Isolation And Phylogenetic Studies Of Viable But Non-Culturable Bacteria From Sewage Treatment Bioreactor Based On Resuscitation Promoting Factor

The cultivated bacteria which are separated from the environment by using traditional pure cultivating technology can only occupy0.01-10percent of all, most bacteria are in viable but non-culturable (VBNC) state. Cells of VBNC bacteria are intact and alive and can resuscitate when surrounding conditions are more favorable. In recent years, much more attention have been paid to the studies on VBNC bacteria. To investigate the composition and phylogenetic relationship of viable but non-culturable bacteria isolated from sewage treatment bioreactor. We used Rpf (resuscitation promoting factor) protein signal molecule which promoted the resuscitation and growth of VBNC bacteria, combined with most probable number (MPN) method and estimated the total number of bacteria of samples based on MPN culture system, used dilution plating procedure and plate separation method to isolate the VBNC bacteria from the biomembrane of a continuous flow soil filter bioreactor and analyzed16S rRNA gene phylogenetic relationships. Applied PCR-DGGE to verify the effect of Rpf and existence of VBNC bacteria in the bioreactor. The main results are summarized as follows:1. In MPN culture system of printing and dyeing wastewater, the amount of culturable bacteria of treatment group (9.3×106cell/g-2.4×108cell/g) were higher than the control group’s (4.3×106cell/g-9.3×106cell/g) which indicated that the total number of culturable bacteria increased from7.5×106cell to2.4×108cell per g of sample by adding Rpf. The effect value VR of Rpf were2.2-32verified that there were VBNC advantage flora that sensitive to Rpf in the dyeing and printing wastewater bioreactor. The OD660of treatment group was higher than control group’s in each dilution of MPN culture system. Which illustrated that Rpf could promote the resuscitation and growth of bacteria.16S rRNA gene sequencing and phylogenetic relationship analysis showed that the major culturable VBNC bacterial strains belonged to the genera: Gordonia, Staphylococcus, Tepidimonas, Ochrobactrum, Moraxella, Enhydrobacter and Cupriavidus. The strains belonged to the genera:Bacillus, Stenotrophomonas and Lysinibacillus showed that the Rpf had stimulating effect on the growth of bacteria. The isolates were with the similarities of96.6-100.0%to those known type species. There were3strains YDWLR2, YDWLR6and YRJ6could be potential novel species from the preliminary analysis of phylogenetic tree.2. In MPN culture system of pharmaceutical wastewater, the amount of culturable bacteria of treatment group (2.4×10-7cell/g-2.4×108cell/g) were higher than the control group’s (1.5×106cell/g-1.1x108cell/g) which indicated that the total number of culturable bacteria increased from4.3×10cell to2.4×10cell per g of sample by adding Rpf. The effect value VR of Rpf were2.2-55.8verified that there were VBNC advantage flora that sensitive to Rpf in the pharmaceutical wastewater bioreactor. The OD66o of treatment group was higher than control group’s in each dilution of MPN culture system. Which illustrated that Rpf could promote the resuscitation and growth of bacteria.16S rRNA gene sequencing and phylogenetic relationship analysis showed that the major culturable VBNC bacterial strains belonged to the genera: Candidimonas, Microbacterium, Gordonia, Xanthobacter, Aminobacter and Leucobacter. The strains belonged to the genera:Comamonas, Alcaligenes and Acidovoraxand showed that the Rpf had stimulating effect on the growth of bacteria. The isolates were with the similarities of96.6-100.0%to known type species. There were4strains ZYM1, ZYM3, ZYZR4and ZYXR1could be potential novel species from the preliminary analysis of phylogenetic tree.3. DGGE samples were collected from the MPN culture system of printing and dyeing wastewater bioreactor ran366days. From DGGE profile analysis, it is noteworthy that more bands were observed in treatment group and Shannon-Weaver diversity index of treatment group also higher than control group’s, indicated that phylotype richness of treatment group was higher than the control group’s obviously and verified that Rpf could stimulate the growth of Rpf bacteria. Combined with the actual isolates and the analysis of nucleotide sequences of major bands in the gel, we believed that strains YRJ1and YRJ6were originally in VBNC state and recovered to be cultivated by the induction of Rpf. They were VBNC bacteria in printing and dyeing wastewater.This research revealed that by adding Rpf we could isolate VBNC bacteria from sewage treatment bioreactor effectively. The culturable VBNC bacteria including actinomycetes, gram positive and negative bacteria. These results can provide important ideas and methods of further study on VBNC bacteria in sewage treatment bioreactor, especially the present state, composition, formation mechanism and recovery mechanism of VBNC bacteria and the advanced degradation process improvemen of printing and dyeing wastewater and pharmaceutical wastewater.