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蜡状芽孢杆菌对水体中镉的吸附特性与机理研究

Study on the Removal of Cd(Ⅱ) from Aqueous Solutions by Bacillus Cereus RC-1: Biosorption Characteristics and Mechanism

【作者】 黄飞

【导师】 党志;

【作者基本信息】 华南理工大学 , 环境工程, 2013, 博士

【摘要】 微生物吸附法因其安全性和经济性等优点,成为水体重金属镉污染修复的潜在技术之一。同时,也引起国内外学者的广泛关注。本论文在展开大宝山矿山水体、悬浮物和沉积物重金属污染分布与评价的基础上,采用课题组前期筛选的耐镉细菌Bacillus cereusRC-1作为微生物吸附剂,对水体中Cd2+的吸附特性与机理进行了初步研究。论文取得的主要研究成果如下:1、开展了大宝山尾矿坝周围水体的重金属污染采样监测工作。大宝山尾矿坝水体、悬浮物和沉积物都遭受了以重金属Cu、Zn、Cd和Pb为主的复合污染,特别是Cd污染尤为严重。水体中Cu、Zn、Cd和Pb最高超标倍数分别达6倍、20倍、35倍和3倍,其中,Cd含量不仅与水体pH值呈负相关,还与水体氧化还原电位有较高的相关系数(R2=0.9387)。此外,在探讨了沉积物中重金属含量分布特征的基础上,采用地累积指数法和潜在生态风险指数法对其重金属污染的生态风险程度进行了评价,发现沉积物中重金属污染程度顺序为Cu> Cd> Zn> Pb。2、以课题组已筛选的耐镉细菌为研究对象,初步探讨了其对Cd2+的耐受性和抗性机制。经过16S rDNA序列同源性分析和NCBI数据库的比对,该菌株属于蜡状芽孢杆菌(Bacillus cereus),GenBank登录号为JF683581。该菌株在含20mg/L Cd2+液体培养基中的生长速率与对照组的相当且最小抑制浓度为200mg/L,预示该菌体在水体镉污染治理方面有很大的应用潜力。此外,采用高温法和SDS法进行质粒消除试验,结果表明处理前后菌株的抗镉性能差异不大即抗性不稳定,同时质粒提取与检测试验也未发现质粒条带。因此,初步认为该菌株的抗性基因可能位于染色体上。3、研究了生长菌株B.cereus RC-1对Cd2+的吸附特性。确定了生长菌株吸附的最佳条件为:pH为7.0、温度为28±2℃、转速为150rpm。另外,Freundlich模型拟合效果较好,表明生长菌体对Cd2+吸附是一个异质面的吸附过程。同时,一级动力学能较好地模拟生物吸附反应的初始阶段,而且预测值与实际值接近;二级动力学能较好的模拟生物吸附反应的整个过程,而且相关系数较高。4、当无营养物质提供时,对比研究了B.cereus RC-1的活细胞和死细胞对Cd2+的吸附。首先,确定了活、死细胞吸附的最佳条件均为:pH为5.0、吸附剂浓度为1.5g/L、转速为150rpm;采用了Langmuir、Freundlich和Redlich-Peterson等三种吸附平衡模型对菌株吸附过程进行模拟。对于活细胞,三个模型都适合拟合其吸附过程,表明活细胞吸附是一个异质面的吸附过程,除了细胞表面吸附之外,其他生物吸附机制如胞内积累等有可能参与了Cd2+的去除过程;对于死细胞,Langmuir模型最适合拟合其吸附过程,而且RL值远远小于活细胞,说明死细胞的吸附过程是一个单分子层吸附并且更加有利进行。其次,对于死菌体吸附,基本在30min内,达到了最大吸附量的93%,而活菌体则需要约50min,而且二级动力学更适合描述两者的动态吸附过程,说明两者是以化学吸附过程为主。最后,通过Zeta测定、TEM、SEM-EDS、FTIR、胞内积累试验和解析试验等,探究了死细胞吸附效果优于活细胞的主要原因是:一方面死细胞表面展示了更多的吸附位点和负电荷量,另一方面活细胞内部对Cd2+积累作用并不显著。5、当有营养物质提供时,探讨了生长菌株B.cereus RC-1对Cd2+吸附机理。首先,SEM-EDS、TEM和AFM结果表明,除了20mg/L Cd2+初始浓度之外,随着Cd2+浓度的增加,菌体表面粗糙度Ra和RMS都显著增加,尤其当高浓度Cd2+时(>20mg/L),生长菌株部分细胞发生了严重变形和细胞质收缩。其次,Zeta测定表明当低浓度Cd2+(≤20mg/L)时,随着菌体的生长时期所起作用的大小也有所不同;而在高浓度Cd2+(>20mg/L)下,静电吸引在生长初期迅速达到了平衡,但生长后期可能通过其他方式来吸附Cd2+。 FTIR分析表明生长菌株表面参与吸附官能团有-OH、C=O、-NH和-CH等,其中-OH和C=O是优先吸附位点,而且在高浓度下,蛋白质中酰胺基在吸附过程中可能起主要作用。最后,胞内外积累试验表明当较低Cd2+浓度(≤20mg/L)时,吸附过程以胞内积累为主;当高Cd2+浓度(>20mg/L)时,吸附过程以胞外吸附为主。综合分析,表明生长菌体在不同镉浓度下的吸附机制有所不同,而且当较低Cd2+浓度(≤20mg/L)时,生长菌体的吸附效果较好。6、探究了生长菌体在不同生长时期的吸附机制。B.cereus RC-1对Cd2+吸附过程初期是一个产酸的过程,后期开始产生碱性物质,这可能是菌体在不同生长时期有不同的吸附机制,同时pH值总体变化不大,说明离子交换不显著。而且,菌体的胞外吸附与胞内积累同菌体处于哪个生长时期有很大关系,对数期细胞的总吸附容量大于稳定期的。此外,当含有ATP酶活性抑制剂DCC的条件下,DCC不仅提高了生长菌体对Cd2+的去除率,而且还阻止了细胞内部Cd2+重新释放到环境中去,进而避免了二次污染。

