Biodegradation of chloroethene compounds under microoxic conditions

The biodegradation of chloroethene compounds under oxic and anoxic conditions is well established. However, the biological reactions that take place under microoxic conditions are unknown. Here, we report the biostimulated (BIOST: addition of lactate) and natural attenuated (NAT) degradation of chloroethene compounds under microoxic conditions by bacterial communities from chloroethene compounds‐contaminated groundwater. The degradation of tetrachloroethene was significantly higher in NAT (15.14% on average) than in BIOST (10.13% on average) conditions at the end of the experiment (90 days). Sporomusa, Paracoccus, Sedimentibacter, Pseudomonas, and Desulfosporosinus were overrepresented in NAT and BIOST compared to the source groundwater. The NAT metagenome contains phenol hydrolase P1 oxygenase (dmpL), catechol‐1,2‐dioxygenase (catA), catechol‐2,3‐dioxygenases (dmpB, todE, and xylE) genes, which could be involved in the cometabolic degradation of chloroethene compounds; and chlorate reductase (clrA), that could be associated with partial reductive dechlorination of chloroethene compounds. Our data provide a better understanding of the bacterial communities, genes, and pathways potentially implicated in the reductive and cometabolic degradation of chloroethene compounds under microoxic conditions.

Under reductive anaerobic conditions, PCE is sequentially degraded by certain microorganisms through halorespiration to TCE, followed by any isomeric forms of DCE, VC, and finally to ethene (a harmless gas) (Dolinová et al., 2017).In this process, chloroethene compounds are used as terminal electron acceptors and hydrogen (H 2 ), usually obtained from organic substrate fermentation, as the electron donor (Harkness & Fisher, 2013;Tiehm & Schmidt, 2011).The extent of the anaerobic degradation of chloroethene compounds depends on the bacterial composition.
The dechlorination capacity of organohalide-respiring bacteria has been applied in water systems to degrade chloroethene compounds.Bacteria under oxic and anoxic conditions have been stimulated with diverse carbon sources (e.g., glucose, emulsified vegetable oil, glycerol, among others), and exogenous inocula have been used for bioaugmentation to enhance the yield of dechlorination processes (Harkness & Fisher, 2013;Lendvay et al., 2003;Robles et al., 2021).However, complete and efficient dechlorination has not been achieved so far under microoxic conditions (between 89.32 and 8.93 µM or 2.86-0.28ppm) (Algeo & Li, 2020;Rullkötter, 2006;Tyson & Pearson, 1991), which are prevalent in most artificial (i.e., water treatments) and natural systems (groundwater systems) (David et al., 2015;Männistö, 2002).Here, we investigated a groundwater sample (O 2 concentration of 62.28 µM or 1.99 ppm on average) collected from a borehole near an undisclosed chemical refinery located in an economic coal deposits area where the relative proportion (soil texture) of sand, silt, and clay particles was 65%, 17%, and 18%, respectively.The groundwater sample contained ca.

| Sampling
The sampling took place in a borehole (37 m deep) near an undisclosed chemical refinery that produces a broad range of petrochemical products.The borehole water was sampled in triplicate (A, B, and C; 4 L per sample) using an Integra Bladder Pump Model 407 (Solinst Canada Ltd).The 200 mL of water per sample was filtered on-site through a 0.2 µm pore size sterile hydrophilic polyethersulfone (PES) membrane (Pall Corporation).The filters were kept in sterile conditions and at 4°C.The remaining water was kept in sterile high-density polyethene bottles (Sigma-Aldrich).The filters and the water samples were transported to the laboratory within 24 h and stored at 4°C in the dark until further analyses.Specifically, the water was used for batch experiments and chemical analysis, while the filters were used for microbial diversity studies.

