【作者】 龙冬艳；

【导师】 陈英旭； 沈超峰；

【作者基本信息】 浙江大学 ， 环境工程， 2013， 博士

【摘要】 铬是一种重要的工业原料,被广泛地应用于印染、电镀、制革、合金生产等行业中。由于对含铬废物的不合理处理处置,导致了严重的土壤铬污染问题。铬在环境中主要以Cr(Ⅵ)和Cr(Ⅲ)两种价态存在。其中,Cr(Ⅵ)具有致畸、致癌等高毒性；而Cr(Ⅲ)化合物的溶解度低,生物毒性小。因此,将高毒性的Cr(Ⅵ)还原为Cr(Ⅲ)是Cr(Ⅵ)解毒和铬污染土壤修复的基本思路。与传统的化学还原法相比,利用微生物还原Cr(Ⅵ)具有经济、环境友好等优势,在大面积、中低浓度铬污染土壤修复中具有广泛的应用前景。由于铬污染土壤多呈碱性、高盐的特点,因此,本论文以高pH、高盐分含量、高Cr(Ⅵ)浓度作为选择压力,筛选获得耐盐耐碱的土著Cr(Ⅵ)还原微生物——解糖假苍白杆菌LY10(Pseudochrobactrum saccharolyticum LY10),并对其还原行为进行了研究。继而采用透射电镜-能谱(TEM-EDS)、X射线吸收光谱(XAS)、电子顺磁共振(EPR)、X射线荧光光谱(XRF)和软X射线谱学显微光束线等现代微结构分析技术系统研究了该微生物的Cr(Ⅵ)还原位置,微生物作用下铬的分子形态变化,以及非饱和生物膜对铬的吸收还原规律。从分子水平上初步阐明了解糖假苍白杆菌P. saccharolyticum LY10还原Cr(Ⅵ)的分子机制,旨在为铬污染土壤微生物修复提供理论依据和技术支撑。研究成果主要包括：(1)筛选获得了耐盐耐碱的土著Cr(Ⅵ)还原微生物。从铬污染土壤筛选获得四株Cr(Ⅵ)还原微生物,分别命名为菌株LY6、LY8. LY10和LY11。根据生理生化特性和16SrRNA基因序列同源性比对,分别鉴定为：不解糖假苍白杆菌、溶血不动杆菌、解糖假苍白杆菌和干丘亮杆菌；首次报道了不解糖假苍白杆菌和解糖假苍白杆菌的Cr(Ⅵ)耐受和还原能力；在四株微生物中,解糖假苍白杆菌LY10表现出耐盐、耐碱、耐Cr(Ⅵ)的综合优势,并具有较强的Cr(Ⅵ)还原能力和细胞存活能力,可耐受400mg L-1Cr(Ⅵ)、60g L-1NaCl、pH11.3,并能在156mg L-1Cr(Ⅵ)胁迫下存活12d以上。菌株LY10为后续研究提供了一种良好材料。(2)考察了菌株LY10的Cr(Ⅵ)还原行为。氧气的存在是菌株LY10生长和进行Cr(Ⅵ)还原的基本所需,其优化的Cr(Ⅵ)还原条件是：培养温度28℃,pH8.3, NaCl添加浓度量20gL-1,菌体接种量1.47×109cells mL-1,在该优化条件下,菌株LY10可在96h内将55mg L-1Cr(Ⅵ)全部去除：1mM Fe(Ⅲ)可促进菌株LY10对Cr(Ⅵ)的还原,1mM Zn(Ⅱ)和Al(Ⅲ)则具有抑制作用；LY10对Cr(Ⅵ)的还原可分为先快后慢两个阶段,初始阶段(0～72h)内的Cr(Ⅵ)还原过程符合一级反应动力学,在最优还原条件下,一级反应速率常数达到最大值(0.0445h-1)。(3)明确了菌株LY10的Cr(Ⅵ)还原位置。菌株LY10主要通过胞外还原的方式去除培养液中Cr(Ⅵ),绝大多数铬存在于培养液中,菌体细胞对铬的固定量较少(5.1%～7.1%)；在亚细胞水平上,胞外多聚物和细胞壁是细胞中铬的主要分布区,仅有少量铬进入到细胞质内；胞外多聚物在菌株LY10耐受Cr(Ⅵ)和还原Cr(Ⅵ)过程中发挥着重要作用,是菌株LY10的主要Cr(Ⅵ)还原位置；在高浓度Cr(Ⅵ)胁迫条件下,LY10分泌出更多的胞外多聚物包裹于细胞外,一方面通过还原作用将高毒性Cr(Ⅵ)进行还原,另一方面通过吸附作用对重金属铬进行拦截。通过细胞壁的进一步截留作用,从而有效阻止毒性铬进入细胞体内。(4)阐明了菌株LY10作用过程中铬的分子形态变化。在菌株LY10的作用下,Cr(Ⅵ)先接受一个电子生成中间产物Cr(Ⅴ),随后再经过两个电子的传递生成稳定产物Cr(Ⅲ)；在单细胞水平,绝大多数Cr(Ⅵ)和Cr(Ⅲ)都分布在细胞周边的胞外多聚物中,仅有少量Cr(Ⅵ)进入细胞并分布于细胞质内,胞外还原产物Cr(Ⅲ)则被截留在胞外多聚物和细胞壁中；菌体中的铬绝大部分为Cr(Ⅲ),其中,53.6%的铬以磷酸铬类似物的形式存在,此外还包含少量的醋酸铬(24.4%)和硫酸铬(19.7%)。(5)揭示了在类似土壤的水份非饱和环境中,LY10非饱和生物膜对铬的吸收还原规律。生物膜对铬的吸收累积是一个快速的过程,在吸收转运前期(10～30min),铬主要分布在生物膜底部靠近培养基层,转运中期(1～12h),铬在生物膜底端和生物膜顶端均有聚集,并随着培养时间的延长,顶端铬富集区逐渐往底部趋近,在吸收转运后期(24h),铬主要在生物膜底部距培养基层10～45um区域内富集；在水平方向,距生物膜中心内径r1=0.4cm、外径r2=1.2cm的环形区域是铬的主要富集区；生物膜对Cr(Ⅵ)的还原迅速而彻底,培养12h时已将吸收转运的铬全部还原为Cr(Ⅲ),还原后的Cr(Ⅲ)主要以磷酸铬形式存在。更多还原

【Abstract】 As an important industrial material, chromium is widely used in pigment manufacturing, electroplating, leather tanning, and alloy production. The inappropriate disposal of Cr(VI)-containing byproducts and uncontrolled release of Cr(VI) wastes have caused serious environmental pollution problems. Although chromium can exist in a variety of valence states, Cr(VI) and Cr (III) predominate in the environment because of their stability. Cr(Ⅵ) is highly soluble, oxidizing, mutagenic and carcinogenic. In contrast, Cr (Ⅲ) compounds are generally sparingly soluble, with low mobility and toxicity. Thus, the reduction of Cr(Ⅵ) to Cr(Ⅲ) can provide a useful method for