CN108046370A - 采用电离辐照去除抗生素抗性基因的方法 - Google Patents
采用电离辐照去除抗生素抗性基因的方法 Download PDFInfo
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- CN108046370A CN108046370A CN201711329143.7A CN201711329143A CN108046370A CN 108046370 A CN108046370 A CN 108046370A CN 201711329143 A CN201711329143 A CN 201711329143A CN 108046370 A CN108046370 A CN 108046370A
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- antibiotic
- discarded object
- resistance gene
- gene
- resistant gene
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
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- C—CHEMISTRY; METALLURGY
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- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
- C10L9/00—Treating solid fuels to improve their combustion
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
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- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
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- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
本文公开了一种采用电离辐照去除抗生素抗性基因的方法,其应用电离辐照技术(包括γ射线和电子加速器产生的高能电子束)处理抗生素菌渣以破坏微生物细胞的DNA,从而实现抗性基因的有效去除,并可同时降解残留的抗生素。所述方法高效、适用面广;辐照可以在常温进行,无需或仅需加入少量化学试剂,不会产生二次污染物。所述方法不仅可应用去除抗生素菌渣中的抗性基因,也可应用于去除水体、土壤和污泥中的抗性基因,在环境领域具有广阔的应用前景。
Description
技术领域
本文属于废弃物处理领域,特别地,本文涉及一种去除制药行业危险固体废弃物-抗生素菌渣中的抗性基因的方法。
背景技术
我国是世界上最大的抗生素生产国和使用国。据统计,2013年我国抗生素的产量为24.8万吨,使用量为16.2万吨。抗生素类药物发酵生产过程中产生大量的固体废弃物-抗生素菌渣。以生产1吨抗生素产生8~10吨湿菌渣计算,每年的菌渣产量高达200万吨以上。菌渣的主要成分为菌体、未利用的培养基、发酵过程的代谢产物以及残留的抗生素和抗生素抗性基因(Antibiotic Resistance Genes)等。尽管菌渣中含有丰富的蛋白质和多糖等营养物质,但残留的抗生素和抗性基因会诱发和传播耐药菌,给生态环境和人类健康造成潜在危害。我国2008年修订的《危险废物名录》明确将抗生素菌渣定为危险废物。抗生素菌渣的安全处置已成为抗生素生产企业急需解决的难题。
