Accepted Article

MR. PRAMESH KUMAR DHUNGANA (Orcid ID : 0000-0001-8497-1143)

PROF. BHESH BHANDARI (Orcid ID : 0000-0001-8800-6295)

Article type : Original Article

The peer review history for this article is available at https://publons.com/publon/10.1111/ijfs.15182

Development of a continuous membrane nanobubble generation method applicable in liquid

food processing
Pramesh Dhungana1, Bhesh Bhandari1*

1 ARC Dairy Innovation Hub, School of Agriculture and Food Sciences, The University of
Queensland, St Lucia QLD-4072, Australia

* Corresponding author; telephone: +61 7 33469192; email: b.bhandari@uq.edu.au

Abstract
A novel continuous nanobubble generation method was developed using a commercial ceramic
membrane as a nanobubble generator. CO2 and air were infused externally across the ceramic
membrane (pore size 50 nm) into the water flowing inside the ceramic membrane tube. Infusion of
each gas was done in a single (20 s) and multiple passes (180 s) at 20-21°C. Results showed that
gaseous nanobubbles could be generated even in a single pass. Low power ultrasound (200 kHz,
30W) was applied for the rapid assessment of nanobubble stability. Single-pass ultrasound
treatment of the samples was carried out at two levels: 2.6 J mL-1 and 5.1 J mL-1. Among the
samples in which gases were infused for multiple passes, air nanobubbles were found more stable
than CO2. In contrast, an opposite trend was observed in those samples in which gases were
infused in the single-pass only (20 s). This process has the potential to be applied to food
processing industries, such as to enhance the products’ sensory attributes, the efficiency of
membrane separation, food drying, and liquid food transportation systems.
1. Introduction
Microbubbles (MBs) ( 10-50 µm) and nanobubbles (NBs) (<200 nm) are tiny gas-filled cavities
within liquids (Temesgen et al., 2017, Agarwal et al., 2011). The popularity of these bubbles has
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 been increasing over time. The primary application areas of microbubbles (MBs) and nanobubbles
Accepted Article
(NBs) include wastewater treatment, mining, agriculture, aquaculture, food processing and
medical applications(Adhikari et al., 2018, Akhavan et al., 2016, Amamcharla et al., 2017, Ebina
et al., 2013, Jannesari et al., 2018, Jannesari et al., 2020, Liu et al., 2008, Liu et al., 2013, Liu et
al., 2012, Ohnari, 2001, Temesgen et al., 2017, Wu et al., 2008). The mechanism of action of NBs
and MBs in food processing could be different depending on their sizes and chemical nature. For
example, gaseous NBs and MBs affect the physical properties of a liquid by decreasing the
interparticle friction of colloidal particles (Liu et al., 2013), leading to a change in the rheological
properties of food. Carbon dioxide NBs and MBs have been claimed to lower the viscosity of the
concentrated dairy dispersions, such as milk protein and whey protein dispersion (Amamcharla et
al., 2017). Another fate of gaseous NBs and MBs affecting food and bioprocessing could be by
enhancing the solubility of gases themselves and mass transfer efficiency. For example, Ogawa et
al. (2012) claimed that air NBs and MBs in seasoning liquid significantly reduced the marination
time of boiled egg to just 5 h from 72 h. The dramatic reduction in margination time is due to
enhanced mass transfer rate by air NBs and MBs. Similarly, Ebina et al. (2013) also demonstrated
a significant increase in O2 content in water from 7.7 to 31.7 mg L-1 when incorporated as O2 NBs
and MBs. In the food processing sector, the use of CO2 is getting more attention than other gases.
This could be due to its high solubility, pH lowering capacity and relatively low reactivity.
Application of CO2 NBs and MBs significantly decreased the melting rate and scooping hardness
and increased the overall acceptability of soft-serve ice cream (Adhikari et al., 2020a). In addition
to alteration of products’ attributes, CO2 NBs and MBs also decreased the churning time during
butter making, ice nucleation time of liquid foods, the crystal size of lactose and anhydrous milk
fat, and increased the ice nucleation temperature of the milk and fruit juice (Adhikari et al., 2018,
Adhikari et al., 2020a, Adhikari et al., 2019, Truong et al., 2018). A detail of gaseous nanobubbles
production methods and their applications is available in a review article by Khanh et al. (2020).

