Carbohydrate Polymers xxx (xxxx) xxx

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Characteristics and function of an extracellular polysaccharide from a green
alga Parachlorella
Mitsuki Sasaki a, Akari Takagi a, Daisaku Sasaki b, Akihiro Nakamura a, c, Munehiko Asayama a, c, *aCollege of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki, 300-0393, Japan
BioX Chemical Industries Co. Ltd., 2-20-11 Inokuchidai, Nishi-ku, Hiroshima, 733-0844, Japanc
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Fuchu, Tokyo, 183-8509, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Algae
Biorefinery
Carbohydrate
Lipid
Polysaccharide
Rhamnan

The green alga Parachlorella sp. BX1.5 is a new strain that can significantly coproduce lipids and polysaccharides,
inside and outside the cells, respectively. This study aimed to investigate the characteristics and functional
properties of the extracellular polysaccharide (EPS). Ethanol-extracted EPS from the cells grown in N-starved
BG11 medium was found to be a novel acidic rhamnan of straight-chain type, with a high molecular mass of >1.75 × 106 daltons. The water-soluble EPS exhibited high viscosity as sol, even at low concentrations, with 0.5 %EPS showing almost constant viscosity at 10–40 ◦ C. Its addition to mayonnaise resulted in improved oil-off,suggesting its possible application as a thickener or food additive. Cells with added EPS were found to exhibit
resistance to environmental stress conditions, such as heat, dryness, and decompression, and ultraviolet rays.
Based on these rheological and biological effects, its unique properties with respect to biorefinery have been
discussed here.

1. Introduction
Microalgae, considered a model microorganism, have strong photo­
synthetic capabilities and are used as a resource in biorefinery (Brennan& Owende, 2010; Caporgno & Mathys, 2018). For example, the green
alga Chlorella cell contains 45 % protein, 20 % lipid, 20 % carbohydrate,and 10 % ash, with vitamins and minerals as other minor components.
The high protein content of Chlorella reflects its promise as a foodresource. Recent reports have indicated microalgal extracts to possibly
show protective effects against antiviral infections and cancer metas­
tasis, immunostimulatory effects, as well as prevention of diabetes.
Microalgal materials have been known not only as nutrients but also as
oil, polysaccharides, photosynthetic pigments, etc.
Polysaccharides from microalgae are mainly known as extracellularpolysaccharides (EPS), their molecular structure and function summa­
rized in Table S1. Sulfated EPS, a branched structure produced from thecyanobacterium Aphanothece sacrum, is known as sacran and consists of
glucose and other sugars, having molecular weight, and is expressed as>1.6 × 107 daltons (Da) (Okajima et al., 2008). Glycosides of sacran,modified with lipids or organic acids, have been reported to possess
properties that make them applicable as food thickener or as antiviral

agents (Kaneko & Kaneko, 2008). Anti-inflammatory effects of sacran onallergic dermatitis in vivo had been reported previously (Ngatu et al.,2012). Acidic EPS, a branched structure derived from the cyanobacte­
rium Arthrospira (Spirulina) sp., is called spirulan and consists ofrhamnose and other sugars; its molecular weight is 2.6 × 104 Da. The
spirulina EPS has been shown to confer protective properties against
tumor metastasis (Hayashi et al., 1997).
The branched-structure EPS from green alga Scenedesmus sp. consistsof galactose and other sugars, with molecular weight of 4.0 × 104 Da.
This EPS contains proteins as glycoproteins, and can function against
tumor metastasis (Ando et al., 1994). The EPS from green alga Mono­stroma nitidum mainly consists of rhamnose (78 % w/w) and abundantsulfate esters, with molecular weight of 8.7 × 105 Da (Karnjanapratum &You, 2011; Lee, Koizumi, Hayashi, & Hayashi, 2010; Mao et al., 2008).
The EPS from green alga Parachlorella kessleri consists of abundantgalactose (67.1 % w/w) and other sugars like mannose (22.5 %), arab­
inose (4.7 %), rhamnose (4 %), and xylose (1.7 %), with molecularweight of 6.5 × 104 Da (Sato & Ri, 2014). This EPS can inhibit colon
carcinoma in mice (Ishiguro et al., 2017). On the other hand, the linear
EPS from non-photosynthesizing microorganism Alcaligenes sp., isknown as curdlan and consists of glucose (approximate 100 %, β-1,Abbreviations: FAMEs, fatty acyl methyl esters; FID, flame ionization detector; TEM, transmission electron microscopy.
* Corresponding author at: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki, 300-0393, Japan.
E-mail address: munehiko.asayama.777@vc.ibaraki.ac.jp (M. Asayama).
https://doi.org/10.1016/j.carbpol.2020.117252
Received 14 August 2020; Received in revised form 21 September 2020; Accepted 13 October 2020
Available online 2 November 20200144-8617/© 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Mitsuki Sasaki, Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2020.117252

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3-bond), with molecular weight > 2.0 × 105 Da (Dobashi et al., 2006).
This EPS has been utilized as food additive and hairdressing fee.
Therefore, microalgal polysaccharides, with molecular mass be­
tween 104 and 105 daltons, exhibit branched carbon chains in mostcases. EPS, for application as food additive (as thickening stabilizer/
gelling agent) or cosmetic moisturizer/emulsifying dispersant, may also
be extracted from plant seed, fruit, sap, and seaweed, representatively
referred to as guar gum, locust bean gum, pectin, Arabic gum, agar, andcarrageenan. Their characteristics of thickening and gelation/solation,
however, greatly depend on the degree of branching. Until date, reports
of polysaccharides with a linear structure are limited. We have suc­cessfully isolated a novel green alga Parachlorella sp. BX1.5 that cansignificantly coproduce lipids (food oils) and acidic polysaccharides,
inside and outside the cells, respectively (Sasaki et al., 2020). In thisstudy, the unique BX1.5 EPS has been characterized and its effective
impacts discussed in terms of algal biorefinery.

