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Novel Substrate (Algal Protein) for Cultivation of Rhodospirillum Rubrum

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Novel Substrate (Algal Protein) for Cultivation of Rhodospirillum Rubrum 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 nitrogen. When grown under these conditions cellular components like lipids, carbohydrates, protein, carotenoids, bacteriochlorophyll 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 bacteria, Phycocyanobilin, Phycocyanin, Protease, Rhodospirillum rubrum Purple non-sulfur bacteria (Rhodospirillaceae) 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 proteobacteria 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. carotenoid 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 . CulturesSpirulina 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-pcPhyco- 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 conditionsR. 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 proceduresCultures 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-pcStudies were carried out using Scanning electron microscopyHarvested 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, caseinExtracellular stubs and gold-coated for 1 min in a JEOL-JFC-1600 protease activity in the culture filtrate of R.
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