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Phylogenetic diversity of airborne microbes in Qingdao downtown in autumn.

青岛市秋季空气微生物群落多样性


采用KC-6120空气综合采样器采集空气微生物样品,通过构建16S/18SrDNA克隆文库方法分析青岛市市区街道秋季空气微生物群落结构特征.结果表明: 空气细菌分布在6大类,分别为变形菌门(78.8%)、厚壁菌门(14.6%)、放线菌门(4.0%)、浮霉菌门(1.3%)、蓝藻门(0.7%)和栖热菌门(0.6%),优势菌属为不动杆菌属(39.7%)、葡萄球菌属(11.3%)、鞘脂单胞菌属(8.6%)和副球菌属(6.0%).空气真菌分布在子囊菌门(97.5%)和担子菌门(2.5%),优势菌属为核腔菌属(76.5%)、炭角菌属(13.6%)和外瓶霉属(2.5%).空气微生物中存在不动杆菌属、鞘脂单胞菌、葡萄球菌等致病菌或条件致病菌,以及引发多种农作物枯萎死亡的麦类核腔菌、团炭角菌和角状平脐疣孢等真菌.
 

To determine the community structure of airborne microbes in Qingdao downtown in autumn, the airborne bacteria and fungi were collected by the KC6120 air sampler and analyzed using the 16S/18S rDNA gene clone library method. Phylogenetic analysis of airborne bacteria showed that they belonged to six major phylogenetic groups: Proteobacteria (78.8%), Firmicutes (14.6%), Actinobacteria (4.0%), Planctomycetes (1.3%), Cyanobacteria (0.7%), and DeinococcusThermus(0.7%). The dominant genera of airborne bacteria included Acinetobacter (39.7%), Staphylococcus (11.3%), Sphingomonas (8.6%),  Paracoccus (6.0%) and Massilia
(5.3%). The main types of airborne fungi were Ascomycota (97.5%) and Basidiomycota (2.5%). Dominant genera of airborne fungi included Pyrenophora (76.5%), Xylaria (13.6%) and Exophiala (2.5%). The pathogens or conditioned pathogens, such as Acinetobacter, Staphylococcus, or Sphingomonas were detected in the airborne bacteria, whereas certain kinds of fungi, such as P. graminea, X. hypoxylon and Zasmidium angulare that could cause a variety of crop diseases were also detected.


全 文 :青岛市秋季空气微生物群落多样性∗
王  琳  宋志文∗∗  徐爱玲  吴等等  夏  岩
(青岛理工大学, 山东青岛 266033)
摘  要  采用 KC⁃6120空气综合采样器采集空气微生物样品,通过构建 16S / 18S rDNA 克隆
文库方法分析青岛市市区街道秋季空气微生物群落结构特征.结果表明: 空气细菌分布在 6
大类,分别为变形菌门(78.8%)、厚壁菌门(14.6%)、放线菌门(4.0%)、浮霉菌门(1.3%)、蓝
藻门(0.7%)和栖热菌门(0.6%),优势菌属为不动杆菌属(39.7%)、葡萄球菌属(11.3%)、鞘
脂单胞菌属(8. 6%)和副球菌属(6. 0%) .空气真菌分布在子囊菌门(97. 5%)和担子菌门
(2.5%),优势菌属为核腔菌属(76.5%)、炭角菌属(13.6%)和外瓶霉属(2.5%) .空气微生物中
存在不动杆菌属、鞘脂单胞菌、葡萄球菌等致病菌或条件致病菌,以及引发多种农作物枯萎死
亡的麦类核腔菌、团炭角菌和角状平脐疣孢等真菌.
关键词  克隆文库; 空气细菌; 空气真菌; 群落结构; 微生物多样性
文章编号  1001-9332(2015)04-1121-09  中图分类号  X172  文献标识码  A
Phylogenetic diversity of airborne microbes in Qingdao downtown in autumn. WANG Lin,
SONG Zhi⁃wen, XU Ai⁃ling, WU Deng⁃deng, XIA Yan ( Institute of Environmental and Municipal
Engineering, Qingdao Technological University, Qingdao 266033, Shandong, China) . ⁃Chin. J.
Appl. Ecol., 2015, 26(4): 1121-1129.
Abstract: To determine the community structure of airborne microbes in Qingdao downtown in au⁃
tumn, the airborne bacteria and fungi were collected by the KC⁃6120 air sampler and analyzed using
the 16S / 18S rDNA gene clone library method. Phylogenetic analysis of airborne bacteria showed
that they belonged to six major phylogenetic groups: Proteobacteria ( 78. 8%), Firmicutes
(14.6%), Actinobacteria (4.0%), Planctomycetes (1.3%), Cyanobacteria (0.7%), and Deino⁃
coccus⁃Thermus ( 0. 7%). The dominant genera of airborne bacteria included Acinetobacter
(39.7%), Staphylococcus (11.3%), Sphingomonas (8.6%), Paracoccus (6.0%) and Massilia
(5.3%). The main types of airborne fungi were Ascomycota (97.5%) and Basidiomycota (2.5%).
Dominant genera of airborne fungi included Pyrenophora (76.5%), Xylaria (13.6%) and Exophiala
(2.5%). The pathogens or conditioned pathogens, such as Acinetobacter, Staphylococcus, or Sphin⁃
gomonas were detected in the airborne bacteria, whereas certain kinds of fungi, such as P. graminea,
X. hypoxylon and Zasmidium angulare that could cause a variety of crop diseases were also detected.
Key words: clone library; airborne bacteria; airborne fungi; community structure; microbial diver⁃
sity.
∗国家自然科学基金项目(31170509)资助.
∗∗通讯作者. E⁃mail: songzhiwen@ qtech.edu.cn
2014⁃05⁃24收稿,2015⁃01⁃15接受.