collected. To the pellet, 5 mL of 99.5 % ethanol was added once again,
vortexed, and 2 mL of the sample subsequently transferred to a 2.5-mLsyringe. Two days later, height of the bed volume of polysaccharide
deposited in the syringe, was measured with a ruler.
2.4. Preparation of BX1.5 EPS extracts
The BX1.5 strain was pre-cultivated in BG11 medium under 2 % CO2air supply in the CO2 cultivation chamber (see the section of Cultivation
conditions). Thereafter, a 5-mL aliquot of culture was collected andsuspended in 50 mL of new BG11-N medium in a 300-mL Erlenmeyer
flask. This culture was kept in the cultivation chamber, supplied with 2%
CO2-air, for 6 days. Subsequently, the cells (from 495-mL culture) were
harvested and transferred into a new tube. This sample was added tothree volumes of ethanol (1485 mL), resulting in 75 % (v/v) concen­
tration. It was vigorously mixed, allowed to stand at room temperature(25 ◦ C) for 30 min, and centrifuged at 6000×g for 10 min. The resultant
pellets were collected and mixed with 495 mL of ethanol. The ethanolbased extractions were performed four times, and finally the extracts
added to 100 mL of acetone and dehydrated overnight. The dried sam­ples were ground in a mortar.

2. Material and methods
2.1. Cultivation conditions
The Parachlorella sp. BX1.5 strain was isolated from an outdoor
culture consisting of natural water collected from a mountainous area in
Hiroshima, Japan (Sasaki et al., 2020). The BX1.5 cells werepre-cultivated in 50 mL of BG11 in 100-mL Erlenmeyer flasks for 5 days.
The flasks were placed in a cultivation chamber CF-415 (TOMY Com­pany Ltd., Tokyo, Japan), supplied with 2 % CO2, and subjected toreciprocating-shaking at 40 rpm under continuous white-light exposure
at 100 μmol photons m-2 s-1. Approximately 5 mL of the cell-culture wascollected therefrom, and the cells suspended into 30 mL or 50 mL of new
BG11 (Rippka, 1989), BG11-N (= BG110 as nitrogen-deficient condition
without NaNO3), BG11 supplied with 2.65 % (w/v) NaCl (= BG11 +NaCl), artificial sea water (= ASW) containing 3.6 % (w/v) of thepowder from a package (MARIN MERIT; Matsuda Co. Ltd., Osaka,
Japan), and BG11-P (as a phosphorous-deficient condition without
K2HPO4) media in 300-mL Erlenmeyer flasks. These flasks were placedin the same cultivation chamber for the main cultivation. After 6 days,
the cells were finally collected. Cells were also cultured under static(standing) conditions, with 0.04 % CO2-air, for 13 days, and used aftercollection, as control.

2.5. Analysis of sugar composition of BX1.5 extracts
Proteins in the BX1.5 extract were assayed by the method of Lowry
et al. (Lowry, Rosebrough, Farr, & Randall, 1951) using bovine serum
albumin as the standard. The total carbohydrate content of the BX1.5
extract was measured by phenol-sulfuric acid method (Dubois, Gilles,
Hamilton, Rebers, & Smith, 1956) using rhamnose as a reference. To
measure the neutral sugar composition of BX1.5 extracts, the groundsamples (0.05 g) were dissolved in 1 mL of 72 % (v/v) sulfuric acid, andallowed to stand for 2 h at room temperature. The resultants were added
to 8 mL of water and hydrolyzed at 105 ◦ C for 20 h to obtain neutralmonosaccharides. After the hydrolysate was neutralized by adding
barium carbonate, the aqueous phase collected was centrifuged at 10,000×g for 10 min, leading to precipitates. The supernatant was filteredthrough a USY-1 filter (Advantec Co. Ltd., Tokyo, Japan), andfreeze-dried. Neutral sugar content was measured by HPLC, using the
Shimadzu 10Avp chromatography system (Shimadzu Co., Ltd., Kyoto,
Japan), consisting of an NH2P-50 4E column (Φ4.6 mm × 250 mm). The
eluent was 80 % acetonitrile (CH3CN) with 250 mM phosphate, at a flowrate of 0.8 mL/min. Thirty microliters of the sample, which had been
dissolved in ultrapure water and filtered through a cellulose acetate
filter (0.45 μm; Advantec Co. Ltd.), was injected into the column.
Relative monosaccharide content (mol%, w/w) was calculated referring
to total monosaccharides as 100 %. Uronic acid content of the sample
was measured by Blumenkrantz method (Blumenkrantz &Asboe-Hansen, 1973) using galacturonic acid as the standard. Uronic
acid composition was measured by HPLC using the Shimadzu 10Avp
chromatography system (Shimadzu Co. Ltd., Kyoto, Japan), consistingof a Shodex SUGAR SH-1821 column (Φ7.6 mm × 300 mm). The eluent
was 5/1,000 N H2SO4 with a flow rate of 1.0 mL/min. Standards ofarabinose (Ara), fucose (Fuc), galactose (Gal), glucose (Glc), rhamnose(Rha), and xylose (Xyl) were obtained from Nacalai Tesque, Inc. (Kyoto,
Japan).