作为抗生素菌渣成分之一的抗性基因是一种新型环境污染物,其产生与抗生素的生产和使用密切相关。菌渣中以及环境中残留的抗生素构成筛选抗性细菌的环境选择压力,促进抗性基因的产生和选择。同一种抗生素可诱导产生多种抗性基因。抗性基因在环境中的存在使微生物对抗生素的抗性不断增加,甚至出现了携带有多种抗性基因的“超级细菌”,导致抗生素治疗无效,给人类健康造成巨大危害。
抗性基因与其他污染物相比,特别是抗生素菌渣中所含的其他污染成分相比,具有独特的性质。一方面,抗性基因作为一种生物遗传物质,与多环芳烃、药品和个人护理品(pharmaceuticals and personal care products,PPCPs)等有机污染物不同。抗性基因产生后并不依存于抗生素,其本身可在各种病原菌、非病原菌微生物,甚至遗传关系较远的微生物之间通过水平转移(horizontal gene transfer)传播。另一方面,抗性基因的去除也与杀灭抗生素菌渣中的菌体不同。研究表明,抗性基因也可以作为裸DNA形式独立于菌体存在于环境中,甚至在某些合适的条件下在环境中自我扩增,从而可以在携带该基因的菌体被杀灭后仍长期存留于环境中。
目前抗生素菌渣的处理处置技术主要有:焚烧、填埋、堆肥、厌氧消化、微波、碱处理等物理化学方法。这些方法在去除抗性基因等方面并不令人满意。例如,焚烧和卫生填埋是传统的处理危险废物的方法。抗生素菌渣的含水率高、热值低,焚烧过程需外加燃料,处理费用高。填埋菌渣占用大量的土地,菌渣腐化液化产生的渗滤液会污染地下水。高温堆肥对抗性基因的去除存在较大差异,还有可能促进抗性基因的增殖。厌氧消化也需在高温下进行才能对抗性基因有一定的削减效果。氯气或紫外消毒工艺对不同抗性基因的去除效果各不相同。
本领域仍需要一种有效去除含抗生素抗性基因的危险废弃物中的抗性基因的改进的方法。
发明内容
在一个方面,本文提供了一种去除含抗生素抗性基因的废弃物中的所述抗生素抗性基因的方法,其包括采用电离辐照技术处理所述废弃物。
本文所使用的术语“抗性基因”或“抗生素抗性基因”是指那些使得细菌得以抵抗抗生素的基因。所述抗性基因所针对的抗生素可以为,例如,β-内酰胺类抗生素如青霉素;大环内酯类抗生素如红霉素、硫氰酸红霉素;氨基糖苷类抗生素例如链霉素;多肽类抗生素例如万古霉素等。已经鉴定出了针对不同抗生素的多种抗性基因,例如,针对β-内酰胺类抗生素的blaCTX-M基因,针对大环内酯类抗生素的ereA、ermA、ermB、ermF、mefA、mphB基因等。
本文所使用的术语“含抗生素抗性基因的废弃物”是指含有抗生素抗性基因的固体或液体物质。例如,含抗生素抗性基因的废弃物包括含抗生素抗性基因的污水厂出水、污泥、抗生素菌渣等等。
优选地,所述含抗生素抗性基因的废弃物是抗生素菌渣。因此,本文特别提供了一种去除抗生素菌渣中的抗生素抗性基因的方法,所述方法包括采用电离辐照处理所述抗生素菌渣。
本文所用术语“抗生素菌渣”是指抗生素生产中所产生的含菌废渣,其含有抗生素抗性基因。抗生素菌渣主要含有未被完全提取的抗生素、其他代谢产物、未被抗生素产生菌完全利用的培养基成分以及抗生素产生菌体本身等。抗生素菌渣中所含的抗生素、菌体以及抗性基因使其成为造成环境污染的危险废弃物。
所述抗生素菌渣可以为采用生物发酵方法生产相应抗生素时产生的菌渣,其含有一种或更多种相应抗生素的抗性基因。例如,青霉素菌渣为采用生物发酵方法生产青霉素时产生的菌渣,其含有一种或更多种青霉素抗性基因。在一些实施方案中,所述抗生素菌渣为红霉素菌渣、硫氰酸红霉素菌渣、青霉素菌渣、链霉素菌渣、头孢菌素菌渣、土霉素菌渣、林可霉素菌渣、螺旋霉素菌渣等等。在一些优选的实施方案中,所述抗生素菌渣选自红霉素菌渣、硫氰酸红霉素菌渣、青霉素菌渣中的一种或者更多种。在一些实施方案中,所述废弃物例如抗生素菌渣中所含的单个抗性基因的含量可以在1×105拷贝/g至1×1010拷贝/g的范围内。
