MICROBIOLOGY
ECOLOGY
ELSEVIER
FEMS Microbiology
Ecology
21 (1996) 87-101
Microflora of a subalpine lake: bacterial populations, size and
DNA distributions, and their dependence on phosphate
D.K. Button a’*, B-R. Robertson a, Friedrich Jiittner b
a Institute of Marine Science, University of Alaska, Fairbanks, Alaska 99775, USA
b Institutjiir Pflanzenbiologie, Limnologische Station, Seestrasse 187, Kilchberg, Swit:erland
Received
12 December 1995; revised 8 May 1996; accepted 9 May 1996
Abstract
In Lake Zirich, a deep subalpine mesotrophic lake, phosphate was low or limiting at 0.2 to 1 FM relative to combined
nitrogen at 50 ~_LM.Heterotrophic bacteria were responsible for 53% of the observed microbial wet biomass in our depth
profile while phytoplankton, largely PZanktoGzrti (Uscillutoriu~
rubescens,
contributed most of the remainder. Most cell
carbon was contributed by this carbon-sufficient
cyanobacterium. A material balance indicated that most of the phosphate
was sequestered by the bacteria due to a higher phosphate content and specific affinity for this nutrient. Size distributions of
the heterotrophic bacteria were narrow; 90% of organisms were from 0.06 to 0.66 km3 in volume. Several subpopulations of
bacteria were resolved by flow cytometry, and bivariate fluorescence (DAPI-DNA) and light scatter (cell-size) histogram
profiles varied with depth. One or two of these subpopulations
appeared to be bacteria with sufficient cytoplasmic
constituents to produce a normal light-scatter signal but retained only a small amount of DNA; an apparent content of 200
kbp or 5% of a usual oligobacterial
genome. These helped increase the oligobacterial population to 6 X lo6 ml-‘.
Application of published specific affinities and measured nutrient concentrations to formulations of system kinetics led to the
conclusion that growth rates of the heterotrophic bacterial fraction were carbon limited with cell size, and thus populations
were controlled by grazing. The depth profile indicated that phototrophs affected phosphate concentrations in a significant
way. Considerations of nutrient uptake kinetics suggested that much potential capacity remained in the dissolved phosphate
pool to support additional phytoplanktonic
biomass. Computations led to the conclusion that, if phosphate is generally
limiting in lakes, then additional mechanisms exist which limit populations of phytoplankton to sufficiently small values to
allow phosphate accumulation to observed levels. Bacterial biomass then depends on the organic carbon from these
phosphate-controlled
organisms.
Keywords:
Bacterial
biomass;
Subalpine
lake; Phosphate
1. Introduction
The bulk of deep-lake biodynamics
is controlled
through carbon fixation by algae and cyanobacteria
with heterotrophic
bacteria remineralizing
most of
* Corresponding
author.
0168-6496/96/$15.00
Copyright
PII SO168-6496(96)00045-l
0 1996 Federation
of European
the organics to complete the cycle. As a group these
transthreptic
microorganisms
depend directly on
membrane transport for sustenance [44]. They remove dissolved nutrients until concentrations decline
below those necessary for efficient collection unless
other factors intervene. Most of the heterotrophic
bacteria are resistant to cultivation [78] and subsist
on concentrations
of dissolved organic compounds
Microbiological
Societies.
Published
by Elsevier Science B.V.
88
D.K. Button et cd. /FEMS
Microbiology
that are particularly dilute. These attributes are distinct from those of the more commonly cultivated
bacteria. Chemoheterotrophic
bacteria able to use
very small concentrations of substrate are sometimes
called oligobacteria [ 141. Ultramicrobacteria
153,741
are thought to be a subgroup of particularly small
oligobacteria. Most reports of these populations rely
on direct microscopic counts and morphometric measurements of the fluorescent
images from fluorophores bound to the nucleic acids of cells. Populations can also be characterized by flow cytometry
[2,19,47,52].
Probes such as fluorescence
from
stained DNA and light scatter from the non-aqueous
constituents may be used to improve the analysis of
most unicellular organisms.
We have used flow cytometry as well as kinetic
analysis to describe the bacteria from Lake Zurich, a
large subalpine, mesotrophic Swiss lake [66]. Samples for depth profiles of nutrients and microorganisms were collected during the autumn when the lake
was stratified to minimize the effects of mixing and
maximize population
stability. Reported here are
population characteristics
with the newly achieved
accuracy of high-resolution
flow cytometry, and descriptions of the status of these populations using
emerging concepts of nutrient uptake kinetics.
2. Materials and methods
2.1. Samples
Water was collected on September 22, 1993, at
mid-lake along a transect between Kilchberg and
Kuesnacht (47”20’ N, 8O34.4’ E) with a 5 I-Friedinger sampler. Samples were immediately
drawn
through 0.2 km pore size polycarbonate
filters by
Nalgene hand pump at 0.8 atm pressure for determination of the inorganic ions. Portions were taken in
triplicate for the microscopic and flow cytometric
measurements,
amended with 35% formaldehyde to
give a final concentration of 0.5% (w/v) and chilled
for subsequent analysis within a month. Preservation
protocol was checked by observing populations which
remained constant between 0 and 39 days in samples
from Blair Lake, Alaska, and between 14 and 60
days from Harding Lake, Alaska. Moreover, populations of Marinobacter
strain T2 remained constant
Ecology 21 (19961 X7-101
over 2.5 years (unpublished
data). Solar radiance
was determined with a LiCor meter fitted with a
Li-2222 SB spherical sensor, and oxygen with a
Oximeter 19 1 Clark-type electrode.