2.2. Cell staining and microscopic observation
To detect the extracellular polysaccharide, a 100-μL aliquot of cell
culture, harvested from the main culture, was suspended in 20 μL of
India ink (Daiso Co. Ltd., Tokyo, Japan), and observed under an optical
microscope (BX53; Olympus, Tokyo, Japan) with differential interfer­
ence contrast (DIC) in half the amount of light and shutter speed of 0.48
s (Sasaki et al., 2020). Based on this observation, inner (Φic) and outer(Φec) diameters of the cells were measured.
2.3. Ethanol precipitation of polysaccharides
After pre-culture, a 5-mL aliquot of culture was collected and thecells suspended into 30 mL of new BG11, BG11-N, BG11 + NaCl, or ASW
in 300-mL Erlenmeyer flasks. These flasks were put in the same culti­
vation chamber as used for pre-cultivation. After 6 days, 10 mL of cul­
ture was collected and transferred to a new tube. To the sample, 40 mLof 99.5 % (v/v) ethanol was added, mixed vigorously, and centrifuged at
6000×g for 10 min to obtain a precipitate fraction. Thereafter, 10 mL ofethanol was added to the pellets. This sample was again mixed, ultra­
sonically disrupted (UD-100; TOMY, Tokyo, Japan: set at an output of 50
for 5 min), and 40 mL of 99.5 % ethanol added. After centrifugation at
6000×g for 10 min, the pellet was collected, 5 mL of 99.5 % ethanol
added, vigorously mixed, and 40 mL of 99.5 % ethanol added again.
After another centrifugation at 6000×g for 10 min, the pellet was finally2.6. Analysis of FT-IR spectrum
The organic functional groups of BX1.5 EPS extracts, involving acidic
rhamnans, were identified by Fourier-transform infrared (FT-IR) spec­
troscopy in the range of 4,000–400 cm− 1 (Lee et al., 2010; Mao et al.,2008). Polysaccharides (1 mg) were adequately dried and ground with
spectroscopic grade KBr powder (10 mg), and pressed into a pellet for
FT-IR measurement using a FT/IR-4200 Fourier-transform infrared
spectrometer (Jasco Co. Ltd., Tokyo, Japan).
2

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Carbohydrate Polymers xxx (xxxx) xxx

2.7. Measurement of molecular weight and distribution

calculated for XG and BX1.5 EPS extract (bxEPS) (n = 3).

Analytical gel-filtration chromatography of BX1.5 acidic rhamnan
was performed in an HPLC system (880-PU; Jasco Co. Ltd.), employing a
TSK guard column PWXL (Φ6.0 mm × 40 mm; Tosoh Co. Ltd., Tokyo,
Japan) and TSKgel G6000PWXL column (Φ7.8 mm × 300 mm, possible
separation range being MW 50–7,000 × 103 Da in a case using dextrans;
Tosoh Co. Ltd.) in 50 mM sodium acetate buffer, pH 5. Aliquots (100 μL)
of 1.0 % (w/v) BX1.5 acidic rhamnan solution, which had been filteredthrough a cellulose acetate filter (0.80 μm; Advantec Co. Ltd.), wasinjected into the G6000PWXL column, and separation carried out at aflow rate of 1.0 mL/min. The BX1.5 EPS sample was detected using a
refractive index detector (Shodex RI-101; Showa Denko Kogyo Co. Ltd.,Tokyo, Japan) and a multi-angle laser light scattering detector with a
He-Ne laser (632.8 nm) as the light source (DAWN EOS; Wyatt Tech­
nology Co. Ltd., Santa Barbara, CA, USA). The absolute molecularweight and radius of gyration were calculated using a multi-angle lightscattering (MALS) detection system. Toluene was used for calibration of
the apparatus and a value of 1.406 × 10− 5 was used as the Rayleighratio. A value of 0.15 mL/g was used as the refractive index increment(dn/dc) for molecular weight calibration (Nakamura, Fujii, Tobe, Ada­chi, & Hirotsuka, 2012).

2.11. Spot assay for stress resistance
The assay was performed as per a previously described method(Sasaki et al., 2020), with slight modifications as follows. After the
pre-cultivation (see the section Cultivation conditions), a 5-mL aliquotof culture was harvested and suspended into 30 mL of fresh BG11 me­dium in a 300-mL Erlenmeyer flask. This was placed in the chamber
CF-415 for 2 days for main cultivation; EPS was not secreted much on
the cell surface. A 100-μL aliquot of cell-culture was harvested andcentrifuged at 10,000 × g for 5 min. Thereafter, the cell pellet wascollected and 0.5 % (w/v) BX1.5 EPS aqueous solution (100 μL) was
either added (+EPS) or not added (control) to the pellet. They were
subjected to heat drying stress (HD), freeze-thaw stress (FT), freeze-dry
stress (FD), and ultraviolet stress (UV), separately. For heat drying
stress, it was left overnight at 42 ◦ C. Sterilized and deionized water (100μL), as controls, were added to the cell pellet when the stress was appliedby ultraviolet irradiation. To these samples, 100 μL of fresh BG11 me­dium were added and mixed well. For freeze-thaw stress, it was natu­
rally thawed after freezing at − 80 ◦ C. This sample was added to 100 μL
of fresh BG11 medium and mixed well. For freeze-dry stress, the sample
was vacuum freeze-dried using V-850 (BUCHI) for 90 min under a 0.002
mBar setting after freezing at − 80 ◦ C. To the dried cell-pellet, 100 μL of
fresh BG11 medium was added and mixed well. For ultraviolet stress,
the sample was transferred to a 96-well plate, and irradiated by UV (260nm) light at 100 μmol photons m-2 s-1 for 5 min at a distance of 4 cm fromthe UV-light source. After each stress treatment, a 5-μL aliquot of thesample was spotted onto the BG11 agar plate. These cells were incubatedunder a 0.04 % CO2-air condition with 30 μmol photons m-2 s-1 of whitefluorescent-light at 30 ◦ C for 15 days. Cells were observed on days 5, 10,
and 15, sequentially.

2.8. Atomic force microscopy
BX1.5 EPS extracts were dispersed into ultrapure water to final
concentrations of approximately 1 mg/mL, using a magnetic stirrer atroom temperature, and then heated at 70 ◦ C for 1 h. They were filteredthrough cellulose acetate filters (0.80 μm; Advantec Co. Ltd.), and thendiluted up to 10 μg/mL with a 10 mM aqueous solution of non-ionicsurfactant Tween-20 (Wako Chemical Co. Ltd., Tokyo, Japan). A 0.5μL aliquot of the sample was immediately deposited onto mica surfaces,and dried at room temperature for 20 min. Atomic force microscope(AFM) imaging was performed in air at 20 ◦ C using an alternating cur­rent cyclic contact mode of a multi-mode imaging unit (SPI4000; Seiko
Instruments Inc., Chiba, Japan) controlled by a probe station (SPI3800
N; Seiko Instruments Inc.). Beam-shaped Si cantilevers (spring constant
20 N/m) were excited at a frequency proximate to a resonant frequencyof 136 kHz. The sample surface was scanned with the probe at a scan­ning frequency of 0.5–1 Hz (Ikeda, Funami, & Zhang, 2005).

