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\begin{document}

\title{Application of microbubble air flotation to harvest Microcystis sp. from
agriculture wastewater: the regulation and mechanisms}



\author[1]{hui liu}%
\author[2]{Jianfeng Ye}%
\author[3]{Zhihao Zhu}%
\author[1]{Zhaofeng Song}%
\author[1]{Huiting Xu}%
\author[1]{Tianchen Xu}%
\affil[1]{Shanghai Academy of Environmental Sciences}%
\affil[2]{Tongji University College of Environmental Science and Engineering}%
\affil[3]{Shanghai Ocean University，999 Hucheng Ring Rd}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The harvesting of microalgae is the main bottleneck of its large-scale
biomass production, and seeking an efficient, green, and low-cost
microalgae harvesting technology is one of the urgent problems to be
solved. Microbubble air flotation has been proved to be an effective
measure, but the generation of the right size of microbubbles and the
mechanism of microbubbles-algal cells attachment are still unclear. In
this study, microbubble air flotation was used as a harvesting method
for \emph{Microcystis} cultured in agricultural wastewater. The process
mechanism of microbubble air flotation harvesting microalgae in
wastewater was fully revealed from three aspects (the design of bubble
formation, the adhesion law, and the recovery rate of microalgae under
different working conditions). The results show that the length of the
release pipe is the main factor affecting the proportion of microbubbles
with particle size less than 50 \selectlanguage{greek}μ\selectlanguage{english}m. In the process of adhesion, when the
particle size of microbubbles is 0.6\textasciitilde{}1.7 times the size
of \emph{Microcystis}, the adhesion efficiency of microbubbles to
\emph{Microcystis} is the highest. Under the conditions of pressure 0.45
MPa, gas-liquid ratio 5\% and release pipe length 100 cm, the harvesting
performance of \emph{Microcystis} was the best. Microbubble air
flotation has better harvesting performance of \emph{Microcystis} with
higher density. By understanding the mechanism of microbubble flotation,
the technical parameters of microbubble flotation for harvesting energy
microalgae are optimized to provide support for the development of
efficient and low-cost devices and equipment for collecting microalgae.%
\end{abstract}%



\sloppy


\textbf{Application of microbubble air flotation to harvest
\emph{Microcystis} sp. from agriculture wastewater: the regulation and
mechanisms}

Jianfeng Ye\textsuperscript{a,b}, Zhihao Zhu\textsuperscript{c},
Zhaofeng Song\textsuperscript{d}, Huiting Xu\textsuperscript{d},
Tianchen Xu\textsuperscript{d}, Hui Liu\textsuperscript{d,*}

(\emph{\textsuperscript{a}College of Environmental Science and
Engineering,Tongji University, 1239 Siping Rd.,} \emph{200092,
Shanghai, China.}

\emph{\textsuperscript{b} State Key Laboratory of Pollution Control and
Resource Reuse,Tongji University, 1239 Siping Rd., 200092, Shanghai,
China.}

\emph{\textsuperscript{c} College of Marine Ecology and Environment,
Shanghai Ocean University,999 Hucheng Ring Rd., 201306, Shanghai,
China.}

\emph{\textsuperscript{d}Water Research Institute, Shanghai Academy of
Environmental Sciences, 508 Qinzhou Rd, 200233 Shanghai, China.} )

* Corresponding author

E-mail: lhsaes@163.com

Tel.: 86-21-64085119

Abstract

The harvesting of microalgae is the main bottleneck of its large-scale
biomass production, and seeking an efficient, green, and low-cost
microalgae harvesting technology is one of the urgent problems to be
solved. Microbubble air flotation has been proved to be an effective
measure, but the generation of the right size of microbubbles and the
mechanism of microbubbles-algal cells attachment are still unclear. In
this study, microbubble air flotation was used as a harvesting method
for \emph{Microcystis} cultured in agricultural wastewater. The process
mechanism of microbubble air flotation harvesting microalgae in
wastewater was fully revealed from three aspects (the design of bubble
formation, the adhesion law, and the recovery rate of microalgae under
different working conditions). The results show that the length of the
release pipe is the main factor affecting the proportion of microbubbles
with particle size less than 50 \selectlanguage{greek}μ\selectlanguage{english}m. In the process of adhesion, when the
particle size of microbubbles is 0.6\textasciitilde{}1.7 times the size
of \emph{Microcystis} , the adhesion efficiency of microbubbles to
\emph{Microcystis} is the highest. Under the conditions of pressure 0.45
MPa, gas-liquid ratio 5\% and release pipe length 100 cm, the harvesting
performance of\emph{Microcystis} was the best. Microbubble air flotation
has better harvesting performance of \emph{Microcystis} with higher
density. By understanding the mechanism of microbubble flotation, the
technical parameters of microbubble flotation for harvesting energy
microalgae are optimized to provide support for the development of
efficient and low-cost devices and equipment for collecting microalgae.

