MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF CYANOBACTERIAL ISOLATES FROM BIOLOGICAL SOIL CRUST OF RAJASTHAN, INDIAHTML Full Text
MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF CYANOBACTERIAL ISOLATES FROM BIOLOGICAL SOIL CRUST OF RAJASTHAN, INDIA
Lokesh Sharma, Shailendra Kumar Singh, Shruti Srivastava and Shanthy Sundaram *
Centers of Biotechnology, University of Allahabad, Prayagraj, Uttar Pradesh, India.
ABSTRACT: The BSC sample was collected at four sites from the arid region of Rajasthan and these isolates were characterized based on morphological and biochemical parameters. We have to isolate both heterocysts and non-heterocysts cyanobacterial species. Oscillatoria tenuis species showed maximum chlorophyll accumulation and Phormium showed maximum carotenoids compared to heterocystous forms. The soluble protein content is high in Non-heterocystous Phormium and Oscillatora curviceps and lowest in heterocystous Nostoc linckia. Heterocystous forms Nostoc commune and Anabaena variabilis were synthesizing high carbohydrates compared to nonheterocytstous forms. Highest nitrogenase activity in Nostoc linckia isolated from Achorl site.
Keywords: BSC-Biological soil crust, Nitrogenase
INTRODUCTION: Cyanobacteria is group of primitive phototrophic prokaryotic organisms whose long evolutionary history dates back to the Proterozoic era. These organisms, endowed with tremendous genome plasticity, are distributed in all possible biotypes of the world. Due to their occurrence in diverse habitats, these organisms are the excellent material for investigation by ecologists, physiologists, biochemists and molecular biologists. These organisms also have tremendous potential in environmental management as soil conditioners, biofertilizers bio monitors of soil fertility and water quality, amelioratory agents in reclamation of saline and usar lands, and in rehabilitation of degraded ecosystems through biosorption of metals, feed for animals and protein supplement 1.
Distribution of these organisms in diverse habitats has always attracted attention of scientists for evolving suitable methods for their ecological investigations. Cyanobacteria dominate the micro-algal populations of hot deserts and comprise a vast majority of micro-flora in arid zones, both in soil crusts 2 and endo-lithic communities. In hot arid zones, these are exposed to more sudden environmental changes because of daily transitions from hot and dry conditions to warm and humid ones when fog and dew may condense at night 3, 4. Cyanobacteria have a remarkable suite of attributes and strategies, which enable them to colonize and survive under extreme habitats. These organisms are able to interact with their niche, develop certain survival mechanisms and either exploit or modify their attributes to make them more suitable under water stress conditions 5, 6.
A host of factors that affect the distribution of cyanobacteria include pH, soil moisture, mineral nutrients, CO2 fixation and combined nitrogen 7, 8, 9. Due to the pivotal role played by these organisms, it was considered worthwhile to examine the existence of cyanobacteria in arid zones of Rajasthan, India, and to analyse their growth parameters and physiological attributes for possible biotechnological applications.
MATERIALS & METHODS:
Soil Sample Collection: BSC sample collected in four sites from Rajasthan. Soil spots randomly selected and scraping about 200 g soil from upper 1cm soil layer from 0.5 ha area had well-developed biocrusts, representing a range of colonization states, from early successional (lightly pigmented: light biocrusts) to late successional (darkly pigmented: dark biocrusts) 10, 11.
Biocrust at each site were collected Fig. 1, using Petri dishes (90 mm diameter, 15 mm deep), allowed to dry completely, sealed in zippered plastic bags, and stored in the dark at room temperature until use 12.
FIG. 1: MICROGRAPHS SITE OF BSC SAMPLE COLLECTION (A) ACHROL (B) VIRATNAGAR (C) ALWAR (D) KUSALGARDH
Cyanobacterial Culture: The enrichment culture technique was utilized for deciphering cyanobacterial populations in the arid zones 13, 14, 15. For the isolation of strains from representative spots, 1 g soil sample was inoculated in 50 mL sterilized BG–11 medium in the absence and presence of nitrogen (1.5 g NaNO3 L–1). Flasks were incubated for 30 days at 28 ± 20C and 3–4 K lux light intensity with cool white fluorescent light tubes under 16/8 h light and dark cycles (Stanier et al. 1971). The cyanobacterial strains were isolated by Serial dilution and pour plate method.
