Response to Comment by Rak & Sedlak

Ananda J. Jadhav and Mostafa Barigou

Summary
Advanced techniques which combine high spatial resolution with chemical sensitivity to directly probe the observed
nano-entities and provide direct evidence that they are truly gas-filled nanobubbles do not exist. Therefore, we focused
in our paper1 on providing multiple varied indirect evidence using a variety of physical and chemical techniques that the
nano-entities are not due to contamination and, hence, they must be nanobubbles. It should be noted that such
techniques require good experimental skills, scientific expertise, reliable equipment and sound protocols. Whilst no
single piece of indirect evidence on its own can be considered as full proof, we estimate that the results presented
combined provide strong evidence that bulk nanobubbles do exist and they are stable. The work presented in our paper 1

is the culmination of a series of reported studies, 2–4 and many authors have either directly or indirectly confirmed our

findings.5–13 Other works so far have tended to follow a piece-meal approach and generally lack objective evidence that
the observed nano-entities are indeed nanobubbles. It is unfortunate, therefore, that Rak & Sedlak have failed to
appreciate the value of our contribution and the fact we are the first authors to have conducted such an extensive and
varied analysis of BNB suspensions and, in their Comment, they reject all of the work we reported. We here address
their comments point by point and show that their criticisms are unfounded.

Abstract
“However, a number of previous works….. procedures are not nanobubbles.” and “In fact, numerous authors …..
nanoobjects as nanobubbles.”

It is very misleading to claim that numerous authors have questioned the existence of BNBs. In fact, only a handful of
papers seem to dispute the existence of BNBs, but none have produced any direct evidence or conclusive indirect
evidence to support their claims. In contrast, we have counted in excess of 180 papers which have reported indirect
experimental and/or theoretical evidence for the existence of bulk nanobubbles (BNBs). Amongst these papers, many
have also reported applications of BNBs in various fields including about a dozen patents. It is, therefore, highly
surprising that Rak & Sedlak are opting to ignore this vast amount of literature which supports the existence of BNBs.

To support their claims, Rak & Sedlak have cited Refs. 11-16 from four research groups. Ref. 11 is a review by
Alheshibri et al. of the literature on the history of surface as well as bulk nanobubbles, and research progress made in
the field; this review does not dispute the existence of BNBs. In addition, Rak & Sedlak have failed to cite a paper
published in the same year by the same group (cited in Ref. 11) in which they produced hydrogen and oxygen-filled
BNBs using electrolysis and used them for surface cleaning. 14 Subsequently, however, the latter group (Refs. 12, 16)
together with the authors of Refs. 13 and 15 as well as Sedlak & Rak (Ref. 14), interpreted the nano-entities observed in
their experiments as oil droplets, solvent contamination, supramolecules or nanoparticles. However, such conclusions
were not based on any direct evidence but rather on disputable experimental work. For example, based on our own
experience with the technique, the use of mass resonance to differentiate BNBs from other nano-entities is unproven
and highly dubious. In addition, the reported findings were not supported by chemical analysis of BNB suspensions to
prove that the observed nano-entities are due to impurities or contamination.

“We have recently investigated in detail particularly processes of ultrasound cavitation and addition of organic
compound (ethanol) to water … with controlled contamination as monitored by HS-SPME GCMS method.”

To support their claims, Rak and Sedlak cite their own work (Ref. 17) on ultrasound cavitation and water-solvent
mixtures. The work reported, however, suffers from flaws in experimental methodology, as follows.

