Screening And Genome Sequencing of a di-n-butyl Phthalate Degrading Bacterium J2 Isolated From Peanut Field Soil

Di-n-butyl phthalate (DBP) is commonly used plasticizers in agricultural plastic lms, and is a priority pollutant due to its toxicity to human health. A newly isolated strain J2, which used DBP as its sole carbon source, was screened from peanut led soil by continuous enrichment cultivation. Based on morphological, physiological characteristics and 16S rRNA gene sequence analysis (GenBank accession No. OK598965), it was identied as Priestia sp. J2. The research results revealed the optimal conditions for DBP degradation as 35 o C and pH 8.0. The strain could effectively degrade 97.6% DBP within 5 days. Substrate tests showed that strain J2 could utilize shorter side-chained PAEs, but could not utilize long-chained PAEs. The whole genome comprises a complete chromosome of 5,067,299 bp and four plasmids of 147,924 bp, 75,940 bp, 11,604 bp, 11,333 bp (GenBank accession No. CP086208-CP086212). This genome harbors 5,585 predicted protein-encoding genes, 130 tRNA genes, and 42 rRNA genes. Gene annotation analyses showed a DBP-degrading gene contained an open reading frame of 930 bp, and the enzyme was named Est-J2-1. The amino acid sequence of the Est-J2-1 exhibited no signicant homology with those of reported DBP-degrading enzymes, suggesting the enzyme is a novel enzyme. The gene of Est-J2-1 was found to be located on the chromosome. This study provided strain resource for DBP removal from farmland and other environments.


Introduction
Plastic lms can maintain soil moisture, regulate soil temperature and restrict weed growth to promote crop yield, which play an indispensable role in modern agricultural production (Sun et al. 2020). Phthalate acid esters (PAEs) are widely added as plasticizers in the production process of plastic lms (Zhang et al. 2021). PAEs are easy to accumulate and migrate in soil and water environment (Vikelsoe et al. 2002; Le et al. 2021). As environmental hormones, they can affect human reproduction and induce genetic aberrations (Surhio et al. 2017). Some PAEs, such as di-n-butyl phthalate (DBP), diethyl phthalate (DEP), Dioctyl Phthalate (DOP), have been classi ed as priority pollutants in USA, European Union and China. DBP has been widely used in agricultural plastic lms, mainly as plasticizer. Because DBP is only physically bound to plastic structures, it tends to escape into soil, water and other environments. Therefore, DBP is be detected in various soil, groundwater, and other environmental samples. In addition, DBP can be absorbed by crops into the food supply, threatening human and animal health (Wang et al. 2016).
DBP removal methods include hydrolysis, photo degradation, and biodegradation. However, because of the ine ciency of the rst two methods, biodegradation plays a major role in the DBP degradation. Biodegradation is considered a promising and environmentally friendly method to eliminate DBP. Many microorganisms capable of degrading DBP were be screened, including Rhodococcus ruber (Li et al. 2006 (Zhang et al. 2014). DBP hydrolase was found in Acinetobacter sp. M673 (Wu et al. 2013).
In this study, a DBP-degrading strain J2 from peanut eld soil was screened and identi ed. Strain J2 could utilize DBP to grow, with high degradation activity. We investigated the DBP degradation characteristics of strain J2, and further sequence the genome to study the DBP-degrading genes. The morphology of strain J2 was observed on LB agar plates after 16 h at 28 o C. The hydrolysis of casein, starch, gelatin, Tween 40, Tween 80 and gelatin was studied as described in previous reports (Romanenko et al. 2013). The strain J2 was identi ed by 16S rRNA gene sequencing. 27F and 1492R primers were used for PCR ampli cation (Frank et al. 2008). PCR ampli cation products were analyzed using 0.8% agarose gel, and were then puri ed. The fragments were linked into pMD19-T, and then were transformed into competent cells. The DNA sequencing was performed using an DNA sequence analyzer ABI 3720 (Applied Biosystems, United States) by Shanghai Personalbio. The 16S rRNA gene sequence was analyzed in Ezbiocloud database. The phylogenetic tree was then constructed using MEGA X (Kumar et al. 2018).

Substrate utilization experiments
In order to study strain J2 to degrade PAEs, the bacterium was inoculated into MSM containing 0.3 g/L of one of PAEs: DBP, DMP, DEP, DOP, DIOP, DINP. Uninoculated media supplemented with each substrate were used negative controls. The suspension was cultured on a rotating shaker at 28 o C. 5 days later, substrate utilization was measured using optical density method.

Biodegradation of DBP by strain J2
Strain J2 was rst cultured in LB medium at 28 o C for 24 h. Strain J2 was harvested and washed three times with phosphate buffer, and then was suspended in the phosphate buffer. Inoculated 1% bacterial solution into 50 mL MSM with 1000 mg/L DBP. In order to study the optical conditions of degradation, the degradation of 1000 mg/L DBP by strain J2 was investigated under pH 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, and different temperature (18, 28, 35, 40, 45 o C). After 5 days, the residue remaining DBP was investigated using HPLC.

Analysis of DBP biodegradation using HPLC
The samples were mixed with ethyl acetate at a 1:1 vol ratio. The aqueous phase was separated using centrifugation at 8,000 g. The aqueous solution samples were extracted twice. The ethyl acetate was evaporated by a rotary evaporator (Buchi, Switzerland). The residue was dissolved in 10 ml methanol, and then ltered. Finally, HPLC was used to analyze the residual concentration of DBP with C18 column. The experiment was carried out at a column temperature of 30 ℃, with a mixture of methanol and water at a ratio of 90:10 (v/v). The concentrations of residual DBP were quanti ed by comparing the integral values of the peak area with the calibration standard curve. DBP degradation e ciency was evaluated, based on the initial and nal concentrations of DBP in the test solution.

