浮游球衣菌单株培养形态学特征的研究

第44卷　第4期2008年7月
南京大学学报(自然科学)
J OU RNAL OF NANJ IN G UN IV ERSIT Y
(NA TU RAL SCIENCES)
Vol.44,No.4
J ulyω2008
浮游球衣菌单株培养形态学特征的研究3
叶　兰33
(安徽省环境监测中心站,合肥,230061)
摘　要:　浮游球衣菌是污水好氧生化处理中的知名菌种,对污水中有机物和重金属有很强的降解和去除作用.本文经分离纯化后,对单个菌株进行连续培养.该菌株在极低营养环境(A培养基)中呈现树枝状向四周辐射的菌落,在普通琼脂平板,呈光滑园形隆起的白色菌落.对数期的细胞10～12min分裂一次;单胞菌体和短丝体阶段均强烈摆动.培养4d后,菌丝达十几个到数十个细胞长时,丝体相互缠结成团,丝体中细胞大量逸出胶鞘.电镜片显示,菌体细胞两端均有纤维状鞭毛并相互连接成丝体,胞外有宽厚的半透明胶鞘.
该菌具有在低营养环境中强力吸取营养的特性,以及快速繁殖和强烈的活动性,与其在污水处理中的卓越效果可能有关联.长丝体缠结成团时,丝体中细胞大量逸出,仅含胶质鞘团块的比重必然减轻,这可能是污泥膨胀的主要原因.在培养后期溶解氧(DO)有明显下降,也可能与污泥澎胀有因果关系.
关键词:　浮游球衣菌,单株培养,形态学特征,电镜分析
中图分类号:　X172
Morphology features cultivating S phaerotil us nat a ns
by single strain research
Ye L an
(Anhui Province Environment Monitoring Centre Station,Hefei,230061,China)
Abstract:　S p haerotil us natans is a well2known strain in the environmental microbiology.In this paper the very low culture concentration is adopted for its isolation and purification(one2tenth of the commonly2used culture medium). The ends of some fine fibrous mycelium formed on the culture medium are picked and readmitted with inoculation needle so that the efficiency of isolation and purification become higher.The strain after purification is continuously diluted and cultured until monocelled strain is obtained.Based on the colony configuration of A and B culture media, through physiological and biochemical tests and analysis of electron microscope,this strain is determined as the S phaerotil us natans.
The S phaerotil us natans splits every10to12minutes during the log phase of growth curve and the unit cell and short filament can make movements.After the unit cell is cultured for2hours and put it into250mL conical flask (with50mL culture fluid inside)where it is cultured at T=30℃and n=150r/min.After24hours,10mL culture fluid is supplemented for each12hours in order to keep the concentration of the culture fluid unchanged.The thallus
3 33基金项目:国家自然科学基金(49672102)收稿日期:2008-01-31
E2mail:jmmdn@
after being cultured for4days has entered into the period of decline,the filament elongated to a dozen of cells,lost the ability for movement and wrapped,then a large number of cells in the filament are escaped f rom the sheath and the specific gravity of that only containing glial lump becomes lighter and float on the liquid level.The electron microscope photo shows that there is semi2transparent sheath with wide and thick coating at the outside of cell and at both ends of the cell there are fibrous flagellums from protoplasm,which are connected with cell.
During log phase and stationary phase of the growth curve,since the S phaerotil us natans has the features of rapid reproduction and vivid movement as well as ability to extract nutrition in the arid nutrition environment and heavy metal,so it has outstanding efficacy for the sewage purification.But after4days when the growth curve reaches the period of decline,its ability of movement disappears and it becomes a lump,large numbers of cells in the filament escape f rom sheath so that the lump floats up,the mud and water are difficult to be separated,which causes the sludge expansion and deteriorates the sewage treatment efficacy.This is caused by the morphologic feature of the S phaerotil us natans during this period of decline and is the primary cause for the f requent occurrence of the sludge expansion in all aerobic biological treatment of sewage.Therefore,to prevent the expansion of sludge,the most important is to let the sludge in the sewage treatment facility drain out prior to the period of decline of S p haerotil us natans has and supplement in large quantity the strains which have been synchronously cultured for24hours and are in the log phase besides going on adjusting many factors in the environment so as to meet the requirements of S phaerotil us natans has.This has put forward a new exploration way to solve this difficult problem of sludge expansion.
