Impact of Ultraviolet-B Radiation on Cyanobactrial Isolates

 

Arumuganainar Suresh^{1, 2}*, Gowrisankar^2,3

¹Department of Biotechnology, School of Life Sciences, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai - 600117, Tamil Nadu, India.

²Department of Microbiology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu 627412, India.

³Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, 30333, USA.

 

ABSTRACT:

Enhanced solar ultraviolet radiation due to ozone depletion is a major stress factor for cyanobacteria in aquatic and terrestrial ecosystems. Therefore to study the impact of UV-B radiation on cyanobacteria, we isolated three genus of blue green algae namely Anabaena spp., Nostoc spp., and Scytonema spp., and were exposed to UV-B at sublethal dose. The UV exposed cyanobacteria were screened analyzed for the specific growth rate, pigments and heterocyst count with wild type strains for a period of 20 days. All UV-B exposed strains were exhibited stunted growth and pigments content except Scytonema spp. It exhibited accelerated growth and increased carotenoids and heterocyst number. This investigation concluded that the UV-B radiation has negative impact on all genus studied except Scytonema spp in which strain improved in many aspects (positive impact). There by high efficiency strains can be generated using UV-B mutation that could potentiate their application as biofertilizer and also for value added products.

 

 

INTRODUCTION:

Cyanobacteria are prokaryotes and have incredible importance to the biosphere by performing photosynthesis using pigments such as chlorophylls a and b and phycocyanin (blue green pigments) which are embedded in the cyanobacterial thylakoid membrane. Pigments absorb wavelengths of light from the visible spectrum of 400 to 700 nm¹ and as it turns out, cyanobacteria are the only known bacteria that produce O₂. Cyanobacteria are major food in ecosystem, giving nitrogen to living organisms because most species are capable of fixing nitrogen, waste water treatment and value added chemicals.² with these essential functions of cyanobacteria in ecosystems, it is important to understand how these microorganisms are adapted to their harsh environment.

 

The ozone-depleting gases in our atmosphere have increased due to anthropogenic emissions recently. With a reduction of the ozone layer, organisms on Earth are being exposed to an increased level of ultraviolet (UV) radiation. UV light has high energy and shorter wavelengths (290-400 nm) than visible light.¹ Exposure to this high-energy irradiation can lead to the damage of proteins and DNA, as well as changes in growth, cellular differentiation, photo orientation, and the motility of cells.³ UV-A (320-400 nm) radiation is not impacted by the presence of the ozone layer, and thus exposure to this form of UV light will not change due to ozone depletion. However, UV-B (290-320 nm) radiations are typically absorbed by the protective ozone layer, so a decrease in ozone will lead to an increased amount of UV-B reaching Earth’s surface.³ This will be of particular importance to cyanobacteria, as these organisms depend on near-constant exposure to sunlight for survival. Consequently, cyanobacteria have been found to poses mechanisms to avoid UV-B rays and defend against them.³ Such as ROS⁴, DNA damage, specific proteins³, and organisms affected by UV-B exposure have thus been shown to grow more slowly and replicate less frequently.^5,6 Moreover, closely related species of cyanobacteria show a large degree of variability in their sensitivity to UV light. For example, UV-absorbing molecules (Mycosporine Amino Acids⁵, extracellular polysaccharides⁶ and Scytonemin⁸) are one way in which cyanobacteria can defend against UV-B radiation. Though they have these mechanisms to defend against UV radiation, further studies on the effects of UV-B light on cyanobacteria will be of great value.⁹ In this context cyanobacterial cultures of Anabaena sp., Nostoc sp., and Scytonema sp., were isolated and analyzed for their response pattern to UV-B exposure. The impact of UV-B rays was analyzed on the basis of changes in specific growth rate, pigments concentration, cell count and heterocyst frequency of above cyanobacterial cultures.

