Using Novel Nanobubble Technology to Control the

Growth of Marine Algal Species

Lisabeth Stewart

St. Mary’s College of Maryland

Advisor: Dr. Emily Brownlee

A St. Mary’s Project Presented to the Faculty of the

Biology Department of St Mary’s College of Maryland

In Partial Fulfillment of the Requirements for the

Degree of Bachelor of Science in Biology

May 3, 2020
Contents

1 Abstract 3

2 Introduction 4

2.1 What is an algal bloom? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.2 How have harmful algal blooms been mitigated? . . . . . . . . . . . . . . . . . . 8

2.3 What are nanobubbles and how do they affect algal blooms? . . . . . . . . . . . 11

2.4 What algal species will be used in this experiment? . . . . . . . . . . . . . . . . 14

2.5 What do we hypothesize? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3 Materials and Methods 17

3.1 Algal Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.2 Standard Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.3 Controlling Nanobubble Concentrations . . . . . . . . . . . . . . . . . . . . . . . 19

3.4 Exponential Growth Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.5 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.6 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4 Results 22

4.1 Standard Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

4.2 Algal Exponential Growth Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 27

4.3 Experimental Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4.3.1 Verification of Nanobubble Presence . . . . . . . . . . . . . . . . . . . . . 29

4.3.2 Algal Growth Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1
 4.3.3 Algal Photosynthetic Efficiency . . . . . . . . . . . . . . . . . . . . . . . 38

5 Discussion 38

5.1 Verification of nanobubble presence . . . . . . . . . . . . . . . . . . . . . . . . . 38

5.2 Analysis of algal growth rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.3 Analysis of algal photosynthetic efficiency . . . . . . . . . . . . . . . . . . . . . 43

5.4 Extensions of this research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

5.5 Environmental impacts of nanobubble technology . . . . . . . . . . . . . . . . . 46

6 Conclusions 48

7 Acknowledgements 48

2
1 Abstract

With increased nutrient inputs to coastal waters from agricultural and urban runoff, the ex-

cessive growth of single-celled plants called algae has also increased. If these algae reach high

enough concentrations, they can produce toxins harmful to humans, fish, and other wildlife, as

well as depleted oxygen levels. While the best way to stop these harmful blooms is prevention,

research is still needed to find the most effective way to control them once they occur. This

research investigates a new method to control algae growth using nanobubbles. Nanobubbles

oxygenate bodies of water and negatively affect algae growth rates, but previously this has only

been examined observationally. This research is the first systematic laboratory study investigat-

ing how nanobubble technology affects the growth rate of marine algal species. P. minimum,

Chaetoceros sp., Isochrysis sp., and Tetraselmis sp. were exposed to five different dilutions of

nanobubbles for six days. The algal growth rates in maximum nanobubble concentration were

significantly lower than the growth rates without exposure to nanobubbles for all four algal

species. No significant differences in the algal species’ photosynthetic efficiencies were found be-

tween any of the treatment groups. Future studies may want to investigate long-term exposure

to nanobubbles on algal species, as well as the differences in algal response if they are exposed

during stationary growth phases, which would be the prevalent phase of algal growth during an

algal bloom. This research can also be expanded into considering other marine algal species,

but also the effect of nanobubbles on freshwater algal species, as well. Nonetheless, this research

indicates nanobubble treatment may be a viable, safe, and environmentally-friendly mechanism

of algal growth control on a variety of algal species.

3
2 Introduction

2.1 What is an algal bloom?

Algae are simple aquatic plants which range in size from microscopic, single-cellular organisms

to macroscopic seaweeds and kelps. Algae serve as crucial primary producers within aquatic

ecosystems. Almost all algae perform photosynthesis and are consumed by various aquatic

organisms such as zooplankton, frogs, fish, and insects. This herbivory oftentimes regulates the

concentration of algae in a body of water and prevents the algae from becoming too thick in the

water column. While algal species are always present in aquatic environments, elevated nutrients

can cause algae to rapidly multiply within a specific area of water and to produce an algal bloom.

This event is often called a harmful algal bloom (HAB). During an algal bloom, algae are so

concentrated in an aquatic environment that they visibly pigment the water. Oftentimes, algal

blooms reach concentrations of several million algae per liter and form patchy collections of algae

visible on the water’s surface (Shumway, 1990). Such blooms can be green, yellow, brown, or

red in accordance with their primary photosynthetic pigments. Assessing the growth and spread

of these algal blooms is crucial in order to maintain a healthy aquatic ecosystem as algal blooms

use up nutrients and pollute the water column.

Researchers have worked to create extensive prediction models for these blooms in order to

understand them, but many have so far been unsuccessful. Aerial monitoring has oftentimes

been used to provide forewarning of larger algal blooms (Shumway, 1990). Because HABs cause

changes in the coloration of the water, satellite imagery has been used to assess and monitor water

quality and algal bloom management (Wang and Shi, 2008). The ability to predict and monitor

4
algal blooms is crucial in order to prevent them from further harming aquatic ecosystems. Algal

blooms can be detrimental to such ecosystems, disrupting both the water and the economies

which rely on those bodies of water. The United States’ economy loses over two billion dollars

each year paying to repair damages that require water treatment as a result of algal overgrowth.

If the prevention of these algal blooms does not occur, problems such as property devaluation

and declines in the value of fish stocks occur and negatively influence the environment (Pelley,

1998).

Algal blooms can form in both freshwater and saltwater environments, and can lead to neg-

ative impacts on either aquatic ecosystem. They form as a result of an excess of nitrogen and

phosphorus, two crucial nutrients for the growth of photosynthetic plants. Large amounts of

research has investigated the importance of nitrogen and phosphorus on causing algal blooms.

Researchers discovered the removal of the phosphorus and nitrogen limitation during the pre-

bloom period of an algal bloom caused the overgrowth of algae into a HAB (Ding et al., 2018).

This was demonstrated through the study of a historically large algal bloom. In 2011, a record-

setting algal bloom formed on Lake Erie. Researchers determined that this large bloom was a

result of warmer conditions, as well as minimal water flow which decreases the removal of excess

nutrients, both nitrogen and phosphorus, from the ecosystem (Michalak et al., 2013). These

excess nutrients lead to optimal bloom conditions.

The sources of excess nutrients include runoff from the land, which often causes a surplus

of nitrogen and phosphorus in the water column (Testa et al., 2017). Since the 1960s, excess

phosphorus has produced nuisance algal blooms and poor water quality in many aquatic envi-

ronments (Dolan, 1993). In order to combat this, the United States and Canada implemented

5
strategies in order to reduce phosphorus loading (De Pinto et al., 1986). This was met with what

appeared to be early successes, yet researchers quickly realized the approach to lower maximum

amounts of phosphorus waste released by water treatment plants and other corporations was

not going to be successful in the long term; algal blooms continued to expand (Michalak et al.,

2013). Even in the Chesapeake Bay, there has been a marked rise in nitrogen and phosphorus

since World War II, leading to larger fluctuations in yearly algal growth (Paerl et al., 2006).

Additionally, weather events such as hurricanes wash more sediment and runoff into the water

column, changing nitrogen and phosphorus levels leading to higher overall algal growth (Paerl

et al., 2006). All of these factors compound the problem, leading to the large and difficult to

control algal blooms.

Many algal blooms are damaging to the environment and require many resources to remove

effectively from the water because they produce neurotoxins. Understanding the ability of algae

to produce neurotoxins is important because at the high concentrations that algae reach within

a bloom, these toxins can have devastating effects on the ecosystem. Research has investigated

the effect of algal blooms on human health, specifically exploring their neurotoxic potential

and mechanism (Mello et al., 2018). Toxin-producing algae are prevalent with three of the

most common groups of phytoplankton (cyanobacteria, diatoms, and dinoflagellates) producing

neurotoxic metabolites (Mello et al., 2018; Ibrahim, 2007). As algal blooms increase in size,

these potentially neurotoxic compounds are created in larger magnitudes, risking the health of

wildlife and humans when they come into contact with affected waters (Mello et al., 2018). The

full extent of these effects may currently be under characterized, as illnesses as a result of algal

blooms in wildlife and humans are oftentimes ascribed to other causes (Carmichael and Boyer,

6
2016).

At high enough levels, such as those found in an algal bloom, toxic by-products can accumulate

in species’ tissues as toxins are ingested. This can cause the toxins to be transferred wildly across

the aquatic food web. For example, in California it has been shown that the ingestion of the

domoic acid-producing algal diatom, Psuedonitzschia australis and its subsequent ingestion by

the Northern anchovy (Engraulis mordax ), has led to the decreased fecundity of adult California

sea lions (Zalophus californianus) and Northern fur seals (Callorhinus ursinus) (Silvagni et al.,

2005). These toxins can influence survival and growth, as well as fecundity of the species that

consume them (Havens, 2008). For example, in the aforementioned California sea lions and

Northern fur seals, seizures, ataxia, head weaving, decreased responsiveness to stimuli, and

scratching behaviour were all observed as clinical signs these animals had been poisoned by

domoic acid (Gulland et al., 2002). In extreme cases, this can lead to an overall loss in aquatic

biodiversity. Due to the impacts of algal blooms, much research has been dedicated to mitigating

them. The study and management of algal blooms is important in order to understand and

maintain the overall health of aquatic ecosystems.