Keywords:Microcystis, Microbubble flotation, Agricultural wastewater,
Mechanisms

1.Introduction

With the gradual depletion of fossil energy and the environmental
pollution caused by the exploitation and utilization of fossil energy,
finding clean and renewable new energy is an urgent problem to be solved
in today's field (Yoro et al., 2021). Energy microalgae, as a renewable
biomass energy source, has become a hot spot because of its short growth
cycle, large biomass per unit area and strong environmental adaptability
(Peter et al., 2022; Siddiki et al., 2022). In the process of
cultivating energy microalgae, it is necessary to provide growth
elements including water, inorganic nutrients and CO\textsubscript{2},
whose cultivation cost accounts for more than 70\% of the total cost of
microbial diesel production (Rawat et al., 2011), which restricts the
development of microalgae biomass energy technology. However, China, as
the largest livestock breeding and consumer in the world (Aravani et
al., 2022), with a large amount of aquaculture wastewater with high
nitrogen and phosphorus content, the microalgae photosynthetic
bioreactor was introduced into the microalgae photosynthetic bioreactor
as the medium for energy microalgae after preliminary treatment, which
could synergistic achieve the goal of deep purification of aquaculture
wastewater and reduction of the culture cost of microalgae biomass
energy (Lopez-Sanchez et al., 2022). However, from the perspective of
large-scale production of energy microalgae, separation from the aqueous
growth medium is difficult and cost as microalgae are small
(3\textasciitilde{}30 \selectlanguage{greek}μ\selectlanguage{english}m) in dilute suspension, and have a specific
gravity similar to that of their medium (Ali et al., 2021). In the
energy microalgae industry chain, the cost consumed in the harvesting
process accounts for about 20\%\textasciitilde{}30\% of the whole
cultivation cost of microalga (Rawat et al., 2011). Therefore,
considering the impact of energy microalgae harvesting technology on oil
extraction and subsequent finishing operation, the method must not
contaminate the biomass or create atoxic medium for recycle (Uduman et
al., 2010).

At present, the harvesting technologies of energy microalgae mainly
include centrifugation, flocculation, filtration, traditional air
flotation, etc. (Zhou et al., 2021). Most current harvesting methods
have either economic or technical limitations, which include high energy
costs, flocculant toxicity, or non-feasible scale-up (Fasaei et al.,
2018; Fuad et al., 2018; Laamanen et al., 2021; Oh et al., 2001; Rawat
et al., 2013). Flotation recently emerging as a promising alternative
based on its good scale-up potential due to technical and economic
parameters, such as lower energy and maintenance costs. Traditionally
flotation is done either by air addition through a diffuser (dispersed
air flotation) or through pressurization (dissolved air flotation)
(Aulenbach et al., 2010). Dissolved air flotation separation is more
efficient than dispersed air flotation, is more commonly used, and is
also proven on a large scale (Christenson \& Sims, 2011).The traditional
air flotation method requires the addition of chemical flocculant
(Laamanen et al., 2021), which has a good harvesting effect, but has a
high cost. Moreover, the quality of energy microalgae will decrease due
to the cytolysis caused by aluminum salt (Teixeira et al., 2010).

It has been shown that smaller bubbles result in higher capture
efficiency (Hanotu et al., 2012). Molina et al. present possibly the
closest technique to micro-flotation for algal harvesting (Molina et
al., 2003). One of the efficient ways of facilitating bubble--particle
interaction in the liquid rather than merely passing the bubbles through
the liquid without it adhering and lifting the particles out of
solution, is to generate the right size of microbubbles. The operational
parameters such as pressure, gas/liquid ratio, and set-up of the release
pipe can affect the size of microbubbles. So far, studies on microalgae
microbubbles flotation have mainly focused on the optimization of
conditions, including hydraulic loading rate, initial algal
concentration, air to solids ratio, pH, salinity, and the type of
flotation. However, the mechanism of micro flotation for harvesting
energy microalgae has not been revealed. The harvesting performance of
the flotation process depends on the attachment of microalgae cells onto
microbubbles. Therefore, a solid understanding of the attachment
mechanism is necessary to perform this harvesting operation.

This work is aimed at performing an experimental investigation of the
interactions between microalgae cells and microbubbles to gain a deep
insight into the mechanism governing microalgae cell to microbubbles
attachment in the flotation process. The morphological characteristics
and motion law of microbubbles under different operational parameters
were analyzed, and the response relationship between the particle size
of microbubbles and the energy microalgae was clarified. The technical
parameters of harvesting energy microalgae by microbubble air flotation
were optimized to provide a basis for the application of microbubble air
flotation harvesting technology in microalgae industrialization.

2.Material and Methods

\textbf{2.1. Wastewater and Microalgae species}

The microalgae selected in this study was \emph{Microcystis} \emph{sp}
.. The effluent from anaerobic fermentation tank of a pig factory in
Fengxian District, Shanghai was used as the culture substrate. Under the
microscope, the main characteristics of \emph{Microcystis}
\emph{sp.}cultured in wastewater were chlorophyll-a (Chla) of 2533
mg/m\textsuperscript{3}, and the density of algae was about
2\selectlanguage{ngerman}×10\textsuperscript{9} cells/mL. The size of algal cells was
10\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, and they were clumped.