3. Results
3.1. Condition for EPS production
The new strain Parachlorella sp. BX1.5 could grow in BG11 liquidmedium under conditions of 0.04 % CO2-air for 13 days, producing asmall amount of extracellular polysaccharide (EPS), as observed in cells
stained with India ink (Fig. 1a). The EPS production was, however,significant under air conditions of 2% CO2, even if the incubation time
was only one day (Fig. S1), thus suggesting high concentration of CO2 tobe effective for EPS overproduction (Fig. 1b). EPS production was mostnotable under conditions of 2 % CO2 and nitrogen-starved BG11
(BG110, BG11-N) medium (Fig. 1c). The overproduction of EPS could
also be observed under conditions of 2% CO2 and phosphate-starved
BG11 (BG11-P) medium (Fig. 1d). No overproduction was observed
when another strain Parachlorella sp. 11h was cultured under the same
conditions (data not shown). This suggested the ability of EPS
overproduction to be specific for the BX1.5 stock. We investigated the
BX1.5 EPS-production status based on values indicated by the ratio of
EPS diameter (Φei) to cell diameter (Φci) under various culture condi­
tions (Fig. 1 bottom, Fig. S1). Results showed the most effective value ofΦei/Φci under BG11-N condition, although other culture conditions
were also tested for EPS production (Fig. S2A). EPS extracts could berecovered using artificial sea water (ASW) while there was almost no
accumulation when BG11+NaCl medium was used (Fig. S2B). There­
fore, we used the BG11-N condition for overproduction and extraction of
EPS in the subsequent experiments.

2.9. Measurement of viscosity
The viscosity of BX1.5 EPS extracts, as 0.5 % (w/v) hydrates, wasmeasured using a corn-plate type viscometer, LV-DV2T, with a spindle
CPA-52Z (AMETEK Brookfield Inc., Middleborough, MA, USA), in thetemperature range of 10–40 ◦ C. Samples of ι- or κ-carrageenans werealso prepared by the same procedure as for standard controls.
2.10. Emulsion stability test
Mixtures were prepared with 13.9 g of water (14 g, if no stabilizer isadded), 4 g of emulsifier (gum arabic), 0.2 g of granulated sugar, 1.4 g of
10 % (w/v) acetic acid, 0.4 g of sodium chloride, and stabilizer [none,
0.12 g of xanthan gum (XG), or 0.12 g of BX1.5 EPS extract]. After
stirring overnight, 20 g of canola oil (J-oil Mills Inc., Tokyo, Japan) wereadded and homogenized using a homogenizer at 15,000 × g for 3 min toprepare egg-free mayonnaise [oil, 50 % (w/w)] (Chivero, Gohtani,
Yoshii, & Nakamura, 2016). After the egg-free mayonnaise had been
stored in the refrigerator (at 4 ◦ C) for 3 weeks, 1-mL drops were spottedon a filter paper (No.1; Advantec Co. Ltd) and heated at 210 ◦ C in anoven (MRO-TS8; Hitachi Co. Ltd., Tokyo, Japan) for 3 min. After heat­ing, area of the oil stain from the egg-free mayonnaise was measured
with a digital microscope (VHX-950F; Keyence Co. Ltd., Osaka, Japan).
The average oil-stained area of egg-less mayonnaise, without any added
stabilizer (none), was considered 100 %; the oil-stained area was also

3.2. EPS extraction
After EPS overproduction, BX1.5 cells were harvested from the
BG11-N medium (Fig. S3a), and EPS samples prepared with several
rounds of ethanol extraction (Fig. S3b, c). The resultant was added to3

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Fig. 1. Sizes of the cells producing exopolysaccharide. Diameters of the cells inside (Φic) and outside (Φec), and the ratio (Φic/Φec) were determined along withstandard deviations from the photographs shown in the top panels. The bar indicates 10 μm. Sample numbers: CO2 0.04 % BG11 (a), n = 17; CO2 2 % BG11 (b), n =28; CO2 2 % -N (c), n = 13, and CO2 2 % -P (d), n = 19, respectively.

acetone and dried for a day (Fig. S3d). The dried EPS extract was
crushed in a mortar and eventually ground (Fig. S3e). It was stored as
EPS extract at room temperature until further use. The extracts weredissolved in water to prepare 0.5 % (w/v) solutions of each (Fig. S3f),
and used for investigating their characteristics and functional proper­
ties. It is noteworthy that the EPS extract was obtained with high yield(approximately 1.6 g/L of BG11-N culture). It, however, contained small
amounts of cells that could not be completely removed despite trying
several extraction methods. This implied that cells are tightly wrappedand protected by EPS. Therefore, we selected this grade of EPS extract
for further experiments.

acidic rhamnan and a novel glycan. Since acidic EPS from the green alga
Monostroma nitidum is referred to as rhamnan sulfate (Karnjanapratum& You, 2011; Lee et al., 2010; Mao et al., 2008), we investigated whether
acidic BX1.5 EPS also possesses sulfate group. The organic functionalgroups of BX1.5 acidic rhamnan were identified by Fourier-transform
infrared (FT-IR) spectroscopy and the results are shown in Fig. 2.
When pullulan was used as a control polysaccharide polymer without
sulfate group, consisting of maltotriose units, also known as α-1, 4- (andα-1, 6-) glucan, a profile with specific peaks was obtained. Whenκ-carrageenan was used as a control polymer, with one sulfate group per
disaccharide, made up of repeating galactose units joined by alternating–O
α-1, 3- and β-1, 4-glycosidic linkages, specific peaks referring to S–
and C–O–S bonds in the sulfate group were identified. BX1.5 EPS did
not contain a sulfate group, and the glycosyl bond pattern (area marked
with a dashed circle) was found to be simpler than in pullulan. There­
fore, the results reconfirmed BX1.5 EPS as a new type of acidic glycan.