【Abstract】 Microbiological biosorption because of its security and economic advantages is one ofthe cadmium pollution restoration potential techniques, and it also causes widespread concernin the domestic and foreign. In this thesis, on the basis of the pollution evaluation of heavymetals for Dabaoshan mine water, suspended particles and sediment, the main object of thisstudy is research on the bisoroption characteristics and mechanism of the cadmium-resistantbacteria in water, which is screened by our team preliminary. The main conclusions are asfollows:1. The heavy metals pollution evaluation of mine tailing from Dabaoshan mine wascarried out. The tailings dam suffered severe water pollution of heavy metals by measuringthe water, suspended particles and sediments, such as Cu, Zn, Cd and Pb, especially Cd.Moreover, the maximum exceeded multiples of the standard reached6,20,35and3timesrespectively. Meanwhile, the Cd(II) concentration in water is not only have a negativecorrelation with pH, but also has a high correlation coefficient with the redox potential of thewater. In addition, based on the discussion of the pollution distribution of heavy metals insediments by using the methods of land accumulation index and potential ecological riskindex, it was found that the degree of pollution of heavy metals in sediments was very high,showing the combination contamination of heavy metals in sediments, such as Cu, Zn, Cd andPb. Furthermore, the order of pollution of heavy metals in sediments was Cu> Cd>Zn> Pb.2. The study on the biological characteristics and the degree of Cd-tolerance was carriedout with a certain cadmium-resistant bacteria existing in our research group. The strainbelongs to Bacillus cereus and its GenBank accession number is JF683581from the analysisof16S rDNA sequence homology and the comparison of NCBI database. The growth rate ofthis strain in liquid medium containing20mg/L Cd2+was quite well by comparison with thecontrol and the minimum inhibitory concentration of strain was200mg/L, which indicatedthat Bacillus cereus RC-1had a great potential application in the cadmium pollutionrestoration. In addition, it was found that the Cd(II)-resistance of this strain was insignificantand showed instability by applying the method of high-temperature and SDS plasmid elimination test, while the test of plasmid extraction and detection didn’t find any plasmidbands. Therefore, we presented a preliminary view of the strain Cd(II)-resistance gene may belocated on the chromosome.3. The biosorption characteristics for the growing cells of B.cereus RC-1wereinvestigated. The optimum conditions of the biosorption was pH=7.0, temperature=28±2℃and rotate speed=150rpm. As for the growing cells, the Freundlich isotherm model was betterin the two kinds of biosorption model, showing the biosorption process of B.cereus RC-1wasa heterogeneous surface. The dynamics simulations of growing cells showed that thefirst-order kinetics could simulate the initial stages of biosorption process and predicatedvalues were very close to the experimental data. Besides, the second-order kinetics had morecapable of simulating the entire biological biosorption process with higher correlationcoefficient.4. The comparative study of biosorption characteristics and mechanisms for the live anddead cells of this strain was carried out. For the both types of cells, the optimum biosorptionconditions was pH=5.0, biosorbent dose=1.5g/L and rotate speed=150rpm. Among the threekinds of biosorption model, such as Langmuir, Freundlich and Redlich-Peterson, all the threemodels were suitable for the simulation of the Cd(II) biosorption by live cells, whichsuggested the biosorption process was a heterogeneous surface, and some other biosorptionmechanisms such as ion exchange may be involved in the