CHAPTER 2

LITERATURE REVIEW

Cyanobacteria, also known as blue-green algae, are photosynthetic gram

negative prokaryotes. They are among some of the oldest known organisms on earth,

with an approximate age of 3 billion years, and they are thought to be responsible for

the oxygenation of the primitive earth atmosphere (Schopf, 2000). Cyanobacteria

produce a wide range of compounds with novel chemistries and pharmacological

effects. For example, sulfolipids with different fatty acid esters were isolated from

Lyngbya lagerheimii and Phormidium tenue, cyanovirin which isolated from an

aqueous cellular extract of Nostoc elipsosporum prevents the in vitro replication and

citopathicity of primate retroviruses, and other compounds have been isolated with

anti-inflammatory, anti-viral, anti-gastric ulcer and anti-cancer activities (Barsanti and

Gualtieri, 2006; Peerapornpisal et al., 2006; Wase and Wright, 2008). Nevertheless

cyanobacteria could be produced a wide range of potent toxins which posed a health

hazard for human and animals (Kuiper-Goodman et al., 1999).

Cyanobacteria are found worldwide, in a wide range of environments,

from the poles to the tropics, from terrestrial to marine environments. In deserts, for

example, they are responsible for helping to stabilise the environment through the

production of extracellular polysaccharides to which sand grains adhere and

subsequently permit other organisms to become established (Wynn-Williams, 2000).

In aquatic environments, under eutrophic conditions, cyanobacteria can “bloom” to

5

form mass populations in the water column and, with the ability of some to form gas

vesicles, can float to the surface of a water body and concentrate into a visible scum.

Furthermore, through the action of gentle winds over a waterbody, such scums can be

further concentrated and will often accumulate on the leeward shore of a lake, near to

where people and animals may use the water (Falconer et al., 1999). Investigations

into the causes of poisoning episodes have historically led to the identification of

specific toxic products of cyanobacteria and research into toxigenic cyanobacteria and

their toxins continues apace.

2.1 Factors of affecting bloom formation

2.1.1 Physical Factors

Temperature

The elevated water temperature is the theory illustrates the

cyanobacterial success (Hyenstrand et al., 1998). Maximum growth rate are attained

by most cyanobacteria at temperatures above 25˚C (Robats and Zohary, 1987). These

temperatures are higher than that for green algae and diatoms (Mur et al., 1999) and

therefore, cyanobacteria can out-compete the other species when subjected to extreme

temperature conditions. This also explains why in temperate and boreal water bodies

most cyanobacteria bloom during summer (Ressom et al., 1994).

Light

Cyanobacteria are primarily photoautotrophs, that is they depend on

light for photosynthesis and hence nutrition growth (Reynolds and Walsby, 1975). In

addition, cyanobacteria have been shown to be both heterotrophic and

6

photohertotrophic and therefore able to survive in conditions of low light intensity. In

general however, cyanobacteria seem to have similar light requirements to other

phytoplankton, although the level at which photosynthesis becomes light-limited is

lower for cyanobacteria than for other algae (Reynolds and Walsby, 1975), due to the

presence of phycobiliprotein. This photosynthetic pigment harvest light with the

lower wavelengths (500-650 nm) in green, yellow and orange part of the spectrum,

which are hardly used by other phytoplankton species (Mur et al., 1999).

Buoyancy

The ability to control their buoyancy is a fundamental and almost

unique feature of the cyanobacteria which gives them a significant advantage over

other phytoplankton. This feature enable them to move up and down in the water

photosynthesizing at the surface layer and nutrient up taking in the deep layer

unavailable to phytoplankton (Ganf and Oliver, 1982). Cyanobacteria are also able to

actively regulate their position in the water column to avoid photoinhibitory light

intensities (Tucker and Hargreaves, 2004).

2.1.2 Chemical factors

Macronutrients

Eutrophication, or enrichment of aquatic systems with dissolved

nutrients, is a common feature and one of the major water quality problems of many

water bodies (Jacoby et al., 2000). The principal elements involved in eutrophication

are phosphorus and nitrogen which are essential for cyanobacterial growth

(Kaebernick et al., 2001; Villareal and Carpenter, 2003). Several researches have

7

shown that, cyanobacteria have higher affinity for nitrogen or phosphorus than other

phytoplankton, so that cyanobacteria can out-compete phytoplankton under conditions

of phosphorus or nitrogen limitation (Kaebernick et al., 2001; Mur et al., 1999).

1. Phosphorus

Phosphorus is vital cellular constituent of all living organisms

and is involved particular in energy dynamics and protein synthesis. Almost all the

phosphorus is released slowly from rock minerals by weathering and erosion. In

natural systems it usually exists as phosphate. The simplest soluble phosphate is

orthophosphate or soluble reactive phosphorus (PO43-). Phosphate is also released as

microbe breakdown organic material in the soil. Phosphorus can cycle many times

through ecosystems; pass through food chains before being recycled by death and

decay. Cyanobacteria can recharge their phosphate stores and then ascend into higher

light intensities suitable for growth and division (Falconer, 2005). Microcystis is one

of the cyanobacteria that can well utilize these advantages, with a large capacity for

phosphorus storage and high and variable buoyancy (Ganf and Oliver 1982;

Kromkamp et al., 1989).

