Optimization of ozone treatment of fresh-cut green leaf lettuce

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烟剂对大棚藜麦菜光合作用和叶绿素含量的影响

第3期（总第411期）2024年3月农业技术与装备AGRICULTURAL TECHNOLOGY&EQUIPMENT No.3烟剂对大棚藜麦菜光合作用和叶绿素含量的影响魏志敏1，王京新1，赵宇1，崔纪菡1，赵文庆1，裴美燕2，李顺国1（1.河北省农林科学院谷子研究所/河北省杂粮研究实验室/国家谷子改良中心，河北石家庄050035；2．邯郸市邯山区农业农村局，河北邯郸050002）摘要在藜麦菜的温室大棚种植中，用12%百菌清烟剂与12%速克灵烟剂防治病害较为常用。

为了研究这两种烟剂对藜麦菜的叶绿素含量和光合作用的影响，施用烟剂后第3天和第5天分别进行藜麦菜叶片叶绿素含量和光合速率的检测。

结果显示，施用烟剂可以提高藜麦叶绿素的含量，提高光合速率，进而提高生产率。

关键词藜麦菜；烟剂；叶绿素；光合速率中图分类号S649文献标志码A doi:10.3969/j.issn.1673-887X.2024.03.060Effects of Fumicants on Photosynthesis and Chlorophyll Content ofChenopodium quinoa Leafy Vegetable in GreenhouseWei Zhimin1,Wang Jingxin1,ZhaoYu1,Cui Jihan1,Zhao Wenqing1,Pei Meiyan2,Li Shunguo1(1.Institute of Millet,Hebei Academy of Agriculture and Forestry Sciences/Hebei Research Laboratoryof Cereals/National Center for Millet Improvement,Shijiazhuang050035,Hebei,China;2.Handan Hanshan Agriculture and Rural Bureau,Handan050002,Hebei,China)Abstract：In the greenhouse planting of Chenopodium quinoa leafy vegetable,12%chlorothalonil fumicants and12%supraline fu‐micants are commonly used for disease control.In order to study the effects of these two fumicants on chlorophyll content and photo‐synthesis of Chenopodium quinoa leafy vegetable,chlorophyll content and photosynthetic rate of Chenopodium quinoa leafy vegeta‐ble were detected on the3rd and5th day after the application of fumicants.The results showed that fumicants application could in‐crease chlorophyll content,photosynthetic rate and productivity of Chenopodium quinoa.Key words：Chenopodium quinoa leafy vegetable;fumicants;chlorophyll;photosynthetic rate藜麦原产于南美洲安第斯山脉，有5000多年的种植历史，籽粒和幼苗均可食用，是印加土著居民的主要食物之一，被印加人称为“粮食之母”“谷物之母”“安第斯山的真金”[1]。

CuOBi_(2)O_(3)光催化耦合过一硫酸盐氧化降解盐酸四环素

'£朽知库环境工程学报第15卷第3期2021年3月Vol. 15, No.3 Mar. 2021Eco-Environmental Knowledge Web C hinese Journal of Environmental Engineering@(010) 62941074文章栏目：水污染防治DOI 10.12030/j.cjee.202008112 中图分类号X701.3 文献标识码A丁丽丹，周家斌，刘文博，等.Cu0/Bi203光催化耦合过一硫酸盐氧化降解盐酸四环素[J].环境工程学报，2021. 15(3): 898- 910.DING Lidan, ZHOU Jiabin, LIU Wenbo, et al. Oxidative degradation of tetracycline hydrochloride by Cu0/Bi203 photocatalysis coupling with peroxymonosulfate[J]. Chinese Journal of Environmental Engineering, 2021, 15(3): 898-910.C u0/B i203光催化耦合过一硫酸盐氧化降解盐酸四环素丁丽丹、周家斌刘文博\陈新2,冯芹芹2,杨玉玲\张天磊11. 西南石油大学化学化工学院，成都6105002. 武汉理工大学资源与环境工程学院，武汉430070第一作者：丁丽丹（1995—)，女，硕士研究生。

研究方向：水处理高级氧化技术。

E-mail: ***************** *通信作者：周家斌（1972—），男，博士，教授。

研究方向：环境功能材料与污染控制：E-mail: ****************摘要利用共沉淀-浸渍法合成了基于可见光响应的Cu0/Bi203复合催化剂，并对其光催化活化过一硫酸盐 (PM S)去抗生素盐酸四环素（TC-HC1)的性能进行了探究。

低温胁迫对类囊体膜脂代谢的影响

植物学通报 2004, 21 (4): 506￣511Chinese Bulletin of Botany低温胁迫对类囊体膜脂代谢的影响①代玉华刘训言孟庆伟② 赵世杰（山东农业大学生命科学学院泰安 271018）摘要类囊体膜主要由膜脂、膜蛋白及一些光合色素等成分组成，它是植物进行光合作用的场所。

低温能通过影响类囊体膜的结构而影响植物的光合作用。

简述了类囊体膜的组成和功能，以及低温胁迫下类囊体膜脂及其脂肪酸组成的变化。

简要介绍了膜脂与光抑制的关系，以及利用分子生物学手段研究三烯脂肪酸与植物抗冷性关系的相关进展。

关键词低温胁迫，类囊体膜，脂肪酸组成Effect of Low Temperature on Lipid Metabolism ofThylakoid MembraneDAI Yu-Hua LIU Xun-Yan MENG Qing-Wei② ZHAO Shi-Jie(College of Life Science, Shandong Agricultural University, Tai’an271018)Abstract Thylakoid membrane, which is the place for photosynthetic reaction, is mainly composed of membrane lipids, membrane proteins and photosynthetic pigments. Low temperature can affect photosynthesis by affecting the construction of thylakoid membrane. In this paper, the function and composition of thylakoid membrane, and its change under low temperature stress are depicted. In addition, we also concerned the relationship between membrane lipids and photoinhibition, and recent studies on the relationship between chilling tolerance and trienoic fatty acids in plants by molecular biology methods.Key words Low temperature stress, Thylakoid membrane, Fatty acid composition低温几乎影响光合作用的所有主要环节，包括气孔导度，类囊体膜上的光合电子传递以及碳同化过程。

英文文献翻译 - 中文

倒置AAO工艺的生产性试验研究陈宏斌1，唐先春1，董斌1，高廷耀1，Martin Wagner21 中国上海同济大学污染控制和资源化研究实验室2 IWAR Institute, Technical University of Darmstadt, 64287 Germany摘要倒置缺氧/厌氧/好氧工艺（倒置AAO工艺）是上世纪90年代中期开发出来的用于脱氮除磷的污水处理工艺。

本文主要阐述了松江污水处理厂（中国）二期工程倒置AAO工艺处理城市污水的运行效果、运行参数以及影响因素。

近两年的运行结果表明：对CODCr 、BOD5、SS、NH3-N和TN具有较好的处理效率，对TN 和NH3-N的处理效率分别达到了0.022 kg TN·kgMLSS-1·d-1和 0.026 kg NH3-N·kgMLSS-1·d-1。

然而，对PO4-P和TP的去除率却并不高。

因此，我们提出了一个除磷效率更高的改进措施。

运行结果表明：倒置AAO工艺不仅适用于新建的污水处理厂的脱氮除磷，同样也适用于现有的脱氮除磷效率不高的污水处理厂改造和扩建。

关键词：硝化反硝化除磷倒置AAO工艺城市污水引言氮和磷是城市污水中导致受纳水体富营养化的主要因素。

生物硝化、反硝化和除磷工艺是经济的可行的控制排放水水质的方法。

生物脱氮除磷技术主要有：AAO工艺系列，氧化沟工艺和序批式活性污泥工艺（SBR法）。

不同菌种脱氮除磷的新陈代谢过程是需要在缺氧，厌氧和好氧条件下进行的。

在AAO工艺和氧化沟工艺中，可通过搅拌、曝气和污泥回流等方法实现上述条件。

SBR工艺是一个以时间顺序实现缺氧，厌氧，好氧的AAO工艺。

在中国，AAO工艺广泛应用于许多污水处理设施。

传统的AAO工艺有很多优点，同时也有两个缺点：1）需要两个回流系统，其总回流比不少于300％；2）从二沉池抽走的剩余污泥有部分没有完全经过厌氧，缺氧和好氧阶段，这可能会减少总磷（TP）的去除率。

