Materials and CL-20 reagents
Activated sludge was collected from the sewage treatment plants of a CL-20 manufacturing factory (Liaoyang City, Liaoning Province, China). The CL-20 samples, with a purity greater than 99%, were obtained from Xi'an Modern Chemistry Research Institute. All other reagents were of analytical grade or chemically purified.
Culture of microbial community and 16s rRNA amplification
The 5 g activated sludge samples were added to a 100-mL conical flask containing 25 mL of sterile Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl, autoclaved at 121 ℃ for 20 min). After incubation in a shaker at 37 ℃ and 120 rpm for 24 h, 1 mL of the culture was taken and transferred to 25 mL of fresh mineral salt medium (MSM) (3.8 g/L Na2HPO4·12H2O, 1.5 g/L KH2PO4, 10 mL of 0.755 g/L CaCl2; 10 mL of filtered sterilized mixed solution: 5.0 g/L MgSO4·7H2O, 0.152 g/L MnSO4·H2O, 0.5 g/L FeSO4·7H2O, and 20% glucose as the carbon source). All reagents mentioned above were autoclaved before use, if not specified otherwise. CL-20 acetone solution (10 g/L) was added to the MSM so that the solution contained 100 mg/L CL-20. The culture was incubated at 28 ℃ and 120 rpm for 7 days. After five successive transfers, five microbial community samples (named MC0 to MC4) were acquired. The microbial composition of each sample was determined using 16s rRNA-based metagenomics analysis. Briefly, a microbial community sample (1 mL) was centrifuged at 12,000 rpm for 5 mins, followed by discarding the supernatant. The centrifugation process was repeated three times. DNA extraction was performed using an E.Z.N.ATM Mag-Bind Soil DNA Kit (OMEGA), and the DNA integrity was checked by agarose gel electrophoresis. Polymerase chain reaction (PCR) amplification was carried out using 16s rRNA primers (F: 5ʹ-CCTACGGGNGGCWGCAG-3ʹ; R: 5ʹ-GACTACHVGGGTATCTAATCC-3ʹ), and the PCR product was purified and subsequently sent to Sangon Biotech Co., Ltd. (Shanghai, China) for metagenomic sequencing.
Bioinformatics analysis of microbial communities
To obtain high-quality datasets, Flash v1.2.3 and Qiime v1.8.0 software were used for sequence splicing, filtering, and processing of clean readouts of the original data. Usearch v5.2.236 software was utilized to calculate operation classification unit (OTU) clustering based on the similarity score. RDP classifier 2.12 was used for annotation of species of representative OTU sequences, and the sequence alignment was performed by the basic local alignment search tool (BLAST). The alpha-diversity, which represents the richness and diversity of the microbial community, was also determined (Bissett and Brown 2018; Wang et al. 2019).
Isolation and identification of the bacterial strain
The microbial community sample was inoculated onto an MSM agar plate containing 100 mg/L CL-20 as the sole nitrogen source. A single colony was picked out and transferred to a liquid MSM culture after incubation for 48 h, followed by shaking at 28 ℃ and 120 rpm for another 48 h. After 4–5 successive rounds of incubation, the bacteria were purified and identified by the morphological characteristics of the colony. A 1-mL aliquot of bacteria was centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and the bacterial pellet was suspended and separately stored in LB medium and MSM at −80 ℃. The identified strain Pseudomonas sp. ZyL-01 (Preservation number CGMCC No. 18373) was deposited in the China General Microbiological Culture Collection Center (CGMCC).
Bacteria in the logarithmic phase of growth were streaked and inoculated into 25 mL of MSM, cultured at 28 ℃ for 48 h, and observed for colony shape, color, viscosity, ridge, and edge morphology. A single colony was selected for Gram staining, followed by observation of the size, shape, flagella, and Gram reaction under a microscope. The isolated strain was identified by 16s rRNA gene sequencing analysis. The obtained sequences were compared with the existing 16s rRNA gene sequences in GenBank by BLAST. The phylogenetic tree was built by the neighbor joining tree method using MEGA-X-10.0.5 software (Kumar et al. 2018).
