to the ini-
tial steady-state level and did not again
decrease when subsequent and even larger
clay additions were made to the feed, die
effect could not have been due to nutrient
removal. The temporary reduction in cell
population was not surprising since biolog-
ical membranes are particularly sensitive
to perturbation when not stabilized with
a buffer such as phosphate or a chelate
ligand. The failure of subsequent additions
of clay to cause similar perturbations can
be explained by the normal capacity of
microorganisms to respond to new situa-
tions after a suitable induction period
(Horecker, Thomas, and Monod 1960).
Thus it seems likely that a chemical change,
such as a rise in concentrations of free
heavy metals, caused the temporary re-
sponse shown in Figs. 2 and 4. Recent
experiments' (unpubh'shed) have shown
similar temporary inhibition by injected
copper at 10~7 M in a medium of low buffer
capacity. Stotzky and Rem (1967) reported
the absence of an inhibitory effect in ex-
periments using a clay culture medium;
this was probably due to the protection
afforded by the yeast extract component
of their basal medium.
Under the various experimental condi-
tions described here, the data show that
10
40
20 30
TIME, HOURS
Fie. 5. Duplicate batch cultures of yeast,
glucose limited at low phosphate concentrations.
Arrow indicates the addition of clay to a level of
100 ing/liter to one of the cultures.
the equilibrium constant between free or-
ganics and those bound to suspended sedi-
mentary material lies far in the direction
of the dissolved state. The experimental
conditions included a range of pH, neutral
and charged substrates, media of high and
low buffer capacity, and different types
of clay preparations and concentrations.
Thus, it seems unlikely that in natural sys-
tems the level of suspended organisms
using small organic molecules is materially
influenced by the level of suspended sedi-
mentary material. The data do show evi-
dence that clays cause temporary reduc-
tion of the growth rates of the organisms
used, which is probably due to the solution
of some component.
REFERENCES
BADEK, R. C., D. W. HOOD, AND J. B. SMITH.
1960. Recovery of dissolved organic matter
in sea-water and organic sorption by particu-
late material. Geochim. Cosmochim. Acta,
19: 236-243.
BUTTON, D. K., AND ]. C. CARVER. 1966. Con-
tinuous culture of Torulopsis ntilis. A kinetic
study of oxygen limited growth. J. Cen.
Microbiol., 45: 195-204.
HENDRICKS, S. B. 1941. Base exchange of the
clay mineral montmorillonite for organic cat-
ions and its dependence upon adsorption due
-------
-48-
100
D. K. BUTTON
to van der waals forces. J. Phys. Chem., 45:
65-81.
HERBERT, D., R. ELSWOHTH, AND R. C. TELLING.
1956. The continuous culture of bacteria; a
theoretical and experimental study. J. Gen.
Microbiol., 14: 601-622.
HORECKER, B. L., J. THOMAS, AND J. MONOD.
1960. Galactose transport in Escherichia
colt. I. General properties as studied in a
Galactokinaseless mutant. J. Biol. Chem.,
235: 1580-1585.
LYNCH, D. L., L. M. WRIGHT, AND L. J. COTNOIR,
JR. 1956. The adsorption of carbohydrates
and related compounds on clay minerals.
Soil Sci. Soc. Am. Proc., 20: 6-9.
MONOD, J. 1950. La technique de culture con-
tinue. Th6orie et applications. Ann. Inst.
Pasteur, 79: 390-399.
NATAHAJAN, K. V., AND R. C. DUGDALE. 1966.
Bioassay and distribution of thiaminc in the
sea. Limnol. Oceanog., 11: 621-629.
SIESKIND, O., AND R. WEY. 1959. Sur 1'adsorp-
tion d'acides amines par la montmoiillonitc-H.
Influence de la position relative des deux
fonctions-NH, et -COOH. Compt. Rend.,
248: 1652-1655.
STOTZKV, G., AND L. T. REM. 1967. Influence
of clay minerals on microorganisms. IV.
MontmonUonite and kaolinite on fungi. Can.
