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中国环境科学 2021,41(12)：5912~5920 China Environmental Science 微生物诱导碳酸盐沉积介导的Cd2+固定朱广森,汪文军,吴峥,司友斌*(安徽农业大学资源与环境学院,农田生态保育与污染防控重点实验室,安徽合肥230036)摘要：实验室条件下,研究了碳酸盐矿化菌施氏假单胞菌(Pseudomonas stutzeri)、枯草芽孢杆菌(Bacillus subtilis)和巴氏芽孢杆菌(Bacillus pasteurii)的生长对Cd2+的耐受性和固定效果,以及羟基磷灰石对3种菌固定Cd2+的影响,通过扫描电镜(SEM)、X射线能谱(EDS)、傅里叶红外光谱(FTIR)和X射线衍射(XRD)对碳酸盐矿化菌的诱导矿化产物进行了表征.结果表明,3种菌生长过程中B. pasteurii的脲酶活性最强,是P. stutzeri和B. subtilis脲酶活性的10倍左右,且P. stutzeri, B. subtilis, B. pasteurii产CO32-的最高浓度分别为588.19, 661.72, 1735.18mg/L,培养体系中pH值均呈现升高的趋势,最高pH值分别为8.23, 9.06, 9.52;Cd2+浓度在0~10mg/L范围内对P. stutzeri的生长影响较小,而当Cd2+浓度高于1mg/L时则会抑制B. subtilis和B. pasteurii的生长.初始Cd2+浓度为0.5mg/L时,培养120h, P. stutzeri, B. subtilis和B. pasteurii对Cd2+的固定去除率分别为96.37%, 99.40%, 97.57%;加入羟基磷灰石能够提高碳酸盐矿化菌对Cd2+的去除率.SEM和EDS结果显示,P. stutzeri和B. subtilis诱导形成的矿化产物多聚集在菌体周围或附着在菌体表面,呈球状或网状结构,表面疏松多孔,B. pasteurii的矿化产物附着在菌体表面,结构致密,呈不规则的球状; FTIR分析表明矿化产物中存在CO32-,结合XRD结果,证实3种菌诱导沉淀矿化产物主要是CaCO3,而Cd2+与Ca2+会以同晶置换的方式形成CdCO3晶体,B. pasteurii在诱导矿化的过程中可将Cd2+以Ca0.67Cd0.33CO3共沉淀的方式固定.关键词：微生物矿化；Cd2+；羟基磷灰石；碳酸盐沉淀中图分类号：X172 文献标识码：A 文章编号：1000−6923(2021)12-5912-09Immobilization of Cd2+ by microbial induced carbonate precipitation. ZHU Guang-sen, WANG Wen-jun, WU Zheng, SI You-bin* (Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University, Hefei 230036, China). China Environmental Science, 2021,41(12)：5912~5920 Abstract：Under laboratory culture conditions, three carbonate mineralization bacteria, Pseudomonas stutzeri, Bacillus subtilis and Bacillus pasteurii were used to study the growth specialities, the resistance and removal of Cd2+ by the bacteria, and the effect of hydroxyapatite on Cd2+ immobilization. The scanning electron microscope (SEM), energy dispersive spectrum (EDS), fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were employed to characterize the biomineralization products. The results showed that the urease activity of B. pasteurii was about 10 times than those of P.stutzeri and B.subtilis. The CO32- was produced and the pH of culture media had an increasing during the growth of the three bacteria. The highest CO32-concentrations