Download INFLUENCE OF HUMIC SUBSTANCES ON THE GROWTH OF

INFLUENCE
OF HUMIC SUBSTANCES ON THE GROWTH
OF MARINE PHYTOPLANKTON:
DINOFLAGELLATES
A. Prakash and M. A. Rashid
Fisheries
Research
Board
Bedford
of Canada and Atlantic Oceanographic
Institute,
Dartmouth,
Nova Scotia
Laboratory,
ABSTRACT
Humic substances, in small amounts, exert a stimulatory
effect on marine dinoflagellates
that is reflected in increased yield, growth rate, and ‘“C uptake. Humic acid was found
to be more active than fulvic acid; in both cases the growth responses were dependent
on concentration.
Of the various fractions isolated, the low molecular weight fractions of
humic acid produced the greatest growth response in unialgal cultures of dinoflagellates.
The positive effect of humic substances on phytoplankton
growth is, for the most part,
independent
of nutrient
concentration
and cannot be attributed
entirely
to chelation
processes. It appears that growth enhancement
in the presence of humic substances is
linked with stimulation
of algal cell metabolism.
Because of their high concentrations
in
coastal waters, humic substances maythus be regarded as an ecologically
significant
entity
influencing
phytoplanktonic
prod&ion.
INTRODUCTION
tivity of soil extract has been attributed
primarily
to the chelating action of the
humic component, resulting in a reduction
of the toxicity or an increase in the availability of trace metals. Soil extract may also
be a source of vitamins, auxins, and other
micronutrients.
Sweeney ( 1954) was able
to replace soil extract with vitamin B12
and cthylenediaminetetraacetate
(EDTA)
which was added to aged seawater. In
several of the completely synthetic media
developed at the Haskins Laboratories,
New York, and at Millport, Scotland, soil
extract has been entirely replaced by organic additives (Provasoli et al. 1957).
We have used one such medium, ASPP
( Provasoli 1963a), for growing several
species of armored dinoflagellates
of the
genus Gonyaulax, but seawater medium
( Erd-Schreibcr)
enriched with soil cxtract has always supported better growth
( Prakash 1967).
In a study of the nutritional
characteristics of Gonyaulax tamarensis of the Bay
of Fundy, natural seawater with high humic
content was able to support reasonable
growth of this dinoflagellate
without further enrichment. Similarly, yellow humusladen river water added to artificial
seawater gave a positive growth response
for this and several other species of dinoflagellates.
Wilson and Collier
( 19%)
Instiobserved an improvement
in growth of
598
Our knowledge of the growth requirements and ecological interactions of marine
dinoflagellates, which show significant seasonal pulses in coastal waters, is fairly restricted. Dinoflagellate maxima in temperate waters usually occur towards the end
of the spring diatom bloom when major
nutrients (N, P, and Si) are in low concentrations. However, the prodigious growths
of dinoflagellates
leading to red-water
conditions have not been convincingly explained. Relatively few species of marine
dinoflagellates have been brought into unialgal and bacteria-free cultures; many of
the successful media have soil extract as
their common ingredient.
While a few
dinoflagellates
can apparently be grown
in inorganic medium, most marine dinoflagellates seem to require some organic
growth factors apparently supplied by soil
extract.
The history of the development of completely artificial seawater media is essentially the history of the elimination of soil
extract and its replacement with vitamins,
chelators, trace metals, and other organic
additives. This aspect has been adequately
reviewed by Provasoli, McLaughlin,
and
Droop ( 1957). The growth-promoting
ac1 Contribution
tu tc.
No. 102 from
the Bedford
EFFECT
OF
HUMIC
SUBSTANCES
Gymnodinium brevis in a seawater medium
to which water from a Florida river was
added, but a much better growth occurred
when both river water and extract of peat
soil were added. Since substantial amounts
of humic matter enter the inshore waters
of the Bay of Fundy from land drainage
just before the onset of the dinoflagellate
maximum, it appeared that runoff was providing natural soil extract which stimulated
the growth of the dinoflagellates.
WC have
examined the nature of these substances
and their influence on the growth of marine dinoflagellates.
