Indian Journal of Experimental Biology Vol. 49, October 2011, pp. 773-780

Novel substrate (algal protein) for cultivation of Rhodospirillum rubrum

T M Vatsala*, R Rekha & R Srividhya Hydrolina Biotech (Pvt) Ltd, TICEL Biopark, Taramani, Chennai 600 113, India Received 20 October 2010; revised 9 July 2011

Rhodospirillum rubrum was grown under light anaerobic conditions with phycocyanin (C-pc) extracted from Spirulina platensis as the sole source of carbon and . When grown under these conditions cellular components like lipids, carbohydrates, protein, , were similar to the one grown with malic acid and ammonium chloride. Growth of R. rubrum increased with increase in concentration of C-pc (200 to 1000 mg/l). R. rubrum also utilized C-pc under dark anaerobic condition. With both malic acid and C-pc as carbon sources C-pc was consumed only after exhaustion of malic acid under light anaerobic condition. No aberration of cell morphology was seen under scanning electron microscope (SEM). R. rubrum utilized both phycocyanobilin and phycoprotein individually as well as in combination. When grown with 1000 mg/l of phycoprotein 450 mg/l of biomass was obtained, and with combination of phycocyanobilin (75 mg/l) and phycoprotein (925 mg/l) 610 mg/l of biomass was obtained. Phycocyanobilin alone did not inhibit the growth of R. rubrum. Utilization of C-pc with protease like activity was observed in plate assay. Protease like activity was also observed as zones around the colonies in plates containing sterilized casein, gelatin and filter sterilized bovine serum albumin. No amino acids were detected in the supernatant when analyzed with ninhydrin. Extracellular protease like activity was highest when C-pc was used as substrate (2.8 U/ml). Intracellular protease like activity was not detected in cell free extracts.

Keywords: Algal protein substrate, Phototropic , Phycocyanobilin, Phycocyanin, Protease, Rhodospirillum rubrum

Purple non-sulfur bacteria () are the cellulose, cotton, silk cotton and condensed corn most studied diverse group of the phototrophic solubles for the growth of R. rubrum were bacteria (PTB). All species grow well under anaerobic demonstrated10-12. Aim of the present study was to conditions in the presence of light, and also have the evaluate the ability of R. rubrum to utilize C-pc a capacity to grow as facultative microaerophilic to potent antioxidant13 as a sole carbon source, and also aerobic chemo-organotrophs1-2. They are ubiquitous nitrogen for its growth and metabolism under both in nature and are abundantly in soil, fresh water lake light and dark anaerobic conditions. To the best of our etc. Rhodospirillum rubrum, a purple non-sulfur knowledge this is the first report on the utilization of a bacteria (PNSB) belonging to the of protein as the source of carbon and nitrogen for the the class C is unique due to the presence of a growth of R. rubrum. spirilloxanthin. R. rubrum ATCC 11170 is a nitrogen fixer and can utilize a number of organic 3 Materials and Methods compounds for its growth and hydrogen production . CulturesSpirulina platensis was provided by The bacterium R. rubrum can also utilize alternative Hydrolina Biotech Private Limited (Chennai, India) as sources of carbon and nitrogen such as ethanol, amino a spray dried powder, and Rhodospirillum rubrum acids and lactic acid containing waste materials for its 4,5 ATCC 11170 was obtained from American Type growth and hydrogen production . Various other Culture Collection (ATCC), Rockville, Maryland, USA. sources including lactate, yoghurt, whey, cassava  waste, and distillery effluent also have been used for Preparation of crude C-pc To 100 g dry powder hydrogen production by R. rubrum6-9. Use of unusual of S. platensis equal quantity of neutral alumina was sources such as microcrystalline cellulose, amorphous added, and the mixture was crushed in a mortar and pestle for 10 min at room temperature (26° ± 2°C). To ______this mixture 200 ml of sodium phosphate buffer (0.1 *Correspondent author M, pH 7.2) was added, and the mixture was kept Telephone: +91 44 24718155; Fax: +91 44 24717066 under static condition overnight at 4ºC. After this, the E-mail: [email protected] mixture was centrifuged at 10,000 rpm for 10 min 774 INDIAN J EXP BIOL, OCTOBER 2011