The taxonomic identification was done following the keys given by Desikachary (1959) and Starmach (1966) 8. The non-heterocystous isolates are maintained in nitrogen enriched (+N), and heterocystous isolates are maintained in a nitrogen deficient (-N) medium in a temperature-regulated culture room under controlled light and dark cycles.
Photosynthetic Pigments Analysis: A cyanobacterial culture was centrifuged and the pellet was suspended in acetone to determine photosynthetic pigments (80 percent) at 4ºC. The cells were incubated overnight. The sample was centrifuged again for 5 minutes at 10,000 rpm 22 and the absorbance of the supernatant for chlorophyll and carotenoids was measured at 665 and 480 nm, respectively, using UV–Vis spectrophotometer (Pharmacia Biotech). The quantity of Chl a was determined using 23 formula, and the total amount of carotenoids was computed using the specific formula Jensen's absorption coefficient as described in 1978. The absorbance of the blue supernatant generated in phosphate buffer was measured using a cyanobacterial pellet and repeated freezing and thawing. (pH of 7.5) at 620 nm and the phycocyanin content was computed in accordance with 24, 25.
Metabolite Content & Nitrogen Fixation Ability: Under controlled circumstances, cultures were grown in nitrogen deficient medium for heterocystous, nitrogen fixing forms, and nitrogen enriched media for non-heterocystous forms to determine chlorophyll accumulation, metabolite content, and nitrogen fixation capacity. All tests were carried out three times. Samples were fully homogenised using a shaker or homogenizer and collected for further examination during the exponential phase of development (15 days incubation). The content of chlorophyll was determined using the hot methanol extraction technique 26, 27.
Total carbohydrates were determined using the anthrone-thiourea technique 28 and soluble proteins were calculated using bovine serum albumin as a reference 29, 30, 31. Solarzano's apprach was used to investigate extracellular ammonia emission. The acetylene reduction test 32, 33 was used to measure nitrogenase activity using a gas chromatograph (Nucon Model GC 5700) with a Porapak N column. Nitrogenase activity was measured using ethylene generated in the gas phase, and the activity was represented as n mole C2H4 mg–1 chl h–134.
RESULTS & DISCUSSION: Morphological characters were identified microscopically based upon the keys given by Desikachary (1959) Table 1. Studies undertaken clearly indicated ubiquitous occurrence of cyanobacteria in water stress habitats. Results showed that Anabaena &Nostoc was predominantly present in the arid areas surveyed. A wide variation was observed among different cyanobacterial strains isolated from arid zones of Rajasthan, India, with respect to chlorophyll accumulation, carotenoids, carbohydrate, soluble protein content and the nitrogen fixing potential Table 2 and Table 3.
TABLE 1: MICROSCOPY IMAGE OF ISOLATED SPP. AND NAME OF SITE
|Site name||Isolated spp.||Microscopy image|
|Achrol (Site1)||Anabaena sphaerica|
|Viratnagar Site 2||Anabaena variabilis|
Chlorophyll content varied in isolated spp. from Achrol, Viratnagar, Alwar and Kushalgarh. Two isolates from Anabaena sphaerica show high chlrophyll content (7.2 ug mL-1) then Nostoc linckia is low (1.3 µg mL–1) but nearly same carotenoid content isolated from the soils of Achrol. Anabaena variabilis isolated from arid zones of Viratnagar, showed highest chlorophyll content (5.4 µg mL–1) whereas Nostoc verrucosum accumulated lowest chlorophyll content (1.1 µg mL–1) and carotenoids content is 4.4 to 3.4 µg mL-1. Isolates from Alwar, Oscillatora curviceps showed highest chlorophyll accumulation (7.3 µg mL–1) indicating it to be a slow growing form and Nostoc commune accumulated chlorophyll very less (1.5 µg mL–1) and carotenoid content 5.3 µg mL-1 to 0.3 µg mL-1 respectively. In Kushalgarh, Phormidium and Oscillatoria tenuis show 7.5 µg mL-1 to 10.3 µg mL-1 chlorophyll and carotenoid is 12.3 µg mL-1 to 5.6µg mL-1 respectively.
