Reconstruction and evaluation of oil-degrading consortia isolated from sediments of hydrothermal vents in the South Mid-Atlantic Ridge

Biodegradability of crude oil by two enrichment cultures

The enrichment culture H7S showed no obvious proliferation in the first five days because sample 7S was a sulphide rock, while H11S showed visible proliferation after the fourth day. After 14 days of cultivation, gravimetric analysis demonstrated that the enrichment cultures H7S and H11S exhibited similar oil-degrading abilities and degraded 54% and 56% of the crude oil, respectively (Fig. 1).

The biodegradation percentages for total n-alkanes (C10–C34) and polycyclic aromatic hydrocarbons (PAHs) were calculated by comparing the two enrichment cultures with the negative controls (Fig. 2). Based on evaluation with C17/pristane and C18/phytane, the degradation efficiencies of the two enrichment cultures were significantly better than those of the negative controls (P < 0.01)²⁵. In detail, the enrichment cultures H7S and H11S exhibited significant n-alkane degradation abilities, degrading 80% and 95% of the n-alkane components respectively. In addition, the enrichment culture H11S also exhibited a superior aromatic compound degradation efficiency (88%) which was much higher than that of the enrichment culture H7S (10%). Clearly, the efficiency of n-alkane removal was higher than that for PAHs, suggesting that the enrichment culture H7S was more inclined to biodegrade n-alkanes than PAHs.

GC–MS analysis confirmed that each enrichment culture was able to use multiple oil components compared with the control group (Fig. 3a). The enrichment culture H7S was more active in degrading short-chain n-alkanes (C10–C19) with the degradation efficiency exceeding 80% and even reaching 93%. However, the ability to degrade long-chain alkanes was unsatisfactory, and the highest degradation efficiency was only 17%. In contrast, the enrichment culture H11S featured a broader degradation range and superior degradation capability. Degradation percentages exceeded 84% even reaching 97% for the n-alkanes within the entire range (C10-C34). It is worth noting that a particularly favourable degradation effect was observed for long-chain (C31–C34) n-alkanes with degradation efficiencies of approximately 91%, 95%, 92% and 92%, respectively. Interestingly, the biomarkers pristane (PR) and phytane (PH) were degraded by the enrichment culture H11S at high degradability levels of 90% and 91% respectively. PR and PH are commonly used to evaluate the biodegradability of oil or determine the source of an oil spill because they are difficult to degrade compared with n-alkanes²⁶. The removal efficiencies for PR and PH by a previously reported bacterial consortium were only 40.1% and 39.5% respectively²⁷. However, our results showing high biodegradability of PR and PH indicated that the enrichment culture H11S offered a superior potential for alkane degradation.

The residual concentration of PAHs in crude oil is shown in Fig. 3b. The enrichment culture H11S played a more active role in the biodegradation of alkylated PAHs (72%-92%) than did the enrichment culture H7S (less than 20%). In particular, the enrichment cultures H7S did not effectively degrade low-molecular-weight polycyclic aromatic hydrocarbons. However, it had excellent degradation effects on high-molecular-weight polycyclic aromatic hydrocarbons, such as alkylated dibenzothiophene (DBT) and alkylated chrysene (CHR). These degradation trends also indicate that increasing the ring number of PAHs, the enrichment culture exerts a positive effect on the biodegradation of the compounds²⁷.

Biodiversity and community structure of two enrichment cultures

QIIME software (version 1.9.1) was used to compare and analyse the alpha diversity of the two enrichment cultures. A total of 81,780 and 90,755 reads were obtained via Illumina MiSeq sequencing of the 16S rRNA gene sequences of the two enrichment cultures H7S and H11S, respectively. At least 97.4% of effective readings were obtained after screening, and the optimized read data showed corresponding lengths of 420 bp and 407 bp, respectively.

The rank abundance curve (Online Resource Fig. S1) indicated that the species richness of the enrichment culture H7S was significantly higher than that of the enrichment culture H11S. In the vertical direction, the smoothness of the curve for the enrichment culture H7S reflected that the species distribution was more uniform. According to statistics, 112 OTUs were gathered from two consortia, and the OTU content of the enrichment culture H7S was much higher than that of the enrichment culture H11S. The Venn diagram of two enrichment cultures H7S and H11S showed that 35 OTUs were shared and exhibited 31% similarity via the RDP Classifier (Online Resource Fig. S2). In addition, the community richness (ACE and Chao) and diversity (Shannon and Simpson) indices of the enrichment culture H7S were higher than those of H11S. The comparison of the Shannon diversity index showed that the microbial community of the enrichment culture H7S was composed of a greater number of phylotypes. The Simpson index also indicated that the favourable characteristics of the enrichment culture H7S were more conspicuous.

