Eco-Physiological Studies on Cyanobacteria in the Sudan A

Eco-Physiological Studies on Cyanobacteria in the Sudan

By:

Eltigani Hamid Shihaba Ali

B.Sc. (Agric.) honours

(Agricultural Biotechnology)

August 2002

A Thesis Submitted to the University of Khartoum in partial fulfillment for the degree of Master of Science (Agric.)

Supervisor:

Prof. Ahmed Ali Ahmed Mahdi

Department of Botany and Agricultural Biotechnology

Faculty of Agriculture

University of Khartoum

February 2010

ﺑﺴﻢ اﷲ اﻟﺮﲪﻦ اﻟﺮﺣﻴﻢ

۩واﷲُ ﺧَﻠﻖ آُﻞَ داﺑﺔٍ ﻣِﻦْ ﻣﺎءٍ ﻓﻤﻨْﻬﻢ ﻣﻦْ ﳝﺸِﻲ ﻋﻠﻲ ﺑﻄْﻨِﻪِ و ﻣﻨْﻬﻢ ﻣﻦ ﳝﺸﻲ ﻋﻠﻲ ِرﺟﻠِﲔ و ﻣﻨْﻬﻢ ﻣﻦْ ﳝﺸِﻲ ﻋﻠﻲ أرﺑﻊ ﻳﺨْﻠُﻖ اﷲُ ﻣﺎ ﻳﺸَﺎءُ إن اﷲَ ﻋﻠَﻲ آُﻞ ﺷﻲءٍ ﻗَﺪِﻳﺮ ۩

اﻟﻨﻮر

ﺻﺪﻕ ﺍﷲ ﺍﻟﻌﻈﻴﻢ

The locomotion type of cyanobacteria is the gliding!!!!

TABLE OF CONTENTS

Item Page No.

Dedication …………………………………………………………...…i

List of Tables ……………………….…………………………………ii

List of Figures ………………………………………………………..iii

List of plates…………………………………………………………..vi

Acknowledgement …………………………………………...... vii

Abstract (English) ………………………………………..…………….ix Abstract (Arabic) ………………………………………… ………….xi

CHAPTER ONE: INTRODUCTION ……………..……….. ………1

CHAPTER TWO: LITERATURE REVIEW ………………..…….6

2. Cyanobacteria…………………………………………………………6

2.1- Definition and characterization ……………………………...……...6

2.2- Biological diversity of cyanobacteria………………………...……...9

2.3- Cyanobacteria and relation with other organisms ………...…..10

2.4- Classification of cyanobacteria………………………...... 13

2.5- Factors affecting growth of Cyanobacteria ……………………....16

2.5.1- Light intensity ………………………………………………..…16

2.5.2-Temperature…………………………………...... …...... 19

2.5.3- Salinity………….……………………………………. . ……...20 2.5.4- pH …………………………………………….……………….22

2.5.5- Phosphorous and Nitrogen ……………………………………22

2.6- Cyanobacteria and Crusting ability………………..…………….23

2.7 -Cyanobacteria and photosynthesis ……………….………………25

2.8 -Cyanobacteria and Nitrogen fixation……………….…………….25

2.9 - Cyanobacterial risks………………………………...…………...27

2.10- Population stability………………………………………...... 29

CHAPTER THREE: MATERIALS AND METHODS………...... 30

3.1- Study area and sampling sites………………………………...……30

3.1- Samples collection, culturing and media …………………………..30

3.3- Isolation and purification ……………………………………...….34

3.4- Identification…………………………………………………….....34

3.5- Physiological studies………………………………………….…...34

3.6- Effects of temperature…………………………………...…………37

3.7- Effects of pH……………………………………….………………37

3.8- Effect of light intensity……………………………….…………….39

3.9- Effects of salinity………………………….………………………39

3. 10- Crusting ability……………………….……………….………….40

CHAPTER FOUR: RESULTS……………………………………….42

Temporal distribution of cyanobacteria……………………………...... 42 Temporal comparison of location……………………………………….42

Spatial distribution of cyanobacteria……………………………………47

Distribution of cyanobacteria on three types of samples………………..47

Frequency of the five cyanobacterial orders…………………………….47

Isolates of the genera of the order Oscillatoriales……….……………...47

Isolates of the genera of the order Nostocales…………………………..53

Isolates of the genera of the order Chroococcales………………………53

Temporal distribution for prevalent orders………………………...... 53

The most frequency cyanobacteria species……………………………..57

Effects of Salinity on cyanobacterial growth………………………...... 57

Effects of Light on cyanobacterial growth ……………………………..57

Effects of pH on cyanobacterial growth ……………………………...61

Effects of temperature on cyanobacterial growth …………………...... 61

Effects of cyanobacteria on sand soils crusting……………………...... 61

Effects of cyanobacteria on clay soils crusting…………………………61

CHAPTER FIVE: DISCUSSION ……………………………….....66

CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS …………………………………………………………………………..82

REFERENCES ……………………………………………………....84

Appendixes.………………………………………………………….114

Dedication

To MY FAMILY:

TO MOTHER AND WIFE,

TO BROTHERS AND SISTERS,

TO THE NEWBORN WHO RESEMPLES HIS FATHER THE MOMENT HEWAS BORN:

MY SON

LIST OF TABLES

Table No. Page No.

1- The principal groups of cyanobacteria …………………………….15

2- Isolates used in the physiological studies………………….……….37

3- The total number of isolates in the two location…………………....43

LIST OF FIGURES

Fig. No. Page No.

1- Total number of cyanobacterial isolates in the 10 sampling points during the period January-November 2008…..………………….43

2- Number of cyanobacterial isolates in each of the 10 sampling points for the months Jan, Mar, May, July Sep and Nov 2008…….………44

3- Total number of cyanobacterial isolates in each sampling point during the period January-November 2008……..………………….……....48

4- Temporal distribution of total isolates of the water samples, dry and wet soil samples……………………………………………………..49

5- Frequency of isolation of members of each of the five cyanobacterial orders in all sampling sites for the months of January, March, May, July, September and November 2008……………………………...... 50

6- Number of isolates of the genera of the order Oscillatoriales in all 10 sampling sites for the period January-November 2008………………51

7- Number of isolates of the genera of the order Nostocales in all 10 sampling sites for the months January-November 2008...…………..54

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8- Number of isolates of the genera of the order Chroococcales in all 10 sampling sites for the months January-November 2008..……………55

9- Temporal distribution of the most prevalent cyanobacterial orders during the period January-November 2008…………………………56

10- Frequency of isolation of the most prevalent cyanobacterial spp. in the sampled areas…………………………………..………………58

11- Effect of salinity on growth of the six selected cynobacterial isolates……………………………………………..………………59

12- Effect of light intensities on the growth of the six chosen cyanobacterial isolates..……………………………………………..60

13- Effect of pH on the growth of the six selected cyanobacterial isolates……………………………………………………………..62

14- Effect of temperature on the growth of the six selected cyanobacterial isolates……………………………………… ……………………..63

15- Sand aggregating activity of the growth of the six selected cyanobacterial isolates...………………………………………...….64

16- Clay aggregating activity of the growth of the six selected cyanobacterial isolates……………………………………………..65

LIST OF PLATES

Plate No. Page No.

1- Map of the Sudan………………….…………………...…………5

2- El-Rahad dry sand site……………………………………….….. 32

3- El-Rahad Turda site………………………………………………33

4- Cultures of cyanobacteria ………………………………………..35

5- Micrographs of hormogona………………………………..…..…36

6- Micrographs of Microcystis sp., Synechocystis sp. and Dermocarpa sp……………………………….………………...……….………38

7- The oscillator apparatus……………………………….…………… 41

8- Micrographs of Lyngbya sp. And Spirulina sp…….………………..46

9- Micrographs of Anabaena sp., Calothrix sp. and Cylindrospermopsis sp……………………………………………….……………….……….52

APPENDIX

1- Mean sequent for the effect of salinity on chosen strains and their interactions on dry weight …………………………………...……..…112

2- Mean sequent for the effect of light on chosen strains and their interactions on dry weight……………… …………………………..112

3- Mean sequent for the effect of pH on chosen strains and their interactions on dry weight ……………………………………...….112

4- Mean sequent for the effect of temperature on chosen strains and their interactions on dry weight ………………………………………….....113

5- Mean sequent for the effect of selected isolates on sand soil particle aggregation….……………..……………………………...……….…114

6- Mean sequent for the effect of selected isolates on clay soil particle aggregation ……………….…………………………………….…...115

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Acknowledgements I would like particularly to express my gratitude to: Prof. Ahmed Ali Mahdi, my supervisor: first, for accepting me as an M.Sc. student at the Dept. of Botany and Agricultural Biotechnology. Second, for his unlimited patience on many challenges providing, many nice discussions and so many ideas. He definitely has taught me a great deal of useful things, ranging from Science to Systems dynamics, and even the simple, but hard mission of smiling at the most rough times. Thank you for all the support and I hope that the time of thanking you would not come soon. A special gratitude to my friends who like brothers to me; Aboalgasim Mohmmed Ahmed, Eltyeb Abd allatif (Hulfawi) and Ismail Ahmed Mohmmed who, played an important role in my work.

All the members of the Department of Botany and Agricultural Biotechnology; Thank you all for the good moments and teamwork spirit, while sharing knowledge with so many colleagues for their precious technical help during my time as lab assistants: always prepared, always very organized, always with a big smile! Thank you! Thanks extend to Biotechnology Commission at the National Center For Research, particularly to Hesien (Hennery), Mu'taz, Eihab, Aiman, Hajo, and all who used smile when we met. Appreciation is expressed to my “better-half”: Ashraf Musa for showing peculiar interest in helping me, for unconditional support from the very beginning, remarkable sense of patience, trust and hope. To my family: Mother, Wife, Brothers, Sisters, and my eldest son Mohmmed Alfatih, who lived without father's kindness during the period of the study. Unending gratefulness goes to all who helped and supported me but are not mentioned here.

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ABSTRACT

This study was carried out in the Biofertilizer Laboratory of the Department of Botany and Agricultural Biotechnology, Faculty of Agriculture, University of Khartoum at Shambat during 2008 - 2009 to investigate the abundance, spatial and temporal distribution of cyanobacteria in Shambat and Omdurman in Khartoum State and El Rahad town in North Kordofan State (Sudan) and to determine the optimal growth conditions and role of some factors affecting cyanobacterial growth.

This study is considered the first of its kind in Sudan on an important microbial group, cyanobacteria, they are found throughout the world with prominent roles in carbon fixation (photosynthesis), nitrogen fixation and many other beneficial effects on soil physical properties in arid environments.

Eight soil and two water samples, from the sampling points in the two locations viz. Khartoum State and North Kordofan State, were inoculated into BG11 broth medium in flasks in the laboratory.

Cyanobacteria was procured from all ten sampling points at all sampling times (January to November 2008). A total of 278 isolates belonging to 46 species were obtained representing the five cyanobacterial orders and 25cyanobacterial genera. Six pure cultures were chosen for studying some physiological traits. The results showed that there were no significant differences in the number of isolates of the samples of the two locations, (136 isolates in the Khartoum State and 142 isolates in Kordofan State). likewise, there were no great significance differences between the wet and dry samples (163and115 respectively). The wide

distributed order was the order Oscillatoriales was more widely distributed than the otherand the most frequently genera were Oscillatoria (isolated 62 times), Anabaena (isolated 57 times), Lyngbya (isolated 31 times) and Microcystis (isolated 27 times).

Generally, cyanobacterial growth decreased with increasing of NaCl concentration. Best growth was obtained in the presence of 2% and least at 5%NaCl. No growth was obtained at 6% NaCl and the differences were not significant. In the temperature range 25°C -45°C,the best growth occurred at 35 °C, and the least at 45°C, the differences were significant. No significant differences were obtained in the pH range 5.5- 8.5. However, best growth was obtained at 6.5 pH. No clear pattern could be discerned from the growth values obtained in the light intensities of 500, 2000, 3000, and 4000 lux. There were high significant differences in the crusting abilities of the tested isolates. Two of the chosen filamentous isolates (Spirulina sp. and Anabaena doliolum) showed best results of soil particle aggregation in the sandy soils approximately (92.7 % and 95.1% soil crusting, respectively), while those for the clay soils were 36.3% and 35.7%, respectively.

اﻟﻤﺴﺘﺨﻠﺺ

أﺟﺮﻳﺖ هﺬﻩ اﻟﺪراﺳﺔ ﻓﻲ ﻣﻌﻤﻞ اﻟﺘﺴﻤﻴﺪ اﻟﺤﻴﻮي ﺑﻘﺴﻢ اﻟﻨﺒﺎت و اﻟﺘﻘﺎﻧﺔ اﻟﺤﻴﻮﻳﺔ اﻟﺰراﻋﻴﺔ، ﺑﻜﻠﻴّﺔ اﻟﺰراﻋﺔ، ﺟﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم ﻓﻲ ﺷﻤﺒﺎت (2008 ⁄ 2009)، ﻟﻠﺘﻌﺮف ﻋﻠﻲ وﺟﻮد اﻟﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮﻳﺎ ودراﺳﺔ ﺗﻮزﻳﻌﻬﺎ اﻟﻤﻜﺎﻧﻲ واﻟﺰﻣﺎﻧﻲ ﻓﻲ ﻣﻮﻗﻌﻴﻦ ﺑﻜﻞ ﻣﻦ وﻻﻳﺔ اﻟﺨﺮﻃﻮم ووﻻﻳﺔ ﺷﻤﺎل آﺮدﻓﺎن(اﻟﺴﻮدان) ﺑﺠﺎﻧﺐ دراﺳﺔ دوز ﺑﻌﺾ اﻟﻌﻮاﻣﻞ اﻟﺘﻲ ﺗﺆﺛﺮ ﻋﻠﻰ ﻧﻤﻮ اﻟﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮﻳﺎ .

