Fast Location and Isolation of Phenanthrene-degrading Halophilic Microbial Community and its Degradation Pathway


 Microbial consortium WZ-4, which could degrade phenanthrene (PHE) as the main carbon and energy source, was isolated from the aerobic sludge of Weizhou wastewater treatment plant. Under the condition of high salinity (3%), the degradation of PHE (100 mg/L) was 87.76% in 7d. Its metabolites, genome sequence and biodegradation pathway were studied. The main metabolites include 1,2-dihydroxynaphthalene, 1-hydroxy-2-naphthalene, 5,6-benzocoumarin and phthalic acid. 12 PHE degrading enzyme genes appeared in the metagenome sequencing of WZ-4, and the genes involved in PHE degradation were included phdE, phdF, phdG, and pcaL. Based on the metabolites detected by GC-MS and the potential PHE-degrading genes identified by BLAST search, biodegradation pathway of PHE by WZ-4 was predicted.


Introduction
Among all kinds of petroleum compounds, polycyclic aromatic hydrocarbons (PAHs) composed from two or more fused aromatic rings had chemical stability and extremely di cult biodegradation 1 . As an common toxic carcinogenic pollutant to human and animals 2, 3 , PAHs threatened human food safety through the accumulation in the food chain 4 . There were large number of PAHs utilizing microbes in high salinity oil polluted sea area and offshore oil eld environment 5,6 . However, few pure culture of extremely halophilic hydrocarbons degration microbes was isolated 7,8 .
Although a halophilic archaeal Phenanthrene (PHE) degrading strain MSNC 14 has been reported, but so far, a single PAH degrading strain adapted to high salinities had never been reported 9 . The informations about the ability of halophilic archaea degrade hydrocarbons in hypersaline environments were limited 9 .
At present, few studies on degradation pathways of PHE by halophilic microbes were reported. And the degradation mechanism is still unclear. Therefore, how to improve the treatment effect of halophilic microbes on PAHs in high salinity environments was the bottleneck.
In this study, the effectiveness of halophilic bacterial consortium (WZ-4) to degrade PAHs was studied under a salinities up to 3% NaCl (w/v). Based on the metabolites detected by GC-MS and the potential PHE degrading genes identi ed by BLAST search, a PHE biodegradation pathway by WZ-4 was predicted.
This study attempts to illuminate the mechanism of action of halophilic microbial consortium in the degradation of PHE.

Sample and material
Aerobic sludge was collected from activated sludge of an aerobic ba ed reactor and sequence batch reactor (ABR-SBR) pond and stored in fridge at -20℃ 10 . The oil eld wastewater was collected from the water inlet of the plant and stored in fridge at 4℃. All the sample was provided by the Weizhou wastewater treatment plant which belongs to Zhanjiang Branch of China National Offshore Oil Corp.
Phenanthrene (purity, ≥ 98%), Chromatographic grade n-hexane, acetone and acetonitrile were purchased from Energy Chemical which belongs to Saen Chemical Technology (Shanghai) Co., Ltd, China. Other reagents were purchased from Sinopharm chemical reagent Beijing Co., Ltd., which are of analytical grade.

Microbes domestication and isolation
The original culturable microbial consortium was enriched from the activated sludge. The method was slightly modi ed according to Zhou and Huang 11,12 . The high salinity beef extract peptone medium (composition in g/L: Beef extract, 3.0; Tryptone, 10.0; NaCl, 30.0; pH 7.0,salinity 3%) was prepared to enrich the microbial community. The activated sludge sample (5 mL) was transferred to a erlenmeyer ask (250 mL) containing sterilized medium (100 mL) and incubated at 35℃ and 150 rpm for 7 days, then centrifuged (4000 g, 10 min) and washed with sterilized normal saline for 3 times to obtain the microbial community. And then transferred to the prepared inorganic salt medium (composition in g/L: KH 2 PO 4 , 0.5; K 2 HPO 4 , 0.5; (NH 4 ) 2 SO 4 , 1.0; Na 2 SO 4 , 1.0; CaCl 2 , 0.1; MgSO 4 , 1.0; NaCl, 30.0; Yeast extract, 1.0; Sodium lactate, 1mL, pH 7.0). In each initial sterilized medium, 20 mg/L was added as the sole carbon source and energy. The inorganic salt medium was cultured at 35℃ and 150 rpm. After 7 days, the bacterial suspension (1 mL) was sampled and transferred to a new medium to continue domestication. The concentration of PHE in the next new medium gradually increased to 40, 60, 80 and 100 mg/mL.
The PHE were evenly distributed in n-hexane solvent, PHE (2 ml) was sprayed on the surface of the inorganic salt agar plate medium. After the solvent volatilized, the diluted of bacterial suspension (0.1 mL) was added and cultured at 35℃ for 2-3 days. The single strain that can grow with great difference in color and morphology were selected and keeped in tube cultures.
NEB Next® Ultra™ DNA Library Prep Kit for Illumina® was used to extract DNA from each sample and construct gene library. polymerase chain reaction (PCR) was performed to amplify 16S rRNA gene.
Universal primer 27F (5′AGAGTTTGATCTGGCTCAG-3′) and 1492R (5′CTACGGCTACCTTGTTACGA-3′) was used to amplify the DNA fragment coding for 16S rRNA gene in PCR. The puri ed PCR products of each strain were used for DNA sequencing by PCR sequencer (ABI-2720, Applied Biosystems, USA). The sequence les were compared with the data in NCBI 16S database by NCBI blast program. 4 strains were isolated, which were all belongs to Halomonas sp..