3.3. Composition of EPS extract
The EPS extract contained proteins at 9.62 wt% and total sugar at
73.8 wt%, with sugar monosaccharide composition being 49 mol%rhamnose, 17 mol% galactose, 13 mol% glucose, 13 mol% xylose, and
9.0 mol% galacturonic acid (Table 1). Arabinose, fructose, or glucuronic
acid was not detected. Since the content profile was apparently different
from what had been known until date, BX1.5 EPS was considered an

3.4. Molecular mass and structure
Molecular mass of BX1.5 EPS was determined by gel-filtration

Table 1BX1.5 EPS information.
Protein(weight %)9.62

Carbohydrate(weight %)73.8

Monosaccharide composition (mol%) *
Rha

Gal

Glc

Xyl

Ara

Fuc

Gal
A

48.5

16.7

13.2

13.1

ND

ND

8.52

*

Absolute molecularweight (kDa)

Radius of
Gyration (nm)

CPS [log (nm)/log(g/mol)] †Length(μm) ‡

1.75 × 103

194.2

0.89

0.85

Rha, rhamnose; Gal, galactose; Ara, arabinose; Fuc, fucose; Xyl, xylose; Glc, glucose; Gal A, galacturonic acid. ND, not detected.
†
CPS, conformation plot slope.
‡
An average value per a linear molecule.
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Carbohydrate Polymers xxx (xxxx) xxx

rhamnans until date, suggesting a novel aspect of BX1.5 EPS. This dis­covery inspired further characterization of BX1.5 EPS properties.
3.5. Viscosity characteristics
Polysaccharides are widely used in food and other industries as
thickening and stabilizing agents; therefore, it was considered importantto characterize the viscosity of BX1.5 EPS. We prepared BX1.5 EPS ex­
tracts at various concentrations, in water, and even 1% (w/v) sample
was found to show a sol state (Fig. 4a). ι- (loose gel) or κ- (gel) carra­
geenan was used as control material for food additive (Fig. 4b, c, Funami
et al., 2007).
The viscosity characteristics are summarized in Table S2, withrespect to other representative food additives. Although polysaccharides
having a straight-chain structure generally tend to have high viscosity,
the viscosity of aqueous BX1.5 EPS solution was found to be the lowest
among all linear polysaccharides, and no gel-formation was observed in
the 0.5–10 % aqueous solutions. Although BX1.5 acidic rhamnan did notform a gel at high concentrations (5–10 %), it did form a highly viscous
sol. On the other hand, polysaccharides having multi-branched struc­ture, with bifurcation or polytomy, have low viscosity that remained at
11.5 mPa £ s (Arabic gum) or 110 mPa £ s (soybean polysaccharides)
even in 10 % aqueous solutions. BX1.5 EPS, as straight-chain rhamnan,
had the same high viscosity of 25,000 mPa £ s in 1% aqueous solution,
thus clarifying BX1.5 acidic rhamnan to be a polysaccharide with uniqueproperties, showing high viscosity at low concentrations while notforming gels. As a preliminary experiment, rhamnan of the present
context had been seen to be soluble in water up to at least 20 % (data notshown).
Temperature dependence of viscosity of 0.5 % (w/v) aqueous solu­
tions was also investigated using a corn-plate type viscometer having aspindle CPA-52Z. The viscosity of 0.5 % aqueous solution of BX1.5 was
9–10 Pa in shear stress (Fig. 5A), thus indicating a non-Newtonian fluid
state, and a plastic-flow, like mayonnaise (Fig. 5A). Viscosity of poly­saccharides is generally temperature-dependent; iota (ι)-carrageenan
and kappa (κ)-carrageenan, commonly used as food additives, show
large changes in viscosity depending on temperature. However, viscos­
ity of BX1.5 EPS hardly changed in the temperature range of 10–40 ◦ C,
and remained stable over a wide range of temperature (Fig. 5B). Its
resistance to change in viscosity over a general temperature range makes
it an excellent candidate as food or cosmetic additive.
3.6. Property for food additives
For the purpose of stabilizing dispersion and improving fluidity ofemulsified oil droplets, mayonnaise-like food were prepared as emul­
sions with polysaccharide extracts of BX1.5 or xanthan gum, generally
used as stabilizer for food additives. When the sample with 0.3 % (w/w)
BX1.5 EPS was prepared, separation of oil (oil-off) and aggregation of oil
droplets were not observed under conditions of 50 % (w/w) oil content(Fig. 6A). Little change was observed in median particle size of oil
droplets and little increase in viscosity was seen, over time, indicating
the mayonnaise-like food with BX1.5 EPS to be stable. On the other
hand, the sample containing 0.3 % (w/w) xanthan gum showed littlechange in the median particle size of the oil droplets and little increase inviscosity over time. However, aggregation of oil droplets was observed(Fig. 6A). Based on these results, BX1.5 acidic rhamnan was concluded
to have an emulsion stabilization function superior to that of xanthan
gum.
When using mayonnaise as a seasoning for the production of food,
like pizza, it is desirable to have no oil separation upon heating, that is,
the emulsified state remains maintained. Therefore, firing resistance
reducing oil-off, for stability during baking, was investigated for themayonnaise-like samples. The mayonnaise-like food with BX1.5 EPS wasseen to maintain an emulsified state even when heated, since there waslittle oil-off (oil-exuded parts shown with arrow heads) caused by

Fig. 2. FT-IR spectrum of BX1.5 acidic rhamnan. Respective peaks referring to
the specific bonds or glycosidic linkage are shown. The BX1.5 acidic rhamnandoes not contain sulfate group, as shown by the dashed circle. As controls,
pullulan (with no sulfate group) and κ-carrageenan (with a sulfate group) wereused and also shown with the area focusing by dashed circles.