\textbf{2.2} \textbf{The device of microbubble air flotation test}

The microbubble air flotation equipment is composed of microbubble
generating device and air floating column. Among them, the microbubble
generation device is composed of gas-liquid mixing pump (Nikuni, 20
FPD04Z, flow rate: 0.9 m\textsuperscript{3}/h), the water inlet tank
(plexiglas, length \selectlanguage{ngerman}× width × height =200×200×500 mm\textsuperscript{3},
thickness =5 mm) and the dissolved gas tank (stainless steel cylindrical
tank, diameter =110 mm, height =810 mm,). The air floating column
(length × width × height =100×30×500 mm\textsuperscript{3}) is made of
plexiglas with the thickness of 3 mm. It has good light transmittance
and is convenient for dynamic observation of microbubbles. Each part of
the test device is connected by PVC pipe (\selectlanguage{greek}φ\selectlanguage{english}=25mm), and the pressure
reducing valve is installed between the microbubble generation device
and the air floating column. The design of flexible pipe joint is
adopted among the above three parts, and the distance between the
pressure reducing valve and the air floating column is adjusted by
changing the length of the release pipe. The whole test device is also
equipped with pressure gauge, gas gauge and flowmeter, which show the
dissolved air pressure, inlet air flow rate and inlet water flow rate,
respectively. The flow chart of the test device is shown in Figure 1,
with the red arrow indicating the flow direction.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1 Flow chart of test equipment (1) Reservoir (2)
Gasometer (3) Gas-liquid mixing pump (4) Dissloved air vessel (5) Air
flotation column (6) Pressure gage (7) Valve (8) Relief valve (9)
Flowmeter (10) Safety valve (11) Union (12) The release pipe (13) Light
source (14) Microalgae (15) injector (16) bubble (17) high-speed
camera)}

\textbf{2.3 Study on the morphology and motion of microbubbles under
different parameters}

A. \textbf{Operational parameters.} Three operational factors including
pressure, gas-liquid ratio and length of the release pipe were selected
for orthogonal test (3\selectlanguage{ngerman}×3) to determine their effects on the particle
size of microbubbles. The specific working conditions are shown in Table
1. The test process is as follows: The microbubble air flotation
equipment was set and operated in turn under 9 kinds of condition
according to Table 1. (1) Start the device. When the liquid level
reaches H=400 mm, the high-speed photography system is started to shoot
the rising process of microbubbles near H=400 mm. Stop flooding when the
liquid level rises to H=450 mm, and the shooting time is 60s. (2) Clean
the device. After one shooting, open the drainage valve at the bottom of
the air floating column to drain the water, and start the device again
to clean the air floating column, so as not to affect the following test
results. The dynamic video was observed, and the particle size of
bubbles was measured and counted. The proportion of bubbles with
particle size \selectlanguage{english}[?]50 \selectlanguage{greek}μ\selectlanguage{english}m was taken as the experimental index to clarify the
relationship between the main factors and the microbubble particle size
distribution and obtain the optimal working condition.

\textbf{Table 1 The orthogonal experiment table}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Condition number & A: Pressure /MPa & B: Gas-liquid ratio B/\% & C
release pipe's length /cm\tabularnewline
\midrule
\endhead
1 & 0.55 & 6 & 100\tabularnewline
2 & 0.55 & 5 & 50\tabularnewline
3 & 0.55 & 8 & 10\tabularnewline
4 & 0.45 & 6 & 50\tabularnewline
5 & 0.45 & 5 & 10\tabularnewline
6 & 0.45 & 8 & 100\tabularnewline
7 & 0.35 & 6 & 10\tabularnewline
8 & 0.35 & 5 & 100\tabularnewline
9 & 0.35 & 8 & 50\tabularnewline
\bottomrule
\end{longtable}

B. \textbf{The diameter of the release pipe.} On the premise of
determining the optimal working condition, change the diameter of the
release pipe (20, 25, 32 mm) and conduct the test (the test process is
the same as A). The influence of the diameter of the release pipe on the
particle size distribution of microbubbles was clarified, and the
diameter of the release pipe when the particle size of microbubbles was
concentrated was determined.

C. \textbf{The shape of the release pipe (straight or elbow).} Select
the optimal parameters determined in the above tests and set straight
type and elbow type release pipe (with the same straight-line length) as
the investigation factor for the test (the test process is the same as
A). The influence of different types of release pipe on the particle
size distribution of microbubbles was analyzed, and the appropriate type
of release pipe was selected.

Based on the test results of A, B and C, the optimal operating
conditions (pressure, gas-liquid ratio, release pipe's length, diameter,
and type) of the device were determined. The dynamic video of the
floating process of microbubbles was observed under the optimal
conditions. The clear single microbubble and microbubble combination
were selected as the capture objects, and the floating process of the
capture objects at the same time was tracked. The kinematic parameter
curves (velocity, acceleration, and displacement) of the capture objects
were obtained by the high-speed photography system. According to the
kinematic parameter curve, the motion state of the single microbubble
and the combination was analyzed, the correlation between the particle
size of the microbubble and the rising rate was revealed, and the
relationship between the particle size of the single microbubble, the
particle size of the combination and the rising rate was established.

\textbf{2.4 Study on the dynamic rule of ``Adhesion--Depolymerization''
between microbubbles and microalgae}

The following tests were carried out under the optimal conditions based
on the above tests. Firstly, a syringe was used to inject 5 mL
wastewater containing \emph{Microcystis} into the air floating column in
the upward direction. When the liquid level reached H=400 mm, the
high-speed photography system was used to shoot the rising process of
microbubbles and microalgae near H=400 mm. The subsequent procedure is
the same as 2.3. In the video, the size and number of microbubbles and
microalgae was observed, and the adhesion rule between microbubble and
microalgae was summarized.