2.2. Chemistry
Ammonia was determined by the phenol-hypochlorite reaction [7 11, nitrite and o&o-phosphate
assays followed standard methods 1281, and total
phosphorus was determined following persulfate oxidation. For nitrate, the UV absorption at 220 nm was
corrected for the value at 275 nm [3]. Method suitability for Lake Zurich water was verified by ion
chromatography. For chlorophyll determinations, 200
ml of lake water were collected on glass fiber filters
(Schleicher and Schuell GF 6) that were ground in
90% acetone and cleared by centrifugation.
Chlorophyll was determined by fluorometry [67] on a Hitachi F-2000 fluorometer with excitation at 430 nm
and detection at 668 nm. Dissolved organic carbon
(DOC) was determined with a Shimadzu TOC 5000
analyzer.
Water
samples
were vacuum-filtered
through polycarbonate
Nuclepore filters (0.2 pm
pore size), acidified with 2 M HCl to pH 2 to 3, and
sparged with oxygen for 10 min to remove CO,.
Potassium hydrogenphthalate
was used as the carbon
standard. The values were corrected for organic carbon leaching from the filters.
To compute phototroph carbon from chlorophyll
a we used a value of 0.07 mg C/Kg chl a. While
carbon-to-chlorophyll
ratios can be depressed by
shading in deeper waters, use of a higher carbon
content is thought to be appropriate only when
deep-mixing occurs [49]. Bacterial carbon was computed from the carbon-to-biovolume
ratio. Values
obtained from Cycloclasticus
oligotruphus
(RM 1,
[ 141) grown at a rate of 0.02 h- ’ on 14C-acetate, and
sized by forward light scatter and by Coulter counter
were 95 mg C (gram wet-weight cells)-’ during late
exponential phase in batch culture (unpublished).
2.3. Phytoplankton
The dominant phytoplankton
and their volume
were identified by epifluorescence microscopy. Lake
water (100 ml) was filtered through polycarbonate
0.2 p,rn pore size filters (Nuclepore) that were cleared
D.K. Button et al. / FEMS Microbiology
with immersion oil and photographed on Agfa Portrait 160 film. The lengths of the cyanobacterial
filament images were measured by calibrated micrometer for volume calculations [S].
2.4. Bacteria
Preserved samples for populations, cell size, and
apparent DNA-per-cell
were filtered through 1 p,m
pore size Nuclepore polycarbonate
filters, stained
with DAPI
(4’,6-diamidino-2-phenylindole)
and
measured with an Ortho 11s flow cytometer as previ-
Ecology 21 (1996) 87-101
89
ously described [19] except that the standard curve
for cell size [63] was refined as follows. Shape of the
standard curve for cell volume V was taken as
V= exp 1.62 X 1O-4 In (k Z>3 + 0.0144 In (k Z)* +
0.680 In (k Z) + 0.274 based on Rayleigh-Gans
theory applied to the conditions used. In the present
application, I is the intensity of UV light scattered
between 0.5” and 20” by cells with an axial ratio of
3: 1 and oriented with a perpendicular
aspect in the
flow stream; k is a constant peculiar to the instrument which must be determined. The axial ratio was
determined
from measurements
of electronmicro-
0
0
-20
-20
-40
-40
-b
1
-60
-80
-100
0
0.0
10203040506070
/JM
Total volume mm3 c’
;.o
1.5
0.5
0
-20
-40
-60
-80
-100
0.0
0.5 PM
012345678
Chlorophyll a, pg r’
1.0
Mean cell size, pm3 cell’
0.1
0.2
0.3
0.0
0
0
-20
-20
-40
-40
-80
-60
-80
-80
-100
-100
01234567
Bacterial population,
0.01
1 O6 ml“
0.1
1
Carbon, mg I-’
and DNA, fg cell’
Fig. 1. Depth profiles of Lake Ziirich. A-C: physical and chemical parameters. D: phytoplankton.
E: population of bacteria, including dim
forms, mean cell volume, and the apparent DNA content per cell. Error bars show one standard deviation. F: distribution of carbon in the
bacteria, phytoplankton and dissolved (total filterable) fractions.
90
D.K. Button et al. /FEMS
Microbiology
Photic zone thickness reflected a mesotrophic mineral-nutrient status with attenuation of light intensity
to 1% of the surface level at 13 m. Other major
nutrient profiles (Fig. 1B and lC> were consistent
with historical records as well. The distribution of
inorganic ions was usual for a stratified mesotrophic
lake with an oxic hypolimnion in late summer. There
was an abundance of combined inorganic nitrogen to
60 p,M as shown (Fig. 1B) as compared to nitrite
and ammonium ion which were in the 0.2 to 1 PM
range. Phosphate levels were up to 200-fold lower
than nitrate, with ortho-phosphate
depleted to 0.2
FM near the surface (Fig. 1C).
graphs of the natural populations. The position of the
curve was set from volumes and forward scatter
intensities of several Coulter-counter
sized bacterial
strains as well as latex spheres, all normalized to the
measured refractive index (1.04) of C. oligotrophus.