THE
ORIGIN
AND
SUBSTANCES
NATURE
IN
OF
HUMIC
ON
599
DINOFLAGELLATES
uble in water; however, recent evidence
shows that certain low molecular weight
fractions of it may be ( Flaig 1960). Fulvic
acid is less complex in structure and more
soluble in water than humic acid and shows
fluorescence in UV light. Humus present
in the dissolved state in natural waters and
aqueous soil extracts is largely composed
of fulvo-hu.mic fractions that are biologacid is an
ically active. Hymatomelonic
alcohol-soluble fraction of humic acid and
is a simpl.er form ( Kukharcnko
1948).
Some workers regard fulvic, hymatomelonic, and humic acids as related compounds
and not as distinct chemical entities (Thiele
and Kettncr 1953).
SEAWATER
The formation of humic substances rcsults from the biochemical transformation
of plant and animal tissues incorporated in
soils or sediments. Lignins, proteins, and
carbohydrates are apparently first broken
down by microorganisms into simpler substances which are then resynthesized into
the more complex molecules of humic matter. The humic material found in coastal
waters is primarily of terrestrial origin and
is present in both dissolved and colloidal
states, imparting a yellowish-brown
color
to the seawater. Planktonic decomposition
products referred to as “gelbstoff” by Kalle
(1966) and as “water humus” by Skopintsev
( 1959) are also a source of yellowish color
in seawater and appear to have characteristics similar to those of humic compounds.
Based on their solubility characteristics
and also on their molecular configurations,
humic substances derived from soil organic
matter have been classified into four main
groups : humins and ulmins, humic, fulvic,
and hymatomelonic acids (Kononova 1966).
Because of the hetcrogenous and dynamic
nature of these compounds, their properties
have not been defined precisely,
Since
humins are insoluble, only humic, fulvic,
and hymatomclonic acids can be rcgardcd
as biologically utilizable by phytoplankton.
It is also unlikely that humic substances
present in the colloidal state in seawater
are efficiently utilized by algal cells. It is
generally held that humic acid is not sol-
MATERIAL
Culture
AND
METHODS
conditions
Bacterized
and bacteria-free
unialgal
cultures of G. tamarensis (Bay of Fundy
and Plymouth strains), G. catenekza, G.
acatenella, and G. monilata were grown
in 125-ml capacity screw-cap erlenmcyer
flasks containing 100 ml of medium. All
cultures except G. monilata were maintained in a constant temperature room at
10 * 1C under constant overhead illumination of 3,000-4,000 lux from banks of 40-w
“cool white” fluorescent lamps. G. moniZata was grown in an illuminated incubator
at 25C. Test cultures were maintained in
a marine synthetic medium ESWA (Table
1) as well as in artificial seawater ( ASW)
that was enriched with only humic additives. The culture medium was sterilized
by filtration through 0.22-p Millipore filters, and all1 glassware was autoclaved at
1.02 atm for 15 min.
All humic additives were filter-sterilized
before introduction
into tither ASW or
ESWA. For growth experiments, an inoculum was usually taken when the parent
culture had reached the stationary phase
of growth.
Growth responses to added
humic
compounds
were measured
as
changes in ccl1 numbers at frequent intcrvals throughout
the growth phase. Cell
counts were made using a sedimentation
method, as well as with a Coultcr counter
(model B ) . Growth was expressed in
600
A.
TABLE 1.
I.
Artificial
Parsons
Preparation
PRAKASII
of marine synthetic
ESWA
seawater
( AS W )-Strickland
( 1960)
Salinity
31%0
NaCl
2.2 g
MgCI,~6HzO
9.7 g
NazSOa
3.7 g
CaCL
1.0 g
KC1
0.65 g
NaHC03
0.17 g
0.02 g
HaI
Distilled
water to 1 liter
2. ES enrichment-Provasoli
Hz0
NaNOs
Naz* glycero PO,
Fe EDTA
P II metals*
*
.
Vitamin BU
Thiamine
Biotin
Tris buffer
pH 7.8
AND
medium
* One
(as
Cl-),
A.
RASHID
TABLE 2.
Growth response of Gonyaulax
arensis in different
media with and without
acid enrichment
tamhumic
and
(unpublished)
100 ml
350 mg
50 mg
2.5 mg
25 ml
10 /-e
0.5 mg
5.0 I-G
500 mg
3. Final concentration:
20 ml of ES enrichment
to each liter of charcoal treated ASW
0.2 mg;
M.
ml of P II metal
mixture
contains:
B (as H,BO,),
Fe (as Cl-),
0.01 mg; Mn (as Cl-),
0.04 mg; Zn
5 pg; Co (as Cl-),
1 pg; Na,-EDTA,
1 mg.
terms of either relative yield or relative
growth constant (K) and mean generation
time ( tg ) represented by the expressions:
1
N2
Ic = ( t2 - tl> log2 xq
and
t, = l/K,
where N1 and N2 are cell numbers at times
tl and t2.