were 588.19, 661.72, 1735.18mg/L, while the highest pH value could reach 8.23, 9.06 and 9.52 for P. stutzeri, B. subtilis and B. pasteurii, respectively. The growth of P. stutzeri was not affected within the Cd2+ concentration of 0~10mg/L, while the growth of B. subtilis and B. pasteurii were inhibited in the Cd2+ concentration beyond 1mg/L. With the initial Cd2+ concentration of 0.5mg/L and biomineralization for 120h, the removal rates of Cd2+ by P. stutzeri, B. subtilis and B. pasteurii were 96.37%, 99.40% and 97.57%, respectively. The addition of hydroxyapatite could enhance the immobilization of Cd2+ by the bacteria to a certain extent. SEM and EDS analysis showed that the mineralized products induced by P. stutzeri and B. subtilis were mostly clustered around or attached to the surface of the bacteria, with spherical, reticulated structure and porous surface, while the mineralized products by B. pasteurii were attached to the surface of the bacteria, with compact structure and irregular spherical structure. FTIR analysis suggested that there was CO32- in the mineralized products. XRD identified, the mineralized products CaCO3 induced by the three bacteria. The CdCO3 crystal were forms by isomorphic substitution of Ca2+ with Cd2+. As an alternative, Ca0.67Cd0.33CO3 co-precipitation were induced by B. pasteurii for Cd2+ immobilization.Key words：microbiological mineralization；cadmium；carbonate precipitation；hydroxyapatite近年来由于铅锌矿的开采、冶炼、大气沉降和农业化学用品的高通量投入等途径的增多,导致重金属镉(Cd)的环境污染问题日益严重[1].重金属Cd 通常存在于土壤,岩石和水体等环境中,经食物链传递到人体中,从而导致不可逆转的慢性肝损伤,骨质疏松症,癌症等疾病[2].据报道,我国Cd污染超收稿日期：2021−04−09基金项目：国家重点研发计划(2019YFC1805203);国家自然科学基金资助项目(42077123);安徽高校协同创新项目(GXXT-2021-061)* 责任作者, 教授,*****************.cn12期朱广森等：微生物诱导碳酸盐沉积介导的Cd2+固定 5913标土壤达7%[3].生物矿化修复因其成本低廉,适应性强,原位无二次污染等特点[4],近年来逐渐成为环境修复领域的热点.微生物诱导碳酸盐沉淀(MICP)是近年来生物矿化领域研究热点之一[4].目前,国内外学者已将MICP成功应用于Pb, Cd, Zn, Cu, Sr和Ni等多种重金属及放射性金属污染修复[5-6].MICP技术关键在于微生物的选择,目前多以从污染土壤中筛选分离出稳定高效的溶脲菌为研究对象[7],例如P. stutzeri Pb12[8], Stenotrophomonas rhizophila A323, Variovorax boronicumulans C113[9]和Enterobacter sp.(CJW-1)[10]等.有研究表明,上述溶脲菌对污染土壤中的Pb, Cd, Zn等重金属具有较高的固定效果.