CHEMICAL
CHANGES INVOLVING
NITROGEN
METABOLISM
IN WATER AND PARTICULA?“F
MATTER
DURING PRIMARY PRODUCTION
EXPERIMENTS
L. Prochdxkomi, P. BlaZka,
Hydrobiological
Laboratory,
Czechoslovak
Academy
of Science,
Praha
5
and
Institute
of Expcrimentnl
Botany,
Czechoslovak
Academy
of Scicncc,
Praha 6
ABSTRACT
The uptake of ammonia and nitrate by particulate
matter during photosynthesis
in large
light and dark bottles was followed
by colorimctric
and mass spectromctric
methods.
Ammonia uptake as determined
by tho 15N isotope was always higher than the parallel
estimate from colorimctric
analysis. The uptake (or release ) of ammonia in the dark was
inversely related to the protein content of the corresponding
sample of particulate
matter.
Utilization
of nitrate in light was inversely proportional
to the concentration
of ammonia
available,
The p,articulate
organic matter contained
50.5% protein in light bottles but
61.7% protein in dark ones. The mean ratio of oxygen changes, as calculated from changes
jr-r protein, carbohydrate,
and lipid to those found experimentally,
was 1.03 in light bottles
as evidence for
(indicating
good agreement)
and -0.34 in dark ones. This is interpreted
some particle formation in the dark that is probably not microbial.
MKTHODS
INTRODUCI’ION
The nitrate
concentration
in the surface
layer of a reservoir is more closely related
to external factors, such as fertilization
and rate of flow (Prochazkova 1966; Prochazkova, Stra3krabova, and Popovslj, in
press), than to primary production. Blaska
( 1966a;) has demonstrated the ro,le of protein metabolism in the production and rcspiration of Cladocera and its relationship to
food concentration and quality. The aim of
this work was to study the role of nitrate
and ammonia as sources of nitrogen for
particulate matter (particularly
algae) and
the facto,rs influencing
its percentage of
proteins.
Dr. P. Javorniclj
(Hydrobiological
Laboratory) kindly supplied counts and volume
estimates of phytoplankton
for Table 1.
Technical assistance of Miss J. Prouskova
and Mr. J. Janou3ek is appreciated. Special
thanks are due toI Dr. J. F. Talling for help
in revising the manuscript.
This paper is a Czcchoslovak contribution
to the International Biological Program.
Dissohed compounds
Ammonia nitrogen was determined spectrophotometrically
as rubazoic acid with
bispyrazolonc reagent ( Prochazkovh 1964 ) .
Nitrate-N was determined after reduction
to nitrite by hydrazine sulfate in alkaline
medium ( Prochazkov& 1959 ) ; the resulting
nitrite was determined according to Rider
and Mellon ( 1946). In several cases, when
the rccovcry of an added standard amount
o,f NO,-N was unsatisfactory, 5 min of boiling after addition osf NaOH solution was
applied ( Brandl, in press).
This gave
better recovery than the Cd-amalgam procedurc (Grasshoff 1964).
Oxygen was dctermincd by the Winkler
procedure.
pH was dctermincd
with a
Lovibond comparator (Salisbury, England)
using Bromthymol Blue and Crcsol Red as
indicators.
Particulate matter analyses
The modified Focrst continuous centrifuge was used to’ concentrate particulate
797
798
L.
l?l\OCII.kZKOV~,
P. BLAhA,
matter. This model worked at 20,000 6,
and the rate of flow was 12 liter/hr.
Direct
counts indicated the complete recovery of
most groups of algae and about 60-70% of
bacteria from natural water; blue-green algae were recovered incompletely.
In some
cases a small amount of particulate matter
was found after the second centrifugation;
we believe that this resulted from dissolved
material leaving the revolving bowl of the
centrifuge-an
origin similar to that described by Baylor and Sutcliffe
(196,3)
after bubbling filtered seawater.
Samples for organic-N
determination
were digested with 1 ml of I12SOII (coacd,
N-free), 1 ml of 10% NaCl solution (Prochazkova 1960), and 1 drop of metallic
mercury (the latter after 15 min digestion,
ca. 0.07 g). After rendering the sample
alkaline before distillation,
1 ml of 40%
Na&03
was added to release NH3 from
After distillation
the
the Hg complex.
resulting NHJ-N was determined colorimetrically
using divided Nessler reagent
( Prochazkova 1960). Organic-N data were
multiplied
by 6.25 to convert them to
proteins.
