Antibacterial Activity of Polymeric Quaternary Ammonium Compounds Tuned by Incorporating
季铵盐与聚季铵盐复配对海生物杀灭性能的评价
季铵盐与聚季铵盐复配对海生物杀灭性能的评价栾安博;周熠;邱美坚;冯璐【摘要】The single chain quaternary ammonium salt (QAS) biocide agent AY1001 and polymeric QAS biocide agent(A and B) have been combined,in order to better control the marine creature pollution problem in the seawater circulation system of power plants,and aiming at their disadvantages,such as high foam,low eliminating rate and apt to produce drug resistance etc. The eliminating capacity of the mixed agent system for marine creatures is evaluated. The results show that the eliminating effect of adding 3 mg/L(AY1001+A) or 3 mg/L(AY1001+B) and reacting for 24 hrs.,is better than that of single biocide AY1001 with the equal concentration.%为了更好控制火力发电厂循环水系统中海生物的污染问题,针对单一非氧化性海生物杀生剂季铵盐类化合物存在泡沫多、杀生率低下、易产生抗药性等缺点,将单链季铵盐杀生剂AY1001与聚季铵盐杀生剂(A和B)进行复配,评价混合复配药剂体系对海生物的杀灭性能.结果表明:投加3 mg/L(AY1001+A)或3 mg/L(AY1001+B)作用24 h,效果优于同等浓度下单一杀生剂AY1001的效果.【期刊名称】《工业水处理》【年(卷),期】2017(037)004【总页数】3页(P51-52,98)【关键词】季铵盐;海生物杀生剂;循环冷却水【作者】栾安博;周熠;邱美坚;冯璐【作者单位】广东省石油与精细化工研究院,广东广州510645;广东省工业表面活性剂重点实验室,广东广州510645;广东省石油与精细化工研究院,广东广州510645;广东省工业表面活性剂重点实验室,广东广州510645;广东省石油与精细化工研究院,广东广州510645;广东省工业表面活性剂重点实验室,广东广州510645;广东省石油与精细化工研究院,广东广州510645;广东省工业表面活性剂重点实验室,广东广州510645【正文语种】中文【中图分类】TQ085+.4采用海水直流冷却技术时海生物会随循环水在管道中附着生长，造成流量降低、管路堵塞、传热效率低下等问题〔1〕。
纳米银在抗菌领域的研究进展
消毒工艺是生活饮用水处理中的一项重要工艺，目标是灭活水中多种病原微生物，对于保障人类的安全和健康有着重要意义。
各个国家均对饮用水的抗菌消毒予以高度重视。
传统的氯化消毒工艺过程中，氯会与水中天然有机物反应生成三卤甲烷和非挥发性的卤代有机物等消毒副产物（DBPs）。
其他的化学消毒工艺如二氧化氯、臭氧消毒等，也可能会使水中生成氯酸盐、亚氯酸盐、溴酸盐等DBPs。
DBPs对人体具有致癌、致畸、致突变的“三致”作用，严重威胁人们健康。
因此，在消毒过程避免DBPs的生成是亟待解决的难题。
而作为一种新型的抗菌消毒材料，纳米银在抗菌方面的优越性，引起了众多学者的研究。
1.纳米材料简介纳米材料是指三维空间中至少一维的尺寸介于1~100 nm之间的材料。
由于尺寸处于纳米级别，纳米材料表现出一些特有的效应，如表面效应、体积效应、量子尺寸效应和宏观量子隧道效应。
此外，纳米材料往往具有非常大的比表面积以及较高的化学活性。
这些性质有利于其抗菌能力的发挥。
常作为抗菌剂的纳米材料主要有两类：碳系纳米材料和纳米金属材料。
碳系纳米材料包括碳纳米管、氧化石墨烯等。
碳纳米材料对水中溶壁微球菌、变异链球菌、沙门氏菌属等均具有抗菌作用。
氧化石墨烯对于大肠杆菌具有很强的灭活能力。
纳米金属材料包括纳米银、纳米铁、纳米氧化锌等。
纳米金属材料由于特有的界面效应，其表面原子缺少临近的配位原子导致化学活性极强，也因此提高了对于细菌的亲和力，易于杀死细菌。
纳米铁即可在氧和无氧的条件下高效的灭活细菌。
纳米银作为最具前景的纳米金属材料之一，其抗菌方面的应用得到了越来越多的关注。
2.纳米银的抗菌研究2.1纳米银抗菌的优势在众多的纳米材料之中，纳米银（nAg）脱颖而出，被广泛研究主要得益于以下特性。
nAg的抗菌活性极高。
银的杀菌能力是锌的上千倍。
银离子对多种革兰氏阴性菌、革兰氏阳性菌、霉菌等均有广谱、强烈的杀灭作用，这是其作为抗菌材料被研究的基础。
许多学者就nAg对细菌的抗菌性能进行了深入研究。
基于网络药理学研究枸杞提取液对肠道致病菌的影响
李倩，廖嘉宁，魏媛，等. 基于网络药理学研究枸杞提取液对肠道致病菌的影响[J]. 食品工业科技，2024，45（2）：12−20. doi:10.13386/j.issn1002-0306.2023060070LI Qian, LIAO Jianing, WEI Yuan, et al. Study on the Effect of Goji Berry Extract on Intestinal Pathogenic Bacteria Based on Network Pharmacology[J]. Science and Technology of Food Industry, 2024, 45(2): 12−20. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023060070· 特邀主编专栏—枸杞、红枣、沙棘等药食同源健康食品研究与开发（客座主编：方海田、田金虎、龚桂萍） ·基于网络药理学研究枸杞提取液对肠道致病菌的影响李 倩，廖嘉宁，魏 媛，王贞香，安 琼*（河西学院医学院，甘肃省河西走廊特色资源利用重点实验室，甘肃张掖 734000）摘 要：目的：采用网络药理学分析方法研究枸杞的抑菌作用机制，并进行体外抑菌实验验证。
方法：用中药系统药理学在线分析数据库TCMSP 筛选出枸杞活性成分及其作用靶点。
以“抑菌”为关键词在GeneCards 、Dis-GeNET 数据库检索相关靶点。
通过Venny 平台获取枸杞和抑菌的交集靶点。
使用STRING 数据库结合Cyt-oscape 软件构建PPI 网络并筛选关键靶点。
运用Metascape 数据库进行GO 功能富集和KEGG 通路富集分析。
枸杞经过80%乙醇提取，浓缩至浸膏，分别用石油醚、二氯甲烷、乙酸乙酯和正丁醇萃取，得到枸杞的不同极性部位，用滤纸片法测定枸杞不同极性部位的抑菌作用。
国内外常见抗菌防霉标准
中国
卫生部《消毒技术规范》-2002版
国家技术法规
在卫生部系统普遍采用。是消毒剂/卫生用品的试验方法标准。
GBT 20944.1-2007纺织品抗菌性能的评价第1部分：琼脂扩散法GBT 20944.2-2007纺织品抗菌性能的评价第2部分：吸收法
纺织品，抗细菌
是ISO 20743/日本JIS L 1902标准的等同采用。
ISO 846-1978（E）塑料在真菌和细菌作用下的行为的测试--用直观检验法或用测量质量或物性变化的方法评价
塑料，抗细菌/防霉
是用来检测霉菌作用对塑料的破坏效果的，但试验方法可用于防霉检测。
2
中国
QB/T 2591-2003抗菌塑料——抗菌性能评价及其测试方法
塑料，抗细菌/防霉
是我国影响很大的抗菌材料抗菌测试标准，被广泛用于坚硬固体表面抗菌性能的评价，是ISO 21996/日本JIS Z 2801标准非等同采用的标准。
国内外常见抗菌防霉标准
序号
国家和组织
标准号和标准名称
适用
范围
备注
1
国际标准化组织
ISO 22196-2007塑料制品表面抗菌性能评价方法Plastics Measurement of antibacterial
塑料，
抗细菌
是日本JIS Z 2801标准和中国QB/T 2591标准的对应国际方法标准。
ASTM G21-96（2002）Standard Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi合成聚合材料防霉（耐真菌）性能测试标准
合成聚合物，防霉
美国材料试验协会标准
新型羊毛织物抗菌防缩功能整理
新型羊毛织物抗菌防缩功能整理俞鑫龙海如(东华大学纺织学院，上海201620)摘要：纺织服装面料的抗菌后整理是当今研究的一个热点。
本文介绍一种新型的羊毛织物抗菌防缩整理，同时研究了整理后毛织物的防缩水和抗菌性能。
实验表明由过一硫酸氢钾复合盐（PMS）和亚硫酸钠预处理的毛织物，在浸染和轧染工艺中，均能与常用杀菌剂聚六亚甲基双胍（PHMB）反应，同时使羊毛织物具有防缩抗菌双重性能。
文章进而研究了PHMB浓度和PHMB处理工艺对于重复洗涤后毛织物防缩和抗菌性能的影响。
关键词：羊毛织物，抗菌整理，防缩水，PMS，亚硫酸钠，PHMB。
A Novel functionally antimicrobial and shrink resistanttreatment on wool textilesYU Xin, LONG Hairu(College of Textiles, Donghua University, Shanghai 201620, China)Abstract:Antibacterial finishing on fabrics and garments is one of hot research topics. In this research, it presented a novel functionally antibacterial and shrink resistant treatment on wool fabrics. The antimicrobial activity and shrink resistance have been investigated. After the pre-treatment with Potassium Monopersulfate（PMS）and sodium sulphite, wool fabrics could react with a common antimicrobial agent Polyhexamethylene Biguanide (PHMB) in both exhaustion and padding procedures. The disposition of PHMB brought wool dual property of shrink resistance and antimicrobial activity. The study further examined the influence of the concentration of PHMB and the treatment procedure on the durable antimicrobial activity and shrink resistant properties after repeated laundering.Key words: Wool, Antibacterial Treatment, Shrink Resistance, PMS, Sodium Sulphite, PHMB.1 背景介绍在适宜的温度和湿度下，天然纤维特别是羊毛纤维能给微生物提供良好生长环境[1]。
抗菌性聚乙烯醇包装材料的研究进展
抗菌性聚乙烯醇包装材料的研究进展梁真真白绘宇*（江南大学化学与材料工程学院，无锡214122，江苏）摘要：抗菌材料能够有效抑制细菌的生长和繁殖，将其应用于生产生活中，对保护人类健康有着十分重要的意义。
安全和环保是目前抗菌包装材料面临的两个主要问题，解决这两个问题将会给包装材料带来更广阔的发展空间。
聚乙烯醇（PVA）是一种水溶性高分子材料，具有可降解、生物相容、安全、无毒和环保等特性，在包装材料领域有着广阔的应用前景。
但是，PVA材料也存在着耐水性差、水蒸气阻隔性差和抗菌活性低等缺点，难以满足实际生产生活的要求。
为了实现PVA在抗菌材料领域的广泛应用，需要对其进行改性以制备具有抗菌性能、高机械性能和耐水性的PVA基复合材料。
本文对近年来抗菌性高性能PVA基复合材料进行综述，该材料有望应用于食品和药物包装等领域。
关键词：抗菌性聚乙烯醇包装Research progress on antibacterial polyvinyl alcohol for packagingLiang Zhenzhen Bai Huiyu*（School of Chemical and MaterialEngineering, Jiangnan University, Wuxi 214122,Jiangsu,China）Abstract：Antibacterial materials can effectively inhibit the growth and reproduction of bacteria. The application of antibacterial materials to life and production is of great significance for the protection of humanhealth. Safety and environmental protection are two main problems for the antibacterial packagingmaterials, and solving these two problems will bring a boarder development space for these materials.Poly(vinyl alcohol) (PVA) is a kind of water-soluble polymer material, which has the characteristicsof biodegradability, biocompatibility, safety, non-toxicity, and environmentally friendly.It has broadapplication prospects in the field of packaging materials. However, PVA materials also haveshortcomings such as poor water resistance, poor water vapor barrier properties, and low antibacterialactivity, which are difficult to meet the requirements of actual production and life. In order tobroadenthe application of PVA in the field of antibacterial materials, it is necessary t o modify it andprepare a PVA-based composite with antibacterial properties, high mechanical properties, and waterresistance. In this paper, the antibacterial high-performance PVA-based composites are reviewed inrecent years, and the composites are expected to be applied in the fields of food and pharmaceuticalpackaging.Keywords：Antibacteria polyvinyl alcohol packaging引言近年来，人类在利用微生物有益性的同时，也十分警惕其作为病原菌的危害。
Chemical Composition and Antibacterial Activity of Essential Oil