    大气环境具有干燥、营养贫瘠、高辐射和扩散能
力强等特点,不适合微生物生长繁殖[1] .但研究表明
空气中不仅存在一定数量的微生物,而且代谢活
跃[2-7] .空气微生物主要附着在灰尘颗粒物上,以生
物气溶胶形式存在,包括细菌、真菌、病毒、原生动物
和其他微小动植物[8] .生物气溶胶可以传播或产生
过敏原和毒素[9],导致人体患呼吸性疾病、过敏反
应、传染病等[10-12] .生物气溶胶还可以作为冰核和
云凝结核,导致云滴和冰晶的形成,从而间接影响全
球气候[13] .
空气微生物研究方法主要包括传统培养法和非
培养法.传统培养法通常采用自然沉降法或空气采
样器采集空气微生物,培养后进行菌落计数,分离和
纯化后,鉴定菌种.非培养法主要包括基于 PCR 技
术的分子生物学法、单一碳源利用法、荧光原位杂交
技术等[14-15] .传统培养法虽然快速有效,但由于空
气微生物中只有大约 1%的微生物能够在培养基上
应 用 生 态 学 报  2015年 4月  第 26卷  第 4期                                                         
Chinese Journal of Applied Ecology, Apr. 2015, 26(4): 1121-1129
形成菌落,因此对群落结构的判定误差较大,不能全
面反映空气微生物群落结构信息[16] .采用分子生物
学技术建立克隆文库进行 RFLP 分析,能有效避免
这一弊端[17-19] .
近年来,分子生物学技术在研究水体、土壤微生
物群落结构方面已发展成为一种成熟有效的技术,
但对空气样品的研究只有少量报道[20-22] .国内学
者[23-25]对于城市空气微生物多样性研究大多采用
传统培养方法,国外学者[26-29]则主要开展室内空气
微生物检测以及废水处理厂、植物堆肥、饲养场等空
气微生物的研究.青岛位于黄海之滨,空气微生物受
内陆与海洋双重影响,研究空气微生物群落多样性,
对于了解沿海城市空气微生物组成以及对大气环
境、人体健康的影响具有重要意义.本文采用 KC⁃
6120空气综合采样器采集空气微生物样品,通过构
建 16S / 18S rDNA克隆文库和 RFLP 方法,分析青岛
市秋季空气微生物群落结构和特征.
1  材料与方法
1􀆰 1  采样地点和采样时间
采样地点选取青岛市市北区抚顺路(36.10° N,
120.37° E).抚顺路是青岛市的交通要道,人流量和
车流量较大,汽车尾气和扬尘污染较严重.采样时间
为 2013 年 10 月 24—25 日,分两次连续采样 20 h.
采样期间天气晴朗,温度 20 ℃左右,采样前一周无
降雨.
1􀆰 2  样品采集方法
将两台 KC⁃6120 空气综合采样器固定于三脚
架上,高度为 1.5 m(人体平均呼吸高度),将无菌滤
膜置于采样器中(滤膜材质为玻璃纤维,直径 80
mm),空气流量为 100 L·min-1,采集后用 0.85%灭
菌生理盐水冲洗滤膜,将滤膜上沉积颗粒物溶于
其中.
1􀆰 3  研究方法
1􀆰 3􀆰 1 DNA提取和目的片段 PCR 扩增   将溶有颗
粒物的生理盐水在离心机中 12000 r·min-1离心 20
min,浓缩于 2 mL离心管中,浓缩物用土壤 DNA 提
取试剂盒(OMEGA)提取 DNA.以提取 DNA为模板,
采用细菌 16S r DNA扩增通用引物 27f / 1500R[30]和
真菌 18 S rDNA 扩增通用引物 EF4f / EF3r[31](引物
序列见表 1)进行 PCR扩增.细菌扩增条件:94 ℃预
变性 5 min;94 ℃变性 45 s;58.5 ℃退火 45 s;72 ℃
延伸 90 s;完成 36 个循环;72 ℃延伸 10 min;4 ℃
保存.真菌扩增条件与细菌唯一区别是退火温度为
表 1  引物序列
Table 1  Sequence of primers
名称
Name
序列
Sequence
27f 5′⁃AGAGTTTGATCCTGGCTCAG⁃3′
1500R 5′⁃AGAAAGGAGGTGATCCAGCC⁃3′
EF4f 5′⁃GGAAGGGGTGTATTTATT Ag⁃3′
EF3r 5′⁃TCCTCTAAATGACCAGTTTG⁃3′
53 ℃ .PCR反应体系:超纯水(ddH2O) 19 μL、上游
引物(5 μmol·L-1)2 μL、下游引物(5 μmol·L-1)2
μL、DNA 2 μL、PCR预混液 (Master mix) 25 μL,总
体积 50 μL.用 1%琼脂糖凝胶电泳检测 PCR产物.
1􀆰 3􀆰 2构建 16S rDNA和 18S rDNA克隆文库  选取
均一且较亮的目的条带进行胶回收,用琼脂糖凝胶
回收纯化试剂盒(OMEGA)进行纯化,纯化后用 T4
连接酶克隆试剂盒(MBI Fermentas)将目的片段与
载体连接,之后转化到 Top10 感受态细胞中[32],涂
板在具有 X⁃gal / IPTG 抗性筛选的 LB 平板培养基
上[33],选取具有氨苄青霉素抗性的白色转化子进行
转板培养构建克隆文库.
1􀆰 3􀆰 3 RFLP 序列分析与测序  通过菌落 PCR 重新
获得 16S rDNA和 18S rDNA,1%琼脂糖凝胶电泳检
测,然后分别用限制性内切酶(HhaI、RsaI)对细菌菌
落和真菌菌落 PCR产物进行酶切[34],用 3%琼脂糖
凝胶电泳检测,获得 RFLP 图谱.分析酶切图谱[35],
在各 OTU中任选一个克隆子制备穿刺管经 16 h 培
养后送生工生物工程(上海)股份有限公司测序.