Carbohydrates were determined as the
acid soluble fraction by anthrone reagent
( Blaika 1966b ) as calorimetric equivalents
of glucose. Lipids were determined by
ethanol-ethyl ether extraction and dichromate digestion of the sample (BlaBka
1966h ) ,
The mass spcctrometric analysis (used
since 1966) generally followed the procedures of Neess et al. (1962) and Dugdale
and Dugdale ( 1965), unless otherwise indicated, Analytical data used for the calculation were corrected for blank values.
Potassium nitra tc and ammonium chloride,
both containing about 50% of the 15N isotope, were supplied by VEB Berlin Chem.
( Adlershof, Germany).
The mass spectrometer (MI 130sUSSR
made) was reconstructed (Kralova, Kyscla,
and Janousck 1967); its error in IBN analysis was 0.5% (coefficient of variation-Kralo& 1967). Samples containing 10-100 pg
N were diluted with unlabeled NH&l and
NIIhF was oxidized with NaOBr. The rela-
AND
M.
KdLOVli
tive error in the determination of the lBN :
14N ratio was 0.5-0.7% (coefficient of variation). Each determination was repcatcd
5 times, and the average was used for furAir standards were run
thcr calculation.
before and after each set of determinations
to check the calibration of the instrument;
their value was 0.369% 15N.
Species composition of phytoplankton
Samples were fixed in concentrated
Lugol’s solution and counted in KolkwitzLund sedimentation cells. Volumes of phytoplankton
cells were calculated
from
measurements and approximation
to g,eometric shapes. Total radiation was rccorded by a thermopile solarimeter (Kipp
and Zoncn, Holland).
MAIN
CEIAMCl~l~ISTIcS
OF LOCALITIES
Water from various localities was used
to get a wide range in the concentration
of chemical constituents, species composition, pH, and illumination,
Neb?ich and
Mastnik are two sampling points at Slapya canyon-type reservoir built for hydroelcctric power production on the Vltava River.
Its maximal depth is 55 m, length 44 km,
area about 10 km2, and mean retention
time about 1 month. Its water is brown,
owing mainly to pollution by paper mills
and partly to sphagnum bogs on the upper
reaches of the river. The sampling point
NebFich is on the main part of the reservoir, 12 km. upstream from the dam (at
the field station of the IIydrobiological
Laboratory).
The euphotic zone is only
3-4 m thick. Mastnik is a bay about 3 km
upstream from Neb?ich; it has a high nutrient input, especially of phosphate, from a
brook. Its phytoplankton
is therefo’re considerably richer than that of the main part
of the reservoir. For a detailed description
see HrbaEek and Stra&raba ( 1966).
Vrane Reservoir is the second small rcscrvoir below Slapy and receives more than
80% of its water from it but is also enriched by both phytoplankton
and phosphate from the Sazava River. Its maximal
depth is about 8 m, length about 6 km,
mean retention time 2.5 days (see Strag-
CIIEMICAL
CHANGES
DURING
kraba and Javornickjr, in press). Korkyn&,
a village pool near the Slapy field station,
is rather small and shallow (ca. 0.2 ha,
maximal depth less than 1 m ) ; it is cnriched by domestric and farm effluents
and by waterfowl.
KliEava is a water supply reservoir about
40 km southwest of Prague, maximum
depth about 27 m, arca 80 ha, mean rctention time slightly less than 1 yr, eupho’tic
zone about 8 m. Use of fertilizers and
pollution
in the drainage arca is rather
limited ( Rozmajzlova-RehGkova
1966).
EXPERIMENTAL
Sampling
procedure and treatment
oj samples
The samples were collected from the
surface layer (O-O.5 m ) with polyethylcnc
bottles. All the water for 1 cxperimcnt was
mixed thoroughly in a PVC pool of 150liter capacity, divided in half, and the respcctivo reagents ( 15N-KNOs, ‘5N-NH&1,
NH&l)
added. The water was filtered
through silk (grade No. 8 or 13) and siphoned into IO-liter light- and dark-glass
bottles. Thcso were exposed in the rescrvoirs with the tops of the bottles at a
depth of 21) cm (and on occasion at 1.2 m).
