Isolation and characterization of acid tolerant ...

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Isolation and characterization of acid tolerant cyanobacterial culture from acid soils of Tamilnadu 8. Tamilselvam, G . Gopalaswamy and S. Kannaiyan Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641 003

ABSTRACT Attempts were made to isolate acid tolerant cyanobacterial cultures from low pH soils of Tamil Nadu. The predominant algae were Anabaena, Westiellopsis, Nostoc and Oscillatoria. Based on the predominance, five forms, Anabaena-AT-TGK-5C10, Nosfoc-AT-TGKdA4, Oscillatoria-AT-TGK5C9, Westiellopsis-AT-TGK-4A7 and Westiellopsis-AT-TGK-5A9 were selected for assessing growth, biomass production and biochemical characterization and compared with a mildly alkaline wet land soil isolate Anabaena-NSTGK-G11. Westiellopsis-AT-TGK-4A7proved to be more acid tolerant. Key words : Acid tolerance, ammonia excretion, chlorophyll-a, Cyanobacteria, nitrogenase activity.

INTRODUCTION Cyanobacteria play a major role in sustained productivity of rice. Their performance under field condition is largely influenced by several ecological factors. Their performance under nonnal and saline soils has been well studied (Venkataraman, 1972; Kannaiyan, 1990). However, very little information is available on the performance of cyanobacteria in acid soils (Aiyer, 1965; Madhusoodanan and Dominic, 1999). Nearly 14 % area under rice in Tamil nadu has acid soils. Development of location specific, efficient and acid tolerant cyanobacterial cultures for these soils is worth attempting. The present study was aimed at isolating and characterizing the cyanobacterial cultures from acid soils of Tarnilnadu with a view to select efficient acid tolerant cyanobacteria for increasing the rice production in acid soils.

MATERIAL AND METHODS Acid tolerant cyanobacterial cultures were isolated by the slightly modified method of S h a m et al. (1992) from the acid soil samples collected randomly

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from blocks A, B and C of Rice Research Station, Ambasamudram, Tamil Nadu. The soil characteristics are listed in Table 1. Table 1. Physico-chemical properties of acid soil of Rice Research Station, Ambasamudram Field

pH range

Nutrient content of soil (Kg acl)

EC(d sm-I) N2

A1-A10 B1-B10 C1-C10

4.8-5.6 4.7-6.1 4.9-6.8

0.25-0.90 0.10-0.27 0.11-0.93

112-132 106-140 120-134

"2O5

11-244 14-30 16-30

K2°

16-35 23-34 32-58

Forty acid tolerant cyanobacteria were isolated and purified by streak plate method using N free BG-11 agar medium and triple antibiotic solution containing penicillin, streptomycin and chloramphenicol. Among these, Anabaena-AT-TGK5C10, Nostoc-AT-TGK-5A4, Oscillatoria-AT-TGK-5C9, Westiellopsis-AT-TGK-4A7 and Westiellopsis-AT-TGK-5A9, were selected based on their pH tolerance. AnabaenaNS-TGK-GI1 isolated from mildy alkaline wetland soil was used as a check. The cultures were characterized by growing in N free BG 11 medium in 250 ml conical flasks and incubated inside a polynet house under light intensity of 3000 lux at 28r1°C. Growth and biomass, ammonia excretion (Solarzano, 1969), chlorophyll-a (Talling and Driver, 1961), phycobilins (Bennett and Bogarad, 1971) nitrogenase activity (Hardy et al., 1969) proteins (Lowry et al., 1987) and polysaccharides (Morris,1949) were estimated at intervals of 10 days. RESULTS AND DISCUSSION

The soils used for isolation of cyanobacteria were acidic in nature having pH range of 4.7-6.25 and low in nitrogen, phosphorus and potassium (Table 1). The predominant genera were Anabaena (50%),Westiellopsis (17.5O/0),Nostoc (15°/~), Oscillatoria (5%) and the unicellular algae like Microcystis and Gloeocapsa (Table 2). Though isolation of cyanobacteria from acid soils has been reported (Madhusoodanan and Dominic, 1995), much of their work was confined to isolation. In the present study, we attempted to characterize better acid tolerant algae for use in acid soils. The cyanobacterial cultures isolated from acid soils showed significant increase in growth and biomass production than the mildly alkaline soil culture Anabaena-NS-TGK-G 11 (Table 3). Among the cultures, Westiellopsis-AT-TGK-4A7 and 5A9 registered maximum growth and biomass production. The growth and

Physiology of acid tolerant cyanobacteriafrom Tamilnadu Table ?.