ﺗﻌﺘﺒﺮ هﺬﻩ اﻟﺪراﺳﺔ اﻷوﻟﻰ ﻣﻦ ﻧﻮﻋﻬﺎ ﻓﻲ اﻟﺴﻮدان ﻟﻤﺠﻤﻮﻋﺔ ﻣﻬﻤﺔ ﻣﻦ اﻷﺣﻴﺎء اﻟﺪﻗﻴﻘﺔ هﻲ اﻟﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮﻳﺎ اﻟﻮاﺳﻌﺔ اﻻﻧﺘﺸﺎر ﻓﻲ آﻞ اﻟﻌﺎﻟﻢ وذات اﻟﺪور اﻟﺒﺎرز ﻓﻲ ﻋﻤﻠﻴﺘﻲ ﺗﺜﺒﻴﺖ اﻟﻜﺮﺑﻮن و اﻟﻨﻴﺘﺮوﺟﻴﻦ و اﻟﺘﺄﺛﻴﺮات اﻟﻤﻔﻴﺪة ﻟﺨﺼﺎﺋﺺ اﻟﺘﺮﺑﺔ اﻟﻔﻴﺰﻳﺎﺋﻴﺔ ﻓﻲ اﻟﺒﻴﺌﺎت اﻟﻘﺎﺣﻠﺔ.

أﺧﺬت ﺛﻤﺎن ﻋﻴﻨﺎت ﻣﻦ اﻟﺘﺮﺑﺔ و ﻋﻴﻨﺘﻴﻦ ﻣﻦ اﻟﻤﺎء ﻣﻦ ﻧﻘﺎط أﺧﺬ اﻟﻌﻴﻨﺎت ﻓﻲ ﻣﻮﻗﻌﻴﻦ ﻓﻲ اﻟﺴﻮدان. (وﻻﻳﺔ اﻟﺨﺮﻃﻮم وو ﻻﻳﺔ ﺷﻤﺎل آﺮدﻓﺎن) و ﻟﻘّﺤﺖ ﻓﻲ ﺑﻴﺌﺔ ﻣﺮق BG11 ﺑﺪوارق ﻓﻲ اﻟﻤﺨﺘﺒﺮ.

ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻲ اﻟﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮﻳﺎ ﻣﻦ آﻞ ﻧﻘﺎط أﺧﺬ اﻟﻌﻴﻨﺎت اﻟﻌﺸﺮ ﻓﻲ آﻞ أوﻗﺎت أﺧﺬ اﻟﻌﻴﻨﺎت (ﻳﻨﺎﻳﺮ إﻟﻲ ﻧﻮﻓﻤﺒﺮ 2008)، و ﻗﺪ أﻣﻜﻦ اﻟﺤﺼﻮل ﻋﻠﻲ 46 ﻋﺰﻟﺔ ﺗﻤﺜّﻞ 25 ﻧﻮﻋﺎ ﻣﻮزﻋﺔ ﻋﻠﻲ اﻟﺮﺗﺐ اﻟﺨﻤﺲ ﻟﻠﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮﻳﺎ. اﺧﺘﻴﺮت ﺳ ﺖّ ﻋﺰﻻت ﻧﻘﻴﺔ ﻟﺪراﺳﺔ ﺗﺄﺛﺮ ﺑﻌﺾ اﻟﺼﻔﺎت اﻟﻔﺴﻴﻮﻟﻮﺣﻴﺔ.

أوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ ﻋﺪم وﺟﻮد ﻓﺮوق ﻣﻌﻨﻮﻳﺔ ﻓﻲ أﻋﺪاد اﻟﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮﻳﺎ ﻓﻲ اﻟﻌﻴﻨﺎت اﻟﻤﺄﺧﻮذة ﻣﻦ اﻟﻤﻮﻗﻌﻴﻦ (142ﻋﺰﻟﺔ ﻓﻲ وﻻﻳﺔ آﺮدﻓﺎن و 136 ﻋﺰﻟﺔ ﻓﻲ وﻻﻳﺔ اﻟﺨﺮﻃﻮم )، آﻤﺎ أﻧﻪ ﻻ ﺗﻮﺟﺪ ﻓﺮوق ﻣﻌﻨﻮﻳﺔ ﻓﻲ اﻷﻋﺪاد ﺑﻴﻦ اﻟﻌﻴﻨﺎت اﻟﺮﻃﺒﺔ و اﻟﺠﺎﻓﺔ (163 ﻋﺰﻟﺔ ﻓﻲ اﻟﻌﻴﻨﺎت اﻟﺮﻃﺒﺔ و 115ﻋﺰﻟﺔ ﻓﻲ اﻟﻌﻴﻨﺎت اﻟﺠﺎﻓﺔ).

آﺎﻧﺖ رﺗﺒﺔ Oscillatoriales هﻲ أآﺜﺮ اﻟﺮﺗﺐ ا ﻧ ﺘ ﺸ ﺎ ر اً، و آﺎﻧﺖ اﻷﺟﻨﺎس اﻷآﺜﺮ اﻧﺘﺸﺎرًا هﻲ Oscillatoria (ﻋﺰﻟﺖ62 ﻣﺮة) و Anabaena (ﻋﺰﻟﺖ 57 ﻣﺮة) و Lyngbya (ﻋﺰﻟﺖ 31 ﻣﺮة) و Microcystis (ﻋﺰﻟﺖ 27 ﻣﺮة).

ﻋﻤﻮﻣﺎ ﺗﻨﺎﻗﺺ ﻣﻌﺪل ﻧﻤﻮاﻟﺴﻴﺎﻧﻮﺑﺎآﺘﻴﺮي ﻣﻊ زﻳﺎدة ﺗﺮآﻴﺰ آﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم وﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻲ أﻓﻀﻞ ﻧﻤﻮ ﻋﻨﺪ اﻟﺘﺮآﻴﺰ 2% و آﺎن أﺿﻌﻔﻪ ﻋﻨﺪ اﻟﺘﺮآﻴﺰ 5% وﺗﻮﻗﻒ اﻟﻨﻤﻮ ﺗﻤﺎﻣﺎ ﻋﻨﺪ اﻟﺘﺮآﻴﺰ

6% و ﻟﻢ ﺗﻜﻦ اﻟﻔﺮ وﻗﺎت ﻣﻌﻨﻮﻳﺔ. ﻓﻲ اﻟﻤﺪي اﻟﺤﺮاري ﺑﻴﻦ 25 إﻟﻲ 45 درﺟﺔ ﻣﺌﻮﻳﺔ آﺎن أﻓﻀﻞ ﻧﻤﻮ ﻋﻨﺪ درﺟﺔ اﻟﺤﺮارة C°35 و آﺎن أﻗﻠﻪ ﻋﻨﺪ درﺟﺔ اﻟﺤﺮارة C°45 اﻟﻔﺮق ﻣﻌﻨﻮي. ﻟﻢ ﺗﻜﻦ هﻨﺎك ﻓﺮوﻗﺎت ﻣﻌﻨﻮﻳﺔ ﻓﻲ ﻣﻌﺪل اﻟﻨﻤﻮ ﻋﻨﺪ ﻣﺪي درﺟﺔ اﻟﺤﻤﻮﺿﺔ و اﻟﻘﻠﻮﻳﺔ ﻣﻦ 5.5 إﻟﻲ 8.5، و آﺎن أﻓﻀﻞ ﻧﻤﻮ ﻋﻨﺪ اﻟﺪرﺟﺔ 6.5.

ﻟﻢ ﻳﻜﻦ ﻟﺸﺪة اﻟﻀﻮء 2000,500، 3000و lux 4000 ﺗﺎﻳﺜﺮ واﺿﺢ ﻋﻠﻲ اﻟﻨﻤﻮ .

آﺎﻧﺖ هﻨﺎك ﻓﺮوﻗﺎت ﻣﻌﻨﻮﻳﺔ ﻓﻲ ﻗﺪرة اﻟﻌﺰﻻت ﻋﻠﻲ ﺗﺠﻤﻊ ﺣﺒﻴﺒﺎت اﻟﺘﺮﺑﺔ و أﻋﻄﺖ اﺛﻨﺘﺎن ﻣﻦ اﻟﻌﺰﻻت اﻟﺨﻴﻄﻴﺔ اﻟﻤﺨﺘﺎرة (.Spirulina sp و Anabaena doliolum) أﻓﻀﻞ اﻟﻨﺘﺎﺋﺞ ﻓﻲ اﻟﺘﺮﺑﺔ اﻟﺮﻣﻠﻴﺔ (92.7 % و95.1 % ، ﻋﻠﻰ اﻟﺘﻮاﻟﻲ) ﺑﻴﻨﻤﺎ آﺎﻧﺖ اﻟﻨﺘﻴﺠﺔ ﻓﻲ اﻟﺘﺮﺑﺔ اﻟﻄﻴﻨﻴﺔ 35.7% و36.3% ﻋﻠﻲ اﻟﺘﻮاﻟﻲ.

xii

CHAPTER ONE

INTRODUCTION

The cyanobacteria ( also known as Blue-Green Algae, Blue-Green Bacteria or Cyanophayta) are an ancient group of prokaryotic organisms that are found all over the world in environments as diverse as Antarctic soils and volcanic hot springs, and often where no other vegetation can exist. Being prokaryotes they share with others of their type the lack of a nucleus and a laminated extracellular wall. Unlike photosynthetic bacteria however, cyanobacteria possess chlorophyll a in common with photosynthetic eukaryotes, and they liberate oxygen during photosynthesis (Bold and Wynne 1985).They are of great antiquity being found in fossil stromatolites dated to 3 billion years BC (Schopf and Walter 1982) and are considered to have been the organisms responsible for the early accumulation of oxygen in the earth’s atmosphere.

The cyanobacteria presently constitute a phylum in the domain Bacteria, embodying five orders (Hoffmann et al. 2005). Traditionally this group of photosynthetic prokaryotes has been classified as a group of algae under the botanic code with a system of classification based on morphological, developmental and ecological characteristics determined on natural samples rather than on pure cultures. This system contains150 genera and about 1000 species and has shown to be inadequate for the classification of cyanobacteria maintained in axenic culture (Stafleu et al.1972).

However, following the unequivocal demonstration of the prokaryotic nature of these organisms, traditional microbiological techniques are now being applied to pure cultures of these organisms in the study of their biological

1 characteristics and classification. As in the traditional botanical taxonomic treatments, morphological and developmental feature form the bases for the description of taxa ( Castenholz 1989a, 1989b,1989c;Waterbury 1989). However, because of the importance of structural and developmental characters for the classification of field and cultured material, it will be possible for the system of classification developed under the bacteriological code (Lapage 1975) to represent an extension and refinement of the classic botanical classification.

Cyanobacteria are the only organisms ever to evolve coupled photosynthesis that harvests electrons from water and produces dioxygen as a byproduct (Knoll 2008). They are photosynthetic prokaryotes typically possessing the ability to synthesize chlorophyll a (Oliver and Galf. 2000). Furthermore, cyanobacteria have also been characterized by their ability to form the phycobiliprotein pigments, viz. phycocyanin, phycoerythrin and allophycocyanin. They sometimes accumulate high concentration of this pigment (phycopiliprotein) under some conditions which leads to the bluish color of the organisms, and hence both of the names by which they are commonly known, cyanobacteria or blue-green algae (Oliver and Galf 2000).

In terms of Earth history, cyanobacteria occupy a privileged position among organisms; as primary producers they play a significant role in Earth’s carbon cycle; as nitrogen fixers, they also figure prominently in the nitrogen cycle. Moreover, they also loom large in our planet’s redox history. In fact, one of the major changes on Earth, the introduction of oxygen into the atmosphere 2450-2320 million years ago, is widely accepted to be attributed to the photosynthetic activity of cyanobacteria. However, different pieces of

2 evidence seem to suggest that cyanobacteria appeared 3000-2700 million years ago (Knoll 2008).

Cyanobacterial ecological plasticity is remarkable and their long evolutionary history is possibly related to some of the reasons for their success in modern habitats. They are mostly found in aquatic, but also in many terrestrial environments. Here, their tolerance to desiccation and water stress is a key factor; they often play a key role in maintaining the stability of the surface crusts of semi-deserts and the fertility of soils used for farming in arid regions. They can even be found growing near the limits for life in the dry deserts, as part of microbial communities below the surface in true deserts, or in many thermal springs. In the aquatic environments (fresh, brackish or saline water), the cosmopolitan distribution is also extraordinary. Reports of dense populations of cyanobacteria forming water-blooms have increased in many countries and some of the factors leading to the formation of such blooms are now being analyzed and carefully characterized. However, the fact that these blooms are often toxic has led to increase awareness and concern over the environmental and health problems associated with them (Oliveira and Lindbland, 2005).

This study was carried out to investigate the abundance and distribution of cyanobacteria in two locations in Sudan; Khartoum State (Shambat) and North Kordofan State (El Rahad town) (plate 1). Very little work has been devoted to investigations on cyanobacteria in Sudan. The fragmentary information available does not exceed cursory observations on cyanobacteria encountered during the course of investigations on other microbial groups.

The main objectives of this study were;

1- Study of the spatial and temporal distribution of these genera and species in the sampled sites.

2- Determination of the factors affecting proliferation and abundance in the different habitats and seasons.