211036564_环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应

生态毒理学报Asian Journal of Ecotoxicology第18卷第1期2023年2月V ol.18,No.1Feb.2023㊀㊀基金项目:农业农村部淡水渔业健康养殖重点实验室开放课题(ZJK202010);中央高校基本科研业务费专项资金(2662021SCPY003)㊀㊀第一作者:孔娟(1995 ),女,硕士研究生,研究方向为生态毒理学,E -mail:*****************㊀㊀*通信作者(Corresponding author ),E -mail:*****************㊀㊀#共同通信作者(Co -corresponding author),E -mail:*********************DOI:10.7524/AJE.1673-5897.20220124001孔娟,范博雅,原居林,等.环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应[J].生态毒理学报,2023,18(1):426-439Kong J,Fan B Y ,Yuan J L,et bined effects of environmental concentration of oxytetracycline and polystyrene microplastics on intestinal tract of juvenile yellow catfish (Pelteobagrus fulvidraco )[J].Asian Journal of Ecotoxicology,2023,18(1):426-439(in Chinese)环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应孔娟1,范博雅1,原居林2,#,余丽琴1,3,*1.华中农业大学水产学院,武汉4300702.浙江省淡水水产研究所,湖州3130003.教育部长江经济带大宗水生生物产业绿色发展教育部工程研究中心,武汉430070收稿日期:2022-01-24㊀㊀录用日期:2022-04-24摘要:近年来,氧四环素(oxytetracycline,OTC)和聚苯乙烯微塑料(polystyrene microplastics,PS -MPs)在水环境中被广泛检出㊂为了探究PS -MPs 与OTC 对鱼类的联合毒性效应,选取黄颡鱼(Pelteobagrus fulvidraco )幼鱼为研究对象,将其暴露于CON(对照)组㊁环境浓度OTC(500ng ㊃L -1)单独组㊁MPs -L(100μg ㊃L -1PS -MPs)单独组㊁MPs -L+OTC(100μg ㊃L -1PS -MPs+500ng ㊃L -1OTC)复合组㊁MPs -H(1000μg ㊃L -1PS -MPs)单独组和MPs -H+OTC(1000μg ㊃L -1PS -MPs+500ng ㊃L -1OTC)复合组中28d ,研究了OTC 和PS -MPs 单独以及联合暴露对幼鱼生长㊁肠道结构和肠道菌群的影响㊂研究结果表明,与对照组相比,OTC 单独暴露和MPs -L 单独暴露对黄颡鱼幼鱼体长㊁体质量及体质量增长率,肠道氧化应激酶(超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT))活性及消化酶(胰蛋白酶(trypsin,TRS)㊁淀粉酶(amylase,AMS)和脂肪酶(lipase,LPS))活性,肠道微生物组成(OTU 数目㊁α多样性㊁β多样性以及门㊁属水平上物种组成的相对丰度)均无显著性影响㊂但MPs -L+OTC 复合暴露导致SOD 和CAT 活性显著升高,引起肠道空泡化㊁肠上皮细胞轻微缺失,变形菌门的相对丰度显著升高,且与OTC 单独暴露组相比,肠道CAT 活性显著性升高㊂MPs -H 单独暴露抑制了黄颡鱼幼鱼体质量和体质量增长率,引起了肠道空泡化,导致其肠道SOD 和CAT 活性显著升高,消化酶TRS 和LPS 活性显著降低,厚壁菌门相对丰度显著降低㊂与MPs -H 单独组和OTC 单独组相比,MPs -H+OTC 组进一步加剧肠道氧化酶活性升高㊁消化酶活性降低㊁肠道损伤和肠道菌群紊乱㊂相关性分析表明,肠道葡萄球菌属和体长显著负相关;鲸杆菌属和SOD 显著正相关;气单胞菌属与LPS 显著负相关,与AMS 显著正相关㊂上述结果显示PS -MPs 高浓度单独以及与OTC 复合暴露可能通过肠道损伤以及肠道菌群的改变,进而影响肠道消化酶活性,导致黄颡鱼幼鱼生长抑制㊂此外,PS -MPs 和OTC 的复合肠道毒性表现出显著的协同效应㊂本实验结果将为水环境中抗生素和微塑料的生态风险评价提供新的视角和理论依据㊂关键词:聚苯乙烯微塑料;氧四环素;黄颡鱼幼鱼;复合暴露;肠道菌群文章编号:1673-5897(2023)1-426-14㊀㊀中图分类号:X171.5㊀㊀文献标识码:ACombined Effects of Environmental Concentration of Oxytetracycline and Polystyrene Microplastics on Intestinal Tract of Juvenile Yellow Catfish (Pelteobagrus fulvidraco )Kong Juan 1,Fan Boya 1,Yuan Julin 2,#,Yu Liqin 1,3,*第1期孔娟等:环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应427㊀1.College of Fisheries,Huazhong Agricultural University,Wuhan430070,China2.Zhejiang Institute of Freshwater Fisheries,Huzhou313000,China3.Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt,Ministry of Education,Wuhan430070,ChinaReceived24January2022㊀㊀accepted24April2022Abstract:In recent years,oxytetracycline(OTC)and polystyrene microplastics(PS-MPs)have been widely detec-ted in aquatic environment.In order to investigate the combined effects of PS-MPs and OTC on intestinal tract of fish,we investigated the effects of OTC and PS-MPs exposure on growth,intestinal structure and intestinal micro-flora of fish.Juvenile yellow catfish(Pelteobagrus fulvidraco)were exposed to500ng㊃L-1OTC(OTC),100(low concentration)(MPs-L)and1000μg㊃L-1(high concentration)(MPs-H)PS-MPs,or their combination(combined MPs-L+OTC,combined MPs-H+OTC)for28d.Results showed that OTC and MPs-L alone exposure had no sig-nificant effect on the growth,intestinal antioxidant enzyme activities(including superoxide dismutase(SOD)and catalase(CAT)),digestive enzyme activities(including trypsin(TRS),amylase(AMS)and lipase(LPS)),or intesti-nal flora(including OTU number,alpha diversity,beta diversity,and relative abundance of species composition at phylum level and genus level)of juvenile yellow catfish.However,combined MPs-L+OTC exposure significantly increased SOD and CAT activities,induced intestinal vacuolation and slight loss of intestinal epithelial cells,as well as significantly increased the relative abundance of Proteobacteria as compared to the control group.Intestinal CAT activity was significantly increased in the combined MPs-L+OTC exposure group as compared to the OTC alone exposure group.Moreover,MPs-H alone exposure inhibited the body weight and weight gain rate of juvenile yellow catfish,induced intestinal vacuolation,significantly increased the activities of SOD and CAT,significantly decreased the activities of digestive enzymes TRS and LPS,and significantly decreased the relative abundance of pared with the MPs-H group and OTC group,the effects on oxidase activities,digestive enzyme activities,intestinal injury and intestinal flora were further exacerbated in the combined MPs-H+OTC group.In ad-dition,correlation analysis showed that a significant negative correlation between Staphylococcus and body length. Abundance of Cetobacterium was positively correlated with the activities of SOD.Aeromonas was negatively cor-related with the activity of LPS while positively correlated with AMS.Thus,high concentration of PS-MPs alone or combined OTC exposure might affect intestinal digestive enzyme activities through intestinal injury and changes in the intestinal flora,resulting in growth inhibition of juvenile yellow catfish.In addition,the combined intestinal tox-icity of PS-MPs and OTC showed significant synergistic effects.The results of this study might provide a new per-spective and theoretical basis for ecological risk assessment of antibiotics and microplastics in aquatic environment. Keywords:polystyrene microplastic;oxytetracycline;juvenile yellow catfish;combined exposure;intestinal flora㊀㊀抗生素(antibiotics)能干扰或抑制致病微生物的存在,广泛使用于人类及动物的疾病防治㊁畜牧及水产养殖等领域[1]㊂据统计,全世界抗生素的使用量可达10~20万t[2],其中我国是抗生素最大的生产国和消费国,2013年我国抗生素的使用量已高达16.2万t[3]㊂大多数的抗生素很难被人体或动物全部吸收,25%~75%的抗生素会以原药或代谢产物的形式通过粪便或尿液排入水环境中,已在各种水体中检测到了其广泛存在[4],主要包括四环素类㊁大环内酯类㊁磺胺类㊁喹诺酮类和氯霉素类[5-8]㊂四环素类抗生素(tetracyclines,TCs)作为我国生产量和实际使用量最大的抗生素,在各种水体中被频繁地检测到[9-12]㊂其中,氧四环素(oxytetracycline, OTC)的检出浓度最高㊂在黄浦江中OTC和TC的最高浓度分别可达479ng㊃L-1和440ng㊃L-1[13]㊂在中国的海陵湾地区水体中检测到了21种抗生素,其中OTC的浓度最高,平均浓度为417.76ng㊃L-1,最高浓度高达15.16μg㊃L-1[12]㊂有关OTC在环境浓度慢性暴露下对水生生物的影响研究较少,近几年的研究发现环境浓度OTC影响鱼类的肠道结构和微生物群落㊂环境浓度OTC(420ng㊃L-1)暴露或者投喂罗非鱼导致其生长受抑制㊁肠道屏障被破坏和428㊀生态毒理学报第18卷肠道菌群失调[14-15]㊂420ng㊃L-1OTC暴露导致斑马鱼抗氧化性显著降低,肠道屏障功能障碍,炎症因子表达量增加,肠道菌群受到干扰[16]㊂在水生态系统中,抗生素对水生生物的毒性效应也会受到一些非生物因素的影响,例如pH㊁温度㊁紫外线以及其他环境污染物(重金属㊁有机污染物等)㊂在众多的水环境污染物中,微塑料不仅能够直接对水生生物产生危害还可以作为载体吸附抗生素[17],因此其对抗生素毒性效应的影响亟待得到关注㊂微塑料(microplastics,MPs)被定义为尺寸<5mm 的塑料颗粒,具有粒径小㊁不易被降解等特点㊂环境调查发现我国水体㊁土壤和沉积物等环境中均有微塑料,且水体是其主要归趋地之一[18]㊂调查表明黄浦江与长江河口交界处微塑料浓度为(4137.3ʃ2461.5)个㊃m-3[19]㊂武汉市湖泊水体大多流动性较小或是静水,其微塑料的累积量高达(8925ʃ1591)个㊃m-3[20]㊂对水环境中微塑料种类的检测发现,聚苯乙烯微塑料(polystyrene