Enhancing Dispersibility of Paramylon Nanofiber Suspensions
through Water-Jet Defibrillation Treatment

Takefumi NARITA1,*, Juna KOIZUMI2, Kasumi WADA2, Ryo NAKADA2,
Ikuhiro TANIDA2, Satoshi OSAWA2

Abstract
In this study, paramylon nanofiber suspensions (PNFSs) with different dispersibility were developed byadjusting the pretreatment conditions, including pretreatment time and concentration of paramylon
granules. The rheological, tribological, and chemical properties of the resulting PNFSs were evaluated.
Two types of paramylon nanofiber (PNF) were prepared through water-jet (WJ) defibrillation: PNF-MP1,
which underwent a short pretreatment time and low concentration and PNF-MP2, which underwent along pretreatment time and high concentration. The defibrillation treatment partially converted PNF-MP1
to fibers, with some granules remaining. In contrast, PNF-MP2 obtained excellent nanofiber formation
owing to the accelerated defibrillation effect. The rheological evaluation revealed that PNF-MP2
exhibited the highest dispersibility and the reversibility of viscosity recovery. Friction property evaluation
showed that PNF-MP2 displayed excellent lubrication effects. Wide angle X-ray diffraction (WAXD)
analysis showed that the defibrillation treatment impaired the microcrystallization and crystal regularity
of PNF. X-ray photoelectron spectroscopy (XPS) surface elemental analysis showed that the defibrillation
treatment exposed hydroxyl groups on the PNF surface, enhancing its hydrophilicity.

Keywords：Paramylon, Nanofiber, Defibrillation, Water-jet, Dispersibility
Received July 11, 2023; accepted July 29, 2023

1)

Industrial Process Technology Group, Tokyo Metropolitan Industrial Technology Research Institute, 2-4-10,
Aomi, Koto-ku, Tokyo 135-0064, Japan2) Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1, Yatsukaho, Hakusan, Ishikawa,
921-8501, Japan*Corresponding author: Takefumi Narita, E-mail: narita.takefumi@iri-tokyo.jp

31

1. Introduction
The effective utilization of biomass is becoming

nanofiber fabrication.

increasingly important toward realizing carbon

a technique for preparing nanofibers using only

neutrality1). Biomass, including cellulose, chitosan,

water and raw materials through WJ treatment. Thisand chitin, has excellent environmental andapproach showcased excellent functionalities of

biocompatible properties and is being explored fornanofibers, such as larger specific surface area andvarious applications in a wide range of fields,

higher biocompatibility compared to cast films

2)

3)

Against this background, we previously reported

4)

including medicine , food , and paints . In recent

obtained with dilute acid when nanofibers are

years, nanofibers derived from biomass resources

converted into a film14). However, paramylon

have emerged as a novel avenue to develop new

granules exhibit oil and water absorption and have5)

applications .

affinity for both hydrophilic andCellulose, the most abundant biomass on earth, ismaterials15).

lipophilic

Therefore, focusing on the unique

derived from plants and wood and can be processed

structure of paramylon and enhancing the water

into nanofibers through physical and chemical

dispersibility of paramylon nanofibers through thistreatments and mechanical crushing and shearing.

fabrication technology using WJ is expected to be

These nanofibers are used as reinforcing fibers6) for

developed as additives in water-based paints andpolymers

and

other

materials7).

Chitinandotherdeacetylated chitosan extracted from crustaceans,applications,

reducing

volatileorganic

compounds.

such as crabs and shrimps, are also being developed

In this study, we attempted to develop highly

as food additives by improving their water

dispersible PNFSs by modifying the pretreatment

dispersibility through nanofiber formation8).

conditions of paramylon granules. The rheological,

Paramylon, a β-1, 3-glucan produced by thetribological, and chemical properties of PNFSs withmicroalga Euglena, shares a similar structure withdifferent dispersibility were evaluated, and detailed

cellulose9).

findings were reported.

