3

CHAPTER 2

LITERATURE REVIEWS

2.1 Introduction to Cladophora

Genus Cladophora Kützing is a branching, filamentous green alga

(Chlorophyta, Cladophorales, Cladophoraceae) in both marine and freshwater

ecosystems. It grows in various habitats and environments. Generally, Cladophora is

an attached alga but may also form free-floating or loose mats on soft substrates

(Prescott, 1975; Dodds and Gudder, 1992; Smith, 1950; Bootsma and Jensen, 2007)

(Figure 1-2).

Figure 1. Cladophora A. The alga attaches on rocks in Nan River B. A

photograph of whole plant

Morphologically, it is characterized by its multinucleate cells, reticulate

chloroplasts, thick cell wall and filamentous-branched thalli. Branching of freshwater

Cladophora consists of uniseriate filaments inserted at oblique to horizontal angles,

and may be sparsely to profusely spaced. Development of this alga is dominated

A B

5 cm 0.5 cm

4

either by apical growth with acropetal organization or intercalary growth with an

irregular organization (Smith, 1950; Dodds and Gudder, 1992; Bootsma and Jensen,

2007; Sze, 1998; Wehr and Sheath, 2003).

Figure 2. Photomicrographs of Cladophora sp.

2.1.1 Taxonomy of freshwater Cladophora

Taxonomic identification within the genus Cladophora is difficult because this

genus exhibits high morphological variation under different ecological conditions

(van den Hoek, 1963; Whitton, 1967; Usher and Blinn, 1990; Dodds and Gudder,

1992; Bergey et al., 1995; Wilson et al., 1999; Ross, 2006). van den Hoek (1963)

established 11 sections, 38 species of the genus Cladophora based primarily on their

morphology; 11 freshwater and 27 marine. Freshwater species are found in six

sections:

Aegagropila

Cladophora aegagropila (L.) Rabenh.

Glomeratae

C. fracta var. fracta (Mull. Ex. Vahl) Kutz.

C. fracta var. intricata (Mull. Ex. Vahl) Kutz.

100 m

5

C. glomerata var. glomerata (L.) Kutz.

C. glomerata var. crassior (L.) Kutz.

Cladophora

C. rivularis (L.) v.d. Hoek

C. surera Brand

Cornuta

C. cornuta Brand

Affines

C. kosterae Hoffm. Tild.

Basicladia

C. basiramosa Schmidle

C. pachyderma (Kjellm.) Brand

In Thailand, two species of freshwater Cladophora have been reported in Nan

river: C. glomerata and Cladophora sp. (Peerapornpisal et al., 2006; Peerapornpisal,

2007). In addition, Chaisuk and Waiyaka (2001) reported C. glomerata from Mekong

river, Chiang Rai.

2.2 Carotenoids

Carotenoids are organic pigments that occur naturally in plants including

algae, some fungi and bacteria. They are divided into two classes, carotenes (contain

no oxygen such as β-carotene, -carotene and lycopene; molecular formula C40H56)

and xanthophylls (contain oxygen such as lutein and zeaxanthin; molecular formula

C40H56O2). In algae, carotenoid normally exist in the chloroplasts, however they can

be in the cell wall and distribute within the chloroplast. Carotenoids assist in taking

up light energy, function as photoprotectants and antioxidants, serve as precursors for

biosynthesis of plant growth regulator, abscisic acid and protect the photosynthetic

apparatus (Goodwin, 1980; Naik et al., 2003; Wikipedia, 2007).

Carotenoids in green algae and plants are produced in two different

compartments and by two different pathways i.e. the acetate-mevalonate pathway and

the phosphoglycerraldehyde-pyruvate pathway. In all organisms, carotenoids are

6

further synthesized from isopentenyl diphosphate and its isomers (Cunningham, 2002;

Goodwin, 1980; Ladygin, 2000).

Carotenoids are powerful antioxidants. Some carotenoids that are beneficial

to human health include beta-carotene (a precursor to vitamin A as well as a cancer-

preventing antioxidant), lutein and zeaxanthin (naturally present in the macula of the

human retina and which protects it by filtering phototoxic blue light and near-

ultraviolet radiation) (Burtin, 2003; Maryland Medical Center, 2006; George Mateljan

Foundation, 2006 ). Previous studies have suggested that carotenoids can prevent or

delay cancer and degenerative diseases in human and animals by contributing to

antioxidative defenses against metabolic oxidative byproduct (Omenn et al., 1996;

Tapiero et al., 2004).