Out of various isolates, the chlorophyll accumulation during exponential phase of growth was highest in Oscillatoria tenuis (10.3 µg mL–1) followed by Phormidium, Oscillatora curviceps and Anabaena sphaerica nearly 7 µg mL-1. Interestingly, another species Phormidium exhibited highest carotenoid accumulation (12.3 µg mL–1) and lowest in Nostoc commune 0.3 µg mL-1.
Soluble Protein & Carbohydrate Content: Anabaena sphaerica, an isolate from Achrol, produced highest soluble proteins (357.2 µg mL–1) and Nostoc linckia produced lowest soluble protein (77.3 µg mL–1). In Viratnagar Anabaena variabilis highest 247.2 µg mL-1 and lowest in Nostoc verrucosum 82 µg mL-1. The non–hetrocystous strain, Oscillatora curviceps isolated from the Alwar soil exhibited maximum soluble protein content (432.5 µg mL–1), followed by heterocystous strain, Nostoc commune (115.0 µg mL–1). Soluble proteins by the isolates of Kushalgarh was markedly high than the ability observed by the strains isolated from Achrol , Viratnagar and Alwar. Out of all isolates from different sites soluble protein content is lowest in Nostoc linckia&Nostoc verrucosum 77.3 µg mL-1 to 82 µg mL-1 respectively and highest in non-heterocyst Phormidium , Oscillatora curviceps and Oscillatoria tenuis 544.1 µg mL-1,432.5 µg mL-1 to 421.3µg mL-1 respectively Carbohydrates content (µg mL–1) examined form different in different arid zones of Rajasthan also showed a variable pattern. Anabaena sphaerica isolated from the soils of Achrol showed maximum carbohydrate (22.2 µg mL–1), followed by the carbohydrate content shown by Nostoc linkia (7.5 µg mL–1).
Anabaena variabilis isolated from the soils of Viratnagar showed highest carbohydrate content (129.5 µg mL–1) and low in Nostoc verrucosum is 12.3ug mL-1. Isolated from the solids of Alwar showed that the carbohydrate contents was low in non-hetrocysts Oscillatora curviceps and high in Nostoc commune that is 102.5 µg mL-1. In Kusalgardh the carbohydrate content was lowest among all the isolates in non-heterocyst Phormidium and Oscillatoria tenuis 2.2 µg mL-1 to 4.4 µg mL-1 respectively.
TABLE 2: CHARACTERIZATION OF CYANOBACTERIAL STRAINS ISOLATED FROM ARID ZONES OF RAJASTHAN, INDIA
|Place/ Name of Isolates||Chlorophyll (µg mL–1) /||Carotenoid (ug mL-1)||Soluble Proteins (µgmL1)||Carbohydrates (µg mL–1)|
*Being non-heterocystous forms not exhibiting nitrogenase activity, NS = nonsignificant
Enzymatic Activity & Extracellular Ammonia Release: In terms of nitrogenase activity, Nostoc linckia (1202.7 n mole C2H4 mg–1 chl h–1) was the most efficient strain from Achrol, whereas Anabaena sphaerica (173.0 n mole C2H4 mg–1 chl h–1) was the least efficient. In Viratnagar Nostoc varrucosum had the highest nitrogenase activity 750 n mole C2H4 mg–1 chl h–1), whereas Anabaena variabilis had the lowest (85.1 n mole C2H4 mg–1 chl h–1). Isolates from Alwar Nostoc commune had 435.2 n mole C2H4 mg–1 chl h– nitrogenase activity.
TABLE 3: ENZYMATIC ACTIVITY OF CYANOBACTERIAL STRAINS
|Place/ Name of Isolates||Nitrogenase Activity (n mole C2H4 mg-1 chl h–1)||Extracellular Ammonia Release (µmole NH4+ mL–1)|
Under aerobic circumstances, Phormidium, Oscillatoria curviceps & Oscillatoria tenuis showed any nitrogenase activity due to their non heterocystous nature. The Strains isolated from soils of Achrol, Viratnagar, Alwar and Kushalgarh showed negligible amount of ammonia release.
The maximum extracellular ammonia release was only 0.04 µmole NH4+ mL–1) by Oscillatoria tenuis from Kushalgarh, an isolate from Achrol showed lowest extracellular ammonia release of 0.003 & 0.004 in Nostoc linckia and Anabena sphaerica respectively.
