Bacteriorhodopsin Producing Halophilic Archaea Isolated from Solar Salt Pan Saline Environment for Conversion of Light Energy Into Electrical Energy

ES Energy Environ., 2021, 13, 57-64 nbslcnls ES Energy & Environment DOI: https://dx.doi.org/10.30919/esee8c444

Bacteriorhodopsin Producing Halophilic Archaea Isolated from Solar Salt Pan Saline Environment for Conversion of Light Energy into Electrical Energy

Pradnya P. Kanekar,1* Snehal O. Kulkarni,1 Prashant K. Dhakephalkar,2 Neha Saxena2 and Habib M. Pathan3

Abstract

Haloarchaea inhabiting hypersaline environments like solar salt pan ecosystem require at least 1.5 M NaCl for their growth, while their optimum growth is observed at 3.5-4.5 M NaCl. A Few haloarchaeal genera possess a unique retinal protein, bacteriorhodopsin (BR) in their cell membrane which is a photochemical material that acts as a light driven proton pump and converts light energy into electrochemical energy in form of Adenosine Tri Phosphate (ATP) and proton gradient to generate electric potential. In the present investigation, haloarchaea isolated from Shiroda solar salt pan ecosystem situated in District Sindhudurga, West Coast of Maharashtra and India were explored for production of BR and its direct application in conversion of light energy into electrical energy. The haloarchaeal strain, Sh2.2 from crude salt of salt pan ecosystem was identified as Halovivax asiaticus and found to produce 161 mg/l of BR. It’s capability of converting light energy into electric potential (34.6 mV) appears to be the first report. The strains Sh3.1, Sh3.2 and Sh5.2 isolated from brine sample and wet soil in the vicinity of salt pan and identified as Halorubrum sodomense were found to produce BR in the range of 26-191 mg/l, which is much higher than the earlier reported one about 12.3 mg/l. Keywords: Solar salt pan saline environment; Haloarchaea; Solar energy; Electrical energy; Bacteriorhodopsin; Halorubrum sodomense; Halovivax asiaticus. Received: 26 November 2020; Accepted date: 25 March 2021. Article type: Research Article.

1. Introduction M NaCl. Extremely halophilic microorganisms i.e. Halophilic archaea are salt loving microorganisms which haloarchaea, bacteria like Salinibacter ruber and algae inhabit mainly extreme saline environments like solar salt Dunaliella salina are isolated from this ecosyatem.[1] pan, natural hyper saline brines in coastal regions, salt lakes, Different marine salterns have been studied throughout the rock salt mines and rarely from sea water. They require at world including salterns in Alicante, Spain, Mexico, least 1.5 M NaCl (approximately 9% (w/v) NaCl) for their SanDiego, California, Netherlands, Israel, Italy, India etc.[3] growth and optimal growth usually occurs at much higher There are 10,000 salt production sites present all over India. concentrations of NaCl i.e. 3.5- 4.5 M NaCl (20-25 % (w/v) A few researchers over the globe have studied isolation and NaCl).[1] They can grow up to saturation (approximately 37% cultivation of haloarchaea from solar salterns,[4,5,3,6,7,8,9,10] salt NaCl concentration).[2] pan sediments[11] crystallizer pond,[12] hypersaline Solar salt pan ecosystem represents one of the extremely environments,[13] hypersaline marshy environments[14] etc.. thalassohaline environments.[3] A very few microorganisms A unique feature of haloarchaea is the production of exist in solar salt pan ecosystems where salinity is upto 3-3.5 chromoprotein, bacteriorhodopsin (BR). Bacteriorhodopsin contains a protein moiety (Bactrio-opsin) and a covalently 1 Department of Biotechnology, Modern College of Arts, Science bound chromophore (retinal) which acts as a light dependant and Commerce, Shivajinagar, Pune – 411005, M. S., India. transmembrane proton pump.[15] BR is a photochemical 2 MACS – Agharkar Research Institute, G. G. Agarkar Road, Pune – material. After excitation by light of a suitable wavelength, a 411004, M. S., India. complex photocycle is initiated in which the Schiff base is 3 Advanced Physics Laboratory, Department of Physics, Savitribai deprotonated and reprotonated. The protonation Phule Pune University, Pune 41107, India. /deprotonation reactions are so arranged that the proton is * Email: [email protected] (P. P. Kanekar) taken up from the cytoplasmic side and released to the