chromatography using an HPLC system (Fig. 3A). Analysis revealed ahigh molecular mass of 1.75 (up to 2.11) × 106 daltons (Da), distributed
uniformly, and a radius of gyration of 194.2 nm in water (Table 1). A
conformation plot showed a slope of 0.89 ± 0.02 log (nm) / log (g/mol),the value calculated using the radius of gyration and absolute molecularweight, thus indicating the BX1.5 EPS structure to be almost linear inwater-soluble form. The shape of EPS was further confirmed to be linear,with average chain length of one molecule estimated as 0.5–1.2 μm byatomic force microscope (AFM) analysis (Fig. 3B, X-, or Y-axis). The highmolecular weight and linear structure of BX1.5 EPS was reconfirmed bycomparing with the AFM results of HM pectin, whose structure re­sembles bifurcated glucan and has smaller molecular size than BX1.5
EPS (Nakamura, Furuta, Kato, Maeda, & Nagamatsu, 2003), as shown in
Fig. S4. There has not been any report of linear structure of acidic
5

M. Sasaki et al.

Carbohydrate Polymers xxx (xxxx) xxx

Fig. 3. Molecular weight and structure of BX1.5 EPS. (A) Analytical gel-filtration chromatography of the BX1.5 acidic rhamnan was performed using an HPLC
system. Profiles of the refractive index (blue dots) and the absolute molecular mass (orange dots) for the effluent are shown. (B) AFM images. The X-Y (upper panel)
and X-Z (lower panel with arrowheads) plots accord to a flat surface and height of the samples on a mica plane, respectively. The section is shown by a linewith arrowheads.

heating (Fig. 6B). The results suggested BX1.5 EPS extract to be easy to
mix with fats/oils, and to remain stable against heat treatment, thus
implying its feasibility of application as a food additive.

added to the BX1.5 cells that did not produce much EPS (Fig. 1a) and thelatter were subjected to various stress conditions. The treated cells (5 μL)
were spotted on a BG11 plate and growth was sequentially observed for
15 days (Fig. 7). Results confirmed that the cells could grow despite
freeze and thaw (FT), with or without EPS, hence indicating the possi­
bility of long-term cryopreservation for the BX1.5 strain. The test groupwith EPS showed better growth than the control group without EPS
under any stress of freeze (vacuum) drying (FD), heat drying (HD), or

3.7. Biological significance in stress tolerance
A spot assay was conducted to elucidate some of the biological
functions of BX1.5 EPS. In this assay, 0.5 % (w/v) BX1.5 EPS extract was
6

M. Sasaki et al.

Carbohydrate Polymers xxx (xxxx) xxx

Fig. 4. Aqueous solution of EPS. Samples were dissolved in water as 1 % (w/v)
solution and treated at 80 ◦ C for 60 min; 2.5 mL of the resultant was left in a
glass tube at 25 ◦ C. (a) BX1.5 EPS (bxEPS) as a sol, (b) ι-carrageenan as loose
gel, and (c) κ-carrageenan as a gel.

Fig. 6. Emulsion stability test using BX1.5 EPS. (A) Egg-free mayonnaise wasadded to BX1.5 EPS [bxEPS, 0.3 % (w/w)] as a stabilizer. Samples withoutbxEPS (none) or with xanthan gum (XG) were used as controls. A photograph ofthe sample after standing for 6 days is shown. (B) Egg-free mayonnaise [1 mL
from panel A, oil content as 50 % (w/w)] was spotted on a filter paper, withoutor with a baking treatment in an oven at 210 ◦ C for 3 min (upper panel). Area ofthe oil-off from egg-free mayonnaise on the filter (arrow heads) was measured
with a digital microscope. The values (n = 3) were evaluated and shown withrespect to the control using “no added stabilizer” as the condition for 100 %(lower panel).

ultraviolet (UV) treatment. Therefore, BX1.5 EPS was found to impart
improved resistance (environmental stress tolerance) to living cells.
Such enhancement of environmental stress tolerance suggested theusefulness of BX1.5 EPS as a cosmetic additive, culture additive, or apharmaceutical additive.
4. Discussion
The green alga Parachlorella sp. BX1.5 was found to coproduce lipids
and polysaccharides abundantly (Figs. 1 and S1). The lipids produced by
BX1.5 were significantly enhanced under nitrogen-starved culture con­ditions (BG110, BG11-N). Those produced in a BX1.5 cell wereapproximately 30–70 % (w/w), and were composed of palmitic acid
(C16:0), oleic acid (C18:1), linoleic acid (C18:2, ω6 oil), and α–linolenicacid (C18:3, ω3 oil) as useful food oil (Sasaki et al., 2020). Dual pro­duction of useful EPS and food oil inside the cells recommend the use ofthis alga for economic production. Since the BX1.5 cells are coated withacidic EPS, they may be cultured in extensive alkaline conditions (pH
10–11) (Sasaki et al., 2020), for which carbon dioxide is effectively
dissolved in liquid culture. It is appropriate for large-scale outdoor

Fig. 5. Viscosity of aqueous BX1.5 EPS solution. A 0.5 % (w/v) bxEPS solution
was prepared and subjected to analysis using a corn-plate type viscometer attemperatures 10–40 ◦ C. Samples of ι-carrageenan or κ-carrageenan were used ascontrols. (A) Effect of shear rate at 20 ◦ C; (B) Effect of temperature at a shear
rate of 1/sec.
7

M. Sasaki et al.

Carbohydrate Polymers xxx (xxxx) xxx

similar to that of xanthan gum. On the other hand, protein in the EPS
extract may work as an emulsifier, similar to soy polysaccharides(Chivero, Gohtani, Yoshii, & Nakamura, 2014). EPS is superior to xan­
than gum in terms of its emulsification activity and emulsion stability(Chivero, Gohtani, Yoshii, & Nakamura, 2015).
The results shown in Table S2 and Figs. 4 and 5 suggest the viscosity
characteristics of BX1.5 EPS to be appropriate for it to be a food additive
and a thickener. Moreover, the results shown in Fig. 6 implied appre­
ciable stabilization of the emulsion even when exposed to high tem­
peratures (210 ◦ C), thus reducing oil-off. This ability could not becomplemented by xanthan gum under the same conditions, indicating
BX1.5 EPS as appropriate for emulsion stabilization, as well as for water
retention as a food additive. Further analyses of properties of BX1.5 EPS
in terms of the shape and composition might explain how the EPS extract
influences its stabilizing capacity. Results shown in Fig. 7 demonstrated
the performance of BX1.5 EPS as environmental stress tolerance
enhancer against freeze (vacuum) drying, heat drying, and ultravioletrays treatment. Therefore, the EPS could be suitable as a culture addi­
tive and/or cosmetic materials.
Taking the above findings into account, BX1.5 EPS might provide
new insights for polysaccharides and be potent to contribute to algalbiorefinery, not only for food and feed but also for cosmetic and medical
considerations. Structural analysis of the sugar-chain-binding mode andfurther functional analysis of BX1.5 EPS are awaited in future.

