Abstracts / Journal of Biotechnology 136S (2008) S558–S576
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VI4-P-013
VI4-P-014
Combinational biosynthesis of a fluorescent alpha-subunit phycocyanin (spirulina platensis) in Escherichia coli
Algicidal activity of Achromobacter sp. (strain YZ) isolated from yellow sea: An assessment with bloom causing cyanobacterium Microcystis aeruginosa
Guan Xiangyu 1,2,∗ , Qin Song 1,2 1
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, PR China 2 Ocean University of China, Qingdao, PR China
Hui Wang ∗ , Zhaopu Liu ∗ , Surya Kant Mehta, Gengmao Zhao Key Laboratory of Marine Biology of Jiangsu Province, Nanjing Agriculture University, Nanjing 210095, China
E-mail address: xiangyu
[email protected] (G. Xiangyu).
E-mail address:
[email protected] (H. Wang).
Among numerous oxidizing species, the reactive oxygen ones play a major part in diverse important pathological processes (Finkel and Holbrook, 2000). Phycocyanin (PC) is a biliprotein of blue–green algae, and a variety of nutritional and pharmacological values of PC have been reported (Romay et al., 1998). A vector harboring two cassettes was constructed: cpcA encoding apo-alpha-PC of Spirulina platensis (Sp) along with cpcE-cpcF of Synechocystis sp. PCC6803 (S6) encoding lyases required for the attachment of bilin chromophores to apo-alpha-PC in one cassette; ho1-pcyA of S6 encoding enzymes required for the conversion of heme to phycocyanobilin (PCB) in another cassette (Tooley et al., 2001; Guan et al., 2007). After induction with IPTG, the transformants with final expression vector pCDF-cpcA(Sp)-cpcE-cpcF, ho1-pcyA exhibited a blue color. A fluorescent holo-alpha-PC of Sp with His-tag (rHHPC) was biosynthesized in Escherichia coli BL21 by only one expression vector. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g/L broth in 5-L bench scale. The recombinant rHHPC was purified by Ni2+ affinity column conveniently. The purified rHHPC showed correct molecular weight on SDS-PAGE gel and emitted orange fluorescence by UV excitation. The maximum peaks of rHHPC in absorbance and fluorescence emission spectrum were at 621 nm and 651 nm, which was similar to those of native C-PC. Furthermore, inhibition effect on hydroxyl and peroxyl radicals makes rHHPC as a potent antioxidant, whose function may be partially responsible to the anti-tumor effect. The IC50 values of rHHPC were 277.526 g/ml against hydroxyl radicals and 20.833 g/ml against peroxyl radicals. This study provides an efficient method for large-scale production of the fluorescent phycobiliproteins. The rHHPC with several unique qualitative and quantitative features exhibits promising applications in therapeutic and fluorescent tagging fields instead of native PC in practice (Sun et al., 2003).
With objective to control algal blooms an algae-lytic bacterium (Zhao et al., 2005), temporarily named as YZ, was isolated from the yellow sea and was test for killing of bloom causing cyanobacterium Microcystis aeruginosa. Based on biochemical and biophysical characteristics (IMCAS, 1978) and 16SrRNA gene sequencing the isolate was identified as Achromobacter sp. The isolate YZ was found very effective in lysing and decomposing laboratory-grown Microcystis. The results showed that initial bacterial and algal cells densities strongly influence the removal rates of chlorophyll-a. The greater the initial bacterial cells density the faster was the degradation of chlorophyll-a. However algal cells density had reciprocal effect on degradation of chlorophyll-a by the bacterium. We further found that lipid peroxidation, measured in term of malondialdehyde content, significantly increased from 0.14098 mol L−1 to 0.252 mol L−1 (78.74%) when alga was co-cultivated with YZ. It suggested that isolate YZ caused lysis of Micorcystis via induction of lipid peroxidation. The growth of alga was strongly inhibited by the bacterial filtrate previously autoclaved and treated by protein K indicating that the algae-lytic substance produced by strain YZ was extracellularly produced, non proteinaceous and thermostable (indirect attack) (Imai et al., 2001). The environmental factors such as temperature, illumination, pH have influence on the killing effects of the bacterium: the best lytic effects were achieved at lower temperatures and in the dark. The ability of YZ to lysis Micorcystis decreased in the following order of pH: 3 > 9 > 5 > 7.
References Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247. Guan, X.Y., Qin, S., Su, Zh.L., Zhao, F.Q., Ge, B.S., Li, F.C., Tang, X.X., 2007. Combinational biosynthesis of a fluorescent cyanobacterial holo-␣-phycocyanin in Escherichia coli by using one expression vector. Appl. Biochem. Biotech. 142, 52–59. Romay, C., Armesto, J., Remirez, D., Gonzalez, R., Ledon, N., Garcia, I., 1998. Antioxidant and anti-inflammatory properties of C-phycocyanin from blue-green algae. Inflamm. Res. 47, 36–41. Sun, L., Wang, S.M., Chen, L.X., Gong, X.Q., 2003. Promising fluorescent probes from Phycobiliproteins. IEEE J. Quantum. Elect. 9, 177–188. Tooley, A.J., Cai, Y.A., Glazer, A.N., 2001. Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host. Proc. Natl. Acad. Sci. U.S.A. 98, 60–65.
doi:10.1016/j.jbiotec.2008.07.1325
References Imai, I., Sunahara, T., Nishikawa, T., 2001. Fluctuations of the red tide flagellates Chatonella spp. (Raphidophyceae) and the algicidal bacterium Cytophaga sp. In the Seto Inland Sea, Japan. Mar. Biol. 138, 1043–1049. Institute of Microbiology of Chinese Academy of Sciences, 1978. The General Method of Bacterial Identification. Science Press, Bejing. Zhao, P.C., Pu, Y.P., Yin, L.H., 2005. Development of research on algicidal bacteria and its evaluation. J. Southeast Univ. 24 (3), 202–206.
doi:10.1016/j.jbiotec.2008.07.1326 VI4-P-015 Carbonylcyanide m-chlorophenylhrazone (CCCP) regulated hydrogen production by marine green algae Platymonas subcordiformis subjected to light-dark-light treatment Yunbin Fu 1,2,∗ , Zhaoan Chen 1 , Zhen Guo 1,2 , Hongbin Lu 1 , Xingju Yu 1 , Wei Zhang 1,2 1 Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100049, China
E-mail address:
[email protected] (W. Zhang). Platymonas subcordiformis was found to produce hydrogen via photolysis of seawater (Guan et al., 2004). In order to improve hydrogen production, the effect of light-dark-light treatment on the duration and yield of hydrogen evolution was investigated in the process reg-
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Abstracts / Journal of Biotechnology 136S (2008) S558–S576
ulated by CCCP. CCCP was found to inhibit the photosystem II (PS II) activity, therefore creating an anaerobic environment essential for the onset of hydrogen evolution (Ran et al., 2006). In our system, a fuel-cell operated by hydrogen was integrated into the photobioreactor for both on-line monitoring of the rate of hydrogen evolution and the reduction of potential hydrogen feedback inhibition by consuming hydrogen in situ. During the first 15 h of hydrogen evolution stage, a period of dark treatment followed the initial light illumination with three testing regimes (9 h/6 h; 6 h/9 h; 3 h/12 h) were investigated, with continuous illumination as the control. After the dark treatment, all the cultures were subjected to continuous light illumination until the hydrogen evolution stopped. Three peaks of hydrogen evolution rate were found under all three lightdark-light treatments, in contrast to only one peak rate observed under continuous illumination. In addition, hydrogen production duration was extended from 40 h under continuous illumination to 120 h. The highest hydrogen yield of 78 ml/l achieved using a light-dark-light treatment of 9 h/6 h/ to the end, was similar to that of continuous illumination. These results indicate that the diurnal alternation of solar energy may be suitable for hydrogen production by marine green algae without sacrificing hydrogen yield, and that artificial night-time illumination may be unnecessary to total cell productivity. References Guan, Y.F., Deng, M.C., et al., 2004. Two-stage photo-biological production of hydrogen by marine green alga Platymonas subcordiformis. Biochem. Eng. J. 19 (1), 69–73. Ran, C.Q., Yu, X.J., et al., 2006. Role of carbonyl cyanide m-chlorophenylhydrazone in enhancing photobiological hydrogen production by marine green alga Platymonas subcordiformis. Biotechnol. Prog. 22 (2), 438–443.
was purified. Through conducting batch and continuous experiments of cultivating Chlorella Vulgaris with human urine, we studied the efficiency of CO2 purification and the O2 production rules, besides, investigated the removal effects of N, P, S, Cl and K from the human urine by Chlorella Vulgaris.