Allowing algal blooms to routinely persist in bodies of water has additional environmental

impacts, as well. Nutrient deficits are commonly seen as a result of an algal bloom’s overuse

of nutrients from the aquatic ecosystem (Arrigo et al., 2014). Other organisms then struggle

to survive with limited nutrients, or are forced to relocate into different, nutrient-rich areas of

water. Algal blooms also lead to low dissolved oxygen levels known as hypoxic regions which

are characterized by oxygen levels below two parts per million. Hypoxic conditions occur when

dissolved oxygen concentrations are low enough to directly harm the animal and plant life in

7
a particular region (Hagy et al., 2004). This has happened chronically in the Chesapeake Bay

where low dissolved oxygen below the pycnocline makes deep waters unsuitable for many aquatic

species (Testa et al., 2017). Much research has gone into developing methods to alleviate these

problems, but many such methods are costly, ineffective, or negatively affect the ecosystem in

other ways.

2.2 How have harmful algal blooms been mitigated?

While extensive research of methods to control harmful algal blooms has been done, many

of those methods cause additional negative impacts on aquatic ecosystems. Chemicals such as

aquatic herbicides and additive oxidants have been successful at controlling algae growth, yet

these compounds can harm other facets of aquatic environments. In Japan, these chemicals

were used in conjunction with clay. While successful at killing 99% of the algae, the chemical

use was nonetheless regarded as controversial since the full extent of potential damage to other

aquatic organisms was not fully known (Pelley, 1998). In contrast, the use of clay alone, which

causes algal species to flocculate and fall out of the water column, has been considered a more

natural alternative. Three bloom-forming phytoplanktons were studied and found the addition

of kaolin clay led to significant decreases in the in vivo fluorescence in all three algal species,

studied (Brownlee, 2005). In some research, clays are further enhanced with chemical flocculants

like polyaluminum chloride to further increase the clay’s effectiveness at removing algae (Sengco

and Anderson, 2004). While this increases cell mortality, future studies still must investigate

the quantification of toxin removal from the addition of clay. Since these chemicals act by lysing

the algal cells, this can lead to a large release of harmful toxins which can be just as detrimental

8
to the aquatic ecosystem. Therefore, the impact of the chemicals used to control HABs on other

aquatic wildlife must also be carefully considered.

Cadmium, copper, and zinc have all been used in order to control the overgrowth of algae

from a river in Argentina (Magdaleno et al., 2014), yet the impacts of such metals being added

into the water was not fully studied on how it would influence other organisms in the ecosystem.

Additionally, cadmium is a known human carcinogen, and at high enough concentrations can

cause irreversible DNA damage in humans (Jin et al., 2003), making it dangerous to add widely

into the environment to control unwanted algal growth. Similarly, the effect of photosensitizers

has been studied on green algae in order to determine if they could be used to control algal

growth, as they act by disrupting photochemical processes in photosynthetic algae (Pohl et al.,

2014). This becomes a problem, though, when they may disrupt other photosynthetic organisms

such as aquatic plants and marshland plants. This disruption of all primary producers could have

long-lasting and detrimental effects on an aquatic ecosystem. Finally, many other chemicals have

been researched for the use of controlling algal growth, yet these chemicals may also negatively

influence ecosystems. Research has indicated, though, that many chemicals are optimal for one

algal species, making it necessary to add many different chemicals in order to effectively control

algal growth (Nagai et al., 2015). Overall, many chemical mechanisms have been used in order to

control the growth of harmful algal blooms, but have unknown and far-reaching environmental

impacts in other areas (Magdaleno et al., 2014). This makes finding an alternative to these

chemical-based algal control mechanisms crucial.

Another class of additives, named biologically derived substances (BDSs), has also been

studied. Oftentimes these compounds have no, or very low, toxicity to humans, making them

9
potentially safer additives (Shao et al., 2013). Some examples of BDSs are extracts from aquatic

and terrestrial plants such as rotted wood and leaf litter. These all have been shown to have

potential in algal control, but may be overall impractical due to their high cost of preparation and

unknown damaging effects to other, non-target aquatic organisms (Shao et al., 2013). Finally,

the use of barley straw as a method of algal control has also been investigated. The predominant

limitation of this method of algal bloom mitigation is that it leads to reduction in algal density in

weeks to months, and does not seem likely to cause immediate reductions in algal bloom densities

(Brownlee et al., 2003). Much more research would need to be done before these methods could

be widely accepted to not cause more damage than good.

In order to prevent the negative side effects of chemically altering the water with additives in

order to reduce algal growth, researchers have also developed methods of physical manipulation

of the water to decrease algae concentrations. Among current treatment methods, sonication has

received increased attention for algal control because of its apparent low impact on ecosystems

and the environment. Not using chemical additives seems appealing, but there is a lack of

information on the upscaling of ultrasonication devices for harmful algal bloom control on larger

bodies of water, which may ultimately render it impractical (Park et al., 2017). While adequate

in a lab setting, it is difficult to work effectively with high water flow rates and in various water

quality conditions. More research would be necessary to ensure this method could be reliably

applied in different field conditions (Park et al., 2017).

Another method of physical manipulation which has been used involves mixing the strata

of the water to destroy the overgrowth of algae (Visser et al., 2016). This method may also

be impractical as it requires high amounts of maintenance, and the results have been shown

10
to be varied in success (Visser et al., 2016). Moreover, other harmful effects of water mixing

have also not been fully studied. Finally, a third physical manipulation has also been studied.

Aeration, the practice of increasing oxygen content in the water usually with bubbles, has been

used to decrease algal concentrations. While this practice is more environmentally friendly than

additives, it does require large amounts of maintenance, labor, and energy use. Aeration of the

water specifically with ozone works well at controlling algal populations, but can create harmful

hypobromous acid byproducts that harm aquatic life at high enough concentrations (Shin et al.,

2017). Overall, aeration may be a viable option as a method to control algal growth, but it will

require more research. In conclusion, there is no “one-size-fits-all” solution to the problem of

algae overgrowth and HABs; many various methods can use more research and improvements

to work effectively in aquatic ecosystems.

2.3 What are nanobubbles and how do they affect algal blooms?

Since the determination of methods for effectively controlling algal blooms is so important

to the overall health of aquatic ecosystems, many novel methods of algal control are being

investigated. Specifically, research looks for novel algal control methods which do not negatively

impact other organisms within the affected body of water, and are conscious of a societies’ limited

resources. One new technology which may fit both of these criteria is nanobubble technology

developed in Japan which is believed to have potential to effectively reduce algal blooms and

aerate low oxygen areas simultaneously. Nanobubbles are about 30 nm high, and have a radius

of curvature between 100-300 nm (Attard et al., 2002). These bubbles form by cavitation: the

formation of an empty space within a solid object or body. While “regular” (macro) bubbles

11
rapidly rise to the surface of water and pop, nanobubbles can remain stable within the water

column for days. This is due to the nanobubbles’ lower interfacial curvature (Agarwal et al.,

2011). Additionally, nanobubbles’ hard hydrogen bonds similar to those found in ice, also

help nanobubbles to remain formed, even at high pressures within the water column (Agarwal

et al., 2011). When nanobubbles do break, they implode under the pressure of the water above

them, which oftentimes requires a great force to do so. These unique characteristics lead to

nanobubbles’ potential usefulness as a method of algal control.

These characteristics have already been investigated in many practical applications. Nanobub-

bles have been used in different aquatic environments in an attempt to improve water quality.

For instance, they have been shown to degrade toxic compounds, disinfect water, and defoul solid

surfaces because of their ability to create free radicals (Agarwal et al., 2011). Nanobubbles have

also been shown to increase dissolved oxygen and the electric conductivity in water, leading to

the ability to purify waters polluted by blue-green algae (Nakashima et al., 2012). This has only

been examined observationally, thus far, though. In the Seto Inland Sea of Japan, nanobubbles

have been used to control Red Tide algal growth (a harmful algal bloom characterized by its

color). Rapid urban development around this sea led to increased concentrations of nitrogen

and phosphorus in the water. Since the sea has a low flow rate into other bodies of water,

harmful toxic algae can easily overgrow in the water column, leading to declining oxygen levels.

Therefore, maintaining adequate levels of oxygen in the sea keeps harmful algae concentrations

low. Nanobubbles have been used to deliver oxygen to this area in order to keep the sea rich

in oxygen to prevent hypoxic, dead zones from forming, and to limit the overgrowth of algae

through the creation of free oxygen radicals (Nanobubble Solutions Limited Ltd, 2019).

12
 Similar observations have been made in other bodies of water in Japan. Lake Suwa, Lake

Hyoko, and Lake Biwa have all had nanobubble treatments to curb harmful algal bloom growth.

In Lake Suwa, it was used because of the high amount of pollutants regularly present in the water.

It was hoped that nanobubbles would be able to help reduce sludge and increase dissolved oxygen

concentrations at the bottom of the lake which were dangerously low due to HAB formation. In

Lake Hyoko, nanobubbles were used in order to decrease sludge at the bottom of the lake. The

sludge in this lake was from excessive rotting algal matter, as well as the decomposition of bird

feces in the water. Since this lake has been a wildlife refuge since 2005, it was crucial that the

methods used to fix this HAB problem were not going to interfere with or damage any of the

wildlife or the surrounding environment. With these constraints, nanobubbles were the solution,

as the small oxygen bubbles have not been shown to disrupt or harm nature in any way. The

bubbles were able to aerate the bottom of this lake and promote decomposition of the sludge,

as well as a reduction of the high algal concentrations causing the sludge to form in the first

place. As the water quality in this lake improved, species of shellfish which had been unable

to live in this water previously were able to inhabit this lake once more. Finally, Lake Biwa

was also treated with nanobubbles in order to reduce algal overgrowth in this lake. This lake

is popular for recreational activities, and the high concentrations of algae had reportedly made

individuals sick. After treating the lake with nanobubbles, a reduction in the algal concentration

and individual illness was seen (Nanobubble Solutions Limited Ltd, 2019).

