The Fermentation Optimization Of Alcaligenes Sp.DN25 Based On The Cost Of Cyanide Biodegradation

A strain DN25 from Alcaligenes sp.,preserved in the lab,shows a high cyanide-degrading activity and thus has a potential of industrial application.Among the cost of treatment which as one of the industrialized indicators,the medium cost occupies mostly in the cells culture.From the perspectives of the cost control in this work,optimization of fermentation medium for Alcaligenes sp DN25 and determination the expression of Cyanide-degradation gene owing to amino acids by constructing RT-PCR system will provide us the base knowledge into industrialized application of DN25.The way using real-time quantitative polymerase chain reaction for detecting the expression level of the cyanide-degrading genes of Alcaligenes sp.DN25 will avoid the light detriment from national standard.The standard DNAs of cdE and house-keeping gene 16S rRNA were established the standard curve with gradient dilution,whose the correlation coeff-icient were 0.9985 and 0.9991 and the amplification efficiency were 1.04 and 0.97 respectively.In this work,culture medium optimization of DN25 was focused on cheap and rich raw materials and then its addictive,replacing glucose,yeast and peptone.In addition,metal irons were added into the medium to investigate the effect on the biomass and activity.The compositions of optimized medium(g/l):corn liqour 10 g,NH4Cl 5 g,NaCl 0.5 g,K2HPO4 2 g,pH=8.0.Compared to minimal medium,the cost reduced 66.5%,and the biomass and activity of optimized medium increased 29.0%and 29.3%respectively.In addition,the incubation time was lessened:the time reached to stationary reduced 4 h and the growth rate increased 2.1 times.Methionine,phenylalanine,alanine,cysteine,histidine,lysine and glutamic were added into the medium to examine which influence on the growth and activity of Alcaligenes sp.DN25.It found that,the consequences were different from each other.Lonely,phenylalanine,methionine,alanine,histidine and cysteine could promote the biomass,respectively increasing 5.5?4.6?3.2?2.0 and 1.3 times.The results analysis of activity presented that although histidine and alanine had no significant differences compared with the control group,the medium added cysteine,methionine and phenylalanine were notably inhibited,respectively reducing 63%?61%and 82%.The result of expression quantity roughly matched the activity,which proved that the real-time PCR was suitable for detecting the expression level of the cyanide-degrading genes of DN25,providing some experimental methods for the future study of industrialized application.In other word,corn liqour was used as nutrition of Alcaligenes sp.DN25,which increasing the biomass and activity while decreasing the medium cost.The result of real-time PCR proved that amino acids could not stimulate the secretion and expression of cyanide enzyme,yet promote the growth of Alcaligenes sp.DN25.