CHEMISTRY&BIODIVERSITY–Vol.7(2010)229Chemical Composition and Antibacterial Activity of Essential Oils fromDifferent Parts of Litsea cubebaby Hongwu Wang*and Yanqing LiuDepartment of Chemistry,Zhaoqing University,Zhaoqing,Guangdong526061,P.R.China (phone:þ86-758-2716366;fax:þ86-758-2716994;e-mail:hwwangcn@)The composition of the essential oils obtained by hydrodistillation of different parts of Litsea cubeba, including roots,stems,leaves,alabastra(flower buds),flowers,and fruits,were investigated by GC(RI) and GC/MS.The antimicrobial activity of the oils was assessed with disc diffusion and microbroth dilution assays.The results showed large variations in the composition among the different oils.The major components in the oils from roots and fruits,from stems,leaves,and alabastra,and from flowers were citral B(neral),b-phellandrene,and b-terpinene,respectively.The inhibition zone(DD)and MIC values for the bacterial strains tested,which were all sensitive to the essential oil of L.cubeba,were in the range of10.1–35.0mm and100–1000m g/ml,respectively.Hence,the oils of the various parts showed moderate activity against the tested bacteria.This investigation showed that the antibacterial activity of L.cubeba was attributed to the essential oils,thus they can be a potential medicinal resource.Introduction.–Litsea cubeba(L our.)P ers.(LC;Lauraceae)is a plant located in Southern China,Japan,and Southeastern Asia.The fruit of LC is known in the traditional Chinese medicine as Chen-Qie-Zi and has been used in the treatment of stomach cold hiccough,gastric cavity crymodynia,cold hernia celialgia,and stagnancy of cold-damp,whereas the fruit of LC has been used as an antibacterial and anti-inflammatory agent[1].The fruit of LC contains alkaloids and sesquiterpenes[2].Some studies have demonstrated that LC possesses bioactivities,such as antioxidant[3]and anti-inflammatory activities[4],and dermal sensitization potential[5].In addition,MeOH extracts of the bark of LC decreased the activity of myeloperoxidase,an enzyme that catalyzes the oxidation of ClÀto HOCl[4],and extracts of LC have shown anti-insect activity[6–7].Variation in the chemical composition of essential oils,in particular,and of extracts of medicinal plants may be observed due to the origin,the environmental conditions, and the developmental stage of the collected plant material.The antimicrobial activity of an essential oil is attributed mainly to its major components,although the synergistic or antagonistic effect of a minor compound of the mixture has to be considered[8]. Therefore,antimicrobial,antioxidant,and other biological activities may vary,based on the variations in the chemical composition[9].LC is widely used in the traditional Chinese medicine,but the active components of LC and their pharmacological effects are still unclear.In the present article,the essential oils were obtained from the root,stem,leaf,alabastrum,flower,and fruit,and their antibacterial activity was investigated against six bacteria with agar disc diffusion2010Verlag Helvetica Chimica Acta AG,ZürichCHEMISTRY&BIODIVERSITY–Vol.7(2010)230and microbroth dilution methods.The compositions of the essential oils from the root, stem,leaf,alabastrum,and fruit of LC were also analyzed by GC(RI)and GC/MS. These results can be used to investigate the optimal harvesting part of this plant for relevant industries.Results and Discussion.–Chemical Composition of the Essential Oil.The essential oils were obtained by hydrodistillation from air-dried parts of L.cubeba and subsequently analyzed by GC(RI)and GC/MS.The identified components with their relative percentages are reported in Table1,and the chemical structures of some lesser known compounds are shown in the Figure.The yields of oils(%,w/w)of the different parts decreased in the order of:flower(3.1%)>alabastrum(2.3%)>fruit(2.1%)> leaf(1.3%)>root(0.31%)>stem(0.29%).In total,33,29,33,27,27,and29 constituents were identified and quantified in the various parts,respectively.Oxy-genated monoterpenes are the major constituents of roots and fruits,and monoterpene hydrocarbons are the major constituents of the other parts(Table1).In the root oil of LC,citral B(neral;21.53%),citronellal(8.57%),linalool(7.45%), isopulegol(6.05%),and b-phellandrene(5.24%)were the main constituents.The essential oil was dominated mainly by oxygenated monoterpenes(60.09%).Thepercentage of monoterpene hydrocarbons,sesquiterpene hydrocarbons,and hydro-CHEMISTRY&BIODIVERSITY–Vol.7(2010)231 Table1.Chemical Composition[%]of the Essential Oils from Different Parts of Litsea cubeba Compounds RI a)Root Stem Leaf Alabastrum Flower Fruit Identification b) a-Thujene9350.37 1.56 3.47 1.74 1.85t RI,MSa-Pinene940 5.41 6.94 3.258.957.51 2.87RI,MS Camphene952 1.91 1.9 2.340.97t c) 3.12RI,MSb-Phellandrene971 5.2118.74 5.2333.74 5.75 1.01RI,MS,CoIb-Pinene979 1.417.12 3.578.337.25 1.37RI,MS Methyl heptenone986 4.320.65 1.23t t 3.54RI,MS6-Methylhept-5-en-2-one9860.87––––0.78RI,MSb-Myrcene991 1.01 2.7 1.71 3.24 3.59 1.21RI,MS3-Carene1009 1.70–0.437.3 2.62t RI,MSa-Terpinene1026 1.25 4.67 1.670.570.27–RI,MSp-Cymene10270.65–0.78–– 2.14RI,MS Cineole1031 1.34t13.9711.2413.680.49RI,MS Limonene1032 4.279.82 6.780.75 5.437.38RI,MSb-trans-Ocimene1036–– 2.15–––RI,MSb-Terpinene1059––– 2.1833.17–RI,MSg-Terpinene1068 1.27 5.23 1.52 1.490.870.17RI,MS Linalool11007.450.87 6.940.17 1.06 1.34RI,MS2,6-Dimethylocta-2,4,6-triene11300.23 1.18–0.18––RI,MSa-Thujanol1133 1.379.430.54 3.39 4.26 1.72RI,MS,CoI Camphor1140 1.700.680.87––t RI,MS(E)-p-Menth-2-en-1-ol1140 1.38 1.310.31 1.280.27t RI,MS Citronellal11538.750.130.10.13t 1.57RI,MS Terpinen-4-ol1179 2.5712.05 2.03 3.99 2.240.82RI,MSa-Terpineol1195 1.27 2.39 1.07 3.8 5.410.32RI,MS Citronellol1230 4.430.38 1.37–t t RI,MSCitral B125421.53 5.27t 1.95t63.57RI,MS,CoI Eugenol12720.720.64 1.24–––RI,MS Geraniol1276 2.250.25–––0.99RI,MSBornyl acetate12890.380.12 5.34–––RI,MS Caryophyllene1296 1.89 2.478.04 2.34 1.9t RI,MS,CoIb-Elemene1333 1.37 1.34 5.870.44t t RI,MSa-Farnesene13530.270.670.710.630.120.21RI,MSSafrol1357––––– 1.12RI,MS Isopulegol1365 6.050.37t t t–RI,MS Geranyl acetate1382–– 4.32t t 1.01RI,MSn-trans-Nerolidol1388–– 3.54––0.73RI,MSg-Elemene1498 2.47–8.27–0.78–RI,MSSelin-6-en-4-ol16850.780.520.210.35––RI,MS Monoterpene hydrocarbons24.0459.8632.1269.4468.3117.13Oxygenated monoterpenes60.0933.1327.225.9526.9270.52Sesquiterpene hydrocarbons 6.00 4.4822.89 3.41 2.800.21Oxygenated sesquiterpenes0.780.52 3.750.35–0.73Others d) 6.94 1.4112.91––8.59Total97.8599.498.8799.1598.0397.18Oil yield(w/w%) 1.7 1.9 2.1 2.3 2.5 2.2No of components332933272729a)RI:Retention index relative to n-alkanes(C–C24)on an RTX-5MS column.b)RI:Comparison of retention6indices with the NIST2005GC/MS library data of the GC/MS system;MS:comparison of the mass spectra with those of the internal reference mass spectral library;CoI:coinjection with an authentic sample.c)t:Trace (<0.1%)d)Others:Hydrocarbons such as alkanes and esters.carbons such as alkanes and esters,classified as others ,were 24.04,6.00,and 6.94%,respectively (Table 1).In addition,root essential oil also contained some oxygenated sesquiterpenes (0.78%).In the stem oil of LC,b -phellandrene (18.74%),terpinene-4-ol (12.05%),limonene (9.82%),a -thujano1(9.43%),and b -pinene (6.94%)were the main components.The major constituents in the essential oil were monoterpene hydrocarbons (59.86%),followed by oxygenated monoterpenes (33.13%),sesquiterpene hydrocarbons (4.48%), others (1.41%),and oxygenated sesquiterpenes (0.52%).As for the leaf oil of LC,the main constituents were cineole (13.97%),g -elemene (8.27%),caryophyllene (8.04%),linalool (6.94%),and limonene (6.78%).Further-more,leaf essential oil was composed of monoterpene hydrocarbons (32.12%),oxygenated