microplastics,PS-MPs)是其主要成分之一[21]㊂MPs极易通过摄食等途径在水生生物体内累积,其在各种鱼类㊁中华白鳍豚和东亚江豚等肠道内均被检测到[22-25]㊂此外,微塑料能够抑制鱼类摄食和生长,引起肠道损伤和炎症,氧化应激毒性和生殖毒性等多种毒性效应[26-29]㊂其中,微塑料对鱼类肠道损伤和微生物群落的影响近年来受到了广泛的关注㊂研究发现PS-MPs暴露导致斑马鱼肠道菌群失调㊁炎症反应和肠道屏障功能障碍[30-32]㊂为了探索PS-MPs和OTC对鱼类的联合毒性效应,本文以黄颡鱼幼鱼为实验生物,探究了环境浓度OTC(500ng㊃L-1)和PS-MPs(100μg㊃L-1和1000μg㊃L-1)单独及联合暴露28d后对黄颡鱼幼鱼的生长㊁肠道氧化应激㊁肠道消化酶活性㊁肠道组织病理及肠道菌群的影响㊂本研究从个体水平㊁器官水平和微生物水平评价OTC和PS-MPs对黄颡鱼幼鱼的肠道影响,进而为评价PS-MPs和OTC的环境健康风险评价提供理论依据㊂1㊀材料与方法(Materials and methods)1.1㊀实验试剂氧四环素,CAS号为6153-64-6,纯度ȡ97%,购自上海麦克林生化科技有限公司,使用去离子水将OTC溶解配制成浓度为50μg㊃mL-1的储备液,储存在4ħ冰箱里㊂聚苯乙烯微塑料(PS-MPs,球形,5μm,纯度ȡ99%)购自天津倍思乐色谱技术开发中心,用去离子水将微塑料原液稀释为1mg㊃mL-1的储备液,储存在4ħ冰箱;PS-MPs储备液使用前需超声20min并用曝气24h的自来水稀释为100μg㊃L-1和1000μg㊃L-1的暴露液㊂1.2㊀黄颡鱼驯养及暴露实验黄颡鱼鱼苗购自武汉市黄优源渔业发展有限公司,将刚出苗3d的黄颡鱼鱼苗转移至室内养殖系统中进行为期2周的暂养㊂在实验开始时,选取规格一致的健康仔鱼(平均体长(13.38ʃ0.62)mm,平均体质量(0.028ʃ0.003)g)分别暴露在CON组㊁OTC (500ng㊃L-1)组㊁MPs-L(100μg㊃L-1PS-MPs)组㊁MPs-H(1000μg㊃L-1PS-MPs)组㊁MPs-L+OTC(100μg㊃L-1PS-MPs+500ng㊃L-1OTC)组和MPs-H+OTC (1000μg㊃L-1PS-MPs+500ng㊃L-1OTC)组共6个浓度组中,每组有3个平行缸,每缸60尾鱼于18L暴露液中㊂暴露养殖水为曝气24h的自来水,暴露过程中持续稳定曝气(防止微塑料颗粒凝聚)㊂保持稳定的水质参数:pH=7~8,水温(26.1ʃ0.8)ħ,溶氧(16.4ʃ0.2)mg㊃L-1,光周期为14h/10h(昼/夜),每天更换一半暴露液㊁暴露时间为28d㊂每天投喂商业饲料3次(10:00㊁16:00和21:00),投喂率为鱼体质量的4%㊂1.3㊀PS-MPs的表征分析表征前,将PS-MPs进行20min超声,采用超纯水制备5mL浓度为1000μg㊃L-1的测试溶液㊂采用透射电子显微镜(TEM,HT-7700,日本日立有限公司)来观察PS-MPs的尺寸;用纳米粒度及Zeta电位分析仪(Zetasizer Nano ZS,英国马尔文公司)测得PS-MPs的Zeta电位㊂1.4㊀生长指标的测定暴露结束后,黄颡鱼禁食24h后浸泡在<4ħ的冰水浴中安乐死㊂每个浓度每个平行取25尾黄颡鱼幼鱼,测量并分析黄颡鱼幼鱼的体质量(body weight,BW)㊁体长(body length,BL)(黄颡鱼幼鱼吻到尾鳍基部)㊁存活率(survival rate,SR)㊁体质量增长率(weight gain rate,WGR)㊁特定生长率(specific growth rate,SGR)(d-1)㊁脑体比(brain somatic index, BSI)和肝体比(hepato somatic index,HSI)㊂计算公式如下:存活率(SR)=(N t/N)ˑ100%体质量增长率(WGR)=(W t-W0)/W0ˑ100%特定生长率(SGR)=[ln(W t/N t)-ln(W/N)]/tˑ100%肝体比(HSI)=(肝脏质量/W t)ˑ100%脑体比(BSI)=(脑质量/W t)ˑ100%式中:W为试验鱼的初始质量(g),W t为试验鱼的第1期孔娟等:环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应429㊀终末质量(g),N和N t为试验开始和试验结束时每组鱼尾数,t为试验时间(d)1.5㊀肠道组织病理分析暴露28d后,随机从每个浓度每个平行缸取4尾黄颡鱼幼鱼用于肠道组织病理学分析㊂取样前禁食24h,解剖取中肠组织(1~2cm)于4%多聚甲醛缓冲液固定24~48h㊂肠组织经乙醇㊁二甲苯透明液脱水,石蜡包埋㊁切片(4μm),苏木精和伊红(H& E)染色切片,使用立体显微镜(Leica,M205FA,德国)的400倍镜头进行组织病理学评估㊂1.6㊀肠道抗氧化酶的活性测定暴露28d后,禁食24h,随机从每个浓度每个平行中取3尾黄颡鱼幼鱼肠道测定酶的活性㊂装入EP冻存管迅速置于液氮冷冻,置于-80ħ冰箱中保存待测㊂使用组织细胞破碎仪(Buller Blender,Tis-sulyser-24,美国Next Advance公司)将肠组织样品和0.86%冷生理盐水均质,4ħ离心15min(2500r㊃min-1)㊂取上清液按照试剂盒说明书测定肠道超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)酶活性㊂蛋白质浓度采用蛋白定量(TP)测定盒(BCA微板法)测定㊂实验所使用酶标仪为Synergy H4(Biotek,VT,USA),使用的商业化试剂盒均购自南京建成研究所㊂1.7㊀肠道消化酶的活性测定暴露结束后,每个浓度每个平行中取3尾黄颡鱼幼鱼用于消化酶活性的测定,肠道的取样方法和前处理方法同1.6㊂测定肠道胰蛋白酶(trypsin, TRS)㊁淀粉酶(amylase,AMS)和脂肪酶(lipase,LPS)活性的方法参照各试剂盒说明书㊂酶液蛋白质浓度采用蛋白定量(TP)测定盒(BCA微板法)测定㊂所使用的商业化试剂盒购自南京建成研究所㊂1.8㊀肠道菌群16S rRNA扩增子测序分析暴露结束后,随机从各实验组的每个平行缸中在无菌操作台上取6尾黄颡鱼幼鱼肠道用于肠道菌群测序检测㊂具体操作如下:冰盘上无菌解剖黄颡鱼肠道并收集,液氮速冻后转移至-80ħ冰箱中待测㊂将取得的36个肠道样本送至广东美立康生物科技有限公司进行测序分析㊂具体分析流程如下:采用两步PCR法,使用通用正向引物515F(5 -GT-GYCAGCMGCCGCGGTA-3 )和反向引物909R(5 -CCCCGYCAATTCMTTTRAGT-3 )扩增微生物16s rRNA基因的V4-V5高变区,扩增条件为94ħ预变性3min,然后进行30个循环的常规扩增(94ħ变性40s,56ħ退火60s,72ħ延伸60s),最后72ħ延伸10min㊂使用1.2%的琼脂糖凝胶电泳后采用sanPrep DNA凝胶回收试剂盒(生工,中国)纯化,所有纯化的DNA等量混合后,对高变区进行PCR扩增后用Illumina Miseq测序平台对PCR扩增产物进行测序,鉴定群落中的微生物组成和其相对丰度㊂采用QIIME1.9.0软件按照序列97%的相似性将序列聚类为OTUs(Operational Taxonomic Units),计算α多样性指数和UniFrac距离矩阵并进行PCoA排序分析㊂1.9㊀数据分析与统计所有数据均以平均值ʃ标准误差 (MeanʃSEM)表示,双因素方差分析(Two-way ANOV A)方法分析试验数据的差异性(IBM SPSS Statistics22)㊂如果差异达到显著水平(P<0.05),选择Duncan法进行多重比较分析㊂运用R(4.1.2)采用相关分析方法分析黄颡鱼幼鱼生长㊁肠道氧化应激水平和肠道消化酶活性与肠道微生物之间的相关性㊂2㊀结果(Results)2.1㊀聚苯乙烯微塑料(PS-MPs)的表征如图1所示,用透射电子显微镜观察到PS-MPs 的尺寸为5μm(图1(a)),用纳米粒度及Zeta电位分析仪(Zetasizer Nano ZS)测得PS-MPs的Zeta电位为-44.3mV(图1(b)),表明PS-MPs在超纯水中,直径为5μm,粒径几乎没有差异㊂2.2㊀OTC和PS-MPs暴露28d后对幼年黄颡鱼生长性能的影响如表1所示,暴露28d后,与对照组相比,OTC 组㊁MPs-L组和MPs-L+OTC组的所有生长指标均无显著性差异;MPs-H组的体质量显著下降15.06%(P< 0.05),体质量增长率显著下降8.41%(P<0.05);MPs-H +OTC组的体质量显著下降15.45%(P<0.05),体质量增长率显著下降17.17%(P<0.05),SGR显著下降7.49%(P<0.05),BSI显著降低25.57%(P<0.05);此外,与MPs-H组相比,MPs-H+OTC组的BSI显著降低了19.79%(P<0.05)㊂2.3㊀中肠组织病理分析如图2所示,黄颡鱼肠组织结构可由黏膜层㊁肌层和浆膜层组成,淋巴细胞常在上皮细胞之间分布,多位于基底部㊂与对照组相比,OTC组和MPs-L组的肠道组织形态学完整,肠黏膜皱襞排列整齐,纹状缘清晰,边缘光滑,没有明显的病理损伤(图2(a)㊁2 (b)和2(d))㊂在MPs-H组中,肠道出现轻微的组织430㊀生态毒理学报第18卷损伤:少量空泡化现象(图2(c))㊂在MPs -L+OTC 组和MPs -H+OTC 组中,组织损伤加剧:部分淋巴细胞轻微向游离面游走,空泡化变性,肠上皮细胞轻微缺失,固有膜结缔组织疏松(图2(e)和2(f))㊂2.4㊀黄颡鱼幼鱼肠道抗氧化酶的活性如图3所示,与对照相比,OTC 组和MPs -L 组未受到显著性影响;MPs -H 组的SOD 活性和CAT活性分别显著提高了35.3%(P <0.05)和100.8%(P <0.05);MPs -L+OTC 组的SOD 活性和CAT 活性分别显著提高了42.7%(P <0.05)和85.5%(P <0.05);MPs -H+OTC 组中SOD 活性和CA T 活性分别显著提高了120.5%(P <0.05)和169.6%(P <0.05)㊂此外,与MPs -H 组相比,MPs -H+OTC 组CAT 活性和SOD 活性分别显著提高了34.3%(P <0.05)和63.7%(P <0.05)㊂图1㊀聚苯乙烯微塑料(PS-MPs )的尺寸和Zeta 电位注:(a)PS -MPs 透射电子显微镜图;(b)PS -MPs 在水溶液中的Zeta 电位㊂Fig.1㊀Dimensions and compositions of polystyrene microplastics (PS -MPs)Note:(a)TEM of PS -MPs;(b)Zeta potential of PS -MPs.表1㊀氧四环素(OTC )和PS-MPs 单独及复合暴露对幼年黄颡鱼生长性能的影响Table 1㊀Effects of oxytetracycline (OTC)and PS -MPs alone or in combination exposureon growth performance of juvenile yellow catfish指标Index CON OTC MPs -L MPs -L+OTC MPs -H MPs -H+OTC存活率(SR)/%Survival rate (SR)/%0.75ʃ0.040.71ʃ0.030.74ʃ0.020.72ʃ0.030.67ʃ0.050.71ʃ0.03体长(BL)/mm Body length (BL)/mm 24.77ʃ0.3324.72ʃ0.4723.99ʃ0.2623.82ʃ0.2624.27ʃ0.3024.31ʃ0.24体质量(BW)/mg Body weight (BW)/mg 279.40ʃ0.01a 274.74ʃ0.02a 245.62ʃ0.01ab 256.10ʃ0.01ab 237.32ʃ0.01b 236.24ʃ0.01b体质量增长率(WGR)/%Growth rate of body weight (WGR)/%897.85ʃ46.31a 881.20ʃ55.32ab 777.21ʃ40.04abc 814.53ʃ36.13abc 762.35ʃ38.08bc 743.70ʃ34.23c特定生长率(SGR)/d -1Specific growth rate (SGR)/d-18.96ʃ0.16a 8.90ʃ0.23a 8.69ʃ0.15ab 8.84ʃ0.14ab 8.43ʃ0.13ab 8.29ʃ0.15b肝体比(HSI)/%Hepato somatic index (HSI)/%2.12ʃ0.33 2.03ʃ0.29 1.85ʃ0.22 1.73ʃ0.17 1.85ʃ0.16 1.96ʃ0.14脑体比(BSI)/%Brain somatic index (BSI)/%3.05ʃ0.19a 2.72ʃ0.18ab 2.89ʃ0.18a 2.67ʃ0.15ab 2.83ʃ0.14a 2.27ʃ0.11b注:同列中标有不同小写字母者表示组间有显著性差异(P <0.05)㊂Note:Different lowercase letters in the same column indicated significant difference among groups (P <0.05).第1期孔娟等:环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应431㊀图2㊀OTC 和PS-MPs 单独及复合暴露对黄颡鱼幼鱼鱼肠道组织的影响(400ˑ,比例尺为50μm )注:柱状上皮细胞(1);杯状细胞(2);固有层(3);淋巴细胞(4);空泡化(ң);肠上皮细胞缺失(һ);淋巴细胞向游离面游走(ʀ)㊂Fig.2㊀Effect of OTC and PS -MPs alone or in combination exposure on gut histologyof juvenile yellow catfish (400ˑ,scale bars 50μm)Note:Columnar epithelial cells (1);goblet cells (2);lamina propria (3);lymphocytes (4);vacuolation (ң);intestinal epithelial cells are absent (һ);lymphocytes migrate to the free surface (ʀ).