3- Identification of the dominant genera and species of cyanobacteria in the studied areas.

4- Determination of optimal growth conditions and some factors affecting cyanobacterial growth.

5- Determination of cyanobacterial ability for soil stabilization and crusting.

Plate 1. The map of the Sudan showing locations of the Study.

CHAPTER TWO

LITERATURE REVIEW

2. Cyanobacteria

2.1 Definition and characteristics

Cyanobacteria are oxygen-evolving phototrophs which are often conspicuous components of aquatic and terrestrial ecosystems (Rippka et al. 1979). Cyanobacteria, (formerly blue-green algae), are relatively simple, primitive life forms closely related to bacteria (both are prokaryotes) that should not be mistaken for true algae. They are referred to in the literature by various names, chief among which are Cyanophyta, Myxophyta, Cyanochloronta, Cyanobacteria, Blue-Green Algae and Blue-Greens. Although they are typically much larger than bacteria, microscopic examination of cells reveals little internal structure. Nearly all are photosynthetic. In addition to the lipid-soluble chlorophylls and carotenoids, cyanobacteria contain characteristic water- soluble pigment called "phycocyanin" which gives the group their blue-green coloration. When cyanobacteria blooms begin to die and disintegrate, this water- soluble pigment may give the water a distinctive bluish color. Depending upon the species, cyanobacteria can occur as single cells or as filamentous in growth (Stainer and Cohen-Bazire 1977).

Although cyanobacteria probably evolved as a group of organisms about 2,000 million years before the advent of eukaryotes, they comprise fewer taxa than eukaryotic microalgae (Bisby 1995). The concept of species in the cyanobacteria has, however, no distinct boundaries. The situation is similar for most organisms, except for those that are sexually reproductive.

Depending on the classification system used, the number of species recognized varies greatly. Based on the International Code of Botanical Nomenclature the class Cyanophyceae, for example, contains about 150 genera and 2,000 species. Chemotaxonomic studies include the use of markers, such as lipid composition, polyamines, caroteneoids and special biochemical features. The resulting data support the more traditional examinations of phenotypic and ecological characteristics (Hoek et al. 1995). Physiological parameters are conveniently studied using laboratory cultures (Packer and Glazer 1988). A molecular approach to the systematics of cyanobacteria may be most fruitful for inferring phylogenetic relationships. Macromolecules, such as nucleic acids and proteins, are copies or translations of genetic information. The methods applied involve direct studies of the relevant macromolecules by sequencing, or indirectly by electrophoresis, hybridisation, or immunological procedures (Wilmotte 1994). Nucleic acid technologies, especially the polymerase chain reaction (PCR), have advanced to the point that it is feasible to amplify and sequence genes and other conserved regions from a single cell. To date, 16S rRNA has given the most detailed information on the relationships within the cyanobacteria (Rudi et al. 1997). However, the molecular results obtained should be integrated with other characteristics as the base for a polyphasic taxonomy (Vandamme et al. 1996).

A considerable morphological, as well as a genotypical, polymorphy exists in the cyanobacteria, although as data from rRNA sequencing indicate they are correlated to a high degree. The phylogenetic relationships of cyanobacteria are the rationale behind the meaningfull systematic grouping. However, it is difficult to set up a system of classification that serves both the everyday need for practical identification, and offers an expression of the natural relationship

7 between the organisms in question (Mayr 1981). Meanwhile, it will be necessary to use the available manuals and reference books to help in these investigations and with the proper identification of the cyanobacteria.

Because they are photoautotrophs, cyanobacteria can be grown in simple mineral media. Vitamin B is the only growth factor that is known to be required by some species. Media must be supplemented with the essential nutrients needed to support cell growth, including sources of nitrogen, phosphorus, trace elements, etc (Sugawara et al. 1993).

Some species of unicellular, heterocystous, and nonheterocystous filamentous cyanobacteria have the ability to fix atmospheric nitrogen (Rippka et al. 1979; Young 1992). Substantial progress has been made in understanding the ecology, biochemistry and molecular biology of nitrogen fixation in cyanobacteria (Gallon and Chaplin 1988; Haselkorn et al.1990; Paerl 1990). Cyanobacterial nitrogen fixation is potentially important in regulating primary productivity in nitrogen-deficient aquatic environments, yet relatively little is known about the ecology of natural populations of nitrogen-fixing cyanobacteria.

Cyanobacteria are unique in that they are Gram-negative prokaryotes and yet perform an oxygenic photosynthesis very similar to that of higher plants. Thus, they may serve as a model system to resolve biological questions difficult to approach in higher plants, and they can be target organisms for research not directly related to photosynthesis. Hence, not only questions associated with the photosynthetic apparatus and function, carbon fixation,

8 light-regulated gene expression, but also cell differentiation and resistance to environmental factors or stress may be easier to address with the power of molecular genetics in cyanobacteria, rather than in higher plants (Cohen and Michael 2006)

2.2 Biological diversity

The diversity of cyanobacteria can be seen in the multitude of structural and functional aspects of cell morphology and in variations in metabolic strategies, motility, cell division, developmental biology, etc. The production of extracellular substances and cyanotoxins by cyanobacteria illustrates the diverse nature of their interactions with other organisms (i.e. allelopathy) (Rizvi and Rizvi 1992).

Cyanobacteria are found throughout the world in terrestrial, fresh water, and marine habitats. However, it is the fresh water habitat that typically experiences a cyanobacterial "bloom". Nutrient- rich bodies of water such as eutrophic lakes, agricultural ponds, or catch basins, may support a rapid growth of cyanobacteria. When conditions are good, a "clear" body of water can become very turbid with a green, blue-green or reddish-brown growth within just a few days. Many species can regulate their buoyancy and float to the surface to form a thin "oily" looking film, or a blue-green scum several inches thick (Carmichael 1988). Cyanobacteria cannot maintain this abnormally high population for long, and will rapidly die and disappear after 1-2 weeks. If conditions remain favorable, another bloom can rapidly replace the previous one. In fact, successive blooms may overlap so that it may appear as if one continuous bloom occurs for up to several months.

2.3 Cyanobacteria and relation with other organisms

Though cyanobacteria are capable of independent existence, they form symbiosis with many organisms (Playfair 1921). Symbiotic interactions between cyanobacteria and other organisms are surprisingly diverse, including fungi (in the formation of lichens), sponges, protists and plants. In these associations, the cyanobacteria provide the different hosts with fixed carbon (e.g. lichens and sponges), with the product of nitrogen fixation (e.g. plants and lichens) or with both products of carbon and nitrogen fixation (e.g. diatoms and lichens) (Costa 2004).

The best studied cyanobacterial associations are those with plants. The presence of chloroplasts in plants is believed to be the consequence of an old cyanobacterial symbiotic event. In fact, chloroplasts are still similar to cyanobacteria, even though most of their genetic material has been transferred to the host nucleus (Rujan and Martin 2001). The most common cyanobacterium found in symbiosis with plants belongs to the filamentous heterocystous genus Nostoc (Dodds and Gudder 1927). Usually, the proportion of heterocysts to vegetative cells is higher in symbiotic forms compared to free-living cyanobacteria and this is determined by the nitrogen status of the environment. A number of cyanobacteria live endophytically in other algae. The small filamentous cyanobacterium Richelia intracellularis lives endophytically in cells of the marine diatom Rhizosolenia sp., whereas Nostoc symbioticum occurs in Geosiphon pyriformis, a siphonous green algae. Species of the cyanobacteria Calothrix, Cyanodictyon, Lyngbya and Phormidium have been reported from the mucilage of other algae. Rhopalodia

10 is a freshwater centric diatom in which a unicellular cyanosymbiont appears intracellularly as inclusion bodies (Plazinsky 1997).

Lichens are symbioses of fungi (ascomycetes and basidiomycetes) with green algae or cyanobacteria. The most common cyanobacteria found in lichens are species of Calothrix, Fischerella, Gloeothece, Nostoc and Scytonema (Hitch and Millbank 1975). The vegetative body of a lichen is termed a thallus and is composed of two organisms, a fungal component (mycobiont) and a green algae or cyanobacterium (phycobiont). Of a total of 18,000 species of lichens, about 8% are composed of nitrogen-fixing cyanobacteria (cyanolichens). The structure of the lichens thallus varies from Collema, where the fungal hyphae and cyanobacterial filaments intermingle through the thallus, to Peltigera canina, where the cyanobacteria are confined to the layer beneath the upper cortex of the fungal hyphae. The cyanobionts, Nostoc calcicola and N. sphaericum occupy mucilage-filled cavities on the ventral side of the gametophyte of the bryophytes, Aneura, Anthoceros, Blasia, Cavicularia, Diplolaena, Notiothylus, Pellia, Riccardia, Riccia and Sphagnum. The cyanosymbionts presumably can penetrate through ‘stomata’or special pores (Peters and Calvert 1983).

Among pteridophytes, by far the best studied cyanom is that of Azolla spp. with Anabaena azollae (Peters and Calvert, 1983). Azolla is a genus of heterosporous aquatic fern that grows on the surface of freshwater ponds, lakes, streams or irrigation channels. Anabaena azollae infects the fronds of the fern at an early stage of plant development and becomes enclosed in a pocket within the frond. The cyanobiont normally remains together with the plant through successive cycles of vegetative and sexual reproduction. Due to

11 high rates of N2 fixation and biomass production, Azolla is an effective green manure for flooded crops and has been used as a biofertilizer in rice-growing countries for centuries ( Nierzwicki and Aulfinger 1990).

As many as 150 species in nine genera belonging to the family Cycadaceae of Gymnosperms, which produce coralloid roots, contain nodule-like structures that are inhabitated by heterocystous cyanobacteria belonging to the genera Anabaena or Nostoc (Grilli-Caiola 1980). The cyanobiont infects coralloid roots, via mucilaginous spaces in the tips, and is usually located intercellularly. In some species both intercellular and intracellular localization occurs in the extra coralloid-root area (Reisser 1984).

In Angiosperms, the best-known symbiotic cyanobacterial association is formed between Gunnera sp. and Nostoc punctiforme, where the cyanobiont is located intracellularly in special stem nodules (Nilsson et al. 2000).

Symbiotic cyanobacteria have been reported in a large variety of marine sponges in which the unicellular cyanobacterium, Aphanocapsa sp. is situated both intercellularly and intracellularly in host vacuoles (Schwemmler and Schenk, 1980). In the green algae Oedogonium oogonia and Codium bursa, different species of filamentous cyanobacteria have been reported (Fogg et al. 1973).

2.4 Classification of cyanobacteria

Cyanobacteria were long classified as blue-green algae under the aegis of the Botanical Code, and it was only in the eighth edition of Bergey’s Manual of

Determinative Bacteriology (Buchanan and Gibbons 1974) that they were first assigned to a separate division of the prokaryotes. Not surprisingly, traditional taxonomic classifications of blue- greens focus on morphology and development, recognizing five principal groups, as described in the second edition of Bergey’s Manual of Systematic Bacteriology (Castenholz 2001):

Subsection I: Unicellular or non-filamentous aggregates of cells held together by outer walls or a gel-like matrix (colonies). Reproduction is by binary fission, in one, two, or three planes, symmetric or asymmetric or by budding (e.g. Gloeocapsa, Gloeobacter, Synechococcus, Synechocystis).

Subsection II: Unicellular or non-filamentous aggregates of cells held together by outer walls or a gel-like matrix (colonies). Reproduction is by internal multiple fissions with the production of daughter cells smaller than the parent; or by multiple plus binary fission (e.g. Cyanocystis, Xenococcus, Chroococcidiopsis, Pleurocapsa).

Subsection III: Filamentous; trichome of cells branched or unbranched, uniseriate or multiseriate. Trichomes composed of cells which do not differentiate into heterocysts or akinetes. Reproduction is by binary fission in one plane (e.g. Crinalium, Spirulina, Lyngbya, Trichodesmium).

Subsection IV: Filamentous; trichome of cells branched or unbranched, uniseriate or multiseriate. One or more cells of trichomes differentiate into

13 heterocysts, at least when the concentration of combined nitrogen in the medium is low; some also produce akinetes. Reproduction is by binary fission in one plane (e.g. Aphanizomenon, Anabaena, Nostoc, Nodularia).

Subsection V: Filamentous; trichome of cells branched or unbranched, uniseriate or multiseriate. One or more cells of trichomes differentiate into heterocysts, at least when the concentration of combined nitrogen in the medium is low; some also produce akinetes. Reproduction is by binary fission, periodically or commonly in more than one plane (e.g. Stigonema, Fischerella, Chlorogloeopsis, Nostochopsis). See table 1.

Table1. The principal groups of cyanobacteria

After Rippka et al. (1979)

2.5 Factors affecting growth of cyanobacteria

Cyanobacteria have a number of special properties which determine their relative importance in phytoplankton communities. However, the behavior of different cyanobacterial taxa in nature is not homogeneous because their ecophysiological properties differ. An understanding of their response to environmental factors is fundamental for setting water and land management targets. Because some cyanobacteria show similar ecological and ecophysiological characteristics, they can be grouped by their behavior in planktonic ecosystems as "ecostrategists" typically inhabiting different niches of aquatic or land ecosystems. Different stresses are limiting factors on the growth and productivity of cyanobacteria. cyanobacteria have developed a number of mechanisms by which they defend themselves against environmental stresses (Allakhverdiev et al. 2001; Rajendran et al. 2007). The physiological bases for the adaptation to high salinity for example in several cyanobacteria species include three main subprocesses: active extrusion of inorganic ions, leading to relatively unchanged internal salt concentrations; accumulation of large internal amounts of organic osmoprotective compounds; and expression of a set of salt stress proteins (Hagemann and Erdmann 1997).