monoterpenes (27.2%),sesquiterpene hydrocarbons (22.89%), others (12.91%),and oxygenated sesquiterpenes (3.75%).In the alabastrum oil of LC,b -phellandrene (33.74%),cineole (11.24%),a -pinene (8.95%),and b -pinene (8.33%)were the main constituents.The alabastrum essential oil comprised monoterpene hydrocarbons (69.44%),oxygenated monoterpenes (25.95%),sesquiterpene hydrocarbons (3.41%),and oxygenated sesquiterpenes (0.35%).In the flower oil of LC,the contents of b -terpinene,cineole,a -pinene,and b -pinene were 33.17,13.68,7.51,and 7.25%,respectively.The flower essential oil was dominated mainly by monoterpene hydrocarbons (68.31%),and the percentages of oxygenated monoterpenes and sesquiterpene hydrocarbons were 26.92and 2.80%,respectively.In the fruit oil of LC,citral B (neral;63.75%)and limonene (7.38%)were the main constituents.Fruit essential oil was composed of oxygenated monoterpenes (70.52%),monoterpene hydrocarbons (17.13%), others (8.59%),oxygenated sesquiterpenes (0.73%),and sesquiterpene hydrocarbons (0.21%).Antibacterial Activity .The diameters of inhibition zones obtained in the disk diffusion assay (DD)and the minimal inhibitory concentrations obtained in the microbroth dilution assay (MIC)of the essential oils of LC against six microorganisms are shown in Table 2.The essential oils of LC revealed antibacterial activity against all six bacteria tested.The DD and MIC values were in the range of 10.1to 35.0mm and 100to 1000m g/ml,pared to the positive antibacterial standard,levofloxacin,the essential oils of LC have moderate antibacterial activity.Conclusions.–The data presented confirmed the antibacterial potential of LC essential oil and suggest that the antibacterial activities of LC can be attributed to essential oil constituents.Hence,the essential oils from the different parts of LC tested represent an inexpensive source of natural antibacterial substances to prevent the growth of bacteria and could be a potential medicinal resource.We thank Vice-Prof.Xiongwei Chen for the identification of plant materials.Experimental PartPlant Material .All parts (root,stem,leaf,floral buds,flowering and seed set)of Litsea cubeba (L our .)P ers.were collected from Southern regions of China by the authors.The plant was identified byCHEMISTRY &BIODIVERSITY –Vol.7(2010)232CHEMISTRY &BIODIVERSITY –Vol.7(2010)233T a b l e 2.A n t i b a c t e r i a l A c t i v i t y o f t h e E s s e n t i a l O i l s f r o m D i f f e r e n t P a r t s o f L i t s e a c u b e b aM i c r o o r g a n i s m sS t e mA l a b a s t r u m L e a f F l o w e r R o o tF r u i tL e v o f l o x a c i nD D a )M I C b )D D M I C D DM I C D DM I C D DM I C D DM I C D D M I CB .s u b t i l i s A TC C 650115.7Æ1.2370012.1Æ1.3290010.1Æ1.27100025.1Æ0.8740033.0Æ1.5215034.5Æ1.0010030.1Æ1.171.7E .c o l i A T C C 2592230.2Æ1.3228034.0Æ1.1220035.0Æ0.7815028.5Æ1.0130025.1Æ0.5150024.1Æ1.0050010.1Æ0.5848.1E .f a e c a l i s A T C C 1575320.5Æ0.7280019.1Æ0.2785020.1Æ1.4580011.7Æ1.03100017.8Æ1.2055018.7Æ1.5060028.0Æ1.015.35M .a l b i c a n s A T C C 6454830.5Æ1.0126033.4Æ1.4118032.0Æ1.0420018.0Æ1.2455015.1Æ1.4267014.1Æ1.1870022.5Æ0.781.12P .a e r u g i n o s a A T C C 2785215.9Æ1.2875017.7Æ1.0765018.1Æ1.5060020.1Æ0.8540018.5Æ1.7862018.5Æ0.8962020.1Æ0.132.15S .a u r e u s A T C C 2592328.3Æ1.0830029.7Æ1.3520030.0Æ0.5118017.1Æ1.2160029.8Æ0.8725030.0Æ0.1220025.3Æ0.860.95a)D D :D i a m e t e r o f i n h i b i t i o n z o n e [m m ]a r o u n d t h e d i s k s (6m m )i m p r e g n a t e d w i t h e s s e n t i a l o i l (3m g /d i s k )o r t h e p o s i t i v e c o n t r o l l e v o f l o x a c i n (5m g /d i s k ).b)M I C :M i n i m u m i n h i b i t o r y c o n c e n t r a t i o n s [m g /m l ].Vice-Prof.Xiongwei Chen ,Zhaoqing University,China.Voucher specimens were deposited with the Herbarium of the Department of Chemistry,Zhaoqing University,China.Isolation of the Essential Oils .The essential oils of all air-dried samples (100g each)were obtained by hydrodistillation on a Clevenger -type apparatus for 3h,according to the method recommended in the Chinese Pharmacopoeia [10].The distill oils were dried (Na 2SO 4)and stored in tightly closed,dark vials at 48until analysis.The oils were yellow in color and had a distinct sharp odor.Anal.GC.Essential oils of LC were analyzed with a GC QP2010system (Shimadzu Ltd .,Japan)equipped with FID and a silica cap.column RTX-5MS (30m Â0.25mm i.d.,film thickness 0.25m m).Oven temp.program:808for 2min,increase to 1208at 28/min,held isothermal at 1208for 1min,increase to 2008at 58/min,and held isothermal at 2008for 2min;injector temp.,2208;detector temp.,2508;carrier gas,N 2(1.0ml/min);injection volume,1.0m l.The percentages of the constituents were calculated by electronic integration of FID peak areas without response factor correction.Anal.GC/MS.Analysis was performed with a GC/MS QP2010Plus system (Shimadzu Ltd .,Japan)equipped with a silica cap.column RTX-5MS (30m Â0.25mm i.d.,film thickness 0.25m m).Oven temp.conditions were as described above.Injector temp.,2508;split flow,50ml/min;carrier gas,He (1.0ml/min).Diluted sample (1.0m l,1:10in Et 2O)was injected manually.MS:at 70eV;mass range 35–425amu;ion source temp.,2008;interface temp.2508.Identification of Compounds .The constituents of the essential oils were identified by calculation of their RI relative to n -alkanes (C 6–C 24)on an RTX-5MS column.Individual compounds were identified by comparison of their mass spectra with those of the internal reference mass spectral library or with authentic compounds and confirmed by comparison of their RI with the NIST 2005GC/MS library data of the GC/MS system and Adams spectral library [11].For the quantification,relative area percentages obtained by FID were used without the use of correction factors.Antibacterial Activity.Agar Disk Diffusion Assay.The agar disc diffusion method [12–15]was employed to determine the diameters of inhibition zones of essential oils [16][17]against the six tested bacteria,viz .,Bacillus subtilis ATCC 6501,Enterococcus faecalis ATCC 15753,Escherichia coli ATCC 25922,Monilia albicans ATCC 64548,Pseudomonas aeruginosa ATCC 27852,and Staphylococcus aureus ATCC 25923.Inoculums (1ml)of the tested bacteria (2Â108colony forming units,CFU/ml)were poured and spread uniformly on the solid media plates.Filter paper discs (6mm diameter)were impregnated with the essential oils (3mg/disk)dissolved in Tween 80(0.5%)and fractions dissolved in DMSO.Levofloxacin (5m g/disk)was used as positive control,and 0.5%Tween 80and DMSO were used as negative controls.The discs were applied on the agar surface,and the plates were incubated at 378for 24h.The diameters of the inhibition zones (DDs)were measured in mm using a Vernier caliper [18].Results are given as mean values of triplicates.Microbroth Dilution Assay .Minimum inhibitory concentrations (MICs),defined as the lowest concentrations of the oils that result in a complete inhibition of visible growth of microorganisms in the broth,were determined according to the method of the NCCLS [19–22].All tests were performed in Mueller–Hinton broth.The essential oils were dissolved in Tween 80and DMSO at a final concentration of 0.5%(v /v ).Serial doubling dilutions of each essential oil were prepared in a 96microtiter plate to get final concentrations ranging from 0.01to 200.0m g/ml,0.01to 6m g/ml,resp.Overnight,broth cultures of each strain were prepared,the final concentration in each positive well was adjusted to 5Â104CFU/ml and the plates were incubated at 378for 24h.The MIC value was defined as the lowest concentration of the samples at which the bacterium did not show any visual growth after macroscopic evaluation.Results are given as mean values of triplicates.REFERENCES[1]Nanjing University of TCM,in Dictionary of Chinese Materia Medica ,2nd edn.,Science andTechnology Press of Shanghai,Shanghai,2006,p.3710.