图3㊀OTC 和PS-MPs 单独及复合暴露对黄颡鱼幼鱼氧化应激酶活性的影响(n =3)注:标注不同小写字母表示组间有显著性差异(P <0.05)㊂Fig.3㊀Effects of OTC and PS -MPs alone or in combination exposure on oxidative stress activityof juvenile yellow catfish gut (n =3)Note:Different lowercase letters indicated significant difference among groups (P <0.05).2.5㊀黄颡鱼幼鱼肠道消化酶的活性如图4所示,与对照组相比,OTC 组㊁MPs -L 组和MPs -L +OTC 组的消化酶未受到显著性影响;MPs -H 暴露组的脂肪酶(LPS)和胰蛋白酶(TRS)活性分别显著下降了35.61%(P <0.05)和48.01%(P <0.05);MPs -H+OTC 暴露组的LPS 和TRS 活性分别432㊀生态毒理学报第18卷图4㊀OTC 和PS-MPs 单独及复合暴露对黄颡鱼幼鱼肠道消化酶活性的影响(n =3)注:标注不同小写字母表示组间有显著性差异(P <0.05)㊂Fig.4㊀Effects of OTC and PS -MPs alone or in combination exposure on gut digestive enzyme activitiesof juvenile yellow catfish (n =3)Note:Different lowercase letters indicated significant difference among groups (P <0.05).显著下降了47.73%(P <0.05)和60.25%(P <0.05)㊂2.6㊀微塑料和氧四环素对黄颡鱼幼鱼肠道菌群的影响2.6.1㊀微塑料和氧四环素对黄颡鱼幼鱼肠道菌群α多样性的影响和主成分分析㊀㊀α多样性中,Chao1指数为描述群落丰富度的指数,指数越大,代表对应的群落其丰富度就越高㊂Shannon 指数和Simpson 指数为描述群落多样性的指数,综合考虑了群落的丰富度和均匀度,其值越高,群落的多样性越高㊂如图5(a)所示,MPs -H +OTC 组OTUs 数目与对照组相比,显著下降(P <0.05),其余组别无显著性影响㊂如图5(b)㊁5(c)和5(d)所示,与对照组相比,所有处理组的Simpson 指数无显著性差异,MPs -H 组的Shannon 指数(菌群的物种多样性)显著性降低(P <0.05),MPs -H+OTC 组的Chao1指数(菌群丰富度)(P <0.05)和Shannon 指数(菌群的物种多样性)显著性降低(P <0.05)㊂如图5(e)所示,从Weighted Unifrac 距离来进行PCoA 分析,一个点代表一个样本,同种颜色的点表示相同的处理组㊂投影分析表明样本之间距离越近,在相应维度中的群落组成越相似㊂与对照组相比,MPs -H +OTC 暴露组发生了分离,其余组均有重合㊂2.6.2㊀微塑料和氧四环素对黄颡鱼幼鱼肠道菌群门㊁属水平的影响在门水平上,共检出53个门(2个古细菌门和51个真细菌门)㊂如图6(b)所示,其中优势门(至少在一个样品的相对丰度超过1%)有变形菌门(Pro -teobacteria)㊁梭杆菌门(Fusobacteria)㊁厚壁菌门(Firm -icutes)㊁蓝藻门(Cyanobacteria)㊁酸杆菌门(Acidobacte -ria)㊁放线菌门(Actinobacteria )㊁拟杆菌门(Bacte -roidetes)㊁浮霉菌门(Planctomycetes)和软壁菌门(Te -nericutes)㊂如图6(a)所示,肠道菌群主要富集在3个门上:变形菌门(Proteobacteria)㊁梭杆菌门(Fuso -bacteria)和厚壁菌门(Firmicutes)㊂对照组中变形菌门㊁梭杆菌门和厚壁菌门的相对丰度分别为38.44%㊁24.22%和15.53%;OTC 组中为33.73%㊁49.65%和13.74%;MPs -L 组中为27.63%㊁5.42%和13.01%;MPs -H 组中为43.56%㊁5.20%和2.52%;MPs -L +OTC 组中为78.57%㊁5.57%和2.65%;MPs -H+OTC 中为15.30%㊁79.32%和0.85%㊂与对照组相比,OTC 组㊁MPs -L 组肠道菌群无显著性变化;MPs -H 组中厚壁菌门的相对丰度显著下降83.77%(P <0.05);MPs -L+OTC 低浓度联合暴露组中变形菌门的相对丰度显著上升104.40%(P <0.05);MPs -H +OTC 高浓度联合暴露组中,变形菌门和厚壁菌门的相对丰度分别显著下降60.20%(P <0.05)和94.53%(P <0.05),梭杆菌门的相对丰度显著上升227.50%(P <0.05);此外,与MPs -H 组相比,MPs -H+OTC 中变形菌门的相对丰度显著下降64.87%(P <0.05),梭杆菌门的相对丰度显著上升了1425.38%(P <0.05)㊂在属水平上,如图6(c)所示,优势属主要有鲸杆菌属(Cetobacterium )㊁埃希氏菌属(Escherichia )和邻单胞菌属(Plesiomonas ),对照组中鲸杆菌属㊁埃希氏菌属和邻单胞菌属相对丰度分别为27.12%㊁25.91%和第1期孔娟等:环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应433㊀图5㊀OTC 和PS-MPs 单独及复合暴露对黄颡鱼幼鱼肠道菌群α多样性指数和β多样性指数的影响注:标注不同小写字母表示组间有显著性差异(P <0.05)㊂Fig.5㊀Effects of OTC and PS -MPs alone or in combination exposure on alpha diversity and beta diversity of intestinal microbiotain juvenile yellow catfishNote:Different lowercase letters indicated significant difference among groups (P <0.05).0.69%,OTC 组中为49.65%㊁0.1%和24.09%,MPs -L 组中为5.23%㊁1.10%和6.63%,MPs -H 组中为5.04%㊁6.83%和20.99%,MPs -L +OTC 组中为5.53%㊁46.57%和1.99%,MPs -H +OTC 组中为79.31%㊁0.05%和14.75%㊂与对照组相比,在MPs -H+OTC 高浓度联合暴露组中,鲸杆菌属的相对丰度显著上升了192.44%(P <0.05);与MPs -H 组相比,MPs -H+OTC 组中鲸杆菌属的相对丰度显著上升了1373.61%(P <0.05)㊂2.6.3㊀微塑料和氧四环素对黄颡鱼幼鱼的生理指标与肠道微生物之间的相关性分析㊀㊀如图7所示,葡萄球菌属(Staphylococcus )与体长成显著负相关(P <0.05)㊂鲸杆菌属(Cetobacterium )与SOD 成显著正相关(P <0.05),与BSI 成显著负相关(P <0.01)㊂气单胞菌属(Aeromonas )与LPS 成显著负相关(P <0.05),与AMS 成显著正相关(P <0.05)㊂3㊀讨论(Discussion )OTC 的滥用导致其在水环境中被频繁检测出,相关研究表明,环境中广泛存在的OTC 能够在水生生物体内累积[33],环境浓度OTC 能够通过PS -MPs 吸附加剧其在生物体内蓄积[34]㊂目前,OTC 和PS -MPs 联合暴露是否影响黄颡鱼幼鱼肠道健康损伤还是未知的㊂本研究通过OTC 和PS -MPs联合暴露黄颡鱼仔鱼28d ,探究其生长指标㊁肠道病理损伤㊁肠道抗氧化酶活性㊁消化酶活性及肠道菌群变化㊂毒理学实验中,生长是评价环境压力对生物影响的重要参数之一[35]㊂本研究中,环境浓度的OTC单独暴露对黄颡鱼幼鱼的生长无显著影响㊂这一结434㊀生态毒理学报第18卷图6㊀OTC 和PS-MPs 单独及复合暴露对黄颡鱼幼鱼肠道菌群组成的影响注:(a)各类群在门水平上的相对丰度;(b)门水平上肠道微生物组成;(c)属水平上的肠道微生物组成;标注不同小写字母表示组间有显著性差异(P <0.05)㊂Fig.6㊀Effect of OTC and PS -MPs alone or in combination exposure on the intestinal microbialcomposition in juvenile yellow catfishNote:(a)Relative abundance of various groups at phylum level;(b)Gut microbiota composition at the phylum level;(c)Gut microbiotacomposition at the genus level;different lowercase letters indicated significant difference among groups (P <0.05).果与之前的研究类似,420ng ㊃L -1OTC 暴露成年斑马鱼42d ,对其生长无显著影响,但引起成年代谢率显著性增加,表明鱼类需要额外的能量来耐受毒理学应激,而不是将其用于生长[35]㊂本实验中,100μg ㊃L -1PS -MPs 对黄颡鱼幼鱼的生长无显著性影响,但1000μg ㊃L -1PS -MPs 显著抑制黄颡鱼幼鱼的体质量增长㊂类似的研究也发现100mg ㊃L -1PS -MPs 暴露50d 后显著抑制了鲫鱼体质量增长[36],这可能是由于PS -MPs 会导致饱腹感,通过影响其进食来抑制其生长㊂本实验中MPs -H+OTC 组黄颡鱼幼鱼会显著降低其体质量,但与对应的MPs -H 和OTC 单独暴露组相比均无显著性㊂在肠道微生物与体长的相关性分析中发现,葡萄球菌属与体长成显著负相关㊂葡萄球菌属是一群革兰氏阳性球菌,其中金黄色葡萄球菌作为一种常见的致病菌,感染严重时能导致坏死性肺炎至肺组织坏死[37],表明金黄色葡萄球菌可能与黄颡鱼生长抑制相关㊂鱼类的肠道是一个重要的器官,不仅参与消化㊁吸收和免疫,而且还能充当有害物质入侵的屏障[32]㊂肠道的形态完整性在一定程度上可以反映肠道的健康状况[33,37]㊂我们的实验结果表明OTC 单独暴露(500ng ㊃L -1)对肠道无显著性病理损伤,但是有研究报道420ng ㊃L -1OTC 暴露斑马鱼48d 减少了斑马鱼肠道中杯状细胞的数量[35]㊂2个研究中结果的不一致可能与抗生素暴露浓度㊁暴露时间不同有关㊂近年来,一些研究探索微塑料暴露对生物肠道上皮细胞的影响[37],值得关注的是5μm 的PS -MPs 可以进入肠道,并穿过黏液屏障,与肠上皮细胞部分直接第1期孔娟等:环境浓度氧四环素与聚苯乙烯微塑料对黄颡鱼幼鱼肠道的联合毒性效应435㊀图7㊀黄颡鱼幼鱼样本的细菌属丰度(>1%)与鱼类健康指数的Pearson相关性分析注:筛选了与鱼类健康指数显著相关的肠道微生物属,蓝色表示正相关,红色表示负相关;*P<0.05和**P<0.01表示该相关性分析具有显著差异;SOD表示超氧化物歧化酶,CAT表示过氧化氢酶, TRS表示胰蛋白酶,LPS表示脂肪酶,AMS表示淀粉酶,HSI表示肝体比,BSI表示脑体比,Length表示体长,Weight表示体质量㊂Fig.7㊀Pearson correlation analysis of bacterial abundance (>1%)and fish health index in juvenile yellow catfish samples Note:The intestinal microbe genera significantly correlated with fish health index were screened,with positive correlation in blue and negative correlation in red;*P<0.05and**P<0.01indicate significant differences in the correlation analysis;SOD stands for superoxide dismutase;CAT stands for catalase;TRS stands for trypsin;LPS stands for lipase;AMS stands for amylase;HSI stands for hepato somatic index;BSI stands for brain somatic index;Length stands for body length;Weight stands for body weight.接触[38]㊂本实验中,MPs-H组造成肠道少量空泡化变性,类似的研究也发现纤维状PS-MPs(10μg㊃L-1)暴露成年斑马鱼21d后,使其肠道产生轻微空泡化变性[27]㊂本研究联合暴露中,MPs-L+OTC组和MPs-H+OTC组肠道中部分淋巴细胞轻微向游离面游走,空泡化变性,肠上皮细胞轻微缺失,固有膜结缔组织疏松,表明OTC与PS-MPs复合暴露加剧了黄颡鱼幼鱼的肠道损伤㊂氧化应激酶可以反映鱼体对外部刺激的反应及其自由基新陈代谢的状态[39]㊂抗氧化酶(包括SOD 和CAT)是对抗生物体中自由基的酶防御机制的第一道防线[40]㊂本实验中,OTC单独组(500ng㊃L-1)对黄颡鱼幼鱼肠道氧化应激酶活性无显著性影响㊂类似研究表明50μg㊃L-1的OTC暴露斑马鱼仔鱼48h后,对SOD和CAT的活性无显著性影响[41]㊂本研究中,MPs-H组中黄颡鱼幼鱼肠道SOD和CAT活性显著升高㊂这一结果与先前的研究类似, PS-MPs作为激活剂能促进体内抗氧化酶(SOD和CAT)的分泌,在这个过程中提高了鱼类清除自由基和减少过氧化氢的能力[39],PS-MPs(5μm,1000μg㊃L-1)暴露斑马鱼21d显著增加SOD和CAT活性[42]㊂本研究MPs-H+OTC组与MPs-H组相比,显著加剧了黄颡鱼幼鱼肠道SOD和CAT活性的升高㊂表明1000μg㊃L-1PS-MPs显著增加了黄颡鱼幼鱼肠道抗氧化能力,而1000μg㊃L-1PS-MPs和500ng㊃L-1OTC复合暴露会给黄颡鱼幼鱼肠道氧化应激抵抗力带来额外的压力,增强对其SOD和CAT活性的促进作用㊂而在肠道微生物与SOD的相关性分析中,鲸杆菌属和SOD呈显著正相关,在MPs-H+OTC组中,PS-MPs与OTC可能通过黄颡鱼幼鱼肠道内鲸杆菌属的显著上升影响其SOD活性的升高㊂鱼类消化酶活性能够直接反应鱼类本身摄食以及消化情况,消化酶活性越高,鱼的生长情况越好[43]㊂本实验中,OTC对黄颡鱼幼鱼肠道淀粉酶(AMS)㊁脂肪酶(LPS)和胰蛋白酶(TRS)活性无显著影响㊂这与肖亮[44]的发现类似,250mg㊃kg-1OTC 饲料喂养异育银鲫28d对其肠道消化酶(AMS㊁LPS 和TRS)活性和生长均无显著影响㊂这表明OTC组肠道消化酶与生长指标结果是相符的㊂本研究中1000μg㊃L-1PS-MPs显著降低了黄颡鱼幼鱼肠道LPS和TRS的活性㊂据报道,PS-MPs进入鱼类肠道,会导致饱腹感,使获取的食物和能量减少,影响其消化性能,MPs浓度越高,对消化性能的抑制作用越大[45],类似研究中PS-MPs(32~40μm,1000μg㊃L-1)暴露孔雀鱼幼鱼28d,显著降低其肠道AMS㊁LPS和TRS活性[46]㊂消化酶在蛋白质㊁脂质和碳水化合物的水解中起着非常重要的作用,从而。

