刘雨,张裕,李赟,朱葆华,潘克厚

• 1:海水养殖教育部重点实验室(中国海洋大学)
• 2:青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室

摘要(Abstract)：

为了解隐秘小环藻(Cyclotella cryptica)共生细菌对其生长的影响，本文运用稀释梯度涂布法分离纯化藻液中的细菌，检测了藻菌共培养液中的溶解有机碳(Dissolved organic carbon, DOC)、溶解无机碳(Dissolved inorganic carbon, DIC)及胞外聚合物(Extracellular polymeric substances, EPS)含量的变化，比较了分离菌株对微藻生物量和脂质的影响，初步探究了藻菌之间的相互作用模式。研究显示，从隐秘小环藻藻液共分离纯化8株可培养细菌，通过比对菌株的部分16S rDNA序列，完成了其分类学鉴定。其中，菌株C-1和C-7为隐秘小环藻的优势促生菌，分别将藻细胞密度提高了27.1%和23.9%。与菌株C-1共培养的隐秘小环藻，脂质含量高达43.53%,脂质产率为16.69 mg·L~(-1)·d~(-1),比无菌纯培养对照组的脂质产率提高了37.37%。碳交换研究结果显示，共培养7 d后，2个促生菌株构建的共培养体系中DOC含量显著低于无菌纯培养对照组；DIC含量显著高于无菌纯培养对照组。与无菌纯培养对照组相比，藻菌共培养导致培养上清液中EPS的多糖和蛋白含量显著降低，而细胞结合态EPS的多糖含量在前4 d高于对照组，之后显著降低，但细胞结合态EPS的蛋白含量始终高于对照组。本研究构建了隐秘小环藻与细菌的共生体系，探究了藻菌之间的互作效应，可为隐秘小环藻的规模化生产提供参考。

