4.1 Degradation of PHE by microbes
In this study, Halomonas sp. was the most abundant genus in PHE experement groups with the relative abundance of 18.68%. Strain identification showed that,YA, YB, NY2 and NY3 belongs to Halomonas sp.. Degradation rates of PHE by YA, YB, NY2 and NY3 were 38.11%, 58.24%, 75.86% and 83.90%, respectively. Different from YA and YB, NY2 and NY3 had obviously degradation rates of PHE (Fig. 2), and shown a homology with Halomonas anticariensis sp. (EA200),with a sequence similarity at 99.3% and 99.8% (Table 1).
Halomonas sp. was usually isolated from high salinity environment and has been proved to viable under different salinity conditions15. On the other hand, Halomonas sp. could be isolated from PAHs contaminated soil and degraded PAHs as the main carbon source. Govarthanan et al. identified strain RM as Halomonas sp., effectively degraded 100 mg/L PHE (67.01%), pyrene (63.21%), naphthalene (60.12%) and benzoapyrene (58.00%) after 7 days of incubation16. Halomonas sp. and Marinobacter sp. could efficiently utilize PHE (90%) in a wide range of NaCl concentrations, from 1% to 17% (w/v)17. These results indicated that Halomonas sp. has a important role in degradation PHE.
These results showed that, upmentioned strains were potential hydrocarbon degrading bacterium, which could be used in PAHs degradation experiments. The results of PAHs degradation experiments showed, the degradation rates of PHE by microbial consortium WZ-4 were 87.04%, which were better than that of all the single bacteria. This result indicating a synergistic relationship between these 4 bacterium, which could increase the degradation depth of PHE. Previous studies reported similar phenomena, the degradation rate of PHE by 5 strains combined increased by 14%, compared with single strain18. PHE biodegradation using the microbial consortium was faster and reached higher degradation value19 In addition, as the PAHs molecular complexity increased, the degradation rates decreasesed.
Based on the retention time in HPLC and molecular weight determined by GC-MS, intermediates metabolic A, B, C and D were inferred to be 1,2-dihydroxynaphthalene, phthalic acid, 1-hydroxy-2-naphthoic acid and 7,8-benzocoumarin. According to Figure 3, 7,8-benzocoumarin was produced 5 hours after inoculation, indicating that WZ-4 has a rapid response in degradation of PHE. Phthalic acid was formed at the 10th hour after inoculation, indicating that WZ-4 can decompose PHE to a high degree in a short time. In addition, at 35h after inoculation, the accumulation of intermediates metabolic E and the stagnation of PHE degradation rate occurred simultaneously. This is probably because a intermediates metabolic has substrate inhibition. This would be an important research direction to further improve the efficiency of WZ-4 degradation of PHE.
4.2 Degradation pathway of PHE by microbial consortium WZ-4
In the process of aerobic PHE degradation, the cracking of a benzene ring usually starts from the hydroxyl containing benzene ring13. According to previous studies, the biodegradation of PHE usually through the double hydroxylation of the C1-C2 pathway or C3-C4 pathway20, 21. In the C1-C2 pathway, dihydroxylation occured at C1 and C2 carbon sites. And then 1,2-dihydroxy-phenanthrene was cleaved to 2-hydroxy-1-naphthoic acid and 5,6-benzocoumarin22. As a secondary metabolite, 5,6-benzocoumarin was considered to be the final metabolite of this pathway, which will accumulate in a large amount during the degradation process. On the C3-C4 pathway, dihydroxylation occurs at C3 and C4 carbon sites. And then 3,4-dihydroxyphenanthrene was cleaved to 1-hydroxy-2-naphthoic acid and naphthol23. In this study, 5,6-benzocoumarin was not detected in the culture medium by GC-MS (Fig. 6), which indicated that the initial oxidation of PHE did not through the C1-C2 pathway. However, 1-hydroxy-2-naphthoic acid and 7,8-benzocoumarin were detected (Fig. 6). Since 7,8-benzocoumarin was a reversible reaction product of 1-hydroxy-2-naphthoic acid, it indicated that PHE was degraded through the C3-C4 pathway.
Subsequently, 1-hydroxy-2-naphthoic acid produced in the previous reaction (C3-C4 pathway) was decomposed into 1,2-dihydroxynaphthalene after decarboxylation reaction24. According to previous studies, 1,2-dihydroxynaphthalene has two possible decomposition pathway: (1) The ortho benzene ring of 1,2-dihydroxynaphthalene could be decomposed into phthalic acid after decarboxylation reaction25. (2) 1,2-dihydroxynaphthalene could be cut off in the intermediate position and decomposed into salicylic acid and endup in tricarboxylic acid cycle26, 27. However, salicylic was not detected (Fig. 6), indicating the degradation of 1,2-dihydroxynaphthalene through the phthalic acid pathway.
Based on the results of genome annotation, potential PHE degradation genes were identified. Genes related to PHE degradation included 3,4-dihydroxy- phenanthrene dehydrogenase gene, 3,4-dihydroxyphenanthrene dioxygenase gene, 1-hydroxy-2-naphtholic acid hydrolase gene and 4-carboxymucate decarboxylase gene28. And downstream genes of PHE degradation were also identified, including protocatechuic acid 3,4 dioxygenase, benzoic acid 1,2 dioxygenase, adipic acid lactone D-Isomerase and dihydroxy-cyclohexanene carboxylic dehydrogenase. The discriminating of these genes indicated that WZ-4 could degraded PHE through the phthalic acid protocatechuic acid pathway.
In summary, the biodegradation process of PHE by WZ-4 was speculated as follows: (1) Under the catalysis of 3,4-dihydroxyphenanthrene dehydrogenase (phdE) and 3,4-dihydroxyphenanthrene dioxygenase (phdF), PHE was dihydroxylated at C3 and C4 carbon sites. (2) Under the catalysis of naphthoic acid hydrolase (phdG), the product was decomposed into 1-hydroxy-2-naphthoic acid. And then, 1-hydroxy-2-naphthoic acid was transformed into 1,2-dihydroxynaphthalene under the action of decarboxylase (pcaL). (3) 1,2-dihydroxynaphthalene was converted into 2-carboxystyrylic acid. And 2-carboxystyrylic acid was converted into 2-Carboxybenzaldehyde by hydrolysis acetal, and then oxidized to phthalic acid by oxidase. (4) Under the catalysis of phthalic acid 3,4 dioxygenase (phtAa and phtAc), the phthalic acid was hydroxylated and converted into 3,4-dihydroxyphthalic acid, and then converted into protocatechuic acid by the action of decarboxylase. (5) After the reaction of protocatechuic acid 3,4 dioxygenase (pacG and pacH), benzoic acid 1,2 dioxygenase (benA-xyL, benB-xyL and benC-xyL), Adipic acid lactone D-isomerase (catC) and dihydroxycyclohexadiene carboxylic acid dehydrogenase (benD-xyL), protocatechuic acid was converted to pyruvate and finally entered the tricarboxylic acid cycle to complete the degradation.