© Engineered Science Publisher LLC 2021 ES Energy Environ., 2021, 13, 57-64 | 57 Research article ES Energy & Environment outside of the cell. Thus the light absorption results in the halophilic archaea, Halostagnicola larsenii RG2.14 and generation of a proton gradient that can be used for Haloferax larsenii RG3D1from rockpit sea water and sea generation of Adenosine Tri Phosphate (ATP) and rock respectively which are low saline environments. Here conversion of light energy into electrical energy. BR is stable we report isolation of halophilic archaea from salt pans in absence of salts, retains the photochemical properties over which are extreme saline environment and therefore with long periods, functions between 0 °C and 45 °C, in the pH possibility of isolation of different haloarchaea containing range of 1-11. It tolerates temperatures over 80 °C when in BR for conversion of light energy into electrical energy. aqueous solution while upto 140 °C when dry. It is reported to be stable to sunlight for years and resists digestion by most 2. Materials and Methods proteases. Production of bacteriorhodopsin (BR) was studied 2.1 Sample collection from solar salt pan ecosystem, till date using haloarchaeal genera namely Halobacterium Shiroda halobium,[16,17,18,19,20] Haloarcula japonica[21] followed by Shiroda salt pans are located in the village Shiroda, Taluka Natrinema,[22] Haloferax larsenii ZJ 206,[23] Halorubrum Vengurla, District Sindhudurga, West coast of Maharashtra, sodomense,[24] Haloferax mucosum.[25] Halostagnicola India. Vengurla is located at 15.87 ºN 73.63 ºE. It has an larsenii and Haloferax larsenii isolated from Rock Garden, average elevation of 11 meters and features a tropical Malvan, West Coast of India were explored for production of monsoon climate. The highest temperature in summer BR and its application in conversion of light energy into reaches 42 ºC while in winter, temperature drops down up to electrical energy.[26,27] Earlier, Der and Keszthelyi[28] have 10 ºC. The annual precipitation is 3,155.3 mm. Arabian Sea described charge motion during the photocycle of BR. water near Vengurla beach is used for salt production in form Solar salt pans situated in Western Maharashtra, India of solar salt pans. especially in Konkan region have not been studied till date. Seven samples namely brine sample from salt pan (Sh.1), Thus to explore bacteriorhodopsin producing halophilic crude salt sample recovered from salt pan (Sh.2) (Fig. 1), archaea, solar salt pans of Shiroda, District Sindhudurga brine from salt crystallizer pond showing pinkish colour were selected. The present paper describes isolation and (Sh.3), brine from salt crystallizer pond showing orange characterization of halophilic archaea from solar salt pan yellow colour (Sh.4), wet soil in the vicinity of salt pan, ecosystem situated in Shiroda, District Sindhudurga, West showing reddish film on the surface (Sh.5), mud in the Coast of Maharashtra, India and reporting their ability for vicinity of salt pan, showing reddish film on the surface production of a novel protein bacteriorhodopsin which (Sh.6) (Fig. 1), and dry soil in the vicinity salt pan showing converts light energy into electrical energy. reddish film on the surface (Sh.7) were collected in sterile Kanekar et al [26,27] have reported production of BR and plastic container and stored at 4 °C until use. conversion of light energy into electrical energy by

Fig. 1 Sample collection sites of salt pan, Shiroda.

