| With the massive exploitation and transportation of oil,the problem of oil pollution in the coastal marine environment is becoming more and more serious.The microbial remediation of petroleum pollution has the advantages of energy saving and high efficiency in a sustainable manner,and has been widely concerned by researchers.Petroleum degrading consortia can promote the rapid degradation and complete oxidation of pollutants,which is the current research hotspot of petroleum bioremediation technology.However,most of the consortia are based on the simple combination of several high-efficient oil-degrading bacteria.Due to the lack of an indepth understanding of the internal interaction mechanism of the bacteria,it is difficult to achieve the expected goal in the practical application.At present,it is still a challenge to control the microbial consortia to build a stable and highly active petroleumdegrading consortium.In this report,we takes initial oil-contaminated seawater(INT),samples stabilized with natural seawater(STA),and samples enriched with diesel oil for cultivation(ST_SW)as research objects,and combines with Bottom-Up and Topdown strategies to construct oil-degrading consortia.The main results are as follows:1.To screen the key consortium capable of oil degradation stably from the coastal oil pollution environment,initial seawater(INT)was used as a starting point to investigate the coastal oil pollution situation.Next,more adaptable consortia were obtained after natural seawater stabilization(STA).Then the diesel was added to enrich the bacterial taxa(ST_SW)which are capable of degrading oil and adapting to the marine environment.The high-throughput sequencing technology was used to analyzed the microbial community structure shifts of petroleum-degrading functional consortia in the three different stages,it showed that there were a large number of potential petroleum-degrading microorganisms in the three samples.Potential petroleumdegrading microbial groups detected in INT involved 27 families and 31 genera.The potential taxonomic groups related to petroleum degradation spanned 21 families and 26 genera in STA.And in ST_SW sample,the potential petroleum degradation-related taxa included 26 families and 32 genera.Furthermore,Flavobacteriaceae and Rhodobacteraceae showed abundance advantages and existed stably in three different treatment stages,indicated that they may play key roles in the process of petroleum degradation and have strong adaptability to the environment.2.To better understand the interactions of the petroleum degrading bacteriaat community level in different stages.Based on the random matrix theory the pMENs network was constructed and the results showed that the keystone taxa for INT are Oceanospirillaceae,Alteromonadaceae,Rhodobacteraceae and Peptostreptococcaceae.Corynebacteriaceae,Oceanospirillaceae,Camobacteriaceae,Hyphomicrob iac eae,Rhodobacteraceae,Caulobacteraceae were identified as keystone taxa in STA.And in the ST_SW sample,Flavobacteriaceae,Rhodospirillaceae,Rhodobacteraceae,Rhizobiaceae,Rhodobiaceae,Erythrobacteraceae,Oceanospirillaceae were detected as keystone taxa.Interestingly,Oceanospirillaceae and Rhodobacteraceae are identified as keystone taxa at different stages and are highly connected to Flavobacteriaceae.And under the pressure of oil enrichment,there are obvious positive and negative correlation modules.The positive correlation module groups are Flavobacteriaceae,Rhodospirillaceae,Erythrobacteraceae,and the positively connected groups are Alcanivoracaceae,Alteromonadaceae and Pseudomonadaceae.3.A total of 52 bacterial isolated from the diesel enrichment system using culturedependent techniques and their taxonomic information was determined.Then the oil plate,plate sublimation and functional gene qualitative methods were used to study their functional characteristics.The results show that 43 strains can degrade petroleum,17 strains can degrade long-chain alkanes,and 10 strains have the potential to produce surfactants.In addition,the strains FM1,S-2.2,Rh-3,FSX-8 and R17 belong to Erythrobacteraceae,Alcanivoracaceae,Rhodospirillaceae,Flavobacteriaceae and Alteromonadaceae,respectively,which are keystone taxa in network analysis and they had co-metabolism in the process of degrading petroleum.Analysis of the whole genomic sequencing and metabolic pathways reconstruction showed that strains FM1,S-2.2,Rh-3,FXS-8,R17 both have complete alkane metabolism pathway,and the degradation rate of 0.5%(V/V)diesel were 26.7%,52%,23.6%,19%,25.4%within 5 days,respectively.Among them,Rh-3 degrades alkanes below C16.FM1 can metabolize alkanes below C28.Strains S-2.2 and R17 can degrade long-chain alkanes above C28.R17 can produce biofilms.Strain FSX-8 has a complete rhamnolipid production pathway,and has strong cell surface hydrophobicity.This shows that they may have interactions in the process of degrading petroleum,which is of great significance for the construction of the oil-degrading consortia.4.The above 5 strains were used to construct oil-degrading consortia based on their complementary characteristics.The results of biomass measurments under diesel pressure show that the consortia grow faster than the single isolate,and the consortia F8:R17,F8:F1:S2,F8:F1:S2:R3 and F8:F1:S2:R3:R17 grew more quickly.It shows that the consortia can shorten the start-up process of degradation.The GC-MS results showed that the degradation rate of consortia was higher than that of single isolate.Consortia of F8:R17 and F8:F1:S2:R3:R17 had the highest degradation rate after 5 days,which were 84.4%and 76.1%,respectively.,indicated that the consortia have a synergistic effect,which can accelerate the degradation efficiency of diesel Combining with the GC-MS degradation spectrum,the main degradation components of strains FM1,S-2.2,Rh-3,FXS-8,and R17 are C8-C18,C8-C16,C8-C24,C8-C14,and C8C20 within 5 days,respectively.The main degradation components of the consortia F8:R17 and F8F1:S2:R3:R17 are C8-C28,indicated that the consortia increase the alkane degradation range. |
PDF Full Text Request