In our study, the microbial OTUs of the two enrichment cultures were identified at different levels via phylogenetic analysis and taxonomic distribution. At the phylum and class levels (Fig. 4a,b), the top 10 most abundant species in the two enrichment cultures are listed as a cylindrical accumulation chart. Proteobacteria is the principal group in environments contaminated by hydrocarbons^28,29, and it also exhibited the highest richness in the two enrichment cultures: 99% and 98% for H7S and H11S, respectively. The abundances of Actinobacteria, Firmicutes, and Bacteroidetes was very low in both enrichment cultures. These phyla have been found to be involved in hydrocarbon biodegradation^30,31,32. Actinobacteria not only constitute one of the most important phylogenetic groups in marine ecosystems but also play a leading role in anaerobic TPH degradation-related bioremediation^30,31. The global biogeography and ecological preferences of Oxyphotobacteria, another common phylum represented by a very low proportion, remain particularly undetected. However, regional studies have shown that Oxyphotobacteria can tolerate high temperature, water stress and ultraviolet radiation³³. The functioning of Oxyphotobacteria needs further study. As shown in Fig. 4b, Gammaproteobacteria was the major class of Proteobacteria in the H7S and H11S enrichment cultures, accounting for 90.9% and 95.6% of the total OTUs respectively, followed by Alphaproteobacteria (4.3% and 8.0%). Some classes belonging to the phylum Actinobacteria existed only in the enrichment culture H7S, including Acidobacteria and Deltaproteobacteria. On the whole, the two enrichment cultures were similar in community structure at the phylum and class levels.

At the genus level, significant differences between the OTUs of the two enrichment cultures were observed (Fig. 4c). The top 10 genera in each enrichment culture are listed in Fig. 4c. The genera with relatively low abundance were merged into other genera. The enrichment culture H7S was mainly composed of Pseudomonas (86.5%), followed by Nitratireductor (7.8%) and Acinetobacter (4.3%). The enrichment culture H11S was dominated by Acinetobacter, whose richness exceeded 95.4%, followed by Nitratireductor, Brevundimonas, and Pseudomonas, and these results were consistent with the proportion at the phylum and class levels. Acinetobacter has the potential to degrade NAP and PHE³⁴. Nitratireductor shows an excellent oil removal capacity³⁵. Brevundimonas plays a key role in the in situ bioremediation of oil spills¹⁸. Pseudomonas is commonly used as a model organism for pollutant degradation³⁶ because it mediates the degradation of certain organic compounds and anthropogenic contaminants^37,38. The abundance ratio of the dominant genera Acinetobacter : Nitratireductor : Brevundimonas : Pseudomonas was 95:4:0.2:0.2. In Fig. 4c, it is shows that the two enrichment cultures contained small components of Pseudonocardia, Paracoccus (able to degrade phenanthrene and fluoropolycyclic aromatics in liquid medium³⁹) and a biosurfactant-producing bacterium: Lactobacillus⁴⁰. Some genera, including Microbacterium and Stenotrophomonas which are typical oil-degrading bacteria⁴¹ and efficient biosurfactant-producing bacteria used for the biodegradation of diesel and engine oil⁴², respectively, only existed in the enrichment culture H7S.

According to the abundance information of all samples, the top 35 genera were selected by cluster analysis and displayed in the heat map (Online Resource Fig. S3). The dominant genera of the enrichment cultures H7S and H11S were obviously different. The dominant genera of H11S were Acinetobacter, Brevundimonas, Paracoccus and an unidentified Clostridiales. The species clustering results showed that the species of H11S were close to each other. However, the dominant genus was Pseudomonas in H7S and the microbial diversity of H7S was higher than that of H11S.

Isolation and identification of strains

The enrichment culture H11S was obtained via approximately two months of enrichment. Six phylogenetically different strains summarized in Table1 have been closely related to a corresponding type strain. The 6 isolated strains showed very close genetic relationships with the corresponding type strain (all above 99%). The bacteria in the enrichment culture H11S belonged to two phyla, Proteobacteria and Actinobacteria. The main bacterial phylum was Proteobacteria, accounting for 80% of the bacterial community H11S. The phylogenetic trees are shown in Fig. 5. The results indicate that the six strains in the enrichment culture H11S belonged to five genera within Proteobacteria and Actinobacteria. The genus Nitratireductor was predominant, and was dominant in the enrichment culture H11S based on the sequencing analysis (Fig. 4c). The 16S rRNA gene sequences of these isolated strains were submitted to the NCBI GenBank database, and the accession numbers are shown in Table 1.