聚乳酸降解菌株筛选鉴定及降解过程优化_林娟

些脂肪族的聚合物 , 如聚乳酸 (Poly lactic acid, PLA)、聚羟烷基酸(PHAs)、聚羧基乙酸内酯(PCL) 和聚丁烯丁二酸盐 (PBS)与传统塑料相比拥有相似的材料性质 , 并兼具可生物降解的特性 [1−2]。在这些可生物降解的材料中 , 聚乳酸 (PLA) 引起了最广泛的关注。 PLA 属于生物可降解脂肪族聚酯, 单体为乳酸(Lactic acid, LA), 以其良好的机械性能、通透
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微生物学通报 Microbiol. China
2013, Vol.40, No.9
性、透明度、耐热性以及环境友好等特性被认为是最有希望的材料
[3−5]
诱导培养基: 含有明胶的基本培养基; 脱脂奶粉培养基(%): 脱脂奶粉 4.0, 琼脂粉 1.5, pH 自然; PLA 降解培养基: 含有 PLA 膜的基本培养基。
Microbiology China
tongbao@
微生物学通报研究报告
Sep. 20, 2013, 40(9): 15601569 © 2013 by Institute of Microbiology, CAS
聚乳酸降解菌株筛选鉴定及降解过程优化
林娟 1,2 周景文 2,3 康振 1,2* 堵国成 2,4* 陈坚 2,5
。虽然 PLA 是聚酯类化合物, 但大部
分报道的 PLA 降解酶都被认为是丝氨酸蛋白。而一些 PLA 的天然类似物 , 如明胶、酪素、蚕丝蛋白和一些肽类等, 因为和 PLA 手性碳在结构的相似性, 对微生物产 PLA 降解酶有诱导作用
[13]
。
为了获得更多关于微生物降解 PLA 的信息、强化 PLA 快速降解过程和揭示降解机理, 本文主要以明胶为唯一碳源来分离筛选得到 PLA 降解微生物, 并系统研究了明胶对于该微生物生长及 PLA 降解的影响。同时, 通过优化降解条件显著提高了 PLA 的降解效率。此外, 初步对降解 PLA 的相关酶进行了分析。

原创英文文献翻译,英文原文名称见介绍(马铃薯在微波和对流干燥过程中的质量和结构变化)

这篇文章是用来测量马铃薯在微波和对流干燥过程中的质量和结构变化。

微波炉经过改良后，选择微波或者对流干燥模式干燥样品。

脱水马铃薯样品的质量品质以抗坏血酸残留量（VC）、复水能力以及具有收缩性的结构为准。

抗坏血酸马铃薯品质的重要指标，且与热变性有关。

抗坏血酸的恶化标志着一级反应情况，进一步的研究表明，取决于空气温度、微波力、湿度含量。

在微波干燥样品中，VC含量破坏减少。

样品的体积皱缩度显示其与湿度的线性关系。

在对流加工过程中，样品自始至终都会出现收缩性，然而，我们却发现微波干燥有两个收缩周期。

微波干燥样品有更高的复水能力。

关键词：对流干燥; 微波干燥; 马铃薯; 复水; 缩水; 维生素C目录1.简介2. 材料与方法3. 结果与讨论3.1.维生素C3.2.收缩性3.3. 复水4.结论参考文献1.简介在微波加工过程中，食物品质是消费者关注的重要指标之一。

微波干燥食品可以提高复杂的化学转换、化学反应。

,这些反应可以导致维生素的分解，脂肪氧化和美拉德反应。

而这些反应机制可以受浓度、温度、水分活度（aw）影响(Bruin & Luyben, 1980)。

经调查研究发现，在微波烹调中维生素会有所减少。

Rosen (1972) 曾研究讨论了微波食品及其相关食料的作用影响，微波量子能在各能级范围内比其他形式的电磁能（X- 和γ-射线）能量都低，也就使得分子和化学集团相互作用从而引起化学变化。

Gerster (1989)把热敏感和水溶性的维生素C 、B1和B2作为指示器来定性分析化学变化。

食品在微波炉中的烫熟、加热以及再加热过程中其维生素残留量可与常规加热方法相比较。

研究发现抗坏血酸的破坏速率随着aw值增加而增加，在解吸附系统中由于粘度的降低破坏速度会大大增加(Labuza, McNally, Gallagher, & Hawkes, 1972)。

Kirk, Dennison, Kokoczka, and Heldman (1977)研究发现，在复水食品体系中，抗坏血酸的稳定性受水分活度、湿度、氧气、贮藏温度的影响。

Journal of Environmental Chemical Engineering

Characterization of nano zero-valent iron (nZVI) and its application in sono-Fenton process to remove COD in palm oil mill efﬂuent
M.R. Taha a, A.H. Ibrahim b,*
This present study highlights the characterization of the nZVI particle and examines the effects of pH, ultrasound intensity (%), and ultrasound duration on the nZVI particle to produce Fe2+. Tests have also been conducted to analyze the application of the nZVI particle as a replacement for FeSO4 in the sono-Fenton process to remove chemical oxygen demand (COD) particularly in palm oil mill efﬂuent (POME).
Journal of Environmental Chemical Engineering 2 (2014) 1–8
Contents lists available at ScienceDirect
Journal of Environmental Chemical Engineering
journal homepage: /locate/jece
The combination of ultrasound with the Fenton process (sono-

溴到硼酸酯

Journal of
Materials Chemistry C
Published on 20 November 2014. Downloaded on 08/12/2016 07:54:22.
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Cite this: J. Mater. Chem. C, 2015, 3, 861
However, these oligouorene functionalized oligomers may suﬀer from the unwanted long wavelength emission under long-term device operation, similar to polyuorene-based macromolecules.34–36
Received 26th September 2014 Accepted 17th November 2014 DOI: 10.1039/c4tc02173h /MaterialsC
Starburst 4,40,400-tris(carbazol-9-yl)triphenylamine-based deep-blue ﬂuorescent emitters with tunable oligophenyl length for solution-processed undoped organic lightemitting diodes†
Introduction
Since 1987, organic light-emitting diodes (OLEDs) have attracted tremendous attention due to their potential applications in at-panel displays and solid-state lightings.1–10 To achieve fullcolor displays, three primary RGB luminescent materials with excellent stability, eﬃciency and color purity are required. With respect to green and red counterparts, it seems to be a big challenge to develop blue emitters, especially deep-blue ones that have a Commission Internationale de l'Eclairage (CIE) y coordinate value of <0.10, because the intrinsic wide bandgap would inevitably result in ineﬃcient charge injection to an emitting layer (EML).11 Therefore, great eﬀorts should be paid to the design of deep-blue emitters to push forward the commercialization of full-color OLEDs.