using centrifuge (C24-BL) REMI at room temperature concentration (1000 mg/l) of 95% pure C-pc was (26° ± 2°C). The blue supernatant was collected. To tested in 50 ml serum bottle. R. rubrum was this, ammonium sulphate (60%) was added and stored inoculated at the as above mentioned concentration. at 4ºC over night, so as to obtain proteins. The Medium with C-pc without inoculum served as a mixture was centrifuged as above and the supernatant negative control. R. rubrum grown using malic acid was discarded. The pellet was suspended in 2.5 mM and ammonium chloride served as a positive control. sodium phosphate buffer (pH 7.0) and dialyzed using R. rubrum was also grown on a mixture of malic acid dialysis membrane (Himedia LA 390) with 3 KDA and C-pc (1000 mg/l each) added together. All molecular weight cut off using the same buffer for experiments were done in triplicates for 10 days with 48 h at 4ºC. This was defined as crude C-pc. Crude C- 12 h photoperiod at 26° ± 2ºC. Growth was studied pc absorbance spectrum was recorded on under dark anaerobic conditions also by wrapping the Spectrophotometer V-630 UV/Visible (Jasco Co., bottles with aluminium foil. Anaerobicity was Tokyo). Amount of C-pc was calculated using maintained by filling the bottles completely. following equation. Cleavage of phycocyanobilin from C-pcPhyco-

14 cyanobilin was cleaved from C-pc following standard PC concentration (mg/ml) = Optical density at 620 nm × 0.1429 17 procedure . Phycocyanobilin was added at increasing Microorganism and culture conditionsR. rubrum concentration ranging from 15, 30, 45, 60 and (ATCC 11170) was grown in Ormerod medium15 75 (mg/l) to the modified Ormerod medium without carbon and nitrogen source. Similarly phycoprotein containing per liter of deionized water, K2HPO4 (900 concentration ranging from 185, 370, 555, 740 and mg), KH2PO4 (600 mg), MgSO4 (200 mg), NH4Cl 925 mg/l was used in Ormerod medium without (100 mg), FeSO4 (12 mg), EDTA (18 mg), CaCl2 (75 carbon and nitrogen source. In yet another experiment mg) and trace metal solution H3BO3 (0.016 mg). both phycocyanobilin and phyco protein was added MnSO44H2O (2.80 mg), Na2MO4. 2H2O .(10 mg), together in the concentration (mg/l) 15 + 185, 30 + ZnSO4 (0.75 mg), CuSO4 (0.24 mg), Biotin (0.001 mg), with 1000 mg/l16 malic acid as a carbon source 370, 45 + 555, 60 + 740, 75 + 925 to the above and 100 mg/l ammonium chloride as a source of mentioned medium. Both served as a source of carbon nitrogen at 26° ± 2ºC in glass bottles stoppered with and nitrogen. R. rubrum was inoculated at the butyl rubber stopper and aluminium crimps, concentration of 60 (mg/l) of dry weight. Growth was illuminated with incandescent 4 X 60 W lamps studied on 0, 5 and 10 days with 12 h photoperiod at 26° ± 2ºC. (Philips) at a light intensity of 2,000-2,500 lux as measured by (LX-102) Lutron light meter, Taiwan. Sampling and analytical proceduresCultures Growth of bacteria was monitored by measuring (5 ml) of R. rubrum were removed aseptically using absorbance at 880 nm (λmax of bacteriochlorophyll) on sterile 5 ml syringe at 2 days interval over 10 days of UV-Vis spectrophotometer. Cells were harvested at period of cultivation. Cells were harvested as above. exponential phase by centrifuging at 10,000 rpm for Both the cells and the culture filtrate were scanned 10 min using centrifuge (C24-BL) REMI at room from 190-1100 nm on a UV-Vis spectrophotometer to temperature (26° ± 2°C), washed and re-suspended in check the growth of R. rubrum and for the presence of th 0.1 M phosphate buffered saline (pH 7.2). The unutilized C-pc. At the end of 10 day cells were concentration was adjusted to 60 (mg/l) dry weight harvested as above and the cells and culture filtrate for using as inoculum for further studies. were analyzed for the following parameters. Growth on C-pcStudies were carried out using Scanning electron microscopyHarvested cells crude C-pc and C-pc of 95% purity gifted by Dr. from malic acid and C-pc grown cultures were fixed Harish Kumar (University of Miyazaki, Japan) as the for SEM as per the procedure of Grekova-Vasileva 18 sole source of carbon and nitrogen for the growth of et al . A 0.2 ml of bacterial suspension was R. rubrum. From a filter sterilized stock solution of centrifuged at 10,000 rpm for 10 min. The supernatant 100 mg/ml of crude C-pc of 200, 400, 600, 800 and was discarded and the pellets were rinsed with sterile 1000 mg were pipetted out into bottles containing 1l distilled water for 30 sec, and then fixed for SEM by sterile modified Ormerod medium16 without carbon the following series of treatments: 2.5% glutaraldehyde and nitrogen source. Sterilization was done by (30 min); 0.15 M phosphate buffer (3 × 15 min); 50% autoclaving at 121°C for 20 min. Only one ethanol (1 × 15 min); 70 ethanol (1 × 15 min); 90% VATSALA et al.: NOVEL SUBSTRATE FOR CULTIVATION OF R. RUBRUM 775