The time of exposure for most casts was
the light part of a day; in some experiments
changes wcrc followed for a longer period
(maximal length was night + day + night).
In such cases, light and dark bottles were
taken at sunrise and sunset, respcctivcly.
Preparation of samples for akndyses
From each bottle, two duplicate lOO-ml
samples were siphoned for oxygen dctermination (200-ml overflow)
and o,ne for
the calorimetric
determination
of nitrate
and ammonia at the beginning and end of
an cxperimcnt. The main volume was centrifugcd, the particulate matter made up to
10 ml with N-free redistilled water, transfcrred into PVC tubes, and kept frozen
(-30C) until analyzed.
Just before an analysis, samples wcrc
thawed, homogenized, and aliquots pipetted into reagents for individual determinations. For organic-N two aliquots of 0.5 ml
were taken. A further 34 ml subsample
PRIMARY
PRODUCITON
799
was used for mass spcctromctric analysis.
After Kjcldahl combustion this portion was
distilled and collected into a 50-ml graduated flask, and 1 ml of distillate was taken
fo’r colorimcric determination of the actual
amount of N&N
by the bispyrazolone
method. The main portion, namely 49 ml
of distillate,
was supplcmentcd
by an
equivalent vo1um.e of 0.01 N sulfuric acid,
cvaporatcd
to dryness under decreased
pressure, and subjected to mass spectrometric analysis.
Ca~lculation and cmsideratian
of errors
Analytical
data for particulate
matter
wcrc recalculated to calories and oxygen
using equivalents given by Brody ( 1945) :
1 g protein+4.6
kcal or 0.95 liter 02; 1 g
glucosc+3.8
kcal or 0.745 liter 02; 1 g
lipid+9.45
kcal or 2.14 liter 02. The sum
of protein, carbohydrate, and lipid was the
basis for the caloric percentage calculation.
Changes in oxygen and ammonia conccntration were obtained by subtracting starting (time, to) from final concentrations,
or in longer expcrimcnts, by subtracting
values of morning and evening samples to
get changes for daytime or night. Data on
nitrogen upt,akc were related to the increase in oxygen concentration in the respective light bottle (but not to primary
production or respiration)
so as toi make
direct comparison with changes in particulate matter possible.
Flow centrifugation
(like filtration)
dots
not permit differentiation
between components of particulate matter. Thcreforc all
considerations refer to, the equilibrium bctwcen free water and particulate
compartments; nothing can be said about the
relations between algae, bacteria, and other
particles.
Because of the relatively high ambient
concentration o,f nitrate ( > 0.5 mg/liter N ),
the co,lorimctric procedure did not ensure
the accuracy necdcd for dctcrmination
of
small Non-N changes. Nitrate uptake was
thcrcfore calculated from mass spcctromctric analysis of particulate matter.
In some calculations
(e.g., NH4 : NO3
uptake ratio) two sets of data were used
Mastnik
Mastnik
KlGava
Mast&k
Vran&
Vrar&
Vran&
Neb?ich
Nebiich
2
3
4
5
6
7
8
9
Locality
1
SO.
Expt
65
65
65
65
65
6.5
66
66
2 VII
7 VII
8 IX
13 IX
28 IX
13 IV
3v
65
14 VI
2 VI
Date
86.7
224.7
192
41.77
7.5
7.9
8.1
8.2
8.0
18.0
17.2
18.2
18.0
17.5
275.3
275.0
6.9
7.3
6.6
16.7
fog
rain
750*
7.8
16.5
170”
8.0
PH
I*
NO,4
(E)P
1.12-1.25
0.50-1.50
(4
1.70
(0)
3.12-3.22
28
(0)
(13:)
1.31
(0)
2.90
0.77
0.23
0.64
2.65
GPP
daytime
(mg OJliter)
1.85-1.90
&
1.42
(0)
added)
of individuaZ
87
(17)f
(ii)
1.75
(0.55)
1.32
(0.26)f
pi)
(22)
(1:;)
(FE)
(A:;
2.45
(0.0)
original
NH,-N
characteristics
(g cal cm-2
(mg/liter
day-l)
Main
14.5
Temp
(“C)
TABLE 1.
