Screening Of Cd?Pb?Mn-tolerant Strain And Preparation Of Compound Microbial Agents

Heavy metal pollution is a widespread and serious problem,affecting the quality of agricultural products and human health.The traditional methods of heavy metal pollution remediation are physical methods,chemical methods and bioremediation methods etc,among which microbial remediation is caused wide concern as a green,economical and recyclable bioremediation technique.In this study,a strain of Aspergillus aculeatus with the tolance to cadmium,lead and manganese?Aspergillus aculeatus?was successfully screened from a manganese ore area in in Hezhou,Guangxi Province,and the fermentation conditions were optimized to shorten the fermentation time and reduce the fermentation cost.The adsorption characteristics of heavy metals by Aspergillus aculeatus was explored through studying the effect factors of pH,adsorption time,cultured temperature and initial added concentrations of heavy metal ions.Then,several kinds of carriers,i.e.single material,compound materials without microorganisms and compound materials with microorganisms were prepared.The reaction surface method was used to optimize the preparation conditions of the bacteria.The optimum mixture formula and the preparation conditions were determined with the least amount of experiment.The main results are as follows:1.In this experiment,one strain of fungi with strong tolerance to cadmium,lead and manganese was successfully screened from a manganese ore area in Hezhou,Guangxi province.By observing the single colony of the strain,it can be found that the fungus belongs to the fungus.The strain has fast growth,large size and flat surface,short mycelium,dense spores,dark gray black,fluffy,unlike the common mold loose structure,the strain is very tight and is not easy to infiltrate from the PDA solid medium.The homology of the strain has higher homology with the gene of the Aspergillus aculeatus in the NCBI database,which reached100%.There is very nearest relationship bewteetn the experimental strain and Aspergillus aculeatus X1 by the analyzed from the phylogenetic tree constructed by MEGA,and both of them belong the same group.The strain was even could grow under the conditions of Cd²⁺=20mg/L,Pb²⁺=4500mg/L and Mn²⁺=7000mg/L.2.The strains showed different characters under different heavy metal stress.The differences of the results within single colony characterization was mainly in colony volume,sporulation and spores color,among which Cadmiun had the greatest impact on the strain,and the strain decreased sharply and the overall performance was white.The results of SEM-EDS were coincided with the morphological observation of single colony,mainly concerned in the form of spores and the thickness of the stalk.The results of scanning electron microscopy showed that the toxicity of cadmium to the strain was the greatest and followed by lead and manganese.Furthermore,The effect of manganese on the Aspergillus sphaericus was the most obvious,followed by cadmium and lead.The mass percentage and atomic percentage of Cd,Pb and Mn in EDS increased significantly.A single factor experiment was used to optimize the fermentation conditions of Aspergillus spaspora.The results showed that the optimum conditions for the growth of Aspergillus spaspora were as follows:carbon source was glucose,nitrogen source was peptone,the temperature was 28-34 C,the initial pH of culture medium was 5.5-6.5,and the rotational speed of the rocking bed was 120-160 r/min.3.The adsorption isotherm,thermodynamics and kinetics experiments showed that the adsorptions of Aspergillus aculeatus for Pb²⁺and Mn²⁺were very rapid,and the adsorption process could be divided into fast adsorption stage and slow adsorption stage.In the pre-10min,the adsorption capacity of Aspergillus aculeatus at different temperatures could reach more than 80%of the equilibrium adsorption capacity,and in the former 20min,the adsorption amount of lead at different temperatures reached more than 85%of the equilibrium adsorption amount,and the adsorption of Pb²⁺and Mn²⁺within 1h were basically reached balance,and the adsorption process was endothermic reaction.The adsorption equation of both Langmuir and Freundlich can well fit the adsorption of Aspergillus aculeatus on Pb²⁺and Mn²⁺,but the fitting effect of Langmuir is better.This shows that the adsorption of lead and manganese is mainly by monolayer adsorption;the order of four kinds of adsorption kinetics is:Pseudo-second order kinetic model>Particle diffusion model>First-order kinetics model>Elovich model,which indicated that the adsorption process of Aspergillus aculeatus on three kinds of heavy metals were controlled by chemical adsorption,and the limiting factors for adsorption process was the active site of adsorbent surface.The fitting curves of the internal diffusion equation for these three kind heavy metals did not pass the original point,it indicated that the intra particle diffusion was not the sole step to control the reaction speed,and the adsorption rate might be influenced by other factors.The adsorption of lead and manganese by Aspergillus aculeatus may both controlled by surface adsorption and intraparticle diffusion.4.The mixture design was used to screen and compound five carriers of diatomite?A?,rice husk?B?,sawdust?C?,peat ash?D?and straw?E?.The results showed that as the amount of A was 57.7%,B was 19.6%,and C was 22.7%,the spore germination was about1.29 x 10⁸,and the removal rates of Cd,Pb and Mn were 105.6%,62.19%and 41.53%,respectively,which were 1.85,1.06 and 1.64 times as that of pure Aspergillus aculeatus.The polynomial regression model of four response values?living bacteria,cadmium removal rate,lead removal rate and manganese removal rate?were all very significant models?P<0.0001?,The correlation coefficients of R² were more than 0.9 except for cadmium removal rate.5.The effects of various factors on the growth of Aspergillus aculeatus at the composite carrier were preliminarily analyzed by Plackett-Burman design.The most important factors were selected.By the analysis of ANOVA,the order of affected factors for the growth of Aspergillus aculeatus in the composite carrier were water material ratio>fermentation temperature>drying temperature>fermentation time>nutrient solution>drying time>pH>inoculation quantity of water.6.Three important factors were concered,i.e.water material ratio,drying temperature and ferm6entation temperature.Using the way of BOX design method,at the same time using the valid number of bacteria as the response value.It is concluded that these three factors had significantly interaction,and the fitting degree of the regression models were all good?P<0.0001?.As the water material ratio was 2.8,the fermentation temperature was 30?,and the drying temperature was 39.5?,the maximum valid living bacteria number was 8.625×10⁷,which was closed to the actual values?8.5×10⁷?,which were 3.2 and 1.029 times higher than that of the lowest and highest living bacteria under the initial condition.The results showed that the model could well predict the actual preparation of bacteria.7.The adsorptions of Pb²⁺and Mn²⁺by composite adsorbents were very rapid,and the adsorption process was divided into fast adsorption stage and slow adsorption stage too.At the first 60min and 30min adsorption time,the adsorption amount of Pb and Mn reached above84%,at different temperatures,and the adsorption reached equilibration within 2 h and 1 h for the element of Pb and Mn,respectively.The fitting effect of these two adsorption kinetics was Pseudo-second order kinetic model>First-order kinetics model.