微生物英文文献及翻译—原文

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic e aerobic sequencing batch reactorZhang Bin a ,b ,Chen Zhe a ,b ,Qiu Zhigang a ,b ,Jin Min a ,b ,Chen Zhiqiang a ,b ,Chen Zhaoli a ,b ,Li Junwen a ,b ,Wang Xuan c ,*,Wang Jingfeng a ,b ,**aInstitute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China bTianjin Key Laboratory of Risk Assessment and Control for Environment and Food Safety,Tianjin 300050,PR China cTianjin Key Laboratory of Hollow Fiber Membrane Material and Membrane Process,Institute of Biological and Chemical Engineering,Tianjin Polytechnical University,Tianjin 300160,PR Chinaa r t i c l e i n f oArticle history:Received 30June 2011Received in revised form 10September 2011Accepted 10September 2011Available online xxx Keywords:Ammonia-oxidizing bacteria Granular sludgeCommunity development Granule sizeNitrifying bacteria distribution Phylogenetic diversitya b s t r a c tThe structure dynamic of ammonia-oxidizing bacteria (AOB)community and the distribution of AOB and nitrite-oxidizing bacteria (NOB)in granular sludge from an anaerobic e aerobic sequencing batch reactor (SBR)were investigated.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and characterize these organisms.The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge.Along with granules formation,the AOB diversity declined due to the selection pressure imposed by process conditions.Denaturing gradient gel electrophoresis (DGGE)and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling e washing out action.Furthermore,DGGE analysis revealed that larger granules beneﬁt more AOB species surviving in the reactor.In the SBR were various size granules coexisted,granule diameter affected the distribution range of AOB and NOB.Small and medium granules (d <0.6mm)cannot restrict oxygen mass transfer in all spaces of the rger granules (d >0.9mm)can result in smaller aerobic volume fraction and inhibition of NOB growth.All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.ª2011Elsevier Ltd.All rights reserved.1.IntroductionAt sufﬁciently high levels,ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica-tion.Accordingly,biodegradation and elimination of ammonia in wastewater are the primary functions of thewastewater treatment process.Nitriﬁcation,the conversion of ammonia to nitrate via nitrite,is an important way to remove ammonia nitrogen.It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB).Aerobic ammonia-oxidation is often the ﬁrst,rate-limiting step of nitriﬁcation;however,it is essential for the*Corresponding author .**Corresponding author.Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China.Tel.:+862284655498;fax:+862223328809.E-mail addresses:wangxuan0116@ (W.Xuan),jingfengwang@ (W.Jingfeng).Available online atjournal homepage:/locate/watresw a t e r r e s e a r c h x x x (2011)1e 100043-1354/$e see front matter ª2011Elsevier Ltd.All rights reserved.doi:10.1016/j.watres.2011.09.026removal of ammonia from the wastewater(Prosser and Nicol, 2008).Comparative analyses of16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti-cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen,2001).However,a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages,and that environmental factors such as process parameter,dis-solved oxygen,salinity,pH,and concentrations of free ammonia can impact certain species of AOB(Erguder et al., 2008;Kim et al.,2006;Koops and Pommerening-Ro¨ser,2001; Kowalchuk and Stephen,2001;Shi et al.,2010).Therefore, the physiological activity and abundance of AOB in waste-water processing is critical in the design and operation of waste treatment systems.For this reason,a better under-standing of the ecology and microbiology of AOB in waste-water treatment systems is necessary to enhance treatment performance.Recently,several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems(Li et al., 2008;Yin and Xu,2009).Currently,the application of molec-ular biotechniques can provide clariﬁcation of the ammonia-oxidizing community in detail(Haseborg et al.,2010;Tawan et al.,2005;Vlaeminck et al.,2010).In recent years,the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy(de Bruin et al.,2004;Liu et al.,2009;Schwarzenbeck et al.,2005;Schwarzenbeck et al.,2004a,b;Xavier et al.,2007). Granules have a more tightly compact structure(Li et al.,2008; Liu and Tay,2008;Wang et al.,2004)and rapid settling velocity (Kong et al.,2009;Lemaire et al.,2008).Therefore,granular sludge systems have a higher mixed liquid suspended sludge (MLSS)concentration and longer solid retention times(SRT) than conventional activated sludge systems.Longer SRT can provide enough time for the growth of organisms that require a long generation time(e.g.,AOB).Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al.,2010;Tsuneda et al.,2003).Other researchers reported that larger granules have been developed with the synthetic organic wastewater in sequencing batch reactors(SBRs)(Li et al., 2008;Liu and Tay,2008).The diverse populations of microor-ganisms that coexist in granules remove the chemical oxygen demand(COD),nitrogen and phosphate(de Kreuk et al.,2005). However,for larger granules with a particle diameter greater than0.6mm,an outer aerobic shell and an inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core.These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth.Some research has shown that particle size and density induced the different distribution and dominance of AOB,NOB and anam-mox(Winkler et al.,2011b).Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems,the information on the dynamics,distribution,and quantiﬁcation of AOB communities during sludge granulation is still limited up to now.To address these concerns,the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seedingﬂocs into granules,and the distribution of AOB and NOB in different size granules from an anaerobic e aerobic SBR.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and char-acterize these organisms.Based on these approaches,we demonstrate the differences in both AOB community evolu-tion and composition of theﬂocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size.It is ex-pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems.2.Material and methods2.1.Reactor set-up and operationThe granules were cultivated in a lab-scale SBR with an effective volume of4L.The effective diameter and height of the reactor was10cm and51cm,respectively.The hydraulic retention time was set at8h.Activated sludge from a full-scale sewage treat-ment plant(Jizhuangzi Sewage Treatment Works,Tianjin, China)was used as the seed sludge for the reactor at an initial sludge concentration of3876mg LÀ1in MLSS.The reactor was operated on6-h cycles,consisting of2-min inﬂuent feeding,90-min anaerobic phase(mixing),240-min aeration phase and5-min efﬂuent discharge periods.The sludge settling time was reduced gradually from10to5min after80SBR cycles in20days, and only particles with a settling velocity higher than4.5m hÀ1 were retained in the reactor.The composition of the inﬂuent media were NaAc(450mg LÀ1),NH4Cl(100mg LÀ1),(NH4)2SO4 (10mg LÀ1),KH2PO4(20mg LÀ1),MgSO4$7H2O(50mg LÀ1),KCl (20mg LÀ1),CaCl2(20mg LÀ1),FeSO4$7H2O(1mg LÀ1),pH7.0e7.5, and0.1mL LÀ1trace element solution(Li et al.,2007).Analytical methods-The total organic carbon(TOC),NHþ4e N, NOÀ2e N,NOÀ3e N,total nitrogen(TN),total phosphate(TP) concentration,mixed liquid suspended solids(MLSS) concentration,and sludge volume index at10min(SVI10)were measured regularly according to the standard methods (APHA-AWWA-WEF,2005).Sludge size distribution was determined by the sieving method(Laguna et al.,1999).Screening was performed with four stainless steel sieves of5cm diameter having respective mesh openings of0.9,0.6,0.45,and0.2mm.A100mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the0.9mm mesh sieve.The sample was subsequently washed with distilled water and particles less than0.9mm in diameter passed through this sieve to the sieves with smaller openings.The washing procedure was repeated several times to separate the gran-ules.The granules collected on the different screens were recovered by backwashing with distilled water.Each fraction was collected in a different beaker andﬁltered on quantitative ﬁlter paper to determine the total suspended solid(TSS).Once the amount of total suspended solid(TSS)retained on each sieve was acquired,it was reasonable to determine for each class of size(<0.2,[0.2e0.45],[0.45e0.6],[0.6e0.9],>0.9mm) the percentage of the total weight that they represent.w a t e r r e s e a r c h x x x(2011)1e10 22.2.DNA extraction and nested PCR e DGGEThe sludge from approximately8mg of MLSS was transferred into a1.5-mL Eppendorf tube and then centrifuged at14,000g for10min.The supernatant was removed,and the pellet was added to1mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules.Genomic DNA was extracted from the pellets using E.Z.N.A.äSoil DNA kit(D5625-01,Omega Bio-tek Inc.,USA).To amplify ammonia-oxidizer speciﬁc16S rRNA for dena-turing gradient gel electrophoresis(DGGE),a nested PCR approach was performed as described previously(Zhang et al., 2010).30m l of nested PCR amplicons(with5m l6Âloading buffer)were loaded and separated by DGGE on polyacrylamide gels(8%,37.5:1acrylamide e bisacrylamide)with a linear gradient of35%e55%denaturant(100%denaturant¼7M urea plus40%formamide).The gel was run for6.5h at140V in 1ÂTAE buffer(40mM Tris-acetate,20mM sodium acetate, 1mM Na2EDTA,pH7.4)maintained at60 C(DCodeäUniversal Mutation Detection System,Bio-Rad,Hercules,CA, USA).After electrophoresis,silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994).These were followed by air-drying and scanning with a gel imaging analysis system(Image Quant350,GE Inc.,USA). The gel images were analyzed with the software Quantity One,version4.31(Bio-rad).Dice index(Cs)of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes(LaPara et al.,2002).This index ranges from0%(no common band)to100%(identical band patterns) with the assistance of Quantity One.The Shannon diversity index(H)was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population.H was calculatedusing the following equation:H¼ÀPn iNlogn iN,where n i/Nis the proportion of community made up by species i(bright-ness of the band i/total brightness of all bands in the lane).Dendrograms relating band pattern similarities were automatically calculated without band weighting(consider-ation of band density)by the unweighted pair group method with arithmetic mean(UPGMA)algorithms in the Quantity One software.Prominent DGGE bands were excised and dissolved in30m L Milli-Q water overnight,at4 C.DNA was recovered from the gel by freeze e thawing thrice.Cloning and sequencing of the target DNA fragments were conducted following the estab-lished method(Zhang et al.,2010).2.3.Distribution of nitrifying bacteriaThree classes of size([0.2e0.45],[0.45e0.6],>0.9mm)were chosen on day180for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules.2mg sludge samples wereﬁxed in4%para-formaldehyde solution for16e24h at4 C and then washed twice with sodium phosphate buffer;the samples were dehydrated in50%,80%and100%ethanol for10min each. Ethanol in the granules was then completely replaced by xylene by serial immersion in ethanol-xylene solutions of3:1, 1:1,and1:3by volume andﬁnally in100%xylene,for10min periods at room temperature.Subsequently,the granules were embedded in parafﬁn(m.p.56e58 C)by serial immer-sion in1:1xylene-parafﬁn for30min at60 C,followed by 100%parafﬁn.After solidiﬁcation in parafﬁn,8-m m-thick sections were prepared and placed on gelatin-coated micro-scopic slides.Parafﬁn was removed by immersing the slide in xylene and ethanol for30min each,followed by air-drying of the slides.The three oligonucleotide probes were used for hybridiza-tion(Downing and Nerenberg,2008):FITC-labeled Nso190, which targets the majority of AOB;TRITC-labeled NIT3,which targets Nitrobacter sp.;TRITC-labeled NSR1156,which targets Nitrospira sp.All probe sequences,their hybridization condi-tions,and washing conditions are given in Table1.Oligonu-cleotides were synthesized andﬂuorescently labeled with ﬂuorochomes by Takara,Inc.(Dalian,China).Hybridizations were performed at46 C for2h with a hybridization buffer(0.9M NaCl,formamide at the percentage shown in Table1,20mM Tris/HCl,pH8.0,0.01% SDS)containing each labeled probe(5ng m LÀ1).After hybrid-ization,unbound oligonucleotides were removed by a strin-gent washing step at48 C for15min in washing buffer containing the same components as the hybridization buffer except for the probes.For detection of all DNA,4,6-diamidino-2-phenylindole (DAPI)was diluted with methanol to aﬁnal concentration of1ng m LÀ1.Cover the slides with DAPI e methanol and incubate for15min at37 C.The slides were subsequently washed once with methanol,rinsed brieﬂy with ddH2O and immediately air-dried.Vectashield(Vector Laboratories)was used to prevent photo bleaching.The hybridization images were captured using a confocal laser scanning microscope (CLSM,Zeiss710).A total of10images were captured for each probe at each class of size.The representative images were selected andﬁnal image evaluation was done in Adobe PhotoShop.w a t e r r e s e a r c h x x x(2011)1e1033.Results3.1.SBR performance and granule characteristicsDuring the startup period,the reactor removed TOC and NH 4þ-N efﬁciently.98%of NH 4þ-N and 100%of TOC were removed from the inﬂuent by day 3and day 5respectively (Figs.S2,S3,Supporting information ).Removal of TN and TP were lower during this period (Figs.S3,S4,Supporting information ),though the removal of TP gradually improved to 100%removal by day 33(Fig.S4,Supporting information ).To determine the sludge volume index of granular sludge,a settling time of 10min was chosen instead of 30min,because granular sludge has a similar SVI after 60min and after 5min of settling (Schwarzenbeck et al.,2004b ).The SVI 10of the inoculating sludge was 108.2mL g À1.The changing patterns of MLSS and SVI 10in the continuous operation of the SBR are illustrated in Fig.1.The sludge settleability increased markedly during the set-up period.Fig.2reﬂects the slow andgradual process of sludge granulation,i.e.,from ﬂocculentsludge to granules.3.2.DGGE analysis:AOB communities structure changes during sludge granulationThe results of nested PCR were shown in Fig.S1.The well-resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig.3).The community structure at the end of experiment was different from that of the initial pattern of the seed sludge.The AOB communities on day 1showed 40%similarity only to that at the end of the GSBR operation (Table S1,Supporting