【作者】 王进；

【导师】 王鹏；

【作者基本信息】 中国海洋大学 ， 食品工程（专业学位）， 2013， 硕士

【摘要】 从腐烂的浒苔中对微生物菌种进行分离筛选和鉴定，开发出了一组性状稳定、高效降解浒苔的微生物菌剂。利用该菌剂发酵浒苔，并将发酵完全的浒苔和其它辅料一起复配成浒苔生物有机肥。研究了该肥料的成分及对小麦、青菜和樱桃萝卜生长的影响。在实验的初筛阶段，共分离出23株细菌和3株真菌，经过进一步复筛验证，获得2株细菌和1株真菌，并分别命名为M-1、M-2、N-1。对这3株菌的16S RNA进行PCR扩增，经过序列测定和在NCBI进行Blast比对。推断M-1、M-2、N-1分别为：蜡状芽孢杆菌(Bacillus cereus strain)、枯草芽孢杆菌（Bacillus subtilisstrain）和米根霉（Rhizopus oryzae）。将筛选出的菌株和实验室保藏的米曲霉复配成EM-W菌剂，并将该菌剂应用于浒苔的固态深层发酵试验。为了探究浒苔的发酵条件，进行了料水比和C/N的单因素试验。结果表明，浒苔发酵的最佳料水比为1:2、C/N为30。该条件下，浒苔腐熟最高温度64℃左右，45℃以上温度维持4-5天。用EM-W菌剂发酵浒苔具有升温迅速、温度高、发酵周期短、组织高度降解、无明显臭味的特点。将发酵完全的浒苔和腐植酸、黄腐酸钾、硅藻土等有机辅料一起复合成浒苔生物有机肥。经过成分测定，该肥料的养分含量为：有机质42%，总养分（N+P2O5+K2O）6%，水分15%，pH值6.8。各项指标均符合国家有机肥相关标准（NY525-2002）。在实验室试验中，将浒苔生物有机肥和对照有机肥应用于小麦的促生长试验，经过对小麦的茎长、叶长、干重、叶绿素和还原糖等指标进行测定。得出，浒苔生物有机肥相对于对照有机肥能够促进小麦的麦叶（增加6.8%）和麦茎（增加4.3%）的生长、干重（增加4.1%）的增加和叶绿素（增加2.8%）的合成。另外，浒苔生物有机肥还可以促进小麦还原糖（增加2.9%）含量的提高。综合表明，浒苔生物有机肥较对照有机肥能促进小麦的生长并提高其营养物质含量。在大田试验中，将浒苔生物有机肥按照2t/hm2、5t/hm2、8t/hm2的施肥量施用在青菜田，经过35天的生长，对采收的青菜进行各项指标的测定。结果表明，浒苔生物有机肥的最佳施肥量为5t/hm2。该施肥量下的试验组比对照有机肥组干重增加4.0%，鲜重增加2.8%，还原糖含量增加5.6%，β-胡萝卜素含量增加2.5%，根系活力增加6.8%，叶绿素含量增加0.1%。结果证明，在青菜种植过程中，施用一定量的浒苔生物肥，能显著提高青菜的生长活力（叶绿素含量和根系活力）、增加青菜的产量（重量）和品质（还原糖、β-胡萝卜素）。再将浒苔生物有机肥应用在樱桃萝卜田，经过33天的生长，对采收的樱桃萝卜进行各项指标的测定。结果表明，浒苔生物有机肥的最佳施肥量为4t/hm2。该施肥量下的试验组比不施肥组在鲜重上增加3.0%，还原糖含量增加0.6%，叶绿素含量增加7.7%。再次证明，在樱桃萝卜种植过程中，施用一定量的浒苔生物肥，能显著提高樱桃萝卜的生长活力（叶绿素含量）、增加樱桃萝卜的产量（重量）和品质（还原糖）。浒苔生物有机肥的开发与利用不仅解决了每年大量打捞的浒苔无法得到有效处理的难题，还为现代农业提供了一种新型肥料，扩大了生物有机肥的原料范围。另一方面，浒苔生物有机肥的使用还可以减少无机化肥的施用量，改善土壤微生物环境，提高土壤有机成分含量，达到充分利用废弃资源，减少环境污染的目的。更多还原