(a) Ultrasound cavitation (Ref. 17)

The authors reported that ultrasonication leads to the generation of fine metal nanoparticles which detach from the
surface of the ultrasound probe. It is well known that this technique generates a lot of heat and can lead to probe
erosion which could mask the existence of BNBs. We had previously discussed this issue in our earlier work, 2 and we
showed that sonication time must be kept short, energy input should be moderate and the sample temperature must be
controlled to minimise such effects. Indeed, such precautions are also part of the standard operating process of
ultrasound sonicators recommended by manufacturers. Finding optimum conditions for using ultrasound cavitation
requires a certain amount of expertise and experimentation. In our present work 1 we used a pulsed mode (5 s ON, 5 s
OFF) as well as a short total exposure time (100 s) and solution temperature was controlled at 20 °C. In contrast, there
were flaws with Rak & Sedlak’s experiments. They sonicated their samples for an extremely long period of time (60
min) and failed to report vital information about operating temperature, temperature control, and sonication intensity .
It is therefore not surprising if their experiments led to the generation of massive amounts of Titanium particles. Hence,
a direct comparison of their work with our experiments is not valid. Independently of our own works, 2–4 other authors

have also been successful in generating BNBs using either ultrasonic or hydrodynamic cavitation. 15,15–18

(b) Water-ethanol mixtures (Ref. 17)

In their work, Rak & Sedlak suggest that the mixing of water with ethanol produces nanoparticles or nanodroplets
originating from so-called mesoscale solubilisation of hydrophobic compounds present in ethanol. However, they do
not state the chemical composition of such impurities. They reported that the density of the observed nano entities was
0.81 gcm-3, and concluded that this fell within the density range of hydrophobic organic compounds. No independent
verification was provided for such measurement. In a separate experiment, they sonicated water-ethanol mixtures for
an excessive time of 60 min without controlling the temperature. The problems arising from such an experiment have
already been discussed above, but they are even more serious in this case given the volatility of ethanol. Consequently,
these sets of experiments seem unreliable.

Rak & Sedlak also cite their earlier work (Refs. 14, 19) which is not particularly relevant to our nanobubble work.
They claim the formation of solvophobicity-driven mesoscale structures (SDMS) in aqueous solutions of inorganic
ionic MgSO4 salt, of organic ionic citric acid, of uncharged organic urea and of tert-butyl alcohol. They also used
ternary mixtures of water-ethanol-octadecane, water-tert-butyl alcohol-decane and ethanol-octadecane-ethylene glycol.
These mixtures some of which contain salts or immiscible organic solvents are very different from our pure water-
ethanol mixtures and, hence, extrapolation of their results to our results is not valid.

Materials
Rak & Sedlak state that the use of analytical grade ethanol (99.9% pure) does not guarantee the absence of
hydrophobic impurities in concentrations sufficient to produce detectable amounts of SDMS upon mixing with water.
However, (i) they do not provide any concrete evidence for the presence of such hydrophobic impurities; (ii) they do
not specify their chemical composition; and (iii) they do not specify at what water-ethanol ratios such hydrophobic
impurities are supposed to be detectable. Furthermore, Rak & Sedlak do not address why in our work 1,4,19 : (iv) the
number per mL of nano-entities (we call BNBs) observed varies strongly with the water-ethanol ratio, such that it
reaches a maximum at an ethanol volume fraction of about 20% and it falls to zero at a fraction of about 60%; and (v)
why these nano-entities do not disappear when ethanol is evaporated in a rotary evaporator. It is also widely known in
the literature that the mixing of ethanol and water leads to the release of gas. 20–23 In fact, other authors recently reported

the formation of BNBs in water-ethanol mixtures. 20,21,24–27 In our follow-up work which studied BNBs in aqueous
solutions of various organic solvents, we confirmed the ethanol results and showed that different solvents behave
differently as the gas solubility of the solvent relative to water as well as its molecular structure are determining factors
in the formation and stability of BNBs.19  Hence, the above comment by Rak & Sedlak appears to be unfounded.