ethanol (1 × 15 min) and 100% ethanol (3 × 15 min). Protease assay Specimens were dried and mounted on aluminum Substrate specificity, caseinExtracellular stubs and gold-coated for 1 min in a JEOL-JFC-1600 protease activity in the culture filtrate of R. rubrum and examined using the SEM model JEOL-JSM- grown with different concentrations of C-pc was 6360. assayed using casein as substrate25,26. The reaction

Determination of dry weight of bacteriaIn order mixture containing 1ml of 0.65% (w/v) casein in 50 to determine cell dry weight, harvested cells were mM potassium phosphate buffer (pH was adjusted to washed with sterile 0.1 M phosphate buffered saline 7.5 at 37ºC using 1.0 ml of 1 M NaOH) and 1 ml (pH 7.2) thrice and dried at 40ºC for 24 h. After enzyme solution (culture filtrate) was incubated at cooling cell dry weight was determined using 37ºC for 1 h. Reaction was stopped by addition of 5 Sartorius analytical balance (AW120 accuracy ±0.1 ml of 110 mM trichloroacetic acid. The tubes were mg). incubated at 37ºC for 30 min, and then centrifuged at Carotenoid determinationCarotenoid was 10,000 rpm for 10 min at 4ºC. Standard tyrosine extracted using 20 mg of the dried cells and 15 ml (concentration 0.010-0.100 mg/ml) was pipetted into (w/v) methanol. This dehydrated the bacteria and different tubes and the volume was made up to 2 ml removed almost all the bacteriochlorophyll. Amount using distilled water. Distilled water (2 ml) served as of bacteriochlorophyll was determined from the blank. To all the tubes, 5 ml of 500 mM sodium absorption coefficient of the solution of pure carbonate and 1 ml of 0.5 mM folin phenol were bacteriochlorophyll at 530 nm which is 28.2 × 10-3 added and incubated at 37ºC for 30 min. The mixture (Ref. 19). The dehydrated residue was extracted with was centrifuged and the supernatant was read at 660 three to four successive portions of benzene. Amount nm using UV-Vis spectrophotometer. One unit of of spirilloxanthin was determined from E value of the enzyme activity was defined as the enzyme quantity 1% benzene extract at 510 nm, (E1cm for the pure that liberates 1 µg of tyrosine per ml of the reaction pigment is 2360)20. mixture per min. Lipid, carbohydrate and protein Synthetic substrate (BAPNA)Protease activity determinationTo estimate lipid content of the cells, was also assayed using a synthetic peptide N-Benzoyl 20 mg of dry cells and 20 mg anhydrous sodium DL-arginine p-nitroanilide (BAPNA) as the sulphate (to remove even traces of moisture) were substrate27. The reaction mixture containing 1.0 ml ground for 5-10 min and then extracted four times 100 mM Tris HCl (pH 7.5), 0.5 ml enzyme solution with diethyl ether. All the fractions were pooled and (culture filtrate of R. rubrum grown in medium with extract was dried in a hot air oven at 50°C for 1 h and highest concentration of C-pc) and 0.5 ml 0.5 mM finally dried to constant weight in a vacuum BAPNA as substrate was incubated at 37ºC for 1 h desiccator21. Dried lipids were determined and the reaction was stopped by addition of 2 ml of gravimetrically using Sartorius analytical weighing ethanol. The liberated p-nitroaniline was read at 405 balance (AW120 accuracy ±0.1 mg). To estimate nm using UV-Vis spectrophotometer. One unit of carbohydrate, 5 ml of ice cold (4°C) sulphuric acid BAPNA enzyme activity (EU) was defined as 1 µM (95%) was added to 20 mg of dry cells mixed well of p-nitroaniline liberated per h. The enzyme activity and 0.1 ml was transferred to 16 × 150 mm screw cap was calculated using a p-nitroaniline molar extinction vials to avoid evaporation, while heating. Anthrone coefficient of 10500 M-1cm-1 at 405 nm. reagent was prepared fresh by dissolving (0.1% v/v) anthrone in ice cold sulphuric acid (95%). Anthrone C-pcCulture filtrate (100 ml) of R. rubrum reagent (4 ml) was added to the mixture in screw cap grown with different concentrations of C-pc was vials mixed well and heated in a boiling water bath concentrated up to 20 ml in a rotovapour at 30ºC. To (95 °C) for 20 min. Samples were removed, cooled to 5 ml of each concentrated samples, 7.5 mg of room temperature 26°±2ºC. The samples were used to phycocyanin was added and their absorbance at 620 th estimate carbohydrate by Dreywood and Morris22,23. To nm was read using a UV-Vis spectrophotometer at 0 estimate protein, 5 ml of 1N NaOH was added to 20 h. The samples were incubated at 12 h photoperiod at mg of dry cells (above mentioned) mixed well and kept 26° ± 2ºC. After 24 h, their absorbance was again read in a boiling water bath for 20 min. The supernatant was at 620 nm to observe the change in concentration of 28 used to estimate protein by Lowry et al 24. C-pc . 776 INDIAN J EXP BIOL, OCTOBER 2011