Cd(II) biosorpiton. As for the deadcells, Langmuir isotherm was the best model for simulating the biosorption process, and theRLvalue is far less than the live cells, suggesting that the dead cells were more favorable forthe Cd (II) biosorption. In addition, the bisorption capacity of dead cells reached the93%ofthe maximum biosorption capacity within30min, while it required approximately50min forthe live cells. Moreover, the second-order kinetic model was more suitable to describe thebiosorption process for both types of cells, and the biosorption was dominated by chemicalbiosorption process. Finally, it could be concluded that the biosorption capacity of dead cellswas higher than that of live cells, which was mainly due to the more biosorption sites on thedead cells surface displayed by heat treatment and the lower intracellular accumulation for the live cells by means of several analysis instruments, such as Zeta potential measurement, TEM,SEM-EDS, FTIR, the test of intracellular accumulation and desorption.5. The study on the biosorption mechanisms of the growing cells was carried out througha variety of analytical methods. Firstly, the biosorption process of the growing cells was aprocess of acid production for the initial biosorption, followed by alkaline production.Therefore, we presented that the growing cells have different biosorption mechanisms fordifferent growth periods, but the overall change in pH of the culture medium is not large. Atthe same time, the electrostatic adsorption played an important role in the biosorption forlower concentration Cd2+(≤20mg/L) and had different impact on the biosorption with differentgrowth periods. However, as for the higher concentration Cd2+(≥50mg/L), the electrostaticadsorption is not significant, suggesting some other mechanisms involved in the removal ofCd(II). Secondly, it had been concluded that the cell surface biosorpiton played a veryimportant role in the Cd(II) biosorption for the growing cells by the measurement ofSEM-EDS, TEM and AFM. Moreover, the RMS and Ra of the growing cells were increasedwith the increase of the Cd2+concentration. When the initial Cd2+concentration was greaterthan or equal to50mg/L, some cells were deformed or even cracked. At this time, the growingcells had resistance to the higher concentration of Cd2+by both extracellular biosorption andintracellular accumulation. Finally, the functional groups on the surface of the growing cells,such as-OH, C=O,-NH and-CH, were involved in the biosorption process, of which-OHand C=O were the preferred adsorption sites. Furthermore, the amides I, II and III bandspectrum peak changed significantly, which indicated that amide group of the protein playedan important role in the biosroption.6. The bisorption mechanisms of the growing cells were studied deeply. The growingcells have some different mechanisms in different growth period under different initial Cd2+concentrations. These results showed that intracellular accumulation was higher thanextracellular biosorption for the lower Cd2+concentration (<20mg/L), while the extracellularbiosorption is far greater than the intracellular accumulation for the higher Cd2+concentration(>20mg/L). In addition, the extracellular biosorption and intracellular accumulation have a great relationship with the growth periods through the test of accumulation for the growingcells. Moreover, the growing cells of logarithmic growth phase showed greater bindingcapacity for Cd(II) than the growing cells of stationary phase. Finally, when the liquidmedium contained DCC inhibiting ATP activities, both types of the growing cells not onlyimproved the removal efficiency, but also prevented intracellular Cd2+re-released into theenvironment, avoiding secondary contamination.

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