Nanobubble technology appears to be a promising endeavor due to its potential at curbing

algal growth and its perceived low-impact on the aquatic ecosystem, low maintenance costs,

and no release of toxins. As such, exploring its ability to increase oxygen concentrations over

13
long periods of time, as well as disrupt algal growth is an exciting new opportunity. Expanding

on the observations of nanobubbles’ effectiveness will allow for the examination of nanobubbles

for controlling algal overgrowth and preventing the formation of algal blooms. Controlled lab-

oratory conditions will allow for the quantification of the changes in algal concentrations as a

result of nanobubbles, without potential confounding factors from external sources in a field

study. Additionally, laboratory-based research has the opportunity to ultimately determine the

potential mechanism of algal control when using nanobubbles. This may occur by examining

the growth rates of algal species, as well as the metabolic and physiological indicator of algal

photosynthetic efficiency (Wang et al., 2017).

2.4 What algal species will be used in this experiment?

In order to characterise the effectiveness of using nanobubbles to control saltwater algal

growth, a total of four variable marine algal species were used in this study. Each of these species

has different characteristics and classifications. Generally, algae are small, aquatic, plants which

function as the main primary producers in many aquatic ecosystems, but ultimately they have

great variation in form and function. A wide variety should be studied when trying to develop

an effective method for HAB control.

For this experiment, two different types of flagellated phytoplankton were studied. The first

one, Isochrysis sp., is a small, brown-gold algae which has two flagella. These flagella allow it

to perform helical movement. It is typically 4-6 µm in diameter, and is an ovoid shape. While

Isochrysis sp. are capable of growing at low phosphorus conditions, they predominantly grow

in high nitrogen conditions (Fernández-Rodrı́guez et al., 2015). This makes them susceptible to

14
overgrowth and the formation of algal blooms in high nitrogen environments. This type of algae

is considered a high-quality food for fish and shrimp because of their large concentrations of

unsaturated fatty acids they contain (Fernández-Rodrı́guez et al., 2015). For this reason, these

algae are often used in aquaculture as food for bivalves, crustaceans, zooplankton, and more.

The second flagellated phytoplankton used was Tetraselmis sp. This green algae varies greatly

in size and shape, but is often between 4-25 µm in diameter and has four flagella allowing

Tetraselmis sp. to easily move within the water column. This motility allows Tetraselmis to

undergo vertical migration, moving up in the water column for increased levels of light in order

to undergo photosynthesis, before moving deeper into nutrient-rich waters (Erga et al., 2003).

Tetraselmis thrives in many different water conditions, including both marine and freshwater.

This versatility also leads to the use of Tetraselmis sp. in many laboratory experiments, due to

its ability to grow quickly, as well as its regular and easily visualized cell division (Trovão et al.,

2019). The inclusion of this algal species provides another unique algal species for this research

to investigate.

Another classification of phytoplankton, dinoflagellates, was also used in this study. Di-

noflagellates are unicellular, with two flagella. Many of these algal species have strains which

contain the ability to produce toxins, which make dinoflagellates environmentally important.

The dinoflagellate used in this study, Prorocentrum minimum, is a medium-sized algae often-

times between 14-22 µm in diameter, with variable shape and is often golden-brown. Addition-

ally, Prorocentrum minimum have trichocysts, specialized filaments used in feeding and defense.

Prorocentrum minimum is metabolically versatile which allows it to be competitive in numerous

marine environments (Mulholland et al., 2018). This makes it an important algal species to

15
consider when conducting research on mechanisms of algal control. Prorocentrum is also an

important dinoflagellate algae due to its large geographic range and potential to harm humans

via shellfish poisoning (Heil et al., 2005). Additionally, it is an annual algal bloom-former within

the Chesapeake Bay and its tributaries (Marshall and Egerton, 2012). Past research has been

done and it is believed that temperature is the primary controlling mechanism of when this

dinoflagellate forms algal blooms in the Bay (Mulholland et al., 2018).

Finally, the fourth algal species investigated was Chaetoceros sp. Chaetoceros sp. are a part

of the largest genus of marine planktonic diatoms. Diatoms have a silica-containing cell wall and

long, thin spines known as setae. They are an important food source within the water column

and form algal blooms which can remain for multiple months because the individual algae can

survive at very low nutrient levels. This algal species has very fast growth and is very nutritious

food for fish and crustaceans in the environment, as well as for aquaculture due to its lipid

accumulation (Lovio-Fragoso et al., 2019). Additionally, Chaetoceros sp. have been shown to be

highly tolerant to viral infection, and in conjunction with the diatom’s fast growth rate suggests

that Chaetoceros sp. may play an important role in maintaining the growth of filter feeders such

as oysters (Tomaru et al., 2018). For these reasons, this algal species was an appropriate model

organism in this study. By working with a diverse set of four algal species, the effectiveness of

the novel nanobubble technology for HAB control can be investigated thoroughly.

2.5 What do we hypothesize?

This research investigates the effect of nanobubble concentrations within algae-rich saltwater

cultures. This study is the first laboratory-based exploration of the effect of nanobubbles on

16
the growth rates of marine algae. It is hypothesized that nanobubbles will significantly reduce

algal growth rates in all four saltwater algal species when compared to cultures not aerated with

nanobubbles at all, based on the reduction of algal presence after nanobubbling lakes and ponds

during field studies on the effect of nanobubbles on algal density (Nanobubble Solutions Limited

Ltd, 2019). Additionally, it is hypothesized that the algal cells will not be made significantly less

metabolically efficient by the nanobubble treatment, as measured by photosynthetic efficiency.

Previous research has indicated algal nutrient availability, as well as light cycle changes influence

photosynthetic efficiency, but it has not been shown to be altered by the physical manipulation

of algal environment (Dubinsky et al., 1990; Sforza et al., 2012; Grobbelaar et al., 1996). It

is believed that the nanobubble exposure will not alter the algal physiology, but instead only

decrease algal growth rates with increasing nanobubble concentrations.

3 Materials and Methods

3.1 Algal Cultures

Isochrysis sp. and Tetraselmis sp. were grown in sterile L1 media without the addition of

silica. The dinoflagellate Prorocentrum minimum, was also grown in sterile L1 media without

added silica, but with the addition of 15 mL of soil supernatant extract (from Carolina Biological

Laboratories) per 1 L of media. Chaetoceros sp. was grown in sterile L1 media, with the addition

of silica. P. minimum, Chaetoceros sp., Isochrysis sp., and Tetraselmis sp. were all obtained

from the Patuxent Environmental and Aquatic Research Laboratory (PEARL) in St. Leonard,

Maryland. Sterile media was made with filtered water from the St. Mary’s River and when

17
needed, the water was diluted with RO water until salinity reached 12ppt. The media was

inoculated separately with the four different algal cultures and were re-inoculated biweekly. All

algal species were grown at 18o C on a 14:10 hour light:dark cycle under white fluorescent light.

3.2 Standard Curves

Before experimental testing began, standard curves of in vivo fluorescence (IVF) vs. cell

density were made for all four species. In vivo fluorescence was measured on a Turner Designs

10–005 field fluorometer. Cell densities were determined using a Sedgewick Rafter Counting Cell

and an Olympus BH-2 compound microscope. Additionally, standard curves of Chlorophyll a vs.

in vivo fluorescence were measured for the four species using a SpectraMax Plus 384 Microplate

Reader Spectrophotometer to measure absorbance. Chlorophyll a extraction protocols involved

adding 1% MgCo3 to 5mL dilutions of algal cultures before the cultures were filtered with

Whatman Gf/F filters. The filter was placed in 90% acetone for 24 hours in the dark at 4o C. The

absorbance of the acetone extract was measured at 663 and 750 nm. Chlorophyll a concentration

was calculated using the following equation (Figure 1). These standard curves were used to

indirectly obtain both cell densities and Chlorophyll a concentration from in vivo fluorescence

for the algal cultures during the experimental testing phase.

Figure 1: Equation used in order to calculate algal Chlorophyll a concentration.

18
3.3 Controlling Nanobubble Concentrations

Nanobubbles were generated from a nanobubbler unit from Nanobubble Solutions Ltd. (Nanobub-

ble Solutions Limited Ltd 2019). The model nanobubbler used in this experiment had a small

nozzle-type diffuser head. The persistence of the nanobubbles from the nanobubbler unit was

quantified before the start of algal experiments.

In order to illustrate nanobubble presence and persistence after nanobubbling media in a

carboy, both qualitative and quantitative methods were used. For this testing, 12 L of media

was placed in a carboy. This carboy was then nanobubbled with oxygen at 29 PSI for 75

minutes using a flow-through pump to move water past the nanobubble nozzle, which was

extruding nanobubbles. The oxygen free radical formation during nanobubbling allows indirect

nanobubble detection via oxidation-reduction potential (ORP) measured with the Accument

AB15 probe from Fisher Scientific, or through hydrogen peroxide concentration using the HYP-

1 Hydrogen Peroxide Test Kit from Hach. ORP is a measure of the free oxygen radicals being

formed via nanobubling, and H2 O2 concentration increases as these radicals react with one

another in water. Therefore, before and after nanobubbling the water, both the oxidation-

reduction potential (ORP) and hydrogen peroxide concentration were measured in order to

quantitatively analyze the presence of nanobubbles. Measurements of H2 O2 concentration were

then taken daily for seven days to assess the persistence and longevity of the nanobubbles in the

media over time. Nanobubble presence was also verified qualitatively using a 532 nm wavelength

laser to visually assess nanobubble presence after bubbling. Since the nanobubbles cause light

to bend and refract, their presence or absence can be seen by the brightness of the laser light as

it passes through the media.