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2.2 Chemical analysis of samples CAA TTC CT-3′).[33] Each reaction mixture contained The temperature of the salt pans was recorded on site. All approximately 10 ng of DNA; 1X PCR buffer (Sigma, USA); samples except crude salt from salt pan (Sh.2) were analyzed 200 μM each dCTP, dGTP, dATP and dTTP, 2 pmol of each, for physicochemical parameters like pH and concentration of forward and reverse primer; and 1 U of Taq DNA ions of chloride (Cl-), sodium (Na+) and magnesium (Mg2+) Polymerase (Sigma, USA) in a final volume of 20 μL. The and the salinity was calculated using standard methods PCR was performed using Applied Biosystems 2720 (Greenberg et al., 1992).[29] Thermal Cycler with a cycle of 95 ºC for 5 min; 30 cycles of 94 ºC, 60 ºC and 72 ºC for 1 min each; and a final extension 2.3 Isolation of halophilic archaea from salt pan of 72 ºC for 20 min, and the mixture was held at 4 ºC. The ecosystem PCR products obtained from above reactions were then Two methods viz. enrichment technique and direct isolation processed for Cycle sequencing reaction (PCR performed by sprinkling/spreading were used for isolation of using only one primer – ARC915r). The ABI Prism Big Dye haloarchaea from salt pan samples. Terminator Cycle Sequencing Ready reaction kit (Applied Enrichment method: 10% (v/v) sample of Sh.1, Sh.3 and Biosystems, Foster City, California) was used for sequencing Sh.4 were inoculated into 90 ml sterile Sehgal and Gibbon’s of the PCR product. The sequencing reaction and template (SG) Medium[30] (Sehgal and Gibbons, 1960) containing 3.42 preparation were performed and purified in accordance with M NaCl. 10% (w/v) Sh.2, Sh.5, Sh.6 and Sh.7 samples were the directions of the manufacturer (Applied Biosystems) and inoculated in SG medium containing 3.42 M NaCl. The subjected to sequencing in an automated sequencer (3100 flasks were incubated in orbital shaker incubator set at 37°C, Avant Gene Analyser, Applied Biosciences, USA). The 16S 150 rpm for 8-10 days. Good growth was observed in first rRNA gene sequences obtained were compared with the enrichment itself in all samples except crude salt (Sh.2). One reference sequences available in the GenBank loopful culture from enrichment was streaked on SG medium (http://www.ncbi.nim.nih.gov/) and Eztaxon-e plates with 3.42 M NaCl and incubated at 37 °C for 8-10 (http://eztaxon-e.ezbiocloud.net) for the identification of the days. Pink pigmented colonies were observed on SG medium isolates. Phylogenetic tree was constructed by using the plates streaked with all salt pan samples (Sh.1 - Sh.7) after 8- neighbor-joining algorithm[34] with the MEGA software 15 days of incubation. v.5.2[35] after alignment of the sequences by CLUSTAL W Direct plating of salt pan samples: Salt pan samples program.[36] namely brine samples Sh.1, Sh.3 and Sh.4 were spread and crude salt sample (Sh.2), wet soil (Sh.5), mud sample (Sh.6) 2.6 Exploration of salt pan isolates for production of BR and dry soil (Sh.7) was sprinkled on SG medium plates The salt pan isolates were explored for production of BR in containing 3.42 M NaCl and incubated at 37 °C for 15 days. SG medium with 3.4 M NaCl. The presence of BR in the Two pink pigmented, mucoid, irregular colonies were isolates was detected by spectrophotometry.[20,26] observed on SG medium plate sprinkled with pinch of crude salt (Sh.2) after 15 days of incubation. From brine samples 2.7 Application of BR: Conversion of light energy into Sh.1 and Sh.3 no growth was observed on SG medium plates electrical energy by the salt pan isolates after one month incubation. From Sh.4, Sh.5, Sh.6 and Sh.7 The salt pan isolates showing BR activity were grown in 200 samples orange, yellow pigmented, cream colonies were ml of SG medium with 3.42 M NaCl for 8 days at 37 °C and observed which were further found to be halotolerant and the liquid culture was exposed to sunlight for 6 h. Growth of hence not studied further. the isolates and generation of electric potential due to production of BR was measured using digital multimeter. [26] 2.4 Cultural and biochemical characterization of salt pan The survival of salt pan isolates after exposure to sunlight for isolates 6 hrs was studied by inoculating the exposed culture on SG Gram staining,[31] motility and halotolerant/halophilic nature medium plates and incubating for 8 days at 37 °C. of pink pigmented cultures isolated from seven sampling sites were performed. Phenotypic tests of isolates were 3. Results performed according to the proposed minimal standards for 3.1 Physicochemical analysis of collected salt pan samples the description of new taxa in the order Halobacterials[32] and The temperature of the brine sample collected from salt pan as described by Kanekar et al.[26,27] was 50 °C. The pH, sodium, chloride and magnesium ion content of collected salt pan samples are detailed in Table 1. 2.5 DNA extraction and 16 S rRNA gene sequencing The colour of brine present in the crystallizer pond was light Genomic DNA was extracted from pure culture of the pink to light orange yellow. The salinity of brine samples isolates by using GenElute Bacterial Genomic DNA Kit collected from crystallizer ponds was 36%. High salinity (Sigma, USA). DNA was amplified using the archaeon- (40.61%) detected in wet soil sample in vicinity of salt pan specific primers GC-ARC344f (5′-ACG GGG YGC AGC showing reddish film on surface might be due to saturation of AGG CGC GA -3′) and ARC915r (5′-GTG CTC CCC CGC soil with brine sample over the years. The salinity of mud