关键词(KeyWords)： 隐秘小环藻;溶解有机碳;溶解无机碳;胞外聚合物;生长;脂质含量;藻际细菌

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(41976118)资助~~

作者(Author): 刘雨,张裕,李赟,朱葆华,潘克厚

DOI: 10.16441/j.cnki.hdxb.20220468

参考文献(References)：

• [1] Sharifah E N,Eguchi M.The Phytoplankton Nannochloropsis oculata enhances the ability of Roseobacter clade bacteria to inhibit the growth of fish pathogen Vibrio anguillarum[J].PLoS One,2013,6(10):e26756.
• [2] Kuo R C,Lin S.Ectobiotic and endobiotic bacteria associated with Eutreptiella sp.isolated from Long Island Sound[J].Protist,2013,164(1):60-74.
• [3] Kazamia E,Czesnick H,Nguyen T,et al.Mutualistic interactions between vitamin B12-dependent algae and heterotrophic bacteria exhibit regulation[J].Environmental Microbiology,2012,14(6):1466-1476.
• [4] Teplitski M,Rajamani S.Signal and nutrient exchange in the interactions between soil algae and bacteria[J].Springer Berlin Heidelberg,2011,23:413-426.
• [5] Cho D H,Ramanan R,Heo J,et al.Enhancing microalgal biomass productivity by engineering a microalgal-bacterial community[J].Bioresource Technology,2015,175:578-585.
• [6] Grossart H P,Levold F,Allgaier M,et al.Marine diatom species harbor distinct bacterial communities[J].Environmental Microbiology,2005,7:860-873.
• [7] Nestor A G,Weber P K,Laura A S,et al.Elevated temperature increases carbon and nitrogen fluxes between phytoplankton and heterotrophic bacteria through physical attachment[J].Multidisciplinary Journal of Microbial Ecology,2017,11(3):641-650.
• [8] Buchan A,LeCleir G R,Gulvik C A,et al.Master recyclers:Features and functions of bacteria associated with phytoplankton blooms[J].Nature Reviews Microbiology,2014,12(10):686-698.
• [9] Kim B H,Ramanan R,Cho D H,et al.Role of Rhizobium,a plant growth promoting bacterium,in enhancing algal biomass through mutualistic interaction[J].Biomass and Bioenergy,2014,69:95-105.
• [10] Durham B P,Sharma S,Luo H W,et al.Cryptic carbon and sulfur cycling between surface ocean plankton[J].Environmental Sciences,2014,112(2):453-457.
• [11] Durham B P,Dearth S P,Sharma S,et al.Recognition cascade and metabolite transfer in a marine bacteria-phytoplankton model system[J].Environmental Microbiology,2017,19(9):3500-3513.
• [12] De-Bashan L E,Bashan Y,Moreno M,et al.Increased pigment and lipid content,lipid variety,and cell and population size of the microalgae Chlorella spp.when co-immobilized in alginate beads with the microalgae-growth-promoting bacterium Azospirillum brasilense[J].Canadian Journal of Microbiology,2002,48(6):514-521.
• [13] Wang R M,Tian Y,Xue S,et al.Construction and characteristics of artificial consortia of Scenedesmus obliquus-bacteria for S.obliquus growth and lipid production[J].Algal Research,2015,12:436-445.
• [14] Amin S A,Hmelo L R,Tol H V,et al.Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria[J].Nature,2015,522(7554):98-101.
• [15] Segev E,Wyche T P,Kim K H,et al.Dynamic metabolic exchange governs a marine algal-bacterial interaction[J].Elife Sciences,2016,5:e17473.
• [16] Viret O,Toti L,Chapela I H,et al.The role of the extracellular sheath in recognition and attachment of conidia of Discula umbrinella (Berk.& Br.) Morelet to the host surface[J].New Phytologist,1994,127(1):123-131.
• [17] Watanabe K,Imase M,Sasaki K,et al.Composition of the sheath produced by the green alga Chlorella sorokiniana[J].Letters in Applied Microbiology,2006,42(5):538-543.
• [18] Brenner K,You L,Arnold F H.Engineering microbial consortia:A new frontier in synthetic biology[J].Trends in Biotechnology,2008,26(9):483-489.
• [19] Kesaano M,Sims R C.Algal biofilm based technology for wastewater treatment[J].Algal Research,2014,5:231-240.
• [20] Subashchandrabose S R,Ramakrishnan B,Megharaj M,et al.Consortia of cyanobacteria/microalgae and bacteria:Biotechnological potential[J].Biotechnology Advances,2011,29:896-907.
• [21] d′Ippolito G,Sardo A,Paris D,et al.Potential of lipid metabolism in marine diatoms for biofuel production[J].Biotechnology for Biofuels,2015,8(1):28.
• [22] Pahl S L,Lewis D M,Chen F,et al.Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica (Bacillariophyceae):Effect of some environmental factors[J].Journal of Bioscience and Bioengineering,2010,109(3):235-239.
• [23] Slocombe S P,Zhang Q,Ross M,et al.Unlocking nature′s treasure-chest:Screening for oleaginous algae[J].European Journal of Phycology,2015,5(1):e09844.