The response of dinoflagellates to humic
additives was also determined by 14C incorporation after various periods of cxposure to the isotope. One ml of 14C labeled
Na2EIC03 solution containing
1 &i
of
activity was added to each test culture and
incubated from l-24 hr under constant
temperature and continuous light. The activity was measured with a thin window
gas-flow counter (Baird Atomic, model
FC-1) . Corrections for dark assimilation
of 14C were made in all experiments.
Medium
Final
yield
(cells /ml
X 103)
ASW
no growth
ASW (autoclaved)
2.1
ASW + humic acid
(5 a/ml)
ESWA
4:;
ESWA + humic acid
(5 b&ml)
14.0
Growth
constant
&&days)-1
Mean
gcncration time
tu(hr)
0.062
117
0.087
0.108
82
67
0.154
46
Isolation and purification
of
humic substances
The soil used for extraction of humic
substances was obtained from wooded
areas of New Brunswick and Nova Scotia,
and samples of marine sediment were collected from Cow Pen Basin on the Scotian
Shelf at a depth of 198 m. The procedure
used for the extraction and purification of
fulvic and humic acid fractions is described
by King ( 1967). The hymatomelonic acid
was extracted according to the method
suggested by Kononova ( 1966). The humic
substances dissolved in the river water
and in aqueous extracts of soil were recovered by evaporation
under reduced
pressure. Concentration
of humic substances was estimated by evaporating to
dryness and weighing known volumes of
purified extracts.
Molecular weight measurements and
fractionation of humic acids
The molecular weight measurements of
humic acids extracted from soil were made
by the Sephadex gel-filtration
tcchniquc
described by Mehta, Dubach, and Deuel
( 1963) and modified by Rashid and King
( 1968). Fractionation of humic acid based
on molecular weight measurements was
accomplished by the same technique using
Sephadex gel G-10, G-15, G-25, and G-50.
The humic acid excluded from the lowest
grade gel was concentrated and introduced
to the next higher grade gel and so on.
The fraction of humic acid absorbed on
EFFECT
OF
Gonyaulax
HUMIC
SUBSTANCES
ON
601
DINOFLAGELLATES
tamarensis
32
pg/ml
13 pg/ml
6pg/ml
Gonyaulax
catenella
0,105
pg/ml
HUMIC
ACID
FIG. 1.
Response of Gonyaulux tamarensis (13
days growth) and G. cuter&a
(6 days growth) to
various concentrations
of humic acid.
Cultures
grown under identical
conditions
of light and
temperature.
Medium
was ASW with different
concentrations
of filter-sterilized
humic acid extractcd from marine scdimcnt.
each gel was elutcd with a Tris buffer
solution of 0.5 M concentration.
Molecular
weight measurements of humic substances
dissolved in aqueous soil extract and river
water were made in the same manner.
RESULTS
Growth
responses to humic acid
enrichment
Test cultures of G. tamarensis were
grown in ASW and ESWA with and without the addition of the humic acid fraction
of soil humus. The growth responses are
shown in Table 2. In media devoid of
humic acid, the yield as well as growth
rates of dinoflagellates
tended to be approximately
30-50% lower than in the
enhanceenriched media. A significant
ment in growth was also seen when ASW,
which by itself is incapable of supporting
any growth, was autoclaved at 1.02 atm
for 15 min and used as a culture medium.
The reason for this rcsponsc is not clearly
understood, but it has been observed by
others (Pramcr, Carlucci, and Scarpino
1963; Jones 1967). Therefore, we have used
only filter-sterilized
ASW for subsequent
enrichment cxperimcnts.
Growth was also enhanced when yellow
humic water from some Nova Scotia and
New Brunswick rivers (avg humic acid
‘\
5
IO
DAYS
\
I
I5
AFTER
20
I
25
I
30
INOCULATION
FIG. 2. [Growth of Gonyacdax tamarensis
ASW enriched
with different
concentrations
humic acid extracted from marine sediment.
in
of
concn, 22 pg/ml)
was added to ASW.
