實驗室先前的研究發現，在將三株由石斑養殖場的Picochlorum sp. strain S1b (簡稱為S1b) 的培養液內純化的三株好氧菌：Labrenzia sp.strain #8、Muricauda sp.strain #50及Arenibacter sp.strain #61一起與無菌的S1b進行共培養時，會產生排除副溶血弧菌的能力，然排除的機制不明。本研究的目的，即在設計實驗，驗證其可能的排除機制。首先，由在分別將三株好氧菌或M1-1弧菌與S1b共培養後，三株好氧菌的生長速率遠高於M1-1的結果，推測三株好氧菌相較於M1-1更能利用來自S1b的營養進行增殖。接著，由螢光顯微鏡的分析中，三株好氧菌更容易貼附在S1b的外圍、形成團聚的結果，推測三株好氧菌更容易貼附在S1b細胞外圍的phycosphere處，與S1b產生緊密接觸，導致不容易接觸的M1-1更容易出現營養排除。緊接著，由與三株好氧菌共培養的S1b在達到生長平緩期時，能夠維持更穩定的細胞密度及較佳的光合作用效率的結果，推測三株好氧菌有助於S1b保持在較健康的狀態。最後，由分析抑制弧菌生長的活性時，抑菌活性集中在三株好氧菌共培養後的S1b藻、菌細胞內，而非培養液的結果，推測S1b與三株好氧菌所產生的藻菌協同體確實含有抗弧菌物質，同時該物質並非持續釋放，而是在出現弧菌後才會被誘發釋放。

Previous research has revealed that the algal-bacterial consortium comprising Picochlorum sp. strain S1b and three aerobic bacteria Labrenzia sp.strain #8, Muricauda sp.strain #50, and Arenibacter sp.strain #61 has a powerful inhibitory effect on Vibrio parahaemolyticus. Our objective in the current study was to identify the mechanisms underlying these inhibitory effects. When S1b was co-cultured with the three aerobic bacteria or the M1-1 strain of Vibrio, the growth rate of the three aerobic bacteria far exceeded that of M1-1. These results suggest that the bacteria were better able to utilize nutrients from S1b for proliferation than was the M1-1. Fluorescence microscopy revealed that the three aerobic bacteria adhered to the periphery of S1b via flocculation, thereby adhering the phycosphere of S1b cells, while remaining in close contact. Note however that M1-1 did not aggregate around S1b, such that it lacked accessibility to nutrients released by S1b. Our results also revealed that the three aerobic bacteria co-cultured with S1b maintained a more stable cell density and superior photosynthesis efficiency to maintain S1b in a healthier state. When co-cultured with S1b, specific antibacterial activity was concentrated in S1b algae and aerobic bacterial cells, rather than in the culture medium. These results indicate that the algae-bacterial consortium indeed contains anti-Vibrio substances, and the production of anti-vibrio substances may be related to algae-bacteria interactions.

黃一城，S1b藻細菌共生體抑制副溶血性弧菌生長機制的研究，國立成功大學生物科技與產業科學系碩士論文，2020。

Amin, S., Hmelo, L., Van Tol, H., Durham, B., Carlson, L., Heal, K., Morales, R., Berthiaume, C., Parker, M., and Djunaedi, B. Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria. Nature 522, 98-101, 2015.

Avendaño, R.E., and Riquelme, C.E. Establishment of mixed‐culture probiotics and microalgae as food for bivalve larvae. Aquaculture Research 30, 893-900, 1999.

Balat, M., Balat, H., and Öz, C. Progress in bioethanol processing. Progress in Energy and Combustion Science 34, 551-573, 2008.

Bell, W.H., Lang, J.M., and Mitchell, R. Selective stimulation of marine bacteria by algal extracellular products 1. Limnology and Oceanography 19, 833-839, 1974.

Bui, T.V., Ross, I.L., Jakob, G., and Hankamer, B. Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae. Public Library of Science 8, e78668, 2013.

Butler, A. Marine siderophores and microbial iron mobilization. Biometals 18, 369-374, 2005.

Campos, J., Otero, L., Franco, A., Mosquera-Corral, A., and Roca, E. Ozonation strategies to reduce sludge production of a seafood industry WWTP. Bioresource Technology 100, 1069-1073, 2009.

Chang, Y.H., Kuo, W.C., Wang, H.C., and Chen, Y.M. Biocontrol of acute hepatopancreatic necrosis disease (AHPND) in shrimp using a microalgal-bacterial consortium. Aquaculture 521, 734990, 2020.

Chen, T. Y., Lin, H.Y., Lin, C.C., Lu, C.K., and Chen, Y.-M. Picochlorum as an alternative to Nannochloropsis for grouper larval rearing. Aquaculture 338, 82-88, 2012.

