Faculty of Resource Science and Technology

Thermal Cyanobacteria from Panchor Hot Spring, Serian

Nur Afiqah binti Abdul Rahim (31980)

Bachelor of Science with Honours (Aquatic Resource Science and Management) 2014

Thermal Cyanobacteria from Panchor Hot Spring, Serian

Nur Afiqah binti Abdul Rahim

This project is submitted in partial fulfilment of the requirement for degree of Bachelor of Science with Honours (Aquatic Resource Science and Management)

Supervisor: Assoc. Prof Dr Norhadi Ismail

Faculty of Resource Science and Technology, UNIVERSITI MALAYSIA SARAWAK 2014

DECLARATION I hereby declare that the work in this project is my own except for quotations and summaries which have been duly acknowledged. No portion of the work referred to in this dissertation has been submitted in support of an application for another degree qualification of this or any other university or institution of higher learning.

……………………………….. Nur Afiqah binti Abdul Rahim Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resources Science and Technology Universiti Malaysia Sarawak

ACKNOWLEDGEMENT ‘‘In the name of Allah, the Most Beneficent, the Most Merciful’’ All praise belongs to Allah S.W.T for the countless blessings without I even asking, make me a stronger person, relieve my burdens and give me strength to finish up this final year project to the fullest. First of all, I would like to express my deep thankful to my great supervisor, Assoc. Prof. Dr. Norhadi Ismail for the support, time spent and patience to assist and guide me to this day. For all the effort in giving comments, suggestion and reading of the manuscript, without which it would not have been possible for me to produce this report in the present form, I’m very thankful. Special thanks goes to Dr Ruhana Hassan, my dearest mentor who are always there to shows me path when I need someone and guide me until I can afford to be independent. My appreciation also goes to other lecturers for all the guidance all this time. Thanks for the support of the Faculty Resource Science and Technology and to the laboratory assistants, Mr. Zaidi bin Haji Ibrahim, Mr. Mohd Norazlan bin Bujang Belly and others. For the permission to conduct this study at Panchor Hot Spring, deep thanks to the head of village, Mr Tapok anak Rayan. Special appreciation to Dr Alan Wilson, an aquatic ecologist from Auburn University, lab director for CyanoPros who helped to verify the identification of cyanobacteria, provide a lot of useful literatures and give comments about the finding in this project. Besides, thanks a lot to my dearest friends, Diyanah Ismail, Izni Arifah, Nor Syuhaidah and Nasnizah for the help and suggestion. For my family especially my beloved parents, Mr. Abdul Rahim Abdul Manaf and Mdm. Faiza Hasan, thanks for all the support and love. Lastly, thanks to all who had assisted and guided me, directly or indirectly to make this final year project completed successfully. I

TABLE OF CONTENTS Acknowledgement

I

Table of Contents

II

List of Abbreviations

IV

List of Tables and Figures

V

Abstract

i

1.0 Introduction & Objectives

1

2.0 Literature Review 2.1 Hot Springs in Malaysia 2.2 Morphology of Cyanobacteria 2.3 Morphological Terms Illustrated/Microscopic Appearances 2.4 Cyanobacteria Studies in Sarawak Freshwaters 2.5 Cyanobacteria of the Thermal Spring 2.6 Microscopic Identification of Cyanobacteria from Hot Spring 2.7 Chlor-Zinc-Iodide (Schultz solution) as staining regent

3 3 4 4 9 9 10 11

3.0 Materials and Methods 3.1 Study Site 3.2 Field Work 3.2.1 Cyanobacteria Samples Collection 3.2.2 Water Quality Parameter 3.3 Microscopic Observation and Identification of Cyanobacteria 3.4 Data Analysis