There are two primary methods for generating NBs and MBs: the cavitation method and the
porous media-assisted method. Gaseous NBs and MBs are generated in aqueous solutions by
creating cavitation through four general mechanisms: hydrodynamic, acoustic, particle, and optical
type. In the case of food, being a complex biomaterial, NBs and MBs generation via some of the
cavitation methods could have detrimental effects on the food itself or its constituents. For
example, the use of electromagnetic waves creates cavitation and affects proteins and fat and

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 generates heat on treated food. A patented methodology (Amamcharla et al., 2017) using
Accepted Article
hydrodynamic cavitation method ( venture tube) claimed to produce NBs and MBs of < 40 µm,
and the authors demonstrated remarkable effects on various dairy food systems. However, this was
a batch type of process. In addition, cavitation based methods have a complex operating system
and lack control over bubble size and uniformity (Ahmed et al., 2018). Our preliminary trials also
found much foaming in dairy protein dispersion when air and CO2 NBs and MBs were
incorporated via a venturi tube system rather than a ceramic membrane-based system. This is also
a reason to choose ceramic membrane as an NBs and MBs generator in this study. Of the porous
media-assisted methods, the fluidic oscillation technique uses a porous membrane to generate
MBs and NBs (Zimmerman et al., 2011). This technique involved high-frequency oscillating gas
pressure, enabling the periodic release of gas bubbles through the membrane pore. On a laboratory
scale, Ahmed et al. (2018) reported that injection of pressurised gas through a ceramic membrane
produced micro and nanobubbles.

The authors reported that an increase in gas injection pressure and a decrease in membrane pore
size reduced the size of bubbles. This is also a batch type process. In a recent publication, Khanh
et al. (2020) reported the continuous generation of fine nanobubbles (Z-Ave 200 to 500 nm) using
a commercial nanobubble generator (Stoney series - Nanobubble Injection System-protected).
Again, this nanobubble generator caused instant foaming on diary protein dispersion when
preliminary trials were carried out in our laboratory. Ahmed et al. (2018) and Khanh et al. (2020)
claimed that the smaller sized nanobubbles were generated at 4 bar gas injection pressure than gas
injection at 3 and 2 bars. Of all the processes described above, the ceramic membrane assisted
method of generating gaseous NBs and MBs is a simple method with the potential to fit into a
continuous system. However, there are no such methods that can generate NBs in a continuous
system. Our proposed ceramic membrane-based system is a continuous system and comprises a
fit-out similar to the normal commercial membrane filtration system. Such fit-out makes this
system much easier to operate.

Besides, the stability of nanobubbles is also an important aspect that helps measure their
functionality in real situations. A low power ultrasound has been reported to create gentle
mechanical agitation without any cavitation (Adhikari et al., 2020a, Santos et al., 2009). Such
agitation would assist the release of dissolved gas and other NBs and MBs as big bubbles. Such a
phenomenon can be utilised for rapid prediction of the stability of NBs and MBs in a real

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 industrial situation where food being process is under the effect of constant vibration or other
Accepted Article
types of mechanical agitation. However, there is limited literature available for continuous
generation of NBs and MBs using ceramic membrane and rapid prediction of their stability. This
report highlights how the commercial ceramic membrane can be used in a continuous system to
generate CO2 and air NBs and MBs and predict their stability using low power ultrasound.
2. Materials and methods
2.1 Nano-bubble generation set-up
A schematic diagram depicting the principle of nanobubble generation by using a ceramic
membrane is presented in Fig 1. A tubular ceramic membrane with 0.25 m length, 0.007 m
internal diameter and effective surface area of 0.005 m2 was used in the NBs generator system
shown in Fig 1. The pore size of the ceramic membrane used for nanobubbles generation was 50
nm. The inlet pressure of the air/gas was kept at 0.4 MPa. No pressure was applied to the liquid
feed except the pressure exerted by the circulatory pump. The water recirculation speed was 17 L
min-1 which corresponds to the velocity of 7.36 m s-1 of water in the tubular ceramic membrane
section. A lab-scale membrane filtration unit fitted with a progressive cavity pump was used to
circulate the water. The filtration unit and tubular ceramic membrane were purchased from
Techniques Industrielles Appliquees (TIA), Bollene, France.