| Chemical analysis
Temperature, pH, ORP (oxidation redox potential), DO (dissolved oxygen), total dissolved solids, electrical conductivity, and redox potential (Eh) were measured on-site using a Multiparameter AP-2000 probe (Aquaread Ltd).The Eh measurements were corrected to the standard hydrogen electrode (Nordstrom & Wilde, 1998) Chloroethene compounds and ethene were allowed to equilibrate between headspace and liquid phases before quantification.Extractions were done using a HeadSpace-Solid Phase Micro Extraction (HS-SPME) method (Chung et al., 2008;Joguet et al., 2023).Ethene quantification was also performed by GC/BID-2010 Plus, as described by Kampbell and Vandegrift (1998).The gas standard for ethene (≥99% pure) was purchased from Afrox.VC was analyzed using GC/MS/HS Agilent Gas chromatograph 6890 with Agilent Mass spectrometer 5973 (Munch, 1995) at Eurofins analytico ® .All the analyses were done in triplicates.

| Batch experiment
Before experiments, tolerance assays were performed to determine the minimum inhibitory concentration (MIC) of PCE.The MIC cut-off value of PCE (more detail in Supporting Information S1: Figure 1) was used in the downstream experiment using the three water samples from the same borehole.Five treatments (Table 1) were prepared inside an anaerobic chamber (Coy Laboratories) in 120 mL serum bottles, sealed with butyl rubber septa, capped, and crimped with aluminum seals.Microoxic conditions (the O 2 concentration of 65 µM) were maintained by initially degassing the empty serum bottles (sealed with butyl rubber septa, capped, and crimped with aluminum seals) for 10 min with oxygen-free nitrogen before conducting the experiments (Wang et al., 2017).A total of 15 samples (5 treatments x 3 water samples) were incubated for 90 days in the dark at 20°C without shaking and under microoxic conditions.
The lactate concentration was stoichiometrically calculated according to the electron donor demand for effective dechlorination (Supporting Information S1: Table 1).Ultra-pure O 2 (99.Sequencing was performed on an Illumina MiSeq, using a 2 × 301 cycle paired-end approach at the University of the Free State.Sequences obtained were deposited at the National Centre for Biotechnology Information Sequence Read Archive.

| Bioinformatics
The quality filtering of the 16S rRNA gene sequences obtained from Illumina sequencing was performed using PrinSeqlite v0.20.4 (Schmieder & Edwards, 2011).Merged paired-end sequences that passed the quality control were analyzed using QIIME v1.9.0 (Caporaso et al., 2010), as described in Cason et al. (2017).Briefly, chimeric sequences were identified using usearch 6.1.544(Edgar, 2010) against the RDP "Gold" database and filtered out by running the identify_chimeric_seqs.py and filter_fasta.pycommands, respectively.Open reference OTU picking was performed, and taxonomy was assigned to representative OTUs using the pick_open_refernece_otus.py script at 97% sequence identity against the SILVA database v128 (Quast et al., 2013).

| Statistical analyses
Statistical analyses were performed using R software (http://www.rproject.org/),version 3.6.1.Alpha diversity indices (Shannon, Observed and Pielou's evenness) were calculated using the vegan R package (Oksanen et al., 2019).T-tests were used to determine the significant differences in the degradation of chloroethene compounds between NAT and BIOST treatments.T-tests were used to determine the significant differences in alpha diversity and water chemistry between GW and NAT, GW and BIOST, and BIOST and NAT.Bray−Curtis distance, after "Hellinger" transformation, was used to generate a dissimilarity matrix.The bacterial community structure was visualized using principal coordinate analysis (Supporting Information S1: Figure 7).

| Metagenome shotgun sequencing
The genomic DNA extract of NAT treatment after pooling the samples at Day 90 was used for metagenome shotgun sequencing.
Briefly, the quality and quantity of the DNA extract were assessed using a NanoDrop 3300 fluorospectrometer (Thermo Fisher Scientific) and agarose gel electrophoresis.The DNA extract of NAT treatment (1 µg) was then used to prepare a DNA library following the Illumina TruSeq DNA library preparation protocol (for the v2 kit).
Briefly, the quality and quantity assessment for the extracted genomic DNA was performed using the 2100 Bioanalyzer Instrument (Agilent Technologies Inc.), using a DNA 12000 Chip.Following the manufacturer's specifications, the sample was sheared on a Covaris S220 Focused-ultrasonicator to ~550 bp.Library quantitation was performed using the Picogreen assay (Invitrogen), and the average library size was determined by running the libraries on a Bioanalyzer DNA 7500 chip (Agilent).Library concentration was normalized to MG-RAST enables phylogenetic and functional annotation and classification.Taxonomic assignments were obtained by aligning the reads against the M5NR protein database, which provides nonredundant integration of many databases.Functional profiling was done using the SEED (Overbeek et al., 2014) and KEGG (Kanehisa, 2000) databases.KEGG pathways were visualized using KEGG Mapper.To identify hits, a maximum E-value cut-off of E < 1e−5 and 60% identity, with a minimum alignment length of 15 bp (taxonomic information) or 15 amino acids (functional information), were used.
The quality-filtered reads uploaded for annotation are accessible on MG-RAST with the ID: mgm4781912.3.