Ninhydrin test for amino acidsRelease of amino separately on above plates. The plates were incubated acids from C-pc was tested in the culture supernatant at 12 h photoperiod for 5 days. Extracellular protease using ninhydrin29. To 0.5 ml of supernatant, 0.5 ml of was detected as zone of clearance by staining the plates 0.1% (w/v) aqueous ninhydrin solution was added and with 0.1% (w/v) amido black in methanol-acetic acid- heated in a water bath at 80ºC for 3 h. The appearance water (30:10:60) for 1 h and then destaining with of bluish purple colour indicates the presence of methanol-acetic acid-water (30:10:60)30. The cell free amino acids. extract of R. rubrum grown with different Intracellular protease assayR. rubrum cells concentrations of C-pc was also checked on plate with grown in modified Ormerod medium with high (1 g/l) 0.5% (w/v) C-pc for zone formation. and low (200 mg/l) concentrations of C-pc were SDS-PAGE followed by Silver stainingSDS- analyzed for the presence of intracellular protease. PAGE (18%) was performed in a vertical slab gel Wet cells (1 g) was used for the assay with and apparatus as described31. culture filtrates and pure without cellulase treatment. Two ml of cellulase [250 C-pc with increase in incubation period were loaded U/ml Texzyme, Texbiosciences (P) Ltd, Chennai] pH on SDS-PAGE. Protein molecular mass standards 5.8 was added to the wet cells and the mixture was (Genei, Bangalore) having 43-6.5 kDa polypeptides incubated overnight at 50°C. At the end of the were used as molecular weight marker. The bands incubation period the reaction mixture was were visualized by silver staining32. centrifuged at 10,000 rpm for 10 min and the pellet ReagentsChemicals used for analyses were was crushed using neutral alumina, and 0.1 M obtained from the following sources. BAPNA and phosphate buffer (pH 7.0). Cells without cellulase ATPMg2+ from Sigma-Aldrich (Steinheim, Germany) treatment were crushed directly using neutral alumina and all the other chemicals were of analytically pure and 0.1 M phosphate buffer (pH 7.0). Both the cell grade and were procured from Loba Chemie, India. free extracts were tested for protease activity using Statistical analysisResults of cellular analysis casein as substrate as above. One unit of enzyme and extracellular protease assay were presented as activity was defined as the enzyme quantity that means ± standard deviations of three replicates liberates 1 µg of tyrosine per ml of the reaction followed by one-way ANOVA by taking P<0.05 as mixture per min. the minimum criteria for statistical significance. Plate assayModified solid medium with 2% agar-agar containing 0.5% (w/v) of C-pc, 0.5% (w/v) Results of Bovine serum albumin, 1% (w/v) of casein and 1% Rapid decrease in the color of C-pc at lower (w/v) of gelatin were prepared separately. Bovine concentration (200 mg/l) was seen within 6 days of serum was filter sterilized and then added to the following inoculation (Fig. 1A). Absorbance of sterile medium. R. rubrum cells grown on highest culture broth increased over the period of cultivation (1000 mg/l) concentration of C-pc was inoculated for all the concentrations of C-pc. that indicated the