1􀆰 3􀆰 4系统发育与克隆文库分析   在 NCBI 中将测
序结果输入 BLAST 程序与数据库中序列进行比
对[36],依据相似度最高的已知菌种序列,运用
MEGA 5.0软件构建系统发育树,建树模型为 Kimu⁃
ra2⁃parameter,Bootstrap值为 1000.计算克隆文库覆
盖度 ( coverage value, C )、 Shannon 多样性指数
(Shannon diversity index, H′)、辛普森指数(Simpson
index, D)、丰富度(species richness)和均匀度(spe⁃
cies evenness, E).具体方法见参考文献[37].
2  结果与分析
2􀆰 1  目的基因的提取及 16S / 18S rDNA扩增
利用土壤 DNA 提取试剂盒(OMEGA)提取总
DNA,DNA产率和纯度能够满足 PCR 扩增要求.以
总 DNA为模板,利用细菌、真菌通用引物进行 PCR
扩增,图 1为 16S rDNA 和 18S rDNA 琼脂糖凝胶电
泳图谱.可以看出,16S rDNA和 18S rDNA PCR扩增
产物片段大小分别为1600和1700 bp左右,与预期
2211 应  用  生  态  学  报                                      26卷
图 1  16S / 18S rDNA PCR琼脂糖凝胶电泳图谱
Fig.1  Agarose gel electrophoresis analysis of 16S / 18S rDNA⁃
PCR result
M:分子标记 DNA marker (BM2000). 下同 The same below. 1)真菌
Fungi; 1′)真菌平行样 Parallel sample of fungi; 2)细菌 Bacteria; 2′)
细菌平行样 Parallel sample of bacteria; 3)空白样 Blank sample.
大小符合,且亮度高,均一性较好.
2􀆰 2  转化子的鉴定
在具有 X⁃gal / IPTG抗性筛选的细菌和真菌 LB
平板上分别随机选取 200和 100个具有氨苄青霉素
抗性的白色转化子,转板标记培养后进行菌落
PCR,1%琼脂糖凝胶电泳分离.空气细菌和空气真
菌 PCR扩增产物片段大小与总 DNA 的 PCR 结果
一致,亮度均一度较高,但也有部分克隆子亮度较弱
且不均一,挑取大小符合要求并且条带均一、亮度高
的克隆子进行酶切反应.
2􀆰 3  RFLP 分析和克隆文库分析
将符合要求的空气细菌和空气真菌菌落 PCR
产物分别经限制性内切酶 HhaI、RsaI酶切后,用 3%
琼脂糖凝胶电泳分离,电泳图谱见图 2.
从图 2可以看出,酶切条带较丰富,条带位置差
异性较大且亮度高,说明选用的限制性内切酶可以
对空气微生物种类进行划分.通过对酶切图谱分析,
将空气细菌 151个克隆子分为 43个 OTU,空气真菌
81个克隆子分为 17 个 OTU,序列多样性分析结果
见表 2.从划分的 OTU数看,青岛市空气细菌与空气
真菌的群落多样性非常丰富.16S rDNA和 18S rDNA
克隆文库覆盖度分别为 93.4%和 93.8%,可包含大
部分空气细菌和空气真菌,比较真实地反映了空气
中微生物的群落特征.构建的 16S / 18S rDNA 克隆
文库的Shannon指数、Simpson指数、丰富度、均匀度
图 2  16S (A) / 18S rDNA(B)菌落 PCR 产物酶切琼脂糖凝
胶电泳图谱
Fig.2  Agarose gel electrophoresis analysis of 16S (A) / 18S
rDNA (B) colonies PCR products.
较高,空气细菌 16S rDNA 克隆文库多样性指数均
大于空气真菌.
空气细菌 16S rDNA、空气真菌 18S rDNA 克隆
文库分析结果见表 3、表 4.构建空气细菌、空气真菌
系统发育树,结果见图 3、图 4.
16S rDNA克隆文库中克隆子与 NCBI中已知菌
种相似性在 81% ~ 99%,其中相似度大于 97%的克
隆子 71 个,占总数的 47.0%,相似度低于 93%的克
隆子 25个,占总数的 16.6%.空气细菌分布在 6 大
类,分别为变形菌门(Proteobacteria, 78.8%)、厚壁
菌门( Firmicutes, 14. 6%)、放线菌门 ( Actinobacte⁃
ria, 4.0%)、浮霉菌门(Planctomycetes, 1.3%)、蓝藻
门(Cyanobacteria, 0.7%)和栖热菌门(Deinococcus⁃
Thermus, 0.7%).变形菌门为优势菌群,其中 α⁃变形
菌纲(Alphaproteobacteria, 29.8%)、β⁃变形菌纲(Be⁃
taproteobacteria, 8. 0%)、 γ⁃变形菌纲 ( Gammapro⁃
teobacteria, 41.1%)占较大比例,此外还有厚壁菌门
中芽孢杆菌纲(Bacilli, 14.6%),放线菌门放线菌纲
(Actinobacteria, 4. 0%)、浮霉菌纲 ( Planctomyceta⁃
cia, 1.3%)、蓝藻门蓝藻纲(Cyanobacteria, 0.7%)和
表 2  克隆文库多样性指数分析
Table 2  Diversity index of 16S / 18S rDNA clone libraries
克隆文库
Clone library
克隆子数
Clone
number
OTUs 覆盖度
Coverage value
Shannon指数
Shannon index
Simpson指数
Simpson index
丰富度
Species richness
均匀度
Evenness
16S rDNA 151 43 93.4% 2.9 0.9 8.4 0.8
18S rDNA 81 17 93.8% 1.9 0.7 3.8 0.6
32114期                              王  琳等: 青岛市秋季空气微生物群落多样性           
表 3  空气细菌 16S rDNA克隆文库分析
Table 3  Analysis of 16S rDNA clone library of airborne bacteria
最相似菌(NCBI登录号)
Most similar(NCBI reference sequence)
相似性
Similarity
(%)
OTUs 丰度
Abundance
(%)
类群
Group
类群百分比
Group percentage
Xanthomonas oryzae pv. oryzae KACC 10331 (NR_074938.1) 97 1 0.7 γ⁃变形菌纲 41.1