Degradation test of PHE
Using PHE as the sole carbon source and energy, 4 single strains and microbial consortium WZ-4 were enriched and cultured in inorganic salt medium at 35℃ and 150 rpm for 7d. And then the bacterial suspension was centrifuged (8000 g, 10 min), and the supernatant was extracted 3 times with an equal volume of solvent (acetone and n-hexane 1:1). The organic phase was extracted and dehydrated by anhydrous Na 2 SO 4 . Than the organic phase was collected and placed in a dry-bath nitrogen gas blower for condensation, and continuously rinsed with acetonitrile. The organic phase was sampled, passed through a 0.22 µm nylon lter, and 1.5 mL was injected into the injection bottle.
The degradation process of PHE was determined by HPLC (Agilent 1260 in nity, Agilent Technologies, USA). The conditions of HPLC were as follows: column temperature was 25℃, mobile phase were acetonitrile and water (3:2), ow rate was 1 mL/min and the detection wavelength was 254 nm.

Intermediate metabolites determination
As experience group, microbial consortium WZ-4 was added into erlenmeyer ask with 30 mL inorganic salt medium and 100 mg/L PHE, and cultured at 35℃ and 150 rpm. 3 bottles added 30 mL inorganic salt medium and 100 mg/L PHE cultured at 35℃ and 150 rpm, as control group. Samples were taken continuously cultured for 0h-40h. Every two hours, 3 bottles sampled, then 0.1mL HgCl 2 was added to terminate the reaction and frozen. All samples were analyzed within 3d. Chromatographic grade n-hexane for solvent.
The intermediate metabolites in the degradation of PHE were determined by GC-MS (Clarus 600, Perkin Elmer, USA). The chromatographic conditions were as follows: the initial column temperature was 70℃, the retention time was 1.5 min, the temperature was raised to 250℃ and the retention time was 10 min. the injection temperature was 250℃, the mode of no split ow was adopted, and the chromatographic column was HP-5MS fused silica column (30 m × 250 µm × 0.25 µm). The mass spectrometry conditions were as follows: ion temperature 200℃, electron energy 70 eV, scanning range (M/z) 50-400, carrier gas helium, ow rate 1 mL/min.

DNA extraction and genome annotation
PowerSoil DNA extraction kit (MoBio Laboratoties Inc.) was used to extract macro DNA. The quality of the macro DNA sample was evaluated by agarose gel electrophoresis on a 1% agarose gel GelRedTM (Biotium Inc.). The Thermo Qubit 4.0 Fluorometer (Thermo Fisher Scienti c Inc.) was used for DNA quanti cation. The establishment of gene library and high-throughput sequencing were commissioned by Shenggong Bioengineering (Shanghai) Co., Ltd. Prodigal was used to predict the ORF of long sequence contigs. Select genes greater than or equal to 100 bp to translate into protein sequences. Prodigal was used to predict the ORF of long sequence contigs. Genes with a length of 100bp are translated into protein sequences. Gene alignment between the obtained protein genes and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were performed, using BLAST algorithm.

Isolation and identi cation of PHE degrading bacterium
PHE degrading strains, marked as YA, YB, NY2 and NY3 were isolated, using PHE as a sole source of carbon and energy. These halophilic strains were culturable. The puri ed 16S rRNA gene PCR products of each strain were sequenced and compared in GeneBank. These strains all belonged to Halomonas sp.. The comparison results was shown in Table 1. The microbial consortium structures of the sampled aerobic sludge form Weizhou wastewater treatment plant ( Fig. 1 WZ1, WZ2 and WZ3) and PHE domesticated sludge (Fig. 1P1) were determined by highthroughput sequencing. The results was shown in Fig. 1.