2.5.1 Light intensity

Like algae, cyanobacteria contain chlorophyll a as a major pigment for harvesting light and conducting photosynthesis. They also contain other pigments such as the phycobiliproteins which include allophycocyanin (blue), phycocyanin (blue) and sometimes phycoerythrine (red). These pigments harvest light in the green, yellow and orange part of the spectrum (500-650 nm) which is hardly used by other phytoplankton species. The phycobiliproteins, together with chlorophyll a, enable cyanobacteria to harvest

16 light energy efficiently and to live in an environment with only green light (Cohen-Bazir and Bryant 1982).

Many cyanobacteria are sensitive to prolonged periods of high light intensities. The growth of Planktothrix agardhii, (formerly Oscillatoria) is inhibited when exposed for extended periods to light intensities above 180 µE m¯² s¯¹ which is lethal for many species (Van Liere and Mur 1980). However, if exposed intermittently to this high light intensity, cyanobacteria grow at their approximate maximal rate (Loogman 1982). This light intensity amounts to less than half of the light intensity at the surface of a lake, which can reach 700-1000 µE m¯² s¯¹.

Cyanobacteria which form surface blooms seem to have a higher tolerance for high light intensities. Paerl et al. (1983) related this to an increase in carotenoid production which protects the cells from photoinhibition. Cyanobacteria are further characterized by a favorable energy balance. Their maintenance constant of light is low which means that they require little energy to maintain cell function and structure (Van Liere et al. 1979). As a result of this, the cyanobacteria can maintain a relatively higher growth rate than other phytoplankton organisms when light intensities are low. The cyanobacteria will therefore have a competitive advantage in lakes which are turbid due to dense growths of other phytoplankton. This was demonstrated in an investigation measuring growth of different species of phytoplankton at various depths in a eutrophic Norwegian lake. The results showed that the diatoms Asterionella, Diatoma and Synedra grew faster than the cyanobacterium Planktothrix at 1 m depth, while the growth rate was about

17 the same for all these organisms at 2 m depth. At the very low light intensities below 3 m only Planktothrix grew (Källqvist 1981).

The ability of cyanobacteria to grow at low light intensities and to harvest certain specific light qualities enables them to grow in the "shadow" of other phytoplankton. Van Liere and Mur (1978) demonstrated competition between cyanobacteria and other phytoplankton. Whereas the green alga Scenedesmus protuberans grew faster at high light intensities, growth of the cyanobacterium Planktothrix agardhii was faster at low light intensities. If both organisms were grown in the same continuous culture at low light intensity, Planktothrix could outcompete Scenedesmus. At high light intensities, the biomass of the green alga increased rapidly, causing an increase in turbidity and a decrease in light availability. This increased the growth rate of the cyanobacterium, which then became dominant after 20 days.

Although cyanobacteria cannot reach the maximum growth rates of green algae, at very low light intensities their growth rate is higher. Therefore, in waters with high turbidity they have better chances of outcompeting other species. This can explain why cyanobacteria which can grow under very poor nutritional conditions often develop blooms in nutrient-rich eutrophic waters. The light conditions in a given water body determine the extent to which the physiological properties of cyanobacteria will be of advantage in their competition against other phytoplankton organisms (Mur et al. 1978).

The zone in which photosynthesis can occur is termed the euphotic zone (Z eu ). By definition, the euphotic zone extends from the surface to the depth at which 1 per cent of the surface light intensity can be detected. It can be estimated by measuring transparency with a Secchi disk and multiplying the Secchi depth reading by a factor of 2-3. The euphotic zone may be deeper or shallower than the mixed, upper zone of a thermally stratified water body, the depth of which is termed the epilimnion (Z m) (Mur et al. 1978).

Many species of planktonic algae and cyanobacteria have little, or only weak means of active movement and are passively entrained in the water circulation within the epilimnion. Thus, they can be photosynthetically active only when the circulation maintains them in the euphotic zone. In eutrophic waters, phytoplankton biomass is frequently very high and causes substantial turbidity. In such situations, the euphotic zone is often more shallow than the epilimnion, i.e. the ratio Zeu/Zm is < 1, and phytoplankton spend part of the daylight period in the dark. Thus, the Zeu/Zm ratio is a reasonable (and easy to measure) approach for describing the light conditions encountered by the planktonic organisms (Chorus and Bartram 1999).

2.5.2 Temperature

Maximum growth rates are attained by most cyanobacteria at temperatures above 25 °C (Robarts and Zohary 1987). These optimum temperatures are higher than for green algae and diatoms. This can explain why in temperate and boreal water bodies most cyanobacteria bloom during summer.

Cyanobacteria are found in environments with quite different temperature ranges. Most cyanobacteria are mesophilic and live in environments where temperature may range from freezing to 40 °C. They typically have growth optima between 20 and 35 °C and maximum temperatures permitting growth are below 45 °C. Cyanobacteria isolated from the open oceans, where temperature ranges are more moderate, often have temperature maxima near 30 °C (Waterbury et al. 1986). Dworkin (2006) reported that one tropical, marine, unicellular, nitrogen-fixing cyanobacterium has a temperature range permitting growth only between 2°C and 32 °C, which is one of the narrowest temperature ranges known for a free-living mesophilic prokaryote. Cyanobacteria are also commonly found in more extreme environments. In Antarctica, they are present as cryptoendoliths in rocks in the cold dry deserts and in the plankton and microbial mats of lakes. With the exception of one strain of Chroococcidiopsis sp. that is a psychrophile, the cyanobacterial isolates from the Antarctic desert rocks are mesophiles, with temperature optima near 35 °C (Waterbury et al. 1986). On the other hand, many of the cyanobacteria isolated from Antarctic lakes are psychrophiles, having maximum temperatures permitting growth at 20 °C (Seaburg et al. 1981). Cyanobacteria, including representatives of each of the five orders, are conspicuous inhabitants of hot springs where they occur at temperatures up to 74 °C. Thermophilic strains of cyanobacteria have growth optima above 45 °C and often fail to grow, but can survive at room temperature (Castenholz 1981).

2.5.3 Salinity

Cyanobacteria occur in habitats of widely differing salinity. Freshwater habitats contain diverse and often prominent populations of cyanobacteria. In marine habitats, cyanobacterial isolates can be divided into two categories

20 based on their major ionic requirements for growth. Some are halotolerant and grow equally well on a medium with either a seawater or freshwater base. Others have obligate requirements for concentrations of sodium, magnesium, calcium, and chloride that reflect the chemistry of seawater. These requirements are not met by supplementing freshwater media with sodium chloride alone (Waterbury et al. 1986). Many cyanobacteria isolated from soils are tolerant to salt concentrations in excess of 1 M NaCl, whereas marine isolates usually fail to grow at this concentration, probably because the salinity of seawater has been relatively constant for long periods. The occurrence of cyanobacteria in hypersaline environments is well documented in the classical descriptive literature (Hof and Frémy 1933) but only a very restricted group is truly halophilic. Individual strains of Aphanothece halophytica isolated from a salt evaporation pond (Yopp et al. 1978), from Great Salt Lake, Utah (Brock 1976), and the Solar Lake, Sinai, (Cohen 1975) each have major ionic requirements for growth that reflect the chemistry of their individual habitats. The isolates from the salt evaporation pond and from Great Salt Lake are truly halophilic. They grow optimally in approximately 2 M NaCl and have minimum NaCl requirements for growth of 0.7 M and 1.0 M NaCl, respectively.

Previous studies indicated that salinity higher than 0.5 to 2 psu is inhibitory for growth and CO2 fixation of the non-N2-fixing toxic, unicellular cyanobacterium Microsystis aeruginosa (Paerl et al. 1983; Sellner et al 1988). However, as a group, cyanobacteria exhibit considerable salinity tolerance (Blumwald and Tel-Or 1982; Reed et al 1986; Apte et al. 1987; Vonshak et al. 1988) and many species are adapted to hypersaline environments (Oren 2000). Since high intracellular concentrations of Na+ are toxic to most

21 biological systems, organisms living in sodium-rich environments must have developed detoxifying mechanisms (Reed et al. 1986).

Growth rates of several cyanobacteria and bacteria decrease under increasing salt concentration, but the extent of growth inhibition can vary (Blumwald and Tel-Or 1982; Vonshak et al 1988; Brabban et al. 1999). Previous studies with eukaryotic algae and cyanobacteria have shown increased photoinhibition and reduced maximum photosynthesis rates under salt stress (Neale and Melis 1989; Zeng and Vonshak 1998).

2.5.4 pH pH is a very important factor in growth, establishment and diversity of cyanobacteria, which have generally been reported to prefer neutral to slightly alkaline pH for optimum growth (Singh 1961; Kaushik 1994). Acidic soils are therefore one of the stressed environments for these organisms and they are normally absent at pH values below 4 or 5; eukaryotic algae, however, flourish under these conditions. Isolates from acidic hot springs and peat bogs grow optimally at neutral pH, indicating that they are mildly acid- tolerant rather than acidophilic (Rippka et al. 1981a). A restricted number of cyanobacteria are characteristic of highly alkaline habitats; for example, Spirulina platensis, a dominant cyanobacterium in highly alkaline lakes, has a pH optimum for growth between 8 and 11(Ciferri 1983).

2.4.5 Phosphorous and Nitrogen

Because cyanobacterial blooms often develop in eutrophic lakes, it was originally assumed that they required high phosphorus and nitrogen concentrations. This assumption was maintained even though cyanobacterial blooms often occurred when concentrations of dissolved phosphate were lowest. Experimental data have shown that the affinity of many cyanobacteria for nitrogen or phosphorus is higher than for many other photosynthetic organisms (Chorus and Mur, 1999). This means that they can out-compete other phytoplankton organisms under conditions of phosphorus or nitrogen limitation. In addition to their high nutrient affinity, cyanobacteria have a substantial storage capacity for phosphorus. They can store enough phosphorus to perform two to four cell divisions, which corresponds to a 4-32 fold increase in biomass. However, if total phosphate rather than only dissolved phosphate is considered, high concentrations indirectly support cyanobacteria because they provide a high carrying capacity for phytoplankton. High phytoplankton density leads to high turbidity and low light availability, and cyanobacteria are the group of phytoplankton organisms which can grow best under these conditions. A low ratio between nitrogen and phosphorus concentrations may favour the development of cyanobacterial blooms. A comparison between the optimum N: P ratios for eukaryotic algae (16-23 molecules N: 1 molecule of P) with the optimum rates for bloom- forming cyanobacteria (10-16 molecules N: 1 molecule P), shows that the ratio is lower for cyanobacteria (Schreurs 1992).

2.6. Crusting ability

Cyanobacteria have been recognized as important agents in the stabilization of soil surfaces primarily through their production of extracellular polysaccharides, which are prominent agents in the process of aggregate

23 formation, and stabilization ( Hu et al. 2003). The beneficial effects of cyanobacteria on soil physical properties in arid environments have been demonstrated through the study of microbiotic crusts (Isichei, 1990; Pérez 1997; Williams et al. 1999; Malam Issa et al. 1999; 2001a). The most abundant microbial constituents of microbiotic crusts are filamentous cyanobacteria that exert a mechanical effect on soil particles as they form a gluing mesh and bind soil particles on the surface of their polysaccharidic sheath material (Belnap and Gardner 1993; Malam Issa et al. 1999; 2001a). Cyanobacteria also excrete extracellular polymeric secretions (Extracellular Polysaccharides Secretions, ESP) mainly composed of polysaccharides (Decho 1990; Hu et al. 2003). Extracellular polymeric secretions ensure the role of binding agent of soil particles (Lynch and Bragg 1985). Microbiotic crusts thus lead to the formation of tough and entangled superficial structures that improve the stability of soil surface and protect it from erosion (Malam Issa et al. 1999; 2001a). Cyanobacterial sheaths and EPS also play a significant role in water storage due to the hygroscopic properties of polysaccharides (Decho 1990). They contribute to increased water retention capacity of soil (Verrecchia et al. 1995; Défarge et al. 1999). It has also been reported that cyanobacteria, as C and N fixers, can improve the nutrient content of soil in arid environments (Mayland and McIntosh 1966; Jeffries et al. 1992; Lange et al. 1994). As photosynthetic organisms they are the main primary producers, enriching the soil with organic matter and favouring biological activity (Lange et al. 1994). They also represent a potential source of nitrogen, which may be beneficial for forthcoming crop production (Rogers and Burns 1994; Zaady et al. 1998; Malam Issa et al. 2001b).

2.7. Cyanobacteria and photosynthesis

Cyanobacteria are a diverse group of oxygenic photosynthetic prokaryotes, exhibiting versatile physiology and wide ecological tolerance that contribute to their competitive success over broad spectrum of environments both planktonic and benthic (Shilo 1989). They are dominant in a broad spectrum of terrestrial habitat. The cyanobacteria and prochloroaceae are the only prokaryotic groups that share the use of photosystems I and II and hence the ability to carry out oxygenic photosynthesis with all photosynthetic eukaryotic organisms (Stanier and Cohen-Bazire 1977). The structure of the reaction center complexes seems to be evolutionarily conserved in all these organisms, but there is a large diversity in their antenna chlorophyll complexes (Glazer 1983). Using reduced electron donors (Cohen 1975; Padan and Cohen 1982;Cohen et al. 1986;DeWit and van Gemerdens 1989; Garcia- Pichel and Castenholz 1990). They also share with many archaebacteria the ability to use elemental sulfur for anaerobic dark respiration (Oren and Shilo 1979).