Pseudomonas mandelii (NR_024902.1) 92 5 3.3
Acinetobacter lwoffii (NR_026209.1) 94 2 1.3
Acinetobacter haemolyticus (NR_026207.1) 98~99 38 25.2
Acinetobacter baumannii ATCC 17978 (NR_074737.1) 91~93 15 9.9
Acinetobacter oleivorans DR1 (NR_102814.1) 93 1 0.7
Amaricoccus macauensis(NR_029202.1) 97 2 1.3 α⁃变形菌纲 29.8
Sphingomonas kaistensis (NR_043171.1) 97 13 8.6
Novosphingobium stygium( NR_040826.1) 96 1 0.7
Novosphingobium naphthalenivorans (NR_041046.1) 98 5 3.3
Caulobacter segnis (NR_074208.1) 89 1 0.7
Paracoccus carotinifaciens (NR_024658.1) 99 9 6.0
Brevundimonas subvibrioides (NR_074136.1) 92 14 9.3
Rubellimicrobium mesophilum (NR_044275.1) 91~92 4 2.7
Staphylococcus epidermidis RP62A (NR_074995.1) 99 17 11.3 芽孢杆菌纲 14.6
Carnobacterium sp. 17⁃4 (NR_074964.1) 93 4 2.7
Exiguobacterium aurantiacum (NR_043478.1) 91 1 0.7
Methylibium petroleiphilum (NR_041768.1) 89 4 2.7 β⁃变形菌纲 8.0
Massilia aurea( NR_042502.1) 95~99 4 2.7
Massilia timonae (NR_026014.1) 96~99 4 2.7
Brachybacterium faecium (NR_074655.1) 95 4 2.7 放线菌纲 4.0
Propionibacterium acnes KPA171202 (NR_074675.1) 99 2 1.3
Schlesneria paludicola (NR_042466.1) 81 2 1.3 浮霉菌纲 1.3
Oscillatoria nigro⁃viridis PCC 7112(NR_102469.1) 90 1 0.7 蓝藻纲 0.7
Truepera radiovictrix (NR_074381.1) 88 1 0.7 异常球菌纲 0.7
表 4  空气真菌 18S rDNA克隆文库分析结果
Table 4  Analysis of 18S rDNA clone library of airborne fungi
最相似菌(NCBI登录号)
Most similar(NCBI reference sequence)
相似性
Similarity
(%)
OTUs 丰度
Abundance
(%)
类群
Group
类群百分比
Group percentage
Zasmidium cellare(NG_016540.1) 99 1 1.2 座囊菌纲 77.7
Pyrenophora phaeocomes (NG_013190.1) 99 62 76.5
Bombardia bombarda voucher F:SMH 3391(NG_013187.1) 96 3 3.7 粪壳菌纲 16.1
Xylaria hypoxylon voucher OSC:100004(NG_013136.1) 95~96 10 12.4
Grifola sordulenta voucher TENN:55054 (NG_013172.1) 88~90 2 2.5 伞菌纲 2.5
Exophiala pisciphila voucher DUKE: W. Untereiner WUC 137 ( NG _
013192.1) 97 2 2.5 散囊菌纲 2.5
Cheilymenia stercorea voucher OSC:100034 (NG_013143.1) 92 1 1.2 盘菌纲 1.2
异常球菌纲(Deinococci, 0.7%)所占比例较小.克隆
文库中与 NCBI中已知菌种相似度大于 93%的克隆
子主要分布在不动杆菌属(Acinetobacter, 39.7%)、
葡萄球菌属(Staphylococcus, 11.3%)、鞘脂单胞菌属
( Sphingomonas, 8. 6%)、 副球菌属 ( Paracoccus,
6􀆰 0%)和马赛菌属(Massilia, 5. 3%),新鞘脂菌属
(Novosphingobium, 4. 0%)、肉杆菌属(Carnobacteri⁃
um, 2􀆰 7%)、丙酸杆菌属(Propionibacterium,1􀆰 3%)、
Amaricoccus(1.3%)和黄色单胞菌属(Xanthomonas,
0􀆰 7%)所占比例较小.
18 rDNA 克隆文库中克隆子与 NCBI 中已知菌
种的相似性在 88% ~ 99%,相似度大于 97%的克隆
子 65个,占总数的 80.3%,相似度低于 93%的克隆
子 3个,占总数的 3.7%.克隆子分布子囊菌门(Asco⁃
mycota, 97.5%)和担子菌门(Basidiomycota, 2.5%).
文库中的克隆子与 NCBI 中已知菌种相似度大于
93%的菌株主要分布在核腔菌属 ( Pyrenophora,
76􀆰 5%)、炭角菌属(Xylaria, 13.6%),外瓶霉属(Ex⁃
ophiala, 2.5%)、平脐疣孢属(Bipolaris, 1.2%)、蚪
孢壳属(Bombardia, 2.5%),核腔菌属占绝对优势.
4211 应  用  生  态  学  报                                      26卷
图 3  青岛市秋季空气细菌系统发育树
Fig.3  Phylogenetic tree based on 16S rDNA clones of airborne bacteria in Qingdao in autumn.
图 4  青岛市秋季空气真菌系统发育树
Fig.4  Phylogenetic tree based on 18S rDNA clones of airborne fungi in Qingdao in autumn.