2.2 Experimental design

Both air and CO2 were used for the generation of nanobubbles. Each gas was infused in deionised
water (DW) via a tubular ceramic membrane for 20 s and 180 s. The term “water” in the rest of the
report represents deionised water. The pressure of the gas at the outer side of the ceramic tube was
kept at 0.4 MPa for all experiments. Six litres of water was kept in the feed tank (Fig 1). The
water circulation rate at the gas diffusor (tubular ceramic membrane) was 17 L min-1 ( DW
velocity = 7.36 m s-1). Water samples without gas infusion were taken as control samples. Firstly,
each gas was infused in water in a single pass. It took 20 s for single-pass infusion. Secondly, each
gas was continuously infused in DW with a multi-pass for 180 s. In the case of CO2 infused
water, the pH of the water became 4.5 and 3.8 in a single pass (20 s) and multi-pass (180 s),
respectively. Similarly, the pH of the water became 5.6 and 5.2 after a single-pass (20 s) and
multi-pass (180 s), respectively. The pH 3.8 was the lowest value that can be reached with CO2 at
the laboratory temperature, i.e., 20-21 °C. Since it was impossible to get the same pH values as in
CO2 infused water, the air was infused in water with the same conditions that required getting pH

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 3.8 and 4.5 in CO2 infused water. pH 5.4 in air-infused water corresponds to the same treatment
Accepted Article
condition (duration and pressure of gas infusion) required to get pH 3.8 in CO2 infused water.
Similarly, pH 5.6 in air-infused water corresponded to the same treatment condition needed to get
pH 4.5 in CO2 infused water.
Low power ultrasound treatment of air and CO2 infused water samples was carried out via
ultrasound transducer with a power of 30 W (200 kHz, UCE-UT-20030PZT-4, UCE Ultrasonic,
PR China, ø: 42 mm). The ultrasound transducer was placed at a point where a cylindrical pipe
with a 13 mm internal diameter was pressed to make a narrow channel having length =19 mm and
height = 6 mm. During ultrasound treatment, the sample flow rate was varied in two levels, 350
mL min-1 and 700 mL min-1. The energy dissipated during low power ultrasound treatment of
water samples were 2.6 J mL-1 and 5.1 J mL-1 at 350 mL min-1 and 700 mL min-1, respectively. A
continuous system for ultrasound treatment is presented in Fig. 2 as a schematic diagram. A
tabulated form of experimental design and abbreviated form of the samples is given in Table 1.
2.3 Size measurement
NBs and MBs' size was measured by using a dynamic light scattering technique in the Zetasizer
(Malvern Zetasizer Nano ZS, Malvern Panalytical Ltd, UK). The bubble size measurement
procedure was adapted from Khanh et al. (2020). In brief, the sample was filled into a disposable
polystyrene cuvette to a recommended level while avoiding bubbles formation. The sample was
left inside the measurement unit for 120 seconds for temperature equilibration. The Refractive
Index of water was set to 1.33, whereas the Refractive Index of particles (bubbles) was set as of
air,1 (Ahmed et al., 2018, Phan et al., 2020, Ushikubo et al., 2010). Triplicate measurement was
done with at least two measurements in each replicate.
3. Results and Discussion
Injection of CO2 and air at 0.4 MPa via a ceramic membrane (pore size-50 nm) showed NBs and
MBs' formation in the water flowing through the ceramic membrane tube (Fig. 3 & 4). Tyndall
effect is seen when light scattering particles present in colloid or in a very fine suspension are
approximately between 40 to 900 nm. In Fig 3, it could be seen clearly the presence of line in the
water infused with CO2. This line is formed due to the Tyndall effect caused by the presence of
MBs and NBs in the water sample. Wang et al. (2019) also demonstrated a thick line in a laser
light scattering image indicating the presence of Nitrogen nanobubbles in water. Ahmed et al.
(2018) reported the formation of nanobubbles using a ceramic membrane in laboratory batch-type