References Bozena, K.S., Izabela, B., Stanisław, M., 2004. Enhancement of photosynthetic O2 evolution in Chlorella Vulgaris under high light and increased CO2 concentration as a sign of acclimation to phosphate deficiency. Plant Physiol. Biochem. 42, 403–409. Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99, 3389–3396. Feng, D., Wu, Z., 2006. Culture of Spirulina platensis in human urine for biomass production and O2 evolution. J. Zhejiang Univ. Sci. B 7, 34–37. Grönlund, E., Klang, A., 2004. Stefan falk sustainability of wastewater treatment with microalgae in cold climate, evaluated with energy and socio-ecological principles. Ecol. Eng. 22, 155–174. Kuang, Q., Tan, Y., 2001. Study on the removal of nitrogen phosphorus and organics by activated algae system. China Environ. Sci. 21, 212–216. Tam, N.F.Y., Wong, Y.S., 1996. Effect of ammonia concentrations on growth of Chlorella Vulgaris and nitrogen removal from media. Bioresour. Technol. 57, 45–50. Yoshitomo, W., Hiroshi, S., 1997. Development of a photobioreactor incorporating Chlorella sp. for removal of CO2 in stack gas. Energy Convers. 38, 499–503.
doi:10.1016/j.jbiotec.2008.07.1328 VI4-P-017 Identify the optimal areal cell density and light path in flat plate photobioreactors for outdoor production of Chaetoceros muelleri
doi:10.1016/j.jbiotec.2008.07.1327
Ning Zou ∗ , Linde Liu, Yan Liang, Donghong Sun
VI4-P-016
School of Life Sciences, Ludong University, Yantai, 264025, China
Reduction of CO2 by a high-density culture of Chlorella Vulgaris in human urine Li Ming 1 , Liu Hong 2,∗ , Luo Chao 1 1
Department of Environmental Engineering, Beijing University of Aeronautics and Astronautics, Beijing, 100083, China 2 Department of Biological Engineering, Beijing University of Aeronautics and Astronautics, Beijing, 100083, China E-mail address:
[email protected] (L. Hong). The photobioreactor cultivating microalgae is a highly efficient biological reactor to convert CO2 into microalgae biomass. Using this kind of microalgal photobioreactor as CO2 buffer system holds practical application value in terms of eliminating CO2 in the waste gas. There is a great deal of nitrogen and phosphorus in human urine, so eutrophication will occur if human urine is discharged into water directly. It is always difficult to process human urine due to its complex components. However, microalgae not only removes nitrogen and phosphorus and other pollutants in human urine efficiently, but also produces algae biomass which can be utilized as feed, fertilizer, biofuel, etc. with high yield. Therefore, human urine can be used as medium to cultivate microalgae. In this study, a special photobioreactor was made to cultivate Chlorella Vulgaris with human urine as medium. CO2 was removed when Chlorella Vulgaris grew, and at the same time human urine
∗ Corresponding author. Fax: +86 10 82339837.
E-mail address:
[email protected] (N. Zou). Chaetoceros muelleri was cultured outdoor in summer, the Negev desert, Israel, in glass flat plate photobioreactors. Temperature was maintained below 29 ◦ C, by spraying cooling water on the reactors. Maximal volumetric cell density (4.3 g ash free dry weight L−1 ) was obtained in 2.6 cm light path reactor. Maximal areal cell density (240 g m−2 ) was obtained in 20.8 cm light path reactor. Maximal areal productivity (35.6 g m−2 d−1 ) was obtained in 20.8 cm reactor when the optimal cell density was maintained at 0.81 g L−1 . The highest chlorophyll/carotenoids ratio (1.95) was obtained in 20.8 cm reactors resulted from the highest average areal cell number (5.7 × 1012 cells m−2 ) and the lowest light cell−1 . Light-path of the reactors was optimized on the basis of areal productivity. Light-path of 1.3 cm is too short to maintain an areal cell density high enough to avoid photoinhibition and contamination. The optimal light path for outdoor production of Chaetoceros muelleri was found to be 20.8 cm among the reactors used (1.3, 2.6, 5.2, 10.4, 20.8 and 30.0 cm). Further increase of the lightpath decreased the area/volume ratio and therefore decreased the efficiency of evaporative cooling. Culture temperature therefore was occasionally over 29 ◦ C in the 30.0 cm light-path reactor, and growth in that reactor could not be optimized when the temperature became growth limiting. Keywords: Areal productivity; Cultivation; Growth characteristics; Microalgae; Pigments content
∗ Corresponding author. Tel.: +86 535 6671053; fax: +86 535 6681121.