Cell mass was corrected using a cell density of 1.07
g cmp3 as determined by Percoll gradient centrifugation.
3. Results
3.1. Temperature
Ecology 21 (19961 87-101
and nutrients
Both the temperature profile and the distinct metalimnic oxygen minimum reflected the expected water-column stability (Fig. 1A). Unpublished
station
records showed that this zone of low oxygen concentration was normally established in mid-summer and
continued until the water turned over in late autumn.
3.2. Phytoplankton
Maximal chlorophyll
concentrations
(Fig. lD>
were at 10 m near the base of the photic zone.
Planktothrix (Oscilla~oria~ rubescens, a filamentous,
nonheterocystous,
gas-vacuolated,
phycoerythrin-
0.6pm
beads -
!
/I
25
Fig. 2. Isogram of DAPI-stained bacteria from 1 m water. Organisms are separated into three subpopulations
forward light scatter. The rounded peak near center contains the bacteria identified as dim.
according
to fluorescence and
D.K. Button et al. /FEMS Microbiology Ecology 21 (1996) 87-101
DNA content, fglcell
containing cyanobacterium,
was the dominant microscopically-visible
phototroph throughout the water
column, and provided much of the chlorophyll. Between 0.06 and 1.5 mm3 1-l of the biovolume was
contributed by this autotrophic bacterium with the
maximum value coinciding with the chlorophyll peak
at 10 m and near the oxygen minimum.
3.3. Heterotrophic
Depth (m)
1
5
30
100
0.01
0.1
1
10
bacterial populations
Numbers of heterotrophic bacteria in the photic
zone approached 7 X lo6 ml-’ (Fig. 1E). Bacterial
size (mean cell volume) was 0.22 km3 (s = 0.018,
n = 33) for 10000 organisms analyzed in each of
triplicate samples. There was a small decrease to
0.20 pm3 (s = 0.0012, n = 9) near the surface and
an increase to 0.24 (S = 0.0016, n = 9), near the
bottom. The apparent DNA content averaged 2 fg
cell-‘, and was remarkably constant with depth as
well. Exceptions included a decrease at 5 m that
correlated directly with cell size and with the inverse
of bacterial population (Fig. 1E) and a general increase in cell size with depth. Part of the heterotrophic bacteria had a particularly low apparent DNA
content and are grouped as a distinct subpopulation
of dim cells (Fig. 1E).
Table 1
Characteristics
91
of the heterotrophic
bacterial
subpopulations
DAPI-DNA
fluorescence intensity
Fig. 3. Dot plot of the data from Fig. 2 used to identify subpopulations. These are isolated in the side-bands where instrument output
data are converted to concentrations by use of standard curves.
3.4. Carbon
Phototrophic
carbon was about fourfold larger
than that from heterotrophic bacteria in the photic
zone (Fig. IF). Because phytoplankton
were present
throughout the 106-m water column, phytoplankton
from selected depths in Lake Erich
*
Subpopulation
properties
lo* organisms
1-l
Volume per
cell (p,m3)
DNA per
cell (fg)
Wet cell
mass (mg l-
Large
Small
Small
Large
Small
Small
Large
Small
Small
Large
Small
Small
13.7
36.4
14.0
14.4
39.8
12.6
6.6
15.0
4.8
1.4
15.7
5.6
0.36
0.12
0.10
0.36
0.12
0.10
0.36
0.12
0.10
0.36
0.12
0.10
3.9
1.3
0.2
3.4
1.3
0.2
3.7
1.3
0.2
3.6
1.2
0.28
0.49
0.43
0.14
0.51
0.48
0.12
0.17
0.18
0.05
0.27
0.18
0.05
bright
bright
dim
bright
bright
dim
bright
bright
dim
bright
bright
dim
’)
% of total
biomass * *
45
42
13
47
43
10
43
45
12
54
36
10
* Values are means for three replicates. Coefficients of variation were 7.5% for population, 0.5% for cell volume, 2.4% for DNA, and 9.2%
for biomass. Total population includes organisms outside those selected in regions 1 to 3 of Fig. 3.
* * Percent of cells occurring in the sum of the three subpopulations.
D.K. Button et al./ FEMS Microbiology Ecolog?; 21 f1996) 87-101
92
carbon integrated over depth exceeded that from
heterotrophic bacteria, 13.8 g m-* as compared to
6.3 g rnp2. Carbon that passed a 0.2 km filter is
labeled dissolved and includes the true dissolved
fraction on which heterotrophic bacteria directly depend. It remained near 1.5 mg l-l, with marginal
increases toward the surface and the sediments.
3.5. Flow cytometry
data
At least three populations of bacteria (peaks 1, 2,
and 3) were distinguished on the basis of organism
size and apparent DNA content (Fig. 2). The first
was a group of larger organisms that were 0.12 to
0.8 pm3 in volume with high apparent DNA concentrations (2 to 8 fg organism-’ > and that comprised
23% of the population. Partially obscured in Fig. 2,
this group was described more clearly by the concentration of spots slanting to the upper right of the dot
_.