Cheremisinoff, N.P. Biological degradation of hazardous wastes. Biotechnology for waste and wastewater treatment. Elsevier, United States of America. 49-57. 1997.

Cho, D.H., Ramanan, R., Heo, J., Lee, J., Kim, B.H., Oh, H.M., and Kim, H.S. Enhancing microalgal biomass productivity by engineering a microalgal-bacterial community. Bioresource Technology 175, 578-585, 2015.

Clarens, A.F., Resurreccion, E.P., White, M.A., and Colosi, L.M. Environmental life cycle comparison of algae to other bioenergy feedstocks. Environmental Science and Technology 44, 1813-1819, 2010.

Croft, M.T., Lawrence, A.D., Raux-Deery, E., Warren, M.J., and Smith, A.G. Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438, 90-93, 2005.

Day, J.G., and Fleck, R.A. Cryo-injury in algae and the implications this has to the conservation of micro-algae. Microalgae Biotechnology 1, 1-11, 2015.

Demirbas, A. Progress and recent trends in biofuels. Progress in Energy and Combustion Science 33, 1-18, 2007.

Dornburg, V., van Vuuren, D., van de Ven, G., Langeveld, H., Meeusen, M., Banse, M., van Oorschot, M., Ros, J., van den Born, G.J., and Aiking, H. Bioenergy revisited: key factors in global potentials of bioenergy. Energy and Environmental Science 3, 258-267, 2010.

Dresselhaus, M.S., and Thomas, I. Alternative energy technologies. Nature 414, 332-337, 2001.

Durham, B.P., Sharma, S., Luo, H., Smith, C.B., Amin, S.A., Bender, S.J., Dearth, S.P., Van Mooy, B.A., Campagna, S.R., and Kujawinski, E.B. Cryptic carbon and sulfur cycling between surface ocean plankton. Proceedings of the National Academy of Sciences of the United States of America 112, 453-457, 2015.

Fernandes, M.S., Calsing, L.C., Nascimento, R.C., Santana, H., Morais, P.B., de Capdeville, G., and Brasil, B.S. Customized cryopreservation protocols for chlorophytes based on cell morphology. Algal Research 38, 101402, 2019.

Fischer, G., and Schrattenholzer, L. Global bioenergy potentials through 2050. Biomass and Bioenergy 20, 151-159, 2001.

Geng, H., Bruhn, J.B., Nielsen, K.F., Gram, L., and Belas, R. Genetic dissection of tropodithietic acid biosynthesis by marine roseobacters. Applied and Environmental Microbiology 74, 1535-1545, 2008.

Guo, W.Q., Yang, S.S., Xiang, W.S., Wang, X.J., and Ren, N.Q. Minimization of excess sludge production by in-situ activated sludge treatment processes-A comprehensive review. Biotechnology Advances 31, 1386-1396, 2013.

Han, P., Lu, Q., Fan, L., and Zhou, W. A Review on the Use of Microalgae for Sustainable Aquaculture. Applied Sciences 9, 2377, 2019.

Hirayama, K. Application of a growth-promoting bacteria for stable mass culture of three marine microalgae. In Live Food in Aquaculture. Springer, Dordrecht. 223-230. 1997.

Jones, C.S., and Mayfield, S.P. Algae biofuels: versatility for the future of bioenergy. Current Opinion in Biotechnology 23, 346-351, 2012.

Kiene, R., Visscher, P., Keller, M., Kirst, G., and Childers, D.L. Biological and environmental chemistry of DMSP and related sulfonium compounds. Estuaries 20, 465-465, 1997.

Kim, D.-H., Yun, H.S., Kim, Y.S., and Kim, J.G. Effects of co-culture on improved productivity and bioresource for microalgal biomass using the floc-forming bacteria Melaminivora jejuensis. Frontiers in Bioengineering and Biotechnology 8, 588210, 2020.

Kim, S., and Dale, B.E. Life cycle assessment of various cropping systems utilized for producing biofuels: bioethanol and biodiesel. Biomass and Bioenergy 29, 426-439, 2005.

Kuo, J.C., Chang, Y.H., Chen, T.Y., and Chen, Y.M. Elucidation of anti-Vibrio factors associated with green alga Picochlorum sp. strain S1b. Journal of Applied Phycology 27, 257-265, 2015.

Lee, J., Cho, D.H., Ramanan, R., Kim, B.H., Oh, H.M., and Kim, H.S. Microalgae-associated bacteria play a key role in the flocculation of Chlorella vulgaris. Bioresource Technology 131, 195-201, 2013.

Ludwig, H.F., Oswald, W.J., Gotaas, H., and Lynch, V. Algae symbiosis in oxidation ponds: I. growth characteristics of" Euglena Gracilis" cultured in sewage. Sewage and Industrial Wastes, Wiley, United States of America. 1337-1355. 1951.