\textbf{2.5 Optimization ofharvesting performance of microalgae under
the regulation of microbubble flotation}

(1) Performance of microbubble flotation for harvesting microalgae under
different conditions

According to the results of 2.3 and 2.4, the length of the release pipe
was the main influencing factors, the following tests were carried out
under different working conditions with the length of the release pipe
of 10 cm (condition 1), 25cm (condition 2), 50 cm (condition 3), 75 cm
(condition 4), and 100 cm (condition 5), respectively (Table S1). The
concentration of Chla of the wastewater which
contained\emph{Microcystis} was firstly analyzed. Then the flotation
device was installed. 400 mL \emph{microcystis} -containing wastewater
was prepared and transferred to the floating column. Start the device
and stop the water inlet when the liquid level raised to H =450 mm. The
air floating time is 60s. After that, the algal residue was scraped from
the top of the air floating column, and the remaining water sample was
removed from the bottom of the air floating column. 100 mL of the
remaining water sample was used to determine the concentration of Chla
after air flotation. After the completion of the first air flotation,
open the drainage valve at the bottom of the air flotation column to
drain the water, and start the device again to clean the air flotation
column, so as not to affect the test results. The above steps were
repeated 3 times under the same condition. When the experiment under
different conditions was complete, the recovery rate of air flotation
was defined based on the change of chlorophyll a concentration before
and after flotation. The harvesting performance of the microbubble
flotation system under different working conditions was compared and
analyzed.

(2) Effects of microalgae densities on the harvesting performance of
microbubble flotation

According to the results of experiment (1), the optimal condition was
selected, and the experimental device was adjusted accordingly. The
mixture with different microalgae density were prepared as following:
400, 800, 1200, 1600 and 2000 mL of microalgae containing wastewater
were put into five large beakers, respectively. Then 1600, 1200, 800,
400 and 0 mL of fresh wastewater was added into different beakers to
form a concentration gradient of 1:2:3:4:5, numbered 1\textasciitilde{}5
respectively. Then the concentration of Chla in different beakers was
determined. The remaining liquids of each beaker were divided into 3
parts with 400 mL volume each. The flotation experiments of different
microalgae density were conducted as described above. According to the
change of Chla concentration, the effect of algae density on the
harvesting performance was investigated.

\textbf{2.6 Methods}

\textbf{2.6.1 Determination method}

See Supporting Information for the determination of microbubble size and
microbubble-algal size. The content of chlorophyll a was determined by
spectrophotometry.

Algal recovery (\%) = (C1-C2) /C1*100

C1: Chla concentration before air flotation (since the ratio of algal
water to dissolved air water is 1:2, the Chla concentration before air
flotation is 1/3 of the Chla concentration of the original water
sample); C2: Chla concentration after air flotation.

\textbf{2.6.2 Data analysis}

The proportion of bubbles with particle size [?]50 \selectlanguage{greek}μ\selectlanguage{english}m is selected as the
test index to analyze the orthogonal experiment results. With the test
index as the dependent variable, and pressure, gas-liquid ratio, and
length of the release pipe as fixed factors, SPSS19.0 analysis software
is used to analyze the orthogonal experiment results of variance
analysis and the effects of key process parameters on the micro bubbles.

3. Result and Discussion

\textbf{3.1 Influence of key process parameters of air flotation device
on microbubble morphology}

\textbf{3.1.1 Morphological characteristics of microbubbles under
different process parameters}

Smaller bubbles have a higher surface area to volume ratio, which leads
to higher probabilities of collision and attachment, a lower detachment
probability, lower ascending rate and higher free surface energy (Garg
et al., 2014; Hanotu et al., 2012). The optimal bubble size has been
reported as approximately 50 \selectlanguage{greek}μ\selectlanguage{english}m, and efficiency drops significantly with
larger bubbles (Cassell et al., 1975). This was attributed to the
reduced number of bubbles, and thus each bubble is required to remove
more cells resulting in decreased efficiency (Henderson et al., 2008).
Thus, the proportion of microbubbles within 50 \selectlanguage{greek}μ\selectlanguage{english}m was analyzed.

The effects of pressure, gas-liquid ratio, and length of the release
pipe on the particle size of microbubbles are shown in Table 2. The
particle size of the microbubbles produced under the working condition
2/3/4/5/7 are all within 100 \selectlanguage{greek}μ\selectlanguage{english}m, and the proportion of microbubbles
within 50 \selectlanguage{greek}μ\selectlanguage{english}m is more than 74\%. The highest (80.58\%) was obtained under
working condition 2. On the contrary, the particle size of microbubbles
produced by working condition 1/6/8/9 exceeds 100 \selectlanguage{greek}μ\selectlanguage{english}m, and more than 50\%
of the microbubbles have a particle size greater than 50 \selectlanguage{greek}μ\selectlanguage{english}m. In
particular, the particle size distribution range of the microbubbles
produced under working condition 6 is the widest, with more than 80\% of
the microbubbles exceeding 50 \selectlanguage{greek}μ\selectlanguage{english}m in size. When the length of the release
pipe is 10 cm, that is, working condition 3/5/7, the proportion of
microbubbles [?]50 \selectlanguage{greek}μ\selectlanguage{english}m is more than 74\%; When the length of the release
pipe is 100 cm, i.e., working condition 1/6/8, the proportion of
microbubbles [?]50 \selectlanguage{greek}μ\selectlanguage{english}m is less than 41\%.