3  讨    论
青岛市秋季空气细菌主要集中在变形菌门,说
明变形菌门细菌比较适合生活在大气环境,与同样
采用分子生物学方法的国内外学者的研究结果一
致[20-22,38-39] .Bowers等[40]在美国科罗拉多州采集空
气样本,研究发现变形菌门占优势,厚壁菌门和放线
菌门比例也较大,并且空气微生物群落结构结果与
天气状况无关.变形菌门细菌属革兰氏阴性菌,分布
广,对低温、辐射等具有很强的抵抗力[40-42] .国外学
者[43-45]在研究空气微生物来源时发现,如果气溶胶
气团的来源为海洋,则革兰氏阴性菌占主导地位,而
当气团来自内陆时则表现为革兰氏阳性菌占优势.
从属水平看,青岛市秋季空气细菌优势菌属不动杆
菌属、鞘脂单胞菌属、葡萄球菌属和马赛菌属,革兰
氏阴性菌远大于革兰氏阳性菌.不动杆菌属为好氧
嗜中温细菌[46],广泛分布在水体和土壤环境中,该
类细菌粘性大,当空气中颗粒物较多时,易粘附于颗
粒物上大量繁殖,潮湿环境下更易生存.鞘脂单胞菌
属在土壤、水体以及各种极端环境中均可以生
存[47-49] .鞘脂单胞菌属可以适应多变的环境,尤其
是营养缺乏环境,可抵抗高温、紫外线、严寒等,与植
物关系密切,说明植被也是青岛市空气微生物的重
要来源[50] .葡萄球菌在自然界分布广泛,主要在哺
乳动物和鸟类的皮肤、皮肤腺体和黏膜上.马赛菌属
以易降解有机物质为底物,属于生长速度较快的富
营养菌,是许多植物土壤根际的优势菌群[51] .
研究发现,采用分子生物学方法和传统培养方
法研究空气微生物,所得结果差别较大.方治国
等[23-24]采用传统培养方法研究表明,北京市空气细
菌优势菌属为芽孢杆菌属、葡萄球菌属、微球菌属.
徐文兵等[25]对青岛市近海秋季生物气溶胶研究表
52114期                              王  琳等: 青岛市秋季空气微生物群落多样性           
明,考克氏菌属 (Kocuria)、短杆菌属 (Brevibacteri⁃
um)和巨大芽孢杆菌(Bacillus megaterium)为优势菌
属,革兰氏阳性菌多于革兰氏阴性菌. 国外学
者[52-59]对城市、农村、沿海、森林等空气微生物群落
结构采用传统培养方法和分子生物学法研究,传统
培养法得到的细菌 70% ~ 90%为革兰氏阳性菌,主
要为芽孢杆菌属、微球属、葡萄球菌属和假单胞菌属
(Pseudomonas)细菌,但采用分子生物学方法得到的
结果为革兰氏阴性菌为优势菌种,尤其以 α⁃变形菌
纲、β⁃变形菌纲、γ⁃变形菌纲及拟杆菌纲较多,常见
菌属为鞘脂单胞菌属和假单胞菌属.
空气真菌优势菌群为子囊菌门,与国内外的研
究结果相同[60-61],从属水平来看,座囊菌纲中的核
腔菌属、粪壳菌纲中的炭角菌属为优势菌属.核腔菌
属主要寄生在植物树叶上,对农作物及果树危害很
大.炭角菌属菌群种类多,分布广,在春末或秋初时
繁殖较快,主要在植物表面产生,说明除水体、土壤
以外,植被也是空气微生物的来源.方治国等[23-24]
采用传统培养方法得到北京空气真菌优势菌属为枝
孢属(Cladosporium) 、链格孢属(Alternaria)和无孢
菌(Sterile moniliaceous);凌琪等[62]对合肥空气真菌
多样性研究显示优势菌属为曲霉属(Aspergillus)和
青霉属(Penicillium);符春兰等[63]对北京、上海、广
州、成都 4城市空气真菌研究表明,优势菌属为枝孢
属、链格孢属、青霉属和曲霉属;国外文献中空气真
菌优势菌属为枝孢菌属、链格孢属、无孢菌、青霉属
以及曲霉属[64-65],与采用分子生物学方法的研究结
果差别较大.
青岛市秋季空气中存在部分致病菌或条件致病
菌.不动杆菌属是条件致病菌,可以引起肺炎、皮肤
和软组织感染、创面感染、泌尿系统感染、继发性脑
膜炎和败血症等疾病[66] .鞘脂单胞菌属可以通过感
染伤口进入人体引起炎症和败血症[67] .葡萄球菌是
常见的化脓性细菌,是医院交叉感染的重要来源.真
菌孢子被发现与人们的过敏有关,其中可导致人体
过敏的真菌主要在子囊菌门中[68] .此外,麦类核腔
菌(P. graminea)、团炭角菌(X. hypoxylon)、角状平
脐疣孢(Z. angulare)与农作物疾病有关,可引起多
种农作物枯萎死亡.
在构建的克隆文库中,细菌克隆子和真菌克隆
子相似度低于 93%的分别占总数的 16.6%和 3.7%.
一般认为,序列相似度若低于 97%即属于不同
种[69],序列相似度小于 93%,则可以认为属于不同
属[23-24] .说明空气中存在部分未知细菌和真菌种类.
另外,在本研究中,16S rDNA和 18S rDNA 克隆文库
的覆盖度分别为 93.4%和 93.8%,虽然已包含大部
分空气细菌和空气真菌,但仍有部分菌种无法涵盖.