1
i10m
.
i
103 ;20m
plot in Fig. 3 (region 1). The second peak (region 2,
Fig. 3) showed bacteria 0.12 km3 in size, with 0.5 to
2 fg DNA cell-’
that accounted for 58% of the
population. The third peak (region 3, Fig. 3) located
the dim bacteria with apparent cellular DNA levels
< 0.2 fg cell-‘, but with cell size nearly the same as
those in peak 2 as shown by the side-band histograms. This dim bacterial subpopulation comprised
about one-fourth of the total population, and was
present at all depths. In some samples, separate
groups appeared in regions 2 and 3 giving a total of
3 to 5 groups resolved.
Peak width reflects the variation within the bacterial subpopulations themselves and is compared with
the narrow peak associated with the internal standard, 0.6 t.r.rn fluorescent microspheres (beads), in
Fig. 2 and the small spot in Fig. 3. Position stability
demonstrated reproducibility
of the forward scatter
signal. Similar l-Km beads used for alignment gave
._
.
.
.:
103
90m
.
1
103 ,100m
“-’ .
DAPI-DNA fluorescence
Fig. 4. Representative
dot plots that characterize
103 105 m
- __.
.
intensity
changes in the bacterial population
with depth.
D.K. Buttonet al./FEMS MicrobiologvEcology 21 (1996) 87-101
coefficients of variation
< 2% for forward scatter
and < 3.5% for fluorescence.
While bacterial biomass decreased with depth
from 1.0 to 0.5 mg l-l, the portion of large, bright
cells increased from 39% to 47%. Mass of dim
bacteria remained constant at 0.05 to 0.14 mg 1-l) or
about 13% of the total (selected data; Table 1).
Cyanobacteria were not observed by flow cytometry. Sample filtration eliminated particles > 1 pm in
size, and signal from unicellular phototrophs such as
Synechococcus
did not appear in the usual location
near the 0.6 pm beads indicating that populations
did not exceed lo4 ml-‘. Based on the characteristics of their marine relatives, small prochlorophytes
would not have been resolved from the heterotrophs
since sample storage at 0-5°C and our use of Triton
X-100 as a permeabilizing
agent would deplete
chlorophyll to undetectable levels in the organisms
(Sheila Frankel, personal communication).
The partially merged pair of subpopulations
of
what appeared to be normal-sized oligobacteria with
low DAPI-DNA
fluorescence
was noteworthy because it corresponded to a DNA content of only 0.1
to 0.7 fg cell-‘. The distribution of this and other
subpopulations
with depth was best visualized from
the dot-plots (Fig. 4). For example, the substantive
population of dim particles at 90 m almost disappeared at 100 m, a small part of which reappeared at
105 or 1 m from the bottom. The broken horizontal
bars at the tops of the panels showed 0.9 pm beads
and their multiples which are above scale, and quantitatively indicate few organisms larger than those
shown. Bars at the bottom were consistent
with
additional fluorescent particles < 0.01 pm3 in volume where viruses should appear.
4. Discussion
4. I. Mineral nutrients
Electron acceptors such as oxygen and nitrate
sustained an oxidative environment
throughout the
water column; however oxygen approached concentrations of 2 mg 1-l which can be limiting in active
systems [17]. The low levels of nitrite and ammonium ion, compared with nitrate, are also consistent
with oxidative conditions. While N:P ratios indicated
93
phosphate deficiency, phosphate concentrations
can
be much lower than reported here. Concentrations
in
Walker Lake, near Alaska’s Endicott Mountains, are
only l-2 pg 1-l [34] as compared to the 12 to 40
lJJg 1-l reported here, and are accompanied by increased transparency and a chlorophyll maximum at
30 m rather than 13 m. Data are consistent with an
influence of phosphate on the biomass of phytoplankton with additional regulation by light.
4.2. Flow cytometry
of bacteria
Detection was based on the blue fluorescence
from DAPI-stained DNA to trigger signal processing
which reduces the processing of light scatter signal
from debris. Forward scatter intensity compares the
dipole-content
of the organisms with the water outside [41]. Small stained phototrophs such as Synechococcus emit blue fluorescence that is less bright
than that from heterotrophic bacteria when stained
because some of the energy absorbed by DAPI-DNA
is transferred to photopigments and lost. In a bivariate histogram such as those shown, the position of
these phototrophs is normally above the bacteria,
near the 0.6 pm3 bead signal; however, in this study
they were either absent or too few to observe, in
agreement with the microscopic observations. Others
could remain undetected with the methods used.
Maximal bacterial populations
were at 5 m, and
differences exceeded standard deviations as shown.
The peak at 90 m could be restricted to a thin layer
since the lines simply connect the data points obtained.
Literature values for the carbon content of bacteria vary widely [56] which is likely related to difficulties in establishing cell size. Values used here are
based on the “C-acetate-content
of C. oligotrophus
and are consistent with refractive index and Coulter
volume measurements
of that small aquatic isolate.
Because it has small specific affinity for acetate but
moderate V,,, , the growth rate can be controlled; at
a growth rate of 0.02 h-r the carbon content was
0.07 g C gram-cells-’
which was used to estimate
bacterial carbon (in preparation).