【Abstract】 Microorganisms were isolated from the decayed Enteromorpha prolifera, andone kind of microbial inoculants with stability and high efficiency to degrade E.prolifera was obtained. The fermentation of E. prolifera with the microorganism inoculantswas studied. Then the biological fertilizer was produced by mixing fermentationproductions and organic accessories. The effects of biological fertilizer on the growthof Wheat, Brassica campestris ssp.chinensis Makino, and Cherry Radish werestudied.In the preliminary screening process,23strains of bacteria and3strains of funguswere isolated. After further tested,2strains of bacteria and1strain of fungus wereobtained, which were named as M-1, M-2, and N-1, respectively. The molecularidentification of microorganisms were performed by extracting, amplifying. Theamplified PCR products were sequenced and compared with BLAST database at theNational Center for Biotechnology Information (NCBI, U.S.A.). Results indicated thatM-1, M-2, and N-1were Bacillus cereus, Bacillus subtili, and Rhizopus oryzae,respectively.The complex microbial inoculants named as EM-W including M-1, M-2, N-1and the strain of Aspergillus oryzae preserved by our laboratory was applied on theexperiment of solid state fermentation of E. prolifera. The effects of material to waterratio and C/N on the E. prolifer fermentation were studied. Results showed the optimumC/N and material to water ration were30:1and1:2, respectively. The fermentation temperatureabove45oC maintained4to5days, and the highest temperature reached64°C. The fermentationprocess has characteristics of rapid warming, high temperature, and short period. The tissue of E.prolifera could be degraded completely with no odor. E. prolifera bio-organic fertilizer wasproduced by mixing the fermentation production and organic accessory including humic acid, potassium fulvic acid, and diatomite. After the determination of itscomposition, the percentage of the organic matter, total nutrient, and water were42%,6%,15%, and6.8, respectively. And the pH was6.8. All kinds of index have reachedthe national standards of organic fertilizer.In laboratory test, the E. prolifera bio-organic fertilizer and ordinary organicfertilizer were applied on the growth-promoting test of Wheat. Parameters includingstem length, leaf length, dry weight, chlorophyll and reducing sugar were measuredrespectively. Results showed that the E. prolifera bio-organic fertilizer was better thanthe ordinary organic fertilizer in promoting the growth of leaf and stem, increase ofdry weight and synthesis of chlorophyll. At the same times, it can also improve thecontent of reducing sugar. All of those indicated that the application of E. proliferabio-organic fertilizer can promote the growth of Wheat and improve the content ofnutrient compared to the ordinary organic fertilizer.In field trials, the E. prolifera bio-organic fertilizer was applied on B. campestrisssp.chinensis Makino with different amounts of2t/hm2,5t/hm2, and8t/hm2.Different index of B. campestris ssp.chinensis Makino’s were measured after the35days culture. Results showed the optimum fertilizer amount was5t/hm2. Compared tothe ordinary organic fertilizer, the E. prolifera bio-organic fertilizer could increase dryweight by4.0%, fresh weight by2.8%, reducing sugar by5.6%, beta carotene by2.5%, root activity by6.8%, and chlorophyll by0.1%. A certain amount of E.prolifera bio-organic fertilizer could significantly improve the viability (chlorophyllcontent and root activity), yield (weight), and quality (reducing sugar and betacarotene) of B. campestris ssp.chinensis Makino.The E. prolifera bio-organic fertilizer was also applied on Cherry Radish. After33days cultures, different index about Cherry Radish were measured. The resultsshowed the optimum fertilizer amount was4t/hm2. Compared to control group, the E.prolifera bio-organic fertilizer could increase fresh weight by3.0%, reducing sugar by0.6%, and chlorophyll by7.7%. Those indicated that a certain amount of E. proliferabio-organic fertilizer could significantly improve the viability (chlorophyll content),the yield (weight), and the quality (reducing sugar) of Cherry Radish. In one hand, the research and utilization of E. prolifera bio-organic fertilizercould not only solve the problem of efficient processing of E.prolifera, but alsoprovided a new kind of fertilizer for the modern agriculture. On other hand, fullutilization of E.prolifera could reduce the environment pollution. Simultaneously,applications of E. prolifera bio-organic fertilizer could bring about the reducing ofchemical fertilizer usage, improvement of soil microbial environment, and increase ofsoil organic component content.更多还原