此外,我们课题组采用实时荧光定量 PCR技术检测
空气 DNA样品,发现可检测出大肠杆菌(Escherichia
coli)、粪肠球菌(Enterococcus faecalis),但这些类群
未在克隆文库中出现,说明克隆文库方法对低含量
微生物灵敏度不够,今后还将采用高通量测序和实
时荧光定量 PCR等方法进行深入研究.
参考文献
[1]  Gandolfi I, Bertolini V, Ambrosini R, et al. Unravelling
the bacterial diversity in the atmosphere. Applied Micro⁃
biology and Biotechnology, 2013, 97: 4727-4736
[2]  Cote V, Kos G, Mortazavi R, et al. Microbial and deno⁃
vo transformation of dicarboxylic acids by three airborne
fungi. Science of the Total Environment, 2008, 390:
530-537
[3]  Fuzzi S, Mandrioli P, Perfetto A, et al. Fog droplets an
atmospheric source of secondary biological aerosol parti⁃
cles. Atmospheric Environment, 1997, 31: 287-290
[4]  Lighthart B. The ecology of bacteria in the alfresco at⁃
mosphere. FEMS Microbiology Ecology, 1997, 23:
263-274
[5]  Sattler B, Puxbaum H, Psenner R, et al. Bacterial
growth in supercooled cloud droplets. Geophysical Re⁃
search Letters, 2001, 28: 239-242
[6]  Tong YY, Lighthart B. Solar radiation is shown to select
for pigmented bacteria in the ambient outdoor atmos⁃
phere. Photochemistry and Photobiology, 1997, 65:
103-106
[7]  Womack AM, Bohannan BJM, Green JL, et al. Biodi⁃
versity and biogeography of the atmosphere. Philosophi⁃
cal Transactions of the Royal Society of London, 2010,
365: 3645-3653
[8]  Naddafi K, Jabbari H, Hoseini M, et al. Investigation of
indoor and outdoor air bacterial density in Tehran sub⁃
way system. Iranian Journal of Environmental Health
Science and Engineering, 2011, 8: 381-386
[9]  Curtis L, Rea W, Smith WP, et al. Adverse health
effects of outdoor air pollutants. Environment Internation⁃
al, 2006, 32: 815-830
[10]  Gorny RL, Reponen T, Willeke K, et al. Fungal frag⁃
ments as indoor air biocontaminants. Applied and Envi⁃
ronmental Microbiology, 2002, 68: 3522-3531
[11]  Fracchia L, Pietronave S, Rinaldi M, et al. The assess⁃
ment of airborne bacterial contamination in three com⁃
posting plants revealed site⁃related biological hazard and
seasonal variations. Journal of Applied Microbiology,
2006, 100: 973-984
[12]  Mandal J, Brandl H. Bioaerosols in indoor environment:
6211 应  用  生  态  学  报                                      26卷
A review with special reference to residential and occu⁃
pational locations. Open Environmental and Biological
Monitoring Journal, 2011, 4: 83-96
[13]  Bauer H, Giebl H, Hitzenberger R, et al. Airborne bac⁃
teria as cloud condensation nuclei. Journal of Geophysi⁃
cal Research, 2003, 108, 4658, doi: 10. 1029 /
2003JD003545, D21
[14]  Wang X⁃D (王晓丹), Li Y⁃H (李艳红). Molecular
biology method applied in the study of microbial ecology
in water. Journal of Microbiology (微生物学通报),
2007, 34(4): 777-781 (in Chinese)
[15]  Liu WT, Marsh TL, Cheng H, et al. Characterization of
microbial diversity by determing terminal restriction frag⁃
ment length polymorphisms of genes encoding
16S rRNA. Applied and Environmental Microbiology,
1997, 63: 4516-4522
[16]  Lighthart B. Mini⁃review of the concentration variations
found in the alfresco atmospheric bacterial populations.
Aerobiologia, 2000, 16: 7-16
[17]  Stubbs SLJ, Brazier JS, Talbot PR, et al. PCR⁃restric⁃
tion fragment length polymorphism analysis for identifi⁃
cation of Bacteroides spp. and characterization of nitroim⁃
idazole resistance genes. Journal of Clinical Microbiolo⁃
gy, 2000, 38: 3209- 3213
[18]  Yavuz E, Gunes H, Bulut C, et al. RFLP of 16S⁃ITS
rDNA region to differentiate Lactobacilli at species level.
World Journal of Microbiology and Biotechnology, 2004,
20: 535-537
[19]  Osbom AM, Moore ERB, Tmmis KN. An evaluation of
terminal⁃restriction fragment length polymorphism
(T⁃RFLP) analysis for the study of microbial community
structure and dynamics. Environmental Microbiology,
2000, 2: 39-50
[20]  Lee T, Grinshpun SA, Martuzevicius D, et al. Relation⁃
ship between indoor and outdoor bioaerosols collected
with a button inhalable aerosol sampler in urban homes.