14.913
9.303
10.498
-
6.683
2.616
-
-
-
Phytoplankton
biomass
(mg/liter)
experiments
Fl
B
Ch
Fl
B
Ch
B
Ch
cy
B
Cr
Ch
cy
B
cy
Cr
Ch
B, Fl
Ch
Fl, B
Ch
Ch
B
Fl
66.9
27.3
5.8
89.5
6.5
4.0
85.2
13.8
1.0
68.0
17.5
14.0
0.5
84
7.1
6.6
2.3
Taxonomic
compositions
( d-e
%1
Cr~tonwnas
reflexa
sp. div.
Crgptomonas
Cgclotellu
Crgptomonas
Pandorina
Prevalent
taxon
Ax, D
A,, B
A,, C
A,, C
A1
A,
A,
G
AZ
Expt
type§
Korkyni:
Nebitich
l\;ebfich
NebZich
NebZich
KliEava
Benson’s
medium
(modified)
10
11
12
13
14
15
16
18 X
5 VI
13 IX
3 VIII
12. VII
28 VI
31 v
Date
67
67
66
66
66
66
66
* Estimated from heliograph records.
t Labeled nitrogen.
$ Ch-Chlorophyceae;
Cr --Cryptophyceae;
B -Bacillariophyceae
( Diatomae) ;
Fl -Flagellates;
CyXyanophyceae.
Locality
Expt
No.
18.1
17.0
20.0
22.2
22.8
19.0
Temp
PC)
7.7
7.1
7.6
7.s
7.3
7.3
?JH
1.
Continued.
1.60
(0.35) t
0.51
(0.24);
1.08
(0.20)f
1.22
(0.35) i
1.38
(0.33)-f
(k&
0.91
(0.10) i
(iii,
&t
(11:)
(8;)
(lo:)
(it)?
(Z)
9 A -time
of exposition;
AI--daytime
only;
AZ-night,
daytime, night;
AS--night,
daytime;
Ah-daytime,
night;
350
252
359
573.0
521.0
180.0
1.47-1.65
0.30-0.26
1.20
1.40-1.39
1.27-1.29
0.90
2.26-2.55
NH,N
GPP
NO,N
11
(g cal cm-a
daytime
day-l)
(mg/liter original added) (mg ,O,/liter)
TABLE
96.6
3.0
0.4
94.4
4.4
1.2
85.2
12.6
1.4
0.8
90.0
8.3
1.7
98.2
1.0
0.8
66.3
33.7
Chlorella
pyrenoidosa
100
B
Fl
Ch
cy
Fl
Ch
B
Fl
Ch
Cy
B
Fl
Ch
B
Ch
Cy
Ch
B
Taxonomic
composition+
( volume To )
C ycibtellu
AdXeTUJ
solitaria
Fragdariu
crotonenk
Fragilariu
crotmnsis
Melosira
granuluta
Scenedesmus
Prevalent
taxon
B -both
calorimetric
and mass spectrometric
nation of NH,-N uptake;
C -two
different concentrations of ammonia
and added);
D*xposition
in two different depths.
6.756
2.983
1.670
5.559
3.774
7.332
14.871
Phytoplankton
biomass
(mg/liter)
(original
determi-
A,, B
Ax, C
Al, C
AZ, Al, C
Al, C
Expt
67x4
802
L.
TABLE 2.
Ammonia
PROCdZKOVli,
absorption
fig N absorbed/ml
Emt
11
No.
(g cal cm-2 day-l)
1
2
5
6
8
8
9
10
11
12
13
14
15
170
750
225
102
275
275
275
180
521
573
359
252
350
3*
fog
4”
7*
9”
rain
87
42
34
in light
0, produced
NH,-N concn NH,-N concn
< 40 ,ug/liter > 60 pg/liter
-
42
78
68
66
22,
3
-
7”
10
98
2#1
18
31
468
70
14
49’
19
37
29
0
Mean
I?. BLAiiKA,
-
109
-
210
640,
-
0
106
Mean
320
* Samples exposed at low light inputs
day-l ) .
(< 100 g cal cm-2
which are not strictly comparable:
analytical for ammonia and isotopic for nitrate
absorption. A much better agreement may
bc expected for analytical and isotopic data
for nitrate than those for ammonia, but
experimental proof would be useful.
No changes in nitrite
concentrations
were recorded during the experiments, and
therefore they were not considered in calculating results.
Chlo~lla pyrenoidusu for expriment No.