2.2.1 Carotenoids in Cladophora and other algal species

Reports of carotenoids and carotenoid composition in Cladophora are few.

Powtongsook (2000) reported 340 µg g-1 of total carotenoid in Cladophora collected

from the Nan River. Whereas, Traichaiyaporn et al. (2007a) reported 840 µg g-1 of

carotenoid in Cladophora culture in 60-100% of canteen wastewater. Yoshii et al.

(2004) reported that carotenoid composition of Cladophora albida, C. coelothrix, C.

glomerata, C. japonica, C. ohkuboana, C. pellucida, C. sericea and C. vagabunda

were as follows: 22, 14, 13, 12, 12, 11, 14 and 16 µg g-1 dry weight (dw) of β-

carotene; 22, 4, 36, 2, 7, 3, 37 and 29 µg g-1 (dw) of lutein, respectively. Dere et al.

(1998) investigated carotene content in C. glomerata by extraction with three solvents

(methanol, diethyl ether and acetone) obtaining 18.8, 19.2 and 20.1 g g-1 fresh

weight, respectively.

Khuantrairong et al. (2009a) studied the effect of phosphorus on pigments

production of Cladophora sp. in mass culture with addition of di-potassium hydrogen

orthophosphate (K2HPO4) at 0-5 mg L-1. The pigments (in g g-1) were observed as

follows: chlorophyll a 148.34–347.97, chlorophyll b 55.58–249.42, total carotenoid

823.23-1,063.16, carotene 44.30–86.36, xanthophyll 778.93–997.29, -carotene

20.01-61.03, lutein 172.80-296.70 and zeaxanthin 24.62-72.17.

In other species, such as blue-green alga Spirulina, Shimamatsu (2004)

reported 4,770 µg g-1 of carotenoid of dried Spirulina powder of Siam Algae

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Company. Whereas, Chainapong and Traichaiyaporn (2009) reported 5,750–6,620 µg

g-1 of carotenoid of S. platensis cultivated under mixotrophic condition. Promya et al.

(2008) reported that -carotene content of S. platensis cultured in 100% kitchen

wastewater and 10% oil-extracted fermented soybean water were 290 and 370 µg g-1,

respectively.

Granodo-Lorencio et al. (2009) stated that zeaxanthin of a green alga

Scenedesmus almeriensis was 340 µg g-1. Whereas, Norziah and Ching (2000)

reported -carotene content of 52 µg g-1 in edible seaweed Gracilaria changgi.

2.2.2 Factors related to carotenoids production in algae

Previous studies showed that phosphorus was effective in carotenoid

production in algae. Brinda et al. (2004) reported that phosphate limitation enhanced

astaxanthin in a green alga Haematococcus pluvialis. In addition, Forján et al. (2007)

suggested that phosphate and sulfur limitation enhanced the production of β-carotene,

zeaxanthin and violaxanthin, whereas nitrogen limitation decreased those carotenoids

in marine microalga Nannochloropsis gaditana. In contrast, Khuantrairong et al.

(2009) suggested that phosphorus supply increased total carotenoid, xanthophylls,

carotene, β-carotene, lutein, zeaxanthin, chlorophyll a and chlorophyll b of

Cladophora sp. Leonardos and Geider (2005) stated that phosphorus and nitrogen

ratio was related to carotenoid and chlorophyll a production in cryptophyte

Rhinomonas reticulata. Latasa and Berdalet (1993) suggested that synthesis of

pigments in dinoflagellate Heterocapsa sp. stopped under phosphorus limitation.

Celekli et al. (2009) reported that phosphate effected biomass and carotenoid

production of Spirulina platensis and the best phosphate concentration was 0.50 g L-1.

Lin (1977) reported that hydrolysis of polyphosphates (reactants in carotenoid

pathway) by C. glomerata in the Milmaukee River was related to pH and dissolved

phosphorus in water.

Orosa et al. (2005) cultured a green microalga Haematococcus pluvialis in

different NaNO3 concentrations and found that nitrate decreased astaxanthin but

increased β-carotene, the optimum concentration of NaNO3 was 0.15 g L-1.

Cifuentes et al. (2003) cultured Haematococcus pluvialis at varying light

intensity, nitrate and acetate, suggesting that these factors affected carotenoid

8

production of this species and light intensity was the best inductive carotenogenic

factor.

