Characteristics of crude oil‐degrading bacteria Gordonia iterans isolated from marine coastal in Taean sediment

Abstract Crude oil is a major pollutant of marine and coastal ecosystems, and it causes environmental problems more seriously. It is believed ultimate and complete degradation is accomplished mainly by microorganisms. In this study, we aim to search out for bacterial strains with high ability in degrading crude oil. From sediments contaminated by the petroleum spilled in 2007, an accident in Taean, South Korea, we isolated thirty‐one bacterial strains in total with potential application in crude oil contamination remediation. In terms of removal percentage after 7 days, one of the strains, Co17, showed the highest removal efficiency with 84.2% of crude oil in Bushnell‐Haas media. The Co17 strain even exhibited outstanding ability removing crude oil at a high salt concentration. Through the whole genome sequencing annotation results, many genes related with n‐alkane degradation in the genome of Gordonia sp. Co17, revealed alkane‐1‐monooxygenase, alcohol dehydrogenase, and Baeyer–Villiger monooxygenase. Specially, for confirmation of gene‐level, alkB gene encoding alkane hydroxylase (alkane‐1‐monooxygenase) was found in the strain Co17. The expression of alkB upregulated 125‐fold after 18 hr accompany with the removal of n‐alkanes of 48.9%. We therefore propose the strain Gordonia iterans Co17, isolated from crude oil‐contaminated marine sediment, could be used to offer a new strategy for bioremediation with high efficiency.

such as oceanic fish and seaweed. The oil pollution affects humans as well. Crude oil spills directly affect the lungs and indirectly affect the whole body following consumption of food derived from marine habitats (Gardner, Yevich, Harshbarger, & Malcolm, 2011;Orisakwe, Akumka, Njan, & Afonne, 2004) .
In this study, we have searched for crude oil high efficiency degrading strains, bacteria Gordonia sp. Co17 was isolated from the oilcontaminated site Hebei Spirit in year of 2007, and the environmental factor for bioremediation efficiency was determined. Through the whole genome de novo sequencing, the genes of Gordonia were annotated. Especially with the alkB gene, which is a key enzyme in the alkane degradation pathway identified in oil-degrading bacteria, we constructed the phylogenetic tree with a closely associated alkB gene family member including bacteria. In addition, the degradation rate was measured and the amount of alkB gene expression was examined by quantitative real-time PCR (qRT-PCR).

| Bacteria isolation and identification
The sample was collected from the oil spill area in Taean, South Korea (36°47′16″N 126°08′37″E) in March 2008. This area was contaminated by crude oil in 2007 because of oil spill accident by Hebei Sprit. Sea water samples were collected from depths of 5 m from surface using sterile water sample bottle. And sediment samples were collected using a sterilized grab along the coastal then sealed in to a plastic aseptic bag. Samples were stored in icebox and transported to the laboratory within 1 day after being collected.
Sea water was filtered using a membrane filter (0.2 μm pore size), and the obtained filtered membrane was cultured in 50 ml of Bushnell Hass media (BH media, 0.2 g/L MgSO 4 , 0.02 g/L CaCl 2 , 1.0 g/L KH 2 PO 4 , 1.0 g/L K 2 HPO 4 , 1.0 g/L KNO 3 , 0.05 g/L FeCl 3 and the pH was adjusted to 7.0 ± 0.2 (Atlas, 2014)). The sediment sample of 1 g weight was cultured the same way. Two percent NaCl within BH media and crude oil were added to the media and incubated at 25°C for 1 week. After obtaining the enriched culture, the suspension was diluted to 10 −6 and spread onto 1.5% agar of BH media, incubated at 25°C.
All isolated strains were conducted of screening of crude oil degradation activity in triplet, and the selected strain was identified using the universal bacterial primers. The pairwise 16S rRNA gene sequence similarity was determined using the Ezbiocloud server (Yoon, Ha, Kwon, & Lim, 2017).

