J. Exp. Mar. Biol. Ecol., 1986, Vol. 104, pp. 143-152
Elsevier
143
JEM 00794
Products
of photosynthesis
by marine phytoplankton
chitin in TCA “protein” precipitates*
:
Richard A. Smucker and Rodger Dawson
Chesapeake Biological Laboratory, Center for Environmental and Estuarine Studies, University of Maryland,
Solomons, MD 20688, U.S.A.
(Received
11 July 1986; revision
received
and accepted
11 September
1986)
Abstract: Evidence is presented
which documents
probable
errors in previous estimates
of ‘%O,
incorporation
and cellular distribution
in estuarine and marine phytoplankton.
Extension of the standard
extraction procedure
for phytoplankton
photosynthetic
carbon pools shows the presence of chitin (poly
N-acetyl-D-glucosamine)
in the previously considered protein pool. Trichloracetic
acid (TCA) precipitates
were processed in several ways including alkali extraction (protein solubilization),
4 and 6 N HCI hydrolysis,
hydrolysis with purified chitinase coupled with HPLC and calorimetric
analysis of amino sugars. Data for
Thalassiosira pseudonana cultures and natural photoplankton
populations
are presented. The results show
the presence of diatom chitin (chitan) in the TCA precipitate which has been previously characterized
solely
as the “protein” fraction.
Key words: Chitin;
Protein
fraction;
Diatoms;
Chitinase;
HPLC;
Chitobiose;
Thalassiosira
INTRODUCTION
Standard procedures for protein determination (Olive et al., 1969; Morris et al.,
1974; Li et al., 1980; Li & Dickie, 1985; Hitchcock, 1986) appear to have a serious
deficiency with respect to marine phytoplankton development and partitioning of photosynthetically fixed CO,. The abundant amino sugar polymer, chitin, is erroneously
included in the hot 5 y0 trichloroacetic acid (TCA) “protein” precipitate as protein and
not considered separately, as chitin. This oversight has significant consequences for
understanding the fundamental chemistry of primary production in the oceans and
estuaries and for dietary implications of planktivores and other heterotrophs.
Nutritional requirements and digestibility of chitin for filter feeders and suspension
feeders has been recently evaluated with respect to phytoplankton, against the larger
background of overall chitin production (Smucker, 1982; Birkbeck & McHenry, 1984;
Smucker & Wright, 1984,1986; Mayasich & Smucker, 1986; Wright & Smucker, 1986).
It is surprising that chitin has not been,previously included in primary productivity
* Contribution
No. 1742 from the Center for Environmental
and Estuarine Studies.
Correspondence
address: Richard A. Smucker, Chesapeake
Biological Laboratory,
Center for Environmental and Estuarine Studies, University of Maryland,
Solomons, MD 20688, U.S.A.
0022-0981/86/$03.50
0
1986 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
144
RlCHARDA.SMUCKER
AND RODGERDAWSON
studies considering that in 1965 McLachlan and co-workers demonstrated 31-38;,;,
chitin in the total cuhured mass of Tha~ssiiosirafru\ti~is, including the fiustules. Their
work was extended to include the C~~ute~l~ cvptica chitin chemistry (McLachlan &
Craigie, 1966). The term chitan was coined to distinguish 100% N-acetylated
/I-14-linked poly-N-acetyl-r>-glucosamine from the chitins of arthropods and fungi
which have a lower degree of N-acetylation in the chitin molecule (McLachlan et al.,
1965; Falk et al., 1966; Brine & Austin, 1981).
Ela~ration
of the diatom chitan fibers occurs whether the cells are under
NJimitation
or CO,-limitation (McLachlan 8c Craigie, 1966). Cell buoyancy in
Thalussiosira and Cyclotella is largely dependent upon the chitan fibers (Walsby &
Xypolita, 1977) which are produced at the base of valve pores (Herth, 1978; Herth &
Barthiott, 1979).
Previous work with chitin from other ~rg~isms suggested that 5% TCA had
negligible effect on chitin, the chitin ultrastructure remaining intact (Smucker & Ptister,
1978). The purpose of this work was to examine the role of poly-N-acetyl-D-glucosamine
in the hot 5 7; TCA insoluble “protein” fraction of pelagic marine phytoplankton. The
hot 5% TCA precipitate (purportedly only protein) was further processed by alkali
extraction to remove protein. The alkali insoluble residues were hydrolyze with 4 N
or 6 N WC1or purifTed chitinase, treatments which revealed the presence of giucosamine
and chitobiose in the residues, respectively.