所述含抗生素抗性基因的废弃物(例如抗生素菌渣)可以以固液混合物(例如湿菌渣,例如含水率高于70%、80%或90%)的形式提供,也可经例如过滤和/或干燥处理后以固体形式提供。
电离辐照是使被作用的物质发生电离的一种辐照,如γ射线或电子加速器等产生的电子束。γ射线和电子束的能量高、穿透力强。受辐射时,体系会产生物理化学效应(如胶体的变性作用)、化学效应(如污染物的辐射分解或氧化作用)及生物学效应(如灭菌、消毒作用)等。电离辐照技术已在水体和污泥处理领域得到应用,以去除有毒的有机污染物等。电离辐照也是去除PPCPs等毒性难降解有机物的一种有效手段。四环素、青霉素以及磺胺类等抗生素都可被电离辐照高效降解。
本发明人出乎意料地发现,电离辐照技术可以以高去除率破坏危险废弃物中的多种抗性基因。在不受理论约束的情况下,认为电离辐照产生的高能射线以及辐照产生的活性粒子作用于抗生素抗性基因,实现了其的高效链断裂和生物功能(例如扩增、转化能力)的破坏等,从而导致了抗生素抗性基因的高效率去除。另外,相较于其他废弃物处理技术,电离辐照技术效率高、适用面广;能够同时去除危险废弃物中的多种危险成分,例如抗性基因以及抗生素;辐照可以在常温进行,无需或仅需加入少量化学试剂,不会产生二次污染物,是一种清洁、可持续利用的技术。
在一些实施方案中,所述去除含抗生素抗性基因的废弃物(例如抗生素菌渣)中的所述抗生素抗性基因的方法包括:将所述含抗生素抗性基因的废弃物放置在γ射线源附近或者高能电子束的扫描靶窗,以进行电离辐照处理。进行电离辐照的装置或设备是本领域公知的。
所述电离辐照可以在任何适宜的温度下进行。在一些实施方案中,电离辐照在常温状态下进行。
在一些实施方案中,所述的γ射线的发射源为Co60或者Cs137。
在一些实施方案中,所述的高能电子束由电子加速器产生。
在一些实施方案中,所述的辐照吸收剂量为大于5kGy,例如10-50kGy,20-50kGy,30-50kGy等。在一些实施方案中,所述的辐照吸收剂量为,例如5、10、15、20、30、40、50kGy。
本文所述的方法能够实现抗性基因的去除。在本发明的上下文中,提及抗性基因时使用的术语“去除”意指与使用根据本发明的方法处理之前所述废弃物中抗性基因的量相比,通过根据本发明的方法处理后抗性基因减少、检测不到或消除。可以通过本领域已知的任何基因检测方法来检测本文所述的方法的抗生素抗性基因的去除效果。例如,所述检测可以是定性检测,例如测序或者聚合酶链反应(PCR)和凝胶电泳等,通过对比辐照前后样品中抗性基因的有无来确定抗性基因的去除。在一些优选的实施方案中,本文所述的抗性基因去除方法导致经辐照的抗生素菌渣中检测不到抗性基因。或者,所述检测可以是定量检测,例如荧光定量PCR,通过例如用辐照前后抗性基因量的差值占辐照前抗性基因的量的百分率来计算去除率。在一些实施方案中,本文所述的抗性基因去除方法能够以高于85%、高于86%、高于87%、高于88%、高于89%、高于90%、高于91%、高于92%、高于93%、高于94%、高于95%、高于96%、高于97%、高于98%、高于99%或以100%的去除率去除所述废弃物中的抗生素抗性基因。在一些实施方案中,通过本申请方法处理后得到的废弃物(例如抗生素菌渣)中所含的单个抗性基因的含量可以在1×104拷贝/g至1×109拷贝/g的范围内。在一些实施方案中,本文所述的方法同时实现了废弃物(例如抗生素菌渣)中其他有害物质(例如抗生素或菌体)的除去或减少。例如,在本文所述的方法中,抗生素的去除率可以为高于40%、高于60%、高于80%、高于90%等。