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 experiments. Ahmed et al. (2018) also reported that it took more than 30 min to get the stability of
Accepted Article
air NBs hydrodynamic diameters when the air was infused at 0.414 MPa. However, a good level
of stability on the average size of MBs and NBs was observed in our study even on the water
samples, which were infused with gases via tubular ceramic membrane for just 20 s at 0.4 MPa.
The improved stability could be attributed to the smaller membrane pore size ( 50 nm) and high
shear force exerted by water flowing at high speed (7.36 m s-1 ). A smaller deviation in bubble size
of consecutive measurements also indicates a contant presence of relatively same size and same
amount of MBs and NBs in water samples. Therefore, the proposed continuous system could
perform better than batch type purposed by Ahmed et al. (2018). The percentage intensity vs size
plot (Fig 4a) indicated that the generation of NBs was more with CO2 than air when gases were
infused for 180 s. However, the distribution is shifted more towards smaller droplet sizes for the
air-infused NBs than the CO2 infused. Higher solubility of CO2 than air in water might have
caused such a trend. At the initial stage of gas infusion, the CO2 NBs might be lost because of
solubilisation. However, on further injection of gas, solubilised CO2 reached the saturation point,
and further dissolution of CO2 slowed down, favouring an increase in NBs concentration.
According to Fick’s law, as CO2 dissolves from MBs and NBs, the concentration gradient
decreases between gas bubbles and water interface, which eventually slowed down the dissolution
of CO2 from NBs (Ahmed et al., 2018, Jaynes and Rogowski, 1983) Fig. 4b showed a significant
amount of NBs in water samples because of gas infusion. Interestingly, the air produced a higher
proportion of smaller MBs and NBs than CO2 in all tested conditions. In addition, average sizes of
the air NBs were smaller than CO2 NBs in both gas infusion times, 20 s and 180 s (Fig. 5).
Application of low power ultrasound ( at 2.6 and 5.1 J mL-1) on the water did not change the size
distribution plot of the water (Fig. 4 c & d). This indicates that the low power ultrasound is not
sufficient to create cavitation and water vapour MBs and NBs. Therefore, all the changes observed
in the size distribution plot of the water sample treated with CO2 and air after ultrasonication are
solely due to changes in the size of CO2 and air NBs only. As expected, a remarkable effect of
ultrasound treatment was observed in both CO2 and air NBs (Fig. 6).
When looking at the intensity vs size graphs of Fig 5 a & c and 6 a & c, and Fig 4, the average
size of CO2 NBs of the samples CO2-180s-2.6J mL-1 and CO2-180s-5.1J mL-1 decreased after
ultrasonication of water sample CO2-180s-0J mL-1, corresponds to pH 3.8 (Fig 5). In contrast, the
average size of CO2 NBs decreased in sample CO2-20s-5.1J mL-1 upon ultrasonication but
increased in sample CO2-20s-2.6J mL-1. Since bigger bubbles have lower Laplace pressure than

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 smaller, bigger MBs and NBs are more sensitive to change in outer pressure than their smaller
Accepted Article
counterparts. Low power ultrasonication just provides uniform mechanical agitation or gentle sock
waves to the water. However, its power is not enough to initiate cavitation (Adhikari et al.,
2020b). The shock waves from low power ultrasound increase the bubble's outer pressure, making
bigger bubble vulnerable to breakage and provides. In the case of water samples that were infused
with CO2 for a longer time (180 s) might have more dissolved CO2 and NBs. And therefore, the
shock wave from the low power ultrasound might cause the formation of numerous MBs and NBs,
facilitated bubble collapse, leading to the formation of relatively larger bubbles and subsequent
loss. But, in the case of water samples that were infused with CO2 for a shorter time (20 s), they
must have less dissolved CO2 and NBs than 180 s CO2 infused samples. The sample CO2-20s-5.1J
mL-1(sample flowing at 350 mL min-1 via ultrasound transducer) received relatively severe
treatment than CO2-20s- 2.6J mL-1 (sample flowing at 700 mL min-1 via US transducer), which
might cause loss of bigger bubbles leaving smaller behind. However, in the sample CO2-20s- 2.6J
mL-1, the shock waves might just facilitate the collapse of existing bubbles or create new bubbles,
but not enough bigger to escape before the time of measurement. Similarly, the average size of air
nanobubbles of the sample at pH 5.2 (AIR-180s-0J mL-1 ) decreased when ultrasonication was
done at 5.1 J mL-1; however, the average size of NBs increased slightly when ultrasonication was
done at 2.6 J mL-1 (Fig. 5).
In the case of the air nanobubbles containing sample AIR-20s-0 J mL-1 (pH 5.6), ultrasound
application at 5.1 J mL-1 did not affect the average size of nanobubbles. Still, it drastically
increased when ultrasonication was done at 2.6 J mL-1. This result is similar to the results obtained
with CO2 infused samples (discussed above). Ultrasonication at a higher feed rate provided shorter
residence time where mechanical shaking of water might have caused the collapse of smaller
bubbles into bigger bubbles but still not big enough to escape quickly. In the case of samples
ultrasonicated at 5.1 J mL-1 (350 mL min-1), the big bubbles formed because of longer residence
time might have escaped before size measurement. Therefore, size distribution curves ( % volume
vs size) of water samples CO2-20s-0J mL-1, AIR-20s-0J mL-1 and AIR-180s-0 J mL-1,
ultrasonicated at 5.1J mL-1 (350 mL min-1 ) were almost flat (Fig. 4 d, 6 d & 7d). In Fig 6, it is
evident that gaseous NBs could remain in a fair amount even after mechanical agitation if the
concentration of gas is high. In addition, air nanobubbles were more resistant to mechanical
shaking than CO2 nanobubbles (Fig 6 a vs 6c and 6b vs 6d) when the concentration of each of CO2
and air was relatively higher. However, at a lower air and CO2 concentration, CO2 NBs were more

