Advances in Bioscience and Biotechnology, 2013, 4, 11-17 ABB
http://dx.doi.org/10.4236/abb.2013.49A002 Published Online September 2013 (http://www.scirp.org/journal/abb/)
Carbohydrate analysis by methanolysis method and
application to compositional analysis of transparent
exopolymer particles
Shigeki Wada1,2*, Kazuo Iseki3, Takeo Hama1
1Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan
2Shimoda Marine Research Center, University of Tsukuba, Shimoda, Japan
3Graduate School of Biosphere Science, Hiroshima University, Hiroshima, Japan
Received 6 July 2013; revised 7 August 2013; accepted 21 August 2013
Copyright © 2013 Shigeki Wada et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Measurement of uronic acids (URAs) which are a
group of acidic sugar, would be useful for the under-
standing of dynamics of bacterial extracellular poly-
meric substances (EPS) in marine environments.
However, the URA analysis using traditional hy-
drolysis method which is used for neutral sugar
analysis poses serious problems in URA that is unsta-
ble under hydrolysis. We developed the methanolysis
method, which deploymerizes polysaccharides while
retaining quantitative information. Our method was
applied to coastal seawater, and the URAs distribu-
tion was compared with that of transparent exopoly-
mer particles (TEP) which are acidic sugar contain-
ing particles. Since the relationship of URA with TEP
was relatively weak, URA-containing polysaccharides
present in bacterial EPS would not participate as a
structural component of TEP.
Keywords: Uronic Acid; Transparent Exopolymer
Particles; Methanolysis; Gas Chromatography Mass
Spectrometry
1. INTRODUCTION
Quantitative and qualitative analyses of carbohydrates in
seawater provide some information on dynamics of or-
ganic matter such as its origin and diagenetic processes
[1-3], as well as its vertical transport and sedimentation
[4-6]. Monosaccharides characterized in these studies
were mainly neutral sugars (NSs), which comprise a
major group of carbohydrates, but there are other minor
sugar groups such as uronic acids (URAs). Although
URA, a carboxylated acidic sugar, has minor contribu-
tion to total carbohydrates in most cases, it is known as
an important component of bacterial extracellular poly-
meric substances (EPSs) (20% - 50% of total carbohy-
drate: [7,8]), and URA measurements are likely to reflect
the dynamics of bacterial EPSs in a water column. EPSs
readily adhere to each other as a result of their pro-
nounced stickiness [9], and constitute amorphous aggre-
gates such as transparent exopolymer particles (TEPs),
which are defined as particles containing acidic sugar
[10,11]. Since TEPs are relevant to sinking particle for-
mation and the supply of food to filter feeders [11-14],
URA measurements should allow us to better understand
the contribution of bacterial EPSs to these processes.
Analysis of neutral sugars has been achieved by using
chromatographic measurements after depolymerization
under acid hydrolysis reaction [15]. On the other hand,
application of acid hydrolysis method to URA analysis
has some problems, because URA, once released after
hydrolysis, forms lactones irreproducibly [16]. To over-
come this, it is necessary to correct the recovery yield of
URA after hydrolysis reaction, or to use other depoly-
merization method. In the present study, we apply the
methanolysis method, which depolymerizes polysaccha-
rides using methanolic HCl. High recovery yields for
authentic standards and some plant materials were
achieved by some previous researchers [17,18], but the
suitability of the methanolysis method for marine envi-
ronmental samples has yet to be examined.
In the present study, we modified a previous metha-
nolysis method for the determination of URA in seawater
samples [17,18]. In addition, we examined the mono-
saccharide composition of natural seawater sample in a
coastal environment using the methanolysis method, and
compared the distribution of URA in the water column
*Corresponding author.

S. Wada et al. / Advances in Bioscience and Biotechnology 4 (2013) 11-17 12
with that of TEP to understand the contribution of URA
to aggregate formation.
2. MATERIALS AND METHODS
2.1. Sample Collection
In 2007, seawater samples were collected from a coastal
region at a station in Suo-Nada (30 m depth, 131,16E,
33,49N) during the fifth cruise of the TRV Toyoshio
Maru of Hiroshima University in July, 2007. Seawater
was collected from 1, 5, 10, 15, 20 and 25 m using a Ro-
sette sampler fitted with Niskin bottles. The samples
were filtered through precombusted (450˚C, 4 h) glass
fiber filters (Whatman GF/F) and the filters were stored
at −20˚C until analysis. In the present study, we carried
out duplicate analyses for the samples at each depth.