Biodegradability of crude oil by the isolated strains

Due to ability for oil degradation, the enrichment culture H11S was chosen for the isolation of oil-degrading bacterial strains. Six isolated strains from different genera were selected and their crude oil removal efficiencies were estimated by the gravimetric method (Table 3). The abilities of the six strains to degrade crude oil were calculated by comparison with uninoculated controls. Among the six strains, Pseudomonas sp. strain H11S-28 exhibited the best crude oil degradation effects, degrading 44.3% of the crude oil, followed by Nitratireductor sp. strain H11S-31 (39.5%) and Acinetobacter junii strain H11S-25 (38.5%).

The results of GC–MS analysis showed that most strains were equally capable of degrading total n-alkanes (C10–C34) and PAHs (Table 3). It is worth noting that the degradation efficiency of n-alkane by the enrichment culture H11S was significantly higher than those for the isolated strains, which may reflect the synergistic effect between bacteria^{43, 44}. The ability to degrade target hydrocarbons by each strain is shown in Online Resource Fig. S4. Specifically, most strains degraded more short-chain (C10–C19) and medium-long-chain (C20–C24) n-alkanes than long-chain (C25–C34) n-alkanes (Online Resource Fig. S4a). Strain H11S-25 showed the highest alkane degradation efficiency of 97.2% for a medium-chain n-alkane (C24). In contrast to the results described in “Isolation and identification of strains”, bacterial strains in the enrichment culture H11S had poor ability to degrade PR or PH, with the highest consumption efficiencies only reaching 70.3% and 43.7% respectively.

In terms of the degradation of PAHs (Online Resource Fig. S4b), all the strains tended to degrade monomethyl-substituted DBT, monomethyl-substituted CHR, and dimethyl-substituted CHR more readily than other methyl-substituted DBTs and CHRs. Strains H11S-28, H11S-35, and H11S-31 showed similar degradation characteristics for monomethyl-substituted DBT, exhibiting high degradation efficiencies of 66.6%, 63.9%, and 56.6% respectively. However, there were significant differences in the degradation of naphthalene (NAP)/phenanthrene (PHE). The isolated strains preferred to degrade tetramethyl-substituted NAP/PHE. This result was consistent with the GC–MS analysis, which revealed that the isolated strains including Acinetobacter junii strain H11S-25, Nitratireductor sp. strain H11S-31 and Pseudomonas sp. strain H11S-28 generally exhibited favourable degradation abilities for n-alkanes and PAHs.

Oil-degrading characteristics of consortium H

The results for the oil degradation efficiency of consortium H on days 3, 9, 15 and 21 are shown in Table 4. The degradation efficiency of consortium H reached an amazing 45.4% after 3 days of degradation, and there was no increase over the next 6 days. Finally, the maximum degradation efficiency reached 57.1% on day 21. Therefore, the first 3 days of degradation by consortium H constituted the high-efficiency degradation period. It suggests that each bacterium in consortium H was able to quickly adapt to a stressful environment and showed excellent synergistic degradation. Another period of degradation enhancement extended from day 9 to day 15, after which the degradation efficiency increased slowly.

The degradation efficiencies of total n-alkanes (C10-C34) and PAHs detected by GC–MS showed the same trend as the results detected by the gravimetric method. The degradation efficiency of n-alkane was 74.2% on the third day, and reached 86.2% on the 15th day. The highest degradation efficiency was 93.3% after 21 days of degradation. In particular, consortium H exhibited an excellent ability to degrade PAHs. The degradation efficiency for PAHs was 65.7% on the third day, and the final degradation efficiency for PAHs reached 79.4% on the 21st day.