Fig. 1Growth of R. rubrum on C-pc. (A) Utilization of phycocyanin; (B) Increase in biomass of R. rubrum over the period of cultivation; and .(C) Degradation of phycocyanin (%) over the period of incubation [Values are mean ± SE of 3 replicates]. VATSALA et al.: NOVEL SUBSTRATE FOR CULTIVATION OF R. RUBRUM 777

growth of R. rubrum (Fig. 1B). This was further biomass was obtained, and the combination of supported by the decrease in absorbance at 620 nm. phycocyanobilin (75 mg/l) and phycoprotein Growth was less under dark condition and optimal (925 mg/l), yielded 610 mg/l of biomass. The cellular with malic acid and ammonium chloride. In the contents like lipids, carbohydrates, protein, medium supplemented with double carbon source, left spirilloxanthin, bacteriochlorophyll in C-pc grown over C-pc was more when compared to all other test cells were similar to the one grown with malic acid samples. Percentage of degradation of C-pc with days. and ammonium chloride (Table 1). Over the period of Complete utilization of C-pc within 6 days of incubation, SDS-PAGE showed decrease intensity of incubation period was seen with pure C-pc (95%; bands particularly between 20 and 14 kDa when 1000 Fig. 1c). Growth of R. rubrum was also observed on mg/l of C-pc was used (Fig. 3). phycocyanobilin (75 mg was obtained from 1000 mg Similar observations were also recorded with all C-pc) and phyco protein added individually or in other concentrations. With pure C-pc, no band could combination to the medium (Fig. 2A, B). The be visible after 6 days of incubation showing chromophore did not inhibit the growth. Biomass complete utilization of C-pc (data not shown). The could not be quantified as the phycocyanobilin added cell morphology of R. rubrum under SEM revealed was only 75 mg/l (0.0075%). Growth inhibition was that cells which were grown in highest concentration seen when 0.01% or more of phycocyanobilin was of C-pc exhibited a spiral and smooth, length 3-10 used. When 1000 mg/l of phycoprotein, 450 mg/l of micrometers, width 0.8-1 micrometer similar to cells grown in malic acid (Fig. 4A, B). Presence of clear zones around the colonies indicated hydrolysis due to enzyme activity (Fig. 5). Zone of hydrolysis with cell free extract increased with increase in concentration of C-pc which was visibly observed without staining (data not shown). Casein (20 mm) and C-pc (15 mm) had the maximum zone of clearance, while gelatin (9 mm) and BSA (9 mm) produced small zones around the colonies. No significant intracellular protease activity was observed using Casein + ATPMg2+. Extracellular protease showed substrate specificity with significant activity on C-pc whereas with casein and BAPNA less activity was observed (Table 2).

Discussion Increase in culture absorbance with a concomitant decrease in C-pc indicates that R. rubrum ATCC11170 was able to utilize C-pc as the sole source of carbon and nitrogen. Moreover, the growth of R. rubrum on C-pc was similar to that obtained on malic acid and ammonium chloride. Increase in cellular components were also similar when the culture was grown on these media indicating that C-pc had no adverse effect on growth and physiology of R. rubrum. This was further confirmed by the viability of C-pc grown cells on transfer to Ormerod medium containing malic acid. Decrease in the intensity of bands with increase in the concentration of C-pc with time as had also been shown by the spectral data of supernatant indicated that R. rubrum was indeed utilizing C-pc as sole source of carbon and nitrogen. This was also proved by rapid Fig. 2(A) Growth of R. rubrum (expressed as mg dry wt/l) on phycocyanobilin + phyco protein added in combination; (B) only utilization of 95% pure C-pc as well as on phycoprotein [Values are mean ± SE of 3 replicates] phycocyanobilin and phycoprotein for its growth and 778 INDIAN J EXP BIOL, OCTOBER 2011

Table 1Cellular contents of Rhodospirillum rubrum grown in different concentrations of phycocyanin

[Values are mean ±SD of 3 replicates]