J. Microbiol., 13: 1535-1550.
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-49-
Organic Matter In Natural Waters, ed D. W. Hood
lust Mar. Sci.. U. of Alaska, Pub. No. 1. 1970
SOME FACTORS INFLUENCING KINETIC CONSTANTS FOR
MICROBIAL GROWTH IN DILUTE SOLUTION
D. K. Button
Institute of Marine Science
University of Alaska
College, Alaska 99701
A growing microorganism reproducing in dilute aqueous environments
such as our natural water systems is faced with the problems of recognizing
and concentrating desired nutrients,' preventing the concomitant entry of
chemical species not required and retaining desired components within itself.
This is successfully accomplished by simple diffusion accompanied by a set
of transport systems, each capable of recognizing a required species and
carrying it across the cell wall to a site of chemical modification so that the
nutrient cannot move back by the same path. The nutrient molecules a
species of microorganism can recognize are genetic parameters and some
transport systems can be produced or omitted in response to the chemical
environment. These systems are sufficiently enzyme-like to behave in a
similar kinetic fashion and can be described with maximum velocities,
apparent Michaelis constants and inhibition constants. These kinetic
constants are a measure of effective collision frequency at the site of the
growth rate limiting step as compared with the growth rate when that step is
saturated.
The rate limiting step for a microorganism grown in dilute solution
can therefore be nutrient diffusion through the aqueous environment to the
cell surface, transport of recognized nutrients through the cell surface into
the organism or a subsequent slow step in the conversion of the nutrient into
useful biochemical forms. The kinetics of each step except the first can be
affected by the external environment of the organism. The following
observations describe how growth velocity can be affected by nutrient
concentration, mixing, temperature, pH and heavy metal concentration.
Growth velocity is normally an increasing function of limiting
substrate concentrations. This can be hyperbolic, linear or logistic depending
on the rate limiting mechanism. The bottom curve of Fig. 1 shows how the
growth velocity of a yeast cell Cryptococcus albidus varies with the vitamin
thiamine at the cell surface. The curve is computed from the
Michaelis-Menton equation, the variation in cell yield with growth rate, the
variation in organism size with growth rate and the apparent Michaelis
constant for growth experimentally determined and reported elsewhere
(Button, 1969). The upper curve is based on a computation of the
concentration of thiamine required in the bulk of the medium to supply
thiamine at the cell surface at the required rate assuming no mixing and
Flickian diffusion. A convenient equation presented by Borkowski and
Johnson (1967) for this purpose is presented below. As one can see, the
effective concentration at the cell surface is about half that in the bulk of
the medium in an unmixed system. Normal growth rates of yeast, bacteria
and algae are indicated to provide a reference scale. These data would
•537-
Reprinted from Organic Matter in Natural Waters, ed. D. W.
Hood, Inst. Mar. Sci., U. of Alaska, Pub. No. 1, 1970, by
permission of the Editor.
-------
-50-
YEAST and BACTERIA
10-18
123456
GROWTH RATE, SECT'XIO8
Fig. 1. The lower curve represents the thiamine concentration at the surface of
the organism, Cryptococcus albidus, computed from the Michaelis Menton
equation, Ks 4.4 x 10"' 3M, and the changes in cell volume and yield with
growth rate. The upper curve represents the thickness of the diffusion film
in a non-mixed solution computed from equation 1.
•538-
-------
-51-
kinetic constants and microbial growth
indicate that in this case of vitamin limited growth, diffusion to the cell
surface is the growth rate limiting step. If that is the case, the thiamine
utilization rate is a function of mixing. We found this to be the case
experimentally (Button, 1969).