Indoor Air, 2005, 16: 37-47
[21]   Polymenakou PN, Mandalakis M. Assessing the short⁃
term variability of bacterial composition in background
aerosols of the Eastern Mediterranean during a rapid
change of meteorological conditions. Aerobiologia, 2013,
29: 429-441
[22]  Despres VR, Nowoisky JF, Klose M, et al. Character⁃
ization of primary biogenic aerosol particles in urban,
rural, and high⁃alpine air by DNA sequence and restric⁃
tion fragment analysis of ribosomal RNA genes. Biogeo⁃
sciences, 2007, 4: 1127-1141
[23]  Fang Z⁃G (方治国), Ouyang Z⁃Y (欧阳志云), Hu L⁃
F (胡利锋), et al. The research progress of the micro⁃
bial community of urban ecological system air. Acta Eco⁃
logica Sinica (生态学报), 2004, 24(2): 315 - 322
(in Chinese)
[24]  Fang Z⁃G (方治国), Ouyang Z⁃Y (欧阳志云), Hu L⁃
F (胡利锋), et al. The summer air microbial communi⁃
ty structure and ecological distribution of Beijing. Acta
Ecologica Sinica (生态学报), 2005, 25(1): 83-88
(in Chinese)
[25]  Xu W⁃B (徐文兵), Qi J⁃H (祁建华), Jin J (金  
川), et al. Qingdao offshore summer, autumn biological
characteristics of aerosol distribution research. Environ⁃
mental Science (环境科学), 2011, 32(1): 9-17 ( in
Chinese)
[26]  D’Amato G. Environmental urban factors (air pollution
and allergens) and the rising trends in allergic respirato⁃
ry diseases. Allergy, 2002, 57: 30-33
[27]  Eames I, Tang JW, Li Y, et al. Airborne transmission
of disease in hospitals. Journal of the Royal Society Inter⁃
face, 2009, 6: 697-S702
[28]  Eduard W, Heederik D, Duchaine C, et al. Bioaerosol
exposure assessment in the workplace: The past, present
and recent advances. Journal of Environmental Monito⁃
ring, 2012, 14: 334-339
[29]  Tang JW. The effect of environmental parameters on the
survival of airborne infectious agents. Journal of the Roy⁃
al Society Interface, 2009, 6: 737-746
[30]  Spröer C, Reichenbach H, Stackebrandt E. The correla⁃
tion between morphogenetic classification of myxobacte⁃
ria. International Journal of Systematic Bacteriology,
1999, 49: 1255-1262
[31]  Smit E, Leeflang P, Glandorf B, et al. Analysis of fun⁃
gal diversity in the wheat rhizosphere by sequencing of
cloned PCR⁃amplified genes encoding 18S rRNA and
temperature gradient gel electrophoresis. Applied and
Environmental Microbiology, 1999, 65: 2614-2621
[32]  Zeng RY, Zhao J, Zhang R, et al. Bacterial community
in sediment from the Western Pacific “Warm Pool” and
its relationship to environment. Science in China Series
D: Earth Sciences, 2005, 48: 282-290
[33]  Hu J (胡   江), Dai X⁃Z (代先祝), Li S⁃P (李顺
鹏). Effects of atrazine and its degrader Exiguobaterium
sp. BTAH1 on soil microbial community. Chinese Jour⁃
nal of Applied Ecology (应用生态学报), 2005, 16
(8): 1518-1522 (in Chinese)
[34]  Jones CM, Thies JE. Soil microbial community analysis
using two⁃dimensional polyacrylamide gel electrophoresis
of the bacterial ribosomal internal transcribed spacer re⁃
gions. Journal of Microbiological Methods, 2007, 69:
256-267
[35]  Diez B, Bauer K, BergmanB. Epilithic cyanobacterial
communities of a marine tropical beach rock (Heron Is⁃
land, Great Barrier Reef): Diversity and diazotrophy.
Applied and Environmental Microbiology, 2007, 73:
3656-3668
[36]  Zhan J (詹  婧), Yang G⁃D (阳贵德), Sun Q⁃Y (孙
庆业). Diversity of nitrogen⁃fixing microorganisms in bi⁃
ological soil crusts of copper mine wastelands. Chinese
72114期                              王  琳等: 青岛市秋季空气微生物群落多样性           
Journal of Applied Ecology (应用生态学报), 2014, 25
(6): 1765-1772 (in Chinese)
[37]  Wang Y (王   英), Teng Q⁃H (滕齐辉), Cui Z⁃L
(崔中利), et al. No⁃tillage paddy soil bacterial diversi⁃
ty and spatial distribution of the research. Acta Pedologi⁃
ca Sinica (土壤学报), 2007, 44(1): 137- 143 ( in
Chinese)
[38]  Cao C, Jiang WJ, Wang BY, et al. Inhalable Microor⁃
ganisms in Beijing’s PM2.5 and PM10 pollutants during
a severe smog event. Environmental Science and Technol⁃
ogy, 2014, 48: 1499-1507
[39]   Maron PA, Lejon DPH, Carvalho E, et al. Assessing
genetic structure and diversity of airborne bacterial com⁃
munities by DNA fingerprinting and 16S rDNA clone li⁃
brary. Atmospheric Environment, 2005, 39: 3687-3695
[40]  Bowers RM, Lauber CL, Wiedinmyer C, et al. Charac⁃
terization of airborne microbial communities at a high⁃el⁃
evation site and their potential to act as atmospheric ice
nuclei. Applied and Environment Microbiology, 2009,
75: 5121-5130
[41]  Amato P, Parazols M, Sancelme M, et al. Microorgan⁃
isms isolated from the water phase of tropospheric clouds
at the Puy de Dôme: Major groups and growth abilities
at low temperatures. FEMS Microbiology Ecology, 2007,
59: 242-254
[42]   Christner BC, Mosley⁃Thompson E, Thompson LG, et
al. Bacterial recovery from ancient glacial ice. Environ⁃
mental Microbiology, 2003, 5: 433-436
[43]  Cho BC, Hwang CY. Prokaryotic abundance and 16S
rRNA gene sequences detected in marine aerosols on the
East Sea (Korea). FEMS Microbiology Ecology, 2011,
76: 327-341
[44]  Fahlgren C, Hagström Å, Nilsson D, et al. Annual vari⁃
ations in the diversity, viability, and origin of airborne