Measurement of DNA-per-cell may be subject to
error due to variation in the AT/GC
among cells
present, cell damage [26], differences in the permeability among even permeabilized
cells, and differ-
94
D.K. Button et al. / FEMS Microbiology
ences in genome conformation [6,25,58], and accessory protein content [54]. For C. oligotraphus
cell
rupture for DNA measurements by spectrophotometry increased the observed value from 2.2 and 2.9 fg
cell - ’ as compared to flow cytometry; such discrepancies are known to vary among species (in preparation). Therefore, the fluorescence observed here is
reported as apparent DNA content but errors are
thought to be minor; both Escherichia coli and C.
oligotrophus
can give integral values for DNA at 1
to 8 chromosomes.
Hoechst 33342 is reported to
improve DNA analyses over DAPI [45,52] because it
produces sharper peaks in marine samples. However,
if the samples are treated with Triton-X 100 and
formaldehyde as specified [19,20], both E. coli and
C. oligotrophus, which can contain groups of cells
with integral numbers of genomes, give extremely
sharp peaks. Also, direct comparisons with isolates
and aquatic samples (unpublished)
show that the
fluorescence from DAPI with Triton X- 100 can be
twice as bright as from Hoechst 33342 as used by
others. Particles which reversibly sorb DNA have
been reported as ghosts [78]; however, with the
smaller concentrations
of DAPI used here, DNA
standard curve intercepts were only about 0.3 fg
DNA cell - ’ , the value which was subtracted to
account for non-specific
staining. Values were reduced to below the lower limit of detection or about
0.06 fg cell-’
for 92% of the organisms from a
culture of C. oligorrophus
by this treatment (in
preparation)
so non-specific
staining amounted to
only 1% to 2% of the bacterial DNA. Cyanine stains
such as TOT0 are also sometimes used [48]. Although insensitive to AT/GC ratio, these dyes bind
to RNA as well and standard curves are not yet
available. Moreover visible light, which produces a
less intense scatter signal is required, so DAPJ
presently appears to be a preferred stain for the study
of oligobacteria.
Coefficients of variation in the size distributions
for C. digorrophus
are equal to those in transmission electron photomicrographs
of pure cultures at
35%, and volumes for various sized particles: Coulter-counter-sized
pseudomonads,
electron
microscope-sized oligobacteria,
and high refractive-index
standard latex spheres, all fit on the theoretical standard curve (in preparation). Therefore, the size distributions shown here are thought to be accurate. Very
Ecology 21 (1996) 87-101
small fluorescing bodies are commonly observed by
epifluorescence
microscopy [77] and considered as
large viruses [30]. Light scatter from viruses is below
our lower threshold at settings used as computed
from Raleigh-Gans
theory. While their detection is
consistent with the horizontal lines of the dot plots in
Fig. 4, they are excluded from the counts presented.
4.3. Churacteristics
of the bacteria
The size distributions
were narrow for a mixed
population. Ninety per cent of the organisms were
between 0.06 and 0.66 pm3 in volume with 3.4% of
the biomass falling above this range and 2.3% below. This corresponds to organisms that are between
0.3 X 0.4 km and 0.5 X 1.5 pm in width and length.
Major populations of bacteria as small as 0.02-0.04
pm3 have been reported in lakes [23,59] which
reflect sizes near the lower extreme of the distributions reported here. Since size measurements
were
dependent on the dimensions of the DNA-containing
region which can be restricted to only part of the
cytoplasm [781, it is probable that size would have
been underestimated for these cells. Acridine orange
measurements
of bacterial size are only slightly
smaller than ours [73]. The forward scatter signal
from dipoles within the cell is taken as a measure of
dry weight. Sizes are based on dry weight, density
and relative refractive index measurements
of the
marine bacterium C. oligotrophus. We assumed that
freshwater bacteria retained similar properties to optimize their surface area to dry-weight ratio when
using this isolate as a size standard. In addition to
being quite small, this organism is quite dilute and
only 19% dry weight as compared to 30% for E.
co/i. The apparent DNA content was also small at
1.7 to 2.2 fg celll’
The AT-dependent
assay assumes the 50% AT/GC ratio of E. coli.
Populations could be separated into at least three
groups according to forward scatter versus DAPIDNA fluorescence histograms and identified as large,
small, and dim. The bacteria in the upper right of the
histograms (Figs. 3 and 4) are most likely large cells
growing at a rate fast enough to contain replicated
DNA; the small cells with intermediate
levels of
DAPI-DNA
fluorescence
are likely those with a
single genome. Only a small portion of the cells in
the high apparent-DNA
content are likely to be
D.K. Button et al. / FEMS Microbiology
dividing cells according to formulae for the frequency of dividing cells [55]; if they were all dividing cells, the unreasonably
high growth rate of IO3
hh’ would result. While size increased with depth,
increased production of organic nutrients is expected
to correlate with the presence of photosynthesis
in
the upper layers, and cell size normally increases
with the rate of growth [62] so larger cells would be
expected near the surface. Thus, size distributions
are consistent with significant control by selective
grazing of the larger cells [36] that would otherwise
appear near the surface. DNA, like size, reflects
growth rate as a larger fraction of the cells replicating are in the ‘D’ phase. Structure of DNA profiles
with the small decrease near the surface is also
observed in marine systems (unpublished).