\textbf{Table 2 Results of particle size distribution of microbubbles}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Condition number & Pressure A/MPa & Gas-liquid ratio B/\% & Length of
the release pipe C/cm & Proportion of microbubbles [?]50 \selectlanguage{greek}μ\selectlanguage{english}m /\% & Size
range of microbubbles /\selectlanguage{greek}μ\selectlanguage{english}m\tabularnewline
\midrule
\endhead
1 & 0.55 & 6 & 100 & 40.97 & 11.29\textasciitilde{}165.63\tabularnewline
2 & 0.55 & 5 & 50 & 80.58 & 8.23\textasciitilde{}85.34\tabularnewline
3 & 0.55 & 8 & 10 & 76.50 & 12.31\textasciitilde{}83.98\tabularnewline
4 & 0.45 & 6 & 50 & 74.95 & 12.16\textasciitilde{}90.99\tabularnewline
5 & 0.45 & 5 & 10 & 78.45 & 11.29\textasciitilde{}84.39\tabularnewline
6 & 0.45 & 8 & 100 & 19.42 & 13.69\textasciitilde{}278.02\tabularnewline
7 & 0.35 & 6 & 10 & 74.76 & 12.42\textasciitilde{}79.8\tabularnewline
8 & 0.35 & 5 & 100 & 39.81 & 14.8\textasciitilde{}109.01\tabularnewline
9 & 0.35 & 8 & 50 & 39.03 & 12.4\textasciitilde{}130.86\tabularnewline
\bottomrule
\end{longtable}

As shown in Fig. 2, the frequency distribution of microbubbles particle
size under 9 working conditions concentrated in the range of
35\textasciitilde{}45 \selectlanguage{greek}μ\selectlanguage{english}m and 55\textasciitilde{}70 \selectlanguage{greek}μ\selectlanguage{english}m. The particle size
of the microbubbles produced in condition 2/3/4/5/7 is concentrated in
the front segment, while the particle size of the microbubbles produced
in condition 1/6/8/9 is concentrated in the rear segment. The main
difference between the two groups mainly lies in the length of the
release pipe (10 cm VS 100 cm). The closer the distance between the
pressure reducing valve and the air floating column, the shorter the
residence time of the released microbubbles in the pipeline. On the one
hand, the collision and merger between the microbubbles are reduced. On
the other hand, the microbubble movement pressure loss is small. The
above two aspects make the microbubbles more stable and the particle
size distribution range is narrow.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2 Frequency distribution of microbubble size under nine
different conditions}

The range analysis of the results shows that the optimal level of each
factor is 0.55 MPa of the pressure, 5\% of the gas-liquid ratio and 10
cm of the length of the release pipe. The order of the range values of
the three factors for the test indexes was length of the release pipe
(43.17) \textgreater{} gas-liquid ratio (21.29) \textgreater{} pressure
(14.82). The length of the release pipe shows obvious influence on the
microbubbles with particle size less than 50 \selectlanguage{greek}μ\selectlanguage{english}m in the floating system.
The P values of pressure, gas-liquid ratio and length of the release
pipe were 0.35, 0.18 and 0.05, respectively. As can be seen from the
F-test results, when the confidence level is 95\%, only the length of
the release pipe is significant to the test index, and there is no
significant difference in the influence of pressure (0.35, 0.45,
0.55MPa) and gas-liquid ratio (5\%, 6\%, 8\%) on the test index.

\textbf{3.1.2 Influence of diameter and shape of the release pipe on the
morphological characteristics of microbubbles}

The effect of the diameter of the release pipe on the particle size
distribution of microbubbles was further explored. It can be seen from
Fig. 3 that when the diameter of the release pipe (25 cm) is the same as
the pipe, the particle size of microbubbles is concentrated between 20
to 60 \selectlanguage{greek}μ\selectlanguage{english}m, and the peak value of the curve appears at 40 \selectlanguage{greek}μ\selectlanguage{english}m. When the
diameter increased to 32 cm, the particle size distribution curve of
microbubbles shifted slightly to the right (20\textasciitilde{}70 \selectlanguage{greek}μ\selectlanguage{english}m),
and the peak value appeared at 45 \selectlanguage{greek}μ\selectlanguage{english}m. When the diameter of the release
pipe decreased to 20 cm, the particle size distribution curve moved to
the right, and the peak value appeared at 60 \selectlanguage{greek}μ\selectlanguage{english}m, and the range of the
particle size became larger (20\textasciitilde{}100 \selectlanguage{greek}μ\selectlanguage{english}m). According to
the cumulative frequency distribution of bubble size (Fig. 3 (b)), the
distribution proportion of microbubbles with particle size less than 50
\selectlanguage{greek}μ\selectlanguage{english}m is 35\% (diameter=20 cm), 71\% (diameter=25 cm) and 67\% (diameter=32
cm), respectively.