Fig. 7. Spot assay for stress tolerance by BX1.5 EPS. BX1.5 cells were preparedand either mixed with 0.5 % (w/v) BX1.5 EPS solution (+EPS) or not (none).
The samples were exposed to stress conditions. An aliquot of 5 μL from thesamples was spotted onto a BG11 plate, incubated for 5, 10, and 15 days toobserve stress resistance. FD, freeze dry; FT, freeze-thaw; HD, heat dry; UV,
ultraviolet-rays irradiation.

CRediT authorship contribution statement
Mitsuki Sasaki: Data curation, Formal analysis, Investigation. Akari
Takagi: Data curation, Formal analysis, Investigation. Daisaku Sasaki:
Data curation, Formal analysis, Investigation. Akihiro Nakamura: Data
curation, Formal analysis, Investigation. Munehiko Asayama: Data
curation, Formal analysis, Investigation.

cultivation without contamination. In fact, we succeeded in cultivating
BX1.5 cells in 10-ton scale in the outdoors. Since BG11 is a medium richin sodium nitrate, we found nitrogen-deficient condition to be one of thebest for EPS-overproduction (Sasaki et al., 2020), promising low cost ofcultivation.
The molecular weight of BX1.5 EPS was determined to be 1.75 (up to2.11) £ 106 Da, which makes it the second largest natural poly­saccharide known until date. The ultra-high molecular weight of BX1.5
EPS was found to be the highest among the natural glucans known until
date, except for sacran (>1.6 £ 107 Da) extracted from the cyanobac­
terium Aphanothece sacrum (Kaneko & Kaneko, 2008). Interestingly, theends of each BX1.5 EPS molecule may be considered to be wound into asphere and have a specific thickness (Fig. 3B, Z-axis). Although Para­
chlorella kessleri can also produce an EPS with branched-type molecular
structure, its molecular weight (6.5 £ 104 Da) and sugar composition(Table S1) (Ishiguro et al., 2017; Sato & Ri, 2014) were totally distinct
from those of BX1.5 EPS (Table 1). Notably, the BX1.5 EPS has a linear
structure (0.5–1.2 μm, Fig. 3B), is water soluble, and shows quite aunique property of solation with high viscosity (7,500 to 24,000 mPa £s) even at low concentrations (0.5–1 %) in water (Table S2, Fig. 4).
Viscosity of the BX1.5 EPS remained constant from 10 to 40 ◦ C (Fig. 5B).
This is the first case of linear-chain EPS reported from microalgae.
Another linear-type EPS, called curdlan, from the non-photosynthetic
microorganism Alcaligenes sp., is well known, containing glucose(approximate 100 %, β1→3 bond), having a molecular weight > 2.0 £105 Da (which is much less than that of BX1.5 EPS), and showing “gel”
formation (Table S2, Dobashi et al., 2006). BX1.5 EPS was evaluated as
an acidic rhamnan containing galacturonic acids (uronic acids, Table 1).
Rhamnan sulfate, an acidic EPS from green alga Monostroma nitidum, ismainly composed of rhamnose [78 wt% (w/w)], and is reported to havea molecular weight of 8.7 × 105 Da (Karnjanapratum & You, 2011; Leeet al., 2010; Mao et al., 2008). Although curdlan and rhamnan sulfate
are soluble in water, resulting in gelation, their properties are appar­
ently different from those of BX1.5 EPS, thus suggesting the novelty of
BX1.5 EPS. Of note, since BX1.5 EPS extract contains proteins (Table 1),
this may influence the unique property of solation with constant highviscosity. EPS suppresses the coalescence of oil droplets by adding vis­
cosity to the aqueous phase of emulsion. This function is considered

Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This work was partially supported by a propulsive project from
Ibaraki University (to MA), Ministry of Agriculture, Forestry and Fish­eries, Japan (to DS and MA), Japan Society for Bioscience, Biotech­
nology, and Agrochemistry (to MA and DS: Academic-Industrial R&D
Support for Small-to-medium-sized Enterprises, SME-3). This work wasalso supported by Japan Science and Technology Agency (to MA and DS:
OPERA, JPMJOP1832).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.117252.
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9

Table S1. Polysaccharides.
PS name(bacteria)
BX1.5 extract
Parachlorella sp. BX1.5(Green alga)

Kessleri extract
Parachlorella kessleri(Green alga)

Rhamnan sulfate
Monostroma nitidum(Green alga)

Glycoprotein
Scenedesmus sp.
(Green alga)

SacranAphanothece sacrum(Cyanobacterium)

Spirulan
Arthrospira platensis(Cyanobacterium)κ-Carrageenan
Kappaphycus cottonii(Red alga)

CurdlanAlcaligenes(Non photosynthesizing bacterium)

Sugar composition *（weight % or mol %）Molecular weight(Da)

Rha : Gal : Glc : Xyl : Gal A= 49 : 17 : 13 : 13 : 9
Uronic acid = 91.75 (〜2.11)
x 106

Gal : Man : Ara : Rha : Xyl= 67.1 : 22.5 : 4.7 : 4 : 1.7(Gal = 50〜79)