Fig. 2 shows how the stagnant film thickness would vary with growth
rate if a concentration of nutrient equivalent to its apparent Michaelis
constant were provided in the nutrient medium. Curves shown were obtained
as described by Borkowski and Johnson (1967). Using the experimentally
determined Michaelis constant for thiamine limited growth of 4 x 10" ' 3 M
(center line) as the concentration at the cell surface, the film thickness at
usual growth rates of 5 x 10s sec"1 is about one micron, which is within the
normal range of stagnant film thicknesses. The upper and lower lines
represent how the film thickness would vary if the apparent Michaelis
constant were either ten times larger or ten times smaller than its actual
value. The divergence suggests the existence of a rational relationship
between the Michaelis constant for a substrate and the amount of substrate
required by a microorganism. This was first noticed in tabulations of
published Michaelis constant data. The resulting relationship js about what
one would expect on the basis of molecular collision frequency in the
Salmonella typhimurium system at published concentrations of sulfate
binding enzyme (Drey fuss, 1964) and assumed values of sulfate flux. The
same relationship can be obtained from the C. a/feidus-thiamine system by
substituting the film thickness at half the maximum growth rate of 5.4 x
10~4 cm, obtained from Fig. 2, into the general equation for relating
diffusion limited nutrient concentration drops presented by Borkowski and
Johnson (1967).
where Cf and Cs are concentrations of nutrient in the bulk of the medium
and at the cell surface; n is the growth rate; x is the distance from the cell
center to the outside of the film in units of r, the cell radius; D is the
diffusivity of the nutrient in the medium; and \ is the yield of cell mass from
nutrient provided. If the experimental values of these constants for the C.
a/bidus-thiamine system are substituted into equation (1) the following
relationship is obtained:
A logarithmic plot of this equation is shown in Fig. 3. Notice that the
experimental values of Wright and Hobbie (1965) and Borkowski and
Johnson (1967) for acetate and oxygen, respectively, fall near this line,
although the yield constants are a factor of 106 lower than the thiamine data
used to formulate the relationship.
Growth velocities of microorganisms when the rate limiting step is
substrate saturated follow the Arrhenius equation as shown in Fig. 4. Pena
(1955) suggested a similar relationship to temperature. Our values of Ks for
•539-
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-52-
14
I'2
cc
SK>
LJ
o
8
n: 2
:cf=f(iOKs)
I 23456
GROWTH RATE, SECT'XIO6
Fig. 2. The center curve (solid line) represents the maximum diffusion film
thickness that will allow the indicated growth rates with the concentration
in the bulk of the unmixed medium equivalent to the Michaelis constant
for thiamine in the thiamine requiring Cryptococcus albidus system. The
darkened lines show the same maximum film thicknesses when the bulk
medium is raised or lowered by a factor of ten.
-540-
-------
C9
O
7
6
5
4
3
2
I
0
-I
I
?
OXYGEN
© S*> ACETATE
o ye
i S i
7 8 9 10 II 12 13 14
-LOG Ks
Fig. 3. Fit of experimental Ks data to equation 5. Points to the left of
the line represent departure from diffusion limited systems.
-------
Tt CENTIGRADE
20 10
C.ALBIDOS
GLUCOSE
R. MUCILAGINO
GLUCOS
i
Ln
Fig. 4. Arrhenius plot of Vmax for R. mucilaginosa (solid circles), and u. Ks vs.
temperature (open circles).
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-55-
kinetic constants and microbial growth
glucose at 1.6 C are shown along with the value of Ks for glucose at 25 C
with C. albidus and Pena's value for glycerol using C. u tilts at 15 C. The
values for these yeast Ks data increase with temperature in the same way as
the maximum velocity of growth. Thus if
Umax = Ae-E*/RT (3)
then
Ks = AVEa/RT (4)
and from (1)
A-e-Ea/RT = _J_^
7xl07X * '
Where growth rate is limited at the cell surface by nutrient transport
the system can be likened to substrate competition for passage through a
matrix of doors. These doors at the cell surface axe chemical in nature and
subject to closing (chemical modification) by inhibitory components of the
medium.
Table 1 shows the maximum growth rate of C. utilis after extended
growth at steady state in continuous culture. Population was regulated by
flow rate at a low value so that the population remained at jumax. The rates
observed were different from the corresponding batch growth rates and
much slower after the first three days. Notice that the rate responds to
glycerol concentration in a different way at pH 4 than at pH 6, indicating a
Michaelis constant of a higher order of magnitude at pH 6. This difference
between batch and continuous culture data also occurred with C. albidus and
Rhodotorula glutmis and is under current consideration. The data show that
long term substrate limited growth at low population can respond to
concentration in a different way than short term substrate uptake.