MATERIALS
AND METHODS
Thu~ussi~sira pseudonuna (Swarm River strain, from I(. Sellner, Philadelphia
Academy of Sciences) was grown in 15-I batch culture. Cells in F/2 medium (Guillard,
1975) were grown under continuous illumination (4150 lux) and aeration at 18 “C.
MFASUR~~~N~OF
PHOTOSYNTHESlS
One integrated subsample from actively growing T. pseudo~uffu was mixed by
inversion and dispensed as 49.5 ml aliquots into 50 ml screw cap test tubes. After 30 min
equilibration, 0.5 ml of stock NaH14C0, (ICN) was added to yield 12.5 PCi 14C and
5 mM NaHCO, (spec. act. 10 &i/mmol). After 4.5 h incubation at 18 “C under
continuous artificial illum~ation (4150 lux) the entire contents of a given tube was
collected on 2.5 cm Whams
GF/F glass fiber filters (Morris & Skea, 1978). Interference by residual inorganic NaH 14C0, was minimized by rinsing the filter with sterile
filtered sea water (12x,) then adding 0.2 ml of 0.1 N HCI to the filter for 40 min
(Hitchcock, 1986). After rinsing the filter with distilled water, the filter pad was placed
in SO”/, ethanol for 40 mm (or stored at - 20 “C until processing) and then processed
for p~itioning.
CHITIN IN PHYTOPLANKTON
TCA “PROTEIN” PRECIPITATES
145
NATURAL POPULATIONS
Carbon p~itioning in situ was estimated from subsurface ph~opl~kton
samples
taken off the Chesapeake Biological Laboratory pier on 20 May 1986. Quadruplicate
250 ml samples were incubated and processed as for the T. pseudonana samples using
the same final added NaH14C0, spec. act. and molar&y. Zooplankton was screened
from incubation bottles using a 100 pm filter. Phytoplankton in the natural water were
identified by C. E. F. Oreo-Dawson;
dominant components were: ~itzschia cZoste~u~,
~haeto&e~~s,~avicuia, Thalassioth~.~, Diploneis, and Thalassiosira. Thalassiosi~a and
Thalassiothrix were most abundant. Sample bottles were attached to a polycarbonate
strip and suspended 0.5 m below the surface for 6.5 h (1030 to 1700). Natural lighting
was characterized by predominantly overcast conditions with occasional full sun.
F~~IONATION
OF CELL CONSTITUENTS
Labeled cells were fractionated using a modification of the methods described by
Morris et al. (1974). For clarity the procedures are outlined in Fig. 1. One of the
modifications consists of 1 N NaOH extraction following TCA precipitation (Brine &
Austin, 1981). After each extraction (in a boiling water bath) the filters (Whatman
RETAINED PARTICULATES
(Whatman GF/F)
Particulates collected
Rinse in 0.1 N HCI/30 mm
Rinse in deionized water
Store filter in 80% ethanol ( - 20 “C)
1
f
Background “‘COa
*
Lipid
,
Hydrolyzed saccharides
Nucleic acid pool
Extraction in 1 N NaOH (3-5 ml)/100 “C/1-23 h
{Repeat previous step as necessary)
Rinse (cold deionized water) (3 x )
i
>
Protein/peptidoglycan
Extraction 4 or 6 N HCI (4-5 ml)/100 “C/12-24 h
Hydrolysis by purified chitinase
Rinse (cold water)
I
,
Chitimresidual
chitiobiose
Extraction 100% ethanol
(3 ml)/100 “C/20 min
Rinse (cold 5% TCA) (3 x )
i
Extraction 5% TCA (3 m1)/100”C/40 min
Rinse (cold 5 % TCA) (3 x )
I
Final retentate
Fig. 1. Sequential extraction procedure.
146
RICHARD A. SMUCKER AND RODGER DAWSON
GF/F) were rinsed three times with the appropriate solvent. The wash fluids, following
each extraction, were pooled with the extraction fluid. In some cases, the sequential
extractions with 1 N NaOH were separated in order to follow the decline of alkali
extractible (protein) content. Radioactivity was determined by placing 1 ml of the
extracts in 9 ml of Scintisol (Isolab, Akron, Ohio). The clear counting solutions showed
80-87 % counting efficiencies.
