Treatment Of Pulp And Paper Effluent By Construction Of Multiple Microorganisms With Inter-kingdom Protoplast Fusion

The pulp and paper effluent is a widespread concern as serious pollution source due to itshigh levels of chemical oxygen demand (COD), low biodegradability and strong toxicity.Biological treatments are considered to be effective method to treat pulp and paper effluent. Ithas the properities of low treatment cost and slight secondary pollution. The effluent gotpurified owing to the function of microbial metabolism which is used to make wastewaterdissolved, degraded into colloidal stable organic pollutants and translated into harmlesssubstance. Recently, researchs mainly focus on the selection and cultivation of singledominant strain which is used to biodegrad effluent. However, reports about inter-kingdomprotoplast fusion are still seldom.In this paper, inter-kingdom protoplast fusion was applied to construct multiplemicroorganisms for improving removal efficiency of pulp and paper effluent. Fusants wereattained via the following three steps. Firstly, the white rot fungi of Psathyrella candolleana,and four bacteria of Bacillus sp., Enterobacter Cloacae, Gordonia sp. and Pseudomonasputida, which were often used in wastewater treatment, were fused, respectively. Secondly,stable engineering strains were screened by antibiotic resistance. Finally, multiplemicroorganisms were constructed by statistical design. The inter-kingdom protoplast fusionprovided new development direction on the treatment of pulp and paper effluent.Protoplasts of Psathyrella candolleana and Enterobacter cloacae were prepared andfused. The optimum protoplast fusion conditions were obtained by Box-Bennken design,response surface analysis and regression analysis. The results showed that the optimumcondition were PEG34.9%,10.4min,36.4℃. The fusants were screened on nystatin andstreptomycin selective medium preliminarily. In comparison with their parental strains andfusants, fusion recombinant PE-9shows higher the lignin-degrading ability which can achieve27.3%after2days incubation.The inter-kingdom protoplast fusion of Psathyrella candolleana and Enterobactercloacae were studied. The laccase enzyme activity of the fusants was measured. The fusantwhich has relatively higher laccase enzyme activity was identified by molecular biology,including total DNA extraction,16srDNA PCR amplification and laccase specific sequence primers screening. By the same method, protoplast fusion of Bacillus sp., Gordonia sp.,Pseudomonas putida and Psathyrella candolleana were discussed respectively. Afterscreening, the morphology of some fusants was observed by scanning electron microscope.The result showed that the total DNA of Psathyrella candolleana, Enterobacter cloacae andtheir fusants had been extracted. It was confirmed by16srDNA PCR amplification that PE-6and PE-9contained the bacterial gene of Enterobacter cloacae. According to functionallaccase gene primers screening, agarose gel electrophoresis of PE-9appeared the bands.However, the specificity of PE-9was not high enough. Further study was needed to identifythe fusant by molecular biology. The laccase enzyme activity of PE-9, PB-3, PG-1and Ppp-3were relative higher. Similar to related research reports, the morphological characteristics ofthe fusants were different from their parental strains.Logistic equation was selected to establish the growth model of PE-3, PE-6, PE-7andPE-9.The model equations of CPE-3(t)=0.0156e0.460t/[1-0.00823(1-e0.460t)],CPE-6(t)=0.0251e0.416t/[1-0.0123(1-e0.416t)],CPE-7(t)=0.0231e0.391t/[1-0.0143(1-e0.391t)] andCPE-9(t)=0.0136e0.505t/[1-0.00695(1-e0.505t)] were obtained, respectively. Through experimentalstudy on the medium of manifold cometabolism nitrogen and carbon sources, the resultshowed that ammonium chloride and sucrose were determined as the suitable nitrogen sourceand carbon source, respectively. The phenomenon of inhibition appeared when the substrateconcentration of lignin reached up to a certain degree. The substrate degradation kinetics ofthem could be expressed by Andrews equation as q(PE-3)=1.61S/(S+48.4+S2/437.2),q(PE-6)=1.80S/(S+51.6+S2/491.1), q(PE-7)=1.42S/(S+41.4+S2/419.0), andq(PE-9)=1.88S/(S+52.8+S2/516.4), respectively. Results showed that the fusant of PE-9had agood potential on research and application about lignin-degrading and treatment of pulp andpaper effluent.In order to enhance the treatment efficiency of pulp and paper effluent, multiplemicroorganisms with9bacterial strains were constructed by statistical design. The statisticaldesign contained Plackett-Burman (PB) design, steepest ascent design, central compositedesign and response surface method. Plackett-Burman design showed that Bacillus sp., PE-9,Pseudomonas putida, Xz6-1were the main affecting strains. The method of steepest ascentwas used to obtain the maximum response region for the central composite design. Finally, the optimal cells concentration (OD600) of each significant factor was determined usingcentral composite design and response surface method analysis. The optimized multiplemicroorganisms contained Bacillus sp.0.35、Enterobacter Cloacae0.2、Gordonia sp.0.3、Pseudomonas putida0.38、PB-30.3、PE-90.41、PG-10.2、Ppp-30.3and Xz6-10.35. Undersuch conditions, the COD of pulp and paper effluent treated by multiple microorganisms wasdecreased by86.9%, which was in good agreement with the predicted one (85.7±0.5)%. Inconclusion, the construction of multiple microorganisms by a statistical experimental des ignhas good reliability.