2. Nitrogen

Nitrogen is present in the environment in a wide variety of

chemical forms including organic nitrogen, ammonium (NH4+), nitrite (NO2

-), nitrate

(NO3-), nitrous oxide (N2O), nitric oxide (NO) or inorganic nitrogen gas (N2). Organic

nitrogen may be in the form of a living organism, humus or in the intermediate

products of organic matter decomposition. The processes of the nitrogen cycle

transform nitrogen from one form to another. Many of those processes are carried out

by microbes, either in their effort to harvest energy or to accumulate nitrogen in a

8

form needed for their growth. Most phytoplankton are able to assimilate and utilize

nitrogen in form of ammonia, nitrate, nitrite or urea, whilst some cyanobacteria have

the additional ability to assimilate molecular nitrogen (Bold and Wynne, 1985).

Nitrogen is an essential component of all living cell and involved primarily in the

synthesis of amino acids and protein. Cyanobacteria can be stored nitrogen in the

form of two proteins, cyanophycin and phycocyanin. The accumulation of

cyanophycin is done under condition of nitrogen availability together with growth

limitation by temperature, low light intensity and low phosphorus or sulfur (Oliver

and Ganf, 2000). Many studies have shown that, low ratio between nitrogen and

phosphorus has also been observed to favor cyanobacteria bloom (Kaebernick et al.,

2001; Villareal and Carpenter, 2003; Falconer, 2005).

Micronutrients

Cyanobacteria have very specific interactions with trace

metals in water. Copper and manganese are toxic at micromolar concentrations, while

iron and zinc have been shown to promote cyanobacterial growth and toxin

production (Reuter and Peterson, 1987; Lukac and Aegerter, 1993). Iron is an

essential component in a number of enzymes and protein complexes of respiratory

and photosynthetic electron transport and nitrate assimilation (Michel et al., 1998).

Excess iron in water is stimulatory and can lead to cyanobacterial growth and

formation of bloom much more rapidly than at the absence or low concentration (≤

2.5 µM) (Lukac and Aegerter, 1993).

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2.1.3 Biological factors

In general, phytoplankton are grazed upon by zooplankton, which in

turn are consumed by fish. Cyanobacteria are either not eaten or pure food for

zooplankton due to their shape and size (Mur et al., 1999). In addition to some

cyanobacteria such as Aphanizomenon or Microcystis have the thick outer layer of

mucilage which is an important factor in selection of food particles by zooplankton.

At least, the toxic cyanobacteria have toxic effect on zooplankton. These mechanisms

may reduce zooplankton growth, reproduction and survival and so, its grazing on

cyanobacteria (Lampert, 1987; Sigee, 2005).

2.2 Ultrastructure of Microcystis (Figure 2.1)

2.2.1 Cell membrane

Cell membrane of Microcystis is multilayered that is consist of three

uniform peptidoglycan layers and outer lipoproteins and lipopolysaccharides layer.

Cell menbrane is responsible for protective coats, molecular sieves and molecule and

ion traps, promoters for cells adhesion and surface recognition and as frameworks,

and maintaining cell shape (Wolk, 1973; Sleyter et al., 1988). Microcystis cells or

colonies are surrounded by polysaccharide mucilage (Wolk, 1973; Sigee, 2005).

2.2.2 Carboxysomes (or polyhedral bodies)

Carboxysomes are used for store of enzyme ribulose-1,5-bisphosphate

carboxylase oxygenase (RuBisCO) which catalyses the photosynthetic fixation of

carbon dioxide (Allen, 1984). Carboxysomes are visible in transmission electron

10

microscope (TEM) as black polyhedral bodies each 200-300 nm in diameter (Berner,

1993).

2.2.3 Cyanophycin granules

Although cyanophycin granules are unique to cyanobacteria, they have

been shown to be absent from some species (Lawry and Siwan, 1982). Cyanophycin

granules consist of co-polymers of arginine and aspartate, which are essential

component in the synthesis of gas vesicles and during nitrogen starvation is firstly

degraded (Berner, 1993; Oliver and Ganf, 2000). Cyanophycin is accumulated when

cells are grown at low temperatures, under condition of phosphorus or sulfur

deficiency, or under light-starved conditions (Allen, 1984).

2.2.4 Glycogen granules

Glycogen granules are located between the thylakoids. They consist of

intracellular polysaccharides, and are used for long-term store of carbon and energy.

They occur as white irregularly spherical form with the measurement 16 x 33 nm,

which are invisible in light microscope (Allen, 1984).

2.2.5 Polyphosphate granules (or volutin or metachromic granules)

The range polyphosphate granules size is 100-400 nm in diameter, which

are form of highly polymerized polyphosphate (Jacobson and Halmann, 1982).

Polyphosphate granules are both phosphate source of synthesis of nucleic acids and

phospholipids, and energy source for ATP synthesis (Allen, 1984). Cyanobacteria are

11

able to store phosphorus, usually in the form of phosphates and these reserves can be

sufficient for several cell doubling (Oliver and Ganf, 2000).