The crystal structure of paramylon

comprises elliptical granules of β-1, 3-glucan with astructure, and the granules are porous10). Paramylon

2. Material and Methods
2.1. Materials. Paramylon powder from Euglena

possesses the advantage of consistent properties andgracilis (E.gracilis) (IRIAL Corporation), sodium

shapesEuglena,dodecyl sulfate (SDS) (FUJIFILM Wako Purefacilitating the production of raw materials with

Chemical Corporation), n-hexane (FUJIFILM Wako

diameter of 1–4 µm, forming a triple-helical crystal

identical

acrossdifferent

properties11).

strains

Previousofstudies

Pure Chemical Corporation) were purchased.

havereported utilizing paramylon for plastic moldings12)

2.2 Isolation of paramylon. In a 50 mL Falcon

and nanofibers13). However, the conversion oftube, 3 g of dry powder of Paramylon wasparamylon into nanofibers has problems in thedispersed in 20 mL of reverse osmosis (RO) water.

manufacturing process, such as the use of strong

To crush the cell membranes of Paramylon, thealkaline solutions containing considerable amounts

Paramylon powder were sonicated in ice-coldof sodium hydroxide, as well as the inevitablewater for 30 min using an ultrasonic homogenizer

inclusion of sodium chloride and other substances(LUH150; Yamato Scientific Co., Ltd.), followed

during neutralization. Therefore, it is desirable toby centrifugation at 6,000 rpm for 5 min using aminimize the use of organic solvents during

centrifuge
32(HimacCF

15R;

Hitachi-Koki

Corporation) to remove the supernatant fluid. The

solution was centrifuged at 6,000 rpm for 5 min to

Falcon tube was then filled with 20 mL of RO

remove the supernatant. This procedure waswater and 10 mL of n-hexane, sonicated for 5 min,performed until the foam in this solution wasand centrifuged again at 6,000 rpm for 5 min. Thiseliminated. The remaining precipitate was heat-purification process was repeated six times todried, and the resulting granules were powdered inincrease the purity of the precipitate. To remove

an agate mortar, which was used in this study as

the oil component, 20 mL of 80 vol% acetone

paramylon granules.

solution was dispersed in this Falcon tube,

2.3 Preparation of PNFSs. The paramylon

sonicated for 5 min, centrifuged again at 6,000

granules obtained in section 2.2 were pretreated

rpm for 5 min, and the supernatant liquid was

before defibrillation. Two different manufacturing

removed. This process was also repeated six times.

processes (MP) were employed as pretreatments for

The precipitate was dispersed in 20 mL of RO

fibers to achieve nanofiber refinement, as shown inwater and filtered through a filter (53 µm aperture).

Fig 1. Nanofiberization was performed using WJ

The filtered precipitate was heat-dried at 80 °C,

treatment on paramylon dispersion with different

and the resulting granules were powdered using an

defibrillation pretreatment methods. PNFSs wereagate mortar. To remove the supernatant liquid,obtained using a tabletop wet atomizer (HJP-

the powder was added to 20 mL of RO water and25001SE; Sugino Machine Co., Ltd.), pressurized atsonicated in ice-cold water for 30 min using an

200 ± 20 MPa, and ejected through a fine nozzle. The

ultrasonicbydispersion impacted ceramic balls multiple times tocentrifugation at 6,000 rpm for 5 min using a

promote defibrillation. Specifically, the PNFSs were

centrifuge. To remove other proteins, 20 mL of 1

obtained by subjecting the dispersion to five impacts

wt% SDS solution was added and washed in awith ceramic balls. The PNFSs obtained using MP1

water bath at 90 °C for 10 min. The solution was

are denoted as PNF-MP1, while those obtained using

centrifuged at 6,000 rpm for 5 min to remove theMP2 are referred to as PNF-MP2. For subsequent

supernatant, and this procedure was repeated threemeasurements, PNFSs adjusted to 5 wt% were used

times. Then, 20 mL of RO water was added to the(PNF-MP1 was adjusted to 5 wt% by collecting the

precipitate to remove any remaining SDS. The

supernatant solution.).

homogenizer,

followed

MP1
Paramylon
0.1 wt%aqueoussolution

Ultrasonichomogenizerin ice-coldwater for
30min

Stirredat 25℃for 1 day

Ultrasonichomogenizerin ice-coldwater for 30
min

Stirredat 25℃for 3 days

Ultrasonichomogenizerin ice-coldwater for 30
min

MP2
Paramylon
5 wt%
aqueoussolution

Ultrasonichomogenizerin ice-coldwater for 30
min

Fig 1. MP of PNFSs by Water-Jet defibrillation treatment.

33

Stirredat 25℃for 4 days

WJ treatment
Jet pressure
200 MPa,
5 times2.4 Morphological analysis through scanningand a scan speed of 2°/min using a divergence slit ofelectron microscope. The samples were observed

1°, scattering slit of 1°, and receiving slit of 0.15 mm.

using a scanning electron microscope (SEM, SEM

2.8 Surface analysis through XPS. X-ray

EDX MP N; High-Technologies Corporation).