2.8 Cyanobacteria and nitrogen fixation

Cyanobacterial nitrogen fixation is potentially important in regulating primary productivity in nitrogen-deficient aquatic environments, yet relatively little is known about the ecology of natural populations of nitrogen-fixing cyanobacteria. A great number of cyanobacterial species have the ability to fix atmospheric nitrogen when facing combined nitrogen depleted conditions, many of them doing so under aerobic conditions. The processes of N2 fixation is carried out by an enzyme called Nitrogenase which is composed of two multisubunit proteins; the MoFe protein encoded by nifD and nifK, and the Fe protein, encoded by nifH, both of protein are conserved among nitrogen-fixing organisms.

One particular important aspect is that nitrogenase is extremely sensitive to oxygen. In order to overcome the compatibility conflict of these two of the carbon fixation and nitrogen fixation processes, several cyanobacteria separate, either spatially or temporarily, the processes of oxygenic photosynthesis and nitrogen fixation. While some filamentous cyanobacteria differentiate specialized cells, named heterocysts, in nitrogen fixation, in which the nitrogenase is confined, some other cyanobacterial strains, unicellular as well as filamentous, express the nitrogenase activity in the dark periods of light-dark growth cycles (Herrero et al. 2001).

On the other hand, Trichodesmium sp., a nitrogen-fixing cyanobacterium of global ecological significance, fixes nitrogen aerobically and expresses nitrogenase in the light periods of light-dark growth cycles, in special, protective cells called diazocytes (Fredriksson and Bergman 1997). Interrelated to the ability of nitrogen fixation, cyanobacteria possess hydrogen metabolism. In fact, the reduction of atmospheric nitrogen to ammonium by the nitrogenase is accompanied by the formation of molecular hydrogen as a byproduct (Tamagnini et al. 2002). The hydrogen is then rapidly consumed by an uptake hydrogenase, an enzyme that has been found in almost all the nitrogen fixing cyanobacteria examined so far, with one reported exception Synechococcus sp. BG 043511 (Ludwig et al. 2006). Additionally, cyanobacteria may contain a bidirectional hydrogenase, an enzyme that is generally present in non-nitrogen fixing species. However, it is absent in Gloeobacter violaceus PCC 7421, a cyanobacterium that possesses a number of unique characteristics such as the absence of thylakoids (Tamagnini et al. 2002).

One of the difficulties in studying natural populations is that nitrogen-fixing microbial communities can be complex mixtures of many species of bacteria and cyanobacteria. Species which develop heterocysts are clearly capable of fixing nitrogen, but other species are less easily identified since nitrogen fixation genes are present in many prokaryotic taxa. Problems with identification of nitrogen-fixing microorganisms in nature are exacerbated by difficulties in culturing microorganisms from the environment (Giovannon et al.1990; Ward et al.1990). The amino acid sequence of Fe protein of the enzyme nitrogenase is very similar among organisms, even those of very different taxonomic groups. Even the alternative nitrogenase Fe protein amino acid sequences are greater than 63% identical to the conventional nitrogenase Fe protein sequence (91% between nifH and vnfH and63% between nifH and anfH).

2.9 Cyanobacrerial risks

Toxic cyanobacteria are found worldwide in inland and coastal water environments. At least 46 species have been shown to cause toxic effects in vertebrates. The most common toxic cyanobacteria in fresh water are Microcystis spp., Cylindrospermopsis raciborskii, Planktothrix (syn. Oscillatoria) rubescens, Synechococcus spp., Planktothrix (syn. Oscillatoria) agardhii, Gloeotrichia spp., Anabaena spp., Lyngbya spp., Aphanizomenon spp., Nostoc spp., some Oscillatoria spp., Schizothrix spp. and Synechocystis spp. Toxicity cannot be excluded for other species and genera. As research broadens and covers more regions over the globe, additional toxic species are likely to be found. Therefore, it is prudent to presume a toxic potential in any cyanobacterial population (Sivonen and Jones 1999).

The most widespread cyanobacterial toxins are microcystins, neurotoxins and cytotoxin. Some species contain neurotoxin and microcystin simultaneously. Field populations of the most common bloom-forming genus, Microcystis, are almost always toxic (Carmichael 1995), but non-toxic strains do occur. Generally, toxicity is not a trait specific for certain species; rather, most species comprise toxic and nontoxic strains. For microcystins, it has been shown that toxicity of a strain depends on whether or not it contains the gene for microcystin production (Dittmann et al. 1996) and that field populations are a mixture of both genotypes with and without this gene (Kurmayer et al. 2002). Experience with cyanobacterial cultures also showed that microcystin production is a fairly constant trait of a given strain or genotype, only somewhat modified by environmental conditions.

While conditions leading to cyanobacterial proliferation are well understood, the physiological or biochemical function of toxins for the cyanobacteria is the subject of many hypotheses as has been shown by (Chorus and Mur 1999), the factors leading to the dominance of toxic strains over non-toxic ones are not known. Worldwide, about 60% of cyanobacterial samples investigated contain toxins. The toxicity of a single bloom may, however, change in both time and space. Demonstrations of toxicity of the cyanobacterial population in a given lake do not necessarily imply an environmental or human hazard as long as the cells remain thinly dispersed. Mass developments and especially surface scums pose the risks. One well documented case of human cyanobacterial toxicity within the tropics occurred in Brazil in 1996, where more than 50 dialysis patients died displaying hepatotoxic and neurotoxic symptoms (Bowling and Baker 1996).

2.10 Population stability

While many planktonic algae are grazed by copepods, daphnids and protozoa, cyanobacteria are not grazed to the same extent, and the impact of grazing by some specialized ciliates and rhizopod protozoans is usually not substantial. Cyanobacteria are attacked by viruses, bacteria and actinobacteria (actinomycetes), but the importance of these natural enemies for the breakdown of populations is not well understood. Because they have few natural enemies, and their capacity for buoyancy regulation prevents sedimentation, the loss rates of cyanobacterial populations are generally low. Thus, their slow growth rates are compensated by the high prevalence of populations once they have been established (Mur et al. 1999).

CHAPTER THREE

MATERIAL AND METHODS

3.1. Study area and sampling sites

Ten samples were collected every two months from two different locations: Khartoum State, most of the samples were obtained from Shambat Khartoum North, 15˚35΄N:32˚31́E and El Rahad town 12˚43 ́ N: 30˚39́ E. (htt://maps.google.com) and its outskirts in North Kordofan State.

Different habitats, viz. wet and dry soils and water bodies were chosen as sampling points in each location. The samples, eight of them were soil, taken from the first five centimeter of soil surface, these samples either wet or dry clay, or wet or dry sand, the other two samples were fresh water taken from the top of water column. The five sampling points in the first location were: (i) dry clay, from the first terrace near the river Nile, (ii) wet clay, from the first terrace near the river Nile (iii) fresh water from the first km of the river Nile after confluence of the two Niles (iv) wet sand from Abo-a’nga rut (v) dry sand from Dar-Elsalaam, western Omdurman. Those of El Rahad town were : (i) wet sand and (ii) wet clay samples from El Rahad-Turda banks, (iii) fresh water from El Rahad-Turda (plate 2), (iv) dry sand from Al-Amara village (30 km North-East of El Rahad town) (plate 3), (v) dry clay samples were taken from Al-Amara village. The first sample set was taken in January 2008; followed by March, May, July, September and November 2008.

3.2. Samples collection, media and culturing

In each case, 300 grams of soil were collected from 4 to 5 sub-locations in the sampling point and were carefully mixed and homogenized. The samples were taken in sterilized polyethylene bags from each sampling site.

Likewise, about 300 ml of water were taken in sterilized bottles from the water sources of each location. All samples were taken to the laboratory of the Department of Botany and Agricultural Biotechnology, Faculty of Agriculture, University of Khartoum at Shambat. About ten grams of samples were inoculated into BG11 broth medium in flasks (100 ml) at room temperature ranged from 25 to35, and light conditions of about 500 lux.

Blue-Green11 (BG11) medium consisted of (per liter) according to Rippka et al. (1979); NaNO3 (1.5g), K2HPO4 (31 mg), MgSO4.7H2O (75mg),

CaCl2.2H2O (36mg), Na2CO3 (20mg), citric acid (6mg), ferric ammonium citrate (6mg), MnCl2 .4H2O (1.81mg), ZnS04.7H40 (220µg) disodium magnesium EDTA (1mg), H3BO3 (2.86mg) and CuSO4.5H20 (80µg). The pH of BG11 medium was 7.5 to 7.6 when it was incubated.

Beside BG11, another medium (BG110) was used. This medium consisted of

BG11 minus NaNO3 but supplemented with 5 mg NaHCO2. For semi-solid media double-strength nutrients were used and 1.5 g/liter agar was added. The nutrients and agar solution were prepared and sterilized separately and then combined according to the method of Allen and Gorham (1981). Both broth and semi-solid media were sterilized by autoclaving at 121˚C for 20 min at 1.08 kg/cm2. Five grams of each sample (5 ml in case of water samples) were inoculated into a hundred ml of BG11 broth medium and incubated at room temperature and light conditions of about 500 lux using cool fluorescent lamps and luxmeter (BBC GOERZ MERTAWATT Germany) for 3-4 weeks to give growth (plate 4).

Plate 2. El-Rahad Turda site.

Plate 3. El-Rahad dry sand site.

3.3. Isolation and purification

Four serial dilutions were prepared from the initial growth of each sample. One ml from each dilution was inoculated onto semi-solid BG11 medium (0.6% agar). Plates of the semi-solid medium were inoculated by adding 5ml from each dilution using the pour plate technique and incubated under light conditions (12 h light) and 28C˚ for 3-4 weeks. Samples were examined under a phase contrast light microscope (A. Krüss. Optironic. Germany) and "unialgal" growth was picked and transferred into a liquid medium and incubated under the same conditions of light and temperature until truly axenic cultures were obtained. The isolates were maintained in the laboratory in BG11 broth media under light condition about 300 to 500 lux and subcultured frequently.

3.4. Identification

All cyanobacterial observed were identified using keys of Rippka et al. (1979). Morphological and physical characteristics of cyanobacteria were used in identification viz. type of cells, length of trichom, shape of terminal cell, the distinction between adjacent sells, gliding and type of the reproduction (plate 5). Moreover, an online identification program was used (http://www-cyanosite.bio.purdue.edu/images/images2.html). Samples under the microscope were compared to the images provided in the online program to be matched for further confirmation of their identity.

3.5. Physiological studies

Six pure cultures representing two unicellular, two heterocystous and two nonheterocystous filamentous cyanobacteria were chosen for physiological studies. Microcystis aerugenosa and Synechococcus buzasii representing

Plate 4. Culturing of cyanobacteria in the laboratory showing some sedimented growth and buoyant growth.

Plate 5: Phase contrast micrographs X400 depicting: a: hormogonia of lyngbya sp.; b: hormogonia of Anabaena sp.; c: hormpgonia of Cylindrospermum sp.

36 unicellular cyanobacteria (plate 6), Anabaena doliolum and Cylindrospermum sp. representing the filamentous heterocystous cyanobacteria, while Lyngbya aerugenosa. and Spirulina sp. representing nonheterocystous cyanobacteria.

Table 2. Isolates used in the physiological studies.

Iso Classification Site of isolation properties

1 Microcystis aerugenosa Khartoum wet clay unicellular

2 Synechococcus buzasii Khartoum wet clay unicellular

3 Spirulina sp. Rahad dry clay nonheterocytous

4 Lyngbya aestuarii Rahad dry clay nonheterocytous

5 Cylindrospermum sp. Rahad wet clay heterocytous

6 Anabaena doliolum Khartoum dry clay heterocytous

3.5.1 Effects of temperature

Each of the six axenic cultures was inoculated by adding one ml of each culture into 30 ml of sterilized BG11 broth medium in bottle and incubated under light conditions of about 500 lux at different temperatures 25˚C, 35˚C and 45˚C for three weeks. The investigation was carried out in triplicates. At the end of the incubation period, all cultures were filtered by using filter papers (Whatman No.1) of known weight, and the dry weight of cyanobacterial growth was calculated using sensitive balance.

37 a

c b

Plate 6: phase contrast micrographs X400 depicting:

a: Microcystis sp. (Chroococcales) isolated from Khartoum Wet Clay in January 2008;

b: Synechocystis sp. (Chroococcales)isolated from Khartoum Wet Clay in May 2008;

c: Dermocarpa sp.(Pleurocapsales) isolated from Khartoum Wet Clay in March 2008.

3.5.2 Effects of pH

Each of the six axenic cultures was inoculated by adding one ml of each culture into 30 ml of sterilized BG11 broth medium in bottles adjusted to the pH values of 5.5, 6.5, 7.5, and 8.5using pH meter, and incubated under light conditions of about 500 lux for three weeks. The investigation was carried out in triplicates. At the end of the incubation period, all cultures were filtered using filter papers (Whatman No.1) of known weight, and the dry weight of cyanobacterial growth was calculated.

3.5.3 Effect of light intensity

Each of the six axenic cultures was inoculated by adding one ml of each culture into 30 ml of sterilized BG11 broth medium in bottles. The bottles were incubated under different light intensities 500, 2000, 3000 and 4000lux, for three weeks. The investigation was carried out in triplicates. At the end of the incubation period, all cultures were filtered using filter papers (Whatman No.1) of known weight, and the dry weight of cyanobacterial growth was calculated.