PHE degradation ability of bacterium
PHE were added into the culture medium of single strains (Y2, Y3, NY2 and NY3) and microbial consortium (WZ-4), and cultured for 7 days respectively. The degradation rates were shown in Fig. 2  Comparing with the standard material, the concentration trend of PHE degradation was determined (Fig. 3 PHE). These results were shown in Fig. 3. According to the concentration of PHE and intermediate product (Fig. 3D), a lag phase about 5h after the inoculation of WZ-4 was presented. Between 4 ~ 32h, the concentration of intermediate products keeps changing and the concentration of PHE keeps decreasing. After 32h, the degradation rate of PHE tends to be stable (Fig. 3 PHE, close to 70%). Similar results from recent study have been reported that strain N4 could only nearly 80% of the PHE had been degraded in 8 days 13 . The concentration of intermediate product E (Fig. 3E) rises to the highest value (close to 45 mg/L), in 40h.

Structural analysis of metabolic intermediates by GC-MS
The metabolic intermediates produced during the degradation of PHE was analyzed by GC-MS. By comparing with the GC retention times of standard sample and retrieving with the mass spectrometry library and characteristic peaks in the reference 14 , four metabolites were identi ed. Their corresponding mass spectra image were shown in Fig. 4. According to the above results, four metabolites (a), (b), (c) and (d) were determined as 1-hydroxy-2-naphthoic acid, 1,2-dihydroxynaphthalene, 7,8-benzocoumarin and phthalic acid respectively.

Genome annotation
28737 genes regulating the pathway of carbon hydrocarbon metabolism were found, which indicating a strong biodegradation potential of WZ-4 in petroleum hydrocarbons. Gene alignment between the found genes and the KEGG database were performed, using BLAST algorithm. Gene annotation of possible metabolic pathways of WZ-4 were performed. Due to the high correlation with PHE degradation, genes related to carbohydrate metabolism in Fig. 5 were emphatically analyzed. Comparing with the KEGG database, genes related to phenanthrene degradation in the metagenomic group of WZ-4 were identi ed.
Genes related to PHE degradation were shown in Table 2. 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 strain 18 . PHE biodegradation using the microbial consortium was faster and reached higher degradation value 19 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-2naphthoic 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 e ciency of WZ-4 degradation of PHE.

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 ring 13 . According to previous studies, the biodegradation of PHE usually through the double hydroxylation of the C1-C2 pathway or C3-C4 pathway 20,21 . In the C1-C2 pathway, dihydroxylation occured at C1 and C2 carbon sites. And then 1,2-dihydroxy-phenanthrene was cleaved to 2-hydroxy-1naphthoic acid and 5,6-benzocoumarin 22 . As a secondary metabolite, 5,6-benzocoumarin was considered to be the nal 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 naphthol 23 . In this study, 5,6benzocoumarin 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 7,8benzocoumarin were detected (Fig. 6). Since 7,8-benzocoumarin was a reversible reaction product of 1hydroxy-2-naphthoic acid, it indicated that PHE was degraded through the C3-C4 pathway.
(2) 1,2-dihydroxynaphthalene could be cut off in the intermediate position and decomposed into salicylic acid and endup in tricarboxylic acid cycle 26,27 . 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 identi ed. Genes related to PHE degradation included 3,4-dihydroxy-phenanthrene dehydrogenase gene, 3,4dihydroxyphenanthrene dioxygenase gene, 1-hydroxy-2-naphtholic acid hydrolase gene and 4carboxymucate decarboxylase gene 28 . And downstream genes of PHE degradation were also identi ed, 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.

Conclusions
In this study, WZ-4, a microbial community that can effectively degrade phenanthrene, was located and separated from aerobic sludge through high-throughput sequencing technology. Based on the metabolic intermediates identi ed by GC-MS and the associated degradation genes revealed by genome sequencing and genomic annotation, the biodegradation pathway of WZ-4 was determined. In the upstream process of biodegradation of WZ-4, C3 and C4 carbon atoms undergo hydroxylation, and the downstream degradation process follows the phthalic acid pathway. The study of potential PHE degrading strains is of great signi cance and prospect for bioremediation of oil contaminated water and soil environment, which could be used for bioremediation of petroleum contaminated environment.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
All the authors declare that they have no con icts of interest.