Cr(Ⅵ) detoxification. Bioremediation, using multifarious microorganisms to reduce toxic Cr(Ⅵ) to Cr(Ⅲ), offers a cost-effective and eco-friendly alternative to traditional chemical method, especially when dealing with Cr(Ⅵ)-containing wastes at low-to-mid concentrations.In this study, high pH, high salinity and high concentration of Cr(Ⅵ) were used as selective pressures to isolate alkaliphilic and halotolerant Cr(Ⅵ)-reducing bacteria. The Cr(Ⅵ)-reducing behavior of a novel potent strain—P.saccharolyticum LY10was studied. Its Cr(Ⅵ)-reducing and immobilizing mechanisms (including the reducing site, the speciation change of chromium, and the chromium accumulation by unsaturated biofilm) were also further investigated by using transmission electron microscopy and energy dispersive X-ray spectroscopy (TEM-EDS), X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR), X-ray fluorescence microprobe (XRF) and soft X-ray spectromicroscopy. Results from this series of studies illustrated the mechanism of Cr(Ⅵ) reduction by Pseudochrobactrum saccharolyticum LY10, and would provide theory basis for microbial bioremediation of chromate-contaminated soil. The main results of this research are as follows:(1) Four indigenous bacteria, named LY6, LY8, LY10and LY11, were isolated from Cr(Ⅵ)-contaminated soil. Based on biochemical analysis and16S rRNA gene sequencing, strain LY6was identified as Pseudochrobactrum asaccharolyticum, strain LY8was identified as Acinetobacter haemolyticus, strain LY10was identified as Pseudochrobactrum saccharolyticum, and strain LY11was identified as Leucobacter aridicollis. It was for the first time the species of Pseudochrobactrum asaccharolyticum and Pseudochrobactrum saccharolyticum were reported for Cr(VI) resistance and removal. Among the four Cr(VI) resistant isolates, strain LY10displayed comprehensive advantages for surperior alkali-tolerance, halotolerance, Cr(VI) tolereace, as well as Cr(VI)-reducing ability and cell viability. Strain LY10could tolerate400mg L-1Cr(Ⅵ),60g L-1NaCl, pH11.3, and even survived at concentrations of156mg L’1Cr(VI) for more than12days. Because of its comprehensive advantages, P. saccharolyticum LY10was used for further studies.(2) The Cr(VI)-reducing behavior of P. saccharolyticum LY10was studied. It was found that oxygen was the basic requirement for bacterial growth and effective Cr(VI)-reducing activity of strain LY10. The optimum conditions for its efficient Cr(VI) reduction were temperature28℃, initial pH8.3,20g L-1NaCl, and1.47×109cells mL-1of cell density. Under these combined opitimal conditions, strain LY10could completely reduce55mg L-1within96h. The Cr(VI)-reducing ability was enhanced by1mM Fe(Ⅲ), but hindered by the presence of1mM Zn(II) and Al(III). The Cr(VI) bioreduction during the long period involved two distinct reaction stages, including an initial rapid reduction process followed by a slower removal stage. Further kinetic analysis demonstrated that the bioreduction during the initial72h could be well described by the first-order kinetic model. Under combined optimal conditions, the maximum reduction rate of (4.45±0.1)×10-2h-1was achieved.