Bacterial populations near 7 X lo6 ml- ’ were at
the high end of the range normally observed in
aquatic systems for surface water, although populations are normally above lo6 ml-‘. Low stainability
of the dim organisms with DAPI would probably
have excluded these organisms from direct counts
and could help account for the high values. Populations, cell size and nutrient concentrations
are all
elevated at 90 m. This anomaly is consistent with
anecdotal reports of deep turbid layers that are
thought to be caused by sinking remnants of algal
blooms during periods of thermal stratification. The
number of dim bacteria in this sample is also elevated, and consistent with our observations
above
and those of others that the portion of DNA-deficient
bacteria increases with activity (see below).
4.4. Dim bacteria
Dim bacteria are taken as bacterial-sized
organisms with an apparent DNA content < 0.3 fg cell- ‘.
These correlated linearly with population over the
depth profile, r = 0.45, n = 11, p = 0.07. The ratio
of dim bacteria to bright was 22% to 30%. ‘Small
dim particles’ have been reported in marine and
estuarine samples at 4 X lo6 to 3 X lo7 ml-‘, and
attributed to very small bacteria and viruses [70],
while 68 to 98% of marine bacteria have been
Ecology 21 (1996) 87-101
9.5
reported as lacking nucleoids [78]. We have observed
populations of dim bacteria in most, but not all, of
our samples from a wide variety of aquatic systems
(unpublished). For example, in Harding Lake, Alaska,
the ratio of dim to bright bacteria was lowest at 0.14
in the dark of January (latitude 64”N), when specific
affinities for amino acid uptake were also low, as
compared to a ratio of 1.OO in July during continuous
light. Preliminary data from C. oligotrophus gives a
steady increase in dims with starvation to 30% in 60
days and they have been seen in old cultures of E.
coli as well. Extension of the staining time gave little
increase in fluorescence so production of forms having a decreased permeability
towards the stain is
unlikely. Normal light-scatter values here as well as
in pure cultures (not shown) show that the dim
organisms have retained most of their internal components with the exception of DNA and have a dry
weight which is usual excluding the possibility that
they are cell ghosts.
4.5. Interdeperldencies
Bacterial abundance can vary with the concentration of chlorophyll [7,22]. However, the correlation
is sometimes weak 1351 and may be better with
primary production [35], organic carbon [69], total
phosphate [22], or with nitrogen and phosphate combined [75]. Predation (top down) control of phytoplankton [50] and bacteria [4,3 1I is reported as well.
Although an N:P ratio of 15 [60] or slightly higher
[72] is regarded as the demarcation point between
nitrogen and phosphorus limitation, small concentrations of substrate require additional expenditures for
ion transport and enzyme synthesis to maintain a
given flux. The absolute concentrations
of limiting
nutrient required are seldom considered.
Major system components are formulated here as
a process at steady state over a short time period for
ease in organizing these concepts. Phosphate is taken
as the limiting nutrient (concentration
S> for biomass
(X). Symbols above the arrows identify the rate
constants associated with the various steps:
D. K. Button et al. / FEMS Microbiology
96
Primary
productivity
Death by
algae-grazers
Secondary
productivity
(CL*)
(d,)
(Kg)
PO, (S,)
+
The rate equations
Algae (X,)
+
Nonliving
Ecology 2 I
(I996187-101
Mineralization
(S,) organic carbon
are:
/_Lx = UXSY
(1)
dX = gXG
(2)
where p is the specific rate of growth (dX/dt)l /X
of algae (phototrophs), X, and bacteria X,, CIis the
specific affinity in 1 g-cells-’
hh’ [12], g is the
corresponding
grazing-rate
constant that describes
the rate of cell death d, G is the quantity of grazers
such as phototroph-consuming
zooplankton and bacteria-consuming
bacterivores and bacteriophages, and
Y is yield, g cells (wet weight) g-substratee’.
The
biomass
term X is retained to emphasize
the
second-order nature of the processes. The specific
affinity is c‘ S-‘, where LJ is the specific rate of
substrate uptake in g substrate g-cells-’
h- ‘. Microbial uptake of substrate is saturable which leads to a
base value of the specific affinity, uos, that is the
initial slope of the [J/S curve. When saturation is
hyperbolic, a?’ at a particular concentration
[S] is
a”,=du dS_‘, and c=a’,
V,,, S(V,,,+
uos S)-’
[18], where V,,, is maximal specific (per g cells)
uptake rate. The material balance equations for a
volume of water are:
;=(dA+k-(T))X+I
where S, is total substrate, X/Y is substrate contained in cell material, S is that outside, A is the
portion of grazed or dead cells liberated as S, k is
the first-order rate constant for leakage, o is the loss
rate due to sedimentation,
and I is the rate per unit
volume of S input from exogenous sources such as
terrestrial run-off. If the rate constants are known
and the system is assumed to be at steady state, Eq.
(4) = 0 and the set can be solved [16]. One conse-
+
Bacteria (X,)
+
PO,
quence is that the measured concentrations
of nutrients, if limiting, should agree with the measured
specific affinities and growth rates of the organisms
that depend on those nutrients.
4.6. Growth rate and specific affinities (Eq. f I ))
Characteristic specific affinities of many algae for
phosphate, a”p, are near 25 1 g-cells-’
hh ’ [l 11. For
example, recent measurements of the related species
Oscillatoria agardhii [61] give a value of 84 1 gcells ’ h- ‘. In this organism, the other kinetic condue to a slow apstant, V,,, , was indeterminate
proach to saturation at high concentrations.