[2]Y.-C.Wu,J.-Y.Liou,C.-Y.Duh,S.-S.Lee,S.-T.Lu,Tetrahedron Lett.1991,32,4169.[3]J.-K.Hwang,E.-M.Choi,J.H.Lee,Fitoterapia 2005,76,684.[4] E.-M.Choi,J.-K.Hwang,Fitoterapia 2004,75,141.CHEMISTRY &BIODIVERSITY –Vol.7(2010)234CHEMISTRY&BIODIVERSITY–Vol.7(2010)235[5]lko,A.M.Api,Food Chem.Toxicol.2006,44,739.[6] A.Amer,H.Mehlhorn,Parasitol.Res.2006,99,478.[7]Z.L.Liu,S.-H.Goh,S.H.Ho,J.Stored Prod.Res.2007,43,290.[8]S.Burt,Int.J.Food Microbiol.2004,94,223.[9] A.Y.Leung,S.Foster,in Encyclopedia of Common Natural Ingredients Used in Foods,Drugs,andCosmetics ,2nd edn.,John Wiley&Sons,New York,1996,p.465.[10] Chinese Pharmacopoeia ,Chemical Industry Press,Beijing,2005,Spl.p.57.[11] Identification of essential components by gas chromatography/quadrupole mass spectroscopy ,3rdedn.,Ed.R.P.Adams,Allured Publishing Corporation,Carol Stream,Illinois,2001.[12]S.A.Burt,R.D.Reinders,Lett.Appl.Microbiol.2003,36,162.[13]M.L.Faleiro,M.G.Miguel,deiro,F.Venaˆncio,R.Tavares,J.C.Brito,A.C.Figueiredo,J.G.Barroso,L.G.Pedro,Lett.Appl.Microbiol.2003,36,35.[14]L.Faleiro,G.Miguel,S.Gomes,L.Costa,F.Venaˆncio,A.Teixeira,A.C.Figueiredo,J.G.Barroso,L.G.Pedro,J.Agric.Food Chem.2005,53,8162.[15]P.R.Murray, E.J.Baron,M.A.Pfaller, F.C.Tenover,R.H.Yolke, Manual of ClinicalMicrobiology ,6th edn.,ASM,Washington,1995.[16]L.Cao,J.Y.Si,Y.Liu,H.Sun,W.Jin,Z.Li,X.H.Zhao,R.L.Pan,Food Chem.2009,115,801.[17]National Committee for Clinical Laboratory Standards(NCCLS), Performance Standards forAntimicrobial Disk Susceptibility Tests ,6th edn.,Approved Standard M2-A6,Wayne,PA,1997.[18]J.Kim,M.R.Marshall,C.Wei,J.Agric.Food Chem.1995,43,2839.[19] C.D.Djipa,M.Delme´e,J.Quetin-Leclercq,J.Ethnopharmacol.2000,71,307.[20]National Committee for Clinical Laboratory Standards(NCCLS), Performance Standards forAntimicrobial Susceptibility Testing ,9th Informational Supplement,M100-S9,Wayne,PA,1999.[21]National Committee for Clinical laboratory Standards(NCCLS), Performance Standards forAntimicrobial Susceptibility Testing ,11th Informational Supplement,M100-S11,Wayne,PA,2001.[22]J.Yu,J.Lei,H.Yu,X.Cai,G.Zou,Phytochemistry2004,65,881.Received December12,2008。
Antibacterial Activity of Fullerene Water Suspensions_
Antibacterial Activity of Fullerene Water Suspensions:Effects of Preparation Method and Particle Size †D E L I N A Y .L Y O N ,*,‡L A U R A K .A D A M S ,‡J O S H U A C .F A L K N E R ,§A N D P E D R O J .J .A L V A R E Z *,‡Department of Civil and Environmental Engineering and Department of Chemistry,Rice University,Houston,Texas 77005Fullerene research in biological systems has beenhindered by the compound’s relative insolubility in water.However,C 60molecules can be made to aggregate,forming stable fullerene water suspensions (FWS)whose properties differ from those of bulk solid C 60.There are many different protocols for making FWS.This paper explores four of these methods and establishes the antibacterial activity of each resulting suspension,including a suspension made without intermediary solvents.The aggregates in each polydisperse suspension were separated by size using differential centrifugation and tested for antibacterial activity using Bacillus subtilis as a test organism.All suspensions exhibited relatively strong antibacterial activity.Fractions containing smaller aggregates had greater antibacterial activity,although the increase in toxicity was disproportionately higher than the associated increase in putative surface area.This suggests the need for improved understanding of the behavior of FWS towards organisms and in the environment to determine how C 60can be safely used and disposed.IntroductionSince the discovery of fullerenes in 1985this third allotrope of carbon has been a promising compound for a number of applications,such as catalysts and sensors (1-3).The cagelike structure of fullerenes allows them to encapsulate other molecules (4).These doped fullerenes can be used as medical therapeutic agents,tissue-specific fullerene-based drugs,and other medical tools (5,6).Fullerenes can also be derivatized or polymerized to tailor them to a purpose (7).For example,nanocomposites incorporating fullerenes are used in non-linear optics and photoelectrochemistry applications (8).Fullerene production has steadily increased since 1990,when a method was first developed for its mass production (9).Frontier Carbon built a plant capable of annually producing 10tons of C 60by 2007(10).The economy of fullerene production indicates that fullerene-containing products will soon be widely available.The manufacture,use,and disposal of these items are not currently regulated,although the U.S.House Scientific Committee has prioritizedlegislation of nanomaterial handling and disposal (11-13).In the interest of making informed decisions,it is imperative to determine the health and environmental implications of fullerene use and disposal.In an attempt to contribute to proactive risk management,this and previous papers have examined some of the potential impacts of fullerene use.Numerous papers on fullerene derivatives show both positive and negative health effects,depending upon the derivative,application,and organism/system of study (14-22).It must be stressed that the effects depend on the specific fullerene derivative;there is no consensus in the literature regarding the toxicity of fullerenes in general.Fewer studies have been performed on the effects of fullerene derivatives on bacteria.Most of these studies found that different derivatives had antibacterial properties,but the level of toxicity again depended on the test organism and the specific derivative (23-31).With a solubility of less than 10-9mg/L,powdered C 60is virtually insoluble in water,and studies examining C 60powder alone did not find antibacterial activity (32-34).Several papers have proposed different methods of making fullerene water suspensions (FWS)(35-39).These methods commonly involve dissolving C 60in a solvent followed by addition of water and subsequent removal of the solvent.A FWS contains nanoscale C 60aggregates in a suspension whose properties differ from those of C 60dissolved in the relevant solvent (34,40).FWS are typically yellow or brown suspensions that do not settle out over time.It has been hypothesized that water molecules stabilize the C 60molecules in the aggregates (41).In the interest of concise and consistent nomenclature in this paper,we call these stable FWS “nC 60”and use an appropriate prefix to designate the production method.One solubilization method used,adapted from Yamakoshi et al.,does not actually produce nC 60but uses a stabilizer common in the pharmaceutical industry,namely poly(vinylpyrroli-done),to encapsulate C 60molecules (38).It has been proposed that nC 60may be the most environmentally relevant form of C 60(34).In the event of a spill of C 60powder or C 60dissolved in a solvent,it is conceivable that nC 60could be formed (42).nC 60has therefore been the subject of several toxicological studies that have received widespread attention (32,34,43-47).Studies have shown nC 60to