Analysis and optimization of single and composite carbon source substrates for acclimation and screening of 1,2-Dichloropropane degrading bacteria

Analysis and optimization of single and composite carbon source substrates for acclimation and screening of 1,2-Dichloropropane degrading bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis and optimization of single and composite carbon source substrates for acclimation and screening of 1,2-Dichloropropane degrading bacteria Fan Jiang, Yujiao Sun, Xueqian Ren, Yujie Sun, Meijun Liu, Guomin Bai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6836071/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 1,2-Dichloropropane (1,2-DCP) is a highly toxic and environmentally persistent chlorinated hydrocarbon pollutant that causes serious contamination of soil and groundwater. This study investigated 1,2-DCP contaminated soil using single and complex carbon source cultivation strategies to screen and acclimate bacterial communities. Through high-throughput sequencing, community structure and functional analysis, and pure culture isolation and identification, the study compared the effects of different carbon substrates on the succession patterns, functional characteristics, and screening advantages of degrading bacterial communities. Results showed that communities acclimated with a single carbon source were dominated by Proteobacteria and gradually diversified, while communities acclimated with complex carbon sources were dominated by Firmicutes and remained relatively stable. Functional prediction revealed that although the two acclimation strategies led to different community structures, functional stability was maintained, with major differences manifested in energy metabolism, ion transport, and DNA repair functions. Four 1,2-DCP degrading strains were isolated and identified, including Microbacterium proteolyticum , Stutzerimonas stutzeri , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Genomic functional prediction suggested that their degradation mechanisms mainly involve monooxygenases, oxidoreductases, and glutathione-related enzyme systems. The study demonstrated that the single carbon source nutrition with pollution pressure has significant advantages in constructing functional bacterial communities, expressing specific functions, and screening specific strains, while complex carbon source cultivation favors the enrichment of microbial communities with broad metabolic capabilities, providing a theoretical foundation and bacterial resources for 1,2-DCP bioremediation. Bioremediation Composite carbon source 1 2-Dichloropropane Degradative strain screening Functional prediction Sole carbon source Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction 1,2-Dichloropropane (1,2-DCP) represents a versatile yet highly toxic chlorinated hydrocarbon contaminant that has been extensively utilized as a solvent, pesticide, and intermediate in organic synthesis[ 1 ]. Due to its volatility, high stability, and environmental persistence, 1,2-DCP has been demonstrated to pose significant ecological risks to soil and groundwater ecosystems, while concurrently presenting substantial threats to human health. Prolonged exposure to 1,2-DCP has been associated with deleterious effects on the central nervous system and visceral organs, and exhibits potential carcinogenicity according to epidemiological and toxicological evidence[ 2 ]. Current remediation technologies for 1,2-DCP contamination primarily encompass physical, chemical, and biological approaches. Physical remediation techniques, such as adsorption and isolation, can rapidly control contaminant migration to a certain extent, but cannot completely remove contaminants[ 3 , 4 ]. Chemical remediation strategies facilitate rapid degradation of contaminants through oxidation-reduction reactions, but may induce secondary contamination and adverse effects on soil ecosystems[ 5 ]. In contrast, bioremediation technologies utilize the metabolic degradation capabilities of microorganisms to reduce 1,2-DCP toxicity, offering significant advantages including cost-effectiveness and environmental friendliness, which has established this approach as a prominent research focus in chlorinated hydrocarbon contamination remediation[ 6 ]. Loffler et al.[ 7 ] conducted enrichment cultures under anaerobic conditions and observed that following a 4-week lag period, 1,2-DCP was transformed into 1-chloropropane and 2-chloropropane, and further converted to propene, indicating that halogenated propanes can be completely dechlorinated by anaerobic bacteria. Maness et al.[ 8 ] demonstrated that Dehalogenimonas alkenigignens and Dehalogenimonas lykanthroporepellens possess the capacity to reductively dechlorinate elevated concentrations of 1,2-dichloroethane, 1,2-DCP, and 1,1,2-trichloroethane. In investigations of microbial degradation of 1,2-DCP, carbon source selection strategies exert substantial influence on degradation efficiency and microbial community acclimation outcomes. In the sole carbon source approach, 1,2-DCP functions as the single carbon and energy source for microbial proliferation, thereby facilitating the screening of functional bacterial strains possessing specialized degradation capabilities. Schmidt et al.