此外,有些菌对环境的适应能力较强,如在10℃的低温条件下, Exiguobacterium undae YR10经过14d培养后可转化污染土壤中90%以上的Cd2+[11].有些研究还进一步证实MICP将可交换态重金属以碳酸盐结合态形式固定[12],或通过形成方解石、六方岩和白云石等生物矿物吸附等途径将重金属固定[10].然而微生物Pseudomonas stutzeri, Bacillus subtilis和Bacillus pasteurii对重金属Cd的固定机制以及化学材料的添加对微生物诱导碳酸盐沉积固定重金属效果的影响研究相对较少,因此,本研究可为环境生物矿化修复技术提供理论参考,也为微生物与化学材料联合修复重金属污染环境提供实践基础.本研究以P. stutzeri, B. subtilis和B. pasteurii 3种碳酸盐矿化菌作为对象,研究碳酸盐矿化菌生长过程中的脲酶活性、产CO32-能力、pH值变化,以及碳酸盐矿化菌对Cd2+耐受性和固定去除效果,探究了羟基磷灰石联合碳酸盐矿化菌对Cd2+固定的影响,并通过SEM, EDS, FTIR, XRD等表征矿化产物的特征,阐明碳酸盐矿化菌的生物矿化及固定Cd2+的作用机制,以期为微生物诱导碳酸盐沉积用于重金属污染土壤与地下水的生物修复提供理论参考.1 材料与方法1.1 试验材料无水氯化镉(CdCl2,分析纯,纯度≥99%),尿素(CO(NH2)2,分析纯,纯度≥99%),乙酸钙(Ca (C2H3O2)2,分析纯,纯度≥98%),均购于上海阿拉丁生化科技股份有限公司;羟基磷灰石(Ca10(PO4)6(OH)2),Ca/P物质的量比1.67,密度3.16g/cm3,粒径4.5μm,溶解度0.04mg/L,购自西安冰禾生物科技有限公司.菌种来源:施氏假单胞菌(P. stutzeri ATCC 17588)由中国微生物菌种保存库提供,枯草芽孢杆菌(B.subtilis A TCC 6633),巴氏芽孢杆菌(B.pasteurii ATCC 11859)由北纳创联生物技术有限公司提供.1.2培养基与菌悬液制备碳酸盐矿化菌培养基(NBU培养基):牛肉膏3.0g,蛋白胨10.0g,氯化钠5.0g,尿素20.0g, pH=(7.0±0.2),蒸馏水定容至1000mL.除尿素外其他组分121℃高压灭菌30min,然后将配制好的尿素溶液过0.22μm一次性无菌滤膜加入其中.菌悬液的制备[13]:在无菌条件下,将保存的P. stutzeri, B. subtilis, B. pasteurii接种到固体NBU 培养基中,在30℃,150r/min条件下培养24h,无菌条件下将碳酸盐矿化菌转移至已灭菌的液体NBU培养基中,培养24h后以4000r/min离心10min,用0.9%无菌生理盐水冲洗2~3次,使菌体重悬于无菌生理盐水.1.3试验方法1.3.1 碳酸盐矿化菌生长过程中脲酶活性,CO32-浓度及pH值变化测定采用电导率法测定微生物的脲酶活性,测量方法:取1mL菌液加入9mL 1.11mol/L的尿素溶液混匀,用电导率测定仪测量混合体系在25℃时5min电导率变化量,记录5min内的电导率平均变化量(mS/(cm⋅min))乘以稀释倍数即为菌液的脲酶活性(mmol/(L⋅min))[14].吸取2mL制备好的菌悬液接种到200mL已灭菌的培养基中,恒温振荡培养箱中30℃,150r/min连续培养72h,分别设置3组平行.培养过程中按照0,2,4,8,12,24,36,48,60,72h连续取样,每次取样5mL, pH计测定溶液pH值.CO32-浓度测定时按0,2,4,8, 12,24,48,72,96,120h连续取样,每次取样5mL,以4000r/min离心10min,然后取1mL,置于50mL锥形瓶中稀释5倍,采用化学滴定法(DZ/T 0064.49-1993)[15]测定[16].1.3.2 碳酸盐矿化菌对Cd2+耐受性试验吸取1mL制备好的菌悬液至Cd2+浓度分别为0,1,3,5,10, 15mg/L的100mL已灭菌的NBU培养基中,每组浓5914 中国环境科学 41卷度梯度设3组平行,30℃,150r/min下振荡培养72h,连续取样时间同1.3.1,每次取样3mL,测定各处理OD600值.其中尿素溶液配制好后过0.22μm一次性无菌滤膜加入培养基.1.3.3 碳酸盐矿化菌对Cd2+的固定试验吸取1mL制备好的菌悬液至含有0.5mg/L Cd2+和25mmol/L Ca2+ 的100mL已灭菌NBU培养基中,对照组接种等量无菌水,设置3组平行.置于30℃,150r/min下振荡培养120h,连续取样时间同1.3.1,每次取样5mL,4000r/min离心15min,利用ICP-MS测定上清液中Cd2+浓度.