Table 1. Physicochemical analysis of collected salt pan samples. SR. Sample pH Salinity (%) Ion concentration (%) No. Sodium chlorides Magnesium 1. Brine from salt pan showing brackish colour 7.88 36.61 0.182 20.27 0.729 (Sh.1) 2. Brine from salt crystallizer pond showing 8.03 36.87 0.168 20.41 0.77 pink colour (Sh.3) 3. Brine from salt crystallizer pond showing 7.84 36.36 0.161 20.13 0.486 orange colour (Sh.4) 4. Wet soil in vicinity of salt pan showing 7.74 40.61 13.26 22.48 1.189 reddish film on the surface (Sh.5) 5. Mud sample in vicinity of salt pan showing 8.04 12.12 6.71 7.57 0.417 reddish film on the surface (Sh.6) 6. Dry soil sample in vicinity of salt pan 7.46 30.56 13.95 16.92 0.958 showing reddish film on the surface (Sh.7) sample was found to be low i.e. 12.12%. The salinity of salt well in presence of chloramphenicol (20 mg/l) while growth pan samples is in good agreement with the salinity of salterns of the isolate was inhibited in presence of sodium in Tamil Nadu.[8] Thus, the Shiroda salt pan ecosystem taurocholate (0.25 g/l). All isolates grew well in absence of comprising of crystallizer ponds, soil and mud represents a amino acids in the growth medium. The salt pan isolates saline environment. showed varied growth response to different concentrations of MgSO4 (Tables S1a, S1b and S1c). Four isolates such as 3.2 Isolation of halophilic archaea from salt pan Sh2.3, Sh4.1, Sh5.2, Sh7.1 could not grow without MgSO4. ecosystem and their characterization All the isolates tolerated high concentrations of MgSO4 i.e. From enrichment of all samples, pink or pink-red pigmented, up to 1 and 1.2 M in the SG medium. All these pinpoint colonies were observed on SG medium plates with characteristics indicated the haloarchaeal nature of the 3.42 M NaCl. From brine sample Sh.1, single type of isolates. colonies were observed which were designated as Sh.1.1. From brine sample Sh.3, two types of colonies were observed 3.3 16 S rRNA gene sequencing and identification of salt such as dark red-pink, mucoid (sh2.2) and faint pink, mucoid pan isolates (Sh.2.3); from brine sample Sh.4, single type of dark pink- Identification of salt pan isolates on the basis of 16 S rRNA red pigmented colony was obtained (Sh4.1); from wet soil gene sequencing and phylogenetic analysis (Fig. 2) revealed sample (Sh.5), two types of colonies were observed namely the presence of three haloarchaeal genera namely dark pink, pinpoint (Sh5.1) and faint pink, mucoid with Halorubrum belonging to the family Halorubraceae, order irregular margin (Sh5.2); from mud sample Sh.6 and dry soil Haloferacales; Halovivax belonging to the family sample Sh.7, single type of pink, pinpoint colonies (Sh6.1) Natrialbaceae, order Natrialbales and Haloferax belonging and (Sh7.1) were observed. From direct isolation method to the family Haloferacaceae, order Haloferacales[37,38] in the using crude salt sample Sh.2, two colonies were observed domain Archaea. such as faint pink, mucoid, irregular and dark pink, irregular The results of identification of eight salt pan isolates which were designated as Sh.2.2 and Sh.2.3. From along with accession numbers are detailed in Table 2. The enrichment method as well as direct isolation method, nine isolate obtained from crude salt sample (Sh2.2) showed 98 % off white, cream, yellow, orange pigmented organisms were similarity to Halovivax asiaticus. Isolates obtained from obtained from all solar salt pan samples which were found to brine in the crystallizer pond of salt pan showing pinkish be halotolerant and hence not considered for further studies. colour (Sh3.1, Sh3.2) and wet soil sample (Sh5.2) showed The isolate Sh1.1 showed very slow and poor growth, thus 100 % and 99 % similarity to Halorubrum sodomense. The not included in further studies. The phenotypic characters of reddish film on the surface of the wet soil might be due to eight salt pan isolates are detailed in Tables S1a, S1b and S1c growth of pink-red pigmented Halorubrum sodomense. The (Supporting information). Some of the peculiar isolate obtained from brine in crystallizer pond of salt pan characteristics of haloarchaea include their low rate of showing pale orange colour (Sh4.1) showed 96.33 % survival in distilled water, resistance to antibiotic similarity to Halorubrum californiense. The isolate obtained chloramphenicol, susceptibility to bile salts, and requirement from mud sample (sh6.1) showed 99.42% similarity to of amino acids and Mg2+ ions for growth. The salt pan Haloferax proahovense. The reddish film on the surface of isolates viz. Sh2.2, Sh2.3, and Sh4.1 could not survive in the mud (Fig. 2) might be due to growth of pink –red distilled water, while isolates Sh3.1, Sh3.2, Sh5.2, Sh6.1 and pigmented Haloferax prahovense. The isolate obtained from Sh7.1 survived in distilled water for 2 days. All isolates grew dry soil sample (sh7.1) showed 99% similarity to Haloferax