[ 9 ] demonstrated aerobic degradation utilizing cis-1,2-dichloroethene as the sole carbon and energy source, establishing a more promising engineered bioremediation approach for cis-dichloroethylene (cDCE) contamination compared to reductive dechlorination or cometabolic degradation pathways. Pan et al.[ 10 ] isolated, purified, and identified a bacterial strain DDT-1 from soil contaminated with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) that could utilize DDT as the sole source of carbon and energy. Compared to single carbon sources, composite carbon source strategies enhance microbial cometabolic degradation of 1,2-DCP through supplementation with readily biodegradable carbon sources including glucose, organic acids, fatty acids, etc., resulting in superior degradation efficiency and accelerated microbial community acclimation kinetics[ 11 ]. Yang et al.[ 12 ] supplemented atrazine-contaminated soil with varying concentrations of biodegradable composite carbon sources; microbial community analyses revealed that the application of composite substrates significantly reduced microbial community richness and diversity, while concurrently enhancing nitrogen metabolism and atrazine degradation efficiency. Dolinov et al.[ 13 ] demonstrated that under aerobic conditions, diverse methanotrophic bacteria, toluene-degrading bacteria, and phenol-degrading bacteria possess the capability to degrade chlorinated ethenes, including perchloroethylene (PCE), trichloroethylene (TCE), cDCE, and vinyl chloride (VC) through cometabolic pathways. Despite the considerable potential of bioremediation, microbial degradation research targeting 1,2-DCP continues to encounter numerous challenges. Primarily, the elevated toxicity and environmental persistence of chlorinated hydrocarbons significantly constrain the degradation efficiency of indigenous microbial communities[ 14 , 15 ]. Additionally, the dynamic succession patterns of microbial communities during 1,2-DCP degradation under diverse cultivation strategies and their ecological mechanisms remain insufficiently elucidated, introducing significant uncertainties for the optimization and practical implementation of microbial remediation approaches[ 16 ]. Furthermore, the mechanistic influence of various carbon substrates on the expression and stability of microbial degradative functional genes remains poorly understood, thereby constraining the identification and application of highly efficient degradative strains. This study aims to address the aforementioned challenges by investigating 1,2-DCP-contaminated soils through acclimatization and enrichment cultivation using both 1,2-DCP as a sole carbon source and combined 1,2-DCP with supplementary organic carbon substrates, thereby evaluating the differential impacts of carbon source compositions on functional microbial consortia development. Integrating high-throughput sequencing and functional analyses, this study comparatively analyzes the successional patterns of microbial community structures, functional characteristics, and the screening advantages of potential remediation strains during the adaptation process to elucidate the influence of medium composition on microbial adaptability. Concurrently, for the highly efficient 1,2-DCP degrading microbial strains screened, their degradation functions and remediation potential will be predicted. This research provides both theoretical foundations and microbial resources for rapid and efficient remediation strategies targeting 1,2-DCP and related halogenated organic contaminants. Materials and Methods Sample Source Soil samples were collected from a 1,2-DCP contaminated site, at depths of 0.5 m and 1.5 m. The collected samples were processed to remove large particles and sieved through a 2 mm mesh, then sealed in zip-lock bags and stored in a refrigerator at 2℃ until use. Culture Media Complex carbon source medium: NaCl 10 g/L, tryptone 10 g/L, yeast extract 5 g/L. Single carbon source inorganic salt medium: Na 2 HPO 4 2440.0 mg/L, KH 2 PO 4 1520.0 mg/L, (NH 4 ) 2 SO 4 500.0 mg/L, MgSO 4 98.0 mg/L, CaCl 2 37.8 mg/L, etc. For solid culture media preparation, 15 g/L of agar was added to the medium. Acclimation and Enrichment of 1,2-DCP Degrading Bacteria Single carbon source acclimation: 20 g of processed soil samples were added to 600 mL of single carbon source medium, with 1,2-DCP (initial concentration of 10 mg/L) as the sole carbon source for acclimation. The mixture was sealed and incubated in a constant temperature incubator at 30℃. Every 5 days, the supernatant was collected and sealed for storage in a refrigerator at 2℃ for future use. Fresh single carbon source medium was replenished to the conical flask, and the concentration of 1,2-DCP was gradually increased (10 mg/L, 20 mg/L, 40 mg/L, and 80 mg/L) to screen for dominant bacterial communities capable of tolerating high pollution concentrations. Complex carbon source acclimation: The procedure for complex carbon source acclimation and enrichment culture was consistent with the above-mentioned single carbon source acclimation. Microbial Community Diversity Analysis To thoroughly investigate the succession changes in microbial communities