Phycocyanin (mg/l) Spirilloxanthin (mg/g) Bacteriochlorophyll (mg/g) Protein (mg/g) Lipids (mg/g) Carbohydrates (mg/g) +ve controla 0.82±0.039* 0.28±0.099* 520.08±0.35* 52.09±0.48* 90.1±0.74* 200 0.839±0.005 0.295±0.06 500.05±0.22 50.1±0.42 87.1±0.73 400 0.906±0.004 0.324±0.071 502.07±0.36 52.08±0.43 87.09±0.73 600 0.976±0.017* 0.38±0.081* 508.1±0.40* 53.08±0.41* 89.09±0.71* 800 0.996±0.002 0.35±0.076 515.08±0.44 53.1±0.60 90.01±0.75 1000 1.0±0.147* 0.350±0.106* 520.09±0.43* 58.1±0.8* 90.1±0.79* M+Pb 1.0±0.104* 0.363±0.127* 520.01±0.3* 58.1±0.80* 90.1±0.75* a Modified Ormerod medium;b Double carbon source . Modified medium (M) +Phycocyanin (P) (1000 mg/l) * Significance at P<0.05

Fig. 3Decrease in the intensity of band of phycocyanin (1 g/l) with time in culture filtrate. Electrophoresis was carried out with 18% polyacrylamide gel. [Lane 1- 0th day, Lane 2- 2nd day; Lane 3-6th day; Lane 4- Molecular weight markers; Lane 5-8th day; Lane 6-10th day and Lane 7- control (1 g/l)]. metabolism. Complete utilization of phycocyanobilin and degradation of tetrapyrrole structures was evidenced by the complete loss of color in C-pc and bilin which were incubated with R. rubrum or culture Fig. 4Scanning electron micrograph of R. rubrum cells grown filtrate. Phycocyanobilin might have had a triggering in (A) malic acid and ammonium chloride; and (B) highest concentration of phycocyanin (1000 mg/l) as only source of activity on enzyme, improving light capturing carbon and nitrogen. efficiency or quenching of oxygen. This was evident in increased biomass when phycocyanobilin and aberration of cells was seen as opposed to aberrations phycoprotein were used together. SEM clearly of cells observed in presence of only D-amino acids33. showed that morphology of R. rubrum was not The clearance of zone clearly showed that R. rubrum affected by phycocyanin or by metabolites. No was able to secrete enzyme outside the cells to VATSALA et al.: NOVEL SUBSTRATE FOR CULTIVATION OF R. RUBRUM 779

could infer that under nutrient limitation R. rubrum adopted a different metabolic pathway for survival. Microorganisms exhibit mechanisms for sensing and responding to changes in their environment. Nutrient limitation or exposure to new substrates involves changes in metabolism of the cells and production of hydrolytic enzymes. A few reports on production of proteases by phototrophic bacteria are available. Proteolytic activity in phototrophic bacteria (R. gelatinosus) is reported35-37and proteolytic activity only in the presence of oxygen towards by R. rubrum has been seen38. The proteome of ATP- dependent Clp protease proteolytic subunit of R. rubrum has been sequenced (http://www.uniprot.org /uniprot/Q2RU45). In the present study in vitro extracellular proteolytic activity was not significant

with casein and synthetic substrate (BAPNA), whereas Fig. 5Extracellular protease production seen as zone of clearance with C-pc significant activity was observed indicating on Modified Ormerod agar with 0.5% (w/v) phycocyanin. substrate specificity. No intracellular ATP dependent

Table 2Extracellular protease activity (U/ml) of supernatant protease activity was observed indicating that the with different substrates mechanism of action on C-pc was different. In

[Values are mean ±SD of 3 replicates] uninoculated control samples because of the presence

Samples Phycocyanin BAPNA Casein of ammonium chloride as nitrogen source in the Ormerod medium no autolysis of C-pc was observed, 1 2.81 ± 0.05* 0.79 ± 0.05* 0.69±0.09* whereas 50% autolysis of C-pc in 24 h has been 2 2.83 ± 0.01 0.8 ± 0.1 0.21±0.02 28 3 1.69 ± 0.05* 1 ± 0.1* 0.41±0.15* reported during nitrogen starvation . The obnoxious 4 2.83 ± 0.01 1.3 ± 0.1 0.27±0.01 odor was completely removed even in the presence of 5 2.18 ± 0.01* 1.6 ± 0.07* 0.2±0.01* 50-60% unutilized C-pc. One report of significant odor 6 3.06 ± 0.01* 1.3 ± 0.1* 0.19±0.01* removal without affecting C-pc is by lactic acid Samples 1-5 Supernatant obtained from cultures containing 200 - bacteria in the presence of glucose added to spirulina 1000 (mg/l) of phycocyanin. Sample 6- Supernatant from double 39 carbon source, Modified Ormerod medium (M) + phycocyanin powder . Mode of action of utilization of C-pc and (1000 mg/l). odor removal is yet to be evaluated. Further studies will