2.2.6 Gas vesicles

Microcystis also contain gas vesicles, formerly termed as gas vesicles

or aerothopes, same as many planktonic cyanobacteria (Komárek, 1991; Komárek and

Anagnostidis, 2005). Gas vesicles are made up of stacks of cylindrical tubes closed at

each end by hollow cone, which are formed by a single wall layer only 2 nm thick, 75

nm in diameter, and up to 1 µm in length (Walsby, 1972; Walsby, 1994). Gas vesicles

have an internal pressure related to the atmospheric pressure but are subject to

hydrostatic pressure, which increases with depth, as well as the turgor pressure of the

cell (Oliver and Ganf 2000). Gas vesicles allow the cells to float at the water surface

and can be adjusted to position cells in optimal light and oxygen condition for growth.

During daytime, high rate of photosynthesis at the surface water lead to decrease

buoyancy causing cyanobacteria to sink down at the end of light period. The

mechanism of decreasing buoyancy can occur in three ways:

1. Carbohydrate accumulation (mainly glycogen) which is a

product of photosynthesis, acts as ballast (contributed by protein, carbohydrate and

lipid) that increases the specific gravity of cells.

2. Photosynthetically – mediated uptake of K+ lead to increase

turgor pressure, which may lead to collapse of gas vesicles and loss of buoyancy.

3. The gvpA and gvpC gene expression which are important in

formation of gas vesicle which, may be inhibited at high light intensity.

12

Cyanobacteria deplete carbohydrate stores by respiration and

conversion to protein at night (at low irradiance), so ballast and turgor pressure are

lost. Formation of new gas vesicles reoccur due to gvpA and gvpC gene expression

and gas diffuses into vesicle at low irradiance. These mechanisms lead to buoyancy

within water bodies at the beginning of next light period (Walsby, 1994; Sigee, 2005;

Zurawell et al., 2005; Barsanti and Gualtieri, 2006).

Figure 2.1 Transmission electron microscope (TEM) image of Microcystis

CB: Carboxysome, CM: Cell membrane, CY: Cyanophycin, GV-c: Gas

vesicle in cross-section, GV-l: Gas vesicle in longitudinal, PP:

Polyphosphate granule

CB

PP

CY

GV-l

GV-c

CM

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2.3 Cyanobacterial secondary metabolites

Cyanobacteria are well known for their ability to produce many diverse

secondary metabolites, compounds that are harmful to animals and its known

potential hazard to human health (Codd et al., 1997; Sivonen and Jones, 1999;

Carmichael, 2001). Microcystis is one of the cyanobacteria that can produce toxic

secondary metabolite toxic and off-flavor compounds.

2.3.1 Microcystins

Toxic cyanobacterial blooms occur frequently in eutrophic reservoirs

and aquaculture ponds all over the world (Dittmann and Wiegand, 2006). Toxins can

occur within the cell (intracellular or cell-bound toxins) or be released from cells to

water (extracellular or dissolved toxins) under certain conditions of growth and/or

external (environmental) stress factors responsible for cell lysis (Codd, 1995).

Microcystis spp. are one of the most harmful algal blooms which have been recorded

from nearly every continent (Yang et al., 2005; Guo and Xie, 2006; Jiang et al.,

2008). Microcystis spp. produce toxic compounds called microcystins which are

hepatotoxin. Microcystins are produced not only by Microcystis but also by some

species of Anabaena, Anabaenopsis, Aphanizomenon, Nostoc, Planktothic

(Oscillatoria) and Hapalosiphon (Carmichael, 1997; Dawson, 1998; Chorus, 2001).

Microcystins are a small monocyclic structure (Figure 2.2) which

consist of seven amino acids (-D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha-),

where X and Z are variable L-amino acids, D-MeAsp is 3-methylaspartic acid, Adda

is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid and Mdha is

N-methyldehydroalanine (Tillett et al., 2000; Mazur and Pliński, 2001; Rodríguez et

14

al., 2008). They are water soluble and can exist outside of the cell for up to 3 weeks

(Kenefick et al., 1993). Although over 70 microcystin variants are known, the most

common is microcystin-LR, which has been identified together with other commonly

found microcystin variants such as microcystin-LA, -RR and -YR in natural water

samples (Falconer et al., 1999; Spoof et al., 2003; Falconer, 2005). Microcystin-LR is

the most toxic microcystin with an LD50 value of 50 mg/kg. Because of the potent

toxicity of microcystin-LR, the World Health Organization (WHO) has set a

provisional guideline value of 1.0 mg/L for microcystin-LR in drinking water (WHO,

1998).

Figure 2.2 Structure of microcystins in which X and Z are variable L amino acid

(Source: Chen et al., 2006)

Hepatotoxins are easily exposed to human and aquaculture product via

consumption and dermal contact. These toxins such as microcystins have caused

detrimental effects in aquatic vertebrates and invertebrates under laboratory and field

conditions. The effects of microcystins on the embryonic, juvenile, and adult stages of

fish include histopathological damage of the liver, kidneys, gills, intestines, heart or

spleen; disrupted osmoregulation, altered serum biochemistry, malformation of

15

embryonic–larval alimentary system, reduced growth rate, induced stress response,

and modified swimming behavior (Smith et al., 2008). Moreover, these toxins have

been reported to cause freshwater and estuarine aquaculture damage (Shumway,

1990; Chen and Xie, 2005). Zimba et al. (2006) reported free microcystin-LR

concentration in dead shrimp hepatopancreas determined by HPLC was 55 μg/g total

shrimp weight, whereas shrimp hepatopancreas from the adjacent pond without

shrimp mortalities had no measurable toxin. Muscle toxin concentration was below

0.1 μg/g. Microcystins can be metabolized by conjugation to glutathione via

glutathione S-transferase in mammals (Kondo et al., 1996). These toxins also inhibit

protein phosphatases 1 and 2A, leading to an imbalance in

phosphorylation/dephosphorylation reactions and eventual loss of cell structure

(Runnegar and Falconer 1986; Carbis et al., 1996), oxidative stress (Ding et al.,

