The Mechanism Of Cr(Ⅵ) Reduction By Pseudochrobactrum Saccharolyticum LY10

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.