This is a
usual situation for microbial uptake, [62] and unpublished data, and one that has impeded use of kinetic
data to describe microbial systems because microbial
kinetics often appear hyperbolic when only a small
range of concentrations is examined. Neglecting saturation at the small (0.02 mg l- ’ ) concentrations of
phosphate [P] present, the specific affinity in Eq. (1)
reduces to a linear rate constant a:” = a”p. Using
a[pP]
= 84 together with a yield of 200 g-cells g-phosphate ’ , the rate equation indicates the presence of
sufficient phosphate for photic zone phototrophs to
growatthefastrateof(841g-cellss’
h-‘)(2X
lo-’
g PO, 1-l) (200 g-cells g-PO;‘)=O.34
hh’, a
doubling time of only 2 h.
Specific affinities ai of > lo3 have been measured for both algae [32] and marine yeasts [ 1 l] so
ample nutrient-sequestering
potential among the organisms is both theoretically possible and observed
to make phosphate non-limiting. The phosphate concentrations observed here, as well as the sequential
existence and coexistence of numerous species among
algae [29], all suggest that competition for phosphate
is only a coequal or secondary factor in setting algal
populations.
Additional possible co-contributors
to
the control of phototrophs in this low-phosphate
D.K. Button et al./ FEMS Microbiology
system include allelopathy [27,38], selective grazing,
and nutrient co-limitation. Numerous volatile organic
compounds from cyanobacteria
are known, and the
more effective inhibitors may be less-easily detected
nonvolatiles [38]. Some have been identified and can
reduce the growth rates of competing species [37].
Reduced growth rate is likely to be reflected by
reduced phosphate uptake which, at constant yield
and phosphate concentration,
means lower effective
affinity. Predation, unrelated to the concentration
of
phosphate, may also affect both the biomass and the
species distribution of phytoplankton [40,46] remaining to compete for limiting nutrient and thereby
disturb what would otherwise be a steady state [9].
For bacteria, typical values for specific affinity
suggest that these organisms can more effectively
eliminate organic carbon from their surroundings,
and leave concentrations
that approach the diffusional limit [l] for required rates of transport. While
these affinities for carbon uptake are near those of
microheterotrophs
for phosphate [ 131, the concentrations of individual organics are 20 or more times
lower [21,64], and cell yields are about lOOO-fold
higher. Thus higher fluxes from those small concentrations are required. In addition, specific affinities
for separately transported species can be additive
[15] or better (unpublished)
which, together with a
high initial value, facilitates the required flux. Accordingly, oligobacteria can coexist with phytoplankton attaining only those populations allowed by the
supply of carbon from phosphate-limited
algae [lo].
The discrepancy between possible phosphate limiting
concentrations
of < 0.01 p,M [62] by yeasts designed to maximize phosphate transport and those
measured here of 0.91 to 0.24 ~_LMis more than
20-fold and can be taken as the effect of either
indirect or non-nutritional
factors. This discrepancy
may be a minimum
since removal from in situ
conditions tends to reduce specific affinities and is
not a source of error in the measurement of ambient
phosphate. Moreover, in situ growth rates are likely
slower than the continuous culture experiments which
should reduce ambient phosphate even further.
4.7. Grazing
Eq. (2) describes the rate of removal of cells by
predation. Thus greater nutrient input increases bac-
Ecology 21 (1996) 87-101
97
teria through Eq. (1) and recycling through Eq. (2)
so that the kinetic effects are an increase in the
growth rate of the bacteria which fosters an increase
in the growth rate and population of grazers. Increased grazing rates require increased bacterial populations at steady state providing an explanation for
the positive correlation between bacterial populations
and primary productivity often observed [23]. One
might expect larger grazing-dependent
variations in
P-limited cyanobacteria
and algae than C-limited
bacteria because there is little reserve capacity in the
small levels of organic carbon as compared with
phosphate. Thus, the heterotrophic bacterial population may not bloom.
The size distributions of the bacteria are consistent with significant bacterivore grazing [42,68] although we have had difficulty establishing
these
rates [ 191 in diluted samples of seawater. The largest
organisms were within a deep, potentially rich zone
of otherwise labile-carbon-poor
water. One might
expect the largest cells a few meters below the
surface where primary productivity produces labile
substrates for bacterial growth [39] because cell size
consistently increases with growth rate. Instead, the
oligobacteria
were reasonably consistent and small
over depth, and decreased slightly near the surface
where photoinhibition
[5,5 11, grazing, or organic carbon depletion by the higher bacterial populations
may have been more intensive. That the largest cells
were in a deep layer may have been due to extra
organic carbon in an area normally deficient in organic carbon due to the absence of primary productivity so that size-dependent
population
control
through bacterivore grazing that depends indirectly
on photosynthate input [39] was restricted.