2.3 Nutritional values of algae

Algae are one of the significant sources of human food and they are high in

nutritional values that are beneficial for supplemental use as human food source and

animal feed. In Thailand, edible freshwater alga Cladophora is well known for

consumption in the Northern part and it is cultured for fish supplemental feed,

especially Mekong Giant Catfish (Traichaiyaporn et al., 2007b). A summary of the

nutritional values of Cladophora has been reported (in percent dw) to compose of

protein 28, fat 6.81, neutral detergent fiber 19.29, acid detergent fiber 19.06, ash

20.80, moisture 13.19, phosphorus 0.36 and carbohydrate 30.34; vitamins (in µg 100g-1

dw): vitamin B1 169.50, vitamin B2 541.10 and vitamin E 4.20; minerals (in mg 100g-1

dw): calcium 943.90, sodium 716.90, magnesium 170.5, manganese 5.36, iron 162.0,

copper 310.00 and zinc 0.65 (Ruangrit et al., 2005; Peerapornpisal, 2007).

Zbikowski et al. (2007) reported mineral content of Cladophora sp. (in mg g-1

dw) collected from Southern Baltic, Gulf of Gdansk and Vistula Lagoon, Poland were

as follows: calcium 4.5, 3.9 and 5.4; magnesium 19.4, 15.0 and 14.9; sodium 37.3,

20.9 and 17.1; potassium 53.4, 38.5 and 30.7; zinc 67.5, 63.0 and 73.1, respectively.

Whereas, Whitton (1970) reported mineral content (in percent) of C. glomerata as

sodium 0.93, potassium 3.0, magnesium 0.43, calcium 0.77 and phosphorus 0.70.

However, Keeney et al. (1976) reported that zinc values in C. glomerata collected

from Deadman Bay and Main Duck, Canada were 23.7 and 8.2 g g-1 dw,

respectively. Elenkov et al. (1996) reported that lipid content of C. vagubunda in

Lake Pomorie, Bulgaria was 1.70-3.17 mg g-1 dw.

Nutritional values of edible freshwater algae in Thailand, Spirogyra spp. (Tao)

(in g 100g-1 dw) were also reported i.e. protein 18.65, fat 5.21, carbohydrate 56.31,

fiber 7.66, ash 11.78 and moisture 8.05. Vitamin and mineral contents (in mg 100g-1

dw) were provitamin A 0.25, vitamin B1 0.04, vitamin B2 0.55, vitamin B6 0.84,

niacin 3.65, iron 33.85, manganese 35.80, magnesium 241.10, potassium 1.19, sodium

1.56, calcium 26.88 and phosphorus 125.76 (Peerapornpisal, 1992)

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Nutritional values (in g 100g-1 dw) of blue-green alga Spirulina were reported

as follows: protein 61.40, fat 8.50, moisture 3.00, fiber 3.00 and ash 7.70; vitamins

(in mg 100g-1): provitamin A 214.00, vitamin B1 1.98, vitamin B2 3.63, vitamin B6

0.59, vitamin B12 0.11 and vitamin E 11.80; minerals (in mg 100g-1 dw): phosphorus

914.00, iron 57.40, calcium 171.00, potassium 1,770, sodium 1,050 and magnesium

257.00 (Shimamatsu, 2004).

Norziah and Ching (2000) reported nutritional contents (in percent dw) of

edible seaweed Gracilaria changgi as follows: protein 6.9, lipid 3.3, fiber 24.7 and

ash 22.7; vitamin and minerals (in mg 100g-1): vitamin C 28.5, calcium 651, iron

95.6, zinc 13.8, copper 0.8 and cadmium 0.3. Whereas, McDermid and Stuercke

(2003) revealed vitamin C content of 3 mg g-1 in Hawaiian seaweeds Enteromorpha

flexuosa.

Gressler et al. (2010) reported nutritional values (in percent dw) of four

species of seaweeds Laurencia filiformis, L. intricate, Gracilaria domingensis and G.

birdiae as follows: soluble protein 6.2, 7.1, 18.3 and 4.6; total lipid 1.3, 1.3, 6.2 and

1.1; and ash 23.8, 22.5, 38.4 and 33.5, respectively. Whereas, nutritional values of

edible seaweed Palmaria palmate and Enteromorpha spp. were reported as follows:

9.7-25.5% of protein in Palmaria palmata; 9-14% of protein and 32-36% of ash in

Enteromorpha spp. (Galland-Irmouli et al., 1999; Aguilera-Morales et al., 2005).