be toxic to bacteria,largemouth bass,and human cell lines (32,43,44).Each of these studies has used nC 60prepared using tetrahydrofuran (THF)as the interme-diate solvent,raising concern that the toxicity is attributable to residual solvent rather than the C 60aggregates themselves (42,48).This research has two aims:first to determine whether nC 60has antibacterial properties,regardless of solvents or solubilizers used during preparation,and second to examine how the morphology of the nC 60aggregate affects its antibacterial activity.We prepared FWS using four different methods,using THF as a solvent (THF/nC 60),sonicating C 60dissolved in toluene with water (son/nC 60),stirring C 60powder in water (aq/nC 60),and using a solubilizing agent (PVP/C 60).We tested each preparation for antibacterial activity toward the Gram-positive bacterium Bacillus subtilis .We then examined fractions of the nC 60aggregates,paying particular attention to how the size and morphology of nC 60affect antibacterial activity.Experimental SectionProducing C 60FWS by Four Different Methods.The four different types of C 60FWS were produced as described in the literature with minor alterations.*Corresponding authors phone:7133484761;fax:7133485203;e-mail:dlyon@ and alvarez@.†This paper is part of a focus group on Effects of Nanomaterials.‡Department of Civil and Environmental Engineering.§Department of Chemistry.Environ.Sci.Technol.2006,40,4360-436643609ENVIRONMENTAL SCIENCE &TECHNOLOGY /VOL.40,NO.14,200610.1021/es0603655CCC:$33.50©2006American Chemical SocietyPublished on Web 04/13/2006THF/nC60.Tetrahydrofuran/nC60,or THF/nC60,was pre-pared by following the method of Deguchi et al.and incorporating the modifications described by Fortner et al. (34,39).Briefly,100mg of99.5%pure C60(SES Research, Houston,TX,or MER Corp.,Tucson,AZ)was dissolved in4 L of spectra-analyzed THF(Fisher Scientific,Houston,TX). The THF was flushed with nitrogen to prevent oxidation prior to the mixture being stirred overnight at room temperature. The solution was then filtered through a0.22µm Osmonics nylon membrane(Fisher Scientific)to remove any undis-solved particles.Five hundred milliliters of the C60-THF solution was stirred vigorously while an equal volume of Milli-Q(Millipore,Billerica,MA)water was added at a rate of1L/min.The THF was removed by evaporation in a heated vacuum system,specifically using a Bu¨chi Rotavapor(Bu¨chi Labortechnik AG,Flawil,Switzerland)complete with hot water bath,refrigerated condenser,and vacuum pump.The C60-THF-water mixture was heated in a round-bottom flask to65°C,allowing the THF to evaporate.The vapor was cooled in the condenser,which was flushed with water at2-10°C, and the liquid THF was collected and disposed of ap-propriately.One liter of the C60-THF-water mixture would yield approximately400mL of THF/nC60prior to concentra-tion and filtration,which is discussed below.Son/nC60.Sonicated nC60,or son/nC60,is produced using the method of Andrievsky et al.(37).This FWS was originally named C60FWS and the aggregates called HyFn,but for the purposes of clarity in this study,we term the suspension son/nC60.A solution of1g/L C60in toluene was prepared, and50mL of this solution was added to500mL of Milli-Q (Millipore)water.This layered mixture was then sonicated using a Sonifier Cell Disruptor(Heat Systems-Ultrasonic Inc., Plainview,NY)at80-100W for15min intervals(allowing the machine to cool for5min between)until all of the toluene had evaporated,or for approximately1015-min intervals. The brown suspension was then filtered sequentially through a1Whatman filter(Fisher Scientific),a0.45µm Osmonics nylon membrane(Fisher Scientific),and a0.22µm nylon membrane to remove aggregates larger than200nm.This suspension was concentrated and filtered further as discussed below.Aq/nC60.Aqueous nC60,or aq/nC60,was prepared by stirring1g of C60powder in1L of Milli-Q water over low heat (40°C)for2-4weeks(36).The brown suspension was filtered sequentially through a1Whatman filter,a0.45µm nylon membrane,and a0.22µm nylon membrane to remove aggregates larger than200nm.This suspension was con-centrated and filtered further as discussed below.PVP/C60.PVP/C60was prepared according to the method of Yamakoshi et al.,in which poly(vinylpyrrolidone)(PVP) was used to encapsulate C60molecules(38).Since PVP/C60 is not composed solely of C60,the term nC60is not applied. One hundred milliliters of a0.31%PVP(k value13-19,Sigma-Aldrich,St.Louis,MO)in chloroform solution was added to 25mL of a1g/L solution of C60in toluene.The mixture was stirred and heated overnight at45°C until the solvents evaporated.The residue was resuspended in500mL of Milli-Q water.The yellow suspension was filtered through a0.45µm nylon membrane and a0.22µm nylon membrane to remove aggregates larger than200nm.This suspension was con-centrated and filtered further as discussed below.Evaporation and Filtration.All FWS were concentrated by evaporating excess water with a Bu¨chi rotary evaporator to a final concentration of between2and15mg/L C60.The FWS were heated in the water bath to75°C under a vacuum, allowing the water to evaporate.The water cooled in the 2-10°C condenser and was collected and disposed of.The concentrated suspensions were filtered-sterilized through a0.22µm cellulose syringe filter or a0.22µm MCE membrane vacuum filter(Fisher Scientific).Bacterial Growth.The Gram-positive,facultative anaer-obe B.subtilis was chosen as a test organism.B.subtilis CB310 (courtesy of Dr.Charles Stewart,Rice University,Houston, TX)was grown as described previously(32).The culture was maintained on Luria-Bertani plates but grown in minimal Davis medium(MD)for experimentation.MD is a variation of Davis medium in which the potassium phosphate con-centration was reduced by90%(49).The medium consists of0.7g of K2HPO4,0.2g of KH2PO4,1g of(NH4)2SO4,0.5g of sodium citrate,0.1g of MgSO4·7H2O,and1g of glucose (added after autoclaving)in1L of Milli-Q water.MD medium was chosen as the antibacterial test medium as previous research shows nC60aggregates precipitate out of suspension in media containing high phosphate concentrations(32).Assessing Antibacterial Activity.The minimal inhibitory concentration(MIC)of each preparation was determined using Clinical and Laboratory Standards Institute(CLSI, formerly NCCLS)methodology as described by Tsao et al.