4.8. Limiting-nutrient
distribution
Eq. (3) specifies the nutrient distribution between
the cells and the water. Phytoplankton
carbon in the
photic zone appears to be restricted by phosphate,
although other factors almost certainly share in control. They accounted for 73% of the particulate
carbon measured while heterotrophic bacteria comprised 27% of the bacteria-phytoplankton
total. Bacteria accounted for about half the biomass due to
their dilute nature, and if cyanobacteria are included,
they provide most of it. However, heterotrophic bac-
98
D.K. Button et al./ FEMS Microbiology
terial carbon amounts to only 10% of that in the
dissolved fraction. This is because much aquatic
DOC is relatively recalcitrant [76] with large molecular weight, molecular diversity, and colloidal properties [65], all of which tend to reduce the affinity of
the bacteria for this fraction.
Heterotrophic
bacteria were an important phosphate sink. Bacterial wet biomass at 38 g m-’ was
approximately equivalent to the 20 g phytoplankton
m -’ present. However, the 2 to 4 mg P gg ’ for
phosphate-sufficient
microheterotrophs
[62] can
greatly exceed the 0.56 mg P g-’ found in algae [9]
so that of the 1.2 g particulate P fraction (2.2 g m-’
total P), more than three-quarters
of it might be
found in the bacteria. This is corroborated by continuous culture data. When supplied with enough phosphate to grow a biomass of 1 mg cells 1-l at
moderate rate, even for a relatively large marine
yeast, 97% of the phosphate can be sequestered by
the cells. For algae under the same conditions, only
69% of the phosphate is sequestered as calculated
from kinetic data [l 11. Although the cells had about
the same surface-to-volume
ratio, the algae left 10
times as much phosphate behind and grew in stable
coexistence with the C-limited heterotrophs. Too few
reliable kinetic data exist to generalize about kinetic
data for various organisms; however, phytoplankton
appear to have lower affinities for substrate than do
bacteria. Furthermore, the phosphorus content of cells
can increase with growth rate in P-limited systems
by an order of magnitude
[ 161, and is generally
higher in heterotrophic bacteria than algae. In the
present system, the cyanobacteria
were taken as P
limited due to the high N:P ratio, while the heterotrophic bacteria having higher specific affinities for
phosphate than Oscillaforia [ 111, are either limited in
part by factors other than phosphate, or are inherently poor at phosphate collection. Thus the C-limited
phosphate-sufficient
bacteria approach the high-end
of their P-content range and sequester most of the
phosphate. Since they approach the phytoplankton in
biomass, they appear to comprise the main phosphorus reservoir in this and other P-deficient
lakes
[24,33].
Biomass would appear to be larger, according to
Eq. (31, when under nutrient limitation rather than
under grazing control because less nutrient is relegated to the dissolved state. However, severely nutri-
Ecology 21 (1996) 87-101
ent-limited
cells are likely to lack vigor, so that
grazed populations might be more sufficient in limiting nutrient and therefore more robust. In deep waters, bacteriophages may become important for rejuvenation [57] when the supply of labile organic
carbon is insufficient
to support adequate populations of bacterivores.
Eq. (4) specifies the rate of change in nutrient
concentration when the system is out of equilibrium.
While simple systems can be stable, such as the
yeast-algae-phosphate
continuous
culture described
above, both models [43] and the observed variations
in natural phytoplankton
populations
[35] specify
oscillation.
These populations
would then vary
around and inversely with the concentration
of S
predicted by nutrient input and output terms in Eq.
(3), together with the inherent affinities of the organisms present. Increases in the equilibrium concentration of S can be caused by either an increase in
grazers (Eq. (2)) or a decrease in the specific affinity
(Eq. cl)), even at steady state. Assuming that the
phytoplankton
population
is well developed
and
growing at a rate of 0.1 dd’, measured conditions
specify a specific affinity for phosphate of only 0.01
1 g-cells-’
hh’, nearly a million-fold less than the
value for a good oligotroph [ 131. A good oligotroph
is defined as an organism with sufficient specific
affinity to grow on dissolved substrates at ambient
concentrations,
typically near 10 1 mg-cells-’
h-’
for the sum of several substrates used simultaneously. Assuming accuracy in this analysis, phytoplankton populations in this low phosphate system
are affected by other factors discussed above. Total
phosphate may well set the upper boundary within
which population variation can occur. Increases in P
input should, according to the equations, affect the
bacterioplankton
through not higher populations, but
higher rates of growth (Eq. (1)).
Bacterial populations
were approximately
constant with depth. and numerous observations from
the literature, as well as our own, are that they are
quite stable across aquatic systems as well. This
stability is consistent with the above equations and
specific
affinities
for individual
biochemical
monomers such as sugars and amino acids either
measured or calculated as above and which approach
10’ 1 g-cells-’
h- ‘. Such consistent and large uptake capacity allows little variation in the S term
D.K. Button et al./FEMS
Microbiology
preventing the heterotrophic equivalent of an algal
bloom. Therefore the rate of bacterial growth should
be the main benefactor of substrate input with some
additional increase due to the larger bacterial populations required for faster growth rates as specified by
taking the biomass as bacterial (X, in Eq. (2)). The
small specific affinities of many phytoplankton
for
phosphate. on the other hand, allow for a cushion of
reserve limiting nutrient which could supply the
resources necessary for spurts of algal growth to
outpace predators.
Acknowledgements
We thank F. Schanz and A. Mechsner for their
physical and chemical determinations
and valuable
comments. Work was supported by grants from the
Ocean Sciences and Metabolic Biochemistry
Sections of the National Science Foundation.
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