在培养120h后取适量样品进行扫描电镜与能谱分析,剩余样品以8000r/min离心15min,弃上清液,置于65℃烘箱中烘干4h后,用玛瑙研钵磨碎过200目筛后,对样品进行傅里叶红外光谱和X射线衍射能谱(XRD)分析.1.3.4 羟基磷灰石对碳酸盐矿化菌固定Cd2+的影响试验将100mg羟基磷灰石(Ca10(PO4)6(OH)2)加入到培养基中灭菌,冷却配制成200mL含1mg/L Cd2+的NBU培养基,再加入2mL制备好的菌悬液,对照组接种等量无菌水,设置3组平行,置于30℃, 150r/min下振荡培养120h, 连续取样时间同1.3.1,每次取样5mL, 4000r/min离心15min,利用ICP-MS 测定上清液中Cd2+浓度.1.4扫描电镜与能谱分析取20mL 1.3.3 中矿化样品于50mL离心管中, 4000r/min离心10min,弃上清液,以2.5%戊二醛固定4~12h,再以0.1mol/L磷酸盐缓冲液淋洗,4000r/ min离心10min,重复2次,弃上清液,依次以不同浓度乙醇梯度脱水,CO2临界点干燥仪干燥10h,镀膜处理后取适量样品进行扫描电镜(SEM,日立S-4800型)及能谱(EDS)分析,观察样品形貌和分析元素组成.1.5傅里叶红外光谱分析采用压片法制备样品,取1~2mg 1.3.3中矿化产物样品在玛瑙研钵中与干燥KBr粉末混合研磨均匀,装入模具内,在压片机上压制成片,进行傅里叶红外光谱(FTIR, Thermo Nicolette is50)分析[17].1.6X射线衍射能谱分析采用X射线衍射仪(XRD)[18]对1.3.3中制备的矿化产物进行分析,用Cu Kα射线检测,设置参数为36kV 和20mA,扫描角度为10°~70°,步长和扫描速度分别为0.02°和0.5°(2θ)/min.1.7数据处理Cd2+的固定去除率计算:R(%)=(C0-C t)/C0×100% (1) 式中:R为Cd2+去除率,%;C0为空白组Cd2+浓度,mg/L;C t为实验组Cd2+浓度, mg/L.采用Origin 9.1软件和Excel 2019进行数据处理、作图,XRD采用MDI Jade6软件分析,数据显著性分析用SPSS 22.0进行统计检验.2结果与分析2.1碳酸盐矿化菌生长过程中脲酶活性,产CO32-浓度及pH值变化不同种类的碳酸盐矿化菌代谢产生的脲酶活性存在较大差异.如图1a所示,3种菌接种至培养基中,最初生长较缓慢,脲酶活性相对较弱, P.stutzeri脲酶活性增加缓慢,24h后逐渐趋于稳定,B. subtilis脲酶活性在第72h达到最高;B. pasteurii前8h脲酶活性迅速增加,第24h达到最高,随后出现一定程度的下降,第48h后逐渐趋于稳定.3种碳酸盐矿化菌生长过程中产生CO32-浓度变化如图1b所示.B. pasteurii产CO32-的能力最强,P.stutzeri和B. subtilis相对较弱.培养初期12h, B. pasteurii产生CO32-的浓度增加很快,48h时达到1735.18mg/L,之后CO32-浓度趋于稳定.P. stutzeri 和B. subtilis在12h后CO32-的浓度开始增加,但增速较为缓慢,72h后均趋于稳定,产生的CO32-浓度分别为588.19和 661.72mg/L.脲酶活性[mmol/(L⋅min)]12期朱广森等：微生物诱导碳酸盐沉积介导的Cd 2+固定 5915C O 32-浓度(m g /L )时间(h)0 8 16 24 3240 48 56 64 72p H 值时间(h)图1 3种碳酸盐矿化菌生长过程中脲酶活性、产CO 32-浓度及pH 值变化Fig.1 Changes in urease activity, CO 32- concentrations and pH during the growth of three carbonate mineralized bacteria3种碳酸盐矿化菌生长过程中pH 值变化如图1c 所示.P . stutzeri 生长过程中pH 值虽在不断升高,但上升较为缓慢,至生长平稳期时pH 值稳定在8.23左右.B.subtilis 在0~8h 出现pH 值降低的现象,可能是由于在最初生长过程中产生乳酸等酸性物质导致其pH 值下降,培养8h 后开始逐渐升高.培养24h 时pH 值上升到8.39,之后逐渐趋于平稳状态,最终pH 值稳定在9.06左右.B. pasteurii 生长过程中pH 值升高迅速,4h 时可达8.67,培养8h 可达9.25,24h 后达到最高pH 值9.52. 