| Detection of crude oil degradation efficiency of strains
Kuwait crude oil was used to conduct bioremediation screening of petroleum. The crude oil (0.1%, v/v) was injected into BH media, and 1.0 g/L (w/v) of single bacteria was inoculated. The bacteria harvested at exponential phase, was washed out with 1X PBS buffer, and then the wet weight was measured after inoculated and cultured at pH 7.0, 25°C for 7 days. After cultivation, the residual oil contaminant was extracted with dimethyl chloride. Dimethyl chloride was injected in half volumes and shaken for 30 min. The aqua-phase was replaced with anhydrous sodium sulfate to remove water in organic-phase. Dimethyl chloride was evaporated, and the residual oil contaminant was redissolved in 2 ml of dimethyl chloride.
The extracted oil contaminant was examined by GC-FID (Gas Chromatography-Flame Ionization Detector) and GC-MS (Gas Chromatography-Mass Spectrometry). The GC-FID column used a capillary DB-1HT, oven condition was maintained at 40°C for 3 min, increased by 12°C/min to 275°C, and held for 12 min. The injector temperature was 280°C, and the detector temperature was 340°C.
Nitrogen gas was used as the carrier gas. The GC-MS analysis was conducted under the following conditions: oven temperature 40°C for 4 min; increased by 5°C/min to 320°C; held for 15 min; and helium gas was used as the carrier gas. The mass spectrum was recorded from m/z 50 to 720 amu, in the total ion chromatography (TIC) mode. The C 8 -C 40 alkane calibration standard (SUPELCO) and total petroleum hydrocarbon (TPH) mixture 1 (SUPELCO, C 10 -C 28 alkane mixture) were used as standard materials.
To calculate the crude oil removal efficiency (R E ), the peak area was used, excluding the Unresolved Complex Mixture (UCM). The R E was calculated according to the modified Equation (1) (Deng, Li, Liang, & Yi, 2014;Zhang, Gai, Hou, & Yang, 2010).
R E , C 0 , and C t denote removal efficiency, control (bacteria no treatment) concentration of crude oil, and the remaining crude oil concentration following bacterial treatment, respectively. (1)

| Effect of environmental variables on crude oil removal efficiency
We investigated crude oil degradation efficiency according to environmental conditions such as temperature and NaCl tolerant during 3 days, the residual oil was extracted and R E value was calculated.

| Whole genome de novo sequencing and analysis
Whole genome sequencing and analysis were conducted in Macrogen. Genomic DNA sequencing was performed on the PacBio RS II platform and Illumina HiSeq platform. By mapping the HiSeq reads to first assembled genome sequence, we can see the mapping result that shows a slight difference from the assembly result. Also, we can get a consensus sequence with higher quality through the self-mapping step. After whole genome or draft genome is assembled, the location of protein-coding sequence, tRNA genes, and rRNA genes were analyzed by ARAGORN v1.2 (Laslett & Canback, 2004) and RNAmmer (v1.2) (Lagesen, Hallin, Rødland, & Staerfeldt, 2007), respectively. Then, their functions are annotated. PacBio sequencing reads were de novo assembled with hierarchical genome assembly process (HGAP, v3.0) and annotation was by Prokka (v1.12b) (Seemann, 2014).
The resulted alkB sequence of Gordonia and other sequences were assembled using SeqMan software (DNASTAR Inc.). The phylogenetic analysis was constructed using the MUSCLE program in MEGA 6, and pairwise distances were calculated with the Kimura 2-parameter model, which was also used to determine the confidence levels of the branches (Felsenstein, 2009;Kimura, 1989;Tamura, Stecher, Peterson, & Filipski, 2013

| Bacterial isolates and the strains with crude oil removal ability
In total, 144 strains were isolated from the sea water and sediment samples contaminated by crude oil (Supporting Information

| Crude oil degradation ability of Gordonia sp. Co17
Through the GC-FID and GC-MS analysis data, the strain G. iterans Co17 exhibited degradation activity (Table 2 and Supporting Information Figure S1). The n-alkanes from octane (C 8 ) to octadecane

| Whole genome de novo sequencing analysis
The resulting genome assembly sequencing (CP027433) of Gordonia sp. Co17 produced one contig with a length of 4,006,485 bp (68.73% GC content), and 3,598 CDS. And genome annotation was conducted 51 of tRNA and 12 rRNA. The general feature of Gordonia sp. Table S3 and circular map showed in Figure 3. Through the annotation results, many genes related with crude oil degradation in the genome of Gordonia sp. Co17 were found in the categories of lipid transport and metabolism (I), inorganic transport and metabolism (P), and amino acid transport and metabolism (E) ( Table 3). The Gordonia sp. Co17 has long-chain hydrogenase such as alkB, alma and has, and 23 gene of short-chain hydrogenase such as P450 cytochrome (Fu, Lai, Dong, & Wang, 2018). Briefly, they have two alkB gene (C6V83_00720 and C6V83_14510) and four almA (C6V83_07075, C6V83_07450, C6V83_11880, and C6V83_15530).