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Fig. 2 Neighbor- joining phylogenetic tree based on 16 S rRNA gene sequences showing the position of salt pan isolates among related species. Accession numbers are shown in parentheses. Bar represents expected changes per site.

Table 2. Identification of eight salt pan isolates along with accession numbers Sr. Source of the isolate Strain GenBank Accession Closest Match Similarity No. No. % 1. Crude salt from salt pan (Sh.2) Sh2.2 KU201277 Halovivax asiaticus 98 2. Crude salt from salt pan (Sh.2) Sh2.3 KU555929 Halovivax asiaticus 97 3. Brine from salt crystallizer pond Sh3.1 KU201276 Halorubrum sodomense 100 showing pink colour (Sh.3) 4. Brine from salt crystallizer pond Sh3.2 KU201278 Halorubrum sodomense 99 showing pink colour (Sh.3) 5. Brine from salt crystallizer pond Sh4.1 KU555930 Halorubrum californiense 96.33 showing orange colour (Sh.4) 6. Wet soil in vicinity of salt pan Sh5.2 KU201279 Halorubrum sodomense 99 showing reddish film on the surface (Sh.5) 7. Mud sample in vicinity of salt pan Sh6.1 KP739993 Haloferax prahovense 99.42 showing reddish film on the surface (Sh.6) 8. Dry soil sample in vicinity of salt Sh7.1 KU215395 Haloferax alexandrinus 99 pan showing reddish film on the surface (Sh.7)

