CN1166291C - 控制靶微生物的方法 - Google Patents
控制靶微生物的方法 Download PDFInfo
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- CN1166291C CN1166291C CNB998037494A CN99803749A CN1166291C CN 1166291 C CN1166291 C CN 1166291C CN B998037494 A CNB998037494 A CN B998037494A CN 99803749 A CN99803749 A CN 99803749A CN 1166291 C CN1166291 C CN 1166291C
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- Prior art keywords
- soil
- microorganism
- serine
- bacterial strain
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Abstract
本发明提供了控制靶微生物的方法,所述方法的特征在于在所述靶微生物和其它微生物共存的培养系统中加入了外源性的环境因子,所述外源性的环境因子为其它微生物提供了有利的生长条件,而为靶微生物提供了不利的生长条件。所述外源性环境因子是可被上述其它微生物同化而不能被靶微生物同化的有机物,其包括糖,氨基酸,低级链烷醇胺和有机酸等。
Description
技术领域
本发明涉及控制特定靶微生物生长的方法,更优选涉及在用微生物纯化污染土壤的纯化处理之后控制人为加入的微生物生长,藉此恢复土著微生物菌群的方法。本发明还涉及控制被病原体微生物污染的土壤中的所述病原体微生物的方法。
背景技术
近年来,人们的注意力集中于生物除污,将其作为纯化污染土壤的技术。在生物除污技术中,培养降解污染物的天然微生物(靶微生物)并将培养的微生物应用于土壤。尽管通过此方法可以纯化土壤,但是很多降解污染物的微生物仍残留在土壤中,这不利于土壤生态系统的平衡。因此,有关除去经处理后仍残留的降解污染物的微生物的方法,下列方法是已知的:例如使用自杀系统(自杀基因)的基因重组技术(将重组体导入生态系统的指南(Mannal for Introduction ofRecombinations into the Ecosystem),1993,农林渔业部(Ministryof Agriculture,Forestry,and Fisheries),p36-47),化学和物理灭菌法(植物生理学辞典(Dictionary of plant physiology),日本植物病理学学会编,Yokendo,1995,p701-719),以及赋予靶微生物营养缺陷型特性的方法。
然而,上述任一种方法都有其自身的问题。例如,由于使用自杀系统的方法基于不适用于野外(开放系统)的重组技术,因此其用途是不确定的。化学和物理灭菌法的缺点是:包括土壤微生物的所有微生物都成为灭菌的目标,因此会扰乱生态系统。另外,在使用营养缺陷型微生物时也有风险,即反向突变的存在可能会使营养缺陷型消失。
发明内容
因此,本发明的目的是提供一种新的恢复土壤中的土著微生物菌群的方法,所述方法不具有常规方法的缺点。
为了解决上述问题,本发明一般性地提供了控制靶微生物的方法,所述方法的特征在于在所述靶微生物和其它微生物共存的培养系统中加入了外源性的环境因子,所述外源性的环境因子为其它微生物提供了有利的生长条件,而为靶微生物提供了不利的生长条件。
上述外源性环境因子是可被上述其它微生物同化而不能被靶微生物同化的有机物。优选有机物是简单的化合物,如糖,氨基酸,低级链烷醇胺和有机酸。有机物取决于靶微生物。例如,当微生物为伯克氏菌(Burkholderia)属的N16-1菌株时,所述有机化合物优选为氨基酸D-丝氨酸。当微生物为Ralstonea solanacearum时,所述有机化合物优选为氨基酸D-丝氨酸或乙醇胺。
本文所用术语“靶微生物”指的是该微生物是降解污染物的微生物或病原体细菌。
附图简述
图1图示了蔗糖对土壤中的NKR菌株生长的控制效应。
