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1 混合菌群发酵马尾藻作为海藻肥料的研究王明鹏 1, 陈蕾 2, 刘正一 2, 王学江 4, 秦松 2 1, 3, 闫培生 (1., ; 2., ; 3., ; 4., ) 摘要 : 从马尾藻原位分离发酵菌群, 采用分子生物学手段分析鉴定菌种组成, 获得发酵菌株 4 株分别为巨大芽孢杆菌 (Bacillus megaterium), 非脱羧勒克氏菌 (Leclerciaadecarboxylata), 鞘氨醇单胞菌 (Sphingomonasleidyi) 以及赖氨酸芽孢杆菌 (Lysinibacillusmacroides); 控制 4 菌株接种比例进行马尾藻发酵, 监测发酵过程中菌种生物量褐藻酸含量及褐藻寡糖含量变化规律, 结果表明, 马尾藻经发酵 36 h 生成聚合度 2-5 的褐藻寡糖, 并伴随菌种二次生长及 ph 下降等易于观察检测的参数变化 ; 含褐藻寡糖的发酵原液稀释 600 倍后, 能够明显促进小麦种子萌发及根生长, 与清水对照相比萌发率提高 26%, 根系长度增长 37% 本研究为微生物发酵法生产海藻肥料提供了可行案例, 证实该方法简单有效, 可操作性强, 有广阔的应用前景, 为将来实现海藻发酵肥工业化生产提供理论支持和数据支撑关键词 : 发酵 ; 马尾藻 ; 褐藻寡糖 ; 促生长中图分类号 : Q815 文献标识码 : A 文章编号 : (2017) DOI: /hykx ,,,, [1],, New AG International, 2012,, 20 ~40, 80, ( ) 2%, [2], , [3],, [4],, 20%~40% 1 4,, [5-6],,,,,,,, : ; : : ( ); (2013BAB01B0); (KFJ-EW-STS-060); ( ); (SKLEC-KF201412); ( ) [Foundation: Science and Technology Development Project of Weihai ( ), National Key Technology R&D Program of China (2013BAB01B0), Science and Technology Service Network Initiative of Chinese Academy of Sciences (KFJ-EW-STS-060), Young Scientists Fund of National Natural Science Foundation of China ( ), Open Foundation of State Key Laboratory of Estuarine and Coastal Research (SKLEC-KF201412), PublicScience and Technology Research Funds Projects of Ocean ( )] : (1988-),,,,, : , wmpdsg@ 126.com;,, yps6@163.com;,, sqin@yic.ac.cn Marine Sciences / Vol. 41, No. 2 /

2 ,,, [7],,,,, [8-10],,,,,,,, [11-13],,,,,, 1 材料与方法 1.1 样品采集与处理,, 4, 60,, (W/V), min, (SWE ), 2%,, 1.2 菌株的筛选与鉴定 3~5 cm,, 2216E, 30, 2~3,,, h,,, 1 min,, 1 min,,, 95%,, 20 s,, 1 min,, 1.2.2, 2216E,, 1 ml DNA, 27F(5 -AGAGTTTGATCCTGGCTCAG- 3 ) 1492R(5 -TACGGTTACCTTGTTACGACTT-3 ) 16SrDNA, PCR 16S rdna MEGA 菌株发酵 E 12~16 h, 600 nm, OD , 4, 1% (SWE ), 30, 200 r/m 4 h, OD 600, ph,, 5 5 ml, 0.3 ml 2.7 ml, 30, h, 1.4 褐藻酸含量检测 [14] 2 ( ), [Cu(Alg) 2 ], ph 8.5 ( ), 447 nm, 1.5 褐藻寡糖的 TLC 检测 (TLC) [15] Silica Gel 60 F254, 2 μl, = 4 5 1(V V V),,, -,, min, 1.6 褐藻寡糖的质谱鉴定 36 h 2 ml 4, r/min 15 min, [16] 118 / 2017 / 41 / 2

1.7 种子萌发及生长实验,, 2000 ( ),, 36 h ( ), 72 h ( ), 200 400 600 800 4, 12 h, 3, 60, 3 180,, 25,, 48 h 12 h,, 25 48 h,,,, 3, 3 9, ( GTOP-310B), 25 12 h, 16 12 h 72 h,, 5, 3 1.8 数据处理 SPSS 19.0 2 结果与分析 2.

