A Culture-dependent Strategy To Capture Large Fractions Of Oil-degrading Bacteria

Using information from culture-independent methods to construct highly active consortiums remains a challenge,due to a lack of key players obtained using culture-dependent methods.Here,we employed culture-before-enrich,a strategy to capture large fragments of culturable microbes from oil contaminated seawater to answer this challenge.First,microbes were cultured as subsets for construction of consortiums from the contaminated site.Second,these subsets were stabilized to enhance degradation activity in consortiums,then,consortiums' activity was evaluated.Finally,we isolated microbes from the consortiums to identify their biodegradation function.The main results were as follows:1.To capture large amounts of culturable oil-degraders,oil contaminated seawater was inoculated on nutrient rich agar and the colonies were washed down as microbial subsets.Illumina sequencing of 16S rRNA gene was employed to quantify the diversity ratio between initial samples and washed colonies as culturable rates.The results exhibited that culturable rates reached?32.8%at OTU level and?34.5%at genus level,with these high rates,40 of 45 genera of well-known oil-degraders from long-term contaminated site were cultured,which highlighted the efficiency of capturing key hydrocarbonoclastic bacteria as consortiums.2.The washed subsets were stabilized in seawater to optimize biodegradation activity,then inoculated into oil-amended seawater to obtain stabilization consortiums.16S rRNA gene sequencing was used for profiling communities in initial samples,washed subsets and stabilized subsets.Oil-degraders' relative abundance,community biomass and oil biodegradation rate of consortiums were measured using 16S rRNA sequencing,qPCR of 16S rRNA gene and GC-MS.The results showed that the stabilized subsets and initial seawater communities clustered together,distinct from washed subsets,exhibiting "auto-organization" process,where aquatic ecosystem putative keystone taxa Rhodobacteraceae and Flavobacteriaceae were promoted as fundamental framework of the consortiums,these keystone microbes then formed active degrading unit with putative oil degraders Alcanivoraceae,Erythrobacteraceae,Alteromonadaceae.Oil-degraders' relative abundance to the presence of oil was higher in stabilization consortiums(up to 83%)than that in non-stabilization consortiums(only up to 54%).Oil degradation rates by stabilization consortiums reached?72%,higher than that by the non-stabilization consortiums which only reached-29%.qPCR indicated community biomass of stabilization consortiums were(108)greater one order of magnitude than non-stabilization consortiums(107).These results propose that enhancement of oil attenuation was achieved by the auto-organization process.3.To obtain oil-degrading bacteria and precisely investigate their role in biodegradation process,microbes were isolated from stabilization consortiums,then oil-plate assay,amplification of alkane degrading(alkB)and biosurfactant producing(srfAA)marker genes were performed.In total,high diversity of 88 isolates belonging to the 33 most abundant genera were isolated,isolates belonging to 14 genera possessed the potential to utilize diesel oil as sole carbon source and 5 of these genera were potentially biosurfactant producers.Of these isolates,oil degraders Marinobacter sp.R17(Alteromonadaceae),Altererythrobacter sp.FM1(Erythrobacteraceae)and keystone taxa Litorivita pollutaquae FSX-11T(Rhodobacteraceae)which made up the active degrading unit,were classified as novel species of oil-degrading and biosurfactant-producing bacteria.Further,TLC results indicated that both FM1 and R17 produced surfactin-like biosurfactant.GC-MS results showed that oil degradation rates were?62%and?70%by strain R17 and FM1.This approach offers new insight on capturing large amounts of culturable oil?degrading bacteria for constructing highly active microbial consortiums.