Mtethiwa, A. H., Munyenyembe, A., Jere, W., and Nyali, E. Efficiency of oxidation ponds in wastewater treatment. International Journal of Environmental Research 2, 149-152, 2008.

Muñoz, R., Alvarez, M.T., Muñoz, A., Terrazas, E., Guieysse, B., and Mattiasson, B. Sequential removal of heavy metals ions and organic pollutants using an algal-bacterial consortium. Chemosphere 63, 903-911, 2006.

Novogrodsky, A., Ravid, A., Rubin, A.L., and Stenzel, K.H. Hydroxyl radical scavengers inhibit lymphocyte mitogenesis. Proceedings of the National Academy of Sciences of the United States of America 79, 1171-1174, 1982.

Piontek, J., Händel, N., De Bodt, C., Harlay, J., Chou, L., and Engel, A. The utilization of polysaccharides by heterotrophic bacterioplankton in the Bay of Biscay (North Atlantic Ocean). Journal of Plankton Research 33, 1719-1735, 2011.

Rico-Mora, R., Voltolina, D., and Villaescusa-Celaya, J.A. Biological control of Vibrio alginolyticus in Skeletonema costatum (Bacillariophyceae) cultures. Aquacultural Engineering 19, 1-6, 1998.

Safonova, E., Kvitko, K., Iankevitch, M., Surgko, L., Afti, I., and Reisser, W. Biotreatment of industrial wastewater by selected algal‐bacterial consortia. Engineering in Life Sciences 4, 347-353, 2004.

Sarker, S.D., Nahar, L., and Kumarasamy, Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42, 321-324, 2007.

Seyedsayamdost, M.R., Carr, G., Kolter, R., and Clardy, J. Roseobacticides: small molecule modulators of an algal-bacterial symbiosis. Journal of the American Chemical Society 133, 18343-18349, 2011.

Seymour, J.R., Amin, S.A., Raina, J.B., and Stocker, R. Zooming in on the phycosphere: the ecological interface for phytoplankton-bacteria relationships. Nature Microbiology 2, 1-12, 2017.

Shibl, A.A., Isaac, A., Ochsenkuhn, M.A., Cárdenas, A., Fei, C., Behringer, G., Arnoux, M., Drou, N., Santos, M.P., and Gunsalus, K.C. Diatom modulation of select bacteria through use of two unique secondary metabolites. Proceedings of the National Academy of Sciences of the United States of America 117, 27445-27455, 2020.

Srivastava, A., Ahn, C.Y., Asthana, R.K., Lee, H.G., and Oh, H.M. Status, alert system, and prediction of cyanobacterial bloom in South Korea. BioMed Research International 2015, 584696, 2015.

Steinrücken, P., Prestegard, S.K., de Vree, J.H., Storesund, J.E., Pree, B., Mjøs, S.A., and Erga, S.R. Comparing EPA production and fatty acid profiles of three Phaeodactylum tricornutum strains under western Norwegian climate conditions. Algal Research 30, 11-22, 2018.

Talaiekhozani, A., and Rezania, S. Application of photosynthetic bacteria for removal of heavy metals, macro-pollutants and dye from wastewater: A review. Journal of Water Process Engineering 19, 312-321, 2017.

Teeling, H., Fuchs, B.M., Becher, D., Klockow, C., Gardebrecht, A., Bennke, C.M., Kassabgy, M., Huang, S., Mann, A.J., and Waldmann, J. Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom. Science 336, 608-611, 2012.

Van Den Hende, S., Claessens, L., De Muylder, E., Boon, N., and Vervaeren, H. Microalgal bacterial flocs originating from aquaculture wastewater treatment as diet ingredient for Litopenaeus vannamei (Boone). Aquaculture Research 47, 1075-1089, 2016.

Von Sperling, M. Complementary items in planning studies. Wastewater Characteristics, Treatment and Disposal. International Water Association Publications, United Kingdom. 277-282. 2007.

Wang, M., Yang, H., Ergas, S.J., and van der Steen, P. A novel shortcut nitrogen removal process using an algal-bacterial consortium in a photo-sequencing batch reactor (PSBR). Water Research 87, 38-48, 2015.

Watanabe, K., Takihana, N., Aoyagi, H., Hanada, S., Watanabe, Y., Ohmura, N., Saiki, H., and Tanaka, H. Symbiotic association in Chlorella culture. Federation of European Microbiological Societies Microbiology Ecology 51, 187-196, 2005.

Yu, Z.W., and Quinn, P.J. Dimethyl sulphoxide: a review of its applications in cell biology. Bioscience Reports 14, 259-281, 1994.