| Physicochemical analysis of groundwater
The physicochemical analysis of the borehole water samples ( respectively, and no ethene was detected in the groundwater (Table 2).
Non-detection of ethene could be due to the negligible ethene concentration below the detection limit.

| Degradation of PCE
The biotransformation of PCE was assessed after amending with PCE (0.6 mM or 99.5 ppm of PCE final concentration) the groundwater with (BIOST, biostimulation) or without lactate (NAT, natural attenuation).
In the abiotic controls (NAT abiotic and BIOST abiotic), no PCE intermediate compounds (Cl − , TCE, DCEs, and VC) were detected, and negligible degradation of PCE was observed (Supporting Information S1: Figure 2).In addition, DO, Eh, and pH were constant throughout the experiment (Supporting Information S1: Figure 3).Likewise, in the non-amended groundwater or biotic control (BioControl), the observed negligible to slight changes might be due to low bacterial metabolic activities promoted due to the organic carbon bioavailable in the water (Supporting Information S1: Figures 4 and 5 and Supporting Information S1: Table 2).
On the other hand, the pH, Eh, and DO values gradually decreased over time in both NAT (GW + PCE) and BIOST (GW + PCE + LAC) treatments during the degradation of PCE and its daughter products (TCE, DCEs, and VC) (Figure 1).The assessment of temporal evolution of the physicochemical parameters showed neutral (pH from 7.73 to 7.16 on average) and microoxic (DO from 67.20 µM or 2.15 ppm to 11.15 µM or 0.36 ppm on average) conditions throughout the experiments and a significant difference was detected between them (p < 0.05).
Throughout the experiment, PCE was partially degraded in both NAT and BIOST treatments (Figure 2).The degradation of PCE might be due to the defense mechanisms of the bacterial communities against the increase in toxic effects of organic pollutants (Katarína et al., 2019;Murínová & Dercová, 2014).Overall, under microoxic conditions, the degradation of PCE was slightly higher in NAT (GW + PCE) (15.14% on average) compared to BIOST (GW + PCE + LAC) (10.13% on average) (t-test p < 0.05) (Figure 2).A similar result was observed when only PCE was supplied as a carbon source rather than PCE plus cosubstrate, such as glucose (Jebakumar & Legge, 2008).(MCL: 2 ppm) is more toxic than the rest of the chloroethene compounds.Therefore, complete degradation of chloroethene compounds to ethene, the harmless gas, is more desirable.