ATR-FT-IR and Raman

Rak & Sedlak question the usefulness of ATR-FT-IR and Raman for detecting nanoscale matter. Vibrational
spectroscopy techniques are used to detect the presence of functional groups in a molecule. 28,29 They are suitable for

detecting nanoscale matter of organic or inorganic nature, and this is well documented in the literature. 30–34 Therefore,
their sensitivity and detection limit could be described as being on the molecular scale which is much smaller than the
nanoscale. At the same time, the number of nano-entities we observed per mL of solution is high (> 10 8), and were
these nano-entities due to contamination molecules, these techniques would have detected them. Hence, the absence of
foreign molecules beside water or solvent in the recorded spectra is proof that there is no contamination present in the
BNB suspensions generated in pure water or in water-solvent solutions. We have already provided in our paper 1 the
spectral range, resolution and wavenumber accuracy which show the high sensitivity used for the detection of
contamination molecules. Other workers have used the same techniques to analyse BNB suspensions. For example,
Ohgaki et.al.35 used attenuated total reflectance infrared spectroscopy (ATR-IR) to show that the surface of bulk
nanobubbles contains hard hydrogen bonds that may reduce the diffusivity of gases through the interfacial film, and
used Raman spectroscopy to confirm the presence of nitrogen gas inside bulk nanobubbles. Jin et. al. 10, on the other
hand, using ATR−FT-IR spectroscopy, detected a stronger hydrogen bond during the shrinking of microbubbles into
bulk nanobubbles.

Interestingly, Rak & Sedlak provide in their Figure 1, FT-IR spectra of 20% aqueous solution of purified ethanol.
Notably, the characteristic peaks that represent the C-O stretch and C-C stretch for ethanol seem to be missing, even
though the ethanol concentration is orders of magnitude higher than the detection limit that they quote. These peaks,
however, are clearly visible in our paper1 (see Figure 3A). Therefore, the FT-IR methodology used by Rak & Sedlak
seems questionable.

Gas Chromatography−Mass Spectrometry

Rak & Sedlak analysed tert-butyl alcohol of 99.7% purity using GC-MS, whereas we analysed 99.9% pure ethanol in
water suspensions. These are widely different systems and direct comparison of results is not valid. In addition, and
contrary to their claim, we used direct injection of our ethanol-water NB suspension and we did observe a very high
peak for ethanol (Figure 3C).1 In conclusion, to prove their point, Rak & Sedlak should have used exactly the same
ethanol-water system as us.

Inductive Coupled Plasma-Mass Spectrometry

Rak & Sedlak are wrong in saying that we did not conduct a digestion procedure in our ICP-MS analysis. This is a
standard procedure which was indeed performed and is described under the “ICP-MS analysis” section of our paper 1.
Moreover, Rak & Sedlak say that our ICP-MS analysis which does not show the presence of Titanium in the NB
suspensions is inconsistent with their own work using acoustic cavitation. We answer this point, as follows: (i) we did
actually observe traces of Titanium but the levels were extremely low in concentration because of our optimised
application of ultrasound cavitation as discussed above under “Ultrasound cavitation”; and (ii) in the same section
above, we discussed the flaws in Rak & Sedlak’s application of ultrasound behind the large amount of Titanium
impurities they observed.

Cryo-Scanning Electron Microscopy

We agree that in Figure 5C-D the size of the cavities should show 5 and 10 m instead of 0.5 to 1 m. However, this
typo mistake does not in any way affect our interpretation of the Cryo-SEM results, contrary to what Rak & Sedlak
seem to imply.

Cryo-SEM experiments were not done for the water-ethanol NB suspensions because etching of the sample is carried
out at -90 °C which is much higher than the freezing point of ethanol (-115 °C). This is a limitation of Cryo-SEM
which does not allow the sample temperature to be maintained below -115 °C for the analysis of water-ethanol samples.

Rak & Sedlak seem to suggest that not doing a composition analysis of the imaged objects, such as EDS analysis which
would have revealed the presence of any metallic elements, makes this work incomplete/invalid. However, they seem
to be oblivious to the fact that we did employ an alternative technique (ICP-MS) which achieves the same objective. In
Cryo-SEM, it is the morphology of the imaged objects which is relevant. Using the same approach, Ohgaki et.al. 35
previously reported a similar image of a BNB.