19
 Additionally, the consistency of the concentrations of nanobubbles within the nanobubbled

water was also measured by way of observing the H2 O2 concentration in triplicate replicates

of nanobubbled media. Using the nanobubbled media from the carboy, 200mL of media was

decanted into each of four 250mL erlenmeyer flasks. The H2 O2 concentration of the decanted

media was measured, and the presence of nanobubbled visually assessed with a 532nm laser. This

was performed to determine if the decanting process altered the concentration of nanobubbles.

3.4 Exponential Growth Assessment

Algal cultures were inoculated in 50 mL culture tubes by combining approximately 45 mL of

the appropriate media with 2 mL of each type of algal stock culture. The cultures were then

allowed to grow at 18o C and on a 14 to 10 hour light/dark cycle. Each day, at the same time,

starting on the day of inoculation, the in vivo fluorescence of the culture was measured until the

stationary phase of growth was reached for each culture. With this data, the time it took each

culture to reach exponential and stationary growth was determined.

3.5 Experimental Design

In preparation for experimental data collection, cultures were inoculated and grown in 300mL

of the appropriate media in 2 L flasks until exponential growth phase was reached.

Once cultures were in exponential growth phase, a carboy filled with 12L of sterile L1 media

was pumped through the nanobubbling apparatus and was nanobubbled with oxygen at 29 PSI

for 75 minutes. This media was then decanted into 50mL culture tubes to create 100%, 75%,

50%, 25%, and 0% dilutions of nanobubbled versus non-nanobubbled sterile L1 media. Three

20
replicate tubes were created for each algal species and each concentration of nanobubbles.

Prior to algal inoculation into the various concentrations of nanobubbles, photosynthetic

efficiency of each initial alga was measured in triplicate with a Turner Designs AquaFlash:

Handheld Active Fluorometer. After photosynthetic efficiency measurements were taken, 1 mL

of the culture was added to each of the media and nanobubble treatments. In vivo fluorescence

of each culture tube was measured at the same time each day after a gentle swirling and inversion

to ensure homogeneity of the sample. In vivo fluorescence measurements were then converted

into measures of algal growth rate using the following exponential growth model (Figure 2).

Additionally, at the end of experimental data collection, photosynthetic efficiency ratios were

again measured for each culture tube.

Figure 2: Equation used in order to calculate algal growth rate.

3.6 Statistical Analysis

All statistical analyses were run in R Studio. Standard curves were made using linear model

regressions. A Shapiro-Wilks test of normality was done prior to any parametric statistical anal-

yses being performed. Additionally, Levene’s test was completed before running any ANOVA

tests. A one-way ANOVA was run in order to determine if there was an effect of nanobubble

concentration on algal cell density or an effect of nanobubble concentration on photosynthetic

efficiency. These analyses were run independently for the four algal species. Statistically signif-

icant p-values after one-way ANOVA analysis were followed by Tukey’s post hoc analyses.

21
4 Results

4.1 Standard Curves

Standard curves illustrating linear relationships between algal cell density and in vivo fluores-

cence were created for all four algal species (Figures 3-6). These standard curves demonstrate a

relationship which can be later utilized to indirectly obtain algal cell density measurements from

the direct measurement of algal in vivo fluorescence. All R-squared values were above 0.970.

Figure 3: The relationship between in vivo cell fluorescence versus cell density for Prorocentrum
minimum. The red line represents the linear regression between these two variables (P < 0.001).

22
Figure 4: The relationship between in vivo cell fluorescence versus cell density for Chaetocerous
sp. The red line represents the linear regression between these two variables (P < 0.001).

Figure 5: The relationship between in vivo cell fluorescence versus cell density for Isochrysis sp.
The red line represents the linear regression between these two variables (P < 0.001).

23
Figure 6: The relationship between in vivo cell fluorescence versus cell density for Tetraselmis
sp. The red line represents the linear regression between these two variables (P < 0.001).

A second set of standard curves were also created which illustrate the linear relationship

between algal extracted Chlorophyll a concentration and in vivo fluorescence. These were created

for all four algal species to demonstrate the linear relationship which can be used to indirectly

obtain algal Chlorophyll a concentrations from the direct measure of algal in vivo fluorescence

(Figures 7-10). All R-squared values were above 0.930.

24
Figure 7: The relationship between in vivo cell fluorescence versus Chlorophyll a concentration
for Prorocentrum minimum. The red line represents the linear regression between these two
variables (P < 0.001).

Figure 8: The relationship between in vivo cell fluorescence versus Chlorophyll a concentration
for Chaetocerous sp. The red line represents the linear regression between these two variables
(P = 0.001).

25
Figure 9: The relationship between in vivo cell fluorescence versus Chlorophyll a concentration
for Isochrysis sp. The red line represents the linear regression between these two variables (P <
0.001).

Figure 10: The relationship between in vivo cell fluorescence versus Chlorophyll a concentration
for Tetraselmis sp. The red line represents the linear regression between these two variables (P<
0.001).

26
4.2 Algal Exponential Growth Analysis

Separately, the four algal species were inoculated and allowed to grow in order to calculate the

time it took them in order to reach exponential growth. It was determined that Prorocentrum

minimum, Chaetoceros sp., and Isochrysis sp. took 3 days after inoculation in order to reach

the beginning of exponential growth (Figures 11-13). For Tetraselmis sp., it was determined

that it takes between 2 and 3 days to reach exponential growth when inoculated under the

same conditions as the other algal species (Figure 14). Additionally, it took P. minimum and

Chaetoceros seven days to reach stationary phase of growth, Isochrysis eight days to reach

stationary phase of growth, and Tetraselmis nine days.

Figure 11: Growth curve for P. minimum under ideal conditions.

27
Figure 12: Growth curve for Chaetocerous sp. under ideal conditions.

Figure 13: Growth curve for Isochrysis sp. under ideal conditions.

28
 Figure 14: Growth curve for Tetraselmis sp. under ideal conditions.

4.3 Experimental Data Collection

4.3.1 Verification of Nanobubble Presence

In order to illustrate the presence of nanobubbles in the carboy of media after being nanobub-

bled, oxidation-reduction potential (ORP), pH, and H2 O2 concentration measurements were

taken. ORP and pH were not found to change greatly as a result of these measurements and

were not used as indirect measurements of nanobubble presence. ORP before and after nanobub-

bling was 214 mV and 213 mV. Likewise, pH did not change greatly after nanobubbling; it was

8.61 versus 8.63 before and after nanobubbling the media. Neither measurement was taken

again, as it was not seen to be a reliable predictor of nanobubble presence.

On the other hand, as described above in the methods, H2 O2 concentration was able to

29
indicate nanobubble persistence. Before nanobubbling, the Hach Hydrogen Peroxide Test Kit

indicated a measure of 0.6 mg L-1 of H2 O2 in algal media, whereas after nanobubbling this value

had increased to 1.2 mg L-1 . H2 O2 concentration remained elevated and persisted at 1.0 mg L-1

for 6 days (Figure 15). This increase and six day elevation in H2 O2 concentration was consistent

for repeated trials of nanobubbling 12L of algal media with oxygen at 29 psi for 75 minutes.

Figure 15: Determination of nanobubble persistence by means of measuring hydrogen peroxide

Qualitative measures were also taken in order to verify nanobubble presence and dilution

before continuing with the experiment using a 532 nm laser. Nanobubbled media had a brighter

laser path through the media than the sample without nanobubbles due to the bubbles bending

and refracting the laser light (Figure 16). Additionally, more highly concentrated nanobubble

dilutions shone brighter than lower nanobubble concentrations when visualized with the 532 nm

laser (Figure 17).

30
Figure 16: Image representing the visualization of media after nanobubbling (left) versus without
nanobubbles (right) using a 532 nm laser.

Figure 17: Image representing the visualization of various nanobubble concentrations before
algal inoculation using a 532 nm laser. Nanobubble concentrations are 25%, 50%, 75%, and
100% in increasing concentrations from left to right.

4.3.2 Algal Growth Rate

After six days of daily in vivo fluorescence measurements, algal growth rates were calculated

using the equation for exponential growth (Figure 2). A decrease in algal growth rates for all

31
four algal species at 100% nanobubble concentrations was seen. Before any ANOVA analysis

was performed, Shapiro-Wilks tests of normality and Levene’s tests of equal variation were run.

All p-values for both tests were found to be greater than 0.05, indicating the datasets were not

abnormally distributed and did not have unequal variances, thus fulfilling the assumptions of

one-way ANOVA analysis.

The mean growth rate of Prorocentrum minimum was highest in 0% nanobubbled media and

lowest in 100% nanobubbled media (Figure 18). There was a significant difference in the growth

rates found (Appendix Figure 27, ANOVA, F4,10 = 4.582, p=0.0135). Tukey’s post hoc analysis

revealed that there was a statistically significant reduction in the growth rate of Prorocentrum

minimum between the culture in 100% nanobubbled media and the culture in 0% nanobubbled

media after six days (Figure 19, p=0.009).

Figure 18: Mean logarithmic growth rates of Prorocentrum minimum at different nanobubble
concentrations between 0 and 100% after six days of growth. Error bars represent ± 1 SEM
(N=3).

32
 Figure 19: Results of Tukey’s post hoc analysis for Prorocentrum minimum.

When considering the growth rate of Chaetoceros sp., as nanobubble concentration increased

in the algal cultures, their growth rates decreased (Figure 20). When statistically analyzed, some

of these decreases in algal growth rate were significant (Appendix Figure 28, ANOVA, F4,10 =

9.585, p=0.002). With Tukey’spost hoc analysis (Appendix Figure 30), it was determined that

there was a significant decrease in the algal growth rate between cultures in 0% nanobubbles

and 75% nanobubbles (Figure 21, p=0.027) as well as 0% nanobubbles and 100% nanobubbles

(Figure 21, p=0.004). There was also a decrease in the growth rates between 25% nanobubbles

and 75% (Figure 21, p=0.030) as well as 25% nanobubbles and 100% (Figure 21, p=0.004).