3.5.4 Effects of salinity

Each of the six axenic cultures was inoculated by adding one ml of each culture to 30 ml of sterilized BG11 broth medium in bottles. The medium of the bottle was adjusted to contain the following NaCl concentrations; zero, 1%, 2%, 3%, 4%, 5%, and 6%. The bottles were incubated under light conditions of about 500 lux, for three weeks. The investigation was carried out in triplicates. At the end of the incubation period, all cultures were filtered

39 using filter papers (Whatman No.1) of known weight, and the dry weight of cyanobacterial growth was calculated.

3.5.5 Crusting ability

Two types of sterilized soil (clay and sand) were used for the investigation on soil crusting. Samples of each soil were sterilized and packaged into sterilized polyethylene bags, then the sterilized soils were saturated by sterilized BG11 broth medium and inoculated by adding one ml of each broth of axenic culture and incubated under light conditions in a glass house. Readings were taken after three, six and nine weeks. The measurements were made on air-dry soil that passed through a sieve of 2.0mm mesh and retained by a sieve of 1.0mm mesh. A quantity of the soil retained by the 1.0mm sieve was placed in a small open container with a fine screen (0.25 mm) at the bottom. This container was placed in distilled water. The sieve container was moved up-and- down in the water through a vertical distance of 1.5 cm at the rate of 30 oscillations per minute (one oscillation is an up -and-down stroke of 1.5 cm in length) plate 7. After a period of 3 min, the sieve containing the aggregates was immersed in a calgon solution (2%) in fresh cans. The aggregates in the sieve were allowed to soak for five minutes while the sieve was moved up and down periodically. Water and calgon solutions were evaporated and weighed. The contents were then removed and visually examined for the extent of breakdown from the original aggregate size. Those materials that showed the least change from the original aggregates had the greatest aggregate stability. Statistical analysis was carried out by applying the analysis of Variance (ANOVA) (Snedecor and Cachran 1976)

Plate 7. The oscillator apparatus with its cans.

CHAPTER FOUR

RESULTS

The present investigations were carried out to study the spatial distribution of the cyanobacterial flora in two locations in the Sudan as well as their temporal variation, and some physiological traits of cyanobacteria in different sampling sites in the two locations, viz. Khartoum State (Shambat and

Omdurman) and El Rahad town (North Kordofan State). Generally, there was no great difference in the total number of isolates between the two locations: Rahad location 142 isolates against 136 1solates in Khartoum location. However, the wet sampling points harbored more cyanobacterial isolates as compared to dry sites as reflected in the number of isolates in each (163 isolates for wet against 115 for dry). Moreover, clay locations yielded more isolates than the sandy locations (127 isolates against 111). Results are shown in Table 2.

With regard to the time of sampling, the month of November yielded the highest number of cyanobacterial isolates, while the least was recorded in March (Fig. 1). Details of these temporal variations are shown in Fig. 2, which clearly shows that the numbers of the November isolates in Rahad dry clay, Khartoum wet sand and Khartoum wet clay were greater than the other sites. Likewise, the numbers of isolates of the month of March were the lowest in Rahad Turda water, Rahad dry sand, Khartoum Nile water, Rahad dry clay and Khartoum dry sand sites.

Table 3. The total number of isolates in the two locations.

type Rahad Khartoum Total

Wet 83 80 163

Dry 59 56 115

Clay 67 60 127

Sand 53 58 111

Total 142 136

Figure1. Total number of cyanobacterial isolates in the 10 sampling points during the period January-November 2008.

Figure 2. Number of cyanobacterial isolates in each of the 10 sampling points for the months Jan, Mar, May, July Sep and Nov 2008.

RWC= Rahad wet clay; RDC= Rahad dry clay, RWS= Rahad wet sand, RDS= Rahad dry sand, RTw= Rahad Turda water, KWC= Khartoum wet clay, KDC= Khartoum dry clay, KWS= Khartoum wet sand, KDS= Khartoum dry sand, KNw= Khartoum Nile water.

45 a

Plate 8: Phase contrast micrographs X400 depicting:

a: Lyngbya sp.(Oscillatoriales) isolated from Rahad Dry Clay in January 2008;

b: Spirulina sp.(Oscillatoriales) isolated from Rahad Dry Clay in January 2008.

As illustrated in Fig. 3, Rahad dry clay harboured the highest number of total isolates (37), followed by Khartoum wet sand (33), then Rahad wet sand and Khartoum dry clay, each of which yielded 31 isolates. On the other hand, Khartoum Nile water yielded the least number of isolates, followed by Rahad Turda water and Rahad dry sand, respectively. Thus, no clear pattern can be depicted for the spatial distribution of cyanobacteria in the two localities. However, it is evident that the two water bodies (the Nile and Turda of Rahad) harboured low total cyanobacterial numbers as compared to wet or dry soil samples.

Figure 4 shows the numbers of isolates obtained from of wet soil samples is bigger than those of dry soil samples and water bodies revealed the least over the months of January, March, May, September and November 2008.

Figure 5 shows the frequency of isolation of members of each of the five cyanobacterial orders. As can be seen, all orders were represented in this study. However, the order Oscillatoriales was the most frequently encountered (44.4%), followed by the order Nostocales (29%), then Chroococcales (16.5 %), Pleurocapsales (7.2 %) while the order Stigonematales stood for just 2.9 %.

The most widely encountered genera in the order Oscillatoriales, in descending order, were Oscillatoria (isolated 62 times), Lyngbya (isolated 30 times), Phormidium (isolated 12 times), Leptolyngbya and Microcoleus (each isolated 5 times), Spirulina (isolated 4 times), Trichodesmium (isolated 3 times) and Schizothrix was encountered only once (Fig. 6).

Figure3. Total number of cyanobacterial isolates in each sampling point during the period January-November 2008.

Fig.4 . Temporal distribution of total isolates of the water samples, dry and wet soil samples.

Fig.5. Frequency of isolation of members of each of the five cyanobacterial orders in all sampling sites for the months of January, March, May, July, September and November 2008.

Figure 6. Number of isolates of the genera of the order Oscillatoriales in all 10 sampling sites for the period January-November 2008.

Plate 9: Phase contrast micrographs X400 depicting: a: Anabaena sp.(Nostocales) isolated from Khartoum Dry Clay in July 2008; b: Calothrix sp. (Nostocales) isolated from Rahad Dry Clay in May 2008; c: Cylindrospermopsis sp.(Nostocales) isolated from Khartoum Wet Sand in May 2008.

The encountered genera in the order Nostocales, in descending order, were Anabaena (isolated 55 times), Aphanizomenon (isolated 10 times), Cylindrospermopsis (isolated 8 times), Nostoc and Scytonema (each isolated twice), then Calothrix, Cylindrospermum, Raphidiopsis and Tolypothrix which was encountered only once (Fig.7).

In the order Chroococcales, the most frequently isolated genera were Microcystis (isolated 27 times), followed by Synechocystis (isolated 8 times), Cyanosarcina (isolated 5 times), Gloeobacter (isolated 4 times) and Gloeocapsa (isolated twice) as shown in Figure 8.

It is noteworthy that the orders Stigonematales and Pleurocapsales were represented by only one genus (Nostochopsis for the Stigonematales, and Dermocapsa for the Pleurocapsales).

Figure 9 depicts the temporal distribution of cyanobacterial isolates over the period January-November 2008. Among the three most prevalent orders, Oscillatoriales was the most ubiquitous during all sampling periods (except November when the order Nostocales was higher). The order Chroococcales showed the least number of isolates during all sampling periods. It is noteworthy that the highest numbers were recorded during the moist period (July-September). It appears that cyanobacterial inocula were not completely wiped whether during the dry period or in the harsh dry sands.

Figure 7. Number of isolates of the genera of the order Nostocales in all 10 sampling sites for the months January-November 2008.

Figure 8. Number of isolates of the genera of the order Chroococcales in all 10 sampling sites for the months January-November 2008.

Figure 9.Temporal distribution of the most prevalent cyanobacterial orders during the period January-November 2008.

Generally, Oscillatoria princeps was the most frequent isolate, followed by Lingbya sp., Microcystis sp., Oscillatoria geminata, Anabeana sperica, Anabaena cardinald and Aphanizomenon sp. as shown in figure 10.

Six isolates were selected for studying the effects of some physiological factors. The six isolates were Microcystis aeruginosa and Synechococcus buzasii (representing unicellular cyanobacteria), Anabaena doliolum and Cylindrospermum sp. (representing filamentous heterocystous cyanobacteria), Lyngbya aestuarii and Spirulina sp., (representing filamentous nonheterocystous cyanobacteria).

Statistical analysis of the results on salinity revealed no significant differences between the six cyanobacterial spp. in their tolerance to salinity (appendix 1). However, Anabeana doliolum and Lyngbya aestuarii showed somewhat good tolerance as shown in Fig.11. They were able to grow at 5% NaCl. All targeted isolates showed similar growth at 1%, 2%, 3% and 4%NaCl, but they couldn't grow in 6%NaCl concentration. The dry weights of isolates ranged between 0.034 and 0.151g.

All the chosen isolates showed approximately the same growth rates in all treatments of light intensity (500, 2000, 3000, and 4000 lux). Spirulina sp. showed relative decrease in the dry weight with increasing light intensity value. Cylidrospermum sp. showed relatively stable growth rates at all treatments. It is noteworthy that Anabaena doliolum which showed a higher growth at light intensity 2000 lux compared to the light intensities as shown in Fig.12, though the statistical analysis revealed no significant difference between the growth rates (appendix 2).

Figure 10. Frequency of isolation of the most prevalent cyanobacterial spp. in the sampled areas.

Figure 11.Effect of salinity on growth of the six selected cynobacterial isolates.

Figure 12. Effect of light intensities on the growth of the six chosen cyanobacterial isolates.

Figure 13 shows the effects of pH on the growth of the chosen cyanobacterial isolates. There were no significant differences in the amount of growth obtained during 21 days in all six isolates (appendix 3). However, Synechococcus buzasii and Lyngbya aestuarii showed a relatively high growth rate in acidic pH (5.5), while Spirulina sp. showed best growth in alkaline pH (8.5).

Although the statistical analysis revealed no significant differences between the growth of the chosen isolates at 25 and 35 C° treatments, there was significant differences in the growth of all isolates at 45C° (appendix 4). Lyngbya aestuarii showed higher dry weight at 35C° as shown in Fig.14.

In the assay for crusting ability, there were significant differences between inoculated and control samples (appendix 5, 6). Aggregate stability gradually increased within the period of the assay in both soil types -sand and clay- (Figs.15 and 16). After three weeks the crusts were clearly visible and by the ninth week the crusting reached a very much advance stage. Anabeana doliolum and Spirulina sp. showed a great potential for soil crusting. These two isolates showed the best ability reaching approximately 95.1% and 92.7% at the ninth week against 62% and 58.8% by the end of the third week, respectively in the sandy soil, and approximately 35.7% and 36.3% by the ninth week against 19.5% and 18.1% by the end of the third week in the clay soil, respectively. In contrast, the unicellular isolates exhibited limited crusting ability.

Figure 13. Effect of pH on the growth of the six selected cyanobacterial isolates.

Figure 14. Effect of temperature on the growth of the six selected cyanobacterial isolates.

Figure 15. Sand aggregating activity of the growth of the six selected cyanobacterial isolates.

Figure 16. Clay aggregating activity of the growth of the six selected cyanobacterial isolates.

CHAPTER FIVE

DISCUSSION

Very little work has been devoted to investigations on cyanobacteria in the Sudan. The fragmentary information available does not exceed cursory observations on cyanobacteria encountered during the course of investigations on other microbial groups (Jagnow, 1964) or in studies devoted to cyanobacteria in other countries (Mahdi, 1993).

The present study was conducted on the Sudan cyanobacteria in locations of semi-desert to poor savanna environment, with variation in temperatures between 11 C˚ (winter) and 45 C˚ (summer) (Khartoum Meteorological Station-2008), with different soil textures (sand to clay), and different moisture levels (completely dry soils to pure water).

In this study cyanobacteria were procured from all sampling points at all sampling times. A total of 278 isolates belonging to 46 species could be observed in the present study. Theses isolates represented all five cyanobacterial orderssections or subclasses. Cyanobacteria are usually classified in five orders, viz.: Chroococcales, Pleurocapsales Oscillatoriales, Nostocales and Stigonematales (Dworkin, 2006). The distribution of cyanobacteria that emerged from the present survey agrees with Castenholz and Waterbury (1989) who stated that cyanobacteria are found in almost every aquatic and terrestrial environments. In arid regions however, crusts remain biologically inactive most of the time (Garcia-Pichel and Belnap 1996). However, when water is available — through precipitation and active biological processes such as photosynthesis and respiration — growth can start within a few minutes.

This finding probably explains the apparent ubiquitous distribution pattern observed in the present study in environments characterized by the lack of moisture.

Cyanobacteria show a remarkable degree of morphological and developmental diversity (Adams and Carr 1981). In the Baltic sea Cox et al. (2004) reported that they found representatives of all five sections (orders) of cyanobacteria, 95% of cyanobacterial genera and 97% of cyanobacterial strains in their samples which corroborates the present findings. The present findings also agree with Wood et al. (2008) who reported that there were significant differences in cyanobacterial community structure encountered over fine scales between two transects located in the Miers valley in Antarctica. In contrast, this study is in contrast with Goh et al. (2009), who reported that the cyanobacteria isolated from Shark Bay, had closest identity to only10 genera.