(31)with a few modifications.Briefly,to calculate the MIC, MD media tubes containing nC60were inoculated with an overnight culture of B.subtilis(to a final OD600of0.002).The concentration of nC60that resulted in no growth was the MIC.Growth was visually assessed by changes in turbidity and quantified by measuring the optical density at600nm (OD600)using a Turner SP-830spectrophotometer135 (Barnstead,Dubuque,IA).Size Separation of nC60Aggregates.The nC60aggregates in suspension were separated into large and small particle size fractions by differential centrifugation.The suspensions were centrifuged for1h at25000g in a Beckman J2-MC centrifuge(Beckman Coulter,Fullerton,CA).The supernatant was removed with a pipet from the bottles while they were still in the centrifuge to maintain the pellet’s integrity.The supernatant was termed the“smaller”fraction.The pellet was resuspended in Milli-Q water to equal the volume of the supernatant,and it was termed the“larger”fraction.Determining the Size and Concentration of the nC60 Suspensions.Particle size range was estimated using a dynamic light scattering(DLS)device(Brookhaven Instru-ment Corp.,Holtsville,NY)and corroborated by transmission electron microscopy(TEM).For the DLS calculation,the mean diameters were weighted according to the number of particles of each size,not by the intensity of the signal.Bright field TEM images were taken on a JEOL-2010transmission electron microscope(JEOL-USA Inc.,Peabody,MA)operating at100kV.TEM grids were prepared by evaporating ap-proximately20µL of nC60solution onto a Ted Pella300mesh grid(Ted Pella Inc.,Redding,CA)with removable Formvar and a5-10nm thick amorphous carbon film.The concentration of nC60was determined by extracting the C60from suspension and analyzing it in an Ultrospec 2100pro spectrophotometer(Amersham Biosciences,Pis-cataway,NJ)at336nm.The extraction was performed by adding1mL of100mM magnesium perchlorate and2mL of toluene to2mL of nC60suspension.The vial was sealed, and the mixture was stirred for2h.The vial was then placed in a-20°C freezer,the aqueous phase allowed to freeze,and the toluene removed for analysis.The absorbance at336nm was compared to a standard curve prepared by dissolving varying amounts of C60in toluene.Both the aq/nC60and the PVP/C60extracted poorly.Their concentrations were determined by measuring their absor-bance at336nm without extraction.These readings were compared to a standard curve made by correlating the concentrations determined by the extraction method above to the absorbance of the nC60suspension itself.Statistical Analyses.All experiments were performed at least in triplicate.Error bars for the MIC values reflect the actual range of values observed.Student t-test was used to VOL.40,NO.14,2006/ENVIRONMENTAL SCIENCE&TECHNOLOGY94361analyze whether there were any statistical differences in the means of the data at a95%confidence interval. Results and DiscussionCharacterization of nC60.The TEM images of THF/nC60,aq/ nC60,and son/nC60show faceted,high-contrast particles characteristic of crystalline aggregates(Figure1).The THF/ nC60and son/nC60aggregates were similar in size and shape to those published previously(34,40).The aq/nC60aggregates have more rounded edges than the either THF/nC60or son/ nC60,indicating overall less crystallinity.The PVP/C60ag-gregates are not expected to be crystalline,due to the coating of PVP,but they are dense enough for visualization.In conjunction with the hydrodynamic diameters determined by DLS,the TEM micrographs indicate that these suspensions have a wide size range,with the THF/nC60particles ranging from50to150nm in diameter and the aq/nC60ranging from 30to100nm.The sizes of the particles are all below200nm, since the suspensions were filtered through0.22µm filters. The son/nC60and PVP/C60suspensions are more uniformly dispersed than the THF/nC60and aq/nC60.The DLS reports their particle diameters at roughly2nm,but the DLS cannot accurately determine the size of particles smaller than10 nm.The TEM images indicate that there are some particles around10-25nm(Figure1B,D)but that the average particle size is below the DLS quantification limit.Antibacterial Activity of Four Different Types of C60FWS. The Gram-positive bacterium B.subtilis was chosen as a test organism because it is a well-studied soil organism that grows easily in the laboratory under both aerobic and anaerobic conditions.Previous studies indicate that the Gram-negative species Escherichia coli behaves similarly to B.subtilis after nC60exposure,so it is assumed that B.subtilis behavior is a good indicator of both Gram-positive and-negative bacterial responses to nC60(32).When grown at37°C in a shaking incubator,B.subtilis initially has the same growth rate in MD medium as in Davis medium,but it does not reach the same density,due to the reduced buffering capacity of the MD medium(data not shown).All four FWS displayed antibacterial activity toward B. subtilis,as reflected by the MIC data(Table1,Figure2). THF/nC60had an MIC of0.09(0.01mg/L,son/nC60had an MIC of0.7(0.3mg/L,aq/nC60had an MIC of0.5(0.13 mg/L,and PVP/C60had an MIC of0.95(0.35mg/L.Other researchers have shown PVP/C60to suppress the growth of B.subtilis at concentrations of about13mg/L in nutrient broth,an undefined medium(50).These data are within the same range as our results.At a95%confidence interval,there is no statistical difference between the antibacterial activity of the son/nC60,aq/nC60,and PVP/nC60preparations(Figure 2).THF/nC60appears to have a more potent antibacterial effect than the other preparations,having an MIC1order of magnitude smaller.This may be due to variability in the extraction procedure and thus the reported concentration of the suspension.Some researchers have argued that the solvents involved in producing the various types of nC60are responsible for the observed antibacterial activity(42,48).There are two pieces of evidence that obviate this concern.First,controls in which the various preparation methods were performed with solvents but without fullerene had either a lower toxicity,as in the case of the sonication preparation method,or they had no observed toxicity,as in the case of the THF and PVP preparation methods(data not shown).This does notmean FIGURE1.TEM micrographs of(A)aq/nC60,(B)son/nC60,(C)THF/nC60,and(D)PVP/nC60.43629ENVIRONMENTAL SCIENCE&TECHNOLOGY/VOL.40,NO.14,2006that those solvents are not toxic to bacteria,but rather that they were not toxic at the concentrations found in these C 60FWS preparations.The second piece of evidence is that the aq/nC 60displayed antibacterial activity at 0.5mg/L,which is similar to the MIC’s of the other FWS prepared with solvents (Table 1,Figure 2).The aq/nC 60preparation involved no solvent or solubilizing agent and would best mimic a spill or disposal scenario in the environment.nC 60Aggregate Size and Morphology Affects Antibacte-rial Activity.Differential centrifugation was used to separate larger and smaller aggregates in each suspension,and the two fractions were tested for antibacterial activity as reflected by their MIC (Table 1).Only the polydisperse suspensions,e.g.,THF/nC 60and the aq/nC 60,were successfully separated by size.Their mean diameters were determined by DLS and corroborated by TEM (Table 1,Figure 3).Unlike the THF/nC 60and aq/nC 60,the son/nC 60particles were below the DLS size detection capabilities and therefore particle diameters could not be determined by this method.TEM images confirm that the two fractions of son/nC 60were not noticeably different (Figure 3C,D).The PVP/C 60suspension was mono-disperse and was therefore excluded from these experiments.In certain images,there appears to be a matrix that encapsulates the particles (Figure 3C).It is unknown whether the matrix is an artifact of drying or if it is part of the FWS.In a survey of different THF/nC 60batches,suspensions with smaller aggregates tended to have a higher level of antibacterial activity than those with larger aggregates (Figure 4).The MIC increases as the mean diameter increases.In one batch,the “small”THF/nC 60MIC was about 80timeslower than the “large”THF/nC 60MIC (Table 1).This increase in antibacterial activity was disproportionate to the increase in putative surface area,which was only about 2.5times higher.The increase in MIC for the “small”versus “large”aq/nC 60was 6.8-fold,while the increase in surface area:volume ratio was approximately 70-fold.Even though the DLS was unable to detect a change in particle size between the “small”and “large”fractions