16 was obtained from the collection of
autotrophic organisms, Czechoslovak Academy of Science, Prague, and was grown
on Benson’s medium’ containing
10 mg
NO:,-N/liter
as the only nitrogen source.
Nitrogen substrates were as indicated in
Table 1. (For more detailed characterization of expcrimcnts see Table I.)
RESULTS
AND
DISCXJSSION
The analytical results indicated that the
mean ammonia uptake in light, with naturally occurring concentrations of ammonia
AND
M.
KRkLOVik
in surface-exposed bottles, was limited by
ambient concentration
of ammonia,
This is demonstrated by the increased uptake after the ammonia concentration was
increased (Table 2). It implies difficulties
in interpretation
of results from bottle cxperiments in which ammonia is declining
throughout the experiment, as can be expectcd where most of the recycling mcchanisms are missing (buIk of zooplankton,
contact with bottom deposits, rain). Therefore very short experiments, or experiments
in which the ammonia concentration is kept
throughout as near as possible to the outside concentration, are required to get data
applicable for detailed nitrogen budgets.
All values in Table 2 exceeding 100 pg
NIId-N absorbed/ml
O2 produced correspond to samples incubated at low light
intensities, indicating high NHh-N rclativc
uptake and possibly also protein synthesis
predominating
over synthesis of carbohydrates and lipids in dim light.
Dark absorption was highly variable; in
nearly half the experiments a reIeasc of
ammonia was observed. Syrett and Fowden ( 1952) described ammonia absorption
in darkness by nitrogen-starved
cells of
Chlorella and related it to the carbohydrate
content of the cells. Fitzgerald
( 1968),
starting from their paper, worked out a
test for N-dcficicncy
in algae and higher
aquatic plants. The correlation between
ammonia dark absorption and carbohydrates for our set of data was insignificant.
The correlation of ammonia dark uptake
(or release) with the percentage of protein
in particulate matter was, however, highly
significant (r = 0.785-Fig.
1). The multiple correlation of these two factors with
ammonia concentration
or a relation of
ammonia absorption to combinations
oE
biomass and length of experiment do not
improve the corrcIation and may cvcn dccrease the r value. The variable ratio of
living cells to detritus and the accumulation
of analytical errors in both complex values
used for this correlation, each of which
comprises three analytical mcasurcments,
probably decrease its closeness. Generally
it supports Syrett’s (1956) view that in
low
CHEMICAL
CIJANGES
DURING
jig NH,- N/ml 0,
+ 00
-501
40
60
50
cd %
90
80
70
FIG. 1. Dark uptake or release of ammonia per
milliliter
of oxygen produced in the corresponding
light bottle, related to caloric percentage of protein
in particulate
matter. Numbers at the points refer
to experiments.
The correlation
coefficient
r =
0.785 is highly significant
and was calculated for
log uptake (release) + 50 and cal “/o of protein.
some algal species lipids may substitute for
carbohydrates as main carbon stores.
So far, ammonia excretion by algae has
not been considcrcd as an ecologically important factosr. But Fig. 1 seems toI demonstrate that it could be the mechanism
controlling the ratio protein : carbohydrate
+ lipid in natural communities,
The behavior of high protein particulate
matter in darkness is similar to that of
planktonic Crustacea or fish having a similar ratio in their tissues ( BlaZka 1966a, c) .
In several experiments both analytical
and isotopic data were obtained (Table 3).
They agree well with the view that the
colorimctric data correspond to the balance
uptake minus rclcase, whcrcas the shortTABLE 3.
Expt
Comparison
No.
Light bottle
Dark bottle
8
8
11
11
11
11
15
15
L
D
L
D
L
D
L
D
of data indicating
PRIMARY
time incubation with tracer gives absorption rate only. Dugdale and Gocring (1967)
judged the underestimate caused by isotopic dilution as less than 10%; the same
is true for our data. They also found for
marinc phytoplankton that the isotopic ammonia absorption rate was linear up to
24-36 hr. In our experiment No. 12, release of NH, was observed in the light
bottle bctwecn 24-31 hr (dark period) of
incubation:
5.0 ,ug NI&-N was released,
and only 1.4 pg ( 28% ) of organic-N was
lost from particulate matter formed from
labeled nitrate during the first 24 hr of
the experiment.