The finding of no significant differences in the diversity of cyanobacterial taxa between sandy and clay soils or dry and wet samples of soil in the present study, can be explained by the fact that cyanobacteria are a diverse group of prokaryotes able to live under a wide range of environmental conditions as has been noted by Stal and Krumbein (1985); Fay, (1992); Whitton and Potts (2000). Also Stal and Krumbein (1985), mentioned that they found microbial mats in sandy as well as in muddy sediments. However, these were dominated (up to 90%) by one or two cyanobacterial genera. Thajuddin and Subramanian (2005) stated that on sandy shores in India, the cyanobacterial population was very poor due to rough tides, absence of substratum, and poor nutrient content of water. In some areas, the stagnated sea water ponds and puddles showed rich populations of cyanobacteria in the form of thick mats , because these habitats remained undisturbed for relatively long periods. Lyngbya confervoides, L. martansiana, Microcoleus chthonoplastes, M.

67 acustissimus, Oscillatoria salina, O. tenuis, Spirulina subsala, Pseudanabaena schemidleii were predominant in these mats.

Kyaruzi et al. ( 2003) who worked in a mangrove ecosystem in Zanzibar town, reported that there was no significant difference in the diversity of the two types of cyanobacterial groups (heterocystous and non-heterocystous) between two stations. Ishizaka et al. (1994) reported that cyanobacteria and prochlorophytes are well-recognized as major contributors to the phytoplankton biomass and primary productivity in the subtropical and tropical Pacific waters. One account estimated that 700 taxa of nonmarine algae are present in Antarctica. The flora is dominated by species of Anabaena, Aphanocapsa, Calothrix, Chroococcidiopsis, Gloeocapsa, Lyngbya, Mastigocladus, Microchaete, Microcoleus, Oscillatoria, Phormidium, Plectonema, Pseudoanabaena, Nodularia, Nostoc, Schizothrix, Scytonema, Stigonema, Synechococcus and Tolypothrix (Broady et al. 1996).

In the present study the order Oscillatoriales was the most frequently encountered (44%), while the order Stigonematales represented the least percentage of isolates (2.9%). This result is in agreement with Hamed et al. (2007) who reported that a total of 86 cyanobacterial taxa, among species and varieties identified in saline alkaline lakes in Egypt, the most frequent order was Oscillatoriales. Also this study is in good agreement with Tang et al. (1997) who reported that the dominant components of polar wetlands are prokaryotic cyanobacteria mainly from the group Oscillatoriales.

However, Masedo et al. (2009) stated that Gloeocapsa and Chroococcus (order chroococcales) and Phormidium (order Oscillatoriales) are the most common cyanobacterial genera on the monuments of the Mediterranean Basin reported on all substrata. Ortega-Calvo et al. (1995), stated that the most

68 common species found on monuments located in Europe, America and Asia belong to the genera Gloeocapsa and Chroococcus (order chroococcales), Phormidium, and Microcoleus (order Oscillatoriales). These genera were ubiquitous and therefore their presence is not strictly related to a specific lithic substratum or climate.

In the Table Mountains of the Guayana shield in northern South America, Budel (1999) reporeted that the corresponding cyanobacterial communities were composed of the following genera : Chroococcus, Gloeocapsa,(Chroococcales) Plectonema (Oscillatoriales), Stigonema (Stigonematales), and Scytonema (Nostocales). The colonial cyanobacterium Gloeocapsa was dominant in the reddish-coloured areas, while in the blackish-green parts Stigonema and Scytonema dominated.

The endolithic cyanobacteria belonging to the genus Chroococcidiopsis also reached a high frequency in the humid savanna areas. The endolithic microhabitat gives protection from intense solar radiation and desiccation, and it provides mineral nutrients, rock moisture and growth surfaces (Friedmann 1982; Bell 1993; Walker et al. 2005). Pentecost (1992) observed that endolithic growth was often obscured by superficial algal growths, and consequently overlooked. Cryptoendolithic cyanobacteria such as Chroococcidiopsis live beneath rock surfaces together with cryptoendolithic lichens, fungi and bacteria. Chroococcidiopsis can survive extreme cold, heat and arid conditions and it may be the single autotrophic organism most tolerant to environmental extremes (Graham et al. 2006). A similar observation has been reported by Stal and Krumbein (1985), who indicated that mat-forming heterocystous cyanobacterial species occur in very low frequencies due to their poorer adaptation to the environmental conditions in the mats, leading to less successful competition with other cyanobacteria.

Tiwari and Singh (2005) reported that in an arid area of Rajasthan in India, they found more heterocystous genera than the non-heterocysous genera. Huber-Pestalozzi (1938) has listed species of the genera Anabaena, Anabaenopsis, Aphanizomenon, Arthrospira, Coelosphaerium, Gloeotrichia, Microcystis, Nostoc, Nodularia, Oscillatoria, Spirulina gomontina and Lyngbya to form water blooms.

Whitton (1987) reported that forms like Gloeocapsa, Gloeothece, Synechococcus, (order Chroococcales), Phormidium, Lyngbya (order Oscillatoriales) and Anabaena (Nostocales) were the most frequent cyanobacterial genera in hot deserts. Microcystis is one of the dominant organisms that is associated with almost permanent blooms in tropical freshwaters, that are exposed to constant sunshine, warmth and nutrients like phosphate, silicate, nitrates, CO2 and lime (Frankelin 1972). Taylor (1954) reported Calothrix and Rivularia as common cyanobacteria inhabiting marine Arctic areas while Gloeocapsa and Nostoc were abundant in freshwaters. Friedmann and Galun (1973) noted that filamentous cyanobacteria such as Lyngbya, Microcoleus, Phormidium, Plectonema (order Oscillatoriales), Anabaena, Nostoc, and Scytonema (order Nostocales) were most frequent on hot desert lithosole.

Budel and Wessel (1991) reported that eleven genera of desert cyanobacteria were recorded from lithic habitats of Africa, North America, Australia and Europe. Thajuddin and Subramanian (2005) reported that the benthic cyanobacteria are abundant in mangrove environments, which are a rich organic muddy substrata with relatively stagnant shallow water conditions, sheltered nature (hence reduced water movement), and optimum salinity conditions (15–30 ppt).

As regards the genera and species composition in this study, genera of filamentous non-heterocystous cyanobacteria that were encountered included Borzia, Lyngbya, Leptolyngbya, Microcholeus, Phormidium, Oscillatoria, Schizothrix Spirulina and Trichodesmium and genera of filamentous heterocystous cyanobacteria were Anabaena, Aphanizomenon, Calothrix, Scytonema, Cylindrospermum, Cylindrospermopsis, Nostoc, Raphidiopsis and Tolypothrix.

Tiwari and Singh (2005) who working in an environmental setting similar to the present environment in four locations at Rajasthan in India, , reported that they recorded only seven genera, five of which were filamentous heterocystous, while the other two genera (Phormidium and Oscillatoria) were filamentous non-heterocystous. In comparison to the present results their findings fall short of what reported here (seven to twenty five gnera). The reason might be due to the unique conditions of each habitat. Kyaruzi et al. (2003), reported that in Tanzania a total of 10 genera of cyanobacteria were encountered; 8 of which were non-heterocystous and 2 heterocystous. Seven cyanobacterial species were isolated from the effluent polluted saline sodic soils from Pugalur, Tamil Nadu, India by Prabu et al. (2007). Among these seven cyanobacterial species three isolates of Nostoc, two isolates of Westiellopsis and two isolates of Oscillatoria were recorded.

In the present study, the genus Oscillatoria was the most common cyanobacterial genus (22%). This finding is in accord with Hamed et al. (2007), who reported that among highly distributed taxa in all investigated lakes and wetlands of Wadi El- Natrun in Egypt, the most frequent species was the Oscillatoria (16.5%). The present study however, is in disagreement with Tiwari et al. (2005), who reported that in an arid area of Rajasthan in India the genus Nostoc was of ubiquitous distribution, which was isolated

71 from all regions surveyed. Also Ozawa et al. (2005), reported that in the Northern Basin of Lake Biwa in Japan only eleven species of cyanobacteria were observed.

Tomaselli et al. (2000) found that the data reported in the literature did not establish a clear relationship between organisms and the nature of the substratum. Nevertheless, these authors contend that Phormidium tenue, Phormidium autumnale and Microcoleus vaginatus prefer siliceous substrata. Friedman and Galun (1973) reported that filamentous cyanobacteria such as Lyngbya, Microcholeos, Phormidium, Plectonema, Anabaena, Nostoc and Scytonema were most frequent in a hot desert Litlosole. Thajuddin and Subramanian (2005) reported as many as 58 species of cyanobacteria belonging to 22 genera in backwaters and mangrove habitats of the southern east coast of India. Cyanobacteria belonging to the order Chroococcales, and families Osillatoriaceae and Nostacaceae occur ordinarily as planktonic forms. Several species grow in abundance and colour the entire body of water, forming the so-called water-blooms. The variation in the frequency of the genera may be due to the variation in the environmental conditions of the sites of isolation. The biological colonization showed a characteristic trend with the microclimate (orientation and presence or absence of trees). Under the most extreme terrestrial climates, such as hot and cold deserts, endolithic cyanobacterial growth can occur, and the cyanobacteria commonly inhabit the outer millimeters to inner centimeters of rocks exposed to such environments (Walker et al. 2005). This assumtion is corroborated by Hamed et al. (2007) who reported that among 8 lakes studied of Wadi El-Natrun (Egypt), each lake had its own cyanobacterial type, depending on the quantitative frequency of the taxon during the study period. This indicated that each habitat has its own cyanobacterial type. The succession and relative abundance of

72 cyanobacterial genera and species is governed by many factors, among these which moisture, temperature and biota stand prominent.

A study in the Northern Basin of Lake Biwa in Japan by Ozawa et al. (2005), showed that eleven species of cyanobacteria dominated the basin. These were : Microcystis aeruginosa, M. ichthyoblabe, M. novacekii, M. wesenbergii, Oscillatoria raciborskii, Anabaena oumiana, A. affinis, A. flos-aquae, A. ucrainica, A. smithii, and A. crassa. On the other hand, a comparison of the epilithic cyanobacterial flora of the tropics and lithophytic floras from temperate regions reveals an astonishing similarity in species (Golubic, 1967). However, a modern and thorough revision of the species concept in cyanobacteria, involving molecular methods, would probably reveal a larger difference in species composition between temperate and tropical cyanobacteria (Budel 1999).

As for the temporal distribution of cyanobacteria in the present environmental settings an increase in the numbers of isolates during the second half of the year (July, September and November) was clear, and this might have been due to an increase in soil moisture due to rainfall, or might be due to the onset of the soil conditions favorable for the growth of cyanobacteria prevailing on these sites at that time. Sommer (1989) and Sterner (1989) reported that seasonal changes in numerical and biomass dominance are often restricted to a much smaller subset of species within the community. The cyanobacterial community composition observed in this study can be compared to that of Lugomela et al. (2001) who conducted a study at Mazizini close to Zanzibar town and that of Borman et al. (2005) who reported that in two soil samples in Fitzory region in Australia, there was only Anabaena circinals in one site in July, while population of the second site was a mixture of spp. dominated by

Aphanizominon, Cylindrospermopsis, Limnothrix, and Planktolyngbya in September.

For physiological studies and characterization, axenic cultures are essential for eubacteria, while they may not be that essential in studying cyanobacteria, as cyanobacterial cultures are rarely free from associated eubacteria in their sheaths or slime. Thus the studies of cyanobacteria are content with "uni- algal" cultures of cyanobacteria for studying many aspects of their biology. The general tendency of cyanobacterial species to grow as "uni-algal" rather than axenic cultures has been noted in the literature and this has been attributed to the fact that the laboratory environment is quite different and limited compared to natural cyanobacterial habitats (Rippka 1981). Information on requirements for optimal growth of cyanobacteria is necessary for the propagation of these species in mass cultures. The variety of biotic interactions that can exist among species may be critically important for the survival of cyanobacterial species.

At the beginning of this study many time consuming manipulations of growth media and some factors affecting cyanobacterial growth have been carried out to arrive at optimal growth conditions. However, only 16 uni-algal cultures (35%) could be obtained through repeated culturing and purification, out of the total of 46 cyanobacterial taxa observed under the microscope, which indicates the difficulty of culturing of most cyanobacteria .

In the present study certain physiological variables have been chosen for studying their effects on the six chosen cyanobacterial isolates. Most of the selected isolates could not grow in media containing above 4% NaCl, and only two isolates (Lyngbya sp. and Anabaena sp.) could grow at 5%, whereas none of them could grow at 6% NaCl. High concentrations of NaCl

74 apparently inhibit growth by ionic (Na+) stress more than by osmotic stress as has been studied by Brownell and Nicolas (1967). Since high intercellular concentrations of Na+ are toxic to most biological systems, organisms living in sodium-rich environments must have developed detoxifying mechanisms. The ability to produce organic osmolytes to cope with ionic and osmotic stresses in the environment is common in nitrogen-fixing cyanobacteria (Reed, et al. 1986). An optimum requirement for moderate salt concentrations (1-4 percentage) may be clearly seen through the present data. Moisander et al. (2002) reported that Anabaena had a similar growth rate at salinities ranging 0 to 10gl¯¹, meaning that there was low absolute requirement for the salt but rather indicating a tolerance toward the presence of the salt; whereas Cylindrospermopsis growth rates were constant between salinities 0 - 2 gl¯¹ . The limits for the salinity tolerance for growth of Anabaena and Cylindrospermopsis were between 15 – 20 gl¯¹ and 2 - 6 gl¯¹, respectively. Overall, Anabaena and Anabaenopsis had higher growth rates and displayed similar growth rates between 2 and 10 gl¯¹ NaCl. Cylindrospermopsis had the ability to grow at 4 g L¯¹ NaCl, suggesting that it can maintain growth and potentially form blooms in low-salinity regions of estuaries and other brackish waters such as the Baltic sea. Growth rates of Cylindrospermopsis in salt-free medium were similar to the growth rates of Anabaena. However, at 2 g L¯¹ it grew significantly more slowly than Anabaena and Anabaenopsis, but at a rate similar to that of Nodularia. In the present study Anabaena could grow at 5%NaCl, while Cylindrospermum could not grow above 4% NaCl.This result is in good agreement with the observations reported above by Moisander et al. (2002). Many fresh water species are reported to withstand high salinities, (Carr and Wyman 1986). Conversely, many marine cyanobacterial forms can survive at low salinities, but for their optimum growth they express specific requirements for additional salts (Rippka et al. 1979). Lehtimaiki et al. (1997)

75 have noted that Aphanizomenon flos-aquae strain 183 isolated from the Baltic sea prefers salinities from 0 to 5%, with declining growth rates at higher salinities.