Photoelectron Spectroscopy (XPS) analysis was

Before observation, the samples were fixed to a

performed with an X-ray photoelectron spectrometer

specimen stand using carbon tape, and then

(ESCA-3400;

gold/palladium alloy was deposited using an ion

measurement conditions involved a vacuum of <sputtering

10−4 Pa, a current of 10 mA, and irradiation withapparatus(E-1010;

HitachiHigh-

Technologies Corporation).

Shimadzu

Corporation).

The

MgKα rays generated at 10 kV.

2.5 Rheological properties of PNFSs. The
3. Results and Discussion

rheological properties of the PNFSs were measured
using a rheometer (MCR302; Anton Paar). The

3.1 Observation of shape of PNFSs. Fig. 2

sensor geometry was selected according to theshows the results of defibrillation of paramylon

viscosity of the sample, and a cone-plate sensor

granules. PNF-MP1 showed granule shape before(diameter = 35 or 50 mm, angle = 1° or 2°) was used.

defibrillation

The measurement temperature was 25 °C. For

remained after WJ treatment (Fig.2 (B)). PNF-MP2

steady-state flow viscosity measurements, the shear

showed expansion of granules by increasing thestress was determined at a shear rate of 0.01 to 1,000

treatment time before defibrillation (Fig.2 (C)). Afters−1. The viscosity recovery was examined for the

defibrillation with WJ treatment, the enlarged

structural recovery measurement when the shear rate

granules were observed to reduce in size, which

was varied in the order of 0.1, 500, and 0.1 s−1.

suggests the possibility of fiber formation (Fig.2(D)).

(Fig.2(A)),

but

many

granules

2.6 Friction properties of PNFSs. The

To confirm the fiber morphology, morphologicalfrictional properties of the PNFSs were measured

observations after dilution of WJ-treated PNF-MP2

using the ball-on-3-pin tribocell setup supplied with

(Fig.2 (D)) to 0.1 wt% with RO water are shown in

the rheometer. The ball was made of SUJ2 with a

Fig. 3. As a result, the fiber morphology ofdiameter of 12.7 mm and a surface roughness of Ra

paramylon was confirmed in Fig. 3(A). In Fig.3(B),

0.1 mm. The pin was a SUJ2 cylinder with afiber diameters of several hundred nm were observed

diameter of 6.0 mm and a thickness of 6.0 mm, andunder high magnification (×11k), indicating thatits surface roughness was Ra 0.1 mm, measured
perpendicular

to

the

sliding

direction.

Themeasurement temperature was 25 °C. The sliding
velocity of friction was measured at a load of 10 N
10 µm

10 µm

(A) PNF MP1-Untreated(B) PNF MP1-WJ treated

a

10 µm

10 µm

monochromatic CuKα line generated at 30 kV and(C) PNF MP2-Untreated(D) PNF MP2-WJ treated(Pmean = 0.85 GPa) and a sliding velocity of 0.000001
to 0.1 m/s.

2.7 Crystal structure through WAXD. WAXD
analysis was performed on an X-ray diffractometer(UltimaIV;

RigakuCorporation)

with

20 mA. The measurement conditions included a

Fig 2. SEM photographs of PNFSs.

scanning range of 2θ = 4°–40°, a 2θ step of 0.02°,34

1000050 µm

Viscosity / Pa・s

1000

3 µm

(B) PNF-MP2(A) PNF-MP2

Fig 3. SEM photographs of diluting WJ-treated
PNF-MP2 to 0.1 wt% with RO water.

100

10
10
1

1

0.1

PNF-MP2 is a nanofiber. It is suggested that the

0.01

longer pretreatment time allowed for higher

0.001

0.1
0.01
0 50 100150200250300350400450500

Time / spenetration into the granule interior, and the higher

Fig 5. 3-step shear rate curve(0.1 s-1 →500 s-1 → 0.1 s-1) of PNFSs.

concentration allowed for a larger surface area of
PNF,

100

Share rate / s-1

Paramylon granules 10000
PNF-MP1
PNF-MP2
Share rate
1000

thereby promoting the effect of thedefibrillation treatment to the interior of the granules.