3 1.7 种子萌发及生长实验,, 2000 ( ),, 36 h ( ), 72 h ( ), , 12 h, 3, 60, 3 180,, 25,, 48 h 12 h,, h,,,, 3, 3 9, ( GTOP-310B), h, h 72 h,, 5, 数据处理 SPSS 结果与分析种菌株的鉴定结果, 4 ( 1), A1 M1, C1 S1 4 16SrDNA,, M1 (Bacillus megaterium) 99.79%, A1 (Lysinibacillus macroides) 99.17%, S1 (Sphingomonas leidyi) 99.06%, C1 (Leclercia adecarboxylata) 99.37% 4, 4 ( 2) 1 Fig. 1 Phenotypic characterization of isolated cultures 2.2 混合菌株发酵马尾藻特性 4, 2216E OD ( 3A) 2216E 4.0( 3B),, 2216E, 36 h, ph ; 2216E,,, 36 h, 40% ( 3C), ( ),, 36 h, 30, ( 3D), 7 h 40%, ; 15 min 2.3 马尾藻发酵液中褐藻寡糖的检测 Marine Sciences / Vol. 41, No. 2 /

4 Fig. 2 2 Phylogenetic analysis of fermentative strain Fig. 3 3 Fermentation characteristics of mixed strains in different media A. ph ; B. 2216E ph ; C. ; D. SFE ; SWE A. The growth and ph curves of mixed strain in seaweed water extract media; B. The growth and ph curves of mixed strain in 2216E media; C. Change of alginate content in seaweed water extract media; D. Detection of alginate lyase activity of fermentation broth SFE stands for seaweed fermentation extract; SWE stands for seaweed water extract 120 / 2017 / 41 / 2

链片段, 同时也是发酵液中含褐藻酸裂解酶的有力延长呈递减趋势, 发酵 40 h 后便检测不到褐藻寡糖证明因此, 我们通过薄层层析(TLC)对马尾藻发酵 (图 4B) 褐藻寡糖出现的时间段与褐藻酸含量突然液进行褐藻寡糖的检测, 从而进一步验证发酵液中下降微生物二次生长的时间段一致由此我们推是否存在褐藻酸裂解酶 TLC 结果显示, 36 h 发酵液测, 在发酵 36 h

的褐藻寡糖, 但褐藻寡糖含量及种类随发酵时间的中富含聚合度 2~5 的褐藻寡糖(图 4C) Fig. 4 图 4 发酵液中褐藻寡糖的 TLC 和 ESI-MS 检测 Detection of alginate oligosaccharides in fermentation broth by TLC and ESI-MS A. 不同时间发酵液中褐藻寡糖的薄层层析图谱; B.

5 链片段, 同时也是发酵液中含褐藻酸裂解酶的有力延长呈递减趋势, 发酵 40 h 后便检测不到褐藻寡糖证明因此, 我们通过薄层层析(TLC)对马尾藻发酵 (图 4B) 褐藻寡糖出现的时间段与褐藻酸含量突然液进行褐藻寡糖的检测, 从而进一步验证发酵液中下降微生物二次生长的时间段一致由此我们推是否存在褐藻酸裂解酶 TLC 结果显示, 36 h 发酵液测, 在发酵 36 h 褐藻酸降解成为褐藻寡糖, 而褐藻中含有褐藻寡糖, 聚合度在 2~8 之间(图 4A); 随后根寡糖作为碳源被菌株利用并出现二次生长现象该据此次点样结果, 调整取样时间(自 24 h 至 48 h, 每 4 h 结果显示发酵 32~36 h 为褐藻寡糖的最佳收获时间取一次样), 结果表明在 28~40 h 这一时段均能检测随后, 36 h 的发酵液经质谱鉴定, 结果显示此发酵液的褐藻寡糖, 但褐藻寡糖含量及种类随发酵时间的中富含聚合度 2~5 的褐藻寡糖(图 4C) Fig. 4 图 4 发酵液中褐藻寡糖的 TLC 和 ESI-MS 检测 Detection of alginate oligosaccharides in fermentation broth by TLC and ESI-MS A. 不同时间发酵液中褐藻寡糖的薄层层析图谱; B. 取样时间缩短后发酵液中褐藻寡糖的薄层层析图谱; C. 36 h 发酵液中褐藻寡糖质谱结果 Control 为寡糖标准品; DP2 为寡聚二糖; DP8 为寡聚八糖 A. The TLC profile of alginate oligosaccharides in fermentation broth with different fermentation time; B. The TLC profile of alginate oligosaccharides in fermentation broth with sampling intervals narrowed; C. The MS result of alginate oligosaccharides in 36 h fermentation broth. Control stands for standard alginate oligosaccharides; DP2 stands for degree of polymerization of 2; DP8 stands for degree of polymerization of 对照及实验组, 验证褐藻寡糖的促生长功能结果如马尾藻发酵液的促生长效果上述 TLC 及 ESI-MS 结果显示, 马尾藻 36 h 发图 5 和表 1 2 所示, 与清水对照相比, 各浓度马尾酵液中含有褐藻寡糖根据文献报道褐藻寡糖对植藻发酵液的促生长效果明显, 小麦萌发率显著提高, 物有促生长及诱导抗性等有益功效 Fig. 5 [17-23] 随后, 设计根系长度明显增加, 并且稀释倍数越大, 其促生长图 5 不同处理最佳浓度下的小麦根长 Root length of wheat under optimum concentration of different treatments Marine Sciences / Vol. 41, No. 2 /