Co17 was indicated in Supporting Information
And confirmed three has genes and eight genes of alcohol dehydrogenase (adh, adhT, and adhB) were also confirmed in Gordonia sp.
Co17. Moreover, single lcfB gene and three FadD genes encoded long-chain fatty acid CoA ligase (EC 6.2.1.3) that converted longchain fatty acid with a complex structure such as aromatic compounds to acyl-CoA. As well as the strain Co17 was found to have acetyl-CoA acetyltransferase, an aromatic compound degrading enzyme, and LysR family transcriptional regulator, a phenolic compound degrading enzyme.

| Phylogenetic analysis of alkane hydroxylase gene and gene expression
The PCR was performed using the primers, alkBF and alkBR, and   The relationship between alkB gene expression and n-alkane R E was monitored by qRT-PCR analysis ( Figure 5). The alkB gene was maximally expressed at 18 hours after the incubation with crude oil, and the expression level of 125 fold was up-regulated. After 24 hr, the expression level of alkB gene gradually decreased. Meanwhile, the R E was most rapidly increased (slope; 4.3) at 9 hr, and the slope of R E was decline to 1.1 at 18 hr. After 48 hr, the slope of R E decreased to 0.6, but it was confirmed that the residual amount n-alkane was decreased continuously.

| D ISCUSS I ON
The strain Co17 exists in the oil phase on culture with crude oil (Supporting Information Figure S2, Olympus, Japan, X 1000), meanwhile did not observed emulsifying phenomena during culture period.
In the year of 2003, reported that Alcanivorax borkumensis, the known to degrade crude oil in marine microorganisms, releases biosurfactant for glucose-lipid and grown at the water/n-hexadecane interphase (Golyshin, Martins Dos Santos, Kaiser, & Ferrer, 2003).
But, newly isolated Gordonia sp. Co17 was grown at the oil phase and this suggests that it has a different bio-surfactant for oil degradation pathway.
The Gordonia strains are known to degrade various kinds of oil.
Gordonia CC-JG39 decomposed 75% of the initial about 2,500 mg-TPH (Total Petroleum Hydrocarbon)/kg after 23 days, and remained about 600 mg-TPH/kg (Liu, Liou, Li, & Su, 2015). Gordonia sp. JG39 was reduced in freshwater with 500 mg-diesel oil/L, 94% of diesel degraded within 11 days (Chen, Whang, Pan, & Yang, 2017 In Dong paper (2012), the expression levels of two long-chain fatty acid CoA ligases (facl1 and facl2) expressed by Geobacillus thermodenitrificans NG80-2 were regulated when crude oil was cultured as a sole carbon source. These genes were related with degrade long-chain alkanes with complex structure such as aromatic compounds. The B. licheniformis Y-1 that isolated from heavy oil-contaminated soil, and the strain could not utility short-chain alkane, but also shown degradation rate with long-chain alkane at high salinity condition (Liu et al., 2016). However, in this study Gordonia sp. Co17 was degraded in short chain and long chain alkane under 12% of salinity condition.
The expression of alkB in Gordonia sp. Co17 was analyzed by qRT-PCR, and it appears that the alkB gene is secreted in the early stage of culture to degrade n-alkane. Based on these results, it is considered that Gordonia sp. Co17 is shown to degrade n-alkane by alkane hydrogenases other than alkB gene after 24 hr.
Although the expression level of alkB gene is decreased, other n-alkane degrading enzymes seem to be involved in the degradation efficiency.
We obtained crude oil degradation bacteria from oil-contaminated sediment, especially the Gordonia sp. Co17 was shown highest efficiency 84.2%. Through the de novo whole genome sequencing, confirmed the alkane degrading related gene such as alkane-1-monooxygenase, alcohol dehydrogenase, and Baeyer-Villiger monooxygenase in Gordonia sp. Co17, and deposited to NCBI as the accession number CP027433. Specially, alkane-1monooxygenase, alkB gene was the highest expressed 125-fold at 18 hr, in during cultivation. Therefore, the Gordonia sp. Co17 might be an applied strain to degrade crude oil for bioremediation of crude oil-contaminated soil as well as sediment in coastal area or deep sea.

ACK N OWLED G M ENT
This work was supported by the National Research Foundation of Korea NRF-2017M3A9B8065734 (Sang-Seob Lee).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflict of interest. The complete genome sequence of Gordonia iterans Co17 has been deposited at Genebank under the accession number CP027433.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
All data are provided in full in the results section of this paper.