12 12 14 14 17 18 19

4.0 Result and Discussion 4.1 Thermophilic cyanobacteria in Panchor Hot Spring 4.2 Systematic Account 4.2.1 Aphanocapsa elachista West & West, 1895 4.2.2 Chroococcus minimus (Keissler) Lemmermann, 1904 4.2.3 Lyngbya martensiana Meneghini, 1837 4.2.4 Lyngbya spirulinoides Gomont ex Gomont, 1890 4.2.5 Oscillatoria anguina Bory ex Gomont, 1892 4.2.6 Oscillatoria formosa Bory ex Gomont, 1827 4.2.7 Oscillatoria granulata Gardner, 1927 4.2.8 Oscillatoria nigra Vaucher ex Gomont, 1803 4.2.9 Oscillatoria princeps Vaucher ex Gomont, 1803 4.2.10 Oscillatoria tenuis C. A. Agardh ex Gomont, 1813 4.2.11 Oscillatoria terebriformis C. A. Agard ex Gomont, 1827 4.2.12 Oscillatoria sp. 4.2.13 Phormidium inundatum Kuetzing ex Gomont, 1892 4.2.14 Planktothrix sp.

20 20 21 21 22 23 26 28 29 31 33 35 36 38 39 40 42

II

4.2.15 Pseudanabaena sp. 4.2.16 Tychonema sp.

43 45

4.3 Composition and Distribution of Cyanobacteria

46

5.0 Conclusion

51

6.0 References

52

7.0 Appendices

59

III

LIST OF ABBREVIATIONS

NH3+

:

Ammonia

ºC

:

Celcius

Chl-a

:

Chlorophyll a

DO

:

Dissolved Oxygen

g

:

Gram

m/s

:

Metre per second

µm

:

Micrometer

mg/L

:

Milligrams per litre

NO-3

:

Nitrate

PO4-3

:

Phosphate

Sp.

:

Species

IV

LIST OF TABLES

Page Table 1:

The structures of cyanobacteria used for cell identification

5

Table 2:

Brief description of the sampling stations

15

Table 3:

The list of thermophilic cyanobacteria found in Panchor Hot Spring

20

Table 4:

The composition and distribution of cyanobacteria in Panchor Hot Spring, Serian

46

Table 5:

The mean water quality parameters (Diyanah Ismail) (2014)

59

LIST OF FIGURES

Page Figure 1:

Map showing the sampling location at Panchor Hot Spring, Serian

13

Figure 2:

Hot spring pools with wooden planks at the floor and the hot water source in Kampung Panchor Dayak, Serian

14

Figure 3:

Illustration of sampling stations at Panchor Hot Spring, Serian

15

Figure 4:

Cyanobacteria growth on substratum

16

Figure 5:

Floating cyanobacteria mats

16

Figure 6:

Micrograph of Aphanocapsa elachista found in the samples

22

Figure 7:

Micrograph of Chroococcus minimus at 1000X magnification

23

V

Page Figure 8:

Micrograph of Lyngbya martensiana found in the samples

25

Figure 9:

Micrograph of Lyngbya spirulinoides found in the samples

27

Figure 10:

Micrograph of Oscillatoria anguina found in this study

29

Figure 11:

Micrograph of Oscillatoria formosa found in the samples

31

Figure 12:

Micrograph of Oscillatoria granulata found in the samples

33

Figure 13:

Micrograph of Oscillatoria nigra found in the samples

34

Figure 14:

Micrograph of Oscillatoria princeps found in the samples

36

Figure 15:

Micrograph of Oscillatoria tenuis found in the samples

37

Figure 16:

Micrograph of Oscillatoria terebriformis found in the samples

39

Figure 17:

Micrograph of Oscillatoria sp. found in the samples

40

Figure 18:

Micrograph of Phormidium inundatum found in this study

41

Figure 19:

Micrograph of Planktothrix sp. found in this study

43

Figure 20:

Micrograph of Pseudanabaena sp. found in this study

44

Figure 21:

Micrograph of Tychonema sp. found in the samples

45

VI

Thermal Cyanobacteria from Panchor Hot Spring, Serian Nur Afiqah binti Abdul Rahim Aquatic Resource Science and Management Programme Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT Cyanobacteria which also known as blue-green algae are ubiquitous, as they can be found in almost all aquatic environments such as marine, freshwater, brackish water and even hot spring. A study was conducted at Panchor Hot Spring, Serian in December 2013, January 2014 and March 2014 for documentation of its species composition of cyanobacteria. Samples of cyanobacteria were qualitatively collected for microscopic observation and identification from the pools of Panchor Hot Spring in the form of microbial mats on the water surface, samples in the water column and samples that attached on the wall of the pools and other substrates present. Live samples were collected for better identification. Results showed 16 species of cyanobacteria were found belonging to eight genera which were Aphanocapsa, Chroococcus, Lyngbya, Oscillatoria, Phormidium, Planktothrix, Pseudanabaena and Tychonema. Systematic accounts for classification, brief description and general environment for each species were discussed and documented. Based on the findings, Oscillatoria terebriformis was considered as dominant species while Oscillatoria nigra was occasionally found at Panchor Hot Spring. Key word: Cyanobacteria, systematic accounts, composition, Panchor Hot Spring

ABSTRAK Sianobakteria atau lebih dikenali sebagai alga biru-hijau boleh dijumpai di hampir semua ekosistem akuatik termasuk laut, air tawar, air payau dan juga air panas. Suatu kajian telah dijalankan di Kolam Air Panas Panchor, Serian pada Disember 2013, Januari 2014 dan Mac 2014 untuk mendokumentasikan komposisi spesies sianobakteria. Sampel sianobakteria dalam bentuk hamparan telah diambil dari permukaan air kolam, dalam turus air dan yang melekat pada beberapa jenis substrat termasuk dinding kayu kolam tersebut. Sampel sianobakteria yang hidup juga telah dikumpulkan untuk proses mengenal pasti dengan lebih baik. Keputusan mendapati 16 spesies sianobakteria daripada lapan genera telah dikenal pasti termasuk Aphanocapsa, Chroococcus, Lyngbya, Oscillatoria, Phormidium, Planktothrix, Pseudanabaena dan Tychonema. Perkelasan sistematik, huraian ringkas dan persekitaran am bagi setiap spesies telah dibincangkan dan didokumentasikan. Berdasarkan hasil kajian, Oscillatoria terebriformis adalah spesies yang dominan sementara Oscillatoria nigra adalah spesies yang jarang dijumpai di Kolam Air Panas Panchor. Kata kunci: Sianobakteria, perkelasan sistematik, komposisi, Kolam Air Panas Panchor

i

1.0 Introduction & Objective Hot springs as defined by Pirajno (2009) are water with temperatures of above 36.7ºC and mean water temperature is higher than mean air temperature. Pirajno (2009) further stated that hot springs can result from regions of anomalous heat flow due to high concentration of radioactive elements in the crust or in high-heat producing granites. However, Patricia and Malcolm (2009) categorised hot springs by water temperature of above 42ºC. Microorganisms that inhabit in the hot spring are extremophiles which were adapted to that condition (Papke et al., 2003). According to Sharma (1986), algae that can be found in very hot waters, where there are no other organisms that can live there are called ‘thermal algae’. Majority of them are members of Cyanophyceae as they have their genetic adaptations to survive in this harsh environment (Brock, 1967). Cyanophyceae is a class for cyanobacteria which are also known as blue-green algae. They are unicellular prokaryotes with a gram-negative cell wall (Prescott, 1978). Cyanobacteria are ubiquitous, as they can be found in marine, freshwater, brackish water and even hot spring (Ruhana et al., 2008). Cyanobacteria grow best in aerobic environment with the availability of sunlight for them to undergo photosynthesis (Richard, 1995). According to Richard (1995), cyanobacteria are capable of producing toxins. Freshwater cyanobacterial toxins are as potent as those produces by marine algae, especially the red tide, which become the most harmful algal blooms (Carmichael, 1994a). According to the World Health Organization (2003), cyanobacterial blooms can occur in marine, estuarine and freshwater ecosystem but its occurrence in freshwater give the greatest public health concern particularly in drinking water reservoir or recreational waters. Carmichael 1