6 表 1 不同发酵液处理下的小麦种子萌发率 Tab. 1 Seed germination rate of wheat under the conditions of different solutions 1/200 1/400 1/600 1/ %±5.09 ab 81.67%±5b cd 74.44%±8.39 ab 73.89%±9.18 ab 71.11%±8.22 a 36 h 84.44%±2.54 cde 88.89%±2.54 cde 92.78%±4.19 e 93.33%±1.67 e 72 h 85.56%±2.54 cde 87.22%±2.54 cde 90%±3.33d e 90.56%±4.19 de %±3.33 bc 83.33%±3.33 cd 87.22%±3.47 cde 88.89%±2.54 cde : 5%. 表 2 不同发酵液处理下小麦根长 Tab. 2 Root length of wheat under the conditions of different solutions 1/200 1/400 1/600 1/ ±0.47 abc 7.56±0.57 def 7.02±0.49 bcd 6.78±0.53 abc 6.48±0.49 ab 36 h 7.7±0.34 efg 8.18±0.30 fg 9±0.4 h 8.32±0.47 g 72 h 7.16±0.48 cde 7.72±0.37 efg 8.28±0.33 g 7.84±0.4 fg ±0.46 a 6.28±0.49 a 6.74±0.4 abc 7.08±0.5 bcd,,,, 讨论与结论,, Uchida,, [24] Shobharani,, [25],,,,,, 72 h ( ),,,,,,, [26, 27],,,, (4 ), 32~36 h, 2-5, ph,, ph,, 122 / 2017 / 41 / 2

7 ,,,,, 600~800, Anisimov [28],,,,, 4, 4, 36 h 2-5, ph, 600,, 26%, 37%, : [1],,. [J]., 1995, 3(4): 4-6. Li Shuqin, Wang Xiaoju, Fan Xiao. Study on the seaweed liquid manure [J]. Marine Sciences, 1995, 3(4): 4-6. [2],,,. [J]., 2014, 3: Yang Fang, Dai Jinquan, Liang Chunchan, et al. Current developing situation of agricultural seaweed and seaweed fertilizer [J].Fujian Agricultural Technology, 2014, 3: [3] FAO. Yearbook of fishery and aquaculture statistics [C]. Rome: Food and Agriculture Organization of the United Nation, [4] Craigie J S. Seaweed extract stimuli in plant science and agriculture[j]. J Appl Phycol, 2011, 23: [5]. [M]. :, Ji Minghou. Seaweed chemistry[m].beijing: Science Press, [6] Michel G, Tonon T, Scornet D, et al.the cell wall polysaccharide metabolism of the brown alga Ectocarpussiliculosus.Insights into the evolution of extracellular matrix polysaccharides in Eukaryotes[J]. New Phytol, 2010, 188: [7],,,. [J]., 2015, 5(4): Wang Mingpeng, Chen Lei, Liu Zhengyi, et al. Progress and prospect of seaweed fertilizer[j]. Current Biotechnology, 2015, 5(4): [8] Adams J M, Gallagher J A, Donnison I S. Fermentation study on Saccharinalatissima for bioethanol production considering variable pre-treatments[j].j Appl Phycol, 2008, 21: [9] Jang J S, Cho Y, Jeong G T, Kim S K. Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharinajaponica[J].Bioprocess Biosyst Eng, 2012, 35: [10] Lee S M, Lee J H. The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminariajaponicautilization[J]. Bioresour Technol, 2011, 102: [11],. [P] , CN A. Zhang Shuqing, Liu Peijing. A method of preparation of seaweed organic liquid fertilizer with composite microorganism[p] , CN A. [12],,,. [P] , CN A. Yuan Liang, Zhao Bingqiang, Li Yanting, et al. A method of producing seaweed fermentation fertilizer and its application[p] , CN A. [13],,,. [P] , CN Shan Junwei, Zhang Junjie, Zhao Zhiqiang, et al. A method of microbial fermentation treatment of Enteromorphaproliferafor seaweed fertilizer production[p] , CN [14],,,. [J]., 2009, 35(5): Wang Zewen, Leng Kailiang, Xing Lihong, et al. The research on the determination of sodium alginate with spectrophotometer[j]. Food and Fermentation Industries, 2009, 35(5): [15],,,. [J]., 2012, 18(8): Nie Ying, Zhang Wenjing, Shi Bo, et al. Establishment of detection methods for alginate oligosaccharides[j]. Food and Nutrition in China, 2012, 18(8): [16] Liu H, Zhang Y H, Yin H, et al. Alginate oligosaccharides enhanced Triticumaestivum L. tolerance to droughtstress[j]. Plant Physiol Biochem, 2013, 62: [17] Hien N Q, Nagasawa N, Tham L X, et al. Growthpromotion of plants with depolymerizedalginates by irradiation[j]. Radiat Phys Chem, 2000, 59: [18] Iwasaki K Matsubara Y. Purification of alginate oligosaccharides with rootgrowth-promoting activity toward lettuce[j]. Biosci Biotechnol Biochem, 2000, 64: [19] Natsume M, Kamo Y, Hirayama M, et al. Isolation and Marine Sciences / Vol. 41, No. 2 /