© Engineered Science Publisher LLC 2021 ES Energy. Environ., 2021, 13, 57-64 | 61 Research article ES Energy & Environment alexandrinus. The reddish film on the surface of the dry soil evidenced by their growth on SG medium plate. might be due to growth of pink pigmented Haloferax alexandrinus. Table 3. Conversion of light energy into electrical energy by salt pan isolates. 3.4 Exploration of salt pan isolates for production of BR Sr. Isolate Growth, Electric potential The growth pattern of the salt pan isolates and production of No. O.D. at 600 generated, BR in SG medium containing 3.42 M NaCl is detailed in Fig. nm milivolts 3. Out of eight salt pan isolates, five isolates showed 1. Sh2.2, 2.341 34.6 production of BR within 8 days of incubation in SG medium Halovivax asiaticus containing 3.42 M NaCl. The isolates obtained from crude 2. Sh2.3, 1.804 20.25 salt sample namely Sh2.2, Sh2.3 identified as Halovivax Halovivax asiaticus asiaticus showed BR production in the range 75-161 mg/l. 3. Sh3.2, 2.010 46.75 Isolates obtained from brine and wet soil sample namely Halorubrum Sh3.1, Sh3.2 and Sh5.2 which were identified as Halorubrum sodomense sodmense showed BR production in the range of 26 mg/l – 4. Sh5.2, 1.893 39.85 191 mg/l. The yield of BR by the strain Sh5.2 of Halorubrum Halorubrum sodomense was much higher (191 mg/l) as compared to 12.3 sodomense mg/l by the isolate Halorubrum sodomense A01.[24] The 5. Uninoculated medium 0.040 0.00 isolates obtained from brine in crystallizer pond of salt pan control showing pale orange colour (Sh4.1), mud sample (Sh6.1) and dry soil sample (Sh7.1) which were identified as Halorubrum 4. Discussion californiense, Haloferax prahovense and Haloferax Solar salterns harbor a diverse community of halophilic alexandrinus did not produce bacteriorhodopsin. microorganisms that represent the three domains of life: archaea, bacteria and eukarya.[39] The archaeal members of 250 2.1 the order Halobacterials, family Halobacteriaceae are significant components of salt pans.[40] Recently, Gupta et 2.05 200 al.[38] have proposed revision of the class ‘Halobacteria’ into 2 three orders namely ‘Halobacteriales’, ‘Haloferacales’ and ‘Natrialbales’. Further the order ‘Halobacteriales’ have been 150 1.95 divided into three families namely ‘Halobacteriaceae’, 100 1.9 ‘Haloarculaceae’ and ‘Halococcaceae’ and order ‘Haloferacales’ into families ‘Haloferaceae’ and

1.85 nm 600 at O.D. ‘Halorubraceae’ while order ‘Natrialbales’ into family BR production mg/l production BR 50 1.8 ‘Natrialbaceae’. 0 1.75 Very few haloarchaeal isolates have been explored for production of BR. In the present studies, five haloarchaeal Sh2.2Sh2.3Sh3.1Sh3.2Sh5.2Sh6.1Sh7.1 isolates viz. Sh2.2, Sh2.3 identified as Halovivax asiaticus Isolates BR mg/l and Sh3.1, Sh3.2, Sh5.2 identified as Halorubrum sodomense showed production of BR in the range from 26 to191 mg/l. The yield of BR in the present study is much higher than the Fig. 3 Growth pattern and production of BR of the salt pan reports of Mohan Raj and Vatsala[24] wherein the yield of BR isolates in SG medium containing 3.42 M NaCl. using Halorubrum sodomense A01 haloarchaeon was 12.3 mg/l while the yield of BR is comparable with the yield by 3.5 Application of BR: Conversion of light energy into Halobacterium halobium (282 mg/l)[20] and Halobacterium electrical energy by the salt pan isolates salinarum PTCC 1685 (197 mg/l).[41] 11 strains of Salt pan isolates namely Sh2.2, Sh2. 3, Sh3.2 and Sh5.2 haloarchaeon Halostagnicola larsenii isolated from rock pit indicated the presence of BR and hence were grown for high sea water, West Coast of Maharashtra, India produced BR in cell mass (200 ml) for detection of electric potential when the range from 35 mg/l to 258 mg/l.[26] Thus the yield of BR exposed to sunlight. The growth of isolates in terms of by haloarchaea isolated from Shiroda salt pan ecosystem is optical density at 600 nm after 8 days of incubation and comparable with the haloarchaon Halostagnicola larsenii electric potential developed after exposure of live culture to isolated from rock pit sea water. Till date the haloarchaeon sunlight for 6 hrs is detailed in Table 3. Halovivax asiaticus has not been reported for production of The salt pan isolates were found to grow well and bacteriorhodopsin. Thus this report presumably is the first generate electric potential in the range of 20.25-46.75 mV. report of production of BR using Halovivax asiaticus isolated All the isolates could survive exposure to sunlight for 6 hr as from crude sea salt sample from solar salterns in India.

