海带表面降油细菌的分离鉴定及降油性能研究

用柴油为唯一碳源的培养基选择分离海带表面具有降解柴油性能的细菌;对分离菌株进行了形态学、生理生化特性以及16SrDNA序列分析并测定其生长特性;测定了接菌不同浓度(7×107 cfu/mL、7×108cfu/mL、7×109 cfu/mL)分离菌对柴油的降解率;另外,测定了加入不同浓度葡萄糖(0.5g/L、1g/L、2g/L、4g/L、6g/L、8g/L、10g/L)作用7d后分离菌对柴油的降解率。结果显示:从海带表面分离到2株降油细菌,编号为:HD-4和HD-6。HD-4菌株菌落形态圆形、直径1.5mm、黄色、不透明、边缘整齐,HD-6菌株菌落圆形,直径1.0mm,浅黄色、透明、边缘整齐。两株菌均为革兰氏阴性短杆菌。生理生化特征和16SrDNA序列分析确定HD-4菌株为假交替单胞菌(Pseudoalteromonas sp.),其16SrDNA同源性为99%;HD-6菌株为交替单胞菌(Alteromonas_sp.),其16SrDNA同源性为98%。2株菌最适生长温度均为15℃。HD-4和HD-6最适生长pH分别为9和8。适宜NaCl浓度分别为2%和4%;在25℃振荡培养(150r/min)7d,接菌量为7×107 cfu/mL、7×108 cfu/mL、7×109 cfu/mL时,HD-4对柴油的初始降解率分别为8.0%,22.1%和27.6%;HD-6初始降解率分别为23.7%、38.8%和43.2%。加入葡萄糖后2株菌的降油率均有所增高,加入4g/L葡萄糖时达最高值,3个接种浓度下HD-4菌株分别为85.4%、82.3%和80.4%,HD-6菌株分别为86.8%、93.7%和89.3%。当接菌量为7×107 cfu/mL,HD-4在葡萄糖含量为4g/L时作用7d,降油率可达到最大值86.72%,HD-6菌株在葡萄糖浓度6g/L达到最大值67.64%。葡萄糖浓度超过4g/L和6g/L时HD-4和HD-6菌株的降油性能有所下降。

Isolation and Degradation Characteristic of Oil Degrading Bacteria f rom kelp

T he experiment isolated strains from the surface of the kelp that grew in intertidal of dalian HeiShiJiao ,And screening two strains named HD -4 and HD -6 which could grow in medium that diesel oil was the sole carbon source .Cellular morphological observations ,biochemical reactions and molecular identifications of 16S rDNA were used to identify strain HD -4、HD -6 ,and the grow th characteristics of HD -4、HD -6 were also studied .Deter mination the initial degradation rate of diesel with different inoculation quantity ,meanwhile the oil -degrading efficiency was also studied when dif-ferent concentrations of glucose was added .The result shows ：HD -4 was yellow ,opaque ,edge neat ,the diameter is 1 .5 mm ;HD -6 was light yellow ,transparent ,regular edge and its diameter is 1 .0 mm ,the above two strains of bacteria were gram -negative bacteria .The sequences of 16SrDNA indicate that HD -4 was related to Pseudoalteromonas sp .E407-2 ,and the homology was 99% ;HD-6 was closely related to A lteromonas sp .HB1 . ,the homology was 98% .The growing characteris-tics of these two strains showed that the optimum growth temperature 、pH and the adaptive of NaCl concentration for HD -4 was 15 ℃ 、pH 9 and 2% ,while HD -6 was 15 ℃ 、pH 8 and 4% respectively . Ultraviolet spectrophotometermeasured HD -4 shows that the initial degradation rate of diesel oil were 8 .0% ,22 .1% and 27 .6% ,in the inoculation quantity of 7 x 107 cfu/mL ,and 7 x 108 cfu/mL ,7 x 109 cfu/mL ,cultured 7 days in constant temperature ,the degradationrate of diesel oilincreased signif-icantly after adding 4 g/L glucose ,reach to 85 .4% ,82 .3% and 80 .4% ,respectively ;In the same situ-ation ,the initial degradation rate of diesel oil of HD -6 were23 .7% ,38 .8% and 43 .2% ,after joining the 4 g/L glucose ,reach to 86 .8% ,93 .7% and 89 .3% ,respectively .Meanwhlie ,in the inoculation quantity of 7 x 107 cell/mL ,adding different quantity of glucose for 0 .5 g/L、1 g/L、2 g/L、4 g/L、6 g/L、8 g/L、10 g/L ,cultured 3 days in constant temperature ,the degrading efficiency of strain HD -4 reach the best w hen adding glucose to a specific concentration of 4 g/L ,about 86 .72% ;and the most efficient concentration of HD -6 is 6 g/L ,reach to 67 .64% .As more glucose was added ,the degrada-tions of these two bacterium were efficiency dropped .The experiments indicated that adding glucose in a appropriate level ,both of them can obviously promote the degradation rate of diesel oil ,but as more glucose was added ,the degradation efficiency dropped .In this paper ,use bacterium isolated from kelp ,provides a theoretical basis and experimental basis by using bacterium to remediate petroleum conta mination .