Studies On Formation,characterization And Transcriptome Analysis Of The Viable But Non-culturable State Of Ochrobactrum Sp.FP1 Under Environmental Stress

Petroleum-degrading bacteria could enter the viable but non-culturable（VBNC）state under various harsh environmental stresses,which could affect their growth and oil-degrading efficiency.Ochrobactrum strains are important petroleum-degrading bacteria and exist widely in the oil contaminated environments.Their survival mechanism in the natural environments remain unclear.In this paper,the effect of low temperature,ampicillin and H₂O₂on the growth of Ochrobactrum sp.FP1 cells were studied and the characteristics of VBNC state cells were observed by spectrophotometry,fluorescence staining-laser confocal microscopy,scanning electron microscopy and transmission electron microscopy.The global transcriptional analysis was used to study gene expression and related biochemical metabolism of Ochrobactrum cells under H₂O₂stress.The main results were as follow.The Ochrobactrum sp.FP1 cells with a concentration of 1.27×10⁸cfu/m L entered into VBNC state when maintained at 4℃for 500 days.The average cell length decreased from 1.59±0.05μm to 0.63±0.05μm,and the average cell width decreased from 0.61±0.04μm to 0.60±0.03μm when compared with that of the cells in exponential phase.The cells contracted into spheres,the periplasmic space increased,and the intracellular material accumulated.Ochrobactrum cells in the VBNC state could be resuscitated to culturable state by temperature up-shift and addition of nutrition.Sodium pyruvate could promote resuscitation of bacterial cells in VBNC state.The number of culturable cells in experimental group with the addition of 50.0 mmol/L sodium pyruvate for 36 h incubation was increased to179.28%compared with that of the control group.The resuscitated Ochrobactrum cells maintained their environmental adaptabilities and oil degradation abilities.The cells grew well at temperature 20.0-40.0℃,p H 5-9,and salinitis of 0.0-1.0%.The oil degradation rates of the resuscitated cells and the original Ochrobactrum FP1 cells were 36.09±11.28%and 49.06±14.27%respectively.The growth of Ochrobactrum sp.FP1 could be inhibited by different concentrations of ampicillin.When the bacterial solution containing a cell count of 9.4×10⁷cfu/m L were maintained with 1.0 mg/m L ampicillin,the cells entered the VBNC state after 24h.The average cell length decreased from 1.59±0.05μm to 1.19±0.04μm,and the cell width decreased from 0.61±0.04μm to 0.60±0.02μm.The cells also shrinked into short rods.There was an increase in the periplasmic space.Ochrobactrum cells in the VBNC state could be resuscitated to culturable by removal of the ampicillin in24h.The number of culturable cells in experimental group with the addition of 50.0mmol/L sodium pyruvate was increased to 204.20%of control group in 36 h,suggesting that sodium pyruvate had a facilitating effect on the resuscitation of bacterial cells in VBNC state.The resuscitated Ochrobactrum cells maintained their environmental adaptabilities and oil degradation abilities.The cells grew well at temperature of 20.0-40.0℃,p H 5-9 and salinities of 0.0-1.0%.The oil degradation rates of the resuscitated Ochrobactrum cells and the original Ochrobactrum cells were38.57±7.42%and 50.61±9.58%respectively.Different concentrations of H₂O₂could inhibit the growth of Ochrobactrum sp.FP1.When 75.0 mmol/L H₂O₂was added to the bacterial solution containing 1.21×10⁸cfu/m L of Ochrobactrum cells,the bacteria cells entered the VBNC state after 36 h.The cell length decreased from 1.59±0.05μm to 0.67±0.02μm,the cell width decreased from 0.61±0.04μm to 0.60±0.02μm.The cells contracted into spheres,the periplasmic space increased,and the intracellular material accumulated.The VBNC Ochrobactrum cells could be resuscitated by removal of the H₂O₂after they entered the VBNC state for 1-60 days.Sodium pyruvate had a facilitating effect on the resuscitation of bacterial cells in the late VBNC state.In the resuscitation experiment on the 60th day,the number of culturable cells in the experimental group with 50.0 mmol/L sodium pyruvate added after 12 h of culture increased by 96.21%compared with the non-added group.The resuscitated Ochrobactrum cells grew well at temperature of 20.0-40.0℃,p H 5-9 and salinities of 0.0-1.0%.The oil degradation rates of the resuscitated Ochrobactrum cells and the original Ochrobactrum cells was38.38±0.42%and 40.88±0.30%respectively.The enzymatic activities of catalase,superoxide dismutase and o-phenyl-1,2-dioxygenase were decreased greatly in the VBNC state Ochrobactrum cell.The catalase and superoxide dismutase were derceased in the resuscitated cells.The o-phenyl-1,2-diphenol dioxygenase activities were almost unchanged,which indicated that the recovered Ochrobactrum sp.cells maintained their environmental adaptabilities and oil degradation abilities.Transcriptome analysis indicated that there were 1051 differentially expressed genes of the VBNC Ochrobactrum cells induced by H₂O₂stress.including 575up-regulated genes and 476 down-regulated genes.The GO enrichment analysis showed that a total of 787 differentially expressed genes were associated with three major processes:biological processes,molecular functions and cellular components.The KEGG enrichment analysis revealed that 9 biochemical metabolism and signaling pathways were involved.It was suggested that under H₂O₂stress the Ochrobactrum cells entered the VBNC state for their survival by controlling many biological processes including protein biosynthesis and protein activity of membrane transporter family protein,biosynthesis and repair of DNA and RNA,biosynthesis and repair of protein,cell division,antioxidants,oxidative phosphorylation,citrate cycle and other functional proteins by regulating gene expression,changed nutrient transport,energy production,biomolecule synthesis,metabolite excretion,and antioxidant mechanisms.