33
Figure 20: Mean logarithmic growth rates of Chaetoceros sp. at different nanobubble concen-
trations between 0 and 100% after six days of growth. Error bars represent ± 1 SEM (N=3).

Figure 21: Results of Tukey’s post hoc analysis for Chaetoceros sp.

34
 For Isochrysis sp., there did not appear to be a distinct trend between algal growth rate and

increasing nanobubble concentration (Figure 22). There were significant differences between the

growth rates of Isochrysis sp. at different concentrations of nanobubbles, though (Appendix

Figure 29, ANOVA, F4,10 = 17.581, p<0.001). Tukey’s post hoc analysis revealed there was a

statistically significant reduction in the growth rate of the Isochrysis sp. for 0%, 25%, 50%, and

75% nanobubbles versus the mean growth rate in 100% nanobubbles (Figure 23, all p-values <

0.005).

Figure 22: Mean logarithmic growth rates of Isochrysis sp. at different nanobubble concentra-
tions between 0 and 100% after six days of growth. Error bars represent ± 1 SEM (N=3).

35
 Figure 23: Results of Tukey’s post hoc analysis for Isochrysis sp.

When considering the growth rate of Tetraselmis sp. there was a trend whereas nanobubble

concentration increased in the algal cultures, growth rate decreased (Figure 24). When statisti-

cally analyzed, it was shown there were significant differences in the Tetraselmis sp. algal growth

rates after six days (Appendix Figure 30, ANOVA, F4,10 = 22.087, p<0.001). Tukey’s post hoc

analysis was performed and showed there was a significant decrease in the algal growth rate of

0% nanobubbles culture versus cultures with 25%, 50%, 75%, and 100% nanobubble concentra-

tions (Figure 25, all p-values < 0.009). Additionally, there were statistically significant decreases

of mean growth rate for 25%, 50%, and 75% nanobubbles versus the 100% nanobubble culture

(Figure 25, all p-values < 0.015). The culture in 100% nanobubbled media had the lowest mean

algal growth rate.

36
Figure 24: Mean logarithmic growth rates of Tetraselmis sp. at different nanobubble concentra-
tions between 0 and 100% after six days of growth. Error bars represent ± 1 SEM (N=3).

Figure 25: Results of Tukey’s post hoc analysis for Tetraselmis sp.

37
4.3.3 Algal Photosynthetic Efficiency

Mean photosynthetic efficiency was calculated for all algal species before and after experi-

mental data collection. There was no trend among the mean photosynthetic efficiencies for each

of the four algal species (Figure 26). No significant differences were found among photosyn-

thetic efficiencies between treatments for each algal species (Appendix Figures 31-34, ANOVA,

all F-statistics5,12 < 3 and all p-values > 0.05.).

Figure 26: Mean photosynthetic efficiency of all four algal species before and after experimental
data collection at varying nanobubble concentrations.

5 Discussion

5.1 Verification of nanobubble presence

The first challenge when designing the experimental protocols for this laboratory study was

how one would be able to quantitatively verify the presence of nanobubbles which are by nature

of their size, invisible to the human eye. Without being able to verify the persistence and

concentration of nanobubbles, one would be unable to efficiently determine if what we believed

the four algal species were being exposed to was in fact the expected independent variable.

In order to address this preliminary question, trials involving Oxidation-Reduction Potential

38
measurements (ORP) were performed. It was expected that as more nanobubbles were pumped

into the algal media, the ORP would increase, since the process of creating nanobubbles also

creates free oxygen radicals (Agarwal et al., 2011). This gathered data was inconclusive, though,

as not all trials after nanobubble exposure led to an increase in ORP. Many cases, in fact, no

change or a decrease in ORP was measured. This prevented accurate data determinants of the

nanobubbles’ persistence from taking place.

It was at this time that we began to utilize a 532 nm wavelength laser in order to qualitatively

determine nanobubble presence. If the nanobobbler was not working as expected and was not

creating nanobubbles, this could have caused the inability to detect differences in ORP before

and after nanobubbling media. With the use of a 532 nm wavelength laser, there were definite

qualitative differences in the media before and after nanobubbling. Before bubbling, the laser

light was dim and shone straight through the media (Figure 16). After nanobubbling, though, the

laser light being shone through the media is much brighter as the curvature of the nanobubbles

scatters and refracts the laser light (Davis, 1955). This verification of nanobubble presence,

while qualitative in nature, provided verification that nanobubbles were being formed, and the

nanobubbler unit was working as expected.

After verifying nanobubbles were being formed, another quantitative measure for the presence

of nanobubbles was sought out. We predicted since oxygen radicals are highly reactive, that the

ORP measurements were not consistent and reliable for what we wanted as the radicals were

immediately reacting with one another and water in the algal media. Instead, we considered the

use of H2 O2 concentration as an indirect measure of nanobubble presence. When the oxygen

radicals are created, they immediately react with water to form O2 and H2 O2 (Halliwell and C.,

39
1984). Knowing this, we began to measure H2 O2 concentration after nanobubbling algal media

as a quantitative measurement of nanobubble presence. This measurement was found to be con-

sistent between trials, and allowed nanobubble consistency and persistence to be quantitatively

measured. After algal media was nanobubbled, H2 O2 concentration jumped to 1.2 mg L-1 and

remained elevated for 6 days (Figure 15), which is how long future experiments with were run

for to ensure continual nanobubble exposure in the algal media.

5.2 Analysis of algal growth rate

After six days of exposure, mean algal growth rates were calculated and analysed with one-way

ANOVA and subsequent Tukey’s post hoc analyses. All four algal cultures showed a significant

reduction in mean algal growth rate in 100% nanobubbled media in comparison to the mean

growth rate without any nanobubbles. This indicates some amount of algal growth control for

all four different species when exposed to 100% nanobubbled media. This aligns with the previ-

ous observational studies which indicated a reduction in algal presence after nanobubbling the

affected area for 24 hours (Nanobubble Solutions Limited Ltd, 2019). For one algal species, Pro-

rocentrum minimum, comparing the mean growth rates of 0% versus 100% nanobubbled media

was the only significant difference seen between all treatment groups (Figure 18). Interestingly,

this indicates Prorocentrum minimum is able to remain unaffected by nanobubbles, except at

the highest concentration of exposure. This may have been seen as Prorocentrum minimum is

known to be a species resistant to many changing environmental parameters and is known to be

a hearty algal species (Tango et al., 2005; Heil et al., 2005). This would align with the results

that the 100% nanobubbled media was the only one to significantly decrease the growth rate of

40
Prorocentrum minimum. Other proposed methods of algal control such as tannic acid (Jeong

et al., 2016) and the oxidizing biocide chlorine (Ebenezer and Ki, 2013) have similarly only been

effective against Prorocentrum minimum at the highest concentrations of exposure tested.

Similarly, the Isochrysis algal species showed that the mean growth rate for the algae in

the 100% nanobubbled media was significantly lower than the mean growth rate for any other

treatment group (Figure 20). There was no effect of 25, 50, or even 75 percent nanobubbled

media in comparison to 0% nanobubbled media on the mean growth rates of Isochrysis, yet at

the final highest concentration of nanobubbles, a reduction in growth rate was seen. Similarly

to Prorocentrum minimum, here we see that Isochrysis remains unaffected by nanobubbles until

exposure to 100% nanobubbled media. Isochrysis sp. are similarly hearty and resistant to many

ecological parameters such as salinity, temperature, and light conditions (Kaplan et al., 1986).

Additionally, research has shown Isochrysis sp. were the most resistant algal species studied

when examining the effectiveness of electrolysis treatment to control algal blooms (Wijesekara

et al., 2006). This algal species was also shown to be susceptible to chloramphenicol, flordenocol,

and thiamphenicol, but only had moderate growth inhibition effects modeled and was not the

most susceptible organism studied (Lai et al., 2009). In this study, while Isochrysis sp. was

significantly affected by the nanobubble exposure, there was no effect of 25, 50, or even 75

percent nanobubbled media in comparison to 0% nanobubbled media on the mean growth rates

of the organism. Similar to past research, Isochrysis sp. is affected by methods of algal growth

control of moderate intensity, making it reasonable this was not the species either most or least

affected by the nanobubble treatment.

Select algal species showed that lower nanobubble concentrations were effective at controlling

41
growth rates. For instance, Tetraselmis mean growth rates in the presence of any nanobubbles

were significantly decreased (Figure 21). Additionally, the mean growth rate of Tetraselmis ex-

posed to 100% nanobubbled media was also significantly different from all other mean growth

rates for this algal species. This illustrates that not only is 100% nanobubbled media signifi-

cantly decreasing Tetraselmis mean growth rates, but that any amount of nanobubbles are also

negatively influencing Tetraselmis mean growth rates. Tetraselmis sp. are not the most adapt-

able organisms compared to Prorocentrum minimum and Isochrysis sp. Though they have the

ability to grow in both freshwater and saltwater, light and nutrient availability may easily limit

their optimal growth (Trovão et al., 2019; Sun and Blatchley, 2017). Additionally, Tetraselmis

sp. have been shown to be effectively controlled and have their growth rate decreased with

moderate amounts of medium-pressure UV irradiation (Liu et al., 2016), chloramphenicol (Lai

et al., 2009), and aeration conditions (Rigobello-Masini et al., 2006). This renders it likely that

aeration via nanobubbling would also negatively affect Tetraselmis sp. growth rates as seen in

this research.