| Bacterial community structure and composition
After quality filtering, removal of chloroplast and mitochondria and rarefaction, 377,514 good quality sequences were obtained.The clustering of these sequences (97% identity level) resulted in 14,836 OTUs, which were used to assess bacterial community structure and composition.Rarefaction curves suggest that the sequencing depth was adequate to capture prokaryotic diversity in each sample except for GW (Supporting Information S1: Figure 6).Alpha-diversity analysis showed that the richness (the number of observed OTUs) decreased drastically in the NAT and BIOST treatments compared with the GW sample (t-test between GW and NAT [p = 0.015], GW and BIOST [p = 0.015]) (Figure 3).No differences in richness were observed between the NAT and BIOST treatments (p > 0.05) after 90 days.Similar trends were found with the Shannon index (Figure 3).effects of some PCE daughter products, such as TCE, cDCE, and VC, on bacterial species have been reported (Matteucci et al., 2015;Mcmurdie et al., 2011;Mohammadi et al., 2021;Olaniran et al., 2008).
Thus, the increment of PCE daughter products observed in NAT and BIOST treatments might also contribute to shaping the bacterial community.
Proteobacteria (63.8 [mean] ± 5.4% [standard deviation] of the reads) was the most predominant phylum across GW (starting inoculum), BIOST, and NAT samples (Figure 4).Interestingly, after 90 days, Firmicutes were overrepresented in the BIOST samples compared to NAT and GW samples.The analysis supports the idea that BIOST was more selective for specific microbial taxa than GW and NAT.
At the genus level, Pseudomonas was the most abundant and prevalent genus in GW and the treatments (Figure 5).NAT bacterial communities were more similar with GW than with BIOST, most likely because most of the bacterial communities in GW were already adapted to certain concentrations of PCE.A number of bacterial genera found here such as Desulfosporosinus, Sporomusa, and Pseudomonas are commonly reported in chlorothene compoundscontaminated environments and are involved in partial reductive dechlorination of PCE, TCE, and DCEs (Robertson et al., 2001;Terzenbach & Blaut, 1994).
db-RDA at the end of the experiments (Figure 6) showed that the changes in the bacterial communities were associated with changes in water chemistry (p < 0.012).
3.4 | Enzyme-coding genes and key microbial taxa potentially involved in the degradation of chloroethene compounds A wide variety of xenobiotic degradation pathways was found in the assembled contigs (Figure 7).
Indeed, several studies have shown that some members of the genus Pseudomonas are particularly active in the cometabolic degradation of PCE daughter products (Guo et al., 2001;Li et al., 2014;Shukla et al., 2012;Zalesak et al., 2017Zalesak et al., , 2021)).Indeed, P. stutzeri OX1 was found to cometabolise PCE during the oxidation of toluene through the activity of a toluene-o-xylene monooxygenase (ToMO) (Doohyun Ryoo et al., 2000).
998% purity) was used to compensate for the O 2 drop in the samples due to microbial activities.Adjustments of O 2 concentration were made after sampling and monitoring the DO.Under microoxic conditions, 8 mL of each sample was withdrawn using a sterile needle and syringe into an autoclaved 10 mL measuring cylinder and the DO, Eh, and pH were measured using a Multiparameter AP-2000 probe (Aquaread Ltd) inside the microoxic chamber.Concomitantly, 4 mL of each sample was withdrawn into a 10 mL headspace vial screwed with a magnetic screw cap (Sigma-Aldrich) to determine the concentrations of PCE, TCE, and DCEs in the experiment at 0, 10, 20, 30, 50, 70, and 90 days of incubation.VC and chloride concentrations were only analyzed at the beginning (Day 0) and the end (Day 90) of the experiments.2.4 | Genomic DNA extraction and Illumina 16S rRNA gene sequencingThe microbial community composition was analyzed in the starting inocula (GW) and NAT and BIOST samples after 90 days.The DNA was extracted using a Powersoil ® DNA isolation kit (Mo BIO Laboratories Inc.) following the manufacturer's instructions.The quality and quantity of the DNA extracts were assessed using a NanoDrop 3300 fluorospectrometer (Thermo Fisher Scientific) and agarose gel electrophoresis.The 16S rRNA genes (V3 and V4 regions) were amplified using forward (5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′) and reverse (5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′) primers(Herlemann et al., 2011;Michailidou et al., 2021).Reactions were performed in a total volume of 25 μL, consisting of 25 ng DNA template, 1.5 μL (0.3 μM) of each primer, 12.5 μL KAPA HiFi HotStart Ready-mix (1X) (ROCHE), and 7 μL nuclease-free water (WhiteSci).The PCR program included an initial denaturation step of 95°C for 3 min followed by 25 amplification cycles consisting of 98°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by a final extension step at 72°C for 5 min.PCR products were viewed with UV light on 2% agarose gels with GelRed™ (Anatech).The PCR products were then used to prepare a DNA library following the Illumina TruSeq ® Nano DNA Sample Preparation protocol (version 2).
Adonis function in the R package vegan was used to perform a PERMANOVA test, which allows assessing differences in the bacterial composition of the treatments.Differences within treatments were further tested using permutational tests of homogeneity of multivariate dispersions (PERMDISP) with betadisper function from the R package vegan v2.4-1.Distance-based redundancy analysis (db-RDA) was used to summarize the variation in microbial community structure in response to chloroethene compound degradation.The environmental variables were selected based on the variance inflation factors (VIFs), and only VIFs of less than 10 were included in the model.A Venn diagram was used to differentiate unique and shared OTUs (Supporting Information S1: Figure 8).DESeq. 2 v1.24.0 was used to test whether bacterial taxa were differentially abundant across treatments (Supporting Information S1: Figures 9-11).