K ey w ords:　S phaerotil us natans,morphology features,single strain cultivating,electron microscope analyse
浮游球衣菌普遍存在于被有机物污染的水体中,对有机物和重金属均有很强的降解和吸附能力,同时还可以积聚β2羟基丁酸(P HB)[1～3]等,在环境微生物学和环境工程中具有重要作用.但由于该菌有胶鞘,极易沾染杂菌,不易纯化;特别是虽然在污水处理初期效果显著,但后期反而又易引发污泥膨胀,因此引起众多专家学者的关注[4,5].美国学者Stokes自20世纪50年代率先对该菌生态特征进行了研究[6],提出了该菌培养和纯化的办法.文献[7, 8]也提出了该菌提纯等一系列方法.
本文从纯化浮游球衣菌开始,对其单细胞菌株进行连续培养,并研究了其生理生态和形态特征,以利于指导其在污水处理中发挥更大的作用.浮游球衣菌在其生长曲线的对数期和稳定期,繁殖速度快速,运动特性活泼,在贫瘠营养环境中能吸取营养和吸附重金属,所以在污水净化中具有卓越的功效.但生长曲线达到衰亡期后,丝体增长,运动能力消失,并结成团状,丝体中细胞大量逸出胶鞘,致使团块上浮,泥水分离困难,造成污泥膨胀,恶化了污水处理效果.这是由于该菌衰亡期的形态学特征所造成的,这也可能就是在所有污水好氧生化处理中,污泥膨胀都常有发生的根本原因.因此,为防止污泥膨胀,除了继续调整环境中诸多因素以适应浮游球衣菌的要求外,最关键之处就是使污泥在菌株衰亡期到来之前排出,并在污水处理设施中大量补充同步培养的处于对数期的菌种,为解决污泥膨胀这一难题提出了新的探索途径.
1　材料与方法
111　材　料　
11111　菌种来源　合肥王小郢污水处理厂. 11112　培养基(质量分数%)　A培养基:蛋白胨0105,葡萄糖011,琼脂2(液体培养不用),蒸馏水配制,p H=710±011.B培养基:蛋白胨015,酵母膏011,乳糖012,K2H PO4
・
2
4
4
・南京大学学报(自然科学) 第44卷
0105,琼脂2,蒸馏水配制,p H=710±011. 11113　主要化学药品试剂及实验仪器　琼脂(食品级),青岛微生物试剂厂.酵母膏(食品级),唐山绿仙生物科技有限公司.蛋白胨(生化试剂),上海医药集团上海化学试剂公司.乳糖(A R),汕头市光华化学厂.葡萄糖(AR),上海医学集团上海化学试剂公司.TDL240B低速离心机,上海安亭科学仪器厂.L R H2250A型生化培养箱,广东省医疗器械厂.SH K299211型恒温摇床,上海实验仪器总厂.FA1104型电子天平,天津天时利电炉有限公司.P HB23型p H 计,上海三信仪表厂.LDO TM溶解氧仪,美国哈希公司.OL YM PU S2H20显微镜,日本OL YM PU S公司.J EM2100CXⅡ透射电镜,日本电子公司.
112　方　法
11211　分离纯化　自活性污泥中,以接种针挑取粘液状菌膜,点播于A培养基平板.30℃培养48～72h后,挑取幅射状菌落末端分枝,反复接种3～4次,可望获得纯菌株.转接种于B 培养基.经观察A、B培养基上的菌落形态、电镜分析菌体、分析其生理生化和革兰氏染色阴性等特征,可确定分离纯化菌株属于鞘细菌类的浮游球衣菌[9].
11212　单胞培养方法　将A培养基中的纯化菌落,转接种于A培养液中,摇床培养(150r/ min)24h,此时菌体生长曲线已是对数期.梯度稀释镜检,至菌胞浓度低于每毫升100个时,转移至凹玻片之凹穴中(每穴只允许一个单胞), 30℃培养并以显微镜连续观察拍片.
11213　菌体生长曲线培养测定方法　从已纯化的浮游球衣菌斜面培养,接种3环至装有50 mL A培养液的250mL三角烧瓶,T=30℃n=150r/min震荡培养,并于24h后补充A营养液10mL.以后每隔12h逐次增加补液量10 mL,尽可能保持培养过程中培养液浓度不变,以模拟污水处理工程中的条件,并定时测定p H、溶解氧(DO).培养物以4000r/min离心15min,收集菌体于80℃烘干至恒重,即得球衣菌干生物量P b.11214　电镜观测方法　用钢网蘸取菌体细胞,于滤纸上室温干燥,透射电镜在加速电压100 kV下观察拍片.