8 characterizationof alginate-derived oligosaccharides with root growth-promoting activities[j]. Carbohydr Res, 1994, 258: [20] Hu X K, Jiang X L, Hwang H M, et al. Promotive effects ofalginate-derived oligosaccharide on maize seed germination[j]. J Appl Phycol, 2004, 16: [21] Tang J C, Zhou Q X, Chu H R, et al. Characterization of alginase andelicitor-active oligosaccharides from GracilibacillusA7 in alleviating salt stressfor Brassica campestrisl[j]. J Agric Food Chem, 2011, 59: [22] Ma L J, Li X M, Bu N, et al. An alginate-derived oligosaccharide enhancedwheat tolerance to cadmium stress[j]. Plant Growth Regul, 2010, 62: [23] Zhang Y H, Zhang G, Liu L Y, et al. The role ofcalcium in regulating alginate-derived oligosaccharides in nitrogen metabolism of Brassica campestris L. var. utilis Tsenet Lee[J]. Plant Growth Regul, 2011, 64: [24] Uchida M, Murata M. Isolation of a lactic acid bacterium and yeast consortium from a fermented material of Ulva spp. (Chlorophyta)[J]. J Appl Microbiol, 2004, 97: [25] Shobharani P, Halami P M, Sachindra N M. Potential of marine lactic acid bacteria to ferment Sargassumsp for enhanced anticoagulant and antioxidant properties[j]. J Appl Microbiol, 2012, 114: [26] Zhang Y, Liu H, Yin H, et al. Nitric oxide mediates alginate oligosaccharides-induced root development in wheat (TriticumaestivumL.)[J]. Plant Physiol Biochem, 2013, 71: [27] Zhang Y, Yin H, Zhao X, et al. The promoting effects of alginate oligosaccharides on root development in Oryza sativa L. mediated by auxinsignaling[j]. Carbohydr Polym, 2014, 113: [28] Anisimov M M, Chaikina E L, Klykov A G, et al. Effect of seaweeds extracts on the growth of seedling roots of buckwheat (Fagopyrumesculentum Moench) is depended on the season of algae collection[j]. Agriculture Science Developments, 2013, 2(8): Studies on fermentation of Sargassum by mixed microorganism utilized as seaweed fertilizer WANG Ming-peng 1, CHEN Lei 2, LIU Zheng-yi 2, WANG Xue-jiang 4, QIN Song 2, YAN Pei-sheng 1, 3 (1. School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin , China; 2. Yantai Institute of Costal Zone Research Chinese Academy of Sciences, Yantai , China; 3. School of Marine Science and Technology, Harbin Institute of Technology, Weihai , China; 4. Worldfull Agricultural Science and Technology Co., Ltd, Yantai , China) Received: Apr. 28, 2016 Key words: fermentation; Sargassum; alginate oligosaccharide; growth promotion Abstract: Fermentative microorganisms present on the surface of seaweed were screened and then identified by 16S rdna sequence analysis. We obtained the following four fermentative strains: Bacillus megaterium, Leclerciaadecarboxylata, Lysinibacillusmacroides, and Sphingomonasleidyi. Using controlled inoculation proportion of these four strains, we obtained a stable fermentation product. We studiedthe changes and mechanisms of seaweed fermentation processto provide theoretical and data support for industrialization production of seaweed fermentation fertilizer. At 36 h in the fermentation process, alginate oligosaccharides with polymerization degrees of 2 5 were generated and accompanied by secondary growth of fermentative strains and a decreased ph, which were the parameters that could be easily observed and monitored. Fermentation broth diluted 600 times could significantly promote the germination and root growth of wheat seeds. Germination rate was 26% higher and root growth was 37% longer than those in the control group, respectively. These results provided a feasible strategy for the production of seaweed fertilizer by microbial fermentation. This simple and effective fermentation method has strong operability and broad application prospects. ( 本文编辑 : 康亦兼 ) 124 / 2017 / 41 / 2

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%