CHROMATOGRAPHY
OF THE CHITIN FRACTION
One of the most assured methods for determining chitin is by enzymic hydrolysis
using the primary end product chitobiose (a dimer of /3-l-Clinked N-acetyl-D-glucosamine) as a quantitative marker. The chitinase (Streptomyces gtieus) used for this work
was purified as described previously (Smucker et al., 1986). High performance liquid
chromato~aphy of the amino sugars was performed under the following conditions:
amino-bonded silica (2 mm x 15 cm) 5 pm packing; Row rate 250 plfmin; detection by
UV absorbance, 205 nm or 210 nm; 10 ~1 sample volume (previously diluted with four
volumes of acetonitrile); acetonitrile : water (v/v) (64 : 26) as the eluting solvent.
Reference standards (glucosamine, N-acetyl-D-glucosamie, chitobiose and chitotriose)
were obtained from Sigma Chemical Company.
RADIOLABELED CHITIN
Radiolabeled [ 3H]chitin (> 95% acetylation) was prepared according to Molano
et al. (1977) and Hirano et al. (1976) and the labeling verified with respect to enzymic
specificity (Smucker & Wright, 1984) and degree of acetylation (Smucker & Kim, 1984).
COLORIMETRIC ANALYSES
Amino sugars were determined by the method of Ohno et al. (1985). The procedure
utilizes indole as a calorimetric agent following hydrochloric acid hydrolysis.
Neutralization or desalting are not required. Amino acids and glucose interfere to a
negligible extent.
RESULTS AND DISCUSSION
Hot 5% TCA had little solubi~zing effect on highly acetylated chitin (Table I), the
chitin remaining as a precipitate. The experiment reported in Table I involved use of
Gelman A/E glass fiber filters (1.0 pm porosity). If Whatman GF/F (0.7 pm porosity)
filters had been used, the percent solubilized figure would most likely have been lower
than the value of 4% shown in Table I. Tritium exchange which would be enhanced in
the highly protonat~ (acidic) en~onment of the TCA treatment may also have contributed to the above figure. Aside from these factors the evidence is that chitin is
practically insoluble in 5% TCA at 100 “C. Chitin is also essentially unaffected by
CHITIN IN PHYTOPLANKTON
147
TCA “PROTEIN” PRECIPITATES
TABLE I
Solubility of [3H]chitin in hot 5% TCA: a, the blank for 5% TCA (cold) extractables was the usual “water
soluble background”, and this has been substracted from the “soluble” values for hot TCA before calculating
the percent solubilized and percent chitin in the insoluble fraction.
Treatment
Original [‘Hlchitin
Cold 5% TCA
Hot 5% TCA
DPM-3H
x (% SE)
Percent
solubilized
179 119 (3, + 2632)
1449 (4, k 69)
9153 (5, f 206)
0.8
4.0
_
Percent chitin
in the insoluble
fraction
_
99.2
96.0
organic solvents and 1 N NaOH treatments at 100 ‘C. For a general review of this topic,
see Muzzarelli (1977).
Data from the lield experiments and from Thalussiosirupseudonana cultures show that
chitin from diatoms is found in the TCA precipitate. This is supported by several lines
of evidence. Repeated alkali extraction (protein solubilization) of the TCA precipitate
leaves a residue which, when hydrolyzed by strong mineral acid (HCl), yields
glucosamine (Table II). The data presented in Tables II and III show the percent of the
incorporated 14C solubilized under the specific incubation conditions. The presence of
glucosamine in the residue remaining after repeated alkali extractions was confirmed by
amino sugar analysis following HCl hydrolysis (Ohno et al., 1985). It is important to
note that glucosamine, GlcNAc, and GlcNAc oligomers are soluble in TCA, even cold
TCA; therefore glucosamine liberated during HCl hydrolysis of the material remaining
after TCA and alkali extraction could only be derived from high molecular weight
GlcNAc oligomers. Acid hydrolysis of the alkali resistant residue from natural phytoplankton showed a significant release of 14C. Hydrolysis of the residue by purified
chitinase confirmed the presence of glucosamine in the residue (Table III). Chitobiose
was the only GlcNAc oligomer released during chitinase hydrolysis. Chromatographic
analysis by HPLC was performed on the same hydrolysis mixture (chitinase) as reported
in Table III. The data shown in Tables II and III and the HPLC traces in Fig. 2 confirm
the presence of diatom chitin (chitan) in the TCA precipitates of cultured and natural
phytoplankton populations. The most convincing evidence of diatom chitin is the
release of only one compound, the dimeric chitobiose, by chitinase hydrolysis (Fig. 2;
Berger & Reynolds, 1958; Jeauniaux, 1982; Lindsay & Gooday, 1985). By contrast
colloidal chitin was hydrolyzed with a characteristic pattern of high molecular weight
oligomers forming first, and only subsequently formation of the dimeric form (Fig. 3).