图2图示了D-丝氨酸对土壤中的NKR菌株生长的控制效应。
图3图示比较了蔗糖和D-丝氨酸的生长控制效应。
图4图示了D-丝氨酸对不同土壤中的NKR菌株生长的控制效应。
图5图示了D-丝氨酸代谢物对土壤中的NKR菌株生长的控制效应。
图6图示了D-丝氨酸对土壤中的R.solanacearum OE-1菌株生长的控制效应。
图7图示了D-丝氨酸对土壤中的R.solanacearum OE1-1菌株生长的控制效应。
图8图示了由图7所述实验测得的土壤总细胞量。
图9图示了乙醇胺对土壤中的R.solanacearum OE1-1菌株生长的控制效应。
图10图示了由图9所述实验测得的土壤总细胞量。
图11用照片显示出在被R.solanacearum OE1-1菌株污染的土壤中加入不同量的D-丝氨酸之后,番茄植株的萌发和生长情况,其中A表示加入0ppm D-丝氨酸得到的结果,B表示加入500ppm D-丝氨酸得到的结果,C表示加入2500ppm D-丝氨酸得到的结果,D表示加入5000ppm D-丝氨酸得到的结果。
发明的实施方案
一般说来,使用本发明可促使多种微生物共存的系统中的其它微生物生长而控制特定微生物的生长,一般在通过生物除污处理被污染的土壤之后使用本发明以恢复天然的微生物系统。或者,也可使用本发明来纯化被病原体微生物污染的土壤。因此,为了纯化被污染物污染的土壤,生物除污包括在欲处理的土壤中接种能同化和/或降解污染物的微生物从而降解并除去土壤中的污染物。结果,在土壤的处理过程完成之后,人为加入土壤中的可降解污染物的微生物也在土壤中定居下来。因此,处理完污染的土壤之后,必需控制处理所用微生物的生长并恢复土著微生物菌群。或者,如果植物病原体微生物在农场大量产生或急剧增加,必需控制所述病原体而维持土著微生物菌群。
因此,根据本发明的方法,在需经纯化处理的土壤中加入了不能被降解污染物的微生物同化但能被土著微生物同化的有机物。这会促进土著微生物的生长,结果,降解污染物的微生物的生长受到控制,从而从土壤中消失。或者,已产生或繁殖的病原体的生长受到控制而消失。
本发明的方法可用于处理被污染物污染的土壤,所述污染物包括例如:有机氯化合物,如三氯乙烯(TCE)和二氯乙烯;石油烃,如酚和重油;农药,如2,4-D(2,4-二氯苯氧乙酸)和多氯酚(pantachlorophenol)。为了处理这些污染物,可使用如白腐菌的真菌,如伯克氏菌属和假单胞菌属的细菌和其它细菌。或者,可使用这些微生物处理病原体微生物大量产生或急剧增加的土壤。不能被靶微生物同化但能被土著微生物同化的有机化合物类型随靶微生物类型而变化,根据本发明公开的内容可以对其进行实验性选择。常规使用的微生物学检测方法可确定特定的微生物是否能同化有机物。
例如,通过在含有受试有机化合物作为唯一碳源或氮源的固体培养基或液体培养基中接种微生物,然后观察菌落的产生或培养基的密度即可进行检测。或者,在含有氧化-还原指示剂和经培养的受试微生物的培养基中加入受试的有机化合物,根据培养基中由氧化-还原指示剂产生的颜色或其生色强度即可测定微生物的同化能力。
然而,本发明中不能总是使用不能被靶微生物同化但能被土著微生物同化的那些有机化合物。例如,如果那些不能被降解污染物之微生物或植物病原体微生物同化的有机物被土著微生物降解,而降解产物或死亡的土著微生物能被降解污染物之微生物或病原体同化的话,所述有机物甚至能促进所述微生物的生长。在这种情况下,如果其降解产物对降解污染物之微生物或病原体微生物的生长促进作用是永久性的而不是瞬时的(更优选后者),它们就不适于用作本发明的有机物。
因此,必需通过使用特定的降解污染物之微生物或病原体微生物进行同化试验来选择(初步筛选)候选有机化合物,然后检测(二次筛选)它们对本发明是否真的有用。根据本发明公开的内容易于进行这一检测试验。例如,收集欲被降解的特定土壤,以几百ppm至几千ppm的浓度在其中加入受试的有机化合物。将受试的微生物(降解污染物之微生物或病原体微生物)接种于土壤中,然后于室温下进行保温。在保温期间或在保温一段时间之后统计受试微生物的数目,以选择导致降解污染物之微生物或病原体微生物数目减少的有机物,根据本发明的公开内容可进行上述试验。