2.2. Derivatization
The experimental scheme is a modification of the meth-
ods of Doco et al. (2001) and [19]. For analysis of par-
ticulate carbohydrates, chopped filter samples were
placed in a 10 ml glass tube, and internal standard (myo-
Inositol) was added. We putted the tube in a vacuum
desiccator with phosphorous oxide (V) for 1 day to re-
move any water completely. After adding 2 ml 0.5 N
methanolic HCl (mixture of 15 ml MeOH with 0.4 ml
acetyl chloride) to the dried pellets, the tubes were soni-
cated for 15 min, sealed tightly and heated at 80˚C for 24
h (methanolysis reaction). After cooling to room tem-
perature, the samples were centrifuged and the super-
natant was transferred to another test tube. MeOH (1 ml)
was added to the tube with chopped filter. The tube
weresonicated for a few s, centrifuged, and resulting su-
pernatant was combined with the previous one (this pro-
cedure was repeated 2×). After addition of 20 μl pyridine
for neutralization of the supernatant, the samples were
dried under an N2 stream at 40˚C and stored in a vacuum
desiccator with phosphorus oxide (V) for 3 days. Since
the reagents for trimethylsilylation are readily decom-
posed by water, complete removal of water from the
samples was checked.
TMSi-H [hexamethyldisilazane/trimethylchlorosilane/
pyridine, 2/1/10 (v/v/v), GL Science] was added (0.2 ml)
to the dried samples and the tubes were heated at 80˚C
for 2 h for the conversion to TMS derivatives. After
cooling to room temperature, the samples were dried
under an N2 stream at 40˚C and the dried pellets were
dissolved in hexane and sonicated for a few s. The tubes
were centrifuged and the supernatant was injected into a
gas chromatograph/mass spectrometer within 24 h after
trimethylsilylation at room temperature, because the de-
rivatized sample becomes unstable a few days after the
reaction. Standard materials for calibration also under-
went methanolysis and trimethylsilyl reactions as well as
the natural samples.
2.3. Analysis with Gas Chromatography/Mass
Spectrometry
GC/MS (QP 2050, Shimadzu) used an HP-1 fused silica
column (30 m × 0.25 mm i.d., 0.25 mm film thickness,
Hewlett Packard) and the electron impact ionization (EI)
mode. The detailed analytical condition were: inlet and
interface temperature 250˚C, ion source temperature
200˚C: column oven temperature programme 50˚C (1
min), to 120˚C at 50˚C· mi n −1, to 145˚C at 1˚C·mi n−1, to
200˚C at 0.9˚C·min−1, to 230˚C (held 10 min) at 10˚C·min−1
in order to separate eight NSs i.e. arabinose (Ara), ribose
(Rib), rhamnose (Rha), fucose (Fuc), xylose (Xyl),
mannose (Man), galactose (Gal) and glucose (Glc), and
two URAs, galacturonic acid (Gal Ac) and glucuronic
acid (Glc Ac) (Table 1). We confirmed the retention time
by measuring authentic standards one by one; D-Arabi-
nose (Wako), D-Ribose (Pfanstiehl Laboratories Inc),
L-Rhamnose monohydrate (Wako), L-Fucose (Pfanstiehl
Laboratory Inc), D-Xylose (Wako), D-Mannose (Wako),
D-Galactose (Wako), D-Glucose (Wako), D-Galacturonic
acid (Wako), and D-Glucuronic acid (Wako).
Table 1. Retention times of each monosaccharide.
Name of Sugar Retention time (min)
Arabinose (1) 14.30 ± 0.021
Arabinose (2) 14.82 ± 0.022
Ribose (2) 15.00 ± 0.020
Ribose (1) 15.70 ± 0.021
Rhamnose (1) 16.01 ± 0.021
Rhamnose (2) 16.33 ± 0.019
Fucose (1) 17.22 ± 0.024
Fucose (2) 18.46 ± 0.023
Xylose (1) 19.78 ± 0.021
Xylose (2) 21.09 ± 0.027
Glucuronic acid (2) 24.88 ± 0.029
Mannose (1) 30.32 ± 0.029
Mannose (2) 32.20 ± 0.031
Galactose (1) 33.51 ± 0.035
Galacturonic acid (1) 34.64 ± 0.029
Galacturonic acid (2) 35.19 ± 0.032
Galactose (2) 36.06 ± 0.028
Glucose (1) 37.69 ± 0.038
Glucuronic acid (1) 39.06 ± 0.036
Glucose (2) 39.90 ± 0.036
Internal Standard (myo-Inositol) 57.74 ± 0.217
Peaks of two isomers were quantified in the present study, and order of peak
intensity between the two isomers was indicated with number in parenthesis
(higher peak: (1), lower peak: (2)).