 

MATERIALS AND METHODS:

Isolation of Cyanobacterial cultures:

The water samples were collected from the paddy field and inoculated in the BG11 and incubated at 26±2°C under 16h light/8hr dark cycle. After 10 days the grown cultures were serially diluted and plated in the BG11 agar plated. The different isolated pure colonies were screened and checked for its identification and contamination under microscopic observation. Finally three genus of cyanobacteria (Anabaena sp., Nostoc sp., and Scytonema sp.,) were identified and maintained in the BG11₀ for further studies.

 

UV-B exposure of cyanobacterial isolates:

Exponentially grown cultures of Anabaena spp., Nostoc spp., and Scytonema spp.,were centrifuged and washed with BG11 medium and homogenized with care not to break the cells with glass beads in a sterilized pestle and mortar. From this 0.1ml of cyanobacterial suspension was taken and spread on BG11 agar plates. The dose of UV was varied by varying the time of exposure with the constant distance of 73 cm being maintained between the UV source and the inoculated plates. After irradiation, plates were kept in dark for 24h to avoid photo reactivation and then incubated in standard light/dark cycle. On the basis of loss of viable growth, lethal and sublethal doses for the cyanobacterial isolates were detected. On the basis of lethal exposure time (LET) cyanobacterial isolates were exposed to UV at sublethal dose (3 min), dark incubated for 24 h and transferred BG11 agar plates and incubated light/dark cycle. Resistant colonies were scored at random and stored for further experimentation.

 

Determination of specific growth rate and cell count:

The freshly grown cyanobacterial mats of wild and UV exposed cultures were taken from 10-15 days old flask. The mats were then transferred into sterile centrifuge tube and centrifuged at 5000 rpm for 10 min at room temperature. After centrifugation the pellets were collected and homogenized using sterile pestle and mortar. Then about 1ml of sterile BG11 medium was added and mixed gently and 0.2 ml was taken and inoculated into the 9.8 ml of BG11 medium in a sterile test tube and incubated with manually shaken 3-5 times daily. At 5 days interval the cyanobacterial cultures were checked for their growth for 20 days by obsorbance at 660 nm using spectronic -20D (Milton Roy) after following centrifugation and homogenization. The specific growth rate constant was calculated using the formula

 

K= specific growth rate constant

N1=cell concentration at the end of the experiment period

N0= cell concentration at the beginning of the experiment period (zero day)

T=days

 

For cell count, 1 ml of 15^th day’s culture was chosen and centrifuged and homogenized separately. The well homogenized cultulture was serially diluted up to 10^-9 in a sterile saline, and 0.1 ml was spread in BG11 agar plates and incubated at 25°C with light/dark cycle for 15 days. The colonies were counted and noted as CFU/mL.

 

Estimation of pigment concentration:

Chlorophyll and carotenoid content of the cyanobacterial isolates were taken as a parameter as this would indicate the positive and negative impact of the test organism to UV-B radiation and followed the methodology of Ritchie, (2006)¹⁰ for chlorophyll and Wellburn, (1994)¹¹ for carotenoid. 1g of (wet weight) of 15 days old cyanobacterial pellet was taken and added 1 ml of 100% methanol and mixed well by vortecxing for 5 min, then centrifuged at 10000 rpm for 10 min. After centrifugation, the supernatant was collected and absorbed spectrophotometer (Spectronic -20D). The chlorophyll_a and carotenoid content was calculated using following formula

 

Determination of heterocyst frequency:

The wild and UV-B exposed cyanobacterial isolates were grown in BG11₀ (without nitrogen) for 20 days and observed microscopically through hanging drop method.  Heterocyst frequency was determined by counting at random the number of heterocyst per hundreds vegetative cells.

 

RESULTS:

Three cynabacteria was isolated from the paddy field and identified through microscopic observation as Anabaena sp., Nostoc sp., and Scytonema sp. All the three strains were exposed to UV-B radiation at different times and the lethal exposure time (LET) was noted. Table 1 shows the clear data on LET and these cyanobacterial isolates have very low LET for UV-B as 6 min.