Chaetoceros mean algal growth rates were also negatively impacted by increasing nanobubble

concentrations. When exposed to 75 or 100 percent nanobubbled media, Chaetoceros sp., dis-

played a significant reduction in mean algal growth rate compared to Chaetoceros growth when

exposed to 0 or 25 percent nanobubbled media (Figure 19). In this instance, nanobubbles are

a potential means of algal control at concentrations of 75 and 100 percent nanobubbles for this

species. Chaetoceros sp. growth has been controlled by the addition of sodium hypochlorite and

ferric in any amount (Deka et al., 2018), as well as sedimented clays (Archambault et al., 2004),

making it less harty to algal growth control methods, aligning with its similar susceptibility to

42
growth rate reduction and algal bloom control via nanobubble technologies.

For all four of the marine algal species studied, nanobubbles at varying concentrations were ef-

fectively able to reduce mean algal growth rates. This aligns with our initial hypothesis given, and

supports the observational results, which indicated a reduction in algal growth after nanobub-

bling in Lake Suwa, Lake Hyoko, and Lake Biwa (Nanobubble Solutions Limited Ltd, 2019).

These results allow us to reject the study’s null hypothesis, that nanobubbles in any concen-

tration have no effect on mean algal growth rate for P. minimum, Chaetoceros sp., Isochrysis

sp., and Tetraselmis sp., opening up the possibility for many future research studies to continue

investigating how this novel nanobubble technology can be used as a method of algal growth

control.

5.3 Analysis of algal photosynthetic efficiency

The null hypothesis was unable to be rejected when considering differences in mean algal

photosynthetic efficiency before and after nanobubble exposure. It was found that none of the

four algal species had significant differences in mean photosynthetic efficiency before or after

experimentation as a result of varying nanobubble concentration (Figure 22). This indicates

that the nanobubbles are not altering a mechanism or physiological pathway which prevents the

algal species from working at maximum metabolic capacity. Instead, the nanobubbles are likely

physically disrupting the algal cell membranes to limit and decrease algal growth rates.

This was expected as physical methods of controlling various algal species have not previously

been shown to alter algal photosynthetic efficiency. Research investigating the effect of aeration

and mixing algae on algal photosynthetic efficiency showed that it only altered photosynthetic

43
efficiency when it simultaneously led to more light availability for the algal species due to moving

the algae towards the surface of a lake or pond, where light was more prevalent (Holmes et al.,

2019; Perin et al., 2019). Other research showed changes in algal photosynthetic efficiency in-

volved altering these algal light cycles, decreased light and increased dark cycles both disrupted

algal ability to photosynthesize (Sforza et al., 2012; Grobbelaar et al., 1996; Sutherland, Monte-

mezzani, Howard-Williams, Turnbull, Broady and Craggs, 2015). Blue light was also shown to

be less effective for algal use in photosynthesis, decreasing photosynthetic efficiency (?). As the

light conditions were held constant for all trials and nanobubble concentrations, no alteration

in photosynthetic efficiency was expected in this study. Additionally, white fluorescent light has

been shown to be efficient for algal growth and was used in this research for that reason.

Photosynthetic efficiency has also been shown to be altered with increased nutrient availabil-

ity (Dubinsky et al., 1990; Sutherland et al., 2014) and increased carbon dioxide (Sutherland,

Howard-Williams, Turnbull, Broady and Craggs, 2015). Both of these things led to an increase

in photosynthetic efficiency, as the algae no longer had nutrient limits to their metabolic capac-

ities. While nutrient availability was also held constant during this experiment, this research

may lead to interesting implications when considering algal blooms. As excess nutrient avail-

ability is what leads to the formation of most algal blooms (Ding et al., 2018), this would lead

to an increase in the photosynthetic efficiency of the algae within algal blooms. Since nutrient

availability and carbon dioxide was not altered in this experiment, photosynthetic efficiency was

hypothesized to not be affected as we saw in this experiment (Figure 22).

Both light and nutrient conditions directly relate to the metabolic capacity of photosynthetic

organisms such as algae, yet other compounds are also able to alter it. In some cases chemicals

44
and compounds able to enter the algal photosystems have disrupted efficient photosynthesis

due to accumulation in the photosystems. Salt (Liu et al., 2012) and microplastics (Zhang

et al., 2017) are two such substances which have been shown to enter and accumulate in algal

photosystens, in both cases reducing algal photosynthetic efficiency. Nanobubbles do not share

the ability to intercalate into the algal photosystems, though, making them unable to alter

photosynthetic efficiency in this experiment. For these reasons, it is concluded that a physical

disruption of algal cell membranes is occurring and leading to a decrease in mean algal growth

rate when exposed to nanobubbles.

5.4 Extensions of this research

Moving forward, there are many other ways the use of nanobubbles can be investigated as

a novel control mechanism for algal overgrowth. Future studies should investigate and develop

methods for longer-term nanobubble exposure for algal cultures. The nanobubbling unit could

only bubble a minimum volume of 12 L, so we were unable to re-nanobubble the individual 50 mL

cultures, which limited the study to 6 days (the amount of time nanobubbles persist). If the algal

cultures were nanobubbled for a longer period of time, we may have seen more drastic decreases

in algal growth rates, or larger effects on growth rate even at lower nanobubble concentrations.

This would require scaling up the size of the cultures being used and nanobubbled, but may

result in a more accurate model of nanobubbling used in a larger lake or pond with a continual

flow-through nanobubbling system.

Additionally, in order to mimic an algal bloom more closely, algal cultures in stationary

phase of growth instead of exponential growth should be examined. When an algal bloom is

45
already established, the algae are not growing exponentially at that point, and have instead

formed a thick film on the surface of the water and in the water column. Once algae have

formed this thick film on a lake or pond, it is often then that the previously done observational

studies nanobubbled the water, and at that point the algae were in a stationary phase of growth

(Nanobubble Solutions Limited Ltd, 2019). Understanding how this alters the results seen in this

experiment would be beneficial, as it would allow the determination of if there are differences in

the nanobubble technology’s effectiveness when the nanobubbling occurs during different phases

of growth for the harmful algal bloom. Finally, expanding this research to more marine algal

species, as well as freshwater algal species would indicate the adaptability of this new technology

to many different bodies of water and types of algal blooms. If this new technology can be used

in a variety of situations and times, it could be seen as even more effective and versatile than

other established methods of algal growth control.

5.5 Environmental impacts of nanobubble technology

While the overall adaptability and usefulness of this new technology to myriad algal species

in both salt and fresh water has not yet been established, this research indicates nanobubble

treatment, which is safe and environmentally-friendly, may be a viable mechanism of algal growth

control. It is important to note that many of the currently used methods of controlling algal

blooms may cause negative and long-lasting impacts on aquatic ecosystems, which have not

been fully studied. Chemical control mechanisms such as additive oxidants, aquatic herbicides,

heavy metals, photosensitizers, and other chemicals have been linked to secondary pollution

and non-target organism toxicity in the aquatic ecosystems (Magdaleno et al., 2014). For these

46
reasons, the minimal environmental impacts of nanobubbling make it an appealing choice for

algal bloom mitigation and control.

Since nanobubbling with oxygen does little more than aerate a body of water with small,

oxygen-dense bubbles, it is not currently believed to have adverse effects on other aspects of

the environment it has been used in. This leaves nanobubbling as a potential harmless method

of algal bloom control, if shown to be effective at reducing algal growth rates on larger scales.

These nanobubbles, while not entirely understood how they mechanistically disrupt algal growth

rates, have been shown to be effective at decreasing the mean growth rate in a variety of marine

algal species, P. minimum, Chaetoceros sp., Isochrysis sp., and Tetraselmis sp.. There is no risk

of harming other wildlife or humans with this technology, making it safe and versatile for algal

overgrowth control.

In fact, this low-impact method of oxygenating bodies of water may even be of benefit to ma-

rine ecosystems in other ways, as well. Low dissolved oxygen negatively affects the development

of many aquatic larvae (Chan et al., 2007), adversely influences predation and food availability

in fish (Breitburg et al., 1994), and has decreased aquatic organisms’ long-term survival (Breit-

burg, 1994). Research has shown that Prorocentrum minimum algal blooms in particular are a

cause of short-term oxygen stress in aquatic ecosystems, which is a stressor on oyster spat and

other living organisms (Brownlee et al., 2005). Other algal bloom-forming species have been

shown to lead to low oxygen conditions and similar environmental stresses, as well (Hagy et al.,

2004). For these reasons, this proposed method of algal bloom mitigation also would alleviate

the predominant negative stressor on the surrounding organisms of low oxygen conditions caused

by algal bloom formation making it an appealing choice. Moving forward, more research needs

47
to investigate the ability of nanobubble technology to effectively control algal bloom formation,

yet these compiled results appear positive for this technology’s future at both controlling algal

overgrowth, as well as alleviating low oxygen conditions via oxygen aeration.

6 Conclusions

In summary, novel nanobubble technology was able to effectively decrease algal growth rates

over the six days of experimental testing. Algal cultures grown in 100 percent nanobubbled media

consistently showed a significant decrease in mean algal growth rate, with some algal species

showing significant decreases in mean growth rates at lower nanobubble concentrations, as well.

Significantly, this was seen in all four different marine algal species which were studied, showing

this technology’s versatility and potential applicability to a wider variety of algal species in order

to effectively contain harmful algal blooms as they form in the water column. Additionally, this

research is important as it provides a safe, environmentally-friendly mechanism of algal growth

control. The nanobubbles are merely small oxygen-filled bubbles, and will not cause long-lasting

effects on the environment or not harm other marine species, unlike other methods of algal

growth control which are currently widely practiced.

7 Acknowledgements

I would like to thank Dr. Brownlee for her guidance and support throughout the duration

of this project, as well as St. Mary’s College of Maryland for financing this research. I would

also like to thank Dr. Kevin Sellner at Hood College for loaning me the nanobubbling unit,

48
as well as their invaluable assistance developing the experimental design and protocols used.