Dinoflagellates
grew reasonably well in
ASW medium diluted up to 50% with
yellow river water, until the change in
salinity became too great to permit growth.
Controls with charcoal-treated river water
failed to show any growth at all.
Dosage effect
Responses of G. tamarensis and G. catenella to various concentrations of humic
acid are shown as dosage-yield curves
(Fig. 1). Similar curves have been obtaincd for other species. At concentrations
less than 2 pg/ml of humic acid in the
medium, the growth was erratic and the
cells tended to be sluggish, enlarged, and
with a high incidcncc of aberrant forms.
On the other hand, at concentrations over
35 pg/ml, a definite decrease in growth
rate as well as yield was noticed.
Another aspect of the response of dinoflagellates to concentrations of humic acid
is seen in Fig. 2. The cultures of G.
tamarensis grown with 6-32 lug/ml of
humic acid in the media showed practically
no change in rate during the exponential
phase of growth, as is evident from identical values for K and ts. However, they
differed considerably in final yields, which
were approximately proportional to the initial concentrations of humic acid in the
602
A. PRAKASH
AND M.
A. RASIIID
TABLE 3. Rc~sponse of Gonyaulax
tamarensis to
various concentrutions
of humic acid as measured
by ‘“C assimilution
PHOSPHATE
Humic
acid
concn
b.WmU
&z-z~=--
Incubation
4
hr
period
6
8
hr
(counts/min)
hr
r-a
/
NITRATE
t
I
10-S 10-4
I
10-3
t
10-Z
I
10-I
CONCENTRATION
I
I
A-
Without
0-
With
HUMIC
HUMIC
ACID
7
14
21
28
430
500
1,220
550’
570
630
1,340
770
870
1,530
680
920
ACID
t
IO
- mg /ml
FIG. 3. Cell yields at different levels of N and
P, with and without 7 PgIml of humic acid.
culture media. Both K and final yields
tended to be significantly reduced in cultures grown in humic acid concentrations
over 32 pg/ml and under 6 pg/ml. These
observations suggest that the difference in
yield under identical K values are related
to differences in duration of the exponential as well as of the lag phase of growth
and are brought about by the presence in
the media of differing amounts of humic
matter.
Uptake of IJC
A definite increase in the rate of 14C
assimilation by G. tamarensis was noticcable when soil humic acid was added to
ESWA. Dinoflagellates
were exposed to
several concentrations of humic acid and
the radiocarbon
uptake measured over
2-8 hr incubation (Table 3). As indicated
by counts per minute, the rate of 14C
assimilation increased with increasing concentration of humic acid, but at concentrations over 21 pg/ml, it tended to be
considerably reduced. This inhibition may
bc due to selective absorption of light by
the yellow of the humic acid, resulting in
rcduccd photosynthesis.
In our growth
experiments discussed earlier, inhibition
was noticed only after the concentration
of humic acid in the medium reached over
35 pg/ml, whereas in 14C experiments, the
inhibition
was detected at the 21 pg/ml
level. This inconsistency can be attributed
partly to the difference between photosynthesis and growth and partly to the different properties of humic acids derived from
marine sediments and from forest soil.
Relative responses to fulvic, humic,
and hymatomelonic acids
Growth responses to differential enrichment of ASW with humic, fulvic, and hymatomelonic acids, as measured by cell
yields, growth constants, and generation
times are shown in Table 4. Growth responses to fulvic acid enrichment were
also dependent on concentration, but for
the same concentrations, humic acid gave
approximately
twice as much growth as
TABLE 4.
melonic
Effect of humic, fulvic, and hymutoacids enrichment on the growth of
Gonyaulax tamarensis
Concn
bglml)
Humic
Fulvic
acid
acid
IIymatomelonic
14.1
;::g
(2!,s
1,179
7.0 1,086
3.5
837
7.5
602
3m.7 451
0.9
283
10.5
5.3
K&lay+-1
0.10
0.10
0.08
0,.07
0.05
0.02
no appreciable
no appreciable
tub9
72.4
72.61
90.3’
103.2
144.5
361.2
growth
growth
EFFECT
OF
HUMIC
SUBSTANCES
FIG. 4. Growth response of dinoflagellates
media at 4 &ml
concentration.