information ),indicating the considerable difference of AOB communities structures between inoculated sludge and granular sludge.Biodiversity based on the DGGE patterns was analyzed by calculating the Shannon diversity index H as204060801001201401254159738494104115125135147160172188Time (d)S V I 10 (m L .g -1)10002000300040005000600070008000900010000M L S S (m g .L -1)Fig.1e Change in biomass content and SVI 10during whole operation.SVI,sludge volume index;MLSS,mixed liquid suspendedsolids.Fig.2e Variation in granule size distribution in the sludge during operation.d,particle diameter;TSS,total suspended solids.w a t e r r e s e a r c h x x x (2011)1e 104shown in Fig.S5.In the phase of sludge inoculation (before day 38),H decreased remarkably (from 0.94to 0.75)due to the absence of some species in the reactor.Though several dominant species (bands2,7,10,11)in the inoculating sludge were preserved,many bands disappeared or weakened (bands 3,4,6,8,13,14,15).After day 45,the diversity index tended to be stable and showed small ﬂuctuation (from 0.72to 0.82).Banding pattern similarity was analyzed by applying UPGMA (Fig.4)algorithms.The UPGMA analysis showed three groups with intragroup similarity at approximately 67%e 78%and intergroup similarity at 44e 62%.Generally,the clustering followed the time course;and the algorithms showed a closer clustering of groups II and III.In the analysis,group I was associated with sludge inoculation and washout,group IIwithFig.3e DGGE proﬁle of the AOB communities in the SBR during the sludge granulation process (lane labels along the top show the sampling time (days)from startup of the bioreactor).The major bands were labeled with the numbers (bands 1e15).Fig.4e UPGMA analysis dendrograms of AOB community DGGE banding patterns,showing schematics of banding patterns.Roman numerals indicate major clusters.w a t e r r e s e a r c h x x x (2011)1e 105startup sludge granulation and decreasing SVI 10,and group III with a stable system and excellent biomass settleability.In Fig.3,the locations of the predominant bands were excised from the gel.DNA in these bands were reampliﬁed,cloned and sequenced.The comparative analysis of these partial 16S rRNA sequences (Table 2and Fig.S6)revealed the phylogenetic afﬁliation of 13sequences retrieved.The majority of the bacteria in seed sludge grouped with members of Nitrosomonas and Nitrosospira .Along with sludge granula-tion,most of Nitrosomonas (Bands 2,5,7,9,10,11)were remained or eventually became dominant in GSBR;however,all of Nitrosospira (Bands 6,13,15)were gradually eliminated from the reactor.3.3.Distribution of AOB and NOB in different sized granulesFISH was performed on the granule sections mainly to deter-mine the location of AOB and NOB within the different size classes of granules,and the images were not further analyzed for quantiﬁcation of cell counts.As shown in Fig.6,in small granules (0.2mm <d <0.45mm),AOB located mainly in the outer part of granular space,whereas NOB were detected only in the core of granules.In medium granules (0.45mm <d <0.6mm),AOB distributed evenly throughout the whole granular space,whereas NOB still existed in the inner part.In the larger granules (d >0.9mm),AOB and NOB were mostly located in the surface area of the granules,and moreover,NOB became rare.4.Discussion4.1.Relationship between granule formation and reactor performanceAfter day 32,the SVI 10stabilized at 20e 35mL g À1,which is very low compared to the values measured for activated sludge (100e 150mL g À1).However,the size distribution of the granules measured on day 32(Fig.2)indicated that only 22%of the biomass was made of granular sludge with diameter largerthan 0.2mm.These results suggest that sludge settleability increased prior to granule formation and was not affected by different particle sizes in the sludge during the GSBR operation.It was observed,however,that the diameter of the granules ﬂuctuated over longer durations.The large granules tended to destabilize due to endogenous respiration,and broke into smaller granules that could seed the formation of large granules again.Pochana and Keller reported that physically broken sludge ﬂocs contribute to lower denitriﬁcation rates,due to their reduced anoxic zone (Pochana and Keller,1999).Therefore,TN removal efﬁciency raises ﬂuctuantly throughout the experiment.Some previous research had demonstrated that bigger,more dense granules favored the enrichment of PAO (Winkler et al.,2011a ).Hence,after day 77,removal efﬁciency of TP was higher and relatively stable because the granules mass fraction was over 90%and more larger granules formed.4.2.Relationship between AOB communities dynamic and sludge granulationFor granule formation,a short settling time was set,and only particles with a settling velocity higher than 4.5m h À1were retained in the reactor.Moreover,as shown in Fig.1,the variation in SVI 10was greater before day 41(from 108.2mL g À1e 34.1mL g À1).During this phase,large amounts of biomass could not survive in the reactor.A clear shift in pop-ulations was evident,with 58%similarity between days 8and 18(Table S1).In the SBR system fed with acetate-based synthetic wastewater,heterotrophic bacteria can produce much larger amounts of extracellular polysaccharides than autotrophic bacteria (Tsuneda et al.,2003).Some researchers found that microorganisms in high shear environments adhered by extracellular polymeric substances (EPS)to resist the damage of suspended cells by environmental forces (Trinet et al.,1991).Additionally,it had been proved that the dominant heterotrophic species in the inoculating sludge were preserved throughout the process in our previous research (Zhang et al.,2011).It is well known that AOB are chemoau-totrophic and slow-growing;accordingly,numerous AOBw a t e r r e s e a r c h x x x (2011)1e 106populations that cannot become big and dense enough to settle fast were washed out from the system.As a result,the variation in AOB was remarkable in the period of sludge inoculation,and the diversity index of population decreased rapidly.After day 45,AOB communities’structure became stable due to the improvement of sludge settleability and the retention of more biomass.These results suggest that the short settling time (selection pressure)apparently stressed the biomass,leading to a violent dynamic of AOB communities.Further,these results suggest that certain populations may have been responsible for the operational success of the GSBR and were able to persist despite the large ﬂuctuations in pop-ulation similarity.This bacterial population instability,coupled with a generally acceptable bioreactor performance,is congruent with the results obtained from a membrane biore-actor (MBR)for graywater treatment (Stamper et al.,2003).Nitrosomonas e like and Nitrosospira e like populations are the dominant AOB populations in wastewater treatment systems (Kowalchuk and Stephen,2001).A few previous studies revealed that the predominant populations in AOB communities are different in various wastewater treatment processes (Tawan et al.,2005;Thomas et al.,2010).Some researchers found that the community was dominated by AOB from the genus Nitrosospira in MBRs (Zhang et al.,2010),whereas Nitrosomonas sp.is the predominant population in bioﬁlter sludge (Yin and Xu,2009).In the currentstudy,Fig.5e DGGE proﬁle of the AOB communities in different size of granules (lane labels along the top show the range of particle diameter (d,mm)).Values along the bottom indicate the Shannon diversity index (H ).Bands labeled with the numbers were consistent with the bands in Fig.3.w a t e r r e s e a r c h x x x (2011)1e 107sequence analysis revealed that selection pressure evidently effect on the survival of Nitrosospira in granular sludge.Almost all of Nitrosospira were washed out initially and had no chance to evolve with the environmental changes.However,some members of Nitrosomonas sp.have been shown to produce more amounts of EPS than Nitrosospira ,especially under limited ammonia conditions (Stehr et al.,1995);and this feature has also been observed for other members of the same lineage.Accordingly,these EPS are helpful to communicate cells with each other and granulate sludge (Adav et al.,2008).Therefore,most of Nitrosomonas could adapt to this challenge (to become big and dense enough to settle fast)and were retained in the reactor.At the end of reactor operation (day 180),granules with different particle size were sieved.The effects of variation in granules size on the composition of the AOBcommunitiesFig.6e Micrographs of FISH performed on three size classes of granule sections.DAPI stain micrographs (A,D,G);AOB appear as green ﬂuorescence (B,E,H),and NOB appear as red ﬂuorescence (C,F,I).Bar [100m m in (A)e (C)and (G)e (I).d,particle diameter.(For interpretation of the references to colour in this ﬁgure legend,the reader is referred to the web version of this article.)w a t e r r e s e a r c h x x x (2011)1e 108were investigated.As shown in Fig.5,AOB communities structures in different size of granules were varied.Although several predominant bands(bands2,5,11)were present in all samples,only bands3and6appeared in the granules with diameters larger than0.6mm.Additionally,bands7and10 were intense in the granules larger than0.45mm.According to Table2,it can be clearly indicated that Nitrosospira could be retained merely in the granules larger than0.6mm.Therefore, Nitrosospira was not present at a high level in Fig.3due to the lower proportion of larger granules(d>0.6mm)in TSS along with reactor operation.DGGE analysis also revealed that larger granules had a greater microbial diversity than smaller ones. This result also demonstrates that more organisms can survive in larger granules as a result of more space,which can provide the suitable environment for the growth of microbes(Fig.6).4.3.Effect of variance in particle size on the distribution of AOB and NOB in granulesAlthough an inﬂuence of granule size has been observed in experiments and simulations for simultaneous N-and P-removal(de Kreuk et al.,2007),the effect of granule size on the distribution of different biomass species need be revealed further with the assistance of visible experimental results, especially in the same granular sludge reactors.Related studies on the diversity of bacterial communities in granular sludge often focus on the distribution of important functional bacteria populations in single-size granules(Matsumoto et al., 2010).In the present study,different size granules were sieved,and the distribution patterns of AOB and NOB were explored.In the nitriﬁcation processes considered,AOB and NOB compete for space and oxygen in the granules(Volcke et al.,2010).Since ammonium oxidizers have a higheroxygen afﬁnity(K AOBO2<K NOBO2)and accumulate more rapidly inthe reactor than nitrite oxidizers(Volcke et al.,2010),NOB are located just below the layer of AOB,where still some oxygen is present and allows ready access to the nitrite produced.In smaller granules,the location boundaries of the both biomass species were distinct due to the limited existence space provided by granules for both microorganism’s growth.AOB exist outside of the granules where oxygen and ammonia are present.Medium granules can provide broader space for microbe multiplying;accordingly,AOB spread out in the whole granules.This result also conﬁrms that oxygen could penetrate deep into the granule’s core without restriction when particle diameter is less than0.6mm.Some mathematic model also supposed that NOBs are favored to grow in smaller granules because of the higher fractional aerobic volume (Volcke et al.,2010).As shown in the results of the batch experiments(Zhang et al.,2011),nitrite accumulation temporarily occurred,accompanied by the more large gran-ules(d>0.9mm)forming.This phenomenon can be attrib-uted to the increased ammonium surface load associated with larger granules and smaller aerobic volume fraction,resulting in outcompetes of NOB.It also suggests that the core areas of large granules(d>0.9mm)could provide anoxic environment for the growth of anaerobic denitriﬁcans(such as Tb.deni-triﬁcans or Tb.thioparus in Fig.S7,Supporting information).As shown in Fig.2and Fig.S3,the removal efﬁciency of total nitrogen increased with formation of larger granules.5.ConclusionsThe variation in AOB communities’structure was remarkable during sludge inoculation,and the diversity index of pop-ulation decreased rapidly.Most of Nitrosomonas in the inocu-lating sludge were retained because of their capability to rapidly adapt to the settling e washing out action.DGGE anal-ysis also revealed that larger granules had greater AOB diversity than that of smaller ones.Oxygen penetration was not restricted in the granules of less than0.6mm particle diameter.However,the larger granules(d>0.9mm)can result in the smaller aerobic volume fraction and inhibition of NOB growth.Henceforth,further studies on controlling and opti-mizing distribution of granule size could be beneﬁcial to the nitrogen removal and expansive application of granular sludge technology.AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation of China(No.51108456,50908227)and the National High Technology Research and Development Program of China(No.2009AA06Z312).Appendix.Supplementary dataSupplementary data associated with this article can be found in online version at doi:10.1016/j.watres.2011.09.026.r e f e r e n c e sAdav,S.S.,Lee, D.J.,Show,K.Y.,2008.Aerobic granular sludge:recent advances.Biotechnology Advances26,411e423.APHA-AWWA-WEF,2005.Standard Methods for the Examination of Water and Wastewater,ﬁrst ed.American Public Health Association/American Water Works Association/WaterEnvironment Federation,Washington,DC.de Bruin,L.M.,de Kreuk,M.,van der Roest,H.F.,Uijterlinde,C., van Loosdrecht,M.C.M.,2004.Aerobic granular sludgetechnology:an alternative to activated sludge?Water Science and Technology49,1e7.de Kreuk,M.,Heijnen,J.J.,van Loosdrecht,M.C.M.,2005.Simultaneous COD,nitrogen,and phosphate removal byaerobic granular sludge.Biotechnology and Bioengineering90, 761e769.de Kreuk,M.,Picioreanu,C.,Hosseini,M.,Xavier,J.B.,van Loosdrecht,M.C.M.,2007.Kinetic model of a granular sludge SBR:inﬂuences on nutrient removal.Biotechnology andBioengineering97,801e815.Downing,L.S.,Nerenberg,R.,2008.Total nitrogen removal ina hybrid,membrane-aerated activated sludge process.WaterResearch42,3697e3708.Erguder,T.H.,Boon,N.,Vlaeminck,S.E.,Verstraete,W.,2008.Partial nitriﬁcation achieved by pulse sulﬁde doses ina sequential batch reactor.Environmental Science andTechnology42,8715e8720.w a t e r r e s e a r c h x x x(2011)1e109。