Fig. 5 shows the response of R. glutmis, a marine pink yeast, to
copper. The yeast was selected because it grows well in continuous culture
with no added chelates or buffers and only substrate quantities of phosphate
(Button, 1969). Under these conditions the rate of growth was sharply
reduced, about 70%, by the addition of copper to a final concentration of
10~6 M. The copper content of the continuous culture reactor decayed at the
rate shown and the population approached its original value of 1.5 x 10"
cells/liter. Succeeding additions of heavy metals had progressively less effect
on the growth rate. However, the steady state population was reduced to
about 1 x 10s cells/liter as shown. This indicates a higher heavy metal
tolerance at the expense of a less efficient transport system which lowers the
total standing crop at a given level of nutrients.
In summary, mixing, temperature, nutrient concentration and
inhibitor concentration all affect the efficiency and rate at which microbial
processes occur and effects can be described in a rational manner.
-543-
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-56-
Table 1. The maximum growth rate of C. utilis after extended growth at
steady state in continuous culture.
Glycerol
(mq/liter)
5
1000
5
1000
pH
4.0
4.0
6.2
6.2
wmax
(hH)
0.425
0.425
0.024
0.144
•544-
-------
-57-
20
r-
'2
x
co 10
o
o
X
ICT6M Cu'
.**
I
240 250 260 270 280
TIME, HOURS
720 730
Fig. 5. Effect of first and second addition of 10~6 M Cu" added to the
reactor of a steady state continuous culture of a marine yeast
(pink 1) growth at0.092/hr.
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-58-
Button
DISCUSSION
BELSER: Dr. Button, Dr. Jones reported alterations in cellular morphology
with the emergence of resistants. Have you observed a similar phenomenon
in these kinds of studies?
BUTTON: This is recent work. We have developed a theory of this over the
past couple of years and this was experiment number one. If I had had things
organized properly, we could have had size distribution prof iles throughout
this copper perturbation. The only yeast that will work for this has turned
out to be tiny and it was below our current facility for measuring size
distribution. We have the facility for answering your question but I do not
have an answer for your question. I think the probability of what you say, or
of what Dr. Jones said, happening, though, is certainly quite great.
JONES: Did the yeast concentrate the metals at all?
BUTTON: The ratio between the copper concentrations and cell mass was
10£; in other words, if all of the copper had gone into the mass of the cell it
would be one part in 106 copper. I do not have data, but I cannot see how
they could do otherwise. One has on the surface the type of material that
has ligands of the type that binds things like heavy metals. As you know, if
you do not have a really clean system and you try inoculating, as Dr.
Jannasch pointed out, with a small inoculum, it is hard to get things going,
but if you use a large inoculum, it is not so hard and this is probably because
the cell surfaces are good complexing agents which, of course, has been
demonstrated by many people.
REFERENCES
BORKOWSKI, J. D., and M. J. JOHNSON. 1967. Experimental evaluation
of liquid film resistance in oxygen transport to microbial cells. Appl.
Microbiol., 15: 1483-1488.
BUTTON, D. K. 1969. Effect of clay on the availability of dilute organic
nutrients to steady-state heterotrophic populations. Limnol.
Oceanogr., 14: 95-100.
BUTTON, D. K. 1969. Thiamine limited steady state growth of the yeast
Cryptococcusalbidus. J. Gen. Microbiol., 57: 777.
DREYFUSS, J. 1964. Characterization of a sulfate and
thiosulfate-transporting system in Salmonella typhimurium. J. Biol
Chem., 239: 2292-2297.
PENA, C. E. 1955. The influence of temperature on the growth of T.
utills. M.S. Thesis, Univ. Wisconsin. Univ. Microfilms, Ann Arbor,
Michigan.
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-59-
kinetic constants and microbial growth
WRIGHT, R. T., and HOBBIE, J. E. 1965. The uptake of organic solutes in
lake water. Limnol. Oceanogr., 10: 22-28.
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