N
Earlier studies on the growth of G.
tamarensis indicated specific requirements
for nitrate, vitamin
B12, and thiamine
(Prakash 1967). H owever, the possibility
that the growth stimulating effect of humic
compounds on dinoflagellates
is due to
the presence of vitamin B12 and thiamine
in humic fractions can bc ruled out because
the extraction and purification
procedures
used to obtain these fractions would result
in destruction of these vitamins.
In order to examinc the possibility that
the growth enhancement was due to increased nitrogen and phosphorus contribution from humic material, test cultures of
G. tamarensis were grown in ESWA with
various concentrations
of sodium nitrate
,and sodium glycerophosphate
for 32 and
11 days respectively.
The yield increased
with the increase in N and P levels, until
603
DINOFLAGELLATES
to the various
fulvic acid. In media enriched with hymatomclonic acid, dinoflagellates remained
viable only for two days and no growth
response could be detcctcd.
Effect on yield at different
and P levels
ON
fractions
of humic
acid
added
to the
at high concentrations an inhibitory effect
became apparent. In cultures with humic
acid cnrichmcnt,
although the response
curves followed the same trend as the
controls, the yields for a given concentration of N and P were considerably higher
( Fig, 3). These experiments suggest that
the growth response of dinoflagellates
in
the presence of humic acid is to a large
extent independent of the N and I? levels
in the medium.
Humic compounds do
not appear to be contributing
significant
amounts of N and P to the organisms as
our fulvic and humic acid fractions never
contained more than 3.4% of nitrogen and
1.2% of phosphorus.
Growth responses to different
molecular weight fractions
Although humic fractions of soil humus
arc generally regarded as compounds of
high molecular weight, there is wide variation in the molecular weight distribution
and this variation is related in some measure to the soil type. In our samples, humic
acid extracted from marinc sediment was
predominantly
represented
by fractions
604
A.
PRAKASH
AND
TABLE 5. Moleculur weight distribution
of humic
acids extracted from various sources
--~Soil
Mol wt
range
<700
700-1,500
1,500-5,000
5,000-10,000
>10,000
extract
(%)
River
water
(%)
Forest
soil
(%I
Marine
sediment
(o/o)
43.8
56.2
-
27.4
7.3
65.3
-
33.2
7.7
11.9
47.2
-
6.5
5.0
5.6,
9.9
73.0
( aqueous
)
having molecular
weights over 10,000,
whereas humic acid cxtractcd from forest
soil was devoid of any fraction over 10,000.
Water soluble humic acids rccovcred from
soil extract and river water were represented almost exclusively
by fractions
lower than 5,000 (Table 5).
To determine the relative effectiveness
in growth stimulation
of each of these
fractions, cultures of three species of Gonyaulax were grown separately in ESWA
and ASW under identical enrichment conditions with different
molecular weight
fractions added (Fig. 4). Although the
three spccics responded somewhat differcntly, in general the greatest response was
found in the lower molecular
weight
fractions.
DISCUSSION
While the role of humic matter in the
development
and growth of terrestrial
plants has been the subject of fairly extensive study, comparable studies on marine algae are almost nonexistent.
That
humic substances may have some stimulating effect on the phytoplankton
production in the sea has been suggested by
a number of authors (Strickland
1965;
Provasoli 1963b), but there is little direct
evidence available. In his study on a marine diatom Skeletonema costatum, Droop
( 1962) found that its growth was enhanced
when humic acid was added to the medium
and that humic fractions were more active
than fulvic fractions. Our results arc in
general agreement with his obscrva tions
except that while he found the maximum
response with the high molecular weight
fraction of humic acid, the maximum activity in our cultures was observed with low
M.
A.
RASHID
molecular weight fractions. Although the
evidence for diatoms is limited, there arc
grounds for believing
that humic substances stimulate growth oE both diatoms
and dinoflagellates and are thus of potential ecological significance in phytoplankton productivity.
The growth-promoting
activity of humic
substances in our experiments cannot be
accounted for in terms of vitamin B12 or
thiamine, as the tcchniqucs used to extract
humic and fulvic acids from soil humus
result in complete destruction
of these
vitamins.
Nor can the growth enhancement in our cultures be attributed entirely
to Fe and other trace metal concentrations
in the humic additives, as most metallic
cations originally
present in the humic
fractions were removed in exchange resin
columns; the ash content was rcduccd to
less than 3%. The N and P contribution
from humic compounds also has limited
effect, since increasing N and P beyond
a certain concentration in the medium did
not proportionately
increase the growth
of the dinoflagellates.
