The Bioremediation Of Heavy Metal Contaminated Soil By Bioaugmentation Of An Active Mutant Bacterium Assisted By Biochar

Bioaugmentation has been receiving much attention recently as an in-situ technology for the remediation of heavy metal polluted soils. The microorganism species and its carrier, used as the nutrition source for microorganism, are the two crucial factors affecting the bioaugmentation efficiency. In this thesis, the immobilization of heavy metals in soil by an active species of microorganism and several carriers was studied. The bacterium used in this study was a mutant species (B38) from Bacillus subtilis obtained by UV irradiation under high cadmium (Cd) concentration. Two types of biochar made of different biomass by pyrolysis were used as the carrier of B38mutant species. One was produced by pyrolyzing corn straw, designated as CBC. The other was produced by pyrolyzing pig manure, designated as PBC. The adsorption behavior of cadmium (Cd), chromium (Cr), mercury (Hg) and lead (Pb) by B38or biochar, and various factors affecting the adsorption, such as the contact time, amount of biomass, pH, temperature and ionic strength of the solution, were investigated. To evaluate the feasibility of bioaugmentation, the pot experiment was conducted. The Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and fluorescent labeling technique were applied to analyze the surface functional group, the morphology, the bulk organic elemental composition, and the growth of native microorganism and B38, respectively, so that the mechanisms of heavy metal immobilization using carried microorganisms could be elucidated. The main results of this thesis are as follows:(1) B38had a higher affinity to heavy metal cations than the anion. Rapid metal binding occurred during the beginning of the adsorption of heavy metal cations by both living and nonliving B38biomass, and then the equilibrium was achieved. A similar behavior was found for heavy metal anion (Cr(Ⅵ)) adsorption by nonliving B38. But the adsorption of Cr(Ⅵ) by living B38was a slow process. This implied that the B38growth was inhibited by the high Cr(Ⅵ) concentrations. The nonliving biomass generally showed greater or similar adsorptive capacities as compared to the living biomass. The sorption kinetics followed the exponential equation for living biomass, and followed the pseudo-first-order and pseudo-second-order model for nonliving biomass for the metal cations and the metal anion, respectively. The equilibrium adsorptive quantity of the heavy metals on B38changed with solution pH. At pH7.0, both of the two sorbents presented the maximum adsorption capacities. However, the effect of the temperature of the adsorption was less significant. It seemed obvious that the adsorption of Hg was not related to ionic strength. But the adsorption quantity of Cd, Cr and Pb decreased with the increase of the ionic strength of solution. This implied that the electrostatic binding of the B38cell wall with the Hg ion was negligible compared to the covalent binding, but important for other ions. The results of binary and ternary sorption experiments indicated that the metals with the higher sorption capacity in the single-component systems showed greater inhibitory effects on the biosorption of other metal ions in the multiple-component systems, but the sorption sites of Hg and Cd or Pb are likely to be different. The FT-IR spectra showed the evidence that some of the functional groups, such as the hydroxyl, carbonyl, carboxyl, and primary amide groups, were involved in the interaction with the heavy metals ions by forming complexes on the cell wall of B38.(2) NovoGro, an industrial by-product, was used as an organic amendment to provide the nutrients necessary for B38growth. In the pot experiment, it was found that B38mutant combined with NovoGro exhibited a high efficiency for the remediation of heavy metal polluted soil. But B38employed alone could not promote the growth of plants. Meanwhile, soil amended with NovoGro alone inhibited plant growth slightly. However, the biomass yields of plants increased significantly when the soil was amended with both B38and NovoGro. Thus, the B38exhibited high activity when assisted by nutrient materials.(3) Extraction by diethylene triamine pentacetate acid (DTPA), Mehlich3(M3), and the first step of BCR (Community Bureau of Reference) sequential extraction method (BCR1) were studied to predict the bioavailability of heavy metal pollutants in soil. The results revealed that DTPA was a good extractant for the predicting of heavy metal bioavailability for leaf vegetables, and M3and BCR1showed good capacity of predicting heavy metal bioavailability for rhizome vegetables and for leguminous plants, respectively.(4) The sorption experiment by biochars showed that CBC had a high affinity to Hg ions, while PBC had a high adsorption capacity for Pb ions. Two combined sorbents between B38and the two biochars had high adsorption capacities and preferences for Pb ions. For the same metal ion, the adsorption capacity of B38combined with PBC was higher than that combined with CBC. The experimental adsorption data by the two biochars were fitted well to the Freundlich isotherms, and the results by the two combined sorbents were fitted well to Langmuir-Langmuir isotherms. Hg ions competed for the same adsorption sites with Pb ions on the biochar surface. But it seemed that the addition of B38altered the physico-chemical properties of biochar surface. The binding sites of Hg ions and Pb ions on the two combined sorbents seemed to be different.(5) The SEM photographs showed significant differences in structures between CBC and PBC. The CBC materials looked like the sieve plates with polyporous structure, while the PBC materials were polyporus structure with lacunose surface. Thus, PBC could provide more adsorption activity sites for heavy metal binding and more room for microorganism growth. The FT-IR and XPS spectra of the two biochar before and after combined with B38indicated that the addition of B38decreased the amount and diversity of the CBC surface, while increased those of the PBC surface. The oxygen-containing functional groups, such as the hydroxyl, carbonyl, carboxyl groups, and the aliphatic ethers groups (C-O-C) in cellulose of CBC were involved in the ion exchange with heavy metal ions. The cation-π interactions involved binding between heavy metal ions and the y-CH group of furan or the β-ring of heterocyclic compounds on the surface of biochar.(6) CBC and PBC were tested as the B38mutant carrier in the remediation of heavy metal contaminated soils by bioaugmentation. Soil amended with B38and PBC increased the plant biomass yield, but soil amended with B38and CBC had no significant effect on the growth of plants. All the four amendments (two biochars and two combined materials of B38and the two biochars) could inhibit the bioaccumulation of heavy metal pollutants by plants, and reduce the bioavailability of heavy metal pollutants in soil. Soil amended with PBC and B38exhibited the maximum remediation efficiency among the four treatments, and the Cd and Pb concentrations in the edible part of plants were below the maximum levels of contaminants in foods.(7) The thesis for the first time used the fluorescent labeling technique for the investigation of the growth of a bioaugmented species in soil. In the soil amended with B38alone, the concentration of B38mutant increased slowly and sustainingly, then reached a steady state. In the soil amended with B38and CBC or NovoGro, the concentration of B38increased sharply, and then achieved the equilibrium state. However, in the soil amended with B38and PBC, the concentration of B38mutant increased first, then decreased. Thus, the B38mutant showed great resilience to the stressful environmental conditions, had great potential for the development, and exhibited a competitive superiority when competed with the native microorganisms. All the three carrier materials could provide nutrients for the growth of both native microorganisms and B38mutant.The present study found that B38, as a high Cd-tolerant mutant strain, had high adsorption capacities to multiple heavy metals. Biochar could not only immobilize the heavy metal contaminants, but also improve and fertilize the soil, and provide necessary nutrient for the microorganism growth. Biochar, especially the one produced from animal wastes, showed to be a good carrier for microorganism. The synergism of microorganisms with biochar is an effective bioaugmentation techonology for the in-situ remediation of heavy metal contamination in soil. Meanwhile, it provided a new concept of the solid waste reuse in agriculture.