during the acclimation process, 50 mL samples were collected from the early (0 d), middle (10 d, 20 d), and late (35 d) stages of acclimation. The bacterial cell pellets were collected by centrifugation at 7000 rcf for 10 min at 4℃, and sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for genomic DNA extraction. Subsequently, PCR amplification of the 16S rRNA gene was performed followed by sequencing. The obtained PE (paired-end) reads were assembled based on overlap relationships, while sequence quality control and filtering were conducted. After sample differentiation, OTU (Operational Taxonomic Unit) clustering analysis, taxonomic classification, and community function prediction were carried out. Screening and Identification of Degrading Bacterial Strains Samples were taken from the acclimation culture when the concentration reached 80 mg/L. The gradient dilution method (1, 10 − 1 , 10 − 2 , 10 − 3 , 10 − 4 , and 10 − 5 ) was used to dilute the culture, which was then spread on selective media containing 80 mg/L of 1,2-DCP. After 48 hours of constant temperature incubation at 30℃, different single colonies were selected based on colony characteristics and purified through streak plate method to obtain pure isolates. The isolates were simultaneously inoculated into liquid medium for expanded cultivation. The obtained strains were sent to Beijing Ruibiotech Biological Technology Co., Ltd. for genomic DNA extraction. The universal primers 27F and 1492R were used to amplify the 16S rDNA sequences of the bacteria through PCR to obtain gene sequences[ 17 ]. BLAST tool was used for comparative analysis in the NCBI database to determine the genus and species of the strains. Construction of Metabolic Models for Degrading Bacterial Strains The genome files of the screened degrading bacterial strains were uploaded to KBase. The RAST database was used to re-annotate the data files and compare them with the annotated microbial genome database. Model SEED was utilized to perform functional analysis on the re-annotated genomic data and construct metabolic models of the strains. Results Microbial Community Changes in Alpha Diversity When acclimating degrading bacteria with a single carbon source, the Rank-Abundance curves (Fig. 1) exhibited a steep downward trend, indicating that species with low OTU rankings had higher abundance, while species with high OTU rankings had lower abundance. The four curves covered a wide range on the horizontal axis, suggesting high species richness throughout the acclimation process. Figure 2 shows the Rank-Abundance curves for the degrading bacteria cultured with complex carbon sources, which similarly displayed steep downward trends. At days 5 and 10, species with low OTU rankings had relatively high abundance in the community. However, as time progressed, at days 20 and 35, the relative abundance of species with high OTU rankings gradually increased. The increase in 1,2-DCP concentration during the acclimation process led to varying degrees of changes in microbial diversity indices in different culture media (Table 1). In the single carbon source medium, as time progressed, the Chao index showed a gradual upward trend, while the Shannon index first decreased and then increased. In the complex carbon source medium, the Chao index also gradually increased over time, while the Shannon index first decreased and then increased, especially at days 20 and 35, when the Chao index exceeded 300. The principal component analysis (PCA) results at the OTU level (Fig. 3) indicate that samples from the same acclimation process were relatively closely distributed in the principal component space, suggesting similar microbial community structures. The significant differences between original samples and acclimated samples demonstrate that the acclimation process induced fundamental structural reorganization of the microbial communities. Samples under different culture medium conditions were clearly separated in the PCA plot, indicating that the type of culture medium had a significant impact on microbial community structure. The community succession trajectory of samples using complex carbon source medium was more consistent with the direction of 1,2-DCP concentration changes, suggesting that their community structure changes might respond more directly to the selective pressure of the degradation substrate. Samples acclimated with single carbon source were concentrated within a smaller range and evolved along different directions, indicating that the microbial community structures among these samples were more consistent, and they adopted different adaptation strategies under nutrient-limited conditions. Comparison of Community Structure The microbial community structure exhibited significant succession characteristics during the acclimation period. Figure 4 shows the community structure bar chart of each sample at the phylum level. In the original samples from day 0, the microbial community displayed a well-balanced and nutritious state, with the relative abundance of major bacterial phyla (such as Proteobacteria , Firmicutes , Actinobacteriota , Bacteroidota , etc.) distributed in a relatively balanced state. In the early samples acclimated with