2.2 碳酸盐矿化菌对Cd 2+的耐受性如图2所示,随着Cd 2+浓度升高,碳酸盐矿化菌生长受到一定抑制作用.P . stutzeri 对Cd 2+的耐受性较强,Cd 2+浓度0~1mg/L 时对其生长有一定促进作用, Cd 2+浓度1~3mg/L 时对其生长影响不显著,Cd 2+浓度5~10mg/L 时,细菌生长减缓,Cd 2+浓度15mg/L 时细菌生长延滞期达到12h,对数生长期相对缩短.B. subtilis 生长过程中,Cd 2+浓度1mg/L 时对数生长期迟滞至24h 后,对数生长期也相对缩短,而Cd 2+浓度3~15mg/L 时该菌几乎不生长.B. pasteurii 生长过程中,Cd 2+浓度0~1mg/L 时其生长受抑制,对数生长期相对缩短,Cd 2+浓度1~3mg/L 时其生长受显著影响,对数生长期迟滞至36h 后,而当Cd 2+浓度5~15mg/L 时该菌几乎不生长.00.30.60.91.21.51.8O D600时间(h)00.40.81.21.62.02.4O D600时间(h)0O D600时间(h)图2 3种碳酸盐矿化菌对重金属Cd 2+的耐受性 Fig.2 The resistance of three carbonate mineralized bacteriato Cd 2+2.3 碳酸盐矿化菌对Cd 2+的固定如图3所示,3种碳酸盐矿化菌均对Cd 2+有较强的固定能力.P . stutzeri 对Cd 2+的去除24h 可达80.65%,120h 可达96.37%; B. subtilis 对Cd 2+的去除24h 即可达98.80%,120h 可达99.40%; B. pasteurii 对Cd 2+的去除24h 可达93.66%, 120h 达97.57%.此外,对照组出现Cd 2+含量降低,可能是由于有氧条件5916 中 国 环 境 科 学 41卷下空气中的CO 2进入含钙培养基中生成碳酸钙,从而与Cd 2+络合形成沉淀.0 16 32 48 64 80 96 112 128C d 2+浓度(m g /L )时间(h)图3 3种碳酸盐矿化菌对Cd 2+的去除效果Fig.3 Removal of Cd 2+ by three carbonate mineralized bacteria2.4 羟基磷灰石对碳酸盐矿化菌固定Cd 2+的影响如图4所示,加入羟基磷灰石(HAP),能够增强3种菌对Cd 2+的固定效果,HAP 联合碳酸盐矿化菌对Cd 2+的固定效果优于单独添加HAP.培养72h 时, HAP 与P . stutzeri , B. subtilis , B. pasteurii 联合作用对Cd 2+的固定去除率分别为85.8%, 87.9%和86.6%, 120h 时Cd 2+的固定率达95%以上,其中HAP 与B. pasteurii 联合固定效果最好.16324864 80 96 11212800.20.40.60.8 1.0C d 2+浓度(m g /L )时间(h)图4 羟基磷灰石联合碳酸盐矿化菌对Cd 2+的固定效果 Fig.4 Immobilization of Cd 2+ effect of hydroxyapatite combined with carbonate mineralization bacteria2.5 碳酸盐矿化菌固定Cd 2+的矿化产物特征分析图5中SEM 和EDS 分析表明,P . stutzeri 和B. subtilis 形成的矿化产物呈现不规则球状或网状结构,表面疏松多孔,并且大多聚集在菌体周围,部分产物附着在菌体表面,这一结构使其对重金属具有良好的物理吸附作用[19];B. pasteurii 的矿化产物结构致密,呈不规则的球状,并附着在整个菌体表面,有的将整个菌体包裹[20].EDS 结果显示,3种碳酸盐矿化菌的诱导矿化产物元素组成均含有C 、O 、Ca 和Cd,表明Cd 2+已被吸附固定.2µm24680102030405060计数(C P S )能量(keV)CdCaOC P . stutzeri2µm02468020406080100120计数(C P S )能量(keV)C OCa CdB. subtilis12期朱广森等：微生物诱导碳酸盐沉积介导的Cd 2+固定 59172µm2468102030405060计数(C P S )能量(keV)COCaCd B. pasteurii图5 3种碳酸盐矿化菌矿化产物SEM 和EDS 分析Fig.5 The SEM and EDS analysis of mineralization products by three carbonate mineralization bacteria4000 3500 3000 25002000 1500 1000500100200300400a P . stutaerib B. subtilisc B. pasteurii透过率(%)波数(cm -1)10561443697876图6 3种碳酸盐矿化菌矿化产物的FTIRFig.6 The FTIR of mineralization products by three carbonatemineralized bacteria球霰石方解石图7 3种碳酸盐矿化菌矿化产物的XRDFig.7 The XRD of mineralization products by three carbonatemineralized bacteria由图6可知,3种菌形成的矿物主要成分为碳酸盐.1443cm -1附近的吸收峰为CO 32-反对称伸缩吸收峰,1056cm -1附近吸收峰为碳氧对称伸缩吸收峰[21],876cm -1附近吸收峰为CO 32-面外弯曲吸收峰,697cm -1附近吸收峰为CO 32-面内弯曲吸收峰[16].