在上述试验中,需要示差计数土壤中降解污染物之微生物或病原体微生物的数目,使用可选择性计数受试微生物的培养基可做到这一点。通过使降解污染物之微生物或病原体微生物变为药物抗性,如抗生素抗性即可容易地实现此目的。例如,通过赋予降解污染物之微生物以普通土著微生物所不具有的抗生素抗性,并在含有所述抗生素的固体培养基或不含抗生素的固体培养基上培养土壤样品,降解污染物的微生物仅在含有抗生素的固体培养基上形成菌落(或大菌落),籍此,通过测定菌落的数目可示差计数微生物的数目。
接着,为了筛选本发明的有机物,根据常规方法可相对较容易地赋予降解污染物的微生物以药物抗性,如抗生素抗性。例如,通过导入质粒或转座子可导入药物抗性基因。或者,可在例如含有抗生素的液体培养基中培养降解污染物的微生物,并在含有较高浓度抗生素的培养基中培养繁殖的微生物,将这种富集传代培养重复多次。或者,在含有一定浓度抗生素的固体培养基上培养降解污染物的微生物以使微生物形成菌落,然后在含有较高浓度抗生素的固体培养基上培养所述菌落以使微生物形成菌落,将这种富集传代培养重复多次。
至于上述外源性环境因子,优选为糖,氨基酸,低级链烷醇胺和有机酸等。糖优选为蔗糖,氨基酸优选为D-氨基酸,如D-丝氨酸,D-丙氨酸,D-半胱氨酸等。低级链烷醇胺优选为具有1至5个碳的链烷醇胺,如乙醇胺。有机酸优选为链烷酸,羟基链烷酸,酮酸或具有1至6个碳的酮酸,如丙酮酸。
作为本发明所用的特例,用于对三氯乙烯所污染的土壤进行生物除污的微生物为伯克氏菌属微生物。在伯克氏菌属微生物中应提及的是N16-1菌株(FERM BP-5504)等。日本专利申请9-04816中具体描述了该微生物,该微生物已被保藏于国立生命科学和人体技术研究所工业技术院,保藏号为FERM BP-5504。
如实施例2所述,伯克氏菌属的N16-1菌株(FERM BP-5504)不能同化多种有机化合物,包括蔗糖(糖),D-丝氨酸(氨基酸)等。当如实施例3所述在其中加入蔗糖时,N16-1菌株瞬时增加,但接着就开始减少,而土著微生物逐渐占据优势。尽管尚不清楚原因何在,但可以假设:蔗糖被土著微生物代谢,其代谢物被N16-1菌株同化,该代谢物瞬时增强了N16-1菌株的生长。
当降解污染物的微生物为伯克氏菌属微生物,尤其是其N16-1菌株时(如实施例4所述),本发明的有机化合物优选为D-丝氨酸。通过加入D-丝氨酸,N16-1菌株的细胞量相对于未加入D-丝氨酸时而言(对照)显著减少。
实施本发明的方法时,例如,可将降解污染物的微生物接种于被污染的土壤中以处理土壤,当污染物被降解之后可加入本发明的有机化合物,如D-丝氨酸。或者,可在被病原体微生物污染的土壤中加入本发明的有机化合物,如D-丝氨酸。尽管所加的有机化合物的量取决于降解污染物之微生物或病原体微生物的类型,但该量一般约为几十ppm至几万ppm,优选为100ppm至10000ppm,更优选为500至5000ppm。有机物可以一次加入,或者可以分成小部分多次加入。控制靶微生物数目所需的时间通常为1至4周。
实施例
下面将参照以下实施例更详细地解释本发明。
实施例1.改良用于细胞计数的菌株(赋予NK1611菌株以利福平抗性)
为了特异性地,准确地计数土壤中的N16-1菌株细胞,发明人开发出一种准确的计数方法,该方法联合使用了其中已导入卡那霉素抗性基因的突变的NK1611菌株,该菌株的选择培养基(1/3LB(按1/3稀释Difco生产的LB)琼脂培养基,其中加入了50ppm卡那霉素和100ppm放线菌酮),和靶向卡那霉素抗性基因的Direct Colony PCR法(日本专利申请9-236452)。在此方法中,必需进行Direct Colony PCR法,因为当仅使用选择培养基时,土著微生物会污染计数。
为了在本发明中进行较简单的细胞计数,使用天然突变赋予NK1611菌株以利福平抗性。因此,将NK1611菌株平铺于1/3LB琼脂培养基(1.5%琼脂)上,所述培养基中含有50ppm利福平,50ppm卡那霉素和100ppm放线菌酮,并于30℃进行培养。在所述培养基的液体培养基中培养天然产生的利福平抗性突变体,然后再次平铺于含有200ppm利福平的相同琼脂培养基上。挑选生长良好的菌落,重复进行单菌落分离以得到50ppm利福平抗性突变体。将该菌株称为NKR菌株。
实施例2.评价NKR菌株同化多种碳源的能力(寻找拮抗物)