Ash (in g 100g-1 dw) and mineral contents (in mg g-1 dw) were reported from five

edible marine seaweeds of Spain as follows: Fucus vesiculosus, ash 30.10, Na 54.69,

K 43.22, Ca 9.38, Mg 9.94, Fe 0.04, Zn 0.04 and Mn 0.06; Laminaria digitata

(Kombu), ash 37.59, Na 38.18, K 115.79, Ca 10.05, Mg 6.59, Fe 0.03, Zn 0.02 and

Mn <0.01; Undaria pinnatifida (Wakame), ash 39.26, Na 70.64, K 86.99, Ca 9.31,

Mg 11.81, Fe 0.08, Zn 0.02 and Mn 0.01; Chondrus crispus (Irish moss), ash 21.08,

Na 42.70, K 31.84, Ca 4.20, Mg 7.32, Fe 0.04, Zn 0.07 and Mn 0.01; Porphyra tenera

(Nori), ash 20.59, Na 36.27, K 35.00, Ca 3.90, Mg 5.65, Fe 0.10, Zn 0.02 and Mn

0.02 (Rupérez, 2002).

Nutritional values of microalgae were reported from Chaetoceros muelleri,

Chaetoceros sp., Isochrysis galbana, Isochrysis sp., Pavlova salina, Pavlova sp.,

Micromanas pusilla, Prasinophyta sp. and Chlorella vulgalis (Martínez-Fernández et

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al., 2006; Ponis et al., 2006; Cai et al., 2007; Janczyk et al. 2007). These species

showed high protein contents suitable for fish larvae feed.

The vitamin contents of five microalgae used for aquaculture in France were

reported. On a dry weight basis, Tetraselmis suecica contained 4,280 μg g−1

provitamin A and 6,323 μg g−1 vitamin E; Pavlova lutheri 1,162 μg g−1 vitamin B12

and 837 μg g−1 vitamin C; Isochrysis galbana 183 μg g−1 vitamin B6 and Skeletonema

costatum 710 μg g−1 vitamin B1 (Roeck-Holtzhauer et al., 1991).

The vitamin C content (in mg g-1 dw) were reported from seaweeds Alaria

valida 0.53, Egregia menziesii 0.04, Fucus evanescens 0.24, Hedophyllum sessile

0.21, Macrocystis pyrifera 0.19, Postelsia palmaeformis 0.09, Agarum fimbriatum

0.02, Costaria costata 0.02, Desmarestia munda 0.01, Laminaria bullata 0.02,

Enteromorpha sp. 0.15, Ulva lactuca 0.46, Gigartina papillata 0.41, Grateloupia

Cutleriae <0.01, Halosaceion glandiforme 0.13, Iridaea sp. 0.26, Porphyra naiadum

0.36, P. nereocystis 0.53, P. perforata 0.60, Prionitis lyallii 0.03, Turnerella pacifica

0.09, Agardhiella tenera <0.01, Anatheca fureata <0.01, Callophyllis sp. <0.01,

Dasyopsis plumosa <0.01, Hymenena sp. <0.01, Opuntiella californica <0.01,

Polyneura latissima <0.01 and Ehodymenia pertusa <0.01 (Norris et al., 1936).

2.4 Biomass production and biomass production rate of Cladophora

The reports on biomass production and growth rate of Cladophora are few.

Pitcairn and Hawkes (1973) reported biomass production (in g m-2 dw) of Cladophora

growth in rivers of UK were as follows: River Arrow 34.6-73.7, River Cole 5.0-50.1,

River Blythe 52.6-60.3, River Tean 20.3-31.6, River Ray 14.5-66.4, River Great Stour

5.0-74.7 and River Darent 62.9. Whereas, Parker and Maberly (2000) observed

biomass production of Cladophora in South Basin and North Basin of Windermere,

UK were 29 and 4.7 g m-2 dw, respectively. The biomass production (in g m-2 dw) of

Cladophora vagabunda were reported from Childs River, 220 and Sage Lot Pond,

USA, 37 (Peckol and Rivers, 1996). Whereas, biomass production (in g m-2 dw) of C.

glomerata were reported from the Neva Estuary, Russia, 95-508 (Gubelit, 2009) and

the Laurentian Great Lake, Canada, 175-200 (Higgins et al., 2008).