Additionally, I would like to recognize Richard Lacouture at PEARL Laboratories for providing

P. minimum, Chaetoceros sp., Isochrysis sp., and Tetraselmis sp. algal cultures, as well as

advice on how to effectively culture those algal species. Finally, I would like to thank Jaimie

Devlin for keeping my algae alive and performing fluorescence measurements while I was away

at grad school interviews, and Shale Beharie for keeping me company while I counted thousands

of algae and ran hundreds of samples in the fluorometer, as well as for when he drove to St.

Mary’s to check on my algae when I was worried they would die over winter break.

49
Appendix

Figure 27: ANOVA table results for analysis of mean growth rates of Prorocentrum minimum
at different nanobubble concentrations.

Figure 28: ANOVA table results for analysis of mean growth rates of Chaetoceros sp. at different
nanobubble concentrations.

Figure 29: ANOVA table results for analysis of mean growth rates of Isochrysis sp. at different
nanobubble concentrations.

Figure 30: ANOVA table results for analysis of mean growth rates of Tetraselmis sp. at different
nanobubble concentrations.

I
Figure 31: ANOVA table results for analysis of photosynthetic efficiency of Prorocentrum mini-
mum at different nanobubble concentrations.

Figure 32: ANOVA table results for analysis of photosynthetic efficiency of Chaetoceros sp. at
different nanobubble concentrations.

Figure 33: ANOVA table results for analysis of photosynthetic efficiency of Isochrysis sp. at
different nanobubble concentrations.

Figure 34: ANOVA table results for analysis of photosynthetic efficiency of Tetraselmis sp. at
different nanobubble concentrations.

II
References

Agarwal, A., W. J. Ng and Y. Liu. 2011. “Principle and applications of microbubble and

nanobubble technology for water treatment.” Chemosphere 84:1175–1180.

Archambault, M. C., V. M. Bricelj and D. M. Anderson. 2004. “Effects of suspended and

sedimented clays on juvenile hard clams, Mercenaria mercenaria, within the context of harmful

algal bloom mitigation.” Marine Biology 144:553–565.

Arrigo, K. R., D. K. Perovich, R. S. Pickart, Z. W. Brown, G. L. van Dijken, K. E. Lowry, M. M.

Mills, M. A. Palmer, W. M. Balch, N. R. Bates, C. R. Benitez-Nelson, E. Brownlee, K. E.

Frey, S. R. Laney, J. Mathis, A. Matsuoka, B. Greg Mitchell, G.W.K. Moore, R. A. Reynolds,

H. M. Sosik and J. H. Swift. 2014. “Phytoplankton blooms beneath the sea ice in the Chukchi

sea.” Deep Sea Research Part II: Topical Studies in Oceanography 105:1–16.

Attard, P., M. P. Moody and J. W. G. Tyrrell. 2002. “Nanobubbles: the big picture.” Physica

A: Statistical Mechanics and its Applications 314(1):696–705.

Breitburg, D. L. 1994. “Behavioral response of fish larvae to low dissolved oxygen concentrations

in a stratified water column.” Marine Biology 120:615–625.

Breitburg, D. L., N. Steinberg, S. DuBeau, C. Cooksey and E. D. Houde. 1994. “Effects of

low dissolved oxygen on predation on estuarine fish larvae.” Marine Ecology Progress Series

104:235–246.

Brownlee, E. F. 2005. “The use of clay to remove algal blooms from Chesapeake Bay waters.”

Proceedings of OCEANS 2005 MTS/IEEE pp. 866–872.

i
Brownlee, E. F., S. G. Sellner and K. G. Sellner. 2003. “Effects of Barley Straw (Hordeum

vulgare) on Freshwater and Brackish Phytoplankton and Cyanobacteria.” Journal of Applied

Phycology 15:525–531.

Brownlee, E. F., S. G. Sellner and K. G. Sellner. 2005. “Prorocentrum minimum blooms:

potential impacts on dissolved oxygen and Chesapeake Bay oyster settlement and growth.”

Harmful Algae 4:593–602.

Carmichael, W. W. and G. L. Boyer. 2016. “Health impacts from cyanobacteria harmful algae

blooms: Implications for the North American Great Lakes.” Harmful Algae 54:194–212.

Chan, H. Y., W. Z. Xu, P. K. S. Shin and S. G. Cheung. 2007. “Prolonged exposure to low dis-

solved oxygen affects early development and swimming behaviour in the gastropod Nassarius

festivus (Nassariidae).” Marine Biology 153:735–743.

Davis, G. E. 1955. “Scattering of Light by an Air Bubble in Water.” Journal of the Optical

Society of America 45:527–581.

De Pinto, J. V., T. C. Young and L. M. McIlroy. 1986. “Great lakes water quality improvement.”

Environmental Science & Technology 20(8):752–759.

Deka, B. J., S. Jeong, S. A. Tabarabai and A. K. An. 2018. “Mitigation of algal organic

matter released from Chaetoceros affinis and Hymenomonas by in situ generated ferrate.”

Chemosphere 206:718–726.

Ding, S., M. Chen, M. Gong, X. Fan, B. Qin, H. Xu, S. Gao, Z. Jin, D. C. W. Tsang and C.

ii
 Zhang. 2018. “Internal phosphorus loading from sediments causes seasonal nitrogen limitation

for harmful algal blooms.” Science of The Total Environment 625:872–884.

Dolan, D. M. 1993. “Point Source Loadings of Phosphorus to Lake Erie: 1986–1990.” Journal

of Great Lakes Research 19(2):212–223.

Dubinsky, Z., N. Stambler, M. Ben-Zion, L. R. McCloskey, L. Muscatine and P. G. Falkowski.

1990. “The effect of external nutrient resources on the optical properties and photosynthetic

efficiency of Stylophora pistillata.” Proceedings of the Royal Society 239:231–246.

Ebenezer, V. and J. Ki. 2013. “Physiological and biochemical responses of the marine dinoflag-

ellate Prorocentrum minimum exposed to the oxidizing biocide chlorine.” Ecotoxicology and

Environmental Safety 92:129–134.

Erga, S. R., M. Dybwad, Ø. Frette, J. K. Lotsberg and K. Aursland. 2003. “New aspects

of migratory behavior of phytoplankton in stratified waters: Effects of halocline strength

and light on Tetraselmis sp. (Prasinophyceae) in an artificial water column.” Limnology and

Oceanography 48(3):1202–1213.

Fernández-Rodrı́guez, M., C. Hidalgo-Lara, A. Jiménez-Rodrı́guez and L. Serrano. 2015.

“Bloom-Forming Microalgae in High-Species Phytoplankton Assemblages Under Light-

Fluctuating and Low Phosphate Conditions.” Estuaries & Coasts 38(5):1642–1655.

Grobbelaar, J. U., L. Nedbal and V. Tichy. 1996. “Influence of high frequency light/dark

fluctuations on photosynthetic characteristics of microalgae photoacclimated to different light

intensities and implications for mass algal cultivation.” Journal of Applied Phycology 8:335–

343.

iii
Gulland, F. M. D., M. Haulena, D. Fauquier, G. Langlois, M. E. Lander, T. Zabka and R. Duerr.

2002. “Domoic acid toxicity in Californian sea lions (Zalophus californianus): clinical signs,

treatment and survival.” Veterinary Record 150:475–480.

Hagy, J. D., W. R. Boynton, C. W. Keefe and K. V. Wood. 2004. “Hypoxia in Chesapeake

Bay, 1950–2001: Long-term change in relation to nutrient loading and river flow.” Estuaries

27(4):634–658.

Halliwell, B. and Gutteridge J. M. C. 1984. “Oxygen toxicity, oxygen radicals, transition metals

and disease.” Biochemical Journal 219:1–14.

Havens, K. E. 2008. “Cyanobacteria blooms: effects on aquatic ecosystems.” pp. 733–747.

Heil, C. A., P. M. Glibert and C. Fan. 2005. “Prorocentrum minimum (Pavillard) Schiller.”

Harmful Algae 4(3):449–470.

Holmes, B., M. B. Paddock, J. S. VanderGheynst and B. T. Higgins. 2019. “Algal photosynthetic

aeration increases the capacity of bacteria to degrade organics in wastewater.” Biotechnology

and Bioengineering 117:62–72.

Ibrahim, A. M. 2007. “Review of the Impact of Harmful Algae Blooms and Toxins of the World

Economy and Human Health.” Egyptian Journal of Aquatic Research 33:210–223.

Jeong, B., E. Jeong, J. M. Malazarte and Y. Sin. 2016. “Physiological and Molecular Response of

Prorocentrum minimum to Tannic Acid: An Experimental Study to Evaluate the Feasibility

of Using Tannic Acid in Controling the Red Tide in a Eutrophic Coastal Water.” International

Journal of Public Health 13:503–516.

iv
Jin, Y.H., A.B. Clark, R.J.C. Slebos, H. Al-Refai, J.A. Taylor, M.A. Kunkel, T.A.and Resnick

and D.A. Gordenin. 2003. “Cadmium is a mutagen that acts by inhibiting mismatch repair.”

Nature Genetics 34:326–329.

Kaplan, D., Z. Cohen and A. Abeliovich. 1986. “Optimal growth conditions for Isochrysis

galbana.” Biomass 9:37–48.

Lai, H., J. Hou and C. Chen. 2009. “Effects of chloramphenicol, florfenicol, and thiamphenicol on

growth of algae Chlorella pyrenoidosa, Isochrysis galbana, and Tetraselmis chui.” Ecotoxicology

and Environmental Safety 72:329–334.