Growth characteristics of the bacteria
A single colony from a solid culture plate was inoculated into 25 mL of LB culture medium and cultured in a shaker at 28 ℃ and 120 rpm until the bacteria reached the logarithmic growth phase. A 25-µL aliquot of culture was then inoculated into 25 mL of fresh LB culture medium and then grown at different temperatures (21 ℃, 28 ℃, and 37 ℃) and pH values (4.0, 5.5, 7.0, 8.5, and 10.0), and the microbial growth of each sample was determined by measuring the optical density at 600 nm (OD600). All the tests were performed in triplicate and repeated three times.
Detection of CL-20 degradation by bacteria
The LB culture at the logarithmic phase of growth (500 μL) was centrifuged at 12,000 rpm for 5 min, followed by removal of the supernatant. The bacteria were resuspended and washed twice in 1 mL of sterile water and then transferred to a vial with 5 mL of liquid MSM containing CL-20 as the sole nitrogen source. The culture was incubated on a shaker at 28 ℃ and 120 rpm; meanwhile, the concentration of CL-20 was determined by high-performance liquid chromatography (HPLC) at different time points. The procedures were performed as described previously (Monteil-Rivera et al. 2004). In brief, a vial of MSM containing culture was left in the fume hood for evaporation overnight, followed by extraction with 10 mL of acetonitrile and ultrasonic treatment for 4 h at 20 ℃ in the dark. After centrifugation at 4,500 rpm for 30 min, a 5-mL sample of supernatant was added to 5 mL of CaCl2-NaHSO4 solution at two different concentrations (5 g/L and 0.2 g/L). The samples were then shaken and left for another 30 min, filtered through a 0.22-µm Millipore filter, and analyzed by HPLC (Waters® e2695 Separations Module, Waters Corp., Milford, MA, USA). The separation was completed on a CORTECS C18 column maintained at 30 ℃. The mobile phase (70% aqueous methanol) was run at 1 ml/min for 8 min. The detector was set to scan from 200 to 350 nm. Chromatograms were extracted at 230 nm, and the injection volume was 20 μL. All the experiments were performed in triplicate and repeated three times.
Biodegradation assay in effluent by ZyL-01
The effluent sample was collected from the Crystal transfer process of CL-20 plant, which contained chloroform (0.374 g/L), ethyl acetate (1.235 g/L) and CL-20 (42.32 mg/L). The bacteria from LB culture (500 μL) at the logarithmic phase of growth was centrifuged and washed for twice, and then transferred to the following 50 mL filtered sterilized effluent samples: a) effluent sample with bacteria; b) effluent sample with glucose (2 g/L) as a carbon source and bacteria; c) effluent sample without bacteria and any supplement as abiotic control. The samples were incubated at 28 ℃ and 120 rpm for 16 days, and the concentration of CL-20 was determined by HPLC as mentioned above. All the experiments were performed in triplicate.
Bioinformatics of the draft genome sequence of Pseudomonas ZyL-01
The sequencing of the selected Pseudomonas sp. ZyL-01 was performed by Sangon Biotech Co., Ltd. (Shanghai, China). The DNA library of Pseudomonas ZyL-01 was constructed with purification by a NEB NextR Ultra™ DNA Library Prep Kit and conventional PCR amplification. The length distribution of the library was determined by an Agilent Technologies 2100 DNA 1000 Kit and used for quality control. The purified DNA pool was sequenced by Illumina HiSeq. The sequencing reads were cleaned up using Trimmomatic and assembled by SPAdes, and then the contig GAP was filled by GapFiller and one scaffold was obtained for further analysis. Finally, the coding genes were predicted using Prokka, while the tRNAs and rRNAs were identified by Aragorn and RNAmmer. The potential functions of the coding genes were predicted using the databases of Swiss-Prot, Cluster of Orthologous Groups of proteins (COGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), NCBI nucleotide sequences (NT), NCBI nonredundant protein sequences (NR), and Conserved Domain Database (CDD).