145

Cyanobacterial occurrence in acid soils of Ambasamudram

Field

Dominant genera

A1-A10

Anabaena, Nostoc, Westiellopsis, Oscillatoria, Microcystis, Arthrospira, Calothrix, Gloeocapsa and Cylindrospermum

I

B1-BIO

Anabaena, Nostoc, Oscillaforia, Gloeocapsa, Westiellopsis and Tolyporthix

I

Cl-C10

Anabaena, Nostoc, Westiellopsis, Tolypothrix, M~crocystis,Gloeocapsa and Oscillatoria.

biomass were found to be interrelated. Chlorophyll a and accessory pigments were maximum in Westiellopsis-AT-TGK-4A7 and 5A9 (Fig. 1).Oscillatoria-ATTGK-5C9 had lowest chlorophyll a and highest phycoerythrin content. Protein and polysaccharide content was maximum in Westiellopsis-AT-TGK4A7. Higher protein and polysaccharide content was related to better growth and nitrogen fixation (Table 4). The production of polysaccharides appeared to be regulated by the water pH and metabolic status of the cells. Increased photosynthesis, higher pigment content and higher nitrogenase activity facilitate increased synthesis of polysaccharides (Yamarnoto et al., 1974). Increased ammonia excretion and nitrogenase activity were observed in the acid tolerant cultures (Table 5). The pattern of ammonia excretion increased 20 days after inoculation, then declined thereafter in all cultures. Maximum ammonia Table 3. Growth and biomass production of the acid tolerant cyanobacterial cultures Cyanobacterial cultures

Biomass Growth (O.D. at 750 mm) 10th day 20th day 30th day 10th day 20th day 30th day

Anabaena-NSTGK-GI1

0.582

0.961

1.480

10.84

31.51

82.32

Anabaena-AT-TGK-5C10

0.634

1.066

1.378

11.01

30.47

80.31

Nostoc-AT-TGK-5A4

0.652

1.100

1.450

10.43

26.81

72.32

Oscillatoria-AT-TGK-5C9

0.680

1.129

1.530

12.11

32.82

79.38

Westiellopsis-AT-TGK-4A7

0.730

1.340

1.730

15.63

38.43

97.52


0.722

1.260

1.642

13.55

33.34

87.25

SED

CD

SED

CD

Days

0.087

0.0177

0.3793

0.7693

Cultures

0.0124

0.0251

0.5364

1.0880

Days x Cultures

0.0177

0.0431

0.9291

1.8846

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Table 4. Protein and polysaccharide content of the cyanobacterial cultures

Cyanobzcterrial cultures

Protein content Polysaccharide content 10th day 20th day 30th day 10th day 20th day 30th day

Anabaena-NS-TGK-GI1

34.26

58.67

85.43

2.443

5.816

8.603

Values in pg ml-'

CD

SED

CD

SED

Days

0.5389

1.0930

0.0558

0.1133

Cultures

0.7621

1.5458

0.0790

0.1602

Days x Cultures

1.3200

2.6774

0.1368

0.2775

Table 5. Ammonia excertion and nitrogenase activity of cyanobacterial cultures

Cyanobacterrial cultures

Ammonia exceretion Nitrogenase activity (nrnoles ml-I) (nmoles C,H,h-'g' dry wt) 10th day 20th day 30th day 30th day

Anabaena-NSTGK-GI1

151.14

183.74

135.60

1167.9

Oscillatoria-AT-TGK-5C9

110.34

151.40

121.62

1014.6


167.30

213.53

150.96

1196.3


160.13

213.62

181.14

1241.6

Days Cultures Days x Cultures

SED

CD

SED

CD

1.2945

2.6257

-

-

Physiology of acid tolerant cyanobacteria from Tamilnadu

147

Fig. 1. Effect of low pH on chlorophyll-a and phycobilins in the acid tolerant cyanobacteria

excretion was observed in Westiellopsis-AT-TGK-4A7 and 5A9. Peters et al. (1980) stated that 35 per cent of the fixed atmospheric nitrogen was excreted into the medium as ammonia by Anabaena azollae. The acetylene reduction assay showed that Westiellopsis-AT-TGK-4A7 and 5A9 registered maximum ethylene production (Table 5). This evidently showed that the nitrogenase activity was not affected by low pH. Stewart et al. (1969) also observed that pH of the medium does not affect the nitrogenase activity.

ACKNOWLEDGEMENT The authors gratefully acknowledge the Department of Biotechnology, New Delhi for providing financial support for this work.

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