• [24] 张裕，刘雨，朱葆华，等.隐秘小环藻无菌化处理研究[J].中国海洋大学学报(自然科学版),2022,52(11):34-42.Zang Y,Liu Y,Zhu B H,et al.Studies on axenic treatment of Cyclotella cryptica[J].Periodical of Ocean University of China,2022,52(11):34-42.
• [25] 程云飞，王路路，李赟，等.盐度对隐秘小环藻生长、沉降及藻壳物理性质的影响[J].中国海洋大学学报(自然科学版),2022,52(9):29-34.Chen Y F,Wang L L,Li Y,et al.Effect of peptides from Antarctic krill on lipid metabolism of Hyperuricemia mice and its underlining mechanism[J].Periodical of Ocean University of China,2022,52(9):29-34.
• [26] Zebua A,Nursyirwani N,Feliatra F.Molecular identification of proteolitic bacteria from mangrove sediment in Dumai marine station[J].Asian Journal of Aquatic Sciences,2020(2):179-188.
• [27] Bligh E G,Dyer W J.A rapid method of total lipid extraction and purification[J].Canadian Journal of Biochemistry and Physiology,1959,37:911-917.
• [28] Niu Y F,Zhang M H,Li D W,et al.Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum[J].Marine Drugs,2013,11:4558-4569.
• [29] Kim B H,Kang Z,Ramanan R,et al.Nutrient removal and biofuel production in high rate algal pond (HRAP) using real municipal wastewater[J].Journal of Microbiology and Biotechnology,2014,24(8):1123-1132.
• [30] Watanabe K,Takihana N,Aoyagi H,et al.Symbiotic association in Chlorella culture[J].Microbiology Ecology,2005,51(2):187-196.
• [31] Zhou Z Z,Li Q,Song K,et al.Exploration of applying growth-promotion bacteria of Chlorella sorokiniana to open cultivation systems[J].Bioprocess and Biosystems Engineering,2021,44(7):1567-1576.
• [32] Stephens E,Ross I L,Mussgnug J H,et al.Future prospects of microalgal biofuel production systems[J].Trends in Plant Science,2010,15(10):554-564.
• [33] Cole J J.Interactions between bacteria and algae in aquatic ecosystems[J].Annual Review of Ecology Systematics,1982,13:291-314.
• [34] Chorazyczewski A M,Huang I S,Abdulla H,et al.The influence of bacteria on the growth,lipid production,and extracellular metabolite accumulation by Phaeodactylum tricornutum (Bacillariophyceae) [J].Journal of Phycology,2021,57(3):931-940.
• [35] Xue L,Shang H,Ma P,et al.Analysis of growth and lipid production characteristics of Chlorella vulgaris in artificially constructed consortia with symbiotic bacteria[J].Journal of Basic Microbiollogy,2018,58(4):358-367.
• [36] Lili X,Xianglong C,Quanxi W.Enhanced Lipid Production in Chlamydomonas reinhardtii by Co-culturing with Azotobacter chroococcum[J].Frontiers in Plant Science,2018,9:741.
• [37] Leyva L A,Bashan Y,De-Bashan L E.Activity of acetyl-CoA carboxylase is not directly linked to accumulation of lipids when Chlorella vulgarisis co-immobilised with Azospirillum brasilensein alginate under autotrophic and heterotrophic conditions[J].Annals of Microbiology,2015,65(1):339-349.
• [38] Croft M T,Lawrence A D,Raux-Deery E,et al.Algae acquire vitamin B12 through a symbiotic relationship with bacteria[J].Nature,2005,438:90-93.
• [39] Amin S A,Green D H,Gardes A,et al.Siderophore-mediated iron uptake in two clades of Marinobacter spp.associated with phytoplankton:The role of light[J].Biometals,2012,25:181-192.
• [40] Hende S V D,Vervaeren H,Desmet S,et al.Bioflocculation of microalgae and bacteria combined with flue gas to improve sewage treatment[J].New Biotechnology,2011,29(1):23-31.
• [41] Munoz R,Kollner C,Guieysse B.Biofilm photobioreactors for the treatment of industrial wastewaters[J].Journal of Hazardous Materials,2009,161:29-34.
• [42] 张圣洁，蔡中华，朱伟胜，等.藻际环境中胞外聚合物的研究进展[J].微生物学报，2020,60(8):1521-1533.Zhang S J,Cai Z H,Zhu W S,et al.Advances in extracellular polymeric substances in phycosphere environment[J].Acta Microbiologica Sinica,2020,60(8):1521-1533.
• [43] Bruckner C G,Bahulikar R,Rahalkar M,et al.Bacteria associated with benthic diatoms from Lake Constance:Phylogeny and influences on diatom growth and secretion of extracellular polymeric substances[J].Applied and Environmental Microbiology,2008,74(24):7740-7749.
• [44] Gardes A,Iversen M H,Grossart H P,et al.Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii[J].The ISME Journal,2011,5:436-445.
• [45] Xiong Y H,Liu Y.Importance of extracellular proteins in maintaining structural integrity of aerobic granules[J].Colloids and Surfaces B:Biointerfaces,2013,112:435-440.

文章评论(Comment)：