在另一个方面,本文提供了通过上文所述的方法得到的去除了抗性基因的废弃物,例如抗生素菌渣。
这样的去除了抗性基因的废弃物可以被再利用,例如用于生产饲料、肥料、能源等等。因此,在又一方面,本文涉及一种生产饲料、肥料和/或能源的方法,其包括:通过本文所述的方法获得经处理的废弃物,以及将所述废弃物用于生产饲料、肥料和/或能源。
具体实施方式
下面结合实施例对本发明的实施方案作进一步说明。但本发明并不限于以下的实施例。
实施例1:
取红霉素菌渣固液混合物(含水率为93%,总悬浮固体含量为72g/L,其中有机固体所占的比例为80%)检测红霉素抗性基因的浓度以及活性物质红霉素A的量。然后将所述菌渣固液混合物放入辐照容器内,采用Co60γ射线(辐射活度3.6×1014Bq),临近中心孔道进行辐照,剂量率约为240Gy/min。通过调整辐照时间控制吸收剂量为5kGy和10kGy。辐照结束后,检测不同吸收剂量下,菌渣中红霉素抗性基因的浓度和残留的活性物质红霉素A浓度。
采用高效液相色谱检测菌渣中残留的红霉素。先用有机溶剂将其从菌渣中提取出来,再采用美国安捷伦公司的高效液相色谱仪(Agilent 1200)检测其浓度。色谱柱为XDB-C18反相柱,柱温:35℃。检测器为紫外检测器,检测波长:215nm;流动相为乙腈和0.01mol/L磷酸氢二钾溶液,混合比例为55/45。
采用荧光定量PCR分析红霉素抗性基因。具体地,采用GENEray DNA提取试剂盒提取DNA,所用电泳仪为美国Major Science公司的Mini Pro 300V电泳仪。所用PCR仪为美国Applied Biosystems公司的ABI7500实时荧光定量PCR仪。PCR反应程序为预变性95℃10min;变性95℃10s,退火延伸60℃34s,95℃15s,循环次数40。所用引物序列如下表1所示:
表1:红霉素抗性基因检测所用引物:
初始菌渣中检测出红霉素抗性基因ermB和ermF,其浓度分别为1.8±0.3×109拷贝/g和8.8±2.9×106拷贝/g。红霉素残留量约为283mg/kg(干固体)。辐照吸收剂量为5kGy和10kGy时,ermB的浓度降为2.2±0.2×108拷贝/g和2.0±0.1×108拷贝/g,去除率在87~89%;ermF的浓度降为3.6±1.6×105拷贝/g和2.2±1.9×105拷贝/g,去除率达到96~98%;红霉素A残留量降为165mg/kg和39mg/kg,去除率分别为42%和86%。
实施例2:
取硫氰酸红霉素菌渣固液混合物(含水率为92%,总悬浮固体含量为75g/L,其中有机固体所占的比例为77%)检测红霉素抗性基因的浓度以及活性物质红霉素A的量。然后将其放入辐照容器内,采用Co60γ射线(辐射活度3.6×1014Bq),临近中心孔道进行辐照,剂量率约为240Gy/min。通过调整辐照时间控制辐照吸收剂量为10kGy,20kGy和30kGy。辐照结束后,检测不同吸收剂量下,硫氰酸红霉素菌渣中残留的红霉素A的浓度以及抗性基因的量。
菌渣中残留的红霉素A先用有机溶剂提取,再采用液相色谱检测。具体检测方法同实施例1。
采用荧光定量PCR仪分析红霉素抗性基因。所用的仪器、试剂盒、引物序列、PCR循环条件同实施例1。
初始菌渣中检测出ereA、ermB、mefA、mphB四种抗性基因,其平均浓度分别为2.3×105拷贝/g,1.2×108拷贝/g,9.0×107拷贝/g和5.1×107拷贝/g。红霉素A残留量约为1588.9mg/kg。吸收剂量为10kGy时,四种抗性基因的浓度降为1.5×105拷贝/g,6.1×107拷贝/g,4.6×107拷贝/g,2.6×107拷贝/g,去除率为34~50%;吸收剂量为20kGy时,四种抗性基因的浓度降为7.1×104拷贝/g,2.8×107拷贝/g,1.4×107拷贝/g,6.1×107拷贝/g,去除率为70~73%;吸收剂量达到30kGy时,四种抗性基因的浓度降为3.3×104拷贝/g,1.0×107拷贝/g,1.1×107拷贝/g,4.7×106拷贝/g,去除率为86~91%,红霉素A残留量降为589.3mg/kg,去除率为63%。