Genome sequencing
The whole genome of strain J2 was sequenced by using PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platform, provided by Shanghai Majorbio Biopharmaceutical Technology Co., LTD. For PacBio sequencing, 20 μg DNA of strain J2 was spun in a Covaris G-tube. The DNA fragments were then puri ed, repaired at the end, and linked with SMRTbell sequencing adapters. The sequence library was constructed by 0.45 x volumes of Agencourt AMPure XP beads. An insertion library of about 10 kb was sequenced on one SMRT cell. For Illumina sequencing, more than 1 μg DNA samples were used for sequencing. According to the manufacturer's protocol, genomic DNA samples were cut into 400-500 bp fragments. The libraries were sequenced on Illumina HiSeq X Ten machine with 2 × 150 bp.

Identi cation of DBP-degrading bacteria
The bacteria were screened from peanut eld soil collected from Laixi Experimental Station of Shandong Peanut Research Institute. After continuous enrichment culture, strain J2 could utilize DBP to grow, with high degradation activity. Therefore, the strain J2 was selected for further study. The colonies of strain J2 appeared on LB agar plates were circular, creamy white, raised (Fig. 1). It is a gram-positive bacterium. The physiological and biochemical of strain J2 are shown in Table 1. It was able to hydrolyze gelatin, casein, starch, urea, but not to hydrolyze Tween 80. It could use glucose, arabinose, mannose, maltose, mannitol and N-Acetyl-glucosamine, but could not assimilate citric acid, proline, malic acid or phenylacetic acid. The 16S rRNA gene of strain J2 was 1513 nt (Fig. 2), and has been submitted to GenBank with the accession number OK598965. Through 16S rRNA gene sequencing and a BLAST homology search, strain J2 shared 16S rRNA gene similarity of 99.93%, 99.80%, 98.78%, 98.17%, 97.76%, 96.07% with P. aryabhattai B8W22 T , P. megaterium NBRC 15308 T , P. exus NBRC 15715 T , P. qingshengii G19 T , P. para exus RC2 T , P. koreensis DSM 16467 T . Phylogenetic analysis clearly indicated that strain J2 was a member of the genus Priestia (Fig. 3). In conclusion, the morphological, physiological and genetic sequence analyses identi ed strain J2 as belonging to the genus Priestia. Therefore, the strain was named Priestia sp. J2. Table 1 Biochemical and physiological characteristics of strain J2

Item
Result

Substrate utilization tests
The substrate utilization test results indicated that the strain has differing abilities for degrading PAEs.
The strain could grow well in media containing DBP, DMP and DEP, but could not grow well in DOP, DIOP or DINP (Table 2). DBP, DMP and DEP were shorter alkyl-chained PAEs, but DOP, DIOP and DINP were longer alkyl-chained PAEs. These results indicated that the side chain of substrates has a signi cant effect on the ability of strain J2 to degrade PAEs, which is consistent with the previous studies. Sphingobium sp. TJ could use DMP, DBP, DEP and MBP, but could use DOP and DIOP (Jin et al. 2013

Characteristics of DBP degradation
The effect of pH on DBP degradation by strain J2 was investigated at 35 o C. The degradation rate of DBP increased with the increase of pH from 5.0 to 8.0. Strain J2 had the highest degradation rate of DBP at pH 8.0. As pH exceeded 8.0, the degradation rate decreased sharply (Fig. 4A). In temperature experiment, when the temperature raised from 18 o C to 35 o C, the degradation rate of DBP increased rapidly (Fig. 4B). Based on the analysis of previous reports, many genera could degrade DBP. This study was the rst report that Priestia genus could degrade DBP.

Genome analysis and DBP-degrading genes analysis
The complete genome of strain J2 contains one complete circular chromosome of 5,067,299 bp, and four circular plasmids (147,924 bp, 75,940 bp, 11,604 bp, 11,333 bp, respectively) (Fig. 5). The genome sequences in this study have been submitted to GenBank under the accession number CP086208-CP086212. As shown in Table 3  The sequence comparison revealed that the sequence of Est-J2-1 shared no intensive homology with the three enzymes. Thus, the results indicated that Est-J2-1 was a novel member of the DBP hydrolase family. The gene of Est-J2-1 was found to be located on the chromosome. Sequence analysis of Est-J2-1 gene indicated that the open reading frame consisted of 930 bp encoding 310 amino acids. The molecular weight of Est-J2-1 was 34700.14 Da and the isoelectric point was 5.04. No signal peptide sequence was found. Est-J2-1 and the three DBP-degrading enzymes had highly conserved functional motifs. The catalytic triad (Ser-Asp-His) and consensus motif (Gly-X-Ser-X-Gly) around the active-site Ser154 are boxed in the black frame (Fig. 6).

Conclusion
In conclusion, the strain J2 isolated from peanut eld soil could use DBP as its sole carbon source. Based on morphological, physiological characteristics and 16S rRNA gene sequence analysis, it was identi ed as Priestia sp. J2. This study was the rst report that Priestia genus could degrade DBP.  Effect of pH and temperature on biodegradation of DBP by Priestia sp. J2.  Multiple-sequence alignment of esterases from Priestia sp. J2 (Est-J2-1, in this study), wastewater treatment plant (DphB) (KC438416), Acinetobacter ap. M673 (DBP hydrolase) (AFK31309), and Sulfobacillus acidophilus (EstS1) (AEW03609). The catalytic triad Ser154, Asp250, and His280 in the Est-J2-1 from Priestia sp. J2, is indicated by arrows. The consensus sequence Gly-X-Ser-X-Gly around the active-site Ser154 is boxed in the black frame.