Traichaiyaporn et al. (2007a) reported the highest biomass production of

Cladophora culture in laboratory with canteen wastewater, 360 m g-2 wet weight.

11

Whereas, biomass production and growth rate of Cladophora in mass culturing were

reported from Cladophora cultured in cement raceway ponds using canteen

wastewater, 253-3,187 g m-2 (wet weight) and Cladophora culture in soil raceway

ponds using fish pond water, 3,117-20,250 g m-2 (wet weight), the highest growth rate

were 1,247 and 1,747 g m-2 week-1, respectively (Traichaiyaporn et al., 2010).

Auer and Canale (1982) indicated that Cladophora growth rate was strongly

related to tissue phosphorus content which was between 1 to 2 µg mg-1, the specific

growth rate was generally between 0.10-0.25 day-1.

2.4.1 Factors related to biomass production and morphology of Cladophora

Many factors, including light intensity, water temperature, pH, water velocity,

suspended solids, and nutrient concentrations were found to influence growth, growth

rate, primary production and morphology of Cladophora (Whitton, 1967; Wong and

Clark, 1976; Birch et al., 1981; Auer, 1994; Graham et al., 1982; Hoffmann and

Graham, 1984; Painter and Kamaitis, 1987; Wilson et al., 1999; Bootsma et al., 2004;

Sandgren et al., 2005; Bootsma et al., 2006; Higgins et al., 2006a; Bootsma and

Jensen, 2007).

Cladophora requires a hard surface for attachment, a relatively high light

environment, ambient pH between 7 and 10, and some degree of water motion. It

grows in oligotrophic to eutrophic ecosystems. Excessive growth is generally

associated with eutrophic water (Whitton, 1970; Higgins et al., 2008).

Reports on nitrogen limiting Cladophora growth from freshwater ecology are

few and a majority of studies indicated phosphorus as limiting nutrient for growth

(Higgins et al., 2008). Pitcairn and Hawkes (1973), Wong and Clark (1976), Birch et

al. (1981), Wharfe et al. (1984), Painter and Kamaitis (1987), Bootsma et al. (2004)

and Higgins et al. (2006a) suggested that phosphorus is a main factor related to

growth and production of freshwater Cladophora. It can also be abundant in habitats

where nitrogen supply limits primary production (Dodds, 1991; Dodds and Gudder,

1992). In addition, Parker and Maberly (2000) stated that nitrogen is not an important

growth limiting factor for Cladophora in North and South Basin of Windermere,

Cumbria, UK, the main factor is phosphate phosphorus.

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Painter and Kamaitis (1987) reported that Cladophora growth in Lake

Ontario, Canada was limited due to low concentrations of phosphorous. The critical

phosphorus level for Cladophora in this lake appears to be below 0.01 mg L-1.

Whereas, research in Lake Michigan showed that Cladophora blooms were related to

high phosphorus levels in water, mainly as a result of human activity such as

agricultural runoff, land fertilizer, poorly maintained septic systems and inadequate

sewage treatments. While phosphorus input from rivers may support Cladophora

production near river outlets, a comparison of estimated phosphorus demand with

river phosphorus inputs suggests that most Cladophora productions is supported by

phosphorus cycling processes within Lake Michigan (Bootsma et al., 2006).

Whereas, C. glomerata bloom in the Neva Estuary, Russia was started in mid-May,

when the water temperatures reached +10 OC. The biomass productions were 95-508

g m-2 dw (Gubelit, 2009).

Higgins et al. (2006b) stated that Cladophora growth in Eastern Lake Erie was

highly sensitive to spatial and temporal variations in soluble phosphorus

concentration. Whereas, Auer (1994) observed that high dissolved phosphorus values

in water induced an increase in stored phosphorus and growth rate of Cladophora.

These were supported by Wharfe et al. (1984). They reported that high dissolved

phosphorus effected the growth rate and hence the accumulation of C. glomerata at

downstream of the River Great Stour, England.

Hagen and Braune (2000) observed that the main environmental factors

controlling C. glomerata growth in the river Ilm, Germany are light intensity, current

velocity, pH, soluble reactive phosphorus and ammonia-nitrogen.