(1998) has reported that freshwater cyanobacteria have higher tendency to produce toxic bloom compare to marine species. The most common cyanobacterial toxins are microcystins and neurotoxins. The peptide toxins in the class microcystins are considered as the most widespread cyanotoxins (Carmichael, 1994b). Several of the cyanobacterial species are known to produce toxin including Anabaena (Sotera-Santos et al., 2008). Research done by Krienitz et al. (2003) found that there are several species of cyanobacteria from hot spring at Lake Bogoria, Kenya, that are capable of producing cyanotoxins. These include Phormidium terebriformis, Oscillatoria willei, Spirulina subsalsa and Synechococcus bigranulatus. According to Brock (1969), the existence of thermophilic cyanobacteria has been extensively documented with the earliest study being carried out at Yellowstone National Park, Wyoming, USA. In several tropical countries, cyanobacteria of hot spring have been studied in Thailand (Krienitz et al., 2003), Kenya, India (Lacap et al., 2005) and Philippines (Sompong et al., 2008). However, published data on thermal cyanobacteria from hot spring are lacking in Malaysia especially in Sarawak. Thus, this is the first study carried out to record the algal community in Panchor Hot Spring in Serian, Sarawak. The main objective of this study was to document species composition of cyanobacteria that inhabit the Panchor Hot Spring. Water quality parameters (dissolved oxygen, pH, temperature, chlorophyll-a, orthophosphate, nitrate and ammonia) of the area in collaboration work with other colleague, Diyanah Ismail (2014) (unpublished data) was used to support the cyanobacteria assemblages there.

2

2.0 Literature Review 2.1 Hot Springs in Malaysia According to Copper and Copper (2009), natural hot springs in Malaysia are volcanic origin. Wehr and Sheath (2003) derived that hot springs are extreme environments in geologically active region with water temperatures ranging from 35 to 110ºC which are affected by the geothermal sources. The water of hot spring is also known to have rich in minerals and have a reputation for being beneficial for health (Sharma, 1986). They are thought to have specific healing power derived from its natural minerals. Different minerals dissolved in the water column can produce different health benefits, colour and smells according to the hot spring’s nature (Sharma, 1986). Pedas Hot Spring in Negeri Sembilan has long been famous for the therapeutic quality of the thermal waters and being visited by people for more than 60 years. There are many locations of undeveloped hot spring in Malaysia and they are mainly used by locals who believe in the healing powers of the water. According to the standard of the World Health Organization (WHO), the most suitable temperature for soaking or bathing is between 35ºC and 45ºC (Zainal et al., 2012). Felda Residence Hot Springs, Sungkai, Perak is one of the most popular hot spring in Peninsular Malaysia. This place is known for its ability to heal disease such as muscle aches, joint pain and improve body circulation. Besides, the residents also believe that the cyanobacteria present are good for their skin when applied and act as algal therapy (Bernama, 2012).

3

2.2 Morphology of Cyanobacteria The size of the cyanobacteria cell varies from less than 1µm in diameter to 2 µm in length (Platt and Li, 1986; Komárek 1999). However, solitary cells that form a cluster or colony may reach a diameter up to 100 µm (Platt and Li, 1986; Komárek 1999). According to Bellinger and Sigee (2010), the unicellular form of Synechococcus to large colonial of Microcystis and Anabaena are barely visible under the light microscope. The cyanobacterial cell walls are mainly made up of protein with a peptidoglycan layer that connects the cytoplasmic membrane. Lipoproteins and lipopolysaccharides are also present (Drew and Weckesser, 1982). Some species of cyanobacteria have gas vacuoles which appear quite dark structure under the light microscope (Bellinger and Sigee, 2010). The gas vacuoles are useful for the planktonic species to control their position in water column (Bellinger and Sigee, 2010). Freshwater cyanobacteria can be divided into four main groups based on their general morphology, presence/absence of specialized cells, and the nature of branching in filamentous forms. These groups form the basis for current taxonomy of this phylum which includes members of order Chroococcales, Oscillatoriales, Nostocales and Stigonematales (John et al., 2002).

