Research On Heavy Metal Biosorption/Accumulation By White-rot Fungi And Fungi Tolerance/Resistance Analysis

Recent industrial and urban activities lead to a rapid expansion of a wide range of contaminants, especially heavy metal discharge and contamination. Parallel to control of harmful effects of contaminants and immediate improvement of human living environment therefore would be necessary. Heavy metal contamination is of great concern because of its high toxic effect, high mobility in the environment and low toxic concentrations. Heavy metals accumulated intracellularly can cause serious damages by thiol-binding and protein denaturation replacing, primary displacement of essential metals involved in biological reactions, or a secondary effect of oxidative stress. Heavy metals are not biodegradable, and can cycle in various environments medium, cause serious damage to the ecological environment. Biosorption technology, especially application of microorganisms as biosorbents, has been suggested as an efficient, cost-effective and eco-friendly alternative to existing treatment techniques. However, environment application in heavy metal biosorption commonly results in a heavy metal induced stress. Numerous studies have revealed that metals can accumulate and redistribute in some microorganisms, causes growth inhibition and even cell death, some microorganisms could survive in the stress exposure, and exhibited tolerance and resistance to heavy metals. The question as how microorganisms may defend themselves against heavy metals is therefore receiving increasing attention. To understand tolerance and maximize the potential application of microorganisms in bioremediation, it is essential to understand the toxicity of the heavy metals and the detoxification mechanisms.White-rot fungi application in biosorption of heavy metals and organic pollutants has been widely reported, taking advantage of favorable heavy metal affinity and good applicability. Hence, we choose Phanerochaete chrysosporium(P. chrysosporium) as biomass strains and the sensitive metal(Cd) as object to study the Cd toxicity(biomass, cell morphology, metabolic activity and reactive oxygen stress) and resistance of P. chrysosporium, to systematic investigate the interaction of heavy metals and P. chrysosporium. The study includes the following four parts:The first part described the adsorption and accumulation of heavy metals by P. chrysosporium and the application of immobilized P. chrysosporium in wastewater treatment. Results found that P. chrysosporium could use as biosorbents for Pb and Cd biosorption, but the biosorption efficiency is limited. MNPs–Ca-alginate immobilized P. chrysosporium biosorbents showed a promising prospect for application in heav y metal-containing wastewater treatment, with great biosorption capacity and even favorable stability. The favorable biosorption application prospect is the most critical basis of study of interaction between heavy metals and P. chrysosporium.The second part focused on the toxicological effect on physiological metabolism and oxidative damage. Long-term exposure of heavy metals during wastewater treatment might result in toxicity and induce stress response, cell injury or damage may affect biosorption efficiency, so the study of toxicity effect is a critical question in our study. Results were conducted at the various Cd concentration s(0, 20, 50, 100 mg/L) during liquid culture of P. chrysosporium. Cd-induced toxicity was observed with growth inhibition and enzyme inactivation(Li P and Mn P) of P. chrysosporium. We further observed the variation of cell morphology in P. chrysosporium, and dose-dependent of heavy metal bioaccumulation and reactive oxygen stress(H2O2 and MDA) were found, results suggested that Cd bioaccumulation led to the toxicity directly. Cd accumulation enhanced generation of ROS can overwhelm cells’ intrinsic antioxidant defenses and exceed the capacity of the ROS suppressing mechanism, thus result in oxidative damage. To understand tolerance and maximize the potential application of P. chrysosporium in bioremediation, it is essential to understand the toxicity of the heavy metalsThe third part emphasized on the tolerance and resistance of P. chrysosporium exposed to Cd.(1) Role of oxalic acid in the Cd uptake and participation in detoxification process in P. chrysosporium has been studied. Cd-induced oxalic acid secretion was observed. The peak value of oxalic acid concentration was observed at 12 d, and initial Cd exposure at 100 mg/L induced a 2.12-fold of oxalic acid increase, meanwhile, negative correlations have been found between oxalic acid concentration and Cd-induced growth inhibition ratios. During the short-term uptake experiments, the uptake of Cd was enhanced and accelerated in the presence of oxalic acid and resulted in alleviated growth and enzyme inhibition ratios. The formation of a metal-oxalate complex therefore may provide a detoxification mechanism via effect on metal bioavailability, whereby many fungi can survive and grow in environments containing high concentrations of toxic metals.(2) Antioxidants and response of P. chrysosporium to Cd exposure were studied. The P. chrysosporium extracts possessed remarkable antioxidant activity, expressing a dose-dependent total antioxidant activity and accompanied with high reactive oxygen radical(O2?–, ?OH and H2O2) scavenging capacity. In addition, it was established that, various compounds, such as antioxidant enzymes(catalase, superoxide dismutase and glutathione peroxidase) and low-molecular-weight antioxidant components(glutathione, phenolics and flavonoids) existed steadily in P. chrysosporium. Linear regression analysis and Pearson correlation coefficient analysis demonstrated that enzymatic and non-enzymatic antioxidants took part in processes and acted as “antioxidant network” in P. chrysosporium by confirming with the significant correlation coefficients among the tested antioxidants. The paper proposes a new validation procedure to specifically validate the admirable tolerance and high efficiency of P. chrysosporium in environmental treatment application, taking advantage of the remarkable antioxidant activity.(3) Pb and Cd induced glutathione(GSH) accumulation and the regulation at the intracellular heavy metal leve l in P. chrysosporium. P. chrysosporium accumulated high levels of GSH, accompanied with high intracellular concentrations of Pb and Cd. Pb bioaccumulation lead to a narrow range of fluctuation in GSH accumulation(0.72–0.84 μmol), while GSH plummeted under Cd exposure at the maximum value of 0.37 μmol. Good correlations between time-course GSH depletion and Cd bioaccumulation were determined( R2>0.87), while no significant correlations have been found between GSH variation and Pb bioaccumulation(R2<0.38). Significantly, concentration-dependent molar ratios of Pb/GSH ranging from 0.10 to 0.18 was observed, while molar ratios of Cd/GSH were at the scope of 1.53–3.32, confirming the dominant role of GSH in Cd chelation. The study also demonstrated that P. chrysosporium showed considerable hypertolerance to Pb ions, accompanied with demand-driven stimulation in GSH synthesis and inconspicuous generation of reactive oxygen stress. GSH plummeted dramatically response to Cd exposure, due to the strong affinity of GSH to Cd and the involvement of GSH in Cd detoxification mechanism mainly as Cd chelators. Investigations into GSH metabolism and its role in ameliorating metal toxicity can offer important information on the application of the microorganism for wastewater treatment.On this basis of the front studies, the fourth part investigated the environmental application of P. chrysosporium in composting treatment of heavy metal-organic contaminants combined polluted sediments. Results demonstrated that composting wa s an economic and effective technology for solid waste treatment. Inocula of P. chrysosporium accelerated the Cd transformation to residual form and contributed to the complete degradation of nonylphenol. After 30 days of composting nonylphenol could be completely degradaded in the sample D(with P. chrysosporium incubation). Results confirmed the environmental application of P. chrysosporium in heavy metal-organic contaminants combined polluted sediments.The study focused on the interaction of heavy metals and P. chrysosporium during the heavy metal treatment process, which could provide available information for the understanding of the tolerance and resistance mechanisms to metal induced toxicity. Investigations into antioxidant response and roles in ameliorating metal toxicity can offer important information on the application of the environmental microorganism for wastewater treatment and bioremediation of sediments.