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Optimizationofozonetreatmentoffresh-cutgreenleaflettuceHülyaÖlmeza,*,MeltemYesilçimenAkbasbaTÜB_ITAKMarmaraResearchCenter,FoodInstitute,POBox21,41470Gebze,Kocaeli,Turkey

bDepartmentofBiology,GebzeInstituteofTechnology,POBox141,41400Gebze,Kocaeli,Turkey

articleinfoArticlehistory:Received19February2008Receivedinrevisedform13July2008Accepted18July2008Availableonline3August2008

Keywords:OzoneGreenleaflettuceOptimizationListeriamonocytogenesOverallvisualqualitySanitizerVitaminCRespirationBrowningChemicaloxygendemand

abstractTheoptimizationofozonetreatmentforfresh-cutgreenleaflettucewasstudiedtodeterminetheeffectsofozoneconcentration(0.5–4.5ppm)andexposuretime(0.5–3.5min)onListeriamonocytogenescountsandtheoverallvisualqualityoflettuce.Priortotheoptimizationstudy,theeffectoftemperatureontheefﬁcacyofozonetreatmentwasevaluatedintherangeof10–26°C.Nosigniﬁcanteffectoftemperatureontheefﬁcacyofozonetreatmentwasobserved.Thequalityandsafetyoflettucesamplestreatedatthedeterminedoptimumozonationcondition(2ppm)werecomparedwiththechlorinatedwater(100ppm),organicacid(0.25g/100gcitricacidplus0.50g/100gascorbicacid),andwatertreatmentsappliedat10°Cfor2min.Sampleswerestoredat4°Cfor12days.Analysisincludeaerobicmesophiliccount,Enterobactericeae,psychrotrophicbacteria,vitaminC,b-carotene,andsensoryquality.Ozonetreat-mentwasfoundtobebetterthanthechlorineandorganicacidtreatmentsinmaintainingthesensoryquality.Ó2008ElsevierLtd.Allrightsreserved.

1.IntroductionTherehasbeenanincreasingdemandforminimallyprocessedproductsinrecentyears.Similartrendsarevalidalsoforminimallyprocessedorganicproducts.Thereisademandforprocessedor-ganicproductswhichhaveashelf-lifeandqualitycomparabletothatoftheconventionalequivalents.Inensuringthesafetyandqualityoffresh-cutvegetablesforadeﬁnedperiodofshelf-life,dis-infectionisoneofthemostimportantcriticalpointsalongthepro-cessingline.Duetotheenvironmentalandhealthrisks(Dychdala,1991),theuseofchlorineisforbiddeninorganicproductioninEur-ope.Moreover,thereisatrendineliminatingchlorinefromthedisinfectionprocess.Thus,thereisaneedforalternativesanitizerstobeusedforfresh-cutvegetablesnotonlyfortheorganicfoodsectorbutalsofortheconventionalfoodprocessors.Therearenumerousstudiesintheliteratureabouttheeffectofozonetreatmentonthesafetyandqualityoficeberglettuce(Beltranetal.,2005;Hassenbergetal.,2007;KosekiandIsobe,2006;Ricoetal.,2006;Singhetal.,2002;Yuketal.,2006).How-ever,mostofthesestudieswereconcentratedmainlyontheanti-microbialefﬁcacyofozonetreatmentandlittlewasmentionedabouttheeffectofozonetreatmentonthenutritionalconstituentsandsensoryqualityoftheproduct.

Intheprocessingindustry,itisimportanttokeeptheappliedozonelevelsaslowaspossible.Itisknownthatthecorrosionpo-tentialofstainlesssteelincreasesabove1ppmozoneconcentra-tionandmostmaterialsarecompatiblewithozoneatmoderateconcentrationsof1–3ppm(Pascualetal.,2007).Forozoneexpo-sureintheworkingenvironment,theTresholdLimitValue(TLV)forlongterm(8h)andforshortterm(15min)exposuresare0.1ppmand0.3ppm,respectively.Itwouldbeeasiertomaintaintheselowlevelsofozoneintheworkingenvironmentiftheappliedozonelevelscanbekeptlow.Moreover,achievinghighdissolvedozoneconcentrationsinwateriscostlyandisdifﬁculttostabilize.Therefore,theuseofthelowestrequiredlevelsofozoneforthedis-infectionoffreshproducethroughoptimizationoftheprocessparametersisimportantandnecessary.Nooptimizationstudywasfoundintheliteratureabouttheozonetreatmentoffresh-cutleafyvegetables,consideringboththeproductqualityandtheantimicrobialefﬁcacyofthetreatment,simultaneously.Therefore,theaimofthecurrentstudywastooptimizetheozonetreatmentofgreenleaflettuceintermsofozoneconcentration,exposuretimeandtemperatureandtoassesstheeffectozonatedwaterwashingonthemicrobiological,nutri-tionalandsensoryqualityparametersofminimallyprocessedgreenleaflettuceincomparisontothecommercialpractices,thataretheaqueouschlorineandorganicacidtreatments.Moreover,thewastewaterqualityparametersofthesedifferenttreatmentswerealsocompared.

0260-8774/$-seefrontmatterÓ2008ElsevierLtd.Allrightsreserved.doi:10.1016/j.jfoodeng.2008.07.026

*Correspondingauthor.Tel./fax:+902626412300.E-mailaddress:Hulya.Olmez@mam.gov.tr(H.Ölmez).

JournalofFoodEngineering90(2009)487–494ContentslistsavailableatScienceDirectJournalofFoodEngineering

journalhomepage:www.elsevier.com/locate/jfoodeng