Liu, L., X. Chu, P. Chen, Y. Xiau and J. Hu. 2016. “Effects of water quality on inactiva-

tion and repair of Microcystis viridis and Tetraselmis suecica following medium-pressure UV

irradiation.” Chemosphere 163:209–216.

Liu, W., Y. Ming and Z. Huang. 2012. “Inhibitory effects of hypo-osmotic stress on extracellu-

lar carbonic anhydrase and photosynthetic efficiency of green alga Dunaliella salina possibly

through reactive oxygen species formation.” Biochemistry 54:43–48.

Lovio-Fragoso, J. P., C. Hayano-Kanashiro and J. A. López-Elı́as. 2019. “Effect of different

phosphorus concentrations on growth and biochemical composition of Chaetoceros muelleri.”

Latin American Journal of Aquatic Research 47(2):361–366.

Magdaleno, A., C.G. Velez, M. T. Wenzel and G. Tell. 2014. “Effects of Cadmium, Copper and

Zinc on Growth of Four Isolated Algae from a Highly Polluted Argentina River.” Bulletin of

Environmental Contamination and Toxicology 92:202–207.

v
Marshall, H. G. and T. A. Egerton. 2012. Bloom and toxin producing algae in Virginia tidal

rivers. In 2012 Oceans. Hampton Roads, VA: IEEE pp. 1–5.

Mello, F. D., N. Braidy, H. Marçal, G. Guillemin, S. M. Nabavi and B. A. Neilan. 2018.

“Mechanisms and Effects Posed by Neurotoxic Products of Cyanobacteria/Microbial Eukary-

otes/Dinoflagellates in Algae Blooms: a Review.” Neurotoxicity Research 33(1):153–167.

Michalak, A. M., E. J. Anderson, D. Beletsky, S. Boland, N. S. Bosch, T. B. Bridgeman, J. D.

Chaffin, K. Cho, R. Confesor, I. Daloğlu, J. V. DePinto, M. A. Evans, G. L. Fahnenstiel, L. He,

J. C. Ho, L. Jenkins, T. H. Johengen, K. C. Kuo, E. LaPorte, X. Liu, M. R. McWilliams, M. R.

Moore, D. J. Posselt, R. P. Richards, D. Scavia, A. L. Steiner, E. Verhamme, D. M. Wright

and M. A. Zagorski. 2013. “Record-setting algal bloom in Lake Erie caused by agricultural and

meteorological trends consistent with expected future conditions.” Proceedings of the National

Academy of Sciences 110(16):6448–6452.

Mulholland, M. R., R. Morse, T. Egerton, P. W. Bernhardt and K. C. Filippino. 2018. “Blooms

of Dinoflagellate Mixotrophs in a Lower Chesapeake Bay Tributary: Carbon and Nitrogen

Uptake over Diurnal, Seasonal, and Interannual Timescales.” Estuaries & Coasts 41(6):1744–

1765.

Nagai, T., K. Taya and I. Yoda. 2015. “Comparative toxicity of 20 herbicides to 5 periphytic

algae and the relationship with mode of action.” Environmental Toxicology 35:368–375.

Nakashima, T., Y. Kobayashi and Y. Hirata. 2012. “Mrthod to exterminate blue-green algae

in a large pond to improve plant growth by micro-nano bubbles in activated water.” Acta

Horticulturae 938:391–400.

vi
Nanobubble Solutions Limited Ltd. 2019. “Nanobubble Solutions Limited Ltd - UK and world-

wide business to business franchise for Anzaikantetsu Co, Ltd.”.

URL: http://nanobubblesolutions.com/tech/tech.php

Paerl, H. W., L. M. Valdes, B. L. Peierls and Lawrence J. W. Adolf, J. E. and. 2006. “Anthro-

pogenic and climatic influences on the eutrophication of large estuarine ecosystems.” Limnol-

ogy and Oceanography 51(12):448–462.

Park, J., J. Church, Y. Son, K. Kim and W. H. Lee. 2017. “Recent advances in ultrasonic

treatment: Challenges and field applications for controlling harmful algal blooms (HABs).”

Ultrasonics Sonochemistry 38:326–334.

Pelley, J. 1998. “What Is Causing Toxic Algal Blooms?” Environmental Science and Technology

32(1):26A–30A.

Perin, G., A. Bellan, A. Bernardi, F. Bezzo and T. Morosinotto. 2019. “The potential of quantita-

tive models to improve microalgae photosynthetic efficiency.” Physiologia Plantarum 166:380–

391.

Pohl, J., I. Saltsman, A. Mahammed, Z. Gross and B. Roder. 2014. “Inhibition of green algae

growth by corrole-based photosensitizers.” Journal of Applied Microbiology 118:47–54.

Rigobello-Masini, M., J. C. Masini and E. Aidar. 2006. “The profiles of nitrate reductase and

carbonic anhydrase activity in batch cultivation of the marine microalgae Tetraselmis gracilis

growing under different aeration conditions.” Microbiology Ecology 57:18–25.

vii
Sengco, M. R. and D. M. Anderson. 2004. “Controlling Harmful Algal Blooms Through Clay

Flocculation1.” The Journal of Eukaryotic Microbiology 51(2):169–172.

Sforza, E., D. Simionato, G. M. Giacometti, A. Bertucco and T. Morosinotto. 2012. “Adjusted

Light and Dark Cycles Can Optimize Photosynthetic Efficiency in Algae Growing in Photo-

bioreactors.” PLoS ONE 7:1–10.

Shao, J., R. Li, J. E. Lepo and J. Gu. 2013. “Potential for control of harmful cyanobacterial

blooms using biologically derived substances: Problems and prospects.” Journal of Environ-

mental Management 125:149–155.

Shin, M., H. Lee, M. S. Kim, N. Park and C. Lee. 2017. “Control of the red tide dinoflagellate

Cochlodinium polykrikoides by ozone in seawater.” Water Research 109:237–244.

Shumway, S. E. 1990. “A Review of the Effects of Algal Blooms on Shellfish and Aquaculture.”

Journal of the World Aquaculture Society 21(2):65–104.

Silvagni, P. A., L. J. Lowenstine, T. Spraker, T. P. Lipscomb and F. M. D. Gulland. 2005.

“Pathology of Domoic Acid Toxicity in California Sea Lions (Zalophus californianus).” Vet-

erinary Pathology 42:184–191.

Sun, Z. and E. R. Blatchley. 2017. “Tetraselmis as a challenge organism for validation of ballast

water UV systems.” Water Research 121:311–319.

Sutherland, D. L., C. Howard-Williams, M. T. Turnbull, P. A. Broady and R. J. Craggs. 2015.

“Frequency of CO2 supply affects wastewater microalgal photosynthesis, productivity and

viii
 nutrient removal efficiency in mesocosms: implications for full-scale high rate algal ponds.”

International Society for Applied Phycology 27:1901–1911.

Sutherland, D. L., M. H. Turnbull, P. A. Broady and R. J. Craggs. 2014. “Effects of two different

nutrient loads on microalgal production, nutrient removal and photosynthetic efficiency in

pilot-scale wastewater high rate algal ponds.” Water Research 66:53–62.

Sutherland, D. L., V. Montemezzani, C. Howard-Williams, M. T. Turnbull, P. A. Broady and

R. J. Craggs. 2015. “Modifying the high rate algal pond light environment and its effects on

light absorption and photosynthesis.” Water Research 70:86–96.

Tango, P. J., R. Magnien, W. Butler, C. Luckett, M. Luckenbach, R. Lacouture and C. Poukish.

2005. “Impacts and potential effects due to Prorocentrum minimum blooms in the Chesapeake

Bay.” Harmful Algae 4:525–531.

Testa, J. M., J. B. Clark, W. C. Dennison, A. W. Donovan, E. C. Fisher, W. Ni, M. Parker, D.

Scavia, S. E. Spitzer, A. M. Waldrop, V. Vargas and G. Ziegler. 2017. “Ecological Forecasting

and the Science of Hypoxia in Chesapeake Bay.” BioScience 67(7):614–626.

Tomaru, Y., K. Toyoda and K. Kimura. 2018. “Occurrence of the planktonic bloom-forming

marine diatom Chaetoceros tenuissimus Meunier and its infectious viruses in western Japan.”

Hydrobiologia 805(1):221–230.

Trovão, M., H. Pereira, J. Silva, J. Páramo, P. Quelhas, T. Santos, J. T. Silva, A. Machado,

L. Gouveia, L. Barreira and J. Varela. 2019. “Growth performance, biochemical composition

and sedimentation velocity of Tetraselmis sp. CTP4 under different salinities using low-cost

lab- and pilot-scale systems.” Heliyon 5(5):1–6.

ix
Visser, P. M., B. W. Ibelings, M. Bormans and J. Huisman. 2016. “Artificial mixing to control

cyanobacterial blooms: a review.” Aquatic Ecology 50(3):423–441.

Wang, L., X. Wang, X. Jin, J. Xu, H. Zhang, J. Yu, Q. Sun, C. Gao and L. Wang. 2017.

“Analysis of algae growth mechanism and water bloom prediction under the effect of multi-

affecting factor.” Saudi Journal of Biological Sciences 24:556–562.

Wang, M. and W. Shi. 2008. “Satellite-Observed Algae Blooms in China’s Lake Taihu.” Eos,

Transactions American Geophysical Union 89(22):201–202.

Wijesekara, S., N. Nomura and M. T. Tsumura. 2006. “Selective control of marine algal species

by electrolysis treatment.” Sri Lanka Journal of Aquatic Science 11:27–42.

Zhang, C., X. Chen, J. Wang and J. Tan. 2017. “Toxic effects of microplastic on marine mi-

croalgae Skeletonema costatum: Interactions between microplastic and algae.” Environmental

Pollution 220:1282–1288.