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 stable than air NBs. Because among the 20 s treated CO2 and air NBs containing samples, only the
Accepted Article
CO2 NBs containing sample showed remarkable size distribution of NBs Fig (Fig. 7a vs 7c and 7b
vs 7d).
4. Conclusion
This research showed a new set up for a continuous generation of CO2 and air nanobubbles in
water. Results proved that a commercial ceramic membrane could be used to generate gaseous
nanobubbles, and it can be fitted into a continuous system. Low power ultrasound treatment of the
water sample containing CO2 and air nanobubbles revealed that gaseous nanobubbles could be lost
due to mechanical shaking depending upon the severity of shaking. Overall, this research could be
a starting point for industrial research and development where a continuous system of gaseous
nanobubbles generation is needed for process and/or product improvement. The developed process
can be used to speed up the food marination process, enhance the sensory quality of food, improve
the membrane separation process of dairy liquids, and speed-up the crystallisation of various
foods. This process could also be a cost-effective alternative for the laboratory scale methods used
by different researchers for infusing CO2 nanobubbles. In an actual situation, a food processing
plant always under some types of vibration or noise. Similarly, liquid foods also need to pass
through various types of liquid handling and processing equipment such as pumps, pasteuriser,
mixer etc. Therefore, the information from this study could be used to predict the loss of MBs and
NBs in the actual industrial scenario by establishing the correlation between industrial vibration
and ultrasonic vibration.
Acknowledgements
This research was supported under Australian Research Council's Industrial Transformation
Research Hub (ITRH) funding scheme (IH120100005). The ARC Dairy Innovation Hub is a
collaboration between the University of Melbourne, the University of Queensland and Dairy
Innovation Australia Ltd (currently disbanded).
Ethical Approval
Ethics approval was not required for this research
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Accepted Article
Table 1. Experimental design showing the types of gas used, gas recirculation time, and
ultrasonication levels. The ultrasound probe has 30W power.

Sample Gas/air water pH Ultrasound Water flow Abbreviated form

-1
type circulation treatment rate (mL min )
time (s) (J mL-1) via US
transducer

Deionised 0 6.3 0 0 DW
water
0 6.3 2.6 700 DW-2.6 J mL-1
(DW)
0 6.3 5.1 350 DW-5.1 J mL-1

CO2 20 4.5 0 0 CO2-20s-0 J mL-1

infused
20 4.5 2.6 700 CO2-20s-2.6 J mL-1

20 4.5 5.1 350 CO2-20s-5.1 J mL-1

180 3.8 0 0 CO2-180s-0 J mL-1

180 3.8 2.6 700 CO2-180s-2.6 J mL-1

180 3.8 5.1 350 CO2-180s-5.1 J mL-1

Air 20 5.6 0 0 AIR-20s-0 J mL-1

infused
20 5.6 2.6 700 AIR-20s-2.6 J mL-1

20 5.6 5.1 350 AIR-20s-5.1 J mL-1

180 5.4 0 0 AIR-180s-0 J mL-1

180 5.2 2.6 700 AIR-180s-2.6 J mL-1

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Fig 1 Schematic diagram of equipment set up for nanobubble generation by using a ceramic
membrane (TIA, France MF/UF unit)
Fig 2 Schematic diagram of equipment set up for continuous nanobubble generation process
using dissolved gas and ultrasound.
Fig 3. The scattering of a laser beam passing through the containers filled with a) water
before CO2 infusion and b) water after CO2 infusion, infused for 180 s at 0.4 MPa (pH 3.8).
Laser wavelength = 532 nm
Fig. 4 Size distribution plot of: CO2 NBs containing samples, a) % intensity vs size (nm), b) %
volume vs size (nm). Air NBs containing samples; c) % intensity vs size (nm), d) % volume
vs size (nm)
Fig 5 Average size of CO2 and air nanobubbles as affected by the application of low power
ultrasound at 2.6 J mL-1 and 5.1 J mL-1
Fig 6 Size distribution plot of: CO2 NBs containing samples, a) % intensity vs size (nm), b) %
volume vs size (nm). Air NBs containing samples; c) % intensity vs size (nm), d) % volume
vs size (nm)
Fig 7 Size distribution plot of : CO2 NBs containing samples, a) % intensity vs size (nm), b) %
volume vs size (nm). Air NBs containing samples; c) % intensity vs size (nm), d) % volume
vs size (nm)