(3) The Cr(VI)-reducing site of the P. saccharolyticum LY10was investigated. Results demonstrated that most of the Cr(VI) was reduced outside the cell. The majority of the Cr remained in the culture solution, and only quite a few were immobilized in the bacterial cell (5.1%～7.1%). Subcellular analysis indicated that most of the immobilized Cr was located in the extracellular polymeric substances (EPS) and cell wall. EPS was of vital importance for the Cr(Ⅵ) tolerance, and was the main Cr(Ⅵ) reduction site of strain LY10. When LY10was treated with high concentration of Cr(Ⅵ), more EPS was produced and encrusted around the cells. On the one side, the toxic Cr(VI) could be reduced by the soluable reduction enzyme in the EPS. On the other side, EPS and cell wall could further immobilized the chromium and prevented the cell from the damage effect caused by toxic Cr(Ⅵ).(4) The molecular speciation change of chromium was studied when Cr(Ⅵ) interact with microorganism. It was found that P. saccharolyticum LY10transiently reduces Cr(Ⅵ) with a one-electron shuttle to form Cr(Ⅴ), followed by a two-electron transfer to generate Cr(III). Most of Cr(Ⅵ) and Cr(Ⅲ) were located in the EPS. Only a few of Cr(Ⅵ) entered the cell and distributed in the cytoplasm. On the contrary, the extracellular reduction product of Cr (Ⅲ) could not enter the wall, and was trapped in the EPS and cell wall. XAFS study further verified that the chromium immobilized by the cells was in the Cr(Ⅲ) state, and most of Cr(Ⅲ) was bond with phosphate (53.6%), acetate (24.4%) and sulphate (19.7%).(5) The chromium accumulation and reduction process were investigated for unsaturated P. saccharolyticum LY10bio film. Results demonstrated that the sorption of chromium by biofilm was rapid. At the initial accumulation stage (10-30min), most of chromium was located at the bottom of the biofilm. Then, chromium was transported to the whole biofilm, and accumulated both at the bottom and the upper side of the biofilm (1～12h). When the biofilm was incubated for24h, chromium precipitated in a10-45um layer near the media-biofilm interface.In the horizontal direction, the ring area with inner diameter (r1) of0.4cm and outer diameter (r2) of1.2cm was the main distribution area for chromium. XAFS results indicated that P. saccharolyticum LY10biofilm could effciently reduce Cr(Ⅵ), with chromium being completely reduced within12h. Most of reduced Cr(Ⅲ) precipitated as chromium phosphate.更多还原