single carbon source (days 5 and 10), the microbial community was predominantly dominated by Proteobacteria . This indicates that Proteobacteria has an advantage in nutrient-poor single carbon source environments, with the ability to quickly adapt to and utilize limited resources. By day 20, the microbial community structure became more diverse. The abundance of Actinobacteria and Firmicutes increased significantly, while other phyla such as Chloroflexi began to appear. By day 35, the community composition further diversified, with a significant increase in the proportion of Firmicutes and a decrease in the proportion of Proteobacteria . In the complex carbon source culture system, the community composition remained relatively simple and stable throughout the cultivation period, with Firmicutes consistently dominating the samples and other species representing smaller proportions. To gain a deeper understanding of the impact of different culture media on microbial community structure during the acclimation process, a comparative analysis of the microbial community structure at the genus level (Fig. 5) was conducted. The results showed that the main dominant genera in the original samples were Acinetobacter and Bacillus , accounting for 23.59% and 11.07%, respectively. Compared with the original samples, the composition of the microbial community acclimated with single carbon source underwent significant changes. The abundance of Delftia and Ralstonia showed an increasing trend in the early acclimation period (W5, W10, and W20), while Curvibacter , which dominated in the initial acclimation stage (W5), gradually decreased in abundance during the acclimation process. In the late acclimation period (W35), as the 1,2-DCP concentration increased, the community structure changed significantly. The abundance of the previously dominant genera rapidly decreased, and Arthrobacter became the most dominant genus, accounting for approximately 19.57%. In the early stages of complex carbon source acclimation (LB5 and LB10), Clostridium and Paraclostridium , which had relatively low abundance in the original samples, increased in abundance and became the absolutely dominant genera. In the late acclimation period (LB20 and LB35), Tissierella and Enterococcus increased significantly and became dominant. As shown in the clustering tree (Fig. 6), samples acclimated with complex carbon source and samples acclimated with single carbon source formed two distinct clusters in terms of microbial community structure, with significant differences in the abundance of species (such as Arthrobacter and Paraclostridium ) between different culture media. Functional Prediction of Bacterial Communities The results of COG functional analysis (Fig. 7) indicate that in the single carbon source culture system, the functional abundance of Category P (inorganic ion transport and metabolism) and Category J (translation and ribosomal structure) slightly increased in the late acclimation period (W35), possibly related to the processing of chloride ions during the dehalogenation process, and increased demand for protein synthesis-related functions[ 18 ]. In the complex carbon source culture system, the functional abundance of Category J (translation and ribosomal structure) and Category K (transcription) significantly increased in the late acclimation period (LB20 and LB35), indicating more active gene expression. The functional abundance of Category L (replication, recombination, and repair) showed a notable increasing trend during the acclimation process, suggesting an increased demand for DNA damage repair[ 19 ]. Meanwhile, Category M (cell wall/membrane/envelope biogenesis) showed an upward trend during the acclimation process, possibly related to cellular defense against the toxicity of 1,2-DCP[ 20 ]. A comparative analysis of community functional distribution was conducted on samples from day 35 of acclimation with complex carbon source and single carbon source (LB35 and W35) (Figs. 8 and 9). The results showed that, except for Category D (cell cycle control, cell division, chromosome partitioning) and Category N (cell motility), the gene abundance of functional categories in the single carbon source acclimated sample was higher than that in the complex carbon source acclimated sample. Among them, the sample acclimated with single carbon source had higher functional abundance in Category C (energy production and conversion) and Category P (inorganic ion transport and metabolism). The sample acclimated with complex carbon source had higher proportions in Category J (translation and ribosomal structure) and Category K (transcription) functions, but relatively lower abundance in Category Q (secondary metabolite biosynthesis) functions, Identification of Degrading Bacteria Through acclimation and enrichment of bacterial communities in chlorinated hydrocarbon contaminated soil, 7 strains were successfully isolated and screened (Table 2), including Enterococcus faecalis , Microbacterium proteolyticum , Stutzerimonas stutzeri , Klebsiella pneumoniae , Pseudomonas aeruginosa , Methylorubrum lusitanum strain, and Sphingomonas sp. Among them, Methylorubrum lusitanum strain and Sphingomonas sp. only appeared in the