Application Of Heavy Metal Hyperaccumulator Endophytes In Heavy Metal Decontamination

Heavy metals pollution by human activities, especially industrial activities, is posing significant threats to human health and the environment due to their high occurrence as a contaminant, low solubility in biota, and the classification of several heavy metals as carcinogenic and mutagenic.Many physicochemical strategies, such as filtration, chemical precipitation, electrochemical treatment, oxidation/reduction, ion exchange, membrane technology, reverse osmosis, and evaporation recovery, have been developed to remove heavy metals. However, most strategies appear to be expensive, inefficient, and labor-intensive, or the treatment process lacks selectivity. Bioremediation which involves the use of potential microbes to remove heavy metals has been considered to be a safe and economic alternative to physicochemical strategies. The crucial constraint of this technology is to obtain promising and useful metal-resistant strains from enormous microbes on earth.Recently, the benefits of endophytes, especially hyperaccumulator endophytes, alone and/or combining with plants have been successfully tried for toxic heavy metal removal from contaminated water and soil. Endophytes refer to the microbes which inhabit in the interior of plant tissues and form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic without causing any harm to the host. Most endophytes are originally from the rhizosphere or phyllosphere, while some may get entrance through the seed. Hyperaccumulator, a plant species that lives in the heavy metals contaminated soil can accumulate exceptionally with high quantities of certain heavy metal. It could also be functioned as'filters'while selecting those promising and useful metal-resistant strains from soil microbes. Based on this theory, an novel technology for obtaining hose promising and useful metal-resistant strains from heavy metal hyperaccumulator endophytes (HMHEs) was developed and operated.In this study, a highly effective new method for isolation of endophytes was established, firstly. Surfactant, SDS and cellulase were applied for extraction endophytes from from their plant hosts. The endopytes of four tested plants (Solanum Nigrum L., alomonia Lour, Chirita fimbrisepala, Schizocapsa plantaginea) were isolated via traditional methods and new methods, respectively. The results indicated that surfactant and cellulase could effectively facilitate the isolation of endophytes by hydrolyzing the cellulose of the plants hosts.Several cadmium hyperaccumulator (Solanum Nigrum L.) plants were collected from the sewage discharge canal bank of Zhuzhou Smeltery (27°52′N, 113°05′E). By using the established isolation method, a total number of 97 endophytes were isolated from Solanum Nigrum L. 34, 36 and 27 endophytes were isolated from the roots, stems and leaves of the plants, respectively. Most of they were bacterial endopytes, only one fungus was isolated from the stem of the plants. The heavy metal resistances (minimal inhibitory concentration, MIC) of all the endophytes were tested. The endophytes from shoot and root demonstrated different degrees of cadmium resistance. These results suggest that different microbial communities may inhabit in the different compartments of the plant.Endophytic Fungus (EF) LSE10 was isolated from the cadmium hyperaccumulator Solanum Nigrum L. It was identified as Microsphaeropsis sp. When cultured in vitro, the biomass yield of this EF was more than twice that of None-endophytic fungus (NEF) Rhizopus cohnii. Subsequently, it was used as a biosorbent for biosorption of cadmium from the aqueous solution. The results showed that the maximum biosorption capacity was 247.5 mg/g (2.2 mmol/g) which was much higher than those of other adsorbents, including biosorbents and activated carbon. Carboxyl, amino, sulphonate and hydroxyl groups on EF LSE10 surface were responsible for the biosorption of cadmium.Heavy metal bioremediation by a multi-metal resistant endophytic bacteria L14 (EB L14) isolated from the cadmium hyperaccumulator Solanum Nigrum L. was characterized for its potential application in metal treatment. 16S rDNA analysis revealed that this endophyte belonged to Bacillus sp. The hormesis of EB L14 were observed in presence of divalent heavy metals (Cu, Cd and Pb) at a relatively lower concentration (10 mg/L). Such hormesis was the side effect of abnormal activities increases of ATPase which was planned to provide energy to help EB L14 reduce the toxicity of heavy metals by exporting the cations. Within 24 h incubation, EB L14 could specifically uptake 75.78%, 80.48%, 21.25% of cadmium, lead and copper at the initial concentration of 10 mg/L. However, nearly no chromium uptake was observed. The mechanism study indicated that its remediation efficiencies may be greatly promoted through inhibiting the activities of ATPase. The excellent adaptation abilities and promising remediation efficiencies strongly indicated the superiority of this endophyte in low concentrations heavy metal bioremediation, which could be useful for developing efficient metal removal system.Bioremediations of cadmium by endophytic bacterium (EB) L14 (Bacillus sp.) in the presence of industrially used metabolic inhibitors (DCC or DNP) were investigated. In the presence of DCC or DNP, the biomass population of EB L14 was greatly inhibited. However, the cadmium removal of EB L14 increased from 73.6% (in the absence of DCC or DNP) to 93.7% and 80.8%, respectively. The analysis of total and intracellular cadmium concentrations during 24 h of incubation indicated that this enhanced cadmium removal was the inhibition effect of DCC or DNP on the cations export resistance system of EB L14. This unique property strongly indicated the superiority of this endophyte for practical application in cadmium bioremediation in the presence of industrially used metabolic inhibitors.This dissertation was the first attempt to directly use heavy metal hyperaccumulator endophytes (HMHEs) which not only possess the excellent adaptability but also have been equipped with various metal-sequestration systems for heavy metal bioremediation. This study offers the ability to fully achieve the application of bioremediation in practical heavy metal decontamination.