As shown in the GC–MS analysis (Table 5), the degradation characteristics of consortium H for short-chain n-alkanes at each time point (days 3, 9, 15 and 21) were almost the same as those determined by the gravimetric method. It was also found that consortium H showed significant degradation of PR on the third day, reaching 84.5%. However, unexpectedly, consortium H did not exhibit an outstanding ability to degrade PH, only reaching 61.6%. Considering the efficiency of PH degradation in the original enrichment culture H11S, it is likely that some bacteria in H11S such as strains of Erythrobacter, preferred to degrade PH⁴⁵. In the process of medium-chain and long-chain n-alkane degradation, consortium H exhibited the same trend as for the degradation of PAHs. The time of degradation by three-strain consortium increased with the increase of alkane chain length. This phenomenon was consistent with the fact that the degradation efficiency of petroleum hydrocarbons decreased as the carbon number of petroleum compounds increased⁴⁶. Consortium H degraded PAHs mainly included monomethyl-substituted and dimethyl-substituted NAPs (Table 6). The degradation efficiency rapidly increased to 79.7% and 73.2% on the 15th day respectively. The degradation efficiency for other PAHs including high-molecular-weight PAHs increased steadily from 2.7% to 15.6%. In particular, consortium H showed excellent degradation efficiency (above 80%) of CHR on the third day and the degradability remained the same thereafter. This result is also consistent with findings of a previous study showing that consortia were able to exert a stronger positive effect on the biodegradation of PAHs with increasing ring number²⁷. In our study, the biodegradation efficiencies for PAHs and high-molecular-weight PAHs were superior to those previously reported of constructed artificial consortia⁴⁷.

GC–MS analysis showed that consortium H degraded most of the total n-alkanes and total PAHs in oil on the third day. The total oil degradation efficiency calculated by gravimetric analysis indicated that the efficiency of oil removal by consortium H could exceed 50% after 15 days of degradation.

Biodegradability of crude oil by the isolated strains and consortia after degradation for 21 days

After 21 days of degradation, the various crude oil degradation characteristics of the isolated strains and consortia were compared. The degradation efficiency of consortium H, which was composed of three different bacterial species, was higher than those for isolated strains and other consortia. The degradation efficiencies of consortia A21, B21 and C21 were not as high as that for Nitratireductor strain H11S-31, which may be due to competition or antagonistic effects between the two artificially combined bacterial strains. Compared with the degradation efficiency of consortium H, Acinetobacter junii strain H11S-25 showed lower degradation efficiency (46.1%) but played a key role in the overall degradation system. This strain probably relieved the competition or antagonism between Pseudomonas sp. strain H11S-28 and Nitratireductor sp. H11S-31.

In addition, GC–MS was used to measure the degradation efficiencies for total alkanes and PAHs. Total alkane and PAHs degradation efficiencies of consortium H reached the highest values of 93.3% and 79.4% respectively. Compared with the corresponding constituent strains, the removal efficiency for total PAHs of consortium A21 improved from 0.9% to 11.4% (Table 4), indicating that strain H11S-25 and strain H11S-31 exerted mutually promoting effects. Perhaps the metabolites produced by the two strains promoted the growth of each other to increase their usage of oil. However, the low total alkane and PAHs degradation efficiencies of consortium B21 (78.2% and 56.2%, respectively) indicated that strain H11S-31 and strain H11S-28 exhibited obvious degradation inhibition effects. The possible reason is that the strains inhibited each other metabolic capacities via producing extracellular substances⁴⁷. The degradation ability for total PAHs of consortium C21 indicated that there was a weak degradation-promoting effect between strain H11S-25 and strain H11S-28.

As shown by the GC–MS analysis (Fig. 6a), consortium A21 showed superior activity (above 89.6%) in degrading short-chain n-alkanes, and this degradation ability was much higher than the performance of strain H11S-25 and strain H11S-31. However, the ability of consortium A21 to degrade PR and PH was unsatisfactory and was lower than that of its constituent strains. The short-chain and medium-chain n-alkane degradation efficiencies of consortium B21 were significantly lower than those of the corresponding individual strains. In addition, the short-chain n-alkane degradation efficiency of consortium C21 was only slightly better than that of strain H11S-28. However, consortium H showed excellent biodegradability for both short- and medium-chain n-alkanes.

The PAHs degradation efficiencies in crude oil are shown in Fig. 6b. Degradation efficiencies for high-molecular-weight PAHs (PHE, DBT, and CHR) of consortia A21, B21, and C21 were not as high as those of the constituent strains. However, the PAHs degradation efficiencies of consortium H exceeded 80.0%, especially for DBT and CHR. That is, with increasing ring number of PAHs, consortium H exhibited a strong positive effect on the biodegradation of those compounds. Our results showed that consortium H had a wider range of degrading capacities for petroleum components. This indicates that the key to constructing an efficient artificial consortium is that the proportion of bacterial strains should strictly follow the proportion of the original enrichment culture.

Source: Ecology - nature.com