early stages of acclimation. Functional Prediction The microbial degradation of 1,2-DCP involves the synergistic action of multiple enzyme systems, forming a complete metabolic network. Through analysis of the screened strains' species information and corresponding genomic data, their potential degradation mechanisms were revealed (Table 3), including monooxygenases, hydrolases, oxidoreductases, and glutathione-related enzymes. Discussion Comparison of Microbial Community Composition Significant differences in microbial community structures under different cultivation conditions. In the single carbon source medium, the initial community was dominated by Proteobacteria , but over time, Actinobacteria and Firmicutes increased significantly, resulting in a more diverse community structure. This succession process reflects how different bacterial species gradually formed a relatively stable diverse structure through competitive and symbiotic relationships. In the complex carbon source medium, Firmicutes consistently dominated, and the community structure remained simple and stable, indicating that nutritional conditions are key factors in shaping microbial community structure. More degradation-functional bacteria were enriched in the single carbon source medium because under low-nutrient conditions, bacteria need to efficiently utilize the target pollutant as a carbon source to maintain growth, thus making it easier to screen out specialized degrading bacteria. Microorganisms in the complex carbon source medium possessed broader metabolic capabilities but were not necessarily highly specialized, and might tend to enrich species that adapt to complex nutritional environments but have relatively weak pollutant degradation abilities. Comparison of microbial community functions Despite the two acclimation strategies leading to distinctly different microbial community structures, they maintained a considerable degree of stability at the functional level, suggesting that microbial community adaptation to 1,2-DCP might be achieved more through adjusting the expression intensity of specific functional genes rather than changing the overall functional composition[ 21 ]. The bacterial community acclimated to the single carbon source exhibited more prominent functions in energy production and conversion and inorganic ion transport and metabolism, indicating that this microbial community could efficiently utilize 1,2-DCP as the sole carbon source and energy source, while developing good ion balance regulation mechanisms to ensure that chloride ions released during the dehalogenation process could be effectively transported out of the cells, thereby avoiding toxic accumulation and maintaining cellular environmental homeostasis[ 20 ].The sample acclimated with complex carbon source had higher proportions in translation and ribosomal structure and transcription functions, but relatively lower abundance in secondary metabolite biosynthesis functions, indicating that this community utilized rich organic substances in the culture medium as carbon and energy sources, maintaining an active basal metabolic state[ 22 ]. Analysis of bacterial degradation function This study successfully isolated and identified four strains with high efficiency in degrading 1,2-DCP. There were significant differences in the dominant degrading bacterial communities screened under different culture medium conditions. The dominant degrading bacteria in the single carbon source medium were mainly Stutzerimonas stutzeri , Microbacterium proteolyticum , and Klebsiella pneumoniae , while in the complex carbon source medium, the dominant degrading bacteria were primarily Pseudomonas aeruginosa and Klebsiella pneumoniae . Klebsiella pneumoniae was enriched in both single carbon source and complex carbon source media, possibly because of its ability to utilize various organic carbon sources and inorganic compounds as nutritional sources, showing strong adaptability and surviving under different culture conditions[ 23 ]. It may play an auxiliary role in the degradation process, for example, by enhancing the degradation rate of certain organic pollutants through co-metabolism. Previous studies have reported various efficient 1,2-DCP degrading bacteria, including Microbacterium proteolyticum , Stutzerimonas stutzeri , Klebsiella pneumoniae , and Pseudomonas aeruginosa isolated in this study. Although Enterococcus faecalis , Methylorubrum lusitanum strain, and Sphingomonas sp. were detected in this study, it is speculated that these strains may not possess the capability to directly degrade chlorinated hydrocarbons, and their presence may be related to symbiotic relationships or the utilization of secondary metabolites. Microbacterium proteolyticum is a facultatively anaerobic or aerobic Gram-positive rod-shaped bacterium that exhibits strong tolerance to various environmental conditions. Previous studies have shown that Microbacterium strains can degrade BTEX compounds (benzene, toluene, ethylbenzene, and xylene) and naphthalene, demonstrating high degradation capacity for pollutants in groundwater and soil environments[ 24 ]. Rybkina et al.