寻找不能被NKR菌株同化的碳源。将NKR菌株接种于Biolog GN平皿(Biolog)中以研究其同化95种碳源的能力。选择不能被同化(氧化还原指示剂未显色)的物质。另外,在含有0.5%碳源的无机盐培养基中证实其生长。以类似的方法评价未包括在Biolog GN平皿中的谷氨酸钠,酒石酸钠和琥珀酸钠。
结果,观察到的未显色的化合物(未被同化的化合物)和显色很浅的化合物(括号中表示的)是:α-环化糊精,糊精,糖原,(纤维二糖),赤藓醇,龙胆二糖,(α-D-乳糖),麦芽糖,β-甲基-D-葡萄糖,(D-阿洛酮糖),D-棉子糖,蔗糖,(D-海藻糖),松二糖,木糖醇,单甲基琥珀酸,乙酸,(α-羟基丁酸),γ-羟基丁酸,衣康酸,(α-酮丁酸),(α-酮戊二酸),(丙二酸),丙酸,(奎宁酸),(D-糖二酸),(癸二酸),(葡糖醛酰胺),(丙氨酰胺),D-丙氨酸,(L-天冬氨酸),甘氨酰-L-天冬氨酸,(L-omithine),D-丝氨酸,(L-丝氨酸),(L-苏氨酸),(尿刊酸),(尿苷),胸苷,苯乙胺,(腐胺),(2-氨基乙醇),2,3-丁二醇,D,L-α-甘油磷酸,葡萄糖-1-磷酸和(葡萄糖-6-磷酸)。
实施例3.加入蔗糖的效应
将5g SB土壤(调节至pH7.0)置于50ml Corning离心管中,在其中接种NKR菌株细胞的水悬浮液。使用含有50ppm利福平,50ppm卡那霉素和100ppm放线菌酮的1/3LB琼脂培养基(按1/3稀释制备的Difco LB,1.5%琼脂)研究细胞数目随时间变化的情况。还使用DNB(按1/100稀释的Eiken’s NB)琼脂培养基(1.5%琼脂)研究总细胞数目的变化情况。培养直至NKR菌株在土壤中建立(细胞数目变得恒定)之后,加入500微升含有150微升无菌水,100微升5×(5倍强度的)M9培养基和250微升10%蔗糖溶液的溶液进行细胞数目的控制处理。
SB土壤是沙质土,其颗粒大小分布为砾石(2-75mm)2%,沙子(75μm-2mm)80%,粉土(5-75μm)13%,粘土(小于5μm)5%,pH为6.5,土壤中的总碳含量为0.06%,可交换的阳离子含量(CEC)为2.6me/100g,密度为2.614g/cm3。
通过菌落形成测定土壤中的总细胞数目和NKR菌株的细胞数目。尽管NKR菌株可在含有200ppm利福平的培养基中生长,但甚至在含有50ppm利福平的SB土壤中也未产生抗性土著菌株,因此,在本实施例中,使用的是含有50ppm利福平的培养基。
结果示于图1。在接种后13天,NKR菌株以107CFU/g土壤的水平被建立。当此时进行细胞数目控制处理时,加入N,P和C源可观察到总细胞数目的增加。另一方面,NKR菌株在瞬时增加之后略有减少。
实施例4.加入D-丝氨酸的效应
在调节了pH的SB土壤中接种NKR菌株之后,于30℃培养2天,在其中加入250微升10%底物溶液和350微升无菌水以通过加入0.5%D-丝氨酸进行细胞数目控制处理。未加入丝氨酸时,可加入100微升无菌水。
如图2所示,当加入D-丝氨酸时,NKR菌株的细胞数目相对于未加入D-丝氨酸的情况而言显著减少。这是因为在NKR菌株在土壤中建立之前加入D-丝氨酸的试验已经开始。如图1所示,在其建立于土壤中之前,NKR菌株的细胞数目显著减少。
上述结果表明:D-丝氨酸对控制土壤中的NKR菌株非常有效。
D-丝氨酸的正规名称为2-氨基-3-羟基丙酸,其包含在生物体中,也可用作食品添加剂,它能很容易地被导入土壤环境中。
实施例5.加入L-丝氨酸的效应
将10g pH值经调节的SB土壤置于50ml Corning离心管中,在其中接种NKR菌株。计数NKR菌株的细胞数目和总细胞数目之后,于20℃培养5天。再在其中加入0.5ml通过在土壤中加入D-丝氨酸,L-丝氨酸或DL-丝氨酸(D型与L型的比例为50∶50)至浓度为5000ppm而制备的各种溶液。未加入丝氨酸时,加入0.1ml无菌水。
如图3所示,当加入D-丝氨酸时,NKR菌株的细胞数目相对于实施例4中未加入D-丝氨酸的情况而言显著减少。然而,当加入L-丝氨酸时,NKR菌株的细胞数目与未加入L-丝氨酸时的几乎相等。另外,当加入DL-丝氨酸时,可观察到对细胞计数的控制作用,但减少的程度较低(图3)。
实施例6.