2001), tumor promotion (Nishiwaki-Matsushima et al., 1991), or in the case of

nodularin, tumor initiation (Ohta et al., 1994). Initial inhibition of phosphatase

activity is through reversible reactions between the hepatotoxins and protein

phosphatases. Microcystins subsequently undergo a secondary reaction with the target

enzyme, forming an irreversible, covalent linkage through its N-

methyldehydroalanine (Mdha) residue.

2.3.2 Off-flavor (Geosmin and MIB)

The term “off-flavor” is used to describe the accumulation of odorous

compounds within water or tissue produced from biological origins. Off-flavor

problems are common in reservoirs, aquaculture ponds and in drinking water which

has been identified in the Americas, Australia, Europe, Africa and Asia (Whelton and

16

Dietrich, 2004). Three common off-flavors consist of geosmin, 2-methylisoborneol

(MIB) and β-cyclocitral (Zimba and Grimm, 2003; Smith et al., 2008). Actinomycetes

are well known for their ability to produce these off-flavors such as Streptomyces and

Nocardia (Zaitlin and Watson, 2006; Jüttner and Watson, 2007). Moreover,

Cyanobacteria are also known as the major producer of off-flavors, including species

from the genera Anabaena, Aphanizomenon, Lyngbya, Microcystis, Oscillatoria,

Planktothrix, Phormidium Pseudanabaena, Symploca and Tychonema (Paerl and

Millie, 1996; Zimba et al., 1999; Jüttner and Watson, 2007; Huang et al., 2008; Smith

et al., 2008).

The majority of all biological odor problems in drinking water

worldwide are caused by microbial production of geosmin and MIB (Jüttner and

Watson, 2007). Geosmin (trans-1,10-dimethyltrans-9-decalol) is typically described

as having an earthy odor with the structure shown in Figure 2.3A. It has a molecular

weight of 182.3 g/mol and Henry’s law constant of 6.6 x 10-5 m3/atm/mol (Lalezary et

al., 1984; Pirbazari et al., 1992). It is produced both intracellular and extracellular and

its release to the water occurs mainly when the algae producing it die and decompose.

Odor threshold for geosmin is 1-10 ng/L at 45oC (McGuire et al., 1981; Rashash et

al., 1997). The 2-methylisoborneol (MIB) is characterized by musty odor with the

structure shown in Figure 2.3B. It has a molecular weight of 168.3 g/mol and Henry’s

law constant of 5.76 x 10-5 m3/atm/mol (Lalezary et al., 1984; Pirbazari et al., 1992).

MIB is produced intracellular and its release to the water occurs mainly when the

algae producing it die and decompose. Odor threshold for MIB is 1-20 ng/L at 45oC

(McGuire et al., 1981; Rashash et al., 1997). Both geosmin and MIB have relatively

moderate hydrophobicity and moderate solubility (Song and O’Shea, 2007).

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Figure 2.3 Structure of off-flavors (A) geosmin and (B) MIB

(Source: Song and O’Shea, 2007)

Off-flavors are receiving widespread attention in drinking water and

especially aquaculture industry as they can compromise the quality of drinking water

and aquatic animals (Izaguirre et al., 1988; Zimba and Grimm, 2003; Schrader, 2005;

Ho et al., 2007a). Off-flavors have been reported to impact a number of commercial

important species. For example, Geosmin in river water affected rainbow trout (Salmo

gairdneri) farms in The UK (Roberson et al., 2006). In wild and farmed freshwater

fish in Asia, the earthy-musty odor was reported (Yamprayoon and Noomhorm,

2000). Zimba and Grimm (2003) reported that geosmin, MIB and β-cyclocitral were

detected in channel catfish (Ictalurus punctatus) production ponds in southern USA.

In addition to Nile tilapia-Oreochromis niloticus (Yamprayoon and Noomhorn, 2000),

shrimp (Whitfield et al., 1988), Atlantic salmon-Salmo salar (Farmer et al., 1995),

catfish species (Lovell et al., 1986; Martin et al., 1987) and white sturgeon-Acipenser

transmontanus (Schrader et al., 2005) also have been reported. The uptake of these

off-flavors by aquatic animals can contaminate the flesh resulting them to be

unpalatable and consequently unacceptable for commercial processing and sale.

Roberson and Lawton (2003) reported that the threshold level of geosmin and MIB

(A) (B)

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was 1.5 µg/kg. The consumers can detect geosmin and MIB in drinking water at

concentrations as low as 5-10 ng/L (Ho et al., 2007a). In Thailand, off-flavor

problems in flesh also have been documented. The accumulation of off-flavor

compounds was found in red tilapia flesh in green water ponds which the abundance

of geosmin and MIB and their levels were higher than those of the threshold

(Whangchai et al., 2008). The troublesome odors; both geosmin and MIB are difficult

to be removed from the water by conventional water treatment methods (Izaguirre, et

al., 1988).