In conclusion, when the diameter of the release pipe is the same as that
of the pipe, the proportion of microbubbles with particle size within 50
\selectlanguage{greek}μ\selectlanguage{english}m is the largest, and the particle size distribution is concentrated.
This is because when the dissolved air water is released at the pressure
reducing valve, the diameter of the release pipe increases, which
weakens the turbulence intensity, the spatial density of microbubbles is
relatively reduced, the collision rate between microbubbles is reduced,
and the microbubbles are relatively stable. However, when the diameter
of the release pipe decreases, the resistance of the microbubbles along
the path increases, and the pressure loss of the microbubbles is great.
At the same time, the spatial density of the microbubbles increases
relatively, and the collision probability between the microbubbles
increases, leading to the merger or rupture of some microbubbles. Both
lead to the larger particle size of the microbubbles.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/758355/articles/732202-application-of-microbubble-air-flotation-to-harvest-microcystis-sp-from-agriculture-wastewater-the-regulation-and-mechanisms}

\textbf{Figure 3. Effect of the diameter and type of the release pipe on
the particle size distribution of microbubbles}

The size distribution of microbubbles in straight and bend pipes with
the same length was shown in Fig. 3(C). The particle size of
microbubbles in the straight release pipe is concentrated between
20\textasciitilde{}60 \selectlanguage{greek}μ\selectlanguage{english}m, and the peak value appears at 40 \selectlanguage{greek}μ\selectlanguage{english}m. While the
microbubbles generated in the bend pipes are concentrated in
40\textasciitilde{}80 \selectlanguage{greek}μ\selectlanguage{english}m, and the peak frequency distribution is 55 \selectlanguage{greek}μ\selectlanguage{english}m.
The cumulative curve of microbubble particle size shows that the
proportion of microbubbles with particle size less than 50 \selectlanguage{greek}μ\selectlanguage{english}m (65\%)
generated in the straight release pipe is 20\% higher than that of the
bend type (Fig. 3(D)). In conclusion, the microbubbles generated by the
straight release pipe have small particle size and concentrated
distribution range. When the microbubble moves in the straight pipe, it
is only subject to the resistance along the way, while when it moves in
the release pipe with an elbow, it is also subject to the local
resistance generated by the elbow in addition to the resistance along
the way, resulting in the loss of microbubble pressure in the pipe,
making the microbubble particle size larger. Besides, the elbow
increases the turbulence intensity of the fluid, which increases the
collision probability in the process of microbubble movement, resulting
in the rupture or merger of microbubbles, and the particle size of
microbubbles becomes larger.

\textbf{3.1.3 The relationship between microbubble size and its floating
rate}

Bubble size was also said to determine the shape and rise pattern of the
bubbles (Edzwald, 2010; Eskanlou et al., 2018).The microbubbles (11.29 [?]
bubble size [?] 86.19\selectlanguage{greek}μ\selectlanguage{english}m, average bubble size = 42.83 \selectlanguage{greek}μ\selectlanguage{english}m) generated under
the conditions of pressure of 0.45 MPa, gas-liquid ratio of 5 \% and
release pipe of 10 cm were photographed. A single microbubble with
bubble size = 45.19 \selectlanguage{greek}μ\selectlanguage{english}m and a combination of two microbubbles with
D\textsubscript{b} = 65.0 \selectlanguage{greek}μ\selectlanguage{english}m (50.97 + 52.19) were selected as targets
for dynamic tracking (948 sec\textasciitilde{}1009 sec). The target is
shown in Fig.S2. (Note: D\textsubscript{b} is calculated based on the
total volume of the two small microbubbles, the same below)

The target microbubbles were observed and analyzed during the tracking
period to obtain their acceleration curve (Fig. S3 and S4). Fig.S4(A)
and (B) represent the acceleration of a single microbubble and a
combination of two microbubbles, respectively. The acceleration
detection curve reflects the movement of the target throughout the
tracking period. The positive and negative in the acceleration detection
curve indicate the direction of the acceleration of the target, and the
value indicates the magnitude of its acceleration. By comparing and
analyzing the acceleration of the single microbubble and the target of
the combination, it is found that the acceleration of the two fluctuates
regularly. Whether it is a single microbubble or a combination of two
microbubbles, the magnitude and direction of the acceleration change
throughout the tracking period, which indicates that the resultant force
of the microbubble during the floating process is a variable force, and
the microbubble performs variable acceleration motion.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

\textbf{Figure 4 The relationship between microbubble size and its
floating rate}

The relationship between the particle size and the average rising rate
is shown in Fig.4. As the particle size of the microbubbles increases,
the average rate increases. The relationship between the average rate
and the particle size of a single microbubble is y = 48.056x + 1814.8
(R\textsuperscript{2}=0.73), and the relationship between the average
rate and the particle size of two microbubbles is y=97.123x-1108.8
(R\textsuperscript{2}=0.77). From the correlation coefficient
R\textsuperscript{2}, in the same water flow environment, whether it is
a single microbubble or a combination of two microbubbles, its average
rate is positively correlated with its particle size, and the effect of
microbubble particle size on the average rate is extremely significant.

\textbf{3.2} \textbf{Adhesion efficiency and adhesion mechanism of
microbubbles to microalgae}

\textbf{3.2.1 Adhesion mode of microbubble-algae aggregates}

The adhesion of microbubbles to algae aggregates is shown in Fig. S5.
The adhesion modes of microbubbles to algae include 1-1 (single
bubble-single algae), 2-1 (double bubble-single algae), n-1
(multi-bubble-single algae) and n-n (multi-bubble-multi-algae). The
first two adhesion modes have one thing in common, that is, algae
aggregates are adhered to the lower half of the microbubbles. According
to the theory of the interaction between microbubbles and particles,
this is because during the floating process of microbubbles, tangential
flow is formed around the microbubbles, so that the particles (algae
aggregates) adhered to them are gathered in the lower half of the
microbubbles.