Therefore, cyanobacteria have been regarded as halotolerant and halophilic forms. For example, Nubel et al. (2000) stated that a halotolerant, euryhaline cyanobacterium (Halospirulina tapeticola) was able to grow at salinities between 3 and 20%, but not at freshwater salinities. It may be noted from earlier literature that although cyanobacteria can adapt to the variations in salinity and other trace metal concentrations but all cyanobacteria are not halotolerant (Yopp et al. 1978; Blumewald and Tel-Or 1982). However, there are strains that can grow well at salinities ranging from 45-99 ppt. as suggested by Thajuddin and Subramanian (2005) and a Nodularia species has been shown to grow in the salinity range of 0-20gL¯¹ (Moisander et al. 2002). Therefore, all cyanobacteria may not be distinctly classified as marine or fresh water forms. In this study it was demonstrated that the native isolates of Anabaena doliolum and Lyngbya aestuarii can be regarded as salt tolerant because they could grow at the concentration of 50 g L¯¹ NaCl. However, it is clear that the rest of the present isolates are mesophilic as far as salinity is concerned. Cyanobacteria have developed a number of mechanisms to defend themselves against environmental stresses (Allakhverdiev et al. 2001; Rajendran et al. 2007). The physiological bases for the adaptation to high salinity in several cyanobacterial species include three main subprocesses: active extrusion of inorganic ions, leading to relatively unchanged internal salt concentrations; accumulation of large internal amounts of organic osmoprotective compounds; and expression of a set of salt stress proteins (Hagemann and Erdmann 1997).

Hof and Fremy (1933) studied the flora of salt waters, and divided cyanobacteria into physiological groups, halotolerant and halophilic. Halotolerant species are not able to grow at NaCl concentration above 3 M (175.5 ppt), e.g. Calothrix scopularum; other examples include salt-water forms such as Microcoleus chthonoplastes and Lyngbya aestuarii. Halophilic species such as Spirulina subsala can grow at salt concentrations above 3 M and therefore occur commonly in salt pans. Feldmann (1951) divided cyanobacteria into euryhaline and stenohaline forms – those living in brines as hypersaline and those in brackish water as hyposaline; Microcoleus chthonoplastes is a euryhaline representative.

Almost all the chosen six isolates in this study showed nearly the same growth rate in all light intensity treatments. The present results agree with many studies indicating cyanobacterial high ability for capturing light, because they contain special pigments. Dworkin (2006) mentioned that, light intensity adaptation is usually characterized by alterations in the photosynthetic apparatus, especially the light-harvesting components. Sinha et al. (2003) recorded that in Nodularia spp. the levels of UV-absorbing compounds (photo-protective compounds) were increased fivefold when the organisms were exposed to the full spectrum of solar radiation for 72 h. Krinsky (1966) reported that Dactylococcopsis salina appeared blue-green in colour when grown under low light intensities, becoming a deep orange colour when grown under high light intensities. This colour change is due to a drastic increase in the carotenoid :chlorophyll a ratio, which suggests a protective role for carotenoids in the adaptation of the organism to high light intensity. Moreover, Jeeji Bai and Seshadr (1980) stated that Arthrospira platensis strains with a tight helical structure could tolerate higher light intensity than strains with loose spirals, and that loose straight filaments could be

77 transformed to tight coiled shapes when the cells were shifted to high-light conditions. The mothers cultures in the laboratory took yellowish colour when they remain long time at low light intensity. This is explained by that the pigmentation changes can occur in response to environmental factors including light intensity, light quality, nutrient availability, temperature and the age of cells. Bartolini et al. (2004) in a study on monuments located on the Appia Antica road (Rome), observed that grey-black patinas were widespread on marble and travertine stone works exposed to sun irradiation, and that green patinas were more frequent on tufaceous materials and mortar in shaded areas.

It should be noted that in the present study just one isolate (Anabaena doliolum) showed higher growth at 2000 lux. This result is in agreement with Budel (1999) who stated that in all cases studied, in the Guayana Uplands of South America, the composition of the crusts showed a typical rarity of members of the non-heterocystous order Oscillatoriales. Garcia-Pichael and Castenholz (1991) explained that this could be due to high light-protecting, intensely coloured sheath, usually not found in the Oscillatoriales, which might be a prerequisite for colonizing such an extreme environment. One of the few members of the Oscillatoriales possessing a coloured sheath is the genus Schizothrix, which can thus be found in highly insolated habitats, often in connection with early soil formation.

Most of tested isolates in the present study showed an optimum growth at pH of 6.5. This result is not in agreement with Rippka et al. (1979) and Rippka et al.(1981a) who reported that the optimum pH for cyanobacterial growth is between 7.5 and 8. In this study only Synechococcus sp. and Lyngbya eastuarii, showed high growth rate at pH 5.5. On the other hand Spirulina sp.

78 showed high growth rate at pH 8.5. This result is in good agreement with Azra Bano and PirzadaI (2004) who reported that, on the basis of chlorophyll content, best growth of Spirulina major was obtained at pH 6.5, whereas highest growth was at pH 8.0. Also this result is supported by results of Buck and Smith (1995) and Burja et al. (2002) who reported that all cyanobacteria were able to grow in acidic (pH 5.5) medium. This reflects that cyanobacteria can adapt to variable pH conditions. Buck and Smith (1995) suggested that the cyanobacteria possess different mechanisms for maintenance of pH homeostasis depending on their natural habitat. Earlier results, however, suggested a complete absence of cyanobacteria in an environment of pH < 4-5 (Rippka et al. 1979). This may suggest that a slightly acidic environment is not deterrent to the growth of cyanobacteria, and in some cases cyanobacteria preferred low pH for higher growth. This conclusion is also supported by the fact that beside, Spirulina major, all other species were also able to grow in pH 6.5 but at lower rates.

All targeted isolates of the present study could grow on the three temperature levels, viz. 25°C, 35°C and 45°C. However, best growth occurred at 35 °C and least growth at 45°C the difference is significant . This result is in good agreement with Dworkin (2006) who stated that cyanobacteria are found in quite different temperature ranges. Hawkins, (1996) reported that Cylindrospermopsis dominated at high temperature in tropical reservoirs with mean surface water temperatures between 24- 32°C, while Anabaena circinals blooms collapse when temperature reaches 26.6°C. It must be noted that the Lyngbya sp. in this study showed best growth at 35°C which is in good agreement with Fogg et al. (1973) who reported that Lyngbya majuscula appears to be eurythermal, with substantial photosynthetic activity between 15 and 35°C. Marine cyanobacteria are generally eurytherms, with temperature optima typically between 25 and 35°C (Fogg et al. (1973). The maximum

79 photosynthetic rate of L. majuscula was observed at 35°C using 14 C incorporation, whereas at 50 °C, there was negligible measurable photosynthetic activity, which would indicate a thermal optimum somewhere between these two temperatures. Determining photosynthetic responses over a finer temperature range at these upper limits might yield a more definite temperature optimum of L. majuscule and other cyanobacterial spp. however, the temperature optimum for nitrogen fixation in a marine Lyngbya sp. has been reported as 35 °C (Jones 1992), which adds substance to the temperature optimum observed in this study.

Soil is a dynamic system comprising physical, chemical and biological components. While the physical and chemical status of the soil has a bearing on the fertility level of the soil, productivity of soil largely depends on the microbial population. In countries like India having tropical climatic conditions as well as Sudan, even though the soil surface is exposed to direct sunlight and is virtually dry during most part of the year, there is sometimes visible growth of blackish-brown soil crusts inhabited by several cyanobacterial forms (Tirkey and Adhikary 2005) . In the present study, almost all inoculated soil samples showed observable soil crusting. However, two of the chosen filamentous isolates (Spirulina sp. and Anabaena doliolum) showed best results in the sandy soils (92.7 %and 95.1% soil crusting, respectively). This result is in good accord with Malam Issa et al. (1999, 2001a) who reported an increase in aggregate stability by two to four times compared to that of un-inoculated samples, six weeks after inoculation. From the first week following inoculation, cyanobacteria and their abundant extracellular polysaccharide secretions (EPS) formed a discontinuous coating over the surface of isolated mineral particles and in some places an organicbridge between contiguous soil particles. In a similar experience, Bailey et al. (1973) obtained an increase of water stability of aggregates by

14% after 6 weeks following inoculation. Rogers and Burns (1994) and Zulpa de Caire et al. (1997) reported an increase in aggregate stability of 18% on average after 300 days and 66% after 365 days following inoculation by Nostoc muscorum into soil. These values are in the same range as those obtained on microbiotic soil crusts undisturbed for four years as reported by Malam Issa et al. (2001a). Malam Issa et al. (2007) stated that after 4– 6 weeks following inoculation a densely covered surface and an intricate network of filaments resulted from further development of cyanobacteria and secretion of EPS.

CHAPTER SIX Conclusions and Recommendations

It thus appears from the present study that the cyanobacterial inocula are present all year round in all sampled sites, indicating that the cyanobacterial potential is there, and it just needs the onset of optimal conditions to take off. All cyanobacterial orders were represented but to varying degrees. More studies are needed to explain the factors affecting the spatio-temporal distribution of cyanobacteria in these and other regions in the Sudan.

Cyanobacterial plasticity enables them to live in a wide range of temperatures, light intensity, pH and salinity. As is known the brightest point in the world is found in the Sudan. The presence and abundance of cyanobacteria at this point is required to be studied.

The present study showed that the soil crusts have considerable cyanobacteria potential, although this is limited by the need for hydration before it becomes functional under hydrated conditions. Substantial quantities of organic matter could be added through primary production, narrowing of the C : N ratio can occur making the soil more fertile and also increase the humus content of the soil. The ecological value of biological crusts is important as they protect soils from wind erosion, and also act as an absorptive organ for moisture/water, which in turn provides germination grounds for seeds of flowering plants.

In addition to morphological diversity and widespread distribution, cyanobacteria reflect a broad spectrum of physiological properties and tolerance to environmental stresses as has been shown in the present study.

The unlimited potential of cyanobacteria must draw concern and attention to be used in the sustainable development, which is needed nowadays.

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APPENDIX

Appendix no 1. Mean sequent for the effect of salinity on chosen strains and their interactions on dry weight

Source of variation Degree of freedom Mean squire Salinity 5 011128178 ns Strains 5 0.0016838 ns salinity*strain 25 0.000931418 ns Error 72 0.000131407 ns Total 107 Ns= non significant at 0.5 and 0.1 % level probability respectively.

Appendix no 2. Mean sequent for the effect of light on chosen strains and their interactions on dry weight

Source of variation Degree of freedom Mean squire Light 2 0.00062205 ns Strains 5 0.00028705 ns light*strain 10 0.00050105 ns Error 36 0.00057805 ns Total 71 Ns= non significant at 0.5 and 0.1 % level probability respectively.

Appendix no 3. Mean sequent for the effect of pH on chosen strains and their interactions on dry weight

Source of variation Degree of freedom Mean squire pH 3 0.000123792 Strains 5 0.00015913905 ns pH*strain 15 0.00023691705 ns Error 48 0.00045902805 Total 71 Ns= non significant at 0.5 and 0.1 % level probability respectively.

* = significant.

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Appendix no 4. Mean sequent for the effect of temperature on chosen strains and their interactions on dry weight

Source of variation Degree of freedom Mean squire Temperature 2 0.000280019 ** Strains 5 0.000454070 ns Temp*strain 10 0.00042174105 ns Error 36 0.00051111105 Total 53 Ns= non significant at 0.5 and 0.1 % level probability respectively.

* = significant.**= High significant.

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Appendix no 6. Mean sequent for the effect of selected isolates on sand soil particle aggregation.

3weeks

Source of variation Degree of freedom Mean squire Strain 6 88.92 Ns Error 14 429.90 Total 20

6weeks

Source of variation Degree of freedom Mean squire Strain 6 418.03 * Error 14 17.29 Total 20

9weeks

Source of variation Degree of freedom Mean squire Strain 6 607.77 ** Error 14 32.98 Total 20

Ns= non significant at 0.5 and 0.1 % level probability respectively

* = significant.**= High significant.

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Appendix no 5. Mean sequent for the effect of selected isolates on clay soil particle aggregation.

3weeks

Source of variation Degree of freedom Mean squire Strain 6 44.13 Ns Error 14 19.05 Total 20

6weeks

Source of variation Degree of freedom Mean squire Strain 6 102.38 * Error 14 15.03 Total 20

9weeks

Source of variation Degree of freedom Mean squire strain 6 129.52 ** Error 14 6.91 Total 20

Ns= non significant at 0.5 and 0.1 % level probability respectively

* = significant.**= High significant.

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