2.4 Impact on biota and human

Many researches have suggested that microcystins affect many organisms,

from microalgae to mammals. Microcystin-LR is able to paralyze the motile green

algae Chalmydomonas reinhadtii, improving its settlement and creating a lake zone

free of competitors for microcystins-producing cyanobacteria (Kearns and Hunter,

2001). The lack of alternative phytoplankton for food when cyanobacteria bloom may

contribute to unfavorable nutritive conditions for zooplankton (Kurmayer and Jüttner,

1999) despite the ingestion of Microcystis colonies by zooplankton which can also be

affected by colony size and/or mucilage and toxic content (Henning et al, 2001).

Some studies have been shown that M. aeruginosa have strong adverse effects on

Daphnia, such as increase mortality, decrease growth rate, delayed maturation and

decreasing offspring production (Hietala et al., 1995; Deng et al., 2008).

Macrophytes such as Ceratophyllum demersum, Elodea canadensis

(Wiegand and Pflugmacher, 2001) and Phragmites australis (Pflugmacher et al.,

2001), have been shown to absorb microcystin-LR. Microcystins also cause a

19

reduction in the number and mass of fronds in the water plants Spirodela oligorrhiza

(Romanowska-Duda and Tarczynska, 2002). Besides, aquatic animals may also

accumulate microcystins. For example, the mussels Mytilus edulis which fed on M.

aeruginosa with high microcystins content for 3 days, accumulated microcystins in

their tissues (Williams et al., 1997). The crayfish Procambarus clarkii accumulates

microcystins in the intestine and hepatopancreas (Vasconcelos et al., 2001). Similarly,

hepatopancreas and kidney of European carp (Cyprinus carpio) were damage due to

low concentrations of microcystins (Fischer and Dietrich, 2000) and the rainbow trout

(Oncorhynchus mykiss) suffers hepatotoxicosis by accumulating microcystin-LR that

lead to changes in cellular morphology, protein phosphatases inhibition, and livers

necrosis (Fischer et al., 2000). The freshwater fish Oreochromis niloticus accumulates

microcystins in the gut, liver, kidneys and muscle tissue (Magalhães et al., 2001;

Mohamed et al., 2003).

Human exposure to microcystins may occur through a direct route such as

drinking water, recreational water or water used for haemodialysis (Kuiper-Goodman

et al. 1999; Zhou et al., 2002; Codd et al., 2005), or through an indirect route such as

food consumption (Magalhães et al., 2001; Codd et al., 2005). Microcystin-LR is a

potent cancer promoter in laboratory animals. So, chronic exposure to low

concentrations of microcystins in drinking water can be a serious problem to public

health, contributing to promotion of cancer in human. Epidemiological studies have

already related the presence of microcystins in drinking water to an increase in the

incidence of colorectal (Zhou et al., 2002) and primary liver cancer (Ueno et al.,

1996). There are groups more sensitive to microcystins poisoning that require special

attention such as B-hepatitis patients, children and old people (Fitzgerald, 2001).

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2.5 Elimination of microcystins and off-flavors

At noted above, cyanobacteria are causing a serious toxic and off-flavors

problems in drinking water and aquaculture ponds. There are many methods such as

physical, chemical and biological treatments for removing these compounds in water.

2.5.1 Air stripping/ Dissolved air flotation

Air stripping is basic method effective to remove some off-flavor

compounds such as hydrogen sulphide (H2S). However, this method is not as efficient

for geosmin and MIB removal because it is only effective for off-flavors having

Henry’s law constant greater than 10-3 m3/atm/mol. Geosmin and MIB have Henry’s

law constants of 6.6 x 10-5 and 5.76 x 10-5 m3/atm/mol, respectively (Youngsug et al.,

1997; Song and O’Shea, 2007). Hargesheimer and Watson (1996) reported that

dissolved air flotation method is not efficient to remove geosmin and MIB supporting

the above claim. For microcystins, this method likewise is not effective as it cannot

remove several dissolved microcystin variants (LR, LY, LW, and LF) (Teixeira and

Rosa, 2006). Conversely, cyanobacterial cells are removed by dissolved air flotation

method depending on the physical properties of cyanobacterial species. The Belgian

dissolved air flotation plant removed Microcystis by 40-80%, Anabaena by 90-100%,

but removed Planktothrix only by 30% (Drikas and Hredey, 1994; Yoo et al., 1995).

2.5.2 Activated carbon filtration

Activated carbon is either applied in its powdered form or as granular

activated carbon by adding directly into water before sedimentation or in a deep bed

filtration. Common sources of activated carbon are coal, coconut, and wood. Both

21

powdered and granular activated carbon are effectively eliminate microcystin from

water. In the case of powdered activated carbon, dosage is an important parameter (10

µg/L toxin: > 200 mg of powdered activated carbon/L), while carbon source of

granular activated carbon is important, probably due to the different pore sizes

relative to the size of microcystins molecule (Donati et al., 1994). Wood based carbon

is more effective in microcystins adsorption than coal based (Huang et al., 2007;

Mohamed et al., 1999). Furthermore, powdered activated carbon can remove geosmin

and MIB with high doses and exhibit reduced efficiency in natural water due to the

presence of other organic matter (Cook et al., 2001; Suffet et al., 1995). Moreover,

the dosage of activated carbon will increase by the rise of toxin, geosmin and MIB

concentrations in natural water (Graham et al., 2000). It is too expensive to treat water

with this method on long periods of time (Nerenberg et al., 2000).