CONCLUSION: In present work we evaluate morphological and biochemical diversity of cyanobacterial species in arid region of Rajasthan. Presence of photosynthetic pigments, biomolecules and nitrogenase enzyme ability shows that biological soil crust have cyanobacterial species and help in soil productivity in arid region of Rajasthan.
ACKNOWLEDGEMENTS: Authors are thankful to the University of Allahabad, Prayagraj, India for providing necessary laboratory facilities. Mr Lokesh Sharma is thankful to the UGC for providing D. Phil. CRET fellowship for the financial assistance. Dr Shailendra Kumar Singh gratefully acknowledges the financial support from the University Grants Commission (UGC) through Dr D. S Kothari Postdoctoral Scheme (Grant Number: EN/19-20/0020).
ACKNOWLEDGEMENT: Authors are grateful to Allahabad University for granting a CRET fellowship from Govt. of India, New Delhi.
- Farid I, Hashem A-N, El-Aty A, Esraa AM, Abbas MHH and Ali M: Integrated approaches towards ameliorating a saline sodic soil and increasing the dry weight of barley plants grown thereon. Environment, Biodiversity and Soil Security 2020; 4(2020): 31-46.
- Sommer V, Karsten U and Glaser K: Halophilic algal communities in biological soil crusts isolated from potash tailings pile areas. Frontiers in Ecology and Evolution 2020; 8: 46.
- Nievola CC, Carvalho CP, Carvalho Vr and Rodrigues E: Rapid responses of plants to temperature changes. Temperature 2017; 4(4): 371-405.
- Bhushan B: Design of water harvesting towers and projections for water collection from fog and condensation. Philosophical Transactions of the Royal Society A 2020; 378(2167): 20190440.
- Mallon CA, Van Elsas JD, Salles JFo. Microbial invasions: the process, patterns, and mechanisms. Trends in Microbiology 2015; 23(11): 719-29.
- Dang H and Lovell CR: Microbial surface colonization and biofilm development in marine environments. Microbiology and Molecular Biology Reviews 2016; 80(1): 91-138.
- Alghanmi HA and Jawad HM: Effect of environmental factors on cyanobacteria richness in some agricultural soils. Geomicrobiology Journal 2019; 36(1): 75-84.
- Singh SK, Rahman A, Dixit K, Nath A and Sundaram S: Evaluation of promising algal strains for sustainable exploitation coupled with CO2 Environmental Technology 2016; 37(5): 613-22.
- Singh SK, Sundaram S, Sinha S, Rahman MA and Kapur S: Recent advances in CO2 uptake and fixation mechanism of cyanobacteria and microalgae. Critical Reviews in Environmental Science and Technology 2016; 46(16): 1297-323
- Bowker MA, Belnap J, Chaudhary VB and Johnson NC: Revisiting classic water erosion models in drylands: the strong impact of biological soil crusts. Soil Biology and Biochemistry 2008; 40(9): 2309-16.
- Belnap J, Weber B, BÃ and del B: Biological soil crusts as an organizing principle in drylands. Biological soil crusts: an organizing principle in drylands: Springer 2016; 3-13.
- Muñoz Martín MÁ, Becerra-Absalón, Itzel, Perona E, FernÃ¡ndezâ€Valbuena L, Garciaâ€Pichel F and Mateo P: Cyanobacterial biocrust diversity in Mediterranean ecosystems along a latitudinal and climatic gradient. New Phytologist 2019; 221(1): 123-41.
- Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 1979; 111(1): 1-61.
- Singh JS, Kumar A, Rai AN and Singh DP: Cyanobacteria: a precious bio-resource in agriculture, ecosystem and environmental sustainability. Frontiers in Microbiology 2016; 7: 529.
- Jung P, Briegel-Williams L, Schermer M, BÃ and del B: Strong in combination: Polyphasic approach enhances arguments for cold-assigned cyanobacterial endemism. Microbiology Open 2019; 8(5): 00729.
- Dahech P, Schlömann M and Ortiz C: Light intensity stimulates the production of extracellular polymeric substances (EPS) in a culture of the desert cyanobacterium Trichormus sp. Journal of Applied Phycology 2021; 33(5): 2795-804.