Both findings support the possibility that
the isotopic data found in exposures of
about 14 hr (range 6-23) can be considered to correspond to absorption. Table 3
indicates further the difficulty of comparing calorimetric and isotopic mcasuremcnts
for ammolnia and the limitations of studies
of equilibria
between cells and medium
using one approach only. But it is highly
probable that ammonia is exceptional in
this respect. With other inorganic-N substrates ( N03, Nz), the first metabolic step
is transformation
to ammonia or to, an
amino group bcforc they are incorporated
into amino acids, proteins, nucleotides, or
nucleic acids. Ammonia is the only compound likely to be released as the nitrogenous waste product, since the excretion of
amino acids is a loss of organic matter
rather than true cxcrction.
Isotopic data for the absorption of NO3
ammonia utilization,
analysis
( daytime)
( dsytimc
( daytime
(daytime)
( day +
( day +
(daytime)
( daytime
based on isotopic
NI”
Exposure
(hr)
11
11
15
15
23
23
16
16
803
PRODUCTION
NC?
(P&liter)
)
)
night)
night)
)
+ By mass spcctrometry determined uptake of NII,-N by particulate matter.
t By bispyrnzolone method dctcrmincd decrease of NII,-N in water.
29.8
1.97
17.7
4.60
23.0
6.90
9.00
6.20
and calorimetric
Nc:N,
26
-18
12
2
16
-1
0.87
-9.10
0.68
0.44
0.70
-0.15
0.78
0.81
804,
TABLE
L.
4.
Isotopic
PROCHtiZKOV&
data on nitrate
light and dark
P. BLASKA,
absorption
in
AND
M.
KR&LOVA
foo-y.f22./1nxl”
IOO-,g
Y
Expt
No.
low
Mean
low
1.81
8
9
9 (1.2 m)
10
10
12
12
13
13
14
14
15
high
D (in dark)
NH,-N*
10.0
22.3
25.3
low
2.65
15.7
-
-
7.08
1.14
23.5
1.79
22.0
1.06
8.35
15.4
-19.6)
8.15
14.40
* High NH,-N concentration
concentration
< 40 @g/liter.
.
o-
10.5
1.84
5.17
80
zo.
,
w
[f6
,
20
,
30
,
40
,
50
,
,
60
?O
NH&.v/l
I
80
,
90
,
fO0
,
f40
,
f20
ioo
f3G
-
2.03
13.0
60
.
-
12.4
40
high
0.68
0.64
-
21.2
[OfSl
L:D
NH,-N *
high
0
20
,ug NO,-N absorbed/ml 6,
produced in light bottle
L (in light)
NH&-N*
fOO-y
% NH&-N
2.88
1.14
3.10
-1.59 1.36
7.29
-13m.29 4.98
1.49
9.13
> 80 ,ug/litcr;
4.96
FIG. 2. The percent share of nitrate (left scale)
or ammonia (right scale) in the total inorganic
nitrogen uptake related to the ambient conccntration of ammonia. X-Natural
populations in freshwater,
with
numbers
of experiments;
O-the
Chlorella
experiment
( No. 16 ) ; 0 -recalculated
data of Goering, Dugdale, and Menzel (1964) for
coefficient
r =
the Sargasso Sea. The correlation
0.98 is highly
significant,
was calculated
for
log ( 100 - y) and log (In x), and for the frcshwater natural populations
only (n = 10).
low NH,-N
in light per ml 02 produced ranged from
1.81-25.3 ,ug NO,-N (Table 4). Dark nitrate absorption was much smaller, the
mean ratio o,f light to dark values being
9.1~close
to the tempcratc region data
of Dugdale and Goering ( 1967). Nitrate
uptake in light was higher at low ammonia
concentration, but dark uptake was hardly
influenced by the ambient ammonia conIn experiment No. 16, using
centration.
Chbrella
( from pure culture), no nitrate
was absorbed in the dark.
The ratio of ammonia to nitrate absorption was highly variable, but in general
indicated a preference for NHh-N, though
the nitrate concentrations were at least 6
times higher than those of ammonia. Pcnnington
(1942), Dugdalc and Dugdale
and
Billaud ( 1968 ) have found
(l%V,
ammonia preference at similar concentrations of both substrates or a surplus of
ammonia, and our data extend the finding
for a surplus of nitrate. Moreover, if the
pcrccntagc of nitrate : total inorganic nitrogcn absorption is plotted against ammonia
concentration, it is evident that the latter
is regulating the ratio (Fig. 2). Extrapola-
tion suggests that at concentrations higher
than 160 pg NH,-N/liter
little or no nitrate
would bc used.