2.5.3 Membrane filtration

Generally, there are four processes that are considered feasible and

more commonly used for membrane treatment in drinking water applications:

microfiltration, ultrafiltration, nanofiltration and reverse osmosis. Both ultrafiltration

and microfiltration have efficiency more than 98% for removing whole cells of

Microcystis aeruginosa but they cannot remove microcystins (Chow et al., 1997).

Several studies reported that more than 82% of microcystins were removed by

nanofiltration (Fawell et al., 1993; Muntisov and Tromboli, 1996; Teixeira and Rosa,

2005). Reverse osmosis also remove both microcystin-LR and –RR which are more

than 96% (Neumann and Weckesser, 1998). In case of off-flavors, nanofiltration and

reverse osmosis can keep off-flavor compounds to more than 70%, but not

22

completely. Muntisov and Trimboli (1996) reported that 60% of geosmin and 45% of

MIB were removed by nanofiltration while Dixon et al. (2011) found that 50-99% of

both geosmin and MIB were removed using nanofiltration and reverse-osmosis. In

contrast, ultrafiltration with cutoffs of approximately 100,000 daltons is not expected

to keep off-flavor compounds, which usually are small compounds of 100-400

daltons. However, ultrafiltration can be combined with powdered activated carbon

which is very effective for off-flavor removal (Bruchet and Laine, 2005).

2.5.4 Oxidation

Oxidation is one of treatment methods for microcystins and off-flavor

removal. The efficiency of oxidation reactions depends on oxidant concentrations,

pH, temperature, and the presence of competing compounds. The oxidants which are

used in this method, include potassium permanganate (Rodríguez et al., 2007),

chlorine, chloramines, chlorine dioxide (Acero et al., 2005) and ozone (Nerenberg et

al., 2000; Hoeger et al., 2002).

2.5.4.1 Potassium permanganate (KMnO4)

Potassium permanganate is an enigmatic chemical used for

oxidizing toxin and off-flavor substances in water. This oxidant is often realized in the

improvement of coagulation through adsorption on the solid manganese dioxide

(MnO2), which is formed by the reduction of permanganate ion (MnO4-). The

efficiency of removal is decreased when natural water has high organic matter.

Rodríguez et al. (2007) reported that a permanganate dosage of 1.25 mg/L was

effective in surface water source with a relatively high organic matter (6.7 mg/L) for

removal of several microcystins variants (microcystin-LR, -RR and –YR), while

23

permanganate has low removal efficiency for geosmin and MIB (McGuire, 1999).

However, permanganate can damage algal cells and lead to a release of intracellular

compounds (Lam et al., 1995; Tung et al., 2004). Permanganate must be used

carefully if applied as a pre-oxidant, especially to water bodies which may be at risk

for toxic cyanobactria.

2.5.4.2 Chlorine (Cl2), chloramines and chlorine dioxide (ClO2)

Chlorine, chloramines and chlorine dioxide are not effective for

removing microcystins, geosmin and MIB (Hitzfeld et al., 2000; Robert et al., 2000).

The efficiency of chlorination seems to depend on the chlorine compounds and the

concentration used. Chlorine at ≥ 1 mg/L removes more than 95% of microcystins

while chloramines at the same dose remove only 40-80% (Nicholson et al., 1994). A

chlorine residual of ≥ 0.5 mg/L is present after 30 min of contact time is a destruction

of cyclic peptides completely (Hoeger et al., 2000). In case of off-flavor, the highest

removal efficiency of geosmin is < 60% and for MIB is 35% under 20 mg/L chlorine

dose (Nerenberg et al., 2000). However, chlorination by-products are generated,

which have been implicated in the subacute toxicity (progressive liver damage) seen

in mice after intraperitoneal injection of chlorinated microcystins (Rositano et al.,

1995). In Thailand, chlorine residual at 30 min of contact time is limited to 0.2-0.5

mg/L in tap water (Department of Health Ministry of Public Health, 2008).

2.5.4.3 Ozone (O3)

Ozone is a very strong oxidant and considered as the most

effective in removing microcystins, geosmin and MIB from water. Tung et al. (2004)

reported that an ozone dosage of 1 mg/L destroyed algal cells and both dissolved and

particulate MIB were completely oxidized in 10 min. However, ozone can produce

24

low molecular weight by-products during oxidization of geosmin and MIB such as

ketones which have other odors in treated water (Masten and Davies, 1994;

Nerenberg et al., 2000). Therefore, removal of geosmin and MIB should use another

process such as granular activated carbon filter or UV after ozonation to remove by-

products from ozone. On the other hand, ozone dosage of 1 mg.L-1 is not enough to

destroy the released toxin (Pietsch et al., 2002; Hoeger et al., 2002). Thus, ozone

concentration should be high enough to be used to remove these toxins and algal cells.