On the other hand, PNF-MP2 showed a monotonous

3.2 Rheological properties of PNFSs. Steadyincrease in shear stress, and no shoulder peak offlow viscosity measurements of PNFSs are shown inshear stress was observed. This means that there

Fig. 4. Paramylon granules showed a significant

were no granule-like solids due to the most advanced

decrease in shear stress at a shear rate of around 10

defibrillation process with the highest pretreatment

s-1, followed by an increase in shear stress again, buteffect. Therefore, PNF-MP2 was found to have theat the lowest value. PNF-MP1 showed shoulder

highest dispersibility.

peaks of shear stress similar to that of paramylon

The thixotropy (viscosity recovery) of the samples

granules at shear rates of 5 s-1 and around 100 s-1,

was investigated when the shear rate was changed

followed by an increase in shear stress again. This

stepwise with respect to the measurement time. Fig.5

indicates that the granules and agglomerates ofshows the change over time in viscosity when thenanofibers are broken down as the shear ratemeasurement was repeated three times, with one

increases. Therefore, the low effect of thecycle consisting of a measurement at a low shear rate

defibrillation process means that more granule-like(0.1 s-1) followed by a measurement at a high shear

solids and agglomerated components were included.

rate (500 s-1) and then again at a low shear rate (0.1s-1). The results show that the viscosity of paramylon

100

granules decreased significantly at high shear, andShare stress / Pa

there was almost no viscosity recovery when the low
shear rate was returned again. In the second cycle,

10

the measurement was interrupted because it fell
below the lower torque limit value. A subsequent
1

0.1
0.01

Paramylon granules
PNF-MP1
PNF-MP2

comparison of PNF-MP1 and MP2 showed that both
samples exhibited structural breakdown after one
cycle of high shear rate, but viscosity was higher for

0.1

1

10

100

PNF-MP2. The same degree of viscosity recovery

1000

Share rate / s-1

was also observed after the second cycle, indicating

Fig 4. Shear stress curves as a function of shear rate
for PNFSs.

that the collapse and recovery of the inter-fiber35

structure continued after structural failure. Thisboundary lubrication zone differ greatly due tosuggests that the high dispersion of nanofibers due todifferent dispersibility. In general, it is known that in

the pretreatment effect of PNF-MP2 resulted in apolymer friction modifiers (PFMs), the PFMs arehigher entanglement effect between nanofibers andadsorbed on the oxide film surface of the contactor,

stabilized structural recovery.

forming an adsorbed molecular film that has a high

3.3 Friction Characteristics of PNFSs. In thefriction reducing effect16). This suggests that theprevious section 3.2, rheological evaluation showedfriction reduction effect is enhanced by thethat PNF-MP2 has high dispersibility in aqueous

adsorption on the contactor surface in aqueous

solution due to the elimination of granules by WJ

environments due to changes in the crystal structure

treatment. PNF-MP1 exhibits lower uniformity dueof paramylon and the chemical properties on theto the mixture of granules and partial fibers in the

PNF surface caused by the PNF-MP2 defibrillation

aqueous solution, while PNF-MP2 indicates thattreatment effect.

nanofibers are uniformly dispersed in the aqueous

3.4 Crystal Structure by WAXD analysis.

solution. Therefore, NF aqueous solutions with

WAXD analysis was performed to clarify change in

different

different

crystal structure due to the defibrillation treatment.

frictional properties. To investigate the frictional

Fig. 7 shows the WAXD of PNF-MP2 with theproperties of nanofibers with different dispersibility

highest dispersion and paramylon granule. The

in an aqueous solution, the coefficient of friction was

major reflections 2θ = 7.0 °, 16.7 °, 19.4 °, 20.7 ° andevaluated. Fig. 6 shows the results of the coefficient24.1 ° are due to (100), (101), (111), (201) and (121)of friction for PNFSs on the SUJ2 contactor.

planes, respectively17). The diffractions become

Paramylon granules showed the highest coefficient

broadened by the defibrillation treatment. Assumingof friction and no lubrication effect. Comparing

the mosaic model, we estimated the crystallite size

PNF-MP1 and MP2, PNF-MP2 showed the lowest(D) normal to the (100) plane using Scherrer's

friction coefficient and the highest lubrication effect.

equation (1).

dispersibility

may

exhibit

In particular, the coefficient of friction issignificantly reduced in the boundary lubrication

D=(Kλ)/(βcosθ) ･･････ (1)zone with a sliding velocity of 10-4 m/s or less. These
results indicate that the frictional properties in thewhere K=shape factor (0.9), λ=wavelength of X-rays
targeted to Cu (CuKα; 1.54Å), β=width at half

0.35

Paramylon granules
PNF-MP1
PNF-MP2

111
201

0.25

100

Intensity / a.u.