Nitrate reductasc activity was probably
not changing during the experiments with
Parallel
experinatural
phytoplankton.
ments with high and low original concentrations of ammonia gave points lying on
at a changed
the same line. Incubation
ammonia concentration for less than 1 day
was probably insufficient for nitrate reducor synthesis (Eppley,
tase degradation
Coatsworth, and Solorzano 1969 ) . Changes
in nitrate reductasc activity might however
be involved in acclimation to very different concentrations of nitrate and ammonia.
Data of Goering et al. (1964) for marine
phytoplankton
acclimated to much lower
N03-N concentration are shifted down, the
points for Chbreltu acclimated to much
higher N03-N are shifted up the curve
( Fig. 2). This suggests that the relationship between percent N03-N utilization
and NH4-N concentration might bc influenced by the previous history of the algae
and perhaps by the methods used but not
so much by species composition and pII
(Table 1).
A few data on the total-N absorption per
-
CHEMICAL
TABLE
Concn
NII,-N
(pg/litcr)
Expt
No.
8
i (1.2 m)
10
10"
12
12*
13"
14
14"
15
Mean
87
28
28
38
105
0
106
88
5
115
13
5.
CHANGES
Uptuke
of total
DURING
PRIMARY
inorganic
fig N absorbed/ml
nitrogen
0, produced
805
PRODUCTION
in light
and dark
pg N ta
2 en up
(fig N particulate matter)-1
(hr)-l
dark
light
dark
light
24.8
28.7
192.0
62.0
110.0
21.2
31.0
36.0
30.5
54.3
25.0
-11.2
-33.7
- 3.5
- 9.8
- 7.1
1.06
2.44
16.1
0.0227
0.0134
0.0162
0.0138
0.0212
0.0115
0.0121
0.0140
0.0179
0.0345
0.0025
-0.0022
-0.0043
-0.0029
0.0005
0.00,14
0.0010
56.0
- 5.7
0.0163
-0.0044
-0.0121
-0.0164
-
[excluding 9 (1.2 m) value, 42.31
* NH,-N
added.
ml O2 produced in light bottles indicate
increased uptake after addition of NHd-N
(Table 5). Values for total dark absorption arc rather scattered as nitrate dark
absorption is low and ammonia absorption
can be either positive or negative. An overall mean for 24 hr ( 15-hr light, 9 hr dark)
would be 34 lug N absorbed/ml 02 produced. An independent estimate of this
value may bc obtained fro’m the ratio of
protein : carbohydrate : lipid (55 : 20 : 25 cal
%-Table 6 and BlaBka 196&z), hence 100
cal in particulate matter+21.65
ml O2 or
55 cal protein; 55 cal protein12.2 mg pro.tein+ 1.95 mg N; about 90 pg N incorporated corresponds to 1 ml O2 produced.
This discrepancy may be caused by incubation of most samples at the surface; in
dim light ( decpcr layers), a considerable
increase of the N : 02 ratio was indicated
(cxpt No,. 9, incubation in 1.2 m).
Total-N uptake per N in particulate
matter and hour-VN
(Table 5)-does not
reflect the difference due to the decreased
light input (depth, cxpt No. 9; turbidity,
cxpt No. 10). These data are, however, of
the same order of magnitude as those of
Dugdalc and Goering (1967) for tempcrate and tropical seas and as the recalculated data of Billaud ( 196S) for an Alaskan
lake. Surprisingly
our mean is closer to
the mean for tropical seas than to that for
temperate ones. One reason for this may
be a somewhat low estimate using analytical data for NHd-N uptake, but this would
change the value only by 30% ( Table 3).
An additional reason is probably the higher
ratio of detritus in freshwater particulate
matter than in that from the sea.
The protein content of particulate matter
is different after exposure in light and
dark bottles, and a similar difference was
found in surface samples taken for cxpcriments at sunset and sunrise (Table 6). This
suggests a preferential synthesis of carbohydrate and lipid o’ver protein during daytime. In the dark, carbohydrate and lipid
are consumed and eventually nitrog,en, particularly ammonia, is absorbed. IIowever
the methods used here do not cnablc us
to differentiate
between the nitrogen absorbed and that incorporated into protein
(Syrctt and Fowden 1952; Syrett 1962).