S. Wada et al. / Advances in Bioscience and Biotechnology 4 (2013) 11-17 13
Three fragment ions (m/z = 204 and 217 in 10 - 24.75
and 25.8 - 100 min, and m/z = 204 and 230 in 24.75 -
25.8 min) commonly found in the mass spectra of TMS
derivatives of carbohydrates when EI is used were moni-
tored in the selective ion monitoring (SIM) mode [19].
The methanolysis reaction theoretically generates 4 - 6
isomers from each monosaccharide, but it is difficult to
quantify all of the isomers because some minor peaks
were too small to quantify. Since the generation patterns
of isomers would be constant if the condition (tempera-
ture and time) of methanolysis reaction does not change
as described in latter section, we choose the highest and
second highest peaks for quantification in most case. For
galactose, the 1st and 3rd peak were quantified in the
present study, because the 3rd highest peak of mannose
coeluted with the 2nd highest one of galactose. The ana-
lytical errors (coefficients of variance between duplicate
analyses) for each monosaccharide were 0.81% - 36%,
and mostly the values were around 10% (Table 2).
2.4. Analyses of Particulate Organic Carbon and
TEP
Particulate organic carbon (POC) concentration was de-
termined using an elemental analyzer (FISONS EA
1108). For analysis of TEP concentration, 70 ml of sea-
water samples were filtered through polycarbonate filter
(Millipore) with pore size of 0.4 μm, and immediately
stained by prefiltered alcian-blue solution (0.2 μm, 0.02%
alcian blue 8 GX in 0.06% acetic acid) which stains
acidic sugar. The filters were transferred into 80% H2SO4
for 2 h, and the absorption at 787 nm was measured. The
TEP concentration operationally defined as alcian blue-
stained particles of size >0.4 μm [10,20], were measured
colorimetrically according to the method of Passow and
Alldredge [20]; the concentration was normalized with a
gum xanthan equivalent per liter (GX equiv. l−1).
3. RESULTS & DISCUSSION
When applying the methanolysis method to seawater
samples, it is essential to take account of the presence of
various organic compounds as concomitants, which can-
not be completely separated from carbohydrates. Appli-
cation of the EI/SIM mode in GC/MS enables us to
minimize the possible effect from non-carbohydrate com-
pounds which overlap with target compounds during the
GC separation, since only the specific fragment ions for
carbohydrates [19] were monitored in the present study,
as described above. We have conducted preliminary
measurements using GC-FID, which detects organic
compounds non-selectively to estimate their concentra-
tions. However, some of the monosaccharides cannot be
quantified due to the overlap with concomitant peaks
(data not shown). Thus, the application of selective de-
tection in the GC/MS would be preferable for the analy-
sis of the concentrations of carbohydrates using the
methanolysis method.
Although 4 - 6 isomers theoretically occur from each
monosaccharide, it is operationally difficult and labori-
ous to detect all the isomers including minor ones. Since
the generation patterns of isomers would be constant
regardless of their initial form such as anomeric or ring
size configurations of the carbohydrates before derivati-
zation [19], we measured only the highest and second
highest peaks in the present study. If such selection of
major peak was appropriate for quantitative analysis,
ratios of peak area of the second highest peak to the first
one would be similar between authentic standards and
environmental samples. Therefore, we also checked the
isomeric ion composition (ratios of second highest peak
against first one) between standard compounds used for
calibration and natural sample. In conclusion, the isomer
composition was similar between them (Table 3).
Concentrations of POC and TCHOs (sum of 8 NSs
and 2 URAs) decreased from 24,000 - 26,000 and 3200 -
4000 nM C in surface layer (1 - 10 m) to 14,000 - 21,000
and 1300 - 2000 nM C in bottom layer (15 - 25 m) (Fig-
ures 1(a) and (b)). The proportions of TCHO to POC
were 8.4% - 16%, and the value was higher in the surface
layer (1 - 10 m: 13% - 16%, 15 - 25 m: 8.4% - 13%).
These results for profiles of TCHO concentration and
contribution of TCHO to POC were generally consistent
Table 2. Error ranges of duplicate samples.
