Construction Of A Novel Two-specie Whole-cell Immobilization System By Marine-derived Fungi And Its Application

It is noteworthy that mycelia pellet possesses a unique structural characteristic, poly-porous network and high surface area, which has been noticeably exploited as a new kind of biological carrier. Using the mycelium pellet as a biological carrier in the immobilized microorganisms, the resulting immobilized pellet has been formed by adopting the "mixed bacteria self-immobilization technology", which establishes a micro-ecology environment made up of mixed bacteria group in a small community, in order that letting all kinds of immobilized microorganisms play their roles together. This sort of immobilized pellet has greatly expanded its appliance in the wastewater treatment. In our research, two strains of marine-derived fungi, isolated by ourselves, which can secrete lignocelluloses, were utilized to construct a novel two-specie whole-cell immobilization system, then applied this system into wastewater treatment.Firstly, we isolated and screened25strains of fungi which are of the capability of degradation of lignin, and8strains of fungi capable to degrade cellulose from sediment, sea-grass and sea-mud samples collected from East China Sea. After further round of screen, a pure culture which secreted higher laccase and another pure culture which produced higher cellulase were picked out. Through the identification of colony morphology and rDNA-ITS, the strain which secreted higher laccase was identified as Pestalotiopsis sp. and named it J63; another strain produced higher cellulase was identified as Penieillium janthinellum, named it P1.Secondly, to the best of our knowledge production of laccase by Pestalotiopsis sp. was rarely documented. So, this work primarily investigated comprehensively the fermentation properties of marine-derived fungus Pestalotiopsis sp. J63, mainly focusing on the effect of production of laccase by the components of medium (including carbon source, nitrogen source, Cu2+concentration, different salinities etc.), cultivation condition (including temperature, initial pH) and extra-added inducers. Results showed that under the optimum fermentation condition the capability of producing laccase can reach at5719.7U/L, which is superior to other fungi’s capability. Additionally, strain J63is a halotolerant microorganism. Therefore, strain J63is considered as a pretty competitive strain to produce a large amount of laccase.Thirdly, in order to fully explore the waste resources, seven agricultural wastes, containing rice straw powder, wheat bran, bean-pod powder, sugarcane bagasse, corn cob powder, water hyacinth and the peel of pomelo, were picked out as substrate to produce laccase under solid-state fermentation. Among them, Water hyacinth is viewed as one of the most harmful grasses in the world. So, there is a far-reaching influence if it can turn wastes into wealth. Results demonstrated that the potential of utilizing agricultural wastes to produce laccase is fairly tremendous, in which the capability of production of laccase by utilization of rice straw powder excelled others, reaching10700U/g. The runner-up is water hyacinth,7593.3U/g. These achievements met the goal of production of laccase in a manner of environmental-friendly means.Fourthly, through UV-mutagenesis and ion implantation mutagenesis for mutation breeding, two of positive mutant strains, A6and B21, were screened after two rounds of screening procedures. Their laccase ability increased38.4%and35.5%in comparison with parent strain J63in the5th day of fermentation respectively. Furthermore, strain A6improved notably either in thermostability or in halotolerancy, whose laccase enzyme activity is5-6fold as high as its parent strain J63. This is very beneficial to apply in practical industrial sectors.Fifthly, marine-derived fungus Penicillium janthinellum P1can form mycelia pellets in a specific liquid culture. Mycelia pellet has been exploited as biological carrier for whole-cell immobilization due to its unique structural characteristic---polyporous and high specific area. This study constructed a novel two-specie whole-cell immobilization system which using P1mycelia pellets as carrier, which was achieved simply by inoculating the marine-derived fungus Pestalotiopsis sp. J63spores into culture medium containing another fungus Penicillium janthinellum P1pre-grown mycelia pellets. This so-constructed immobilization system possesses the ability not only to degrade lignin but also to degrade cellulose. Optimum conditions of immobilized procedure for maximum biodegradation capacity were determined using orthogonal design at biomass of P1pellets,10g (wet weight); concentration of J63spore, 2×109/mL; and immobilization time,2d.Sixthly, our work investigated the effect of biological wastewater treatment by utilization of immobilized pellets, mainly concentrating on different concentrations of extra carbon source and nitrogen source, different dosages of immobilized pellets, temperature and initial pH. Results demonstrated that immobilized pellets can be fairly effective to biodegrade paper mill effluent in a ten-hour batch run. Biodegradation capacity exceeded99%. Furthermore, biodegradation capacity still retained96.4%after6batches of consecutive treatment. This exhibited that immobilized pellets are capable to maintain very active for dealing with effluent efficiently and persistently for a longer time. During wastewater treatment, re-growth phenomenon of immobilized pellets occurred. The average diameter of immobilized pellets treated by6batches of semi-successive effluent treatment, was3.7mm, which is1.6fold as large as those untreated immobilized pellets. After treatment, the wastewater, originally feculent, turned out to be clarified and odourless, without visible particles.At last, immobilized pellets and P1mycelia pellets were applied to treat simulated dye solution Azure B respectively. It was revealed that both P1mycelia pellets and immobilized pellets can effectively decolorize Azure B. However, the decolorization capability of immobilized pellets improved notably, by26%, compared with mycelia pellets during a10-day treatment. This further confirmed that this co-immobilization system was more advantageous than single mycelia pellets in industrial application.