- Saxena S, Singh BV, Tiwari S and Dhar DW: Physiological characterization of cyanobacterial isolates from Orissa and West Bengal. Indian Journal of Plant Physiology 2007; 12(2): 181
- Anahas AMP and Muralitharan G: Isolation and screening of heterocystous cyanobacterial strains for biodiesel production by evaluating the fuel properties from fatty acid methyl ester (FAME) profiles. Bioresource Technology 2015; 184: 9-17.
- Thiyagu R and Sivarajan P: Isolation and characterization of novel bacterial strain present in a lab scale hybrid UASB reactor treating distillery spent wash. Environmental Technology 2019; 40(25): 3351-7.
- Desikachary TV: Cyanophyta New Delhi: Indian council of agricultural research 1959; 2:
- Anand N, Thajuddin N and Dadheech PK: Cyanobacterial taxonomy: Morphometry to molecular studies. Cyanobacteria: Elsevier 2019; 43-64.
- Chakraborty S and Mishra AK: Mitigation of zinc toxicity through differential strategies in two species of the cyanobacterium Anabaena isolated from zinc polluted paddy field. Environmental Pollution 2020; 263: 114375.
- Parida S, Dash S and Rath B: In-vitro antimicrobial and antioxidant activities of certain brackish water cyanobacteria from Chilika Lake, India. Vegetos 2022; 35(1): 38-50.
- Purohit A, Kumar V, Chownk M and Yadav SK: Processing-independent extracellular production of high purity C-phycocyanin from Spirulina platensis. ACS Biomaterials Science & Engineering 2019; 5(7): 3237-45.
- Naaz H, Yasin D, Afzal B, Sami N, Khan NJ and Fatma T: Exogenous salicylic acid mediated herbicide (Paraquat) resistance in cyanobacterial biofertilizer Microchaete sp. NCCU-342. Environmental Science and Pollution Res 2021; 1-11.
- Mackinney G: Absorption of light by chlorophyll solutions. Journal of Biological Chemistry 1941; 140(2): 315-22.
- Sarkar S, Manna MS, Bhowmick TK and Gayen K: Extraction of chlorophylls and carotenoids from dry and wet biomass of isolated Chlorella Thermophila: Optimization of process parameters and modelling by artificial neural network. Process Biochemistry 2020; 96: 58-72.
- Herbert D, Phipps PJ and Strange RE: Chapter III chemical analysis of microbial cells. Methods in microbiology. Elsevier 1971; 209-344.
- Lowry OH, Rosebrough NJ, Farr AL and York. Randall RJ: Protein measurement with folin phenol reagent. Journal of Biology and Chemistry 1951; 193: 262–75.
- Shanmugam A, Sigamani S, Venkatachalam H, Jayaraman JD and Ramamurthy D: Antibacterial activity of extracted phycocyanin from Oscillatoria sp. Journal of Applied Pharmaceutical Science 2017; 7(3): 62-7.
- Deepachandi B, Weerasinghe S, Andrahennadi TP, Karunaweera ND, Wickramarachchi N and Soysa P: Quantification of soluble or insoluble fractions of Leishmania parasite proteins in microvolume applications: a simplification to standard lowry assay. International Journal of Analytical Chemistry 2020; 2020.
- Saiz E, Sgouridis F, Drijfhout FP and Ullah S: Biological nitrogen fixation in peatlands: comparison between acetylene reduction assay and 15N2 assimilation methods. Soil Biology and Biochemistry 2019; 131: 157-65.
- Soper FM, Simon C and Jauss V: Measuring nitrogen fixation by the acetylene reduction assay (ARA): is 3 the magic ratio. Biogeochemistry 2021; 152(2): 345-51.
- Mohan A and Kumar B: Plant growth promoting activities of cyanobacteria growing in rhizosphere of agriculturally fertile soil. J Biotechnol Biochem 2019; 5: 28-36.
How to cite this article:
Sharma L, Singh SK, Srivastava S and Sundaram S: Morphological and biochemical characterization of cyanobacterial isolates from biological soil crust of Rajasthan, India. Int J Pharm Sci & Res 2023; 14(2): 891-97. doi: 10.13040/IJPSR.0975-8232.14(2).891-97.
All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Lokesh Sharma, Shailendra Kumar Singh, Shruti Srivastava and Shanthy Sundaram *
Centers of Biotechnology, University of Allahabad, Prayagraj, Uttar Pradesh, India.
10 June 2022
27 July 2022
04 August 2022
01 February 2023