Bellis and McLarty (1967) suggested that light and temperature are very

important ecological factors with respect to growth and periodicity of C. glomerata in

Southern Ontario, Canada. Whereas, Cheney and Hough (1983) reported that

productivity of C. fracta in Shoe Lake, Michigan correlated most strongly with total

alkalinity and pH when phosphorus and nitrogen were above the limiting

concentrations. Higgins et al. (2008) stated that C. glomerata blooms in the

Laurentian Great Lake, Canada related to ecosystem level changes in substratum

availability, water clarity and phosphorus recycling.

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Study on the cultivation of Cladophora using canteen wastewater had been

done by Traichaiyaporn et al. (2007a; 2010). They suggested that the optimal

phosphate concentrations of canteen wastewater on algal growth in laboratory cultures

were 0.11-1.88 mg L-1. Whereas, the optimal phosphate concentration of canteen

wastewater on algal growth in mass culture was 0.01-14.78 mg L-1. They concluded

that biomass production was strongly correlated to temperature, light intensity and

nutrient concentrations and this alga can improve water quality with decreased BOD,

COD and nutrients of wastewater.

Laboratory experiments by Auer and Canale (1982) indicated that Cladophora

growth rate was strongly related to tissue phosphorus content. They observed that

tissue phosphorus content was between 1 to 2 µg mg-1, the specific growth rate was

generally between 0.10-0.25 day-1. This was supported by Bootsma et al. (2006), who

suggested that tissue phosphorus content of Cladophora was positivly correlated to

growth rate and biomass. van den Hoek (1963) observed that C. glomerata

apparently requires eutrophic conditions, where the pH is rather high (7.5-8.5).

Pitcairn and Hawkes (1973) cultured Cladophora in flasks with synthetic

media containing phosphorus from 0.1 to 7 mg L-1 and found no significant increase

in growth when phosphate was above 1 mg L-1, but it was significant when phosphate

was below 1 mg L-1.

Whitton (1967) cultured C. glomerata in flasks with a modified CHU No. 10

medium at varying temperatures and light intensities and found that rapid growth

occurred between 15 and 25 °C, whilst 6 and 30 °C were the lower and upper limits of

detectable growth. The algal growth rate increased when the light intensity was up to

7,500 lux. It was found that light intensity had a marked effect on the growth form at

all temperatures tested (15-25 °C) and that higher light intensity induced a greater

degree of branching. Bellis (1968) found that cultures of C. glomerata were killed at

initial pH values less than 7.0 and above 10.0.

Robinson and Hawkes (1986) studied the growth of C. glomerata in flasks

with continuous flow culture. They revealed that optimal specific growth rate

occurred at 20 OC, light intensity 6,000 lux, photoperiod 24 h, ammonia-nitrogen 0.18-

0.20 mg L-1, nitrite-nitrogen 0.077-1.057 mg L-1, nitrate-nitrogen 7.2-15.2 mg L-1 and

phosphate-phosphorus 0-1.9 mg L-1.

14

Hoffmann and Graham (1984) revealed that temperature was the first factor

affecting dry weight production of Cladophora in laboratory culture. The maximum

dry weight production occurred at 25 OC. The second and third factors were light

intensity and photoperiod, the highest dry weight was observed at light intensity 125

mol m-2 s-1 and photoperiods 16 h. In addition, the photoperiod is the primary factor

influencing zoosporogenesis, 8 h light:16 h dark photoperiods elicited the greatest

number of zoosporangia.

Laboratory experiments of Cladophora showed that photosynthetic production

of dissolved oxygen decreased significantly below 25 °C, the maximum

photosynthetic rate was observed at 25 °C to 31 °C (Graham et al., 1982).

Cladophora grown in nutrient-depleted media do not produce any branches,

but generate long cells (Wilson et al., 1999). In natural environments, exposure to

strong sunlight can decrease the diameter of Cladophora cells (Bellis and McLarty,

1967). Rönnberg and Lax (1980) reported reduced cell lengths in filaments of

Cladophora exposed to high wave action in the littoral region of the north Baltic. van

den Hoek (1963) showed a trend for increased branching of Cladophora with

increased water velocity.