of the son/nC 60,there was a 4-fold increase in antibacterial activity for the “small”fraction.There is a lack of a linear relationship between toxicity and particle size (Figure 5),indicating that other factors besides an increase in surface area are likely respon-sible for the higher antibacterial activity of the smaller aggregates.Furthermore,differently prepared suspensions containing aggregates of the same size did not necessarily have the same MIC (Table 1),possibly due to differences in the production methods (e.g.,reagents,temperature,rate of addition of water,rate of stirring)or discrepancies in the determination of the concentration of the suspension.While all the suspensions possess antibacterial activity,their morphologies are different,suggesting that there are influential differences in surface chemistry and/or morphol-ogy.The separated nC 60suspensions contain both crystalline and amorphous aggregates (Figure 3).Following centrifuga-tion,the aggregates in the “smaller”size fraction had a higher level of amorphous aggregates than the “larger”size fraction,as evident in the TEM images.The small aggregates inherently appear amorphous,due to a lack of repeating structure that is needed to establish a defined crystal.Aggregates with diameters of 2-3nm have been calculated to contain only 4-13molecules of C 60(41).It is unclear whether the greater antibacterial activity of the “smaller”size fraction is attributed directly to the smaller size itself or with the presence of an amorphous structure.Alternative Factors Affecting Antibacterial Activity.Previous studies have shown that THF/nC 60attaches to bacterial and human cells (32,45),but it is not clear whether contact is necessary for antibacterial activity.It has been proposed that nC 60aggregates have an outer layer of hydrated or hydroxylated C 60molecules (34,41).It has also been previously demonstrated that hydroxylated C 60(C 60(OH)22-24)does not display antibacterial activity at solubility limits (32).If the nC 60clusters are composed of an outer shell of nonbactericidal particles,this is an indication that direct interaction of the nC 60surface with the bacterial cell is not involved in the antibacterial mechanism.Indeed,PVP/C 60has an outer coating of PVP molecules (which themselves did not exhibit antibacterial properties,data not shown)that shield the cell from the C 60molecule.Yet,PVP/C 60has antibacterial properties,which highlights that direct contact may not be necessary for the antibacterial mechanism.While the mechanisms behind the antibacterial activity have not been determined,reactive oxygen species (ROS)are believed to be responsible for eukaryotic cell membrane disruption and eukaryotic lipid peroxidation (43-45).It is unclear whether ROS are produced by nanomaterials them-selves or by the eukaryotic cells immune response to nanomaterials.ROS have been implicated in the antibacterial mechanism of PVP/C 60(50),but they have not been confirmed as the main factors responsible for the bactericidal activity of any of the FWS.In a previous study of nC 60with bacteria,toxicity was not affected by the presence or absence of light that is needed to stimulate ROS production by nC 60(32).nC 60was also able to kill bacteria under anaerobic and fermentative conditions (results not shown)where O 2,a critical ROS precursor,was absent.All these lines of evidence indicate that photocatalyzed ROS production is probably not the sole antibacterial mechanism associated with nC 60.Thus,further research on the relationship between nC 60surface chemistry,morphology,and reactivity is warranted toTABLE 1.MIC’s of the Four Different Preparations and Their Constituent Small and Large Aggregate Fractions aMIC (mg/L)mean diameter (nm)surface area:volume ratio bTHF/nC 600.08-0.1075.60.079THF/nC 60small 0.008-0.01039.10.153THF/nC 60large 0.6-0.897.40.062son/nC 600.4-0.6∼2c 3son/nC 60small 0.15-0.20∼2c 3son/nC 60large 0.6-0.8∼2c 3aq/nC 600.4-0.674.90.080aq/nC 60small 0.1-0.23∼2c 3aq/nC 60large 0.75-1.5142.30.042PVP/C 600.6-1.0∼2c3a“Large”refers to particles that were pelleted by the differential centrifugation method,and “small”refers to the supernatant.b Surface area and volume were calculated by assuming the mean diameter given is a good reflection of the population and that the aggregates are spherical.c Theoretical detection limits of the DLS are from 10nm to 1µm.FIGURE 2.Minimal inhibitory concentrations (MIC’s)of the various FWS preparations.Error bars represent the range of the measured MIC values (n )5).VOL.40,NO.14,2006/ENVIRONMENTAL SCIENCE &TECHNOLOGY94363elucidate toxicity mechanisms and identify manufacturing and derivatization processes that decrease toxicity.Environmental Relevance of nC 60.C 60powder has not been shown to have detrimental environmental or health effects (32-34).In a study using the same methods as this paper,C 60powder did not display antibacterial activity against either B.subtilis or E.coli (32).However,disposal or a spill of C 60as a powder or in a solvent could result in nC 60formation,and the full repercussions are as yet unclear.A comparison of the MIC’s for B.subtilis presented in this paper with the MIC’s for the antibiotic vancomycin shows that vancomycin has less antibacterial activity than nC 60(51).This indicates that nC 60is a potent antibacterial agent that warrants further investigation for both its implications in environmental health and for its application as an antibiotic or disinfectant.This study addressed the antibacterial activity of four different FWS with a pure culture of bacteria in MD,a lowsalt medium.The ionic strength of MD is about 0.04M,which is within the 0.01-0.1M typical range for freshwater (52).Results for one laboratory strain in MD may not give an accurate reflection of the behavior of nC 60in aquatic environments with mixed cultures and higher ionic strength,where coagulation of the nC 60aggregates could occur (40,42,53).This coagulation results in the aggregates precipitating out of suspension and the loss of the antibacterial activity (32).Microbial activities are important to the health of all known ecosystems,and the observed bactericidal effects of nC 60suggest the need for caution against accidental releases and disposal of fullerenes.AcknowledgmentsThe authors thank Matthew Hotze and Jonathan Brant for helpful discussion.This research was funded by NSF (BES-0508207),NSF through the Center for Biological andEnvi-FIGURE 3.TEM micrographs of size-separated nC 60:(A)large aq/nC 60,(B)small aq/nC 60,(C)large son/nC 60,(D)small son/nC 60,(E)large THF/nC 60,and (F)small THF/nC60FIGURE 4.Size-separated THF/nC 60suspensions show that increas-ing aggregate size is associated with decreasing antibacterial activity.For each suspension of “small”,“large”,and noncentrifuged nC 60,the median particle size,as determined by DLS,is compared to the MIC of thatsuspension.FIGURE 5.Relationship between MIC and aggregate surface area.There is no linear relationship between the mean MIC and the surface area to volume ratio calculated,indicating that the difference in surface area alone does not account for the difference in MIC between the small and large aggregates.43649ENVIRONMENTAL SCIENCE &TECHNOLOGY /VOL.40,NO.14,2006ronmental Nanotechnology at Rice University(EEC-0118007), and EPA-STAR(91650901-0).Literature Cited(1)Kroto,H.W.;Heath,J.R.;O’Brien,S.C.;Curl,R.F.;Smalley,R.E.C60-Buckminsterfullerene.Nature1985,318,162-163.