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nM and validated by qPCR on a ViiA-7 real-time thermocycler (Applied Biosystems) using qPCR primers recommended in Illumina's qPCR protocol and Illumina's PhiX control library as standard.The library was then sequenced on an Illumina MiSeq sequencer at a read-length of 301 bp paired-end with MiSeq v3 reagents at the Centre for Proteomics and Genomics Research.The unassembled DNA sequences were annotated with the Metagenomic Rapid Annotation using subsystems Technology, MG-RAST server v3.0(Keegan et al., 2016) with default parameters.
The relative amounts of daughter products fluctuate throughout the experiments, reaching concentrations below the initial values at the end of the experiments in the case of TCE, cis-1,2-DCE, and trans-1,2-DCE.The degradation of TCE and cis-1,2-DCE in BIOST was higher than in NAT (t-test p < 0.05).The opposite trend was observed in trans-1,2-DCE.Only 1,1-DCE was accumulated during the experiments with higher NAT values (t-test p < 0.05).Consequently, there was an accumulation of VC as well as chloride (Cl − ) in both NAT and BIOST; VC accumulation was higher in NAT (4.16E−03 mM or 0.26 ppm) than in BIOST (1.37E−03 mM or 0.09 ppm) but Cl − accumulation was 2.35 times higher in BIOST (0.73 mM or 25.88 ppm) than in NAT at the end of the experiments (Supporting Information S1: Figure 3).Accumulation of VC is due to the degradation of PCE to TCE and TCE to DCEs (reductive dechlorination).The accumulation of Cl − probably resulted from the cometabolic degradation of chloroethene compounds; this indicated that the in situ microorganisms actively cometabolised and reductively dechlorinated chloroethene compounds in BIOST and NAT.
The degradation of PCE or TCE under microoxic conditions to cis-1,2-DCE or trans-1,2-DCE is of interest because these DCEs are less toxic than PCE and TCE.Hence, the maximum contaminant levels (MCLs) of DCEs (cis 1,2-DCE: 70 ppm and trans-1,2-DCE: 100 ppm) in drinking water as stipulated by the US EPA (2023) are higher than the MCLs of PCE and TCE (5 ppm each).In contrast, VC The dramatic loss of alpha diversity in treatments after 90 days of exposure to PCE (NAT treatment) and PCE plus lactate (BIOST treatment) indicates a strong selection for specific microbial taxa.The key selective factor was likely the addition of external carbon sources (PCE or PCE + LAC) in the treatments.This phenomenon was also reported byCui et al. (2016), where carbon sources induced a shift in both bacterial community diversity and composition.The toxic F I G U R E 1 Physicochemical properties of the NAT (GW + PCE) and BIOST (GW + PCE + LAC) treatments.Each treatment is composed of one experiment in triplicate.PCE, tetrachloroethene.F I G U R E 2 Biotransformation of PCE and its secondary metabolites in NAT and BIOST treatments.The reported values represent the mean of three values ± SE (standard error).y-and x-axes of each graph represent concentrations (mM) and time (days).Red dots: BIOST and blue dots: NAT.PCE, tetrachloroethene.
identified in Bin 73 (Pseudomonas) genes coding for phenol hydrolase P1 oxygenase (also known as phenol-2-monooxygenase) and catechol-2,3-dioxygenase (Figures8 and 9), involved in the degradation of xylene and toluene and the cometabolic degradation of F I G U R E 3 Alpha diversity measures (observed number of OTUs [richness], Chao1 and Shannon diversity index) of GW (starting inocula) and BIOST (Day 90), GW and NAT (Day 90), and NAT (Day 90) and BIOST (Day 90).The line inside each box represents the median, while whiskers represent the lowest and highest levels within the 1.5 interquartile range (IQR).Sample data for experiments (borehole water: in triplicates) in GW, NAT, and BIOST were used for the analysis.