图7中XRD 分析表明,P . stutzeri 在生长过程中产生的矿物主要是球霰石,B. subtilis 形成的矿化产物主要为方解石和球霰石,而B. pasteurii 矿化产物为方解石,XRD 特征吸收峰比较尖锐,表明形成矿物中含杂质少,结晶度好[22].经XRD 主要衍射峰与标准PDF 卡片检索发现,3种菌对Cd 2+固定的生物矿化产物主要存在形式为CdCO 3,其中B. pasteurii 的矿化产物以稳定的Ca 0.67Cd 0.33CO 3共结晶的形式产生共沉淀[23]. 3 讨论3.1 诱导碳酸盐沉积的微生物诱导碳酸盐沉积的微生物广泛分布在土壤和水体中.这些微生物主要包括芽孢杆菌属,假单胞菌属,克雷伯菌属和芽孢八叠球菌属等[24],而不同种类的微生物在诱导碳酸盐沉积过程中的产脲酶能力及脲酶活性,碳酸根的产量也存在较大差异[25].例如,Dhami 等[26]从印度安得拉邦石灰性土壤中分离到B. megaterium SS3 和B. thuringiensis ,其产脲酶分别为690和620U/mL,产生碳酸根能力分别为 1870和1670mg/L;Li 等[27]研究表明Terrabacter tumescens A12和B6的脲酶活性分别为14.81 和1.25U/mL.本研究选取的3种碳酸盐矿化菌,B. pasteurii 的脲酶活性最强[28],而P . stutzeri ,B. subtilis 相对较弱;脲酶活性的强弱从侧面反映出3种碳酸盐矿化菌产CO 32-能力的强弱,B. pasteurii 产CO 32-浓度最高可达1735.18mg/L,P . stutzeri 和B. subtilis 产生的CO 32-浓度分别为588.19和 661.72mg/L.3.2 微生物诱导碳酸盐沉积介导Cd 2+固定机制微生物诱导碳酸盐沉积可通过尿素水解[29]、光5918 中国环境科学 41卷合作用[30-31]、硫酸盐还原[32]、反硝化作用[33]等途径实现,其中微生物水解尿素诱导碳酸盐沉淀因其机理相对简单,过程消耗少,对环境友好等优点而被广泛研究.微生物诱导碳酸盐沉淀固定重金属的基本原理是,通过微生物代谢产生脲酶,水解尿素产生NH4+和CO32-,NH4+释放NH3使pH值升高,从而在碱性环境下,足够浓度的Ca2+与CO32-结合,在细菌表面形成CaCO3沉淀,进而将Cd2+以类质同象置换方式占据Ca2+位置而固定[34],或Cd2+与CaCO3形成共结晶的方式被固定[35].本研究中3种碳酸盐矿化菌在水解尿素过程中产生OH-会提高环境pH值和平衡碳酸氢盐,从而形成CO32-,重金属Cd2+与游离的CO32-形成CdCO3晶体,从而降低有效态Cd的含量,这与Fujita等[36]研究结果一致.也正是上述固定机制的存在,3种碳酸盐矿化菌对培养环境中的Cd2+表现出高效的去除效果.基于微生物诱导碳酸盐沉积固定Cd2+的机理,对3种碳酸盐矿化菌固定Cd2+的次生矿物进行表征.SEM分析发现3种菌产生的矿化产物主要位于菌体表面或者菌体周围,这可能是由于菌体表面带有更多的负电荷,有更多的非均相成核位点[37],使Ca2+被大量吸附在菌体表面,在CO32-和碱性环境中,会沉淀形成CaCO3结晶[38].FTIR研究证实在1443cm-1附近特征吸收峰为碳酸盐官能团的对称伸缩振动产生,在1100~1000cm-1范围内是碳氧伸缩键的特征吸收峰[39],纯方解石和球霰石的典型振动带处于876,848,745cm-1[40],本研究中的FTIR图谱呈现出相似的结果,也进一步表明3种菌形成的次生矿物主要是碳酸盐.XRD图谱分析显示, B. pasteurii产生的次生矿物中检测到方解石型CaCO3和Ca0.67Cd0.33CO3,而P. stutzeri的次生矿物主要是球霰石型CaCO3和CdCO3,B. subtilis的次生矿物为方解石型CaCO3,球霰石型CaCO3共存和CdCO3.含Cd 矿物形成的可能机制是,Cd2+通过同构取代Ca2+而形成CdCO3或是与方解石的固溶体[41-42],之前的研究报道中也出现过类似的化学成分和晶体形式[43].3.3 羟基磷灰石对碳酸盐矿化菌固定Cd2+的影响机制当培养环境中加入化学材料羟基磷灰石(HAP)时,在一定程度上会减缓微生物诱导碳酸盐沉积的速率,但HAP和菌体共存体系提高了Cd2+的去除效果.HAP(Ca10(PO4)6(OH)2)因其较大的比表面积,微溶于水,有良好的吸附特性,而被广泛应用于重金属治理.本研究中加入HAP,除自身结构会对Cd2+吸附外[44],其特殊的空间结构,良好的生物相容性亦可作为微生物的吸附剂[45],为菌体诱导碳酸盐沉淀提供成核载体.本研究中可能存在的机制为:根据沉淀溶解平衡,HAP会有一部分Ca2+ 溶解到环境中,以此作为钙源,被吸附在HAP上的菌体会诱导CaCO3沉淀,进而对Cd2+生物矿化固定.