Data from Wilson et al. (1999), Usher and Blinn (1990) and Salovius and

Bonsdorff (2004) indicated that high suspended sediment decreased biomass and cell

length of C. glomerata, but increased cell width. Bergey et al. (1995) reported a

reduction of branch number and fragmentation of C. glomerata in high water

velocities. Shyam (1980) observed that the morphology of C. callicoma in natural

and cultural conditions were similar. Whereas, Khuantrairong and Traichaiyaporn

(2008) stated that addition of phosphorus in standard media decreased cell width and

cell length of Cladophora sp. under standing water cultures.

Cladophora is usually absent in fast flowing water. It is abundant in water

with appropriate flow rate of 20 cm s-1 (Pitcairn and Hawkes, 1973). Zimmermann

(1961) studied the effect of flow rate and concentration of sewage on various algal

growth, found that growth of C. glomerata in the presence of the highest sewage

concentration tested occurred at flow rates of 20 cm s-1 and 80 cm s-1.

Furthermore, Menendez et al. (2002) cultured a green macroalga

Chaematomorpha linum in different phosphorus and nitrogen concentration and found

15

that these nutrients affected biomass production, the highest biomass was observed

when both phosphorus and nitrogen were added.

2.5 Molecular study of Cladophora

Identification of Cladophora is difficult because of its morphological

variations in different ecosystems (van den Hoek, 1963; Whitton, 1967; Usher and

Blinn, 1990; Dodds and Gudder, 1992; Bergey et al., 1995; Wilson et al., 1999; Ross,

2006). Therefore molecular studies were performed for taxonomic and phylogenetic

studies within the genus Cladophora and often amplified in the ribosomal internal

transcribed spacer (ITS) region (Ponsen and Looijen, 1995). ITS refers to a piece of

non-functional RNA situated between structural ribosomal RNA (rRNA) on a

common precursor transcript. This polycistronic rRNA precursor transcript contains

the 5’ external transcribed sequence (5’ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2,

28S rRNA and 3’ETS (Wikipedia, 2008).

Studies on nucleotide sequences of rRNA ITS region in Cladophora albida by

Bakker et al. (1992) indicated that the ITS sequences of C. albida within Atlantic and

Pacific regions had similarity of 99% and 99.5%, respectively, whereas between these

regions similarity sequence was 79%. Moreover, Bakker et al. (1995a) studied the

phylogeographic relationships of C. vagabunda from the Atlantic and Pacific oceans

based on these nucleotide sequences. They suggested that C. vagabunda was closely

related to C. albida and this species from Pacific region is a monophyletic group.

Bakker et al. (1994) explored some of the diversity within the generic

complex Cladophora and its siphonocladalean allies using 18S rRNA gene sequences

and confirmed that there is no basis for the independent recognition of the

Cladophorales polyphyletic. Studies of nuclear rRNA ITS sequences in the 13

species of Cladophora albida/sericea clade showed six ITS sequence types, four of

which exhibited virtually no within-type sequence divergence (Bakker et al., 1995b)

Ross (2006) amplified the ITS region of freshwater Cladophora from North

America, including cultures of C. glomerata var. glomerata, C. glomerata var.

crassior and C. fracta var. fracta from Europe. The results indicated that the

sequences of these species had high similarity (98%). Whereas, Marks and

16

Cummings (1996) reported the DNA sequences variation in the ITS region of C.

glomerata, C. albida, C. columbiana and C. vagabunda.

Bot et al. (1991) analyzed the reassociation kinetics of the DNA from C.

pellucid and indicated that the genome of this alga comprised of approximately 75%

repetitive sequences and no significant divergence was observed between the single-

copy sequences of this species isolated from the East Atlantic coast and

Mediterranean Sea. Whereas, Báez et al. (2005) suggested that Cladophora in the

western Mediterranean Sea and the Adriatic Sea were significantly similar in

distribution patterns (Chorotypes).

Hanyuda et al. (2002) performed molecular phylogenetic analyses using

nuclear 18S rRNA gene sequences to reveal the relationship between Aegagropila

linnaei and Cladophora sp. They stated that A. linnaei from two localities (Lake

Akan and Lake Dannemora) showed identical nucleotide sequences and there was

0.9% divergence between A. linnaei and Cladophora sp.

Leliaert et al. (2007) studied the molecular phylogeny of the Siphonocladales,

Clodophorophyceae based on partial large subunit (LSU) and small subunit (SSU)

rRNA of 166 samples. The results revealed that nine siphonocladalean clades were

observed and all siphonocladalean architectures may be derived from a single

Cladophora-like ancestor.