On the contrary,
while at high concentrations of N and P,
both the growth rate and the total yield
decreased, our results suggest that humic
acid is capable of removing the negative
effect of high dosage of N and P. The
identical phenomena have been reported
by Chaminade (1958) who grew rye grass
in various concentrations of nitrogen with
and without sodium humate.
Since the positive growth response of
marine phytoplankton
in the presence of
humic substances cannot be adequately
explained on the basis of N, P, trace metals,
and vitamin requirements alone, it appears
that other processes are involved.
The
most plausible suggestion is that humic
and fulvic components of soil humus, in
small amounts, act as stimulants for marine
algal cells and may be involved in cellular
metabolic processes in the same manner
that has been suggested for terrestrial
plants [ Prozorovskaya ( cited in Kononova
1966) ; Khristeva 1951; Chaminade 19561.
However, the mechanism underlying
the
physiological
stimulation
by humic sub-
EFFECT
OF IIUMIC
SUBSTANCES ON DINOFLAGELLATES
stances and the pathways leading to growth
enhancement are little undcrs tood.
A limited amount of experimental work
suggests that humic substances act as
specific sensitizing agents enhancing the
permeability
of the plant cell membrane
and thereby increasing the uptake of nutrients from the surrounding medium. IIumic
compounds may also act as respiratory
catalysts since oxygen is absorbed more
intensively by plant tissue in the presence
of humic acid (Biber and Magaziner 1951;
Khristeva 1953; Smidova 1960). Prjt and
Pospisil ( 1959) and P&t, Smidova, and
Cincerova ( 1961) using 14C-labeled humic
acid showed that both humic and fulvic
acids are capable of penetrating the plant
cell, but the latter is more efficient bccausc
of its smaller molecular size.
Since coastal waters rcceivc significant
quantities of humic substances and other
nutrients as a result of river discharge <and
runoff, it is reasonable to assume that these
substances may influence the production
and succession of phytoplankton
species.
The ecological significance of humic substances in coastal waters might not be
rcstrictcd to stimulation of processes within
the algal cell. Chelation could also be an
important factor; De Kock (1955) showed
that iron became readily available to the
plant cell in the presence of humic acid
and stimulated growth, and Gran (1933)
linked the seasonal abundance of phytoplankton in the Gulf of Maine with iron
concentration, nutrients, and humic compounds washed from the shore. Apparently, algal excretion and decomposition
products are biologically
similar to terrestrial humic substances, Nordli ( 1957)
found that aqueous extracts of benthic
algae stimulated growth of a number of
marinc dinoflagellates.
In one expcrimcnt,
we found that decomposition products of
Nitzschia stimulated growth of Gonyaulax,
and the growth response was conccntration dependent
( Prakash, unpublished ) ,
Yentsch and Reichert (1962) observed an
increase in oxygen uptake by bacteria
when yellow filtrate from algal cultures
was added to raw seawater.
605
The stimulatory cffcct of freshwater runoff on dinoflagellate production in coastal
waters could be due to two indcpendcnt
mechanisms. In addition to bringing in
ecologically significant amounts of humic
substances, the freshwater runoff creates
low salinity conditions which favor faster
division rates among marine dinoflagellates
(Braarud 1951; Prakash 1967). It is possible thus to account for the development
of dinoflagellate
blooms, often leading to
red-tide conditions in coastal areas. There
is sufficient indirect evidence to suggest
that heavy rainfall or land drainage is a
prerequisite to most, if not all, dinoflagcllate blooms in coastal waters and that
the intensity of the bloom may be related
to humic or other nutritional factors cntering the cnvironmcnt
(Slobodkin
1953;
Wilson 1966; Burkholdcr, Burkholder, and
Almadovar 1967). The biologically active
ingredients of humic matter that stimulate
growth of marine phytoplankton
are perhaps as varied as they are complex and
offer a fertile field for critical enquiry.
REFERENCES
BIBER, V., AND K. MAGAZINER.
1951. The effect
of humic and fulvic acids on the respiration
Dokl. Akad. Nauk
of isolated plant tissues.
SSSR, 76: 609.
BRAARIJD, T.
1951.
Salinity
as an ecological
factor
in marine
phytoplankton.
Physiol.
Plantarum,
4 : 28-34.
BUIKEIOLDER, P. R., L. M. BUIXKHOLDER, AND
1967.
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