2.3 Morphological Terms Illustrated/ Microscopic Appearances There are various morphological characteristics that can be made to differentiate the genus or species of the cyanobacteria. Some of the morphological characteristics that usually used are based on the present of heterocyst and akinetes to differentiate between the order Nostocales and Stigonematales with the order Oscillatoriales (John et al., 2002). 4

Table 1: The structures of cyanobacteria used for cell identification according to Anagnostidis and Komárek (1986; 1988; 1989; 1991); Bellinger and Sigee (2010); Deka and Sarma (2011); Prescott (1962) and Wehr and Sheath (2003).

Illustrations

Definitions

1. Trichome

A term referring to a filament or thread-like series of cells without its sheath, usually found in filamentous cyanobacteria.

2a. Akinete

a. A spore or thick-walled cell produced from

2b. Heterocyst

a vegetative cells attached to filaments which as an asexual resting stage, concentrated food reserve and usually resistant to harsh a

condition.

b

b. A thick-walled, multi-layered, specialised cell and weakly pigmented cell in some filamentous which enable fixation of gaseous nitrogen to ammonium.

5

3. Apex (Apical) cell

Cell positioned at the end or tip of a filament or thallus.

4. Capitate

With a distinct head which swollen at one end or at both ends.

6

5. Attenuated/ Tapered

Narrowed or tapering toward the ends.

6. Calyptra

A thickened or enlarged tip of a trichome in filamentous cyanobacteria.

a

a. Round-shaped b

b. Cone-shaped

Without calyptra

7

7. Sheath

A covering envelope which are sometimes thin and composed of mucilage enclosing a filament or group of cells.

8. Cross Wall

A partition or dividing wall which divide each

a.

cell. a. Present of cross wall b. Absent of cross wall c. Granular at cross wall

b.

c.

2.4 Cyanobacteria Studies in Sarawak Freshwaters 8

Ruhana et al. (2008) studied the composition of cyanobacteria in Sungai Semadang and Sungai Bengoh in Kuching, Sarawak. They found 38 genera of cyanobacteria where Lyngbya, Oscillatoria, Stigonema and Spirulina were the common genera (Ruhana et al., 2008). Nasarudin and Ruhana (2011a) reported 43 species of cyanobacteria belonging to 30 genera in selected aquatic ecosystems including aquaculture ponds, cage cultures, waterfall and artificial lake in Serian, Bau and Batang Ai. The most widely distributed genera found were Chroococcus, Lyngbya, Nostoc and Oscillatoria (Nasarudin and Ruhana, 2011a). Nasarudin and Ruhana in (2011b) had further studied cyanobacteria in empurau (Tor tambroides) aquaculture ponds at IFRPC Tarat. They found that Synechocystis, Oscillatoria, Chroococcus, Nostoc and Pleurocapsa were the common cyanobacteria found in these ponds.

2.5 Cyanobacteria of the Thermal Spring Cyanobacteria can undergo photosynthesis and tolerate well in high temperature and in other extreme environments. Cyanobacteria can occur abundantly in almost photic habitats. As stated by Ward and Castenholz (2000), cyanobacteria that are dominating in thermal waters include masses of coccoid species (Aphanocapsa, Chroococcus, Cyanobacterium and Synechococcus) and filamentous species (Mastigocladus, Oscillatoria and Phormidium). Cyanobacterial accumulations may form mat of several centimeters thick and have quite high rates of primary production (Castenholz and Wickstrom, 1975). Akmar et al. (2011) in their study at Ulu Legong Hot Spring, Baling, Kedah stated that the organisms that forming microbial mat are usually cyanobacteria, diatoms and bacteria that compete for 9

available nutrients (nitrogen, phosphorus, iron and carbon). Studies made by Sompong et al. (2008) in six hot spring in Thailand found that there are 14 distinct species of cyanobacteria according to their morphological structure. Thermophilic blue-green algae are particularly concentrated in hot spring waters with a pH of > 6 where they form conspicuous and often unicellular algae mat-like covering over submerged substrate. According to Castenholz (1969), the shallowness and the clarity of most thermal waters, in addition to high light intensities exposure, the thermophilic organisms should consider a various types of heat adaptation. Moreover, they can also adapt to high concentration of ions present in the water column (Castenholz, 1969). Algae present at the thermal environment are attached, at least initially to the substratum. Finally, they may form large floating surface mats which can be buoyed up by the oxygen produced during photosynthesis (King, 1988). According to Health Canada (2002), cyanobacterial mat are formed mostly at night, when they are unable to adjust their buoyancy and finally become over buoyant especially when there are no wind and water circulation.