以107cfu/g湿土的量将NKR菌株接种于10g Kuroboku(黑)土A(野地土),Kuroboku(黑)土B(熟地土(developed land soil)),沙地土A(熟地土),沙地土B(熟地土),沙地土C(熟地土)或沙地土D(草地非根围土)之后,加入500微升10%D-丝氨酸溶液(至终浓度为5000ppm)。在接种有微生物的各种土壤中加入500微升无菌水以用作对照。
如图4所示,在除Kuroboku(黑)土A之外的所有土壤中,NKR菌株的细胞数目与对照样品相比显著减少,这显示出控制效应。
Kuroboku(黑)土A是典型的Kuroboku(黑)土,它比Kuroboku(黑)土B吸附的有机物更多,其微生物菌群是特征性的,因此认为未观察到控制效应。
实施例7.
以3.5×107cfu/g湿土的量将NKR菌株接种于10g SB土壤之后,各加入500微升无菌的10%底物溶液(至终浓度为5000ppm)。所用底物是D-半胱氨酸,D-α-丙氨酸,甘氨酸,乙胺或丙酮酸。这些物质是从被认为是D-丝氨酸在活体内的代谢物的物质中随机选择的。在接种有微生物的土壤中加入500微升无菌水以用作对照。
如图5所示,通过加入这些物质,NKR菌株的细胞数目与对照样品相比有所减少,这与加入D-丝氨酸的结果相同。
尤其是,当加入D-半胱氨酸,D-α-丙氨酸,乙醇胺或丙酮酸时,NKR菌株的细胞数目与对照样品相比显著减少,这显示出控制效应。
从上述结果可以看出:D-丝氨酸对控制土壤中的NKR菌株非常有效,而L-丝氨酸对控制NKR菌株的细胞数目几乎没有什么作用。另外,由实施例7可以看出,D-半胱氨酸,D-α-丙氨酸,乙醇胺或丙酮酸也表现出控制效应。
实施例8.
以1.6×108cfu/g湿土的量将番茄萎蔫病致病菌(Ralstoniasolanacearum OE1-1菌株)接种于10g收集自Aichi-ken农业研究中心野地的土壤之后,加入50mg D-丝氨酸,D-苯丙氨酸,肌醇,麦芽糖,乳糖,D(+)-棉子糖或D-山梨糖醇(至终浓度为5000ppm)。将未加入微生物的土壤用作对照。
如图6所示,通过加入D-丝氨酸,OE1-1菌株的细胞数目与对照相比显著减少,表现出控制效应。
实施例9.