Due to the high density of algae in the experimental water sample
(10\textsuperscript{9}/ mL), the algae cells in the water sample exist
in the form of algae aggregates, and it is difficult to count when the
microbubbles adhere to the algae aggregates in the n-n way. Therefore,
only the proportion of three adhesion modes of 1-1, 2-1 and n-1 was
counted (sample number 1000). When microbubbles adhered
to\emph{Microcystis} , the 1-1 mode accounted for 94.6\%, the 2-1 mode
accounted for 4.7\%, and the n-1 mode accounted for less than 1\%. When
microbubbles adhere to \emph{Microcystis} , the main adhesion mode is
1-1, that is, one microbubble adheres to one algae cell most easily.

\textbf{3.2.2 Size distribution of microbubble and Microcystis}

When the microbubbles adhere to \emph{Microcystis} , the size
distribution of microbubble-algae aggregates is shown in Fig.5. The
particle size of microbubbles in the microbubble-algae complex was in
the range of 10\textasciitilde{}170 \selectlanguage{greek}μ\selectlanguage{english}m, and the size
of\emph{Microcystis} was in the range of 10\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m.
Among them, the proportion of microbubbles with a particle size of
70\textasciitilde{}125 \selectlanguage{greek}μ\selectlanguage{english}m is as high as 87\%, and 85\%
of\emph{Microcystis} are concentrated in 50\textasciitilde{}200 \selectlanguage{greek}μ\selectlanguage{english}m. In
the size-frequency distribution curve of adhered \emph{Microcystis}
(Fig. 5(b)A), the peak frequency of \emph{Microcystis} appeared at 100
\selectlanguage{greek}μ\selectlanguage{english}m (8.5\%), that is, \emph{Microcystis} in the range of
90\textasciitilde{}100 \selectlanguage{greek}μ\selectlanguage{english}m accounted for 8.5\% of the
total\emph{Microcystis} . In the particle size distribution curve of
microbubbles (total microbubbles) adhering to \emph{Microcystis} (Fig.
5(b)B), the peak frequency of microbubble size appeared at 90 \selectlanguage{greek}μ\selectlanguage{english}m
(12.0\%), that is, microbubbles with particle size between
80\textasciitilde{}90 \selectlanguage{greek}μ\selectlanguage{english}m accounted for 12.0\% of the total microbubbles.
In the particle size distribution of microbubbles generated under this
working condition (see Fig. 5(b)C), 45.8\% of the microbubbles were
distributed between 40 and 60 \selectlanguage{greek}μ\selectlanguage{english}m, and the proportion of microbubbles
adhering to \emph{Microcystis} (80\textasciitilde{}90 \selectlanguage{greek}μ\selectlanguage{english}m) was less than
1\%. The frequency distribution peak of the particle size of the adhered
microbubbles is like that of the microalgae. It was reported that
morphology of algae including shape and size also have impacts on the
performance of flotation process when considering the bubble-cell
attachment, in addition, spherical (\emph{Microcystis} ) and oval
(\emph{Chlamydomonas} ) configures had greater flotation efficiencies
than filamentous shape (\emph{Phormidium} ) (Bui et al., 2015).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

\textbf{Figure 5 (a) Distribution curve of microbubble size
and\emph{Microcystis} sp. Size (b) Distribution curve of microbubble
size and \emph{Microcystis} sp. frequency}

\textbf{3.2.3 Response relationship between microbubble size and
Microcystis size}

The particle size ratio distribution of adhered \emph{Microcystis} and
microbubbles is shown in Fig.6 A). The particle size ratio
of\emph{Microcystis} and microbubbles is in the range of
0\textasciitilde{}4.2. When the ratio of the two is between 0 and 1.2,
with the increase of the ratio, the proportion of
microbubble-Microcystis in this range increases from 0.5\% to 22.1\%.
When the ratio increases from 1.2 to 4.2, the proportion of
microbubble-Microcystis in this range gradually decreases. With the
ratio exceeding 2.7, the proportion is less than 2\%. From the
cumulative percentage, the cumulative ratio in the range of
0.6\textasciitilde{}1.5 is as high as 63\%, and the cumulative curve
rises slowly when the ratio is greater than 1.5. When the size
of\emph{Microcystis} is 0.6\textasciitilde{}1.5 times the size of
microbubbles, \emph{Microcystis} is easily adhered by microbubbles, and
the adhesion rate is as high as 63\%. Among them, the probability of
adhesion is the highest when the ratio is between
0.9\textasciitilde{}1.2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

\textbf{Figure 6. (A) ratio of \emph{Microcystis} sp. size to
microbubble size; (B) cluster analysis of \emph{Microcystis} sp. and
microbubble particle size}

The cluster analysis results of the size ratio of \emph{Microcystis} and
microbubble are shown in Fig. 6(B). Class II analysis were carried out
and the clustering centers were 1.03 and 2.24. The proportion of cases
in each clustering center was 76\% and 23\%, respectively. The cluster
centers of the Class III analysis were 0.9,1.67 and 2.89, accounting for
58\%, 34\% and 8\%, respectively. Based on the results of the two types
of analysis, it is known that in the formed microbubble-algae
combination, the proportion of Microcystis/microbubble size ratio of
1.03 and 0.9 is the highest, and both exceed 50\%. When the size
of\emph{Microcystis} is equivalent to the size of microbubbles, the two
are most likely to adhere.