 

Table 1 Determination of lethal esposure time of UV-B to Cyanobacterial isolates

 

The Figure 1 shows the growth of Ananabae sp on BG11 agar plate and the more exposure the growth is reduced. Morphological changes were observed on UV- B radiation on cyanobacterial isolates as microscopic observation (Figure 2) and heterocyst count (Figure 3). The microscopic observation revealed very interesting impact as the chains of Anabana sp became straight and the wild strain was curved, while Nostoc sp shown lots of extracellular excretes around the strains in UV exposed strains. In the case of Scytonema sp., the heterocyst was stout and lucid in UV exposed whereas in wild strain heterocyst was slim and shrinked in shape (Figure 2).  Moreover the heterocyst number (Figure 3) was increased (10%) in UV-B exposed Scytonema sp than the wild but in other culture the count was reduced. Based LET, we used 3 min as sublethal dose for these strains to study UV-B impact in them.

 

Figure 1. Cyanobacterial isolates of Anabaena sp. showing the growth after UV-B exposure in BG11 medium

 

Figure 2. Cyanobacterial isolates of wild (A) and UV-B  exposed (B) cultures under microscope (100x)

 

Figure 3 Heterocyst frequency of wild and UV-exposed cultures  of cyanobacterial isolates in BG11₀

 

Both the wild and UV exposed strains were checked for the specific growth rate (Figure 4). In the case Anabaena sp., and Nostoc sp., UV-B exposed cultures have exhibited lower growth rate than the wild strain, while UV-B exposed Scytonema sp., observed marginally higher K value (0.28) over wild strain (0.24), respectively.  This was further confirmed by cell count as Scytonema sp., shown more cell number than the wild stain (Table 2) whereas other cyanobacterial isolates shown reduced count.

Table 2 Cell count of wild and UV-B exposed cultures of cyanobacterial isolates on 15^th days of incubation

 

In order to understand the impact of UV-B on pigmentation of cyanobacteria, the pigment profile (Chlorophyll_a and carotenoid) of the wild and the UV-B exposed strains were analyzed (Figure 5)The retardation of growth rate can be extrapolated with the data as the pigment concentration in the UV-B exposed strain was significantly low compare to wild strains. As with the observation of K value, even in the pigment concentration, UV-B exposed Scytonema sp., had higher concentration of pigment over wild strain in Chlorophyll_a and carotenoid.

 

Figure 4  Specific growth rate trend of wild and UV-B exposed cyanobacterial isolates of Anabaena sp. (A), Nostoc sp. (B), and Scytonema sp. (C).

 

Figure 5 Chlorophyll_a and carotenoid content of wild and UV-B exposed cyanobacterial isolates of Anabaena sp. (A), Nostoc sp. (B), and Scytonema sp. (C).

 

DISCUSSION:

Cyanobacteria are a group of microorganisms that exhibit remarkable metabolic versatility. Owing to this, an increase attention is being directed towards this class of microorganisms. Cyanobacteria have gained importance into various fields that includes agriculture as biofertilizer¹², food supplements, source of antioxidents (medicine)² and also in bioenergy production⁹. Through this study, an attempt was made to explore the impact of UV-B on cyanobacterial strains for further improvement. Being photosynthetic in nature these strains were recorded to be highly vulnerable to UV-B radiation that was evident from the low LET of UV-B (6 min) when compared to heterotrophic bacteria such as Escherichia coli (over 15 min). On the basis of this, 3 min was chosen as sub lethal dose and all the three strains were exposed to UV-B in order to study the impact on growth analysis. Specific growth rate was calculated and the UV-B exposed strains of Anabaena sp., and Nostoc sp., were found to have lower K value when compared to wild strains. In support of this, Kumar and Kumar (1990)¹² reported marked reduction in the growth, heterocyst count and nitrogen fixation in Nostoc linckia.