Hoeger et al. (2002) reported that ozone concentrations of ≥ 1.5 mg/L were required

to provide enough oxidation potential to destroy the toxin present in 5 x 105

Microcystis aeruginosa cells/mL. However, the confounding factors of ozone

treatment for cyanotoxins and off-flavors are sensitive to alkalinity and temperature

and especially oxidation of the dissolved carbon competes with the destruction of

toxins and off-flavors (Westrick, 2008). One disadvantage of ozonation is the

formation of hazardous oxidation by-products, especially in waters containing

bromide ion (Br-) where carcinogenic bromated ion (BrO3-) is formed (Song and

O’Shea, 2007).

2.5.5 Biodegrading bacteria

Although several chemical treatments of water are proposed, it is

possible that chemical treatments sometimes produce carcinogenic substances and

other mutagens. Biodegradation is one of the safest and mildest treatments for

removing cyanobacterial toxins and off-flavors from water (Ho, et al., 2007a; Ishii et

al., 2004). There are many isolated bacteria reported as having the capacity to degrade

microcystin in water. The ability of Sphingomonas to degrade microcystins was

25

studied in several water resources. Sphingomonas sp.B9 which was isolated from

Lake Tsukui Japan, was able to degrade microcystin-LF, -LR and –RR (Harada et al.,

2004; Imanishi et al., 2005). Another Japanese Sphingomonas, 7CY which was

isolated from Lake Suwa, was shown to degrade a wider range of microcystins,

including MC-LR, -RR, -LY, -LW and –LF (Ishii et al., 2004). In Murrumbidgee

River, Australia, Sphingomonas sp.ACM-3962 degraded microcystins-LR and –RR

(Jones et al., 1994; Bourne et al., 1996) while Valeria et al. (2006) described

Sphingomonas sp.CBA4 which was isolated from San Roque Reservoir, Argentina

was capable of degrading completely MC-RR (200 µg/L) within 36 h. Ji et al. (2009)

reported that the removal efficiencies of Pseudomonas spp. and Bacillus spp. of total

microcystins-RR and -LR extracellular microcystins-RR and -LR were 67.0%, 40.5%,

40.0% and 66.0%, respectively. Ho et al. (2007b) showed that a novel bacterium,

Sphingopyxis sp. LH21 capable of degrading microcystin-LR and -LA was isolated

from a biological sand filter which was previously shown to effectively remove these

toxins from source waters. Holst et al. (2003) and Hyenstrand et al. (2003) confirmed

microcystins biodegradation with 14C-microcystin-LR. Holst et al. (2003) described

that aerobic as well as anaerobic microorganism in sediments of a water recharge

facility can efficiently remove microcystins, while, Hyenstrand et al. (2003) found

carbon dioxide as a major end product of microcystins degradation.

The biodegradability of MIB and geosmin in water indicates that there

is potential for using biological filtration processes as a viable treatment option for

removing these compounds. Of the biological filtration studies conducted on MIB and

geosmin, the majority have used sand or granular activated carbon media (Yagi et al.,

1988; Hrudey et al., 1995; Elhadi et al., 2004; Ho et al., 2007a). Yagi et al. (1988)

26

showed that more than 72% geosmin (1.5 mg/L in raw water) and 56% MIB (1.7

mg/L in raw water) absorbed into activated carbon which contained Bacillus subtilis

were degraded. Tanaka et al. (1996) collected Pseudomonas sp. and Enterobacter sp.

in backwashed water from biological filter at Lake Biwa, Japan and they degraded

MIB to 2-methylcamphene and 2-methylenebornane. Then another bacterium from

Enterobacter sp. oxidatively decomposed 2-methylenebornane to camphor and

lactone. Many bacterial species can degrade MIB including Bacillus fusiformis,

Bacillus sphaericus (Lauderdale et al., 2004), Pseudomonas aeruginosa (Egashira et

al., 1992), Pseudomonas putida (Oikawa et al., 1995) and Flavobacterium

multivorum (Egashira et al., 1992). Ho et al. (2007a) reported that Pseudomonas sp.,

Alphaproteobacterium, Sphingomonas sp. and Acidobacteriaceae member were

identified as microorganisms most likely involved in the biodegradation of geosmin

within the sand filters and also the bioreactors. Another bacterial species can degrade

geosmin consist of Bacillus cereus (Narayan and Nunez, 1974), Arthrobacter

atrocyaneus, Arthrobacter globiformis, Chlorophenolicus strain N-1053 and

Rhodococcus moris (Saadoun and El-Migdadi, 1998).

2.6 Ultrasonic quantities

Sound is the rapid motion of molecules. It propagates in waves that

transport energy from one medium such as gas, liquid or solid to another. The sound

which human can hear (audible sound) is an approximate frequency range between 20

Hz and 20 kHz (Figure 2.4). The ultrasound frequency range starts at 20 kHz.