\textbf{3.3 Effects of the microbubble flotation parameters on
microalgae harvest performance}

The performance of microbubble air flotation on
\emph{Microcystis}harvesting under different working conditions is shown
in Fig.7(A). The concentration of Chla was 844 mg/m\textsuperscript{3}
before air flotation, and the concentration of Chla decreased
significantly after microbubble air flotation. Among them, the lowest
Chla concentration after flotation in condition 5 was 308 mg/
m\textsuperscript{3}, and the highest Chla concentration after flotation
in condition 1 was 532 mg/m\textsuperscript{3}. The recovery rate of
\emph{Microcystis} by microbubble air flotation under different working
conditions was sorted from high to low as follows: working condition 5
(63.5\%) \textgreater{} working condition 4 (52\%) \textgreater{}
working condition 2 (50\%) \textgreater{} working condition 3 (41\%)
\textgreater{} working condition 1 (37\%). According to the content of
3.2.1, the distribution peak of \emph{Microcystis} is
90\textasciitilde{}100 \selectlanguage{greek}μ\selectlanguage{english}m, and the adhesion rate is the largest when the
particle size of microbubbles is equal to the size of \emph{Microcystis}
. The proportion of bubbles with a particle size greater than 80 \selectlanguage{greek}μ\selectlanguage{english}m
produced by condition 5 is the largest, and its algae harvesting
efficiency is the highest. The proportion of bubbles with a particle
size of more than 80 \selectlanguage{greek}μ\selectlanguage{english}m produced by condition 1 is the smallest, and its
algae collection rate is the lowest. The proportion of bubbles with a
particle size of more than 80 \selectlanguage{greek}μ\selectlanguage{english}m produced by condition 3 is greater than
that of condition 2, but its algae collection efficiency is 9\% lower
than that of condition 2, which may be caused by the floating of
multiple large bubbles observed during the experiment. Because the
fast-floating speed of large bubbles disturbed the adhesion of
microbubbles to the algae, or caused the desorption of the
microbubble-microcystin aggregates that have adhered, thereby reducing
the algae collection rate. In the process of harvesting algae by
microbubble air flotation, the rate of algae harvesting depends not only
on the adhesion of microbubbles to algae, but also on the influence of
water flow environment.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image17/image17}

\end{center}
\end{figure}

\textbf{Fig. 7} \textbf{Effect of micro-bubbles flotation on
\emph{Microcystis} sp. harvesting under different conditions(A) and
density(B)}

The results of microbubble air flotation treatment of
\emph{Microcystis}with different density under the condition of
condition 5 are shown in Fig. 7(B). It can be seen from the diagram that
with the increase of Chla concentration from 85 mg/m\textsuperscript{3}
to 844 mg/m\textsuperscript{3}, the recovery rate of microbubble air
flotation to \emph{Microcystis} also showed an increasing trend. After
flotation, the Chla concentration of density 1 decreased to 54
mg/m\textsuperscript{3}, and the Chla concentration of density 5
decreased to 126 mg/m\textsuperscript{3}. The lowest algae collection
rate of microbubble air flotation for density 1 was 35.6\%, while the
algae collection rate for density 5 was as high as 63.5\%. The analysis
shows that under low concentration conditions, the number
of\emph{Microcystis} colonies is small, and the probability of adhesion
by microbubbles is small; when the algae concentration increased to 844
mg/m\textsuperscript{3}, the number of \emph{Microcystis} colonies in
the water increased, the probability of microbubbles colliding with them
increased, and the adhesion probability also increased. Microbubble air
flotation has a better harvesting effect on higher
\emph{Microcystis}density.

4.Conclusion

Microbubble air flotation is an effective way for microalgae harvesting.
During the process of microbubble generation, the proportion of
microbubbles with particle size less than 50 \selectlanguage{greek}μ\selectlanguage{english}m is optimal with the
pressure of 0.55 MPa, the gas-liquid ratio of 5 \% and the length of the
release pipe of 10 cm, and the length of the release pipe is the main
influencing factor, followed by the gas-liquid ratio. No matter a single
microbubble or a combination of two microbubbles, they all undergo
variable acceleration motion during the floating process. In the process
of adhesion, when the size of \emph{Microcystis} was
0.6\textasciitilde{}1.5 times of the size of microbubbles, microbubbles
could adhere to 63\% of\emph{Microcystis} . When the size of
\emph{Microcystis} was equivalent to the size of microbubbles (ratio of
1.03 to 0.9), the adhesion probability of the two was the largest (both
more than 50\%). That can also explain that the best the harvesting rate
of \emph{Microcystis} (63.5\%) was obtained under the working condition
5 (pressure 0.45 MPa, gas-liquid ratio 5\% and length of the release
pipe 100 cm) due to the similar size distribution of the microbubbles
and the \emph{Microcystis} in this study. The harvesting performance was
also affected by the\emph{Microcystis} density and the water flow
environment. More investigations would need to be undertaken for
application in large scale with varied concentrations and other
dissolved organic matters.

Acknowledgement

This work was financially supported by the Key Technologies Research and
Development Program (2017YFE0135500); the National Natural Science
Foundation of China (51908355); the Natural Science Foundation of
Shanghai (19ZR1443700); and the Scientific and Innovative Action Plan of
Shanghai (19DZ1204500).

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