The release of a single dimer was also observed with T. pseudonunu cultures prewashed
only with ethanol prior to enzyme hydrolysis (data not shown). Using light microscopy
Lindsay & Gooday (1985) observed that only the apices of T.fluviutiZis chitan spines
were hydrolyzed, an observation consistent with the present work where enzymatic
hydrolysis successively removes the dimeric form and results in the single HPLC dimer
Replicate
II. TCA
15795
(5%)
11392
(3.8%)
16155
(5%)
9270
(3.4%)
I. Ethanol
146139
(46%)
145910
(48.4%)
137000
(42.5%)
150024
(55%)
2nd
_
29386
23716
1st
119232
(37.6%)
102828
(34.1%)
118044
74616
III. NaOH
4th
6960
3870
(NaOH total = 49%)
5446
2910
(NaOH total = 39%)
_
3rd
Extraction step”
Percent of total 14C incorporated
_
6N
2460
667gb
(HCl total = 2.8%)
2717
3648
(HCI total = 2.3%)
4N
IV. HCl”
36355
(11.4%)
41476
(13.8%)
1592
(0.5%)
635
(0.23%)
Filter
residue
14C0, patitioning in Thalussiosira pseudonana: a, details of methods are in the text; values are expressed as DPM solubilized by the given extraction step;
repeated extractions began with ethanol and ended with alkali or HCI; b, presence of glucosamine in the HCl hydrolysates was confirmed with the specific
method of Ohno et al. (1985).
TABLE II
CHITIN IN PHYTOPLANKTON
TCA “PROTEIN” PRECIPITATES
149
III
TABLE
“C02 partitioning in natural population of phytopl~kton: a, extraction steps I-IV were carried out at
100 “C; Step V. at 43 “C; detaiis in the text; b, this sample was monitored by HPLC for GlcNAc
oligosaccharides (Fig. 2); the dimer of GlcNAc, but not other oligomers, is the only product formed by
enzymic hydrolysis from diatom chitin (chitan); this was confirmed as the pattern formed from the S. meus
chitinase treatment.
DPM and percent of total i4C incorporated
Extraction step”
IV. HQ
Replicate
1. Ethanol
II. TCA
1.
12805
(26.6%)
11856
9000
(18.8%)
8830
15330
(32%)
14847
4275
(29.3%)
13195
(30.5 %)
(22%)
10050
(23.3%)
(36.7%)
16107
(37.3%)
(10.6%)
3325
(7.7%)
2.
3.
III. NaOH
V. Chitinase
7128b
(14.8%)
_
-
Filter
residue
-3729
(7.8%)
612
(1.7%)
520
(1.2%)
18 hr
I
t
G2
Fig. 2. Purified chitinase hydrolysis of chitin in the TCA precipitate from a natural ph~opla~ton
population: the HPLC profiles (amino bonded column) show the presence of chitobiose released from
diatom chitan; only the dimer (C,) was observed during the entire 18-h hydrolysis period; this result is
indicative of the expected hydrolysis pattern of diatom chitan by purified chitinase.
150
RICHARD A. SMUCKER AND RODGER DAWSON
C2
COLLOIDAL
i’ ii’
9
N
Pa,
b-fv)
Lo
ELUTION
CHITIN
STANDARDS
In
b
TIME (mid
Fig. 3. HPLC analysis of GlcNAc oligomers released from colloidal chitin by purified chitinase: the
unresolved higher order of GlcNAc oligomers at the early hydrolysis times is characteristic of the enzymic
hydrolysis of colloidal chitin; chitobiose develops as the predominant oligomer after several hours
hydrolysis; this is in contrast to the release of chitobiose at all stages of hydrolysis with highly crystalline
chitin such as diatom chitan shown in Fig. 2; standards are shown for glucosamine (C), N-acetyl glucosamine
(C,), chitobiose (C,), and chitotriose (C,).
peak (Fig. 2). The occurrence of a single HPLC peak (chitobiose) from the enzymatic
chitinase hydrolysis of diatom chitin appears to be characteristic of the highly crystalline
form of chitin.
The present results from short term 14C0, incorporation experiments show that
chitan may contribute up to 33% of the TCA precipitate, previously considered to be
the protein fraction (Morris & Skea, 1978). This clearly indicates the need for a more
comprehensive profile of chitin in the “protein” fraction of natural primary production
and of diatoms in particular. One of the outstanding questions is the variability of the
protein : chitan ratio as a function of the cell division cycle.
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