2.6 Microscopic Identification of Cyanobacteria from Hot Spring Lukavský et al. (2011) had found eight taxa of cyanobacteria in a hot spring of Panharevo, Sofia, Bulgaria. The mats of the cyanobacteria were differentiated according to their position with respect to the outlet of the hot water. The species found were Gloeocapsa gelatinosa, Leibleinia epiphytica, Phormidesmis molle, Phormidium corium, Symploca thermalis, Lyngbya thermalis, Calothrix thermalis and Mastigocladus laminosus.

10

Previously, microscopic analysis of cyanobacteria from the Wonder Lake geothermal springs, Laguna, Phillippines by Lacap et al. (2005) had recorded Fischerella and Oscillatoria in their study site. Arif (1997) had recorded that Synechococcus and Oscillatoria to be the common cyanobacterial genera in the Asian hot springs. The author also noted that Synechococcus spp. usually occurred as a thin mat. Oscillatoriales has been reported as the most conspicuous species of cyanobacteria that inhabit the hot springs of temperature between 40ºC to 66ºC (Pentecost, 2003). Chorus and Bartram (1999) documented that the filamentous Oscillatoriales are well present in sulphur-rich environments. Their filamentous structure and polysaccharides structure may be the reason for them to become the backbone of the microbial mat (Van Gemerden, 1993). 2.7 Chlor-Zinc-Iodide (Schultz solution) as staining reagent Members of Oscillatoriales family especially the genus Oscillatoria were seldom get confused with genus Phormidium. Therefore, observation and cell identification are much better to be done by staining them with an aqueous dye or chlor-zinc-iodide (Prescott, 1962). The protocol to prepare this staining solution was discussed by Drouet (1937) which composed of potassium iodide (KI), anhydrous zinc chloride (ZnCl2) and iodine crystals. The chlor-zinc-iodide is used to stain the sheaths of cyanobacteria hence different species of sheathed cyanobacteria may be differentiated. The solution should be mounted accordingly and less than a minute is required for the production of the distinct blue colour in the sheaths. 3.0 Materials and Methods 3.1 Study Site Sampling was carried out at Panchor Hot Spring which is situated at N01⁰15’16.6’’, E110⁰26’51.3’’, Serian, Sarawak (Figure 1). This hot spring is managed by the villagers of 11

Kampung Panchor Dayak under the supervision of The Ministry of Tourism Sarawak. The surrounding area of the hot spring is swampy covered by secondary forest (Roslan, 2006). There are eight pools divided by wood planks (Figure 2). The floor of each pool is also made up of wood planks. Thus the water is freely flowing from the source of the hot spring to other pools and finally discharges in the channel that flows through the swamp into Sungai Kepayang. Four of the pools are shaded under a roof while the other four are exposed to direct sunlight.

12

(Google Maps, 2014)

N

Panchor Hot Spring

1º 15” N

110º 26” E Figure 1: Map showing the sampling location at Panchor Hot Spring, Serian (Director of National Mapping, Malaysia, 2010)

13

Figure 2: Hot spring pools with wooden planks at the floor and the hot water source (arrow) in Kampung Panchor Dayak, Serian.

3.2 Field work 3.2.1 Cyanobacteria Samples Collection This study was conducted in three sampling times which were on December 2013, January 2014 and March 2014. Samples of cyanobacteria were qualitatively collected from the pools of Panchor Hot Spring. Six out of eight pools were selected at the sampling stations and the brief description of the sampling station were recorded as in Table 2, where three pools are under shaded area and the other three from the exposed area (Figure 3). Additional samples were collected from the water discharge drainage. Triplicate samples were taken from each station at the periphery (Figure 3) and from the middle of the pools.

14