Study On Tolerance Mechanisms Of S. Cerevisiae And P. Stipitis Toward Cd²⁺ And Its Application In Ethanol Fermentation

In recent years, heavy metals contamination of cultivated land is becoming increasingly serious. How to manage the cultivated land and deal with the biomass contaminated by heavy metals has become the main task of environmental management. The root and stalk of plants have strong abilities in absorption of heavy metals. After physical and chemical pretreatments of lignocellulosic biomass, heavy metals can be removed from food chain through microorganism fermentation, which can convert the biomass into biofuels and biomaterials. This way can not only convert wastes into available materials and reduce the air and soil pollutions, but also can solve the energy problem. Hence, it can be considered as a very potential method for disposal of lignocellulosic biomass. At present, however, the disposal of straw is mainly focused on the bioconversion of uncontaminated straw. To investigate the application of the bioconversion to soil remediation, it is neccessary to study the effect of pretreatment and saccharification on the release of heavy metals, the effect of heavy metals on the growth of microorganism and efficiency of fermentation, and the removal of heavy metals. At the basis of the background, S. cerevisiae, P. stipitis and anti-cadmium engineered yeast were taken as the objects for studying the effect of cadmium(Cd²⁺) on yeast growth and ethanol fermentation to provide a theoretical basis for the application of straw biotransformation to soil remediation.Firstly, the relationship between Cd²⁺ and glutathione（GSH） synthesis was evaluated, finding the positive correlation between Cd²⁺ and GSH synthesis. GSH synthesis could be enhanced by increasing the amount of yeast extract（YE）, resulting in the increase in tolerance of Saccharomyces cerevisiae toward Cd²⁺. It was found that corn steep liquor（CSL） and YE played different roles in GSH accumulation in cell even though both of them could alleviate the inhibition by Cd²⁺. Intracellular GSH decreased with increasing calcium(Ca²⁺) concentrations, and high Ca²⁺ concentrations rendered the yeast more tolerant to Cd²⁺ stress than the nitrogen sources did. When the mole ratio of Ca²⁺ to Cd²⁺ was 100:1, S. cerevisiae tolerated 1,000 μmol/L Cd²⁺ with no decrease in efficiency in ethanol production. As a result, the use of Ca²⁺ allowed a significant saving of high-cost nutrient YE with an efficient ethanol production.Next, the effects of glucose concentration on the tolerance of S. cerevisiae toward Cd²⁺ and on ethanol fermentation were investigated. The presence of Cd²⁺ inhibited cell growth and ethanol fermentation under any conditions, especially in the cases of high concentration of glucose. Due to Cd²⁺ stress, the inhibitory effect of initial concentration of glucose on ethanol fermentation became more serious so that high concentration of ethanol could not be obtained by increasing glucose concentration. To obtain higher concentration of ethanol, the effect of Ca²⁺ on the tolerance of S. cerevisiae toward Cd²⁺ with different concentration of glucose was investigated. Ca²⁺ could alleviate Cd²⁺ toxicity and improve efficiency in ethanol production even at high glucose concentration. The assays of Cd²⁺ and GSH in broth and cell demonstrated that due to the addition of Ca²⁺ the concentrations of Cd²⁺ and GSH in cell markedly dereased. Taken together, detoxification of Cd²⁺ in S. cerevisiae might be as following: without Ca²⁺, GSH is synthetized to chelated with Cd²⁺, resulting in decrease in toxicity of Cd²⁺; when Ca²⁺ is present, the uptake of Cd²⁺ would be lower due to the transport competition between Cd²⁺ and Ca²⁺.To discuss the feasibility of conversion of hemicelluloses in contaminated biomass into ethanol, the effect of Cd²⁺ on ethanol fermentation by P. stipitis was investigated. As compared to S. cerevisiae, Cd²⁺ had a stronger inhibitory effect on the P. stipites: inhibitory effect on xylose metabolism was greater than the inhibition of glucose metabolism. When Cd²⁺ was present, xylose reductase（XR） and xylitol dehydrogenase（XDH） activities decreased in different degrees. Similar to S. cerevisiae, Ca²⁺ can improve the tolerance of P. stipites toward Cd²⁺ and efficiency in ethanol fermentation. When the mole ratio of Ca²⁺ to Cd²⁺ was 100:1, the growth of P. stipitis and efficiency of ethanol fermentation significantly increased, i.e., glucose and xylose were completely consumed and the maximum concentration of ethanol was 1.15 times higher than that without Ca²⁺ addition. However, the maximum concentration of ethanol was only 63% of that obtained in the control fermentation(without Cd²⁺). The assay of Cd²⁺ in cell indicated that the addition of Ca²⁺ could not completely limit the absorbtion of Cd²⁺ by P. stipitis. On the other hand, even though manganese(Mn²⁺) had much lower effect on the tolerance of P. stipites toward Cd²⁺ than Ca²⁺, The co-use of Ca²⁺ and Mn²⁺ significantly increased the efficiency in ethanol fermentation, i.e., there were little decreases in the cousumption rates of glucose and xylose and the maximum concentration of ethanol relative to those obtained in the the control fermentation(without Cd²⁺). After the assay of intracellular Cd²⁺, it showed that Cd²⁺ concentration in the cells was maintained at very low levels when Ca²⁺ and Mn²⁺ were added at the same time.The mechanism of this phenomenon remains to be further studied.Lastly, the application of DvCRP1 gene to ethanol fermentation was investigated. DvCRP1 gene is a Cd²⁺-resistance gene that induces Cd²⁺ accumulation in microbial and plant cells. S. cerevisiae was used as the model system. Through response surface method combining factor analysis and regression analysis, the effects of DvCRP1 on both Cd²⁺ detoxification and ethanol production were investigated using OD600 value, the concentrations of Cd²⁺, GSH, ethanol and glucose as the factors. Without the transformation of DvCRP1, Cd²⁺ had significant inhibitory effect on ethanol fermentation, i.e., glucose consumption and ethanol production decreased 23%-89%and 17%-92%, respectively. On the other hand, the transformation of DvCRP1 led to the decreases of 9%-65%for glucose consumption and 5%-64% for ethanol production. The presence of DvCRP1 could reduce the inhibitory effect of Cd²⁺ effectively and the mitigation effect was more obvious at higher Cd²⁺ concentrations and lower initial OD600. DvCRP1 played a more important role than initial OD600. The Cd²⁺ detoxification of yeast only improved 50 μmol/L by increasing the initial OD600 from 0.02 to 0.2, whereas the Cd²⁺ detoxification of yeast with DvCRP1 could be double from 150 μmol/L to 300 μmol/L Cd²⁺...