实施例3:
取青霉素菌渣固液混合物(含水率为88%,总悬浮固体含量为116g/L,其中有机固体所占的比例为87%)检测青霉素抗性基因的浓度以及青霉素的量。然后将所述菌渣固液混合物放入辐照容器内,采用Co60γ射线(辐射活度3.6×1014Bq),临近中心孔道进行辐照,剂量率约为240Gy/min。通过调整辐照时间控制辐照吸收剂量为5kGy和10kGy。辐照结束后,检测不同吸收剂量下青霉素菌渣中残留的青霉素浓度和进行抗性基因的定性检测。
对于菌渣中残留的青霉素先用有机溶剂提取,再采用液相色谱检测。其中,所用液相色谱仪为美国安捷伦公司的高效液相色谱仪(Agilent 1200),色谱柱为XDB-C18反相柱,柱温:25℃。检测器为紫外检测器,检测波长:220nm;流动相为乙腈和0.1%甲酸水溶液,混合比例为1:1。
采用普通PCR分析青霉素抗性基因。所用PCR仪为北京东胜创新生物科技有限公司的普通PCR仪,PCR反应条件为50℃2min;95℃15min,94℃15s,58℃1min,45个循环。青霉素抗性基因的引物序列如下表2所示:
表2:青霉素抗性基因检测所用引物:
初始菌渣中检测出blaCTX-M青霉素抗性基因,青霉素残留量约为262mg/kg。吸收剂量到10kGy时,blaCTX-M检测为阴性,青霉素残留量降至53mg/kg,去除率为80%。
实施例4:
取干燥后的硫氰酸红霉素菌渣固体(10mL)检测红霉素抗性基因的浓度。然后将其放入辐照容器内,采用Co60γ射线(辐射活度3.6×1014Bq),临近中心孔道进行辐照,剂量率约为240Gy/min。通过调整辐照时间控制辐照吸收剂量为30kGy,40kGy和50kGy。辐照结束后,检测不同吸收剂量下,硫氰酸霉素菌渣中的红霉素抗性基因。使用实施例3所述的方法和实施例1所述引物采用普通PCR分析红霉素抗性基因。
初始菌渣中检测出的四种抗性基因ereA、ermA、mefA、mphB,当吸收剂量为30kGy时,ereA、ermA为阴性;吸收剂量为50kGy时,ereA、ermA、mefA、mphB为阴性
最后应说明的是,上述实施例仅是为清楚地说明本发明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围之中。
Claims (9)
1.一种去除含抗生素抗性基因的废弃物中的抗生素抗性基因的方法,所述方法包括采用电离辐照处理所述废弃物。
2.根据权利要求1所述的方法,其中所述电离辐照使用γ射线或高能电子束进行。
3.根据权利要求2所述的方法,其中所述γ射线由放射性同位素Co60或者Cs137衰变产生。
4.根据权利要求2所述的方法,其中所述高能电子束由电子加速器产生。
5.根据前述权利要求中任一项所述的方法,其中所述废弃物为抗生素菌渣,优选地,所述抗生素菌渣选自红霉素菌渣、硫氰酸红霉素菌渣和青霉素菌渣中的一种或更多种。
6.根据前述权利要求中任一项所述的方法,其中所述辐照处理的吸收剂量为大于5kGy,优选地,吸收剂量为10~50kGy。
7.根据前述权利要求中任一项所述的方法,其中所述废弃物的形式为固体或固液混合物。
8.通过权利要求1-7中任一项所述的方法得到的经处理的废弃物。
9.一种生产饲料、肥料和/或能源的方法,其包括:通过权利要求1-7中任一项所述的方法获得经处理的废弃物,以及将所述废弃物用于生产饲料、肥料和/或能源。
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