Depth(m) Ara Rib Rha Fuc Xyl Glc AcGal AcMan Gal Glc TCHO
1 11.7 30.2 20.3 17.6 11.7 4.93 5.89 4.38 4.37 8.62 0.808
5 ND ND ND ND ND ND ND ND ND ND ND
10 4.08 21.2 5.51 4.10 3.75 6.15 4.46 1.99 1.07 0.697 5.40
15 5.01 16.8 8.74 7.00 10.5 10.1 9.34 16.5 0.909 11.0 11.3
20 6.55 1.02 13.9 11.6 5.51 12.1 17.9 24.6 10.9 1.58 13.1
25 16.0 5.82 36.2 25.6 18.7 3.24 3.77 10.1 0.810 2.73 11.4
Values are coefficient of variance between duplicate analysis. ND means not determined because single data was obtained for the sample at 5 m depth.

S. Wada et al. / Advances in Bioscience and Biotechnology 4 (2013) 11-17 14
Table 3. Isomer ratios of each monosaccharide.
Standard (n = 7) Samples (n = 6)
Arabinose 60 ± 12 57 ± 2.2
Ribose 11 ± 2.1 17 ± 3.4
Rhamnose 7.6 ± 1.1 26 ± 2.4
Fucose 38 ± 2.5 50 ± 3.3
Xylose 49 ± 2.0 49 ± 1.5
Mannose 7.1 ± 1.0 7.3 ± 0.63
Galactose 34 ± 2.8 38 ± 2.6
Glucose 38 ± 1.0 39 ± 2.7
Galacturonic acid 42 ± 6.7 67 ± 9.8
Glucuronic acid 38 ± 1.2 39 ± 8.8
The values are peak area ratios of 2nd abundant isomer to 1st one for each
monosaccharide. The column of standard indicates the values of authentic
standard which is measured for calibration. Samples indicate the average
values of among the samples from all the depths (1, 5, 10, 15, 20 and 25 m).
with those in previous findings [21-25]. The carbohy-
drate concentrations decreased more sharply with depth
than bulk POC (Figures 1(a) and (b)), indicating that
carbohydrates are relatively reactive components, as
shown in other studies [1-3,26,27].
Concentrations of Gal Ac and Glc Ac in POM from
surface seawater (1 - 10 m depth) were 22 - 30 and 29 -
40 nM C, respectively, slightly higher than those in the
bottom layer (15 - 25 m; Gal Ac and Glc Ac, 16 - 26 and
14 - 25 nM C, respectively) (Figure 1(c)). Total URA
(Gal Ac and Glc Ac concentrations) were 30 - 70 nM C,
accounting for 0.19% - 0.29% and 1.5% - 2.5% of POC
and particulate TCHO, respectively. In the previous
studies, distributions of URA in POC have been investi-
gated with the colorimetric method (1.4% - 4.5%, 4.7% -
23% [28,29]), being comparable with those in the present
study. The trends in vertical profiles in the mol percent-
ages of URA species in TCHO differed between Gal Ac
and Glc Ac. Gal Ac in particulate TCHO accounted for
0.67% - 0.79% in the surface layer (1 - 10 m depth), rela-
tively lower than that in the bottom layer (0.90% - 1.3%).
On the other hand, the contribution of Glc Ac to particu-
late TCHO was 0.80% - 1.2%, with no distinctive verti-
cal trend in Suo-Nada (Figure 2(a)).
The dynamics of NS components were also different
among each component (Figures 1(d) and (e)). Glucose
fractions in the particulate TCHO decreased with depth
from 27% - 34% (1 - 10 m) to 20% - 23% (15 - 25 m)
(Figure 2(b)), while other monosaccharides components
were mostly constant or increased with depth (constant:
Rha and Gal, increase: Ara, Rib, Fuc, Xyl and Man)
(Figures 2(b) and (c)). Most of the glucose would origi-
nate from glucan, a carbohydrate reserved in phyto-
plankton [30]. Since glucan is considered as one of the
most bio-labile components of OM derived from phyto-
plankton [3,31], it is conceivable that the glucan pro-
duced in surface layers by phytoplankton is rapidly de-
composed during export to depth.