(2)Sherigara,B.S.;Kutner,W.;D’Souza,F.Electrocatalytic proper-ties and sensor applications of fullerenes and carbon nanotubes.Electrocatalysis2003,15,753-772.(3)Goldshleger,N.F.Fullerenes and fullerene-based materials incatalysis.Fullerene Sci.Technol.2001,9,255-280.(4)Forro,L.;Mihaly,L.Electronic properties of doped fullerenes.Rep.Prog.Phys.2001,64,649-699.(5)Wilson,L.J.Medical applications of fullerenes and metallo-fullerenes.Electrochem.Soc.Interface1999,24-28.(6)Da Ros,T.;Prato,M.Medicinal chemistry with fullerenes andfullerene mun.1999,663-669.(7)Karaulova,E.N.;Bagrii,E.I.Fullerenes:Functionalisation andprospects for the use of derivatives.Russ.Chem.Rev.1999,68, 889-907.(8)Innocenzi,P.;Brusatin,G.Fullerene-based organic-inorganicnanocomposites and their applications.Chem.Mater.2001, 13,3126-3139.(9)Kratschmer,W.;Lamb,L.D.;Fostiropoulos,K.;Huffman,D.R.Solid C60s A new form of carbon.Nature1990,347,354-358.(10)Tremblay,J.-F.Mitsubishi chemical aims at breakthrough.Chem.Eng.News2002,80,16-17.(11)Hogue,C.Regulating chemicals:Concerns regarding REACH,nanomaterials,biomonitoring voiced at GlobalChem meeting.Chem.Eng.News2005,83,53-58.(12)Jones,R.M.Nanotechnology legislation on fast track.FYI2003,38.(13)Eilperin,J.Nanotechnology’s big question:safety,some saymicromaterials are coming to market without adequate controls.The Washington Post,October23,2005;p A11.(14)Bullard-Dillard,R.;Creek,K.E.;Scrivens,W.A.;Tour,J.M.Tissue sites of uptake of14C-labeled C60.Bioorg.Chem.1996, 24,376-385.(15)Foley,S.;Curtis,A.D.M.;Hirsch,A.;Brettreich,M.;Pelegrin,A.;Seta,P.;Larroque,C.Interaction of a water soluble fullerenederivative with reactive oxygen species and model enzymatic systems.Fullerenes Nanotubes Carbon Nanostruct.2002,10, 49-67.(16)Tsuchiya,T.;Oguri,I.;Yamakoshi,Y.;Miyata,N.Novel harmfuleffects of[60]fullerene on mouse embryos in vitro and in vivo.FEBS Lett.1996,393,139-145.(17)Tokuyama,H.;Yamago,S.;Nakamura,E.Photoinduced bio-chemical activity of fullerene carboxylic acid.J.Am.Chem.Soc.1993,115,7918-7919.(18)Yang,X.;Fan,C.;Zhu,H.Photo-induced cytotoxicity of malonicacid[C-60]fullerene derivatives and its mechanism.Toxicol.in Vitro2002,16,41-46.(19)Kamat,J.P.;Devasagayam,T.P.;Priyadarsini,K.I.;Mohan,H.Reactive oxygen species mediated membrane damage induced by fullerene derivatives and its possible biological implications.Toxicology2000,155,55-61.(20)Kim,J.E.;Lee,M.Fullerene inhibits beta-amyloid peptidemun.2003,303,576-579.(21)Tabata,Y.;Ikada,Y.Biological functions of fullerenes.Pure Appl.Chem.1999,71,2047-2053.(22)Wharton,T.;Kini,V.U.;Mortis,R.A.;Wilson,L.J.New non-ionic,highly water-soluble derivatives of C-60designed for biological compatibility.Tetrahedron Lett.2001,42,5159-5162.(23)Babynin,E.V.;Nuretdinov,I.A.;Gubskaia,V.P.;Barabansh-chikov,B.I.Study of mutagenic activity of fullerene and some of its derivatives using His+reversions of Salmonella typh-imurium as an example.Genetika2002,38,453-457. (24)Bosi,S.;Da Ros,T.;Castellano,S.;Banfi, E.;Prato,M.Antimycobacterial activity of ionic fullerene derivatives.Bioorg.Med.Chem.Lett.2000,10,1043-1045.(25)Mashino,T.;Okuda,K.;Hirota,T.;Hirobe,M.;Nagano,T.;Mochizuki,M.Inhibition of E.coli growth by fullerene derivatives and inhibition mechanism.Bioorg.Med.Chem.Lett.1999,9, 2959-2962.(26)Mashino,T.;Nishikawa,D.;Takahashi,K.;Usui,N.;Yamori,T.;Seki,M.;Endo,T.;Mochizuki,M.Antibacterial and antipro-liferative activity of cationic fullerene derivatives.Bioorg.Med.Chem.Lett.2003,13,4395-4397.(27)Mashino,T.;Usui,N.;Okuda,K.;Hirota,T.;Mochizuki,M.Respiratory chain inhibition by fullerene derivatives:hydrogen peroxide production caused by fullerene derivatives and a respiratory chain system.Bioorg Med.Chem.2003,11,1433-1438.(28)Sera,N.;Tokiwa,H.;Miyata,N.Mutagenicity of the fullereneC60-generated singlet oxygen dependent formation of lipid peroxides.Carcinogenesis1996,17,2163-2169.(29)Tsao,N.;Kanakamma,P.P.;Luh,T.Y.;Chou,C.K.;Lei,H.Y.Inhibition of Escherichia coli-induced meningitis by carboxy-fullerene.Antimicrob.Agents Chemother.1999,43,2273-2277.(30)Tsao,N.;Luh,T.;Chou,C.;Wu,J.;Lin,Y.;Lei,K.Inhibition ofgroup A streptococcus infection by carboxyfullerene.Antimicrob.Agents Chemother.2001,45,1788-1793.(31)Tsao,N.;Luh,T.;Chou,C.;Chang,T.;Wu,J.;Liu,C.;Lei,H.Invitro action of carboxyfullerene.Antimicrob.Agents Chemother.2002,49,641-649.(32)Lyon,D.Y.;Fortner,J.D.;Sayes,C.M.;Colvin,V.L.;Hughes,J.B.Bacterial cell association and antimicrobial activity of a C60water suspension.Environ.Toxicol.Chem.2005,24,2757-2762.(33)Jia,G.;Wang,H.;Yan,L.;Wang,X.;Pei,R.;Yan,T.;Zhao,Y.;Guo,X.Cytotoxicity of carbon nanomaterials:single-wall nanotube,multi-wall nanotube,and fullerene.Environ.Sci.Technol.2005,39,1378-1383.(34)Fortner,J.D.;Lyon,D.Y.;Sayes,C.M.;Boyd,A.M.;Falkner,J.C.;Hotze,E.M.;Alemany,L.B.;Tao,Y.J.;Guo,W.;Ausman, K.D.;Colvin,V.L.;Hughes,J.B.C60in water:Nanocrystal formation and microbial response.Environ.Sci.Technol.2005, 39,4307-4316.(35)Heymann,D.Solubility of C60and C70in seven normal alcoholsand their deduced solubility in water.Fullerene Sci.Technol.1994,4,509-515.(36)Cheng,X.;Kan,A.T.;Tomson,M.B.Naphthalene adsorptionand desorption from aqueous C60fullerene.J.Chem.Eng.Data 2004,49,675-683.(37)Andrievsky,G.V.;Kosevich,M.V.;Vovk,O.M.;Shelkovsky,V.S.;Vashchenko,L.A.On the production of an aqueous colloidal solution of mun.1995,1281-1282. (38)Yamakoshi,Y.N.;Yagami,T.;Fukuhara,K.;Sueyoshi,S.;Miyata,N.Solubilization of fullerenes into water with polyvinylpyr-rolidone applicable to biological tests.J.Chem.Soc.,Chem.Commun.1994,4,517-518.(39)Deguchi,S.;Rossitza,G.A.;Tsujii,K.Stable dispersions offullerenes,C60and C70in water.Preparation and ngmuir2001,17,6013-6017.(40)Andrievsky,G.V.;Klochkov,V.K.;Karyakina,E.L.;Mchedlov-Petrossyan,N.O.Studies of aqueous colloidal solutions of fullerene C60by electron microscopy.Chem.Phys.Lett.1999, 300,392-396.(41)Prilutski,Y.I.;Durov,S.S.;Yashchuk,V.N.;Ogul’chansky,T.Y.;Pogorelov,V.E.;Astashkin,Y.A.;Buzaneva,E.V.;Kirghisov, Y.D.;Andrievsky,G.V.;Scharff,P.Theoretical predictions and experimental studies of self-organized C60nanoparticles in water solution and on the support.Eur.Phys.J.D1999,9,341-343.(42)Brant,J.;Lecoanet,H.;Hotze,M.;Wiesner,parison ofelectrokinetic properties of colloidal fullerenes(n-C60)formed using two procedures.Environ.Sci.Technol.2005,39,6343-6351.(43)Oberdo¨rster,E.Manufactured nanomaterials(fullerenes,C60)induce oxidative stress in the brain of juvenile largemouth bass.Environ.Health Perspect.2004,112,1058-1062.(44)Sayes,C.;Fortner,J.;Guo,W.;Lyon,D.;Boyd,A.;Ausman,K.;Tao,Y.;Sitharaman,B.;Wilson,L.;Huges,J.;West,J.;VL,C.The differential cytotoxicity of water-soluble fullerenes.Nano Lett.2004,4,1881-1887.(45)Sayes,C.M.;Gobin,A.M.;Ausman,K.D.;Mendeza,J.;West,J.L.;Colvin,V.L.Nano-C60cytotoxicity is due to lipid peroxidation.Biomaterials2005,26,7587-7595.(46)Weiss,R.Nanoparticles toxic in aquatic habitat,study finds.InThe Washington Post,March29,2004;p A.02.(47)Feder,B.J.Study raises concerns about carbon particles.In TheNew York Times,March29,2004.(48)Andrievsky,G.;Klochkov,V.;Derevyanchenko,L.Is C60fullerenemolecule toxic?!Fullerenes,Nanotubes,Carbon Nanostruct.2005,13,363-376.(49)Atlas,R.M.Handbook of Microbiological Media;CRC Press:Boca Raton,FL,1993.(50)Kai,Y.;Komazawa,Y.;Miyajima,A.;Miyata,N.;Yamakoshi,Y.[60]Fullerene as a novel photoinduced antibiotic.Fullerenes,Nanotubes,Carbon Nanostruct.2003,11,79-87.VOL.40,NO.14,2006/ENVIRONMENTAL SCIENCE&TECHNOLOGY94365。