Rak & Sedlak keep alluding to the presence of oil contamination throughout their comments, but they fail to identify
any plausible sources for such oil contamination. They are also choosing to overlook all our clean experimental
protocols and dismiss all the results from our GC-MS analysis which confirm that the NB suspensions were free of oil.
Cryo-SEM images of oil droplets or solid particles would look different from our images where the NBs are identified
by craters, i.e. bowl-shaped depressions. This is in fact illustrated in the very reference (Lee et al. 36) which has been
cited by Rak & Sedlak to support their argument. In this case, the reported images show that the oil droplets have a
dome shape and the craters which are hollow are similar to our NB images. Such dome shapes are not observed in our
images which again confirms that the observed objects are not oil droplets. Observations of dome-shaped droplets are
supported by ample Cryo-SEM results in the literature. 37–43

Encapsulation of Nanobubbles
Rak & Sedlak’s interpretation of our TEM images is incorrect. In the case of air-filled particles, the core region does
appear brighter than the shell. This fact is well known in the literature and has also been independently confirmed using
SEM analysis.44–55 Furthermore, one of us (Jadhav) has had extensive experience with producing hollow air-filled

nanoparticles and using TEM and SEM analysis. 58 In the case of encapsulated nanodroplets, it is not usually possible to
distinguish the shell from the inside of the droplet. In our images, however, the air-filled nanobubbles can be clearly
distinguished from the surrounding solid shell.

It is unfortunate that Rak & Sedlak are ignoring our detailed description and discussion, so we feel compelled to repeat
a lot of what is already explained in the paper. 1 The HSRS device is not a commercial nanobubble generator. This
device is simply a continuous high-shear mixer which we adapted for this purpose. The flow loop is clean and contains
no lubricating oil or oil seals. Nonetheless, the ultrapure water (Type-I) used was analysed using a combination of
chemical analytical methods and no oil molecules were detected.

Freezing and thawing of nanobubble suspensions

Given their comments, it is quite clear that Rak & Sedlak have failed to understand some crucial aspects of this part of
our work. We were the first to suggest freezing and thawing as a way of qualitatively distinguishing NBs from solid
nanoparticles and droplets.2–4 The freezing temperatures were selected based on the freezing point of each NB
suspension, which is a sensible scientific approach. For NBs in pure water -18 °C was more than sufficient to ensure
that all of the water was completely frozen. Similarly, for the water-ethanol mixture, we used -180 °C, which is well
below the freezing temperature of pure ethanol (-115 °C). NBs in pure water vanished after freezing and thawing. NBs
in water-ethanol suspensions, however, did not disappear after freezing and thawing, and the possible reasons are
discussed in our paper.1 To test our interpretation of the phenomenon, after thawing, the ethanol was completely
removed in a rotary evaporator. The NBs were not affected by the evaporation of ethanol and were now suspended in
pure water. Now, freezing this suspension again at -18 °C gave the same results as for NBs originally generated in pure
water. In other words, all NBs disappeared on thawing in the same way, which confirmed our interpretation of the NB
generation processes in pure water and in water-ethanol mixtures.

Drying of nanobubble suspensions

Rak & Sedlak seem not to understand the two different drying experiments described in our paper: 1 In the first
experiment, ethanol (solvent) only was evaporated, leaving the observed nano-entities intact in water, contrary to Rak &
Sedlak’s assertion. The non-disappearance of the NBs shows that the observed nano-entities cannot be due to so-called
“solvophobicity-driven mesoscale structures”. In the second experiment, NB suspensions generated in pure water and
NB suspensions generated in water-ethanol mixtures were dried up completely, which resulted in the complete
disappearance of the nano-entities with no residue remaining. Therefore, Rak & Sedlak’s arguments are incorrect.

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