Low light promoted a preferential synthesis of protein over carbohydrate and lipid;
the same effect is used in the preparation
of high protein cultures of algae (Taub
and Dollar 1965). There is no conclusive
evidence in our results that particulate
matter from a low ambient concentration
of ammonia has a lower percentage of
protein, in spite of incrcascd nitrogen up-
806
TABLE
ulate
L.
PROCdZKOVri,
P. BLAiiKA,
6. Caloric percentage of protein in particmatter in light and durk bottles and in to
(initial)
samples
AND
TABLE
~.
M.
KRiiLOVli
7. The ratio A 02 calculated : A 02 exparimental for light and dark bottles
- -_
Expt
ENa”’
Light
45.4”
48.2
64.3”
55.8
1
2
3
z.7
42.7”
43.5
42.5
48.1
4
61.7
32
62m.9
9
z5
10
54.5
55.1
57.6
53.6
61.8
53.8”
45.4
50.2
42.7
-
12
13
Mcnn
Mean t
(light “= dark,
Whitney)
p <
t.OO.l-U-test
Light
1
1.52
0.78
0.34
1.64
1.30
0.82
0.44
1.37
1.63
1.44
0.59
1.12
1.84
1.96
0.55
1.00
0.57
0.64
0.90
0.82
1.26
0.61
0.61
5
6
9
50.5
25
51.5
51.6
38.3
35.3
No.
2
3
4
59.3
51.2”
58.0
60.5”
43.1
48.8
z.5
63.9
z.8
6
11
57.8
61.0
z.2*
63.5
60.7”
51.9
63.4”
74.7
63.0
77.7”
65.8
56.9,
62.9”
70.9
69.1
59.7”
60.0
59.6
57.0”
65.8
56.0
68.1
61.9
76.7”
6’5.4
66.6
5
8
Dark
10
11
12
13
Mean
(light =
Whitney)
of % arm and
.-
* ta samples. Those t&en at sunset incorporated into the
light-bottle
group, those at sunrise into the dark-bottle
group.
take after the ammonia concentration
is
incrcascd ( Table 5).
The oxygen concentration decreased in
all dark bottles, but in many of them an
increase in particulate matter (rather than
a decrease) was observed (13 out of 21
values ), In all light bottles, both oxygen
and particulate matter concentrations increased. For a more quantitative
cvalua-
dark,
p <
1.03
O.OOl-U-tat
Dark
-0.07
0.31
0.50
0.63
-0.26
-0.08
0.46
-0.77
-0.24
-1.26
0.99
0.92
-0.83
-4.80
-0.53
-1.80
1.19
-0.24
0.27
-0.47
-0.80
-0.34
of Mann
and
tion, changes in particulate
matter wcrc
recalculated to their O2 equivalents and
the ratio O2 calculated : 02 expcrimcntal
was determined. This mean ratio for light
is 1.03, but it is -0.34 for dark (Table 7).
This indicates that in light bottles the increase in particulate matter was proportional to the photosynthetic
activity
of
algae, although in a great number of dark
bottles, particulate matter was produced by
some other mechanism.
Bacterial production does not seem to be
It would have to be
the explanation.
equivalent to about 300 ,ul 02/U hr at
NebEch ( Slapy Reservoir ) to correct the
ratio to near 1. But actual mean increase
in bacterial biomass is equivalent to, 15 ~1
02/12 hr in KM-ml bottles for the same
station and the period April-September
1966 ( V. Straikrabova, personal communication) and probably even less in the loliter bottles used in our experiments. This
finding is therefore interpreted rather as
CILEMICAL
CHANGES
DURING
evidence for the aggregation into’ particles
of dissolved organic matter in the dark.
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E. R., AND W. H. SUTCLIFX~Z.
1963.
Dissolved
organic matter in seawater as a
source of particulate
food.
Limnol.
Occanogr. 8 : 369-371.
BILLAUD,
V. A. 1968. Nitrogen fixation and the
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of other inorganic nitrogen sources
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J. Fish. Res. Bd. Can.
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BLA~KA,
P. 1966~. Metabolism
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related to
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Int. Ver. Theor. Angcw. Limnol. Verh. 16: 380-385.
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