5〜8 x 104

Ishiguro et al., 2017
Sato & Ri, 2014

8.7 x 105

Karnjanapratum &You, 2011
Lee et al., 2010
Mao et al., 2008

4.0 x 105

Ando et al., 1994

2.0 x 106〜1.6 x 107

Kaneko & Kaneko,
2008

Rha : Rib : Man : Fru : Gal :Xyl : Glc : Glc A : Gal A= 60 : 3 : 1 : 46 : 3 :0.8 : 6 : 10 : 5

2.6 x 105

Hayashi et al., 1997

Gal
= 100

7.1 x 105

Funami et al., 2007

Glc
= 100

>2.0 x 105

Dobashi et al., 2006

Rha : Gal : Glc : Xyl= 79 : 5.3 : 10 : 5.2
Uronic acid = 7.62
Sulfate ester = 28.2
Gal : Glc : Man : Rha :Fuc: Glcs : Xly= 29 : 18 : 16 : 16:
14 : 4 : 3
Ara : Fuc : Rha : Xly : Man :Gal : Gal A : Glc A : Glc : Gals
= 1 : 4 : 15 : 17 : 10.5 :12 : 5 : 5 : 29 : 2

References

This study
Sasaki et al., 2020

* Rha, rhamnose; Gal, galactose; Glc, glucose; Xyl, xylose; Man, mannose; Fuc, fucose; Ara,arabinose; Gal A, galacturonic acid; Glc A, glucuronic acid; Gals, galactosamine; Glcs, glucosamine;Fru, fructose.

Table S2. Polysaccharide property.
PS name

Structure

Viscosity (mPa・sec) *Shear rate
(1/sec)

0.1%

0.2%

0.5%

1%

Gelation†

5%

10%

BX1.5 extract

Linear0.1

151,800 7,500 25,000

UM

UM

－

CurdlanLinear0.1

320

38,400

UM

UM

UM

UM

＋

κ-Carrageenan

Linear

0.1

20

4,600

UM

UM

UM

UM

＋

Guar gum

Bifurcation

0.1

120

10,500

UM

UM

UM

UM

－

PectinBifurcation

0.1

8

80

750

6,500

UM

UM

＋

Xanthan gum

Bifurcation

0.4

140

744

2,325

5,907

UM

UM

－

Soybean PS

Polytomy

100

1.2

2.5

5.4

12

64.5

110

－

Pullulan

Polytomy

100

0.7

0.9

1.1

1.9

12.8

92.5

－

Arabian gum

Polytomy

100

1.1

1.9

2.8

5

9.5

11.5

－

* water soluble of polysaccharides [% (w/v)]; UM, unmeasurable cases under high viscosity. † +,gelation ; -, non gelation.

BG11

-N
12

12

1.66

10
8

1st day

8

5.63

6

4

4

2

2
0

0
4

6

8

2

10

4

6

8

8
6

8

8

4

4

2

2

2

6

8

10

12

2

4

6

8

1.84

8

3.22

8

8

4

4

2

2

2

0

0

4

6

8

10

12

2

4

6

8

10
8
6

8

8

4

4

2

2

2

6

8

10

8

10

6

8

10

0

0
4

6

6.53

6

4

2

4

1.78

10

4.25

6

0

10

12

3.24

10

5.08

2

10

12

2.16

8

6.00

6

4

2

6

2.21

10

4.58

6

0

4

12

10

4.96

6

2

10

12

10

10

0

0
4

8

6.16

6

4

2

6

1.80

10

4.76

6

0

4

12

2.81

10

5.29

2

10

12

1.96

10

5.56

6

2

12

6th day

8

5.54

6

2.88

10

4

2

3rd day

12

2.16

10

0

2nd day

-P

2

4

6

8

10

2

4

Figure S1. Sizes of the cells producing exopolysaccharide. After pre-culture, each culture wascollected on the 1st, 2nd, 3rd, and 6th days of the main culture, and diameters inside (Φic) andoutside (Φec) of the cells were measured. X-axis of the graph indicates the diameter inside (Φic,µm), and Y-axis indicates the ratio (Φic/Φec). The blue dots show values measured from 20
randomly chosen cells, and the red square shows the average value corresponding to Φic (redframe) and Φic/Φec (blue frame). This experiment was performed as independent session, as
shown in Fig. 1.

A

BG11
-N

BG11

B

BG11
+NaCl

ASW

BG11

BG11
-N

BG11
+NaCl

ASW

29%

62%

0.65%

51%

100%

Figure S2.

BX1.5 cells and ethanol precipitation. (A) Microscopic observation of the
BX1.5 cells, stained without (upper) or with (lower) India ink, under different culture
conditions. (B) Ethanol precipitation shown with relative bed volume (%, v/v); white-colorpellet contains EPS.

a

c

e

Figure S3.

b

d

f

Preparation of BX1.5 EPS extracts. A 55-mL cell culture of BX1.5, grown in
the BG11-N medium in the incubation chamber (supplied with 2% CO2 gas) for 6 days (a),was harvested and transferred into a new tube. The sample was added to ethanol at 70%(v/v) concentration, vigorously mixed, allowed to stand at room temperature for 30 min(b), and centrifuged at 6,000 × g for 10 min. After this, the pellet fraction was collected
and ethanol (50 mL) was added. This sample was again mixed, and centrifuged; theethanol extractions were performed four times. Thereafter, 10 mL of acetone was added
to the pellets (c) and dehydrated overnight (d). The dried samples were ground in amortar (e). An aliquot of the sample from (e) was dissolved in water at 0.5% (w/v),
treated at 95 ˚C for 10 min, and cooled back to room temperature (f). See more details in
the text.

1
0.8
0.6
[µm]
0.4
0.2
0
0
0.00

Figure S4.

0.2

0.4

0.6
[µm]

0.8

1

[nm]

2.96

AFM image of HM pectin. The bifurcation glucan, of average molecularmass 3.8 x 105 Da, is composed of α-1,4-bonds of polygalacturonic acid. A color gradationof the bar indicates height of the polysaccharide on the mica.