(
2004) reported cometabolic degradation of TCE and phenol by a phenol hydroxylase found in Ralstonia taiwanensis.Similarly,Li et al. (2014) found that the enzyme catechol-1,2-dioxygenase was involved in the cometabolism of TCE and phenol.Nevertheless, further studies are needed to confirm the roles of these enzymes in the MAGs.RdhA reductases (RdhA) and chlorate reductase (ClrA) were among the enzymes listed to produce or consume chlorinecontaining compounds(Ismaeil et al., 2017).On the other hand, it F I G U R E 4 Relative abundance of taxa (phylum level) for BIOST, GW, and NAT.Each treatment is composed of one experiment in triplicate A, B, and C (AB, BB, and CB [BIOST]; AN, BN, CN [NAT], and the starting inoculum [GW] contains AG, BG, and CG).

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I G U R E 5 Relative abundance of taxa (genus level) for BIOST, GW, and NAT.Each treatment is composed of one experiment in triplicate A, B, and C (AB, BB, and CB [BIOST]; AN, BN, CN [NAT], and the starting inoculum [GW] contains AG, BG, and CG).F I G U R E 6 Microbial community composition change (at the end of 90 days) estimated using db-RDA based on Bray−Curtis dissimilarity.Black arrows indicate chloroethene compounds and red arrows depict bacterial genera that were correlated with the microbial communities in the ordination space.Green dots refer to BIOST bacterial communities, brown dots to GW bacterial communities, and blue dots to NAT bacterial communities.db-RDA, distance-based redundancy analysis.F I G U R E 7 Mapping of assembled reads on xenobiotic biodegradation and metabolism pathways present in the KEGG database.

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I G U R E 8 KEGG pathway analysis from RAST annotation showing enzymes required for toluene degradation.1.14.13.7 (phenol-2monooxygenase) and1.13.11.2 (catechol-2,3-dioxygenase).Green boxes indicate enzymes required for toluene degradation could be encoded by genes found in NAT assembled contigs; no shading indicates no match was found.F I G U R E 9 KEGG pathway analysis from RAST annotation showing enzymes required for p-xylene degradation.The blue (catechol-2,3dioxygenase) box indicates an enzyme required for p-xylene degradation that could be encoded by the gene found in NAT-assembled contigs.Boxes with red and black outlines indicate no match was found.established.ClrA found in NAT metagenome might drive the reductive dechlorination of DCE to VC and finally to ethene.4 | CONCLUSIONS Here, we showed that chloroethene compounds were partially biodegraded under microoxic conditions, most likely through the cooccurrence of cometabolic and reductive processes.Enzymes coded by genes such as dmpL, catA, dmpB, todE, and xylE were possibly involved in cometabolism, whereas ClrA (encoded by clrA) was possibly in charge of reductive dechlorination.Our data analysis also provides evidence of the dominance of Proteobacteria taxa in these processes.Overall, this highlights the importance of several bacterial community members and different pathways in the biodegradation of chloroethene compounds under microoxic conditions.AUTHOR CONTRIBUTIONS Abidemi O. Ojo designed the study and conducted the experiment and data analysis.Castillo J., Cason E., and Valverde A. supervised the interpretation of the results.Abidemi O. Ojo prepared the manuscript and all authors provided critical feedback that shaped the manuscript.
T A B L E 2Note: Eh, ORP (meter reading) against the Ag/AgCl reference electrode + 208.The reported values represent the mean of three values ± SE (standard error).Abbreviations: GW, groundwater; ND, not detected; PCE, tetrachloroethene; TCE, trichloroethene; TDS, total dissolved solids; VC, vinyl chloride Blue boxes indicate the presence of specific genes in the NAT metagenome.