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Haloarchaeon Halobacterium sodomense sp. nov. (Now [2] W. D. Grant, M. Kamemkura, T. J. McGenity, A. Ventosa, classified as Halorubrum sodomense) was reported to Class III Halobacteria class, nov. In Bergey’s Manual of produce bacteriorhodopsin in light under reduced oxygen systematic Bacteriology, Vol. 1, 2nd edition ed. D.R. Boone, tension.[42] The members of Halorubrum and Haloquadratum, R.W. Castenholz, G.M. Garrity, New York, NY: Springer 2001 were found to present a significant fraction of solar saltern pp. 294–334. microbial communities in Solvenian and Spanish crystallizer [3] S. Dave, H. Desai, Curr. Sci., 2006, 90, 497-500, ponds. In the shallow crystallizer ponds where salt http://www.jstor.org/stable/24088939. precipitation occurs, the lowest concentration of oxygen is [4] S. Benlloch, S. Acinas, J. Anton, A. Lopez-Lopez, S. Luz, F. observed. The dominance of these genera under such Rodriguez-Valera, Microb. Ecol., 2001, 41, 12-19, doi: conditions hints that bacteriorhodopsin based energy 10.1007/s002480000069. generating photosystems may play an important role in the [5] K. Asha, A. Denslin, G. Sehgal, W. Manjusha, N. Sukumaran success of survival of these organisms in such ecosystems.[42] J. Selvin Int. J. Med. Microbiol., 2004, 1, Thus our finding of dominance of Halorubrum sodomense in http://ispub.com/IJMB/1/2/8605. salt pan ecosystem, Shiroda, and production of BR by these [6] X. Xu, Y. Wu, C. Wang, A. Oren, P. Zhou, M. Wu, Int. J. haloarchaeal isolates is in complete agreement with earlier Syst. Evol. Microbiol., 2007, 57, 717-720, doi: reports. 10.1099/ijs.0.64573-0. The salt pan isolates namely Halovivax asiaticus (Sh2.2, [7] A. Akolkar, G. Deshpande, K. Raval, D. Durai, A. Nerurkar, Sh2.3) and Halorubrum sodomense (Sh3.2, Sh5.2) were A. Desai, J. Basic Microbiol., 2008, 48, 421-425, doi: found to generate electric potential in the range of 20.25- 10.1002/jobm.200800012. 46.75 mV. The ability of 11 strains of haloarchaeon [8] M. Manikandan, V. Kannan, L. Pasic, World J. Microbiol. Halostagnicola larsenii for conversion of light energy to Biotechnol., 2009, 25, 1001-1017, doi: 10.1007/s11274-009- electrical energy was reported in the range of 0.7-44.2 mV.[26] 9980-y. Generation of photocurrent using purple membrane sheets [9] S. Sabet, L. Diallo, L. Hays, W. Jung, J. G. Dillon, containing BR and BR film systems was reported.[43,44,45] Thus Extremophiles, 2009, 13, 643-656, doi: 10.1007/s00792-009- the present investigation first time reports the generation of 0247-1. electric current using live culture of haloarchaeal strains [10] P. Karthikeyan, S. Bhat, M. Chandrasekaran, Saudi J. Biol. namely Halovivax asiaticus and Halorubrum sodomense. Sci., 2013, 20, 205-212, doi: 10.1016/j.sjbs.2013.02.002. In summary, halophilic archaea belonging to genera [11] N. Ahmad, S. Sharma, F. Khan, R. Kumar, S. Johri, M. Halovivax, Halorubrum and Haloferax have been isolated Abdin, G. Qazi, Curr. Microbiol., 2008, doi: 10.1007/s00284- from salt pan ecosystem of Shiroda, District Sindhudurga, 008-9167-z. West Coast of India. The haloarchaeal cultures Sh2.2 and [12] D. Burns, H. Camakaris, P. Janssen, M. Dyall-Smith, Appl. Sh2.3 identified as Halovivax asiaticus is reported for the Environ. Microbiol., 2004, 79, 5258-5265, doi: first time for production of a unique membrane protein, 10.1128/AEM.70.9.5258-5265.2004. bacteriorhodopsin and demonstrated to convert light energy [13] T. Ochsenreiter, F. Pfeifer, C. Schleper, Extremophiles, into electric potential in milivolts. Likewise, the yield of BR 2002, 6, 247-274, doi: 10.1007/s00792-001-0253-4. by the strain Sh5.2 of Halorubrum sodomense was much [14] M. Thomas, K. Pal, R. Dey, A. Saxena, S. Dave, Curr. 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