以1.6×108cfu/g湿土的量将番茄萎蔫病致病菌(Ralstoneasolanacearum OE1-1菌株)接种于10g收集自Aichi-ken农业研究中心野地的土壤中以制备12份污染土壤,在5份试验样品中加入0ppm(未加入),500ppm,1000ppm,2000ppm或5000ppm D-丝氨酸,在7份试验样品中加入0ppm(未加入),500ppm,500ppm×2500ppm×3100ppm,1000ppm×2或1500ppm乙醇胺。上述500ppm×2指的是在第0周和第1周各加入500ppm乙醇胺。500ppm×3指的是在第0,1和第2周各加入500ppm乙醇胺。
如图7和8所示,通过加入D-丝氨酸,OE1-1菌株的细胞数目与0ppm(对照)相比有所减少,但总细胞数目几乎没有变化。这表明D-丝氨酸的加入特异性地控制了R.solanacearum OE-1菌株。
另外,如图9和10所示,一次加入500ppm乙醇胺产生的效果与0ppm时差不多,但当连续加入500ppm或者加入1000ppm或更多的乙醇胺时,与0ppm时相比OE1-1菌株的细胞数目有所减少。由于无论加入的浓度为多少,总细胞数目都维持不变,显然乙醇胺的加入特异性地控制了R.solanacearum OE-1菌株。
实施例10.通过D-丝氨酸处理被R.solanacearum OE-1菌株污染的土壤
制备4份各含200g被R.solanacearum OE-1菌株污染的土壤的样品。在这些试验样品中加入0ppm(未加入),500ppm,2500ppm或5000ppm D-丝氨酸,1周后,在每份试验土壤中植入20粒番茄种子。播种4天后,在未加入D-丝氨酸的土壤和加入500ppm D-丝氨酸的土壤中观察到番茄种子的萌发。播种6天后,未加入D-丝氨酸的土壤中的一些番茄幼苗开始死亡,第10天,除了一株未加入D-丝氨酸的土壤中的幼苗以外所有幼苗都已死亡。相反,加入500ppm D-丝氨酸的土壤中的所有番茄幼苗都能正常生长。结果示于图11,图11中的A表示加入0ppm D-丝氨酸的结果,B表示500ppm的结果,C表示2500ppm的结果,D表示5000ppm的结果。
从上文实施例9和10可清楚地看出:加入较高浓度(500ppm或更高)的针对R.solanacearum OE-1菌株的外源性环境因子,如D-丝氨酸和乙醇胺可以控制R.solanacearum OE-1菌株,而对于茄科生物家族之植物的生长而言,过多地加入针对R.solanacearum OE-1菌株的外源性环境因子是不合适的,500ppm至2500ppm的范围是适当的。
在本发明的实施例中,番茄被用作茄科作物的例子,但本发明对于除番茄以外的植物也是有用的,所述植物如茄子,青椒,烟草,马铃薯和辣椒。
Claims (4)
1.一种非直接降低靶微生物数量的方法,包括下列步骤:
(1)选择一种对靶微生物无毒而且可以被本土微生物菌落吸收但不能被靶微生物吸收的物质,其中所述靶微生物是降解土壤中污染物的微生物或者是土壤中的致病微生物,并且其中靶微生物与本土微生物菌落在土壤中共存;
(2)将步骤(1)所获得的物质以足以引起本土微生物菌落生长而降低或消除靶微生物生长的量加入土壤中;
其中所述土壤是生物整治后的土壤,并且所述靶微生物是降解土壤中污染物的微生物伯克氏菌(Burkholderia)属N16-1菌株;或者其中所述土壤是被致病微生物污染的,并且所述靶微生物是致病微生物Ralstonia solanacearum OE-1菌株;
其中向土壤中加入的物质是D-丝氨酸。
2.根据权利要求1的方法,其中所述靶微生物是伯克氏菌(Burkholderia)属N16-1菌株。
3.根据权利要求1的方法,其中所述靶微生物是Ralstonia solanacearum OE-1菌株。
4.根据权利要求1的方法,其中向土壤中加入的D一丝氨酸的量为500ppm-5000ppm。
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1999
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- 1999-03-05 US US09/623,196 patent/US6861235B1/en not_active Expired - Fee Related
- 1999-03-05 CA CA002321534A patent/CA2321534C/en not_active Expired - Fee Related
- 1999-03-05 CN CNB998037494A patent/CN1166291C/zh not_active Expired - Fee Related
- 1999-03-05 EP EP99937833A patent/EP1060668A4/en not_active Withdrawn
- 1999-03-05 JP JP2000534050A patent/JP3531610B2/ja not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
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JP3531610B2 (ja) | 2004-05-31 |
WO1999044422A1 (fr) | 1999-09-10 |
CN1292639A (zh) | 2001-04-25 |
EP1060668A1 (en) | 2000-12-20 |
CA2321534A1 (en) | 1999-09-10 |
EP1060668A4 (en) | 2004-12-15 |
CA2321534C (en) | 2005-11-29 |
US6861235B1 (en) | 2005-03-01 |
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