Friction coefficient / -

0.30

0.20
0.15

0.10

101

121

Paramylon
granules

0.05

PNF-MP2

0.00
0.000001 0.00001

0.0001

0.001

0.01

0.1

0

Sliding velocity/ m/s

5

10

15

20
25
2θ / deg

30

35

40

Fig 7. WAXD patterns of Paramylon granules and
PNF-MP2.

Fig 6. Frictional properties of PNFSs.

36

maximum, and θ=Bragg angle.

interior of the granules, which promotes nanofiber

The D100 for paramylon granules and PNF-MP2

Table 1. Area ratio of binding mode of C1s
spectrum

were approximately 176 Å and 68 Å, respectively.
This indicates that the crystallite size in the fibers

Binding
mode
C-C
C-O-H
C-O-C

radial direction decreased due to the disassembly offibers perpendicular to the C-axis (fiber length
direction). The WAXD profile of PNF-MP2 issignificantly broader compared to that of paramylon
granules, suggesting that the crystalline regularity

Paramylon
granules
44.1
38.5
17.4

PNF-MP2

46.5
42.3
11.2

was also lost as the defibrillation process progressed,
formation during the WJ defibrillation process andleading to amorphization.

further exposes the hydroxy groups on the PNF

3.5 Surface Analysis by XPS. In the previous
section 3.4, it was found that the crystalline

surfaceregularity

themicrocrystallization and crystal regularity are

defibrillation treatment. In order to clarify the

impaired, thereby improving the hydrophilicity ofdifference in the surface properties of PNF due to thethe material. Therefore, it is thought that the material

defibrillation treatment, Fig. 8 shows a narrow scan

became highly dispersible without solid componentsof the C1s of paramylon granules, PNF-MP2. The

such as granules, resulting in higher water

peak area ratios of the binding modes of the C 1s

dispersibility. The above results indicate that the

spectra from the results of Fig. 8 are shown in Table

rheological properties of the PNF material indicate

1. The results show that the peak area ratio of thethat the water dispersibility is increased, and thehydroxy group (OH) of PNF increased compared tolubricating effect is also increased due to the higherthat of the paramylon granules. This suggests that thepolarity.

is

lost

and

amorphized

by

to

the

material

surfacewhen

pretreatment effect of MP2 penetrates into the
4. Conclusion
In this study, PNFSs with different dispersibility

Paramylon
granuleswere developed by adjusting the pretreatment
conditions,

including

pretreatment

time

and

Intensity / a.u.

concentration. Through defibrillation treatment,
PNF-1 exhibited limited fiber formation, with
granules still present, while PNF-MP2 demonstrated
an accelerated defibrillation effect, resulting in the

PNF-MP2

formationofwell-dispersed

nanofibers.

Therheological evaluation showed that PNF-MP2
exhibited

292

288

284

the

highest dispersibility

and

the

reversibility of viscosity recovery. among the280

samples. The friction property evaluation showed

Binding energy / eV
C-C
C-O-H
C-O-C
Envelopethat PNF-MP2 has a high lubrication effect. WAXD
analysis showed that the defibrillation treatment

Fig 8. Narrow scan spectrum of the C1s region of
Paramylon granules and PNF-MP2.

substantially affected the microcrystallization and37

crystal regularity of PNF, while XPS surface

8) H. Tanaka, M. Egusa, Y. Takemura, Y. Iwata, T.

elemental analysis revealed an increased exposure ofNagae, S. Ifuku, H. Kaminaka, Nippon Shokuhinhydroxyl groups on the PNF surface, resulting in
improvedhydrophilicity.

The

Kagaku Kogaku Kaishi, 2016, 63, 18-24.

defibrillation

9) Y. Kawahara, Carbohydr. Polym., 2014, 112,

treatment effect enhanced the hydrophilicity of PNF

73-76.

was improved by exposing hydroxyl groups on its10) K. Kobayashi, S. Kimura, E. Togawa, M.

surface. These findings indicate that paramylon
nanofibers, produced through the WJ fiber treatment

Wada, S. Kuga, Carbohydr. Polym., 2010, 80,

without organic solvents, have excellent water

491-497

dispersibility.

11)M. Shibakami, J of Oleo Science, 2023, 2, 87-Consequently,

they

hold

great

potential for applications in water-based paints and93

related industries.

12) M. Shibakami, G. Tsubouchi, M. Hayashi,
Carbohydr. Polym., 2014, 105, 90-96．

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