2　结　果
211　单胞培养显示浮游球衣菌的分裂周期为10～12min(图1a～d,×600)
.
图1　浮游球衣菌的分裂周期(×600)
Fig.1　The split cycle of S phaerotilus natans(×600) a.开始时,单胞菌株;b.10min时,1分为2个细胞;c.22min 时,2分为4个细胞;d.35min时,4分为8个细胞
212　菌落形态在贫瘠营养的A培养基上,呈现由中心向四周辐射的白色绒毛状分枝,菌苔极薄(图2a).在B培养基上,形成白色圆形隆起的菌落,菌落表面光滑,菌苔厚实(图2b). 213　菌液培养各项指标分析结果　从表1中可知,p H和培养液温度在培养5d中无显著变化,仅DO有明显下降.
214　菌体生长曲线培养测定结果　该菌培养在前4h处于延滞期,4h后即开始处于对数期.特别在18h以后生物量急剧增长,直至54 h达到最大值.此后过渡到生长曲线稳定期,并延续至84h,从90h起其生物量即逐渐下降,进入衰亡期,特别在96h以后下降速率更高.其生长曲线见图3.
图4～6为处于对数期及衰亡期的菌体形态特征.
215　电镜分析　从电镜照片(图7),可以清楚地见到菌体细胞外被宽厚半透明胶鞘,细胞两
・
3
4
4
・
　第4期叶　兰:浮游球衣菌单株培养形态学特征
表1　菌液震荡培养参数测定
T able 1　T raining parameters of B acilli shocks
时间(h )
p H DO (mg/L )T (℃)补液量(mL/L )
0712212107301050127126111052918502471171210430106036712611150301570487135111053012806071241015730109072710610105301010084711710156291811096712391613010120108713281823010130120712681652918140132711981063010150144712771642916160156710271243010170168710971032915180180618761642918190192619461223010200204710761242916210216711761263010220228710261063015230240619461073010240252710451863010250264
6193
5168
2918
260
端胶鞘厚度和细胞横径几近相等.细胞两端均具有自原生质体伸出的纤维状鞭毛,伸至胶鞘
外缘,丝体中的细胞由鞭毛相互连接
.
图2　浮游球衣菌在A 、B 培养基上的菌落形态
Fig.2　The colony conf iguration on A and B
media
图3　浮游球衣菌的生长曲线
Fig.3　G row th curve of S phaerotilus natans
・
444・南京大学学报(自然科学) 第44卷
3　讨　论
311　浮游球衣菌的分离纯化,很多学者作过成功的研究[7,8,10,11].本文由分离纯化产生的单细胞菌株,进行单株同步培养,研究其生长曲线中各个时期的形态学特征,则尚未见报导.
312　该菌快速的繁殖率,强烈的运动特性,以及在贫瘠营养环境中吸取营养物能力,可能与其在污水处理中的卓越功效有关.
313　污水好氧处理工程突出的问题是以浮游球衣菌为主体的活性污泥膨胀,污泥悬浮水中不能沉降,泥水分离困难,使水处理效果恶化.此乃环境工程中的难题之一,又是环境微生物学上的理论问题,至今仍未有定论.有关浮游球衣菌引起污泥膨胀的报导颇多,提出导致污泥膨胀的原因通常有水温过高、p H值或DO下降等,但这些参数等对污泥膨胀的影响常缺乏实质根据,报道结果也并不一致[6,7,12,13].
314　该菌生长曲线的对数期至稳定期,对污水的降解净化效果极佳.但其菌龄达到96h时,则已处于衰亡期.衰亡期的菌体形态特征是:菌丝缠绕成团,丝体中大量细胞游离出丝体外鞘,致使丝体成为空鞘,整个丝体比重下降,所以造成污泥膨胀,恶化污水处理效果.污水好氧处理池是敞开系统,系统内外的物化环境无法精确控制,一旦多数菌体衰亡期同时发生,污泥膨胀
・
5
4
4
・
　第4期叶　兰:浮游球衣菌单株培养形态学特征
就不可避免.这就是为什么在所有曝气池中,污泥膨胀都常有发生的根本原因.因此,为防止污泥膨胀发生,在持续调整环境中的诸多因素以适应球衣菌的要求外,关键之处,是使污水处理设施中的污泥在达到球衣菌衰亡期前夕,即时排出,同时,将同步培养24h的正处于对数期的菌种,大量补充至污水处理设施中.本文对浮游球衣菌形态学特征的研究,为解决避免污泥膨胀这一污水处理工程中的难题,指出了新的探索途径.