上述机制仅是根据本研究所做出的推断, HAP和菌体联合作用过程,以及对多种重金属的固定产物及其机理还需更深入细致的研究.4结论4.1碳酸盐矿化菌P. stutzeri, B. subtilis, B. pasteurii生长过程中,B. pasteurii d的脲酶活性和产CO32-能力最强,同时,3种菌的代谢均使培养体系pH 值升高.4.2Cd2+浓度在0~10mg/L范围时对P. stutzeri生长影响较小,而Cd2+浓度高于1mg/L时则会抑制B. subtilis和B. pasteurii的生长.P. stutzeri对Cd2+的耐受能力比B. subtilis和B. pasteurii强.4.33种碳酸盐矿化菌均对0~1mg/L Cd2+有较强的固定能力,120h时Cd2+的固定率可达95%以上;加入羟基磷灰石能够增强碳酸盐矿化菌对Cd2+的固定效果.4.43种碳酸盐矿化菌的诱导矿化产物是碳酸盐.P. stutzeri的主要产物是球霰石,B. subtilis的矿化产物为方解石和球霰石共存,而B. pasteurii矿化产物以方解石为主;Cd2+与Ca2+同晶置换以CdCO3的形式沉淀,部分以稳定的Ca0.67Cd0.33CO3共结晶的形式存在.参考文献：[1] Zang F, Wang S L, Nan Zhongren,et al. 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山东农业大学学报(自然科学版),2024,55(1):076-083Journal of Shandong Agricultural University ( Natural Science Edition )VOL.55 NO.1 2024 doi:10.3969/j.issn.1000-2324.2024.01.011Trametes hirsuta漆酶的分离纯化及其对活性染料脱色研究刘飞，李治宏，张仕豪，刘璇，郑晓晴，焦若若，朱友双*济宁医学院生物科学学院，山东日照 276800摘要：漆酶是一种含铜的多酚氧化酶，在生物检测、工业染料脱色、有机农药降解、纸浆漂白及食品饮料等领域具有广泛的应用价值。
本研究使用实验室自主筛选鉴定的漆酶高产菌株粗毛栓菌（Trametes hirsuta），液态发酵后，培养液经硫酸铵分级沉淀、DEAE Sepharose FF 阴离子交换层析分离纯化，酶活总得率57.2%，纯化倍数6.0倍，比活力为758.5 U/mg，漆酶的分子量约为50 kDa。
利用粗毛栓菌粗酶液分别对结晶紫、溴酚蓝、孔雀石绿、詹姆斯绿B进行脱色，同时研究了染料浓度、脱色温度、pH和NaCl对溴酚蓝和孔雀石绿脱色率的影响。
结果表明，溴酚蓝和孔雀石绿浓度分别为40 mg/L 和50 mg/L时脱色率较高；在脱色温度为50 ℃时，漆酶对溴酚蓝和孔雀石绿的脱色率较高，最高脱色率分别为68.51%和83.06%；溴酚蓝在pH 3.5时脱色率最高达到72.61%，而孔雀石绿的脱色率在pH 4.5时最高达到83.49%；NaCl对Trametes hirsuta漆酶催化染料脱色有一定的抑制作用。
本研究表明Trametes hirsuta漆酶在染料脱色中具有较大的应用前景，在工业废水的处理中具有良好的应用潜力。
关键词：粗毛栓菌；漆酶；分离纯化；染料脱色中图法分类号：Q939.5文献标识码： A文章编号：1000-2324（2024）01-0076-08 Isolation and Purification of Laccase from Trametes hirsuta and Its Application in Reactive Dye DecolorizationLIU Fei, LI Zhi-hong, ZHANG Shi-hao, LIU Xuan, ZHENG Xiao-qing,JIAO Ruo-ruo, ZHU You-shuang*School of Biological Science/Jining Medical University, Rizhao 276800, ChinaAbstract: Laccase is a copper-containing polyphenol oxidase with a wide range of application, including bio-detection, industrial dye decolorization, organic pesticide degradation, pulp bleaching, and the food and beverage industries. We utilized a high-yield laccase strain of Tramete hirsuta identified in the laboratory. The laccase was separated and purified through ammonium sulfate precipitation