 

Whereas UV-B seems to have generated a potentiated strain in Scytonema sp., where the K value was significantly higher than the wild strain. In fact, cell counts were also revealed the high in UV-B treated than the wild strain. The improved growth pattern in response to UV treatment could very well be due to inductive changes in the genetic segments that encode for pigment component. This could be true as significant increase in chlorophyll_a and carotenoid concentrations was recorded in the UV-B exposed Scytonema sp. Further evidence for the metabolic potentials of the UV-B exposed Scytonema sp., found elevated heterocyst count as 10% than the wild strain. This clearly demonstrate that the strain improvement can be employed for generating potentiated biofertilizer and pigment production as this strain would ensure enhanced rate of nitrogen fixation and carotenoid leading to better value added products.² Stress related changes in pigment and biomass was found in marine cyanobacteriaum Phormidium tenue by Palaniselvam et al., (1998)¹³, where chlorophyll was reduced in response to salinity stress, nevertheless, an increase in caroteoids was reported. It is accordance to our research data on UV-B stress. Stress condition need not result in the generation of potentiated strains. It can also result in the alteration of genome giving raise to organisms that have certain character compromised as we observed in Anabaena sp., and Nostoc sp., they shown reduced pigments. Despite the numerous ways in which cyanobacteria can avoid and defend against UV-B irradiation, the constant exposure of these organisms to sunlight means that damage does sometimes occur. The relative positive impact of UV-B on Scytonema sp., could be attributed to the induction of enzyme system for more pigment production such as scytonemin and some other molecules such as Mycosporine Amino Acids (MAA)⁵ and antioxidants enzymes.⁴ Scytonemin is a pigment that absorbs a maximum wavelength of 370 nm³, enabling this compound to serve a similar sunscreen function as MAA and the role of scytonemin as a sunscreen has been observed in the terrestrial cyanobacterium Chlorogloeopsis sp⁸.

 

A combination of scytonemin and MAA in cells appears to optimize protection against UV-B photons.³ Interestingly, UV-B exposed the Nostoc sp., shown lots of extracellular excretes and it may be the extracellular polysaccharides (EPS) around the cells but that was not observed in wild strain. The same observation was reported by Ehling-Shulz (1997)³ in Nostoc commune and said increase in UV-B irradiation can stimulate the production of EPS in order to defend the cyanobacterium from UV-B damage. In addition, a previous study reported that the concentration of pigment increase when the cell exposed to UV. Scherer et al., (1988)¹⁴ remarked the occurrence of such UV-B absorbing pigment in Nostoc commune. On the basis of overall response of UV-B exposed this study may very well be supportive of strain improvement techniques with simple agents such as UV radiation. This agent can generate hyper activated cells that elaborate their attribute to the survival, which we can take advantage in value added products in agriculture and medicine for our human welfare.

 

CONCLUSION:

Cyanobacteria have a wide range of strategies to avoid and protect against UV-B irradiation form the sun but how and which means. In support of that question, In this study of UV-B on cyanopbacterial isolates revealed Anabaena sp., and Nostoc sp., shown stunned growth on UV-B treatment as reduced pigments (chlorophyll_a and carotenoid) and heterocyst count, while Scytonema sp., shown better adaptation as increased pigments and heterocyst count for their survival. These mechanisms (increases pigments concentration and its morphology) likely evolved early in Earth’s history and have helped cyanobacteria to become one of the most dominant microbial phylums seen in ecosystems today. More importantly, adaptations to UV-B irradiation may have played a major role in allowing life to have better characters. This investigation is indicative of relevance of UV-B in the strain improvement of cyanobacteria (not all) thereby high efficiency strains can be generated that could potentiate their application as value added products. Still further studies needed on the effects of UV-B radiation on cyanobactera in molecular level will be of great value.

 

ACKNOWLEDGEMENTS:

The authors hereby acknowledge the support given by Centre for Biotechnology, Manonmanium Sundaranar University for using photomicroscope.

 

CONFLICT OF INTEREST:

Conflict of interest declared none.

 

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