R eferences
[1]　Qin Y C,Guan X H,Wang L W,et al.Bio2
sorption of S phaerotil us natans to the heavy
metal ions Pb2+,Cu2+,Zn2+and Cd2+.Envi2
ronmental Pollution and Control,2005,27(9):
648～650.(秦玉春,关晓辉,王立文等.浮游球
衣菌对Pb2+、Cu2+、Zn2+、Cd2+的吸附性能研
究.环境污染与防治,2005,27(9):648～
650).
[2]　She C X,Xu X P,Shen X X,et al.Biosorption
and tolerance of S phaerotil us natans to heavy
metal ion.Chinese Journal of Applied and Envi2
ronmental Biology,2005,11(1):90～92.(佘
晨兴,许旭萍,沈雪贤等.浮游球衣菌对重金
属离子的耐受性及其吸附能力.应用与环境生
物学报,2005,11(1):90～92).
[3]　Chen J F,Xu X P,Li H Z.G eneral situation in
S p haerotil us study.Environmental Science and
Technology,2002,25(6):43～46.(陈接锋,
许旭萍,李惠珍.球衣菌属的研究概况.环境
科学与技术,2002,25(6):3～46).
[4]　Pellegrin V J,Stefan J,Michael W,et al.
Morphological and biochemical properties of a
S p haerotil us sp.isolation f rom paper mill
slimes.Applied and Environmental Microbiolo2
gy,1999,65(1):156～162.
[5]　Ta Kada M,Fumio N.Isolation and chemical
composition of the sheathed of S p haerotil us na2
tans.Bioscience,Biotechnology,and Biochem2
istry,1998,62:1138～1143.
[6]　Stokes J L.Studies on the filamentous sheathed
iron bacterium S p haerotil us natans.The Jour2
nal of Bacteriology,1954,67:278～291.
[7]　Xu Y X,Liu S W.Isolation and study on bio2
logical characteristics of S p haerotil us natans.
Journal of Microbiology,2002,22(1):20～
21.(徐艳霞,刘世旺.球衣细菌的分离和生物
特性研究.微生物学杂志,2002,22(1):20～
21).
[8]　Weng S Y,Ke J K,Ji B J,et al.Study of
S phaerotil us natans2the Sphaerotilus of isola2
tion,identification and culture preservation.
Journal of East China Normal University(Nat2
ural Siciences),1980(3):77～84.(翁酥颖,柯
嘉康,寂蓓静等.球衣菌的研究———球衣菌的纯
种分离、鉴定和保存.华东师范大学学报(自然
科学),1980(3):77～84).
[9]　EBuchanan R,G ibbons N E.Bergey’s Manual
of Determinative Bacteriology.Microbial Re2
search Institute of Chinese Academy of Sci2
ences.The8th edition.Beijing:Science Press,
1984,157～160.(布坎南R E,吉本斯N E.伯
杰氏细菌鉴定手册.中国科学院微生物研究
所.笫8版.北京:科学出版社,1984,157～
160).
[10]　Li H Z,Xu X P,Lin Y X,et al.Isolation iden2
tification and habitat conditions of Fe-Mn oxi2
dized sheathed bacteria.Acta Hydrobiologica
Sinica,1999,23(4):311～315.(李惠珍,许
旭萍,林跃鑫等.铁锰氧化鞘细菌的生境条件
及分离鉴定.水生生物学报,1999,23(4):
311～315).
[11]　Liu S W,Deng Y X.Isolation and preservation
of S p haerotil us natans.Journal of Huanggang
Normal University,1997,17(4):38～42.(刘
世旺,邓元修.球衣菌的分离和保藏.黄冈师
专学报,1997,17(4):38～42).
[12]　Ding F,Wang S Y,Li X W,et al.Activated
sludge bulking due to lack of nutrient and its
control.Journal of Harbin University,2001,34
(5):59～63.(丁　峰,王淑莹,李秀玮等.缺乏
营养物质(N、P等)引起的污泥膨胀及其控制.
哈尔滨工业大学学报,2001,34(5):59～63). [13]　Xu X L,Shen X Y.Analysis of the cuse of fla2
ment Bulk2ing in activated sludge and control2
ling mothodology.Chinese Journal of Environ2
mental Science,1992,13(6):57～61.(许晓
路,申秀英.活性污泥丝状菌膨胀起因分析及
控制措施.环境科学,1992,13(6):57～61).
・
6
4
4
・南京大学学报(自然科学) 第44卷。