bacteria. Applied and Environmental Microbiology,
2010, 76: 3015-3025
[45]  Urbano R, Palenik B, Gaston CJ, et al. Detection and
phylogenetic analysis of coastal bioaerosols using culture
dependent and independent techniques. Biogeosciences,
2011, 8: 301-309
[46]  Ai M⁃Q (艾明强), Li H (李   慧), Liu X⁃B (刘晓
波), et al. Bacterial community structure in production
water from oil reservoirs in Daqing Oilfield. Chinese
Journal of Applied Ecology (应用生态学报), 2010, 21
(4): 1014-1020 (in Chinese)
[47]  Berg G, Ballin G. Bacterial antagonists to Verticillium
dahliae Kleb. Journal of Phytopathology, 1994, 141:
99-110
[48]  Adhikari TB, Joseph CM, Yang G, et al. Evaluation of
bacteria isolated from rice for plant growth promotion and
biological control of seedling disease of rice. Canadian
Journal of Microbiology, 2001, 47: 916-924
[49]  Pinhassi J, Berman T. Differential growth response of
colony forming and proteobacteria in dilution culture and
nutrient addition experiments from Lake Kinneret ( Isra⁃
el), the eastern Mediterranean Sea, and the Gulf of
Eilat. Applied and Environmental Microbiology, 2003,
69: 199-211
[50]  Li Y, Kawamura Y, Fujiwara N, et al. Sphingomonas
yabuuchiae sp. nov. and Brevundimonas nasdae sp.nov.,
isolated from the Russian Space Laboratory Mir. Interna⁃
tional Journal of Systematic and Evolutionary Microbiolo⁃
gy, 2004, 54: 819-825
[51]   Ofek M, Hadar Y, Minz D. Ecology of root colonizing
Massilia (Oxalo⁃bacteraceae). PLoS One, 2012, 7(7):
e40117
[52]  Brodie EL, DeSantis TZ, Parker JPM, et al. Urban
aerosols harbor diverse and dynamic bacterial popula⁃
tions. Proceedings of the National Academy of Sciences of
the United States of America, 2007, 104: 299-304
[53]  Shaffer B, Lighthart B, et al. Survey of culturable air⁃
borne bacteria at four diverse locations in Oregon:
Urban, rural, forest, and coastal. Microbial Ecology,
1997, 34: 167-177
[54]  Bovallus A, Bucht B, Roffey R, et al. Three⁃year inves⁃
tigation of the natural airborne bacterial flora at four lo⁃
calities in Sweden. Applied and Environmental Microbiol⁃
ogy, 1978, 35: 847-852
[55]  Fang ZG, Ouyang ZY, Zheng H, et al. Cultural air⁃
borne bacteria in outdoor environments in Beijing, Chi⁃
na. Microbial Ecology, 2007, 54: 487-496
[56]  Zweifel UL, Hagström Å, Holmfeldt K, et al. High bac⁃
terial 16S rRNA gene diversity above the atmospheric
boundary layer. Aerobiologia, 2012, 28: 481-498
[57]  Maron PA, Mougel C, Lejon DPH, et al. Temporal var⁃
iability of airborne bacterial community structure in an
urban area. Atmospheric Environment, 2006, 40: 8074-
8080
[58]  Fahlgren C, Bratbak G, Sandaa RA, et al. Diversity of
airborne bacteria in samples collected using different de⁃
vices for aerosol collection. Aerobiologia, 2011, 27:
107-120
[59]  Di Giorgio C, Krempff A, Guiraud H, et al. Atmospher⁃
ic pollution by airborne microorganisms in the city of
Marseilles. Atmospheric Environment, 1996, 30: 155 -
160
[60]  Safatov AS, Teplyakova TV, Belan BD, et al. Atmos⁃
pheric aerosol fungi concentration and diversity in the
south of western Siberia. Atmospheric and Oceanic Op⁃
tics, 2010, 23: 73-79
[61]  Sunghee L, Bora C, Seung MY, et al. Characterization
of microbial community during Asian dust events in
Korea. Science of the Total Environment, 2009, 407:
5308-5314
[62]  Ling Q (凌  琪), Wang Y⁃P (王晏平), Wang L (王
莉), et al. The preliminary study on the distribution
8211 应  用  生  态  学  报                                      26卷
feature of Hefei air microorganisms. Journal of Environ⁃
ment and Health (环境与健康杂志), 2007, 24(1):
40-43 (in Chinese)
[63]  Fu C⁃L (符春兰), He W⁃H (何文华), Jia J⁃H (贾建
华), et al. Fungi special investigation four cities in our
country. Journal of Microbiology (微生物学通报),
2000, 27(4): 264-269 (in Chinese)
[64]   Chang CW, Chung H, Huang CF, et al. Exposure of
workers to airborne microorganisms in open⁃air swine
houses. Applied and Environmental Microbiology, 2001,
67: 155-161
[65]  Shelton BG, Kirkland KH, Flanders WD, et al. Profiles
of airborne fungi in building and outdoor environments in
the Unites Stated. Applied and Environmental Microbiolo⁃
gy, 2002, 68: 1743-1753
[66]  Dijkshoorn L, Nemec A, Seifert H, et al. An increasing
threat in hospitals: Multidrug⁃resistant Acinetobacter
baumannii. Nature Reviews Microbiology, 2007, 5:
939-951
[67]  Hu J (胡   杰), He X⁃H (何晓红), Li D⁃P (李大
平), et al. The research progress of Sphingomonas. Chi⁃
nese Journal of Applied and Environmental Biology (应
用与环境生物学报), 2007, 13(3): 431 - 437 ( in
Chinese)
[68]  Chew F, Lim S, Shang H, et al. Evaluation of the aller⁃
genicity of tropical pollen and airborne spores in Singa⁃
pore. Allergy, 2000, 55: 340-347
[69]  Brodie EL, DeSantis TZ, Parker JP, et al. Urban aero⁃
sols harbor diverse and dynamic bacterial populations.
Proceedings of the National Academy of Sciences of the
United States of America, 2007, 104: 299-304
作者简介  王  琳,女,1988 年生,硕士研究生.主要从事环
境微生物研究. E⁃mail: 154316701@ qq.com
责任编辑  肖  红
92114期                              王  琳等: 青岛市秋季空气微生物群落多样性