and DEAE Sepharose FF anion exchange chromatography. The enzyme activity yield was 57.2%, with a purification fold of 6.0 and a specific activity of 758.5 U/mg. The molecular weight of laccase was about 50 kDa. The crude enzyme solution from Tramete hirsuta was used to decolorize crystal violet, bromophenol blue, malachite green, and Janus green B. The effects of dye concentration, temperature, pH and NaCl on the decolorization rate of bromophenol blue and malachite green were also investigated. The decolorization rates were higher when the dye concentration was 40 mg/L for bromophenol blue and 50 mg/L for malachite green. The decolorization rates of laccase on bromophenol blue and malachite green were 68.51% and 83.06%, respectively, at the temperature of 50℃. Bromophenol blue exhibited the highest decolorization rate of 72.61% at pH 3.5, while malachite green showed the highest decolorization rate of 83.49% at pH 4.5. NaCl had an inhibitory effect on the dye decolorization catalyzed by Trametes hirsuta laccase. Our study showed Trametes hirsuta laccase has a great application potential in dye decolorization and industrial wastewater treatment.Keywords: Trametes hirsuta; laccase; isolation and purification; dye decolorization漆酶是一种古老的含铜多酚氧化还原酶，最早发现于日本漆树中(Rhusvernicifera)[1]，属于铜蓝氧化酶。
广东药学院硕士学位论文γ-谷氨酰转肽酶酶学性质及其催化应用研究姓名：***申请学位级别：硕士专业：病原生物学指导教师：***2011-05-谷氨酰转肽酶酶学性质及其催化应用研究中文摘要-谷氨酰转肽酶在生物体内广泛存在，是谷胱甘肽代谢的关键酶之一，随着生物技术发展的快速发展，利用?-谷氨酰转肽酶制备系列?-谷氨酰基类化合物的研究已成为生物催化领域的热点。
目前，本实验室筛选到一株?-谷氨酰转肽酶高产菌株，并已探索了以菌体作为酶源催化合成茶氨酸的生产工艺。
在此基础上，我们作了进一步的研究。
目的以Bacillus licheniformis C12菌株为?-谷氨酰转肽酶制备的菌种来源，通过对其产酶发酵条件优化、提取工艺、酶学性质及固定化该酶催化底物生产茶氨酸进行系统研究，为固定化该酶工业化生产茶氨酸打下基础。
方法1. 产酶发酵的诱导条件研究：采用底物诱导作用，通过不同底物（甘氨酸、谷氨酸、谷氨酰胺）对菌体产酶发酵进行诱导，提高菌体的产酶量。
2. 酶的提取及酶学性质研究：采用溶菌酶法、反复冻融法、超声波法破碎细胞壁，高速冷冻离心后获得?-谷氨酰转肽酶粗酶液，经硫酸铵分级沉淀及透析脱盐初步纯化后，得到酶的提取物。
采用?-谷氨酰转肽酶常规测定方法检测其酶学性质。
3. 固定化酶制备及催化条件研究：采用海藻酸钠包埋戊二醛交联法，制备固定化酶，通过正交试验对固定化酶条件进行优化。
通过采取调整底物浓度、反应温度、pH 以及添加酶促进剂提高固定化酶催化生产茶氨酸量，并采用正交试验得到最佳的固定化酶催化生产茶氨酸的工艺条件。
结果1. 产酶发酵的诱导条件研究：产酶发酵24h后添加谷氨酰胺至浓度5mmol/L，诱导2h后，其酶活力最高为7.0U/ml，是对照组的2.26倍。
2. 酶提取及酶学性质研究：菌体细胞破碎的较佳方法为溶菌酶破碎法，其操作条件选择为每克湿菌体加入2.5mg溶菌酶，35℃酶解20min，冷冻离心得粗酶液。
