Construction Of Whole-cell Lipase And Its Application For Oil Waste Water Treatment

oil wastewater with high fat content、COD and BOD caused series of environmental problems, which has been the concern of society and the scientific community.The ways of treatment oil wastewater are mainly physical, chemical and biological at present, Compared with those methods, biological methods are very effective method, To improve the biodegradability, a variety of microorganisms and lipases have been used in oily wastewater treatment processes. These microorganisms and lipases are usually used in the pre-treatment, so that some especial equipment and process must be added, implying excess wastewater pre-treatment process, long wastewater treatment cycle and high cost.In this study, to combine the advantage of the oleaginous yeast Yarrowia lipolytica with the high activity of some fungal lipases for oily wastewater treatment, three effective lipase-displaying arming yeasts were constructed, which are whole-cell lipases.the whole-cell lipases were added into open activated sludge bioreactors directly, they exhibited remarkable oil removal and COD degradation effect compared with the control samples. The experiments and conclusions are as follows:1) Construction of whole-cell lipaseThe fusion gene fragments of FLO-His-LipRS、FLO-His-Y7and FLO-His-Y8were obtained by PCR, then they were cloned into the expression vector pINA1296and pINA1297, the recombinant plasmid was successfully constructed.The recombinant plasmid pINA1297-F-LipY7、 pINA1297-F-LipY8and pINA1296-F-LipRS were linearized with NotI and transformed into Yarrowia lipolytica polg、 polh as described by Madzak et al. Transformants were obtained by MD selective medium plates and incubated at28℃for36～48hours. The lipase display in the yeast cell surface successfully by indirect immunofluorescence assay. The whole-cell lipase activity measured by spectrophotometry were as follows:polg-pINA1296-F-LipRS is293U/dry cell, polh-pINA1297-F-LipY7is340U/g dry cell and polh-pINA1297-F-LipY8is312U/g dry cell. 2) Application of the whole-cell lipase in oily wastewater treatmentCollected activated sludge from Wastewater Treatment Plant of Shahu in Wuhan, Synthetic oily wastewater containing5xlO3mg/L oil was used in each experiment.the cultivation of whole-cell lipase (500mL, about108/mL) are added into the activated sludge reactor. Oil content reduced from5000mg/L to about200~400mg/L after72h, the removal rate91%to96%, the COD was reduced from1.5x103mg/L to300-800mg/L, COD removal efficiency reached92%to97%, The whole-cell lipase exhibited remarkable oil removal and COD degradation effect compared with the control samples, examplifying its application potential.