* Significance at P<0.05 following ANOVA test be focused on this aspect.

breakdown C-pc. The activity increased with increase Acknowledgement in concentration of C-pc indicating that it was inducible We would like to express our thanks to Dr Harish by the amount of substrate present. Appearance of zone Kumar (University of Miyazaki, Japan) for providing in plate with casein and gelatin might be due to pure C-pc and to Dr Manimaran for helping us on denaturation of these proteins during sterilization, statistical analysis.We extend our sincere thanks to while filter sterilized BSA produced zone only around Hydrolina Biotech Private Limited for supporting our the colonies as the protein remained intact due to filter work. sterilization. This is the first report of phototrophic bacteria References utilizing a protein isolated from cyanobacteria for its 1 Pfennig N & Triiper H G, The Phototrophic bacteria, in Bergey’s Manual of Determinative Bacteriology, vol. 8, edited growth and metabolism. Tests with ninhydrin were by R E Buchanan, and N E Gibbons (Williams & Wilkins, negative indicating that either no free amino acid was Baltimore) 1974, 24. formed or that they were consumed immediately. 2 Pfennig N, Phototrophic green and : A Growth studies have been carried out with many comparative, systematic survey, Annu Rev Microbiol, 31 33 (1977) 275. individual L-amino acids and L-glutamic acid which 34 3 Vatsala T M, Influence of nutritional factors on hydrogen require the presence of CO2 for growth , but no other production by R. rubrum ATCC 11170, J Microb Biotechnol, study on utilization of protein has been reported. One 2 (1987) 127. 780 INDIAN J EXP BIOL, OCTOBER 2011

4 Zurrer H & Bachofen R, Hydrogen production by the 21 Goodwin T W & Osman H G, Studies in carotenogenesis. photosynthetic bacterium Rhodospirillum rubrum, Appl Environ 10. Spirilloxanthin synthesis by washed cells of Microbiol, 37 (1979) 789. Rhodospirillum rubrum, Biochem J, 56 (1954) 222. 5 Van Niel C B, The culture, general physiology, morphology, and 22 Dreywood R, Qualitative test for carbohydrate material, Ind classification of the non-sulfur purple and brown bacteria, Eng Chem Anal Ed, 18 (1946) 499. Bacteriol Rev, 8 (1944) 1. 23 Morris D L, Quantitative determination of carbohydrates 6 Weetall H H, Sharma B P & Detar C C, Photometabolic with Dreywood’s anthrone reagent, Science, 107 (1948) 254. production of hydrogen from organic substrates by free and 24 Lowry O H, Rosebrough N J, Farr A L & Randall R L, immobilized mixed cultures of Rhodospirillum rubrum and Protein measurement using folin-phenol reagent, J Biol Klebsiella pneumoniae, Biotechnol Bioeng, 23 (1980) 605. Chem, 193 (1951) 265. 7 Reungsang A, Sangyoka S, Chaiprasert P & Imai T, Factors 25 Anson M L, The estimation of Pepsin, Trypsin, Papain and affecting hydrogen production from cassava wastewater by a co- Cathepsin with Hemoglobin, J Gen Physiol, 22 (1938) 79. culture of anaerobic sludge and Rhodospirillum rubum, Pak J 26 Folin O & Ciocalteu V, On Tyrosine and Tryptophane Biol Sci, 10 (2007) 3571. determinations in Proteins, J Biol Chem, 73 (1927) 627

8 Venkataraman C & Vatsala T M, Hydrogen production from 27 Panicker L M, Usha R, Roy S & Mandal C, Purification and whey by phototrophic bacteria. W.H.E.C. Hydrogen Energy characterization of a serine protease (CESP) from mature Progress VIII, 2 (1990) 781. coconut endosperm, BMC Research Notes, 2 (2009) 81.