Transparent exopolymer particles (TEPs) are opera-
tionally defined as those above 0.4 μm when stained by
alcian blue, a binding dye for acidic sugars, including
URAs [10,11]. Since TEPs are likely to promote sinking
particle formation and bacterial colonization in water
columns, their biogeochemical and ecological impor-
tance in marine environments has been intensively stud-
ied [11,14,32]. In the present study, the depth profiles of
TEPs showed that their concentrations ranged from 20 to
100 μg GX equiv. l−1 (Figure 3), and decreased from the
surface with depth. The concentrations and depth profiles
are comparable to those from previous studies of coastal
0
5
10
15
20
25
30
02000 4000 6000
0
5
10
15
20
25
30
010000 20000 30000
0
5
10
15
20
25
30
0 204060
0
5
10
15
20
25
30
0100 200 300 400 500
0
5
10
15
20
25
30
05001000 1500
(a) (b)
(c) (d)
(e)
Depth (m)
Concentration ( nM)
Figure 1. Vertical profiles of concentrations of POC, total car-
bohydrates, uronic acids (Gal Ac and Glc Ac) and neutral sug-
ars. The ordinate and abscissa indicate depth (m) and concen-
trations (nM). The profiles of POC (a), total carbohydrates (b),
each uronic acid (galacturonic acid (●) and glucuronic acid (○))
(c), each neutral sugar (arabinose (■), ribose (□), rhamnose (▲),
△ fucose (), xylose (◆), mannose (◇), galactose (●), and glu-
cose (○)) (d and e) were shown. The abscissa of the fifth figure
(e) is zoomed to show the minor components.

S. Wada et al. / Advances in Bioscience and Biotechnology 4 (2013) 11-17 15
ecosystems (Monterey Bay and Santa Barbara Channel,
6 - 270 μg GX equiv. l−1; [20]).
We had hypothesized that the concentrations of TEPs
and particulate URAs have a close mutual relationship,
given that the amounts of binding dye would be propor-
tional to acidic sugar content [33]. However, the concen-
tration of TEPs showed a relatively weak relationship
with those of particulate URAs when compared with NSs
in Suo-Nada (Table 4), even though URAs are one of the
well-known acidic sugar groups. This suggests that the
contribution of URAs to TEPs is a lesser one, making it
essential to consider the contribution of other acidic
sugar components such as sulfated carbohydrates. There
have been just a few studies on the comparison between
URA and sulfated carbohydrates so far [34,35], and they
had found abundant formation of TEP, high stickness of
aggregates, and higher contribution of sulfated carbohy-
drates compared with URA. Considering these finding
together with our results, sulfated carbohydrate could be
a major acidic sugar component in TEPContents of URA
and sulphates in EPS would be variable among source
organisms. Since it has been suggested that bacterial
EPSs are relatively abundant in URA compared with
phytoplanktonic EPSs [8], URA-containing polysaccha-
rides present in bacterial EPS don’t participate as struc-
tural components of TEP.
Table 4. Relationships of each monosaccharide and TEP concentrations.
r
2
Arabinose 0.0018 Not significant
Ribose 0.619 Not significant
Rhamnose 0.740 <0.05
Fucose 0.688 <0.05
Xylose 0.733 <0.05
Mannose 0.738 <0.05
Galactose 0.720 <0.05
Glucose 0.726 <0.05
Galacturonic acid 0.239 Not significant
Glucuronic acid 0.539 Not significant
TCHO 0.728 <0.05
POC 0.323 Not significant
0
5
10
15
20
25
30
00.511.5
0
5
10
15
20
25
30
02040
0
5
10
15
20
25
30
024681
Mol percentages (%)
Depth (m)
(a)(b) (c)
0
Figure 2. Vertical profiles of proportions of each monosaccharide. The ordinate and abscissa indicate depth (m)
and mol percentages of each monosaccharide component. The profiles of each uronic acid (galacturonic acid (●)
and glucuronic acid (○)) (a) and each neutral sugar (arabinose (■), ribose (□), rhamnose (▲), fucose (△), xylose
(◆), mannose (◇), galactuose (●) and glucose (○)) (b and c). The abscissa of the third figure (c) is zoomed to
show the minor components.

S. Wada et al. / Advances in Bioscience and Biotechnology 4 (2013) 11-17 16
0
5
10
15
20
25
30
050100 150
Concentrati on(GX equiv.l
‐1
)
Depth(m)
Figure 3. Vertical profile of the concen-
tration of TEP. The ordinate and abscissa
indicate depth (m) and concentrations
(GX equiv. l−1) of TEP.
4. ACKNOWLEDGEMENTS
We wish to thank the crews for the Toyoshio-Maru research cruise
(University of Hiroshima). The study was supported by grants from the
Ministry of Education, Culture, Sports, Science and Technology, Japan
(Nos. 14340166 and 19310003) and a Sasakawa Scientific Research
Grant from the Japan Science Society. We are also grateful to the staff
of Chemical Analysis Center (University of Tsukuba) for their help
with GC/MS analysis.
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