27

Figure 2.4 Approximate frequency range corresponding to ultrasound

(Source: O’Brien Jr., 2007)

History of ultrasonic technology began in 1916-1917, Paul Langevin and

Chilowski developed the first submarine detector using thin quartz crystals at

approximate frequency of 150 kHz (O’Brien Jr., 2007). Another decade passed,

ultrasonic technology was applied to the steel industry, initial with flaw detection and

later joined by wall thickness measurement. Ultrasound was used for physical therapy

in the 1920s and 1930s, and in 1942, Dr.Karl Dussika, a neurologist and a psychiatrist

from Austria, was the first physician to use ultrasound for medical diagnosis. The

procedure was called “Hyperphonography”. He attempted to locate brain tumors and

cerebral ventricles by measuring the transmission of the ultrasound beam through the

head (O’Brien Jr., 2007; Orenstein, 2008). Subsequently, many researchers developed

diagnostic and therapeutic technology in medical field using ultrasound such as

detection of gallstones, obstetric sonography, cleaning teeth in dental hygiene.

Ultrasound is known to have harmful effects on the structure and

functional state of organisms (Phull et al., 1997; Rott, 1998). When ultrasound

applied to water, the compression and decompression is generated alternatively, and

the pressure of decompression form cavities or bubbles; this phenomenon is called

cavitation (Honda Electronics Co., Ltd., 2007) (Figure 2.5). The size of the bubble

1 Hz 100 Hz 10 kHz 1 MHz 100 MHz

Infrasound Audible sound Ultrasound

28

depends on ultrasonic frequencies. The higher frequencies have the resonant radius of

a bubble smaller than low frequencies, and therefore fewer acoustic cycles are

required before the bubble reaches its resonant size (Honda Electronics Co., Ltd.

2007; González-García et al., 2010). With a greater number of acoustic cycles per unit

time at higher frequencies, rectified diffusion occurs more rapidly. Thus, a greater

number of nuclei can reach resonance more quickly than at lower frequencies. This

implies that higher frequencies will be favored for the degradation of the chemicals

which undergo pyrolysis inside the bubble (González-García et al., 2010). The inner

part of the bubbles is filled with liquid vapor or gas dissolved in liquid. With the

change of pressure the bubble grows and collapses which generates both physical and

chemical effects (Joyce et al., 2003; Koda et al., 2009). The physical effects involve

intense shock waves and shear forces produced by the bubble collapse and acoustic

streaming. These effects are known to break down biological cell membranes, for

many years these have been used in microbiology laboratories for the release of cell

content and used for cleaning such as, cleaning jewelry, small metal component and

heavy duty cleaning (Frizzell, 1988; Honda Electronics Co., Ltd., 2007; Cameron et

al., 2008; Mason et al., 2011). The chemical effects cause the formation of OH• and

H• radicals, from the decomposition of water vapour within the collapsing bubble.

Each cavitation bubble collapse will result in extreme conditions involving high

temperatures (>5000 K) and high pressure (>1000 atm), this combination is easily

capable of fragmenting water into radicals. These radicals can attack cell membranes

leading to lysis of cell contents (Petrier et al., 1998; Mason and Peters, 2002; Koda et

al., 2009).

29

Figure 2.5 The relation between the change of ultrasonic acoustic pressure and

the change in the size of bubbles

(Source: Honda Electronics Co., Ltd., 2007)

Many studies have developed environmental remediation by ultrasound

irradiation because it is considered to be a green technology using sound energy and

requires either no additional chemicals or much reduced quality (Adewuyi, 2001).

Doosti et al. (2012) used 20, 28, 40, 45 and 200 kHz at 40 W for 1 hour to treat

turbidity and total suspended solid. The highest turbidity reduction is at 28 kHz of

frequency with 76%. Besides, ultrasound irradiation was used for control

cyanobacterial growth. Hao et al. (2004) examined the effects of 5 minute exposure to

ultrasound of suspended samples of cyanobacteria at different frequencies (20 kHz,

200 kHz and 1.7 MHz) at 40 W. It should be note that 5 min ultrasonic irradiation at

20 kHz reduced the initial biomass by 44%. Similarly, Wu et al. (2012) reported that

low frequency; 20 kHz ultrasound with high intensity (0.0403 W/cm3) is effective for

30

inactivation of Microcystis aeruginosa cells. However, Zhang et al. (2006a) found

that high ultrasound power and long irradiation caused microcystins to increase. The

ultrasound power of 80 W at 80 kHz sonication for 5 min increased the extracellular

microcystins concentrations from 0.87 µg/L to 3.11 µg/L. Although sonication may

lead to release of microcystins due to cell lysis, Ma et al. (2005) has been reported

that the removal rate of microcystins reached 70% at 150 kHz and 30 W for 20 min.

2.7 Quantification of chemical reaction

When water is sonicated, it produces various kinds of free radicals such

OH•, H• and HO2• (Hart and Henglein, 1985; Petrier et al., 1998). The free radicals

were detected using method of Hart and Henglein (1985) which used an oxidation

reaction involving an aqueous potassium iodide (KI) solution. These free radicals can

react with potassium iodide in the solution resulting in iodine liberation. Lee (2000)

reported that the various reactions, which appear in the liquid phase, are given as

2KI + 2OH• 2KOH + I2

OH• + OH• H2O2

2HO•2 + 2KI 2KOH + I2 + O2

HO•2 + HO•2 H2O2 + O2

2O + 2I- + 2K + 2H2O I2 + O2 + 2KOH + H•2

The products formed from above reactions are I2 and H2O2. The H2O2 also

oxidize iodide ion to iodine as follow.

H2O2 + 2KI 2KOH + I2

I2 + I- I3-