9 Vatsala T M, Degradation of cellulose by phototrophic 28 Nanni B, Balestreri E, Dainese E, Cozzani I & Felicioli R, bacterium Rhodospirillum rubrum ATCC 11170, Indian J Exp Characterization of a specific Phycocyanin-hydrolysing Biol, 27 (1989).963. protease purified from Spirulina platensis, Microbiol Res,

10 Vatsala T M & Seshadri C V, Phototrophic breakdown of solid 156 (2001) 259. cellulose from silk cotton to hydrogen and volatile fatty acids by 29 McDade J J & Weaver R H, Rapid methods for the detection Rhodospirillum rubrum, Curr Sci, 58 (1989) 173. of gelatin hydrolysis, J Bacteriol, 77 (1959) 60. 11 Vatsala T M, Mohanraj S & Manimaran A, A pilot-scale study 30 Vermelho A B, Meirelles M N L, Lopes A, Petinate S D G , of biohydrogen production from distillery effluent using defined Chaia A A & Branquinha M H, Detection of extracellular Int J Hydrogen Energy, bacterial co-culture, 33 (2008) 5404. proteases from microrganisms on agar plates, Mem Inst 12 Smith R l, West T P & Gibbons W R, Rhodospirillum rubrum: Oswaldo Cruz, 91 (1996) 755. Utilization of condensed corn solubles for poly-(3- 31 Laemmli U K, Cleavage of structural proteins during the hydroxybutyrate –co-3-hydroxyvalerate) production, J Appl assembly of the head of bacteriophage T4, Nature, 227 Microbiol, 104 (2008) 1488. (1970) 680. 13 Madhyastha H & Vatsala T M, Cysteine rich cyanopepetide β2 32 Blum H, Beier H & Gross H J, Improved silver staining of from Spirulina fussiformis exhibits plasmid DNA pBR322 plant proteins, RNA and DNA in polyacrylamide gels, scission prevention and cellular antioxidant activity, Indian J Electrophoresis, 8 (1987) 93. Exp Biol, 48 (2010) 486. 14 Narayan A V & Raghavarao K S M S, Extraction and 33 Tuttle A L & Gest H, Induction of morphological aberrations , purification of C-phycocyanin from Spirulina platensis in Rhodospirillum rubrum by D-amino acids, J Bacteriol 79 employing aqueous two phase systems, Int J Food Eng, 3 (2007) (1960) 213.

1. 34 Hoover T R & Ludden P W, Derepression of nitrogenase by 15 Ormerod J G, Ormerod K S & Gest H, Light – dependent addition of malate to cultures of R. rubrum grown with utilization of organic compounds and photoproduction of glutamate as the carbon and nitrogen source, J Bacteriol, 159 molecular hydrogen by phtotosynthetic bacteria; relationships (1984) 400.

with nitrogen metabolism, Arch Biochem Biophys, 94 (1961) 35 Klemme J H & Pfleiderer C, Production of extracellular 449. proteolytic enzyme by phototrophic bacteria, FEMS 16 Vatsala T M, Influence of nutritional factors on hydrogen Microbiol Lett, 1 (1977) 297.

production by Rhodospirillum rubrum ATCC11170. J Microb 36 Hyun M S, Okuda S & Izaki K, Purification and Biotechnol, 2 (1987) 127. characterization of a neutral serine protease from non-sulfur 17 Beuhler R J, Pierce R C, Friedman L & Siegelman H W, purple photosynthetic bacterium, Curr Microbiol, 18 (1989) Cleavage of phycocyanobilin from C-phycocyanin, J Biol Chem, 379. 251 (1976) 2405. 37 Oda K, Tanskul S, Oyama H & Noparatnaraporn N. 18 Grekova-Vasileva M, Popov I, Vassilev D & Topalova Y, Purification and Characterization of Alkaline serine Isolation and characterization of microbial strain Azo29 capable proteinase from photosynthetic bacterium, Rubrivivax of azo dye decolourization, Biotechnol & Biotechnol EQ, 23 gelatinosus KDDS1 Biosci Biotechnol Biochem, 68 (2004) (2009) 318. 650. 19 French C S, The Pigment-protein compound in Photosynthetic 38 Bhuiyan H, Lindblad A & Nordlund S,. Studies on a bacteria II. The absorption curves of Photosynthin from several proteolytic activity towards Nitrogenase in Rhodospirillum species of bacteria, J Gen Physiol, 22 (1939) 483 . rubrum. In: : From molecules to crop 20 Polgar A, Van Niel C B & Zeckmiester L, Studies on the productivity, Curr Pl Sci Biotechnol Agric, 38 (2006) 61. pigments of the purple bacteria. II. A spectroscopic and 39 Sasakibara M, Fukuda Y, Sekiya A, Nishihashi H & stereochemical investigation of spirilloxanthin, Arch Biochem, 5 Hirahashi T, Process for treating spiriluna. US Pat 7, 326, (1944) 243. 558 B2. Feb 5 (2008).