[ 25 ] isolated Microbacterium sp. B51 from soil contaminated with chemical industrial waste, which was capable of oxidizing the ortho-chlorinated rings of 2,2'-DCB and 2,4'-DCB, as well as the para-chlorinated ring of 4,4'-DCB. This strain possesses highly active HOPDA hydrolase (BphD), which exhibits dioxygenase activity toward the carbon atoms at positions 2 and 3 of the ortho-chlorinated ring, cleaving the chlorine atoms. Stutzerimonas stutzeri is an obligate aerobic Gram-negative rod, non-spore-forming, using oxygen as the final electron acceptor. Dijk et al.[ 26 ] studied that Pseudomonas stutzeri JJ could initially degrade 2-chloroethanol to acetaldehyde under denitrifying conditions, further oxidize it to glycolic acid, then metabolize it to glyoxylic acid, and finally enter the tricarboxylic acid (TCA) cycle, achieving complete mineralization. Ryoo et al.[ 27 ] reported that the bacterium Pseudomonas stutzeri OX1 aerobically degraded 0.56 µmol of 2.0 µmol PCE within 21 hours through the expression of toluene-o-xylene monooxygenase (ToMO). Klebsiella pneumoniae is a Gram-negative bacterium, non-spore-forming and non-flagellated, with a relatively thick capsule, widely distributed in various environments and possessing strong adaptability. Li et al.[ 28 ] isolated Klebsiella pneumoniae from underground sediments that could dechlorinate carbon tetrachloride to chloroform. Setlhare et al.[ 29 ] enriched and isolated Klebsiella KZNSA from activated sludge samples, which could utilize 2,4-DCP as the sole carbon and energy source. It possesses catabolic genes encoding enzymes involved in the degradation of 2,4-DCP via the ortho pathway. Pseudomonas aeruginosa is an obligate aerobic Gram-negative bacterium, non-spore forming, capable of forming capsules. Verce et al.[ 30 ] found that Pseudomonas aeruginosa MF1, cultivated with vinyl chloride and dichloroethylene as the main substrates, demonstrated strong co-metabolic capability and could degrade vinyl chloride and dichloroethylene. Stancu[ 31 ] discovered that the genome of Pseudomonas aeruginosa IBBCt8 contains alkB (870 bp) and rhlAB (216 bp) genes, exhibiting high tolerance and degradation efficiency for hexadecane, decane, and cyclohexane. Pseudomonas aeruginosa possesses enzymes involved in the oxidation of alkanes to fatty acids, including alkane hydroxylase, alcohol dehydrogenase, and aldehyde dehydrogenase, which can hydroxylate the C-H bonds in chlorinated hydrocarbons and gradually oxidize them[ 32 ]. Additionally, Pseudomonas aeruginosa can produce abundant rhamnolipid biosurfactants, increasing the water solubility of chlorinated hydrocarbons, making them more accessible for microbial contact and degradation[ 33 ]. Prediction of bacterial strain function Based on genomic functional prediction of the isolated strains, their degradation mechanisms mainly involve monooxygenases, oxidoreductases, and glutathione-related enzyme systems. The FMNH 2 -dependent monooxygenase likely plays a key role in the initial transformation of 1,2-dichloropropane, utilizing FMNH 2 as a cofactor to introduce oxygen atoms into organic molecules, promoting dechlorination reactions. Rudolph et al.[ 34 ] found that flavin-dependent monooxygenases can hydroxylate natural phenols at carbon atoms with hydrogen substituents, transforming pentachlorophenol into the highly active product tetrachlorobenzoquinone. Iwakiri et al.[ 35 ] genetically engineered a Rhodococcus strain KF711, which expresses modified camphor monooxygenase and hybrid dioxygenase, capable of completely degrading pentachloroethane. Oxidoreductases such as F 2+ :NAD + oxidoreductase likely provide essential electron transfer support during the degradation process, facilitating the removal of chlorine atoms and subsequent oxidation reactions[ 36 ]. Glutathione (GSH) is an important antioxidant in cells, playing a crucial role in maintaining redox homeostasis and preventing oxidative stress[ 37 ]. The degradation of 1,2-dichloropropane may produce cytotoxic intermediates or reactive oxygen species, and glutathione-related enzyme systems such as glutathione:hydrogen-peroxide oxidoreductase may play a protective role during the degradation process, participating in detoxification mechanisms with GSH as a co-substrate, and regulating cellular redox homeostasis through maintaining thiol/disulfide balance[ 36 ]. Conclusion This investigation elucidated the influence patterns of carbon source complexity on the succession and functional expression of 1,2-DCP degrading bacterial communities. The single carbon source acclimation strategy is more conducive to screening efficient and specific degrading functional bacteria, suitable for isolating specialized strains targeting specific pollutants. In contrast, the complex carbon source acclimation strategy is more favorable for enriching microbial communities with broad metabolic capabilities, appropriate for bioremediation of complex contaminated environments. This research provides theoretical foundation and bacterial resources for optimizing bioremediation technologies for organic halogenated hydrocarbon pollutants such as 1,2-DCP. Statements & Declarations Data availability No datasets were generated or analysed during the current study. Funding This work was supported by National Natural Science Foundation of China (51978056). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Fan Jiang. The first draft of the manuscript was written by Fan Jiang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Field, J.A., Sierra-Alvarez, R. (2004). Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds. Reviews in environmental Science and Bio/technology, 3, 185-254. Yamada, K., Kumagai, S., Nagoya, T., Endo, G. (2014). Chemical exposure levels in printing workers with cholangiocarcinoma. journal of Occupational Health, 56, 332-338. Mackenzie, K., Battke, J., Koehler, R., Kopinke, F. (2005). Catalytic effects of activated carbon on hydrolysis reactions of chlorinated organic compounds: Part 2. 1, 1, 2, 2-Tetrachloroethane. Applied Catalysis B: Environmental, 59, 171-179. Tseng, H., Su, J., Liang, C. (2011). Synthesis of granular activated carbon/zero valent iron composites for simultaneous adsorption/dechlorination of trichloroethylene. Journal of hazardous materials, 192, 500-506. Wang, C., Zhang, W. (1997). Synthesizing nanoscale iron particles for rapid and complete dechlorination of TCE and PCBs. Environmental science \& technology, 31, 2154-2156. De Wildeman, S., Verstraete, W. (2003). The quest for microbial reductive dechlorination of C 2 to C 4 chloroalkanes is warranted. Applied microbiology and biotechnology, 61, 94-102. Loffler, F.E., Champine, J.E., Ritalahti, K.M., Sprague, S.J., Tiedje, J.M. (1997). Complete reductive dechlorination of 1, 2-dichloropropane by anaerobic bacteria. Applied and environmental microbiology, 63, 2870-2875. Maness, A.D., Bowman, K.S., Yan, J., Rainey, F.A., Moe, W.M. (2012). Dehalogenimonas spp. can reductively dehalogenate high concentrations of 1, 2-dichloroethane, 1, 2-dichloropropane, and 1, 1, 2-trichloroethane. Amb Express, 2, 1-7. Schmidt, K.R., Augenstein, T., Heidinger, M., Ertl, S., Tiehm, A. (2010). Aerobic biodegradation of cis-1,2-dichloroethene as sole carbon source: Stable carbon isotope fractionation and growth characteristics. Chemosphere, 78, 527-532. Pan, X., Lin, D., Zheng, Y., Zhang, Q., Yin, Y., Cai, L., Fang, H., Yu, Y. (2016). Biodegradation of DDT by Stenotrophomonas sp. DDT-1: characterization and genome functional analysis. Scientific reports, 6, 21332. Ma, Q., Yi, J., Tang, Y., Geng, Z., Zhang, C., Sun, W., Liu, Z., Xiong, W., Wu, H., Xie, X. (2024). Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals. Biotechnology Advances, 73, 108380. Yang, Z., Lou, Y., Pan, H., Wang, H., Yang, Q., Sun, Y., Zhuge, Y. (2023). Reinforced Bioremediation of Excessive Nitrate in Atrazine-Contaminated Soil by Biodegradable Composite Carbon Source. Polymers, 15, 2765. Dolinov A, I., V S Trojsov A, M., V C Ern I K, M., N V E Me V C Ek, J., Mach A V C Kov A, J.V.R.I., V S Evc Uu, A. (2017). Microbial degradation of chloroethenes: a review. Environmental Science and Pollution Research, 24, 13262-13283. Juwarkar, A.A., Singh, S.K., Mudhoo, A. (2010). A comprehensive overview of elements in bioremediation. Reviews in Environmental Science and bio/technology, 9, 215-288. Zhang, M., Yoshikawa, M. (2016). An overview of remediation technologies for sites contaminated with volatile organic compounds. Geo-Chicago 2016, 295-301. Yin, H., Meng, L., Li, L., Xiao, J., Liang, L., Huang, N., Shi, Y., Zhao, A., Hou, J. (2025). Polydopamine-modified biochar supported polylactic acid and zero-valent iron affects the functional microbial community structure for 1, 1, 1-trichloroethane removal in simulated groundwater. Chinese Chemical Letters, 36, 110313. Sun, Y., Sun, Y., Ren, X., Xuan, Y., Liu, M., Bai, G., Jiang, F. (2025). Enhancement of volatile fatty acids to extremely high content in fermentation of food waste: Optimization of conditions, microbial functional genes, and mechanisms. Bioresource Technology, 416, 131735. Thomas, P.C. (1973). Microbial protein synthesis. Proceedings of the Nutrition Society, 32, 85-91. Sirbu, B.M., Cortez, D. (2013). DNA damage response: three levels of DNA repair regulation. Cold Spring Harbor perspectives in biology, 5, a12724. Arjoon, A., Olaniran, A.O., Pillay, B. (2013). Co-contamination of water with chlorinated hydrocarbons and heavy metals: challenges and current bioremediation strategies. International Journal of Environmental Science and Technology, 10, 395-412. Chen, Y., Geng, N., Hu, T., Baeyens, J., Wang, S., Su, H. (2022). Adaptive regulation of activated sludge's core functional flora based on granular internal spatial microenvironment. Journal of Environmental Management, 319, 115714. Feng, G., Xie, T., Wang, X., Bai, J., Tang, L., Zhao, H., Wei, W., Wang, M., Zhao, Y. (2018). Metagenomic analysis of microbial community and function involved in cd-contaminated soil. BMC microbiology, 18, 1-13. Pu, D., Zhao, J., Chang, K., Zhuo, X., Cao, B. (2023). “Superbugs” with hypervirulence and carbapenem resistance in Klebsiella pneumoniae: the rise of such emerging nosocomial pathogens in China. Science Bulletin, 68, 2658-2670. Hao, P., Wu, S., Zhang, X., Gou, C., Wang, Y., Wang, L., Zhu, Y., Basang, W., Gao, Y. (2022). Characterization and Degradation Pathways of Microbacterium resistens MZT7, A Novel 17β-Estradiol-Degrading Bacterium. International Journal of Environmental Research and Public Health, 19, 11097. Rybkina, D.O., Plotnikova, E.G., Dorofeeva, L.V., Mironenko, Y.L., Demakov, V.A. (2003). A new aerobic gram-positive bacterium with a unique ability to degrade ortho-and para-chlorinated biphenyls. Microbiology, 72, 672-677. Dijk, J.A., Gerritse, J., Schraa, G., Stams, A.J.M. (2004). Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions. Archives of Microbiology, 182, 514-519. Ryoo, D., Shim, H., Canada, K., Barbieri, P., Wood, T.K. (2000). Aerobic degradation of tetrachloroethylene by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1. Nature Biotechnology, 18, 775-778. Li, X.M., Zhou, S.G., Li, F.B., Wu, C.Y., Zhuang, L., Xu, W., Liu, L. (2009). Fe(III) oxide reduction and carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17. Journal of Applied Microbiology, 106, 130-139. Setlhare, B., Kumar, A., Aregbesola, O.A., Mokoena, M.P., Olaniran, A.O. (2021). 2, 4-dichlorophenol Degradation by Indigenous Pseudomonas sp. P KZNSA and Klebsiella pneumoniae Kp KZNSA: Kinetics, Enzyme Activity and Catabolic Gene Detection. Applied Biochemistry and Microbiology, 57, 656-665. Verce, M.F., Gunsch, C.K., Danko, A.S., Freedman, D.L. (2002). Cometabolism of cis-1,2-Dichloroethene by Aerobic Cultures Grown on Vinyl Chloride as the Primary Substrate. Environmental Science & Technology, 36, 2171-2177. Stancu, M.M. (2022). ALIPHATIC HYDROCARBONS BIODEGRADATION BY A Pseudomonas STRAIN. AgroLife Scientific Journal, 11, Vandecasteele, J.P., Blanchet, D., Tassin, J.P., Bonamy, A.M., Guerrillot, L. (1983). Enzymology of alkane degradation in Pseudomonas aeruginosa. Acta biotechnologica, 3, 339-344. Reis, R.S., Pereira, A.G., Neves, B.C., Freire, D.M. (2011). Gene regulation of rhamnolipid production in Pseudomonas aeruginosa--a review. Bioresource technology, 102, 6377-6384. Rudolph, J., Erbse, A.H., Behlen, L.S., Copley, S.D. (2014). A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum. Biochemistry, 53, 6539-6549. Iwakiri, R., Yoshihira, K., Futagami, T., Goto, M., Furukawa, K., Others (2004). Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase. Bioscience, biotechnology, and biochemistry, 68, 1353-1356. Yakovlev, G., Reda, T., Hirst, J. (2007). Reevaluating the relationship between EPR spectra and enzyme structure for the iron--sulfur clusters in NADH: quinone oxidoreductase. Proceedings of the National Academy of Sciences, 104, 12720-12725. Couto, N., Wood, J., Barber, J. (2016). The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radical Biology and Medicine, 95, 27-42. Tables Table 1 Alpha diversity indices Sample OTUs Chao Shannon Coverage W5 274 237.13 3.16 1 W10 231 264.91 3.06 1 W20 259 256.79 2.82 1 W35 244 291.59 3.74 1 LB5 295 157.24 2.86 1 LB10 152 217.43 2.73 1 LB20 200 301 3.16 1 LB35 295 302.37 3.3 1 Table 2 Identification of 1,2-DCP degrading bacteria Strain No. Closest Related Strain NCBI Reference Sequence Sequence Similarity（%） Carbon Source Substrate 1 Enterococcus faecalis strain SR5 PP218401.1 100 Single Carbon Source, Complex Carbon Source 2 Microbacterium proteolyticum strain TC01-40 MW928414.1 100 Single Carbon Source 3 Stutzerimonas stutzeri strain XQ-3 MT471993.1 100 Single Carbon Source 4 Klebsiella pneumoniae subsp. ozaenae OQ405432.1 99.32 Single Carbon Source, Complex Carbon Source 5 Pseudomonas aeruginosa strain XL1 PP758286.1 100 Complex Carbon Source 6 Methylorubrum lusitanum strain 13635N EU741086.1 100 Single Carbon Source, Complex Carbon Source 7 Sphingomonas sp. strain Rb-3 OP390782.1 99 Single Carbon Source, Complex Carbon Source Table 3 Key Enzymes in 1,2-DCP Degradation Category Enzyme Name Source direction deltag Monooxygenases FMNH2-dependent monooxygenase (butanesulfonate) S. stutzeri 、 K. pneumoniae 、 P.aeruginosa > -71.9142 FMNH2-dependent monooxygenase (methanesulfonate) S. stutzeri 、 K. pneumoniae 、 P.aeruginosa > -76.97 FMNH2-dependent monooxygenase (sulfoacetate) S. stutzeri 、 K. pneumoniae 、 P.aeruginosa > -78.98 FMNH2-dependent monooxygenase (ethanesulfonate) S. stutzeri 、 K. pneumoniae 、 P.aeruginosa > -80.79 Hydrolases propane-1,2-diol hydro-lyase S. stutzeri 、 K. pneumoniae > -9.17 Oxidoreductases Fe²⁺:NAD⁺ oxidoreductase M. proteolyticum 、 S. stutzeri 、 K. pneumoniae 、 P.aeruginosa - 50.3 hydrogen:ferredoxin oxidoreductase M. proteolyticum 、 S. stutzeri 、 K. pneumoniae 、 P.aeruginosa - 22.94 Ferredoxin:NADP + oxidoreductase M. proteolyticum 、 S. stutzeri 、 K. pneumoniae 、 P.aeruginosa > 14.8 Glutathione-related Enzymes glutathione:hydrogen-peroxide oxidoreductase M. proteolyticum 、 S. stutzeri 、 K. pneumoniae 、 P.aeruginosa > -74.15 glutathione:NADP+ oxidoreductase S. stutzeri 、 K. pneumoniae 、 P.aeruginosa - 2.92 glutathione gamma-glutamylaminopeptidase M. proteolyticum 、 S. stutzeri 、 K. pneumoniae 、 P.aeruginosa - -0.46 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6836071","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481492124,"identity":"ff5a9451-064e-40bc-88cb-29549d3c7f31","order_by":0,"name":"Fan Jiang","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Jiang","suffix":""},{"id":481492125,"identity":"c91c0698-b9ea-4b53-853f-0b4caf12abee","order_by":1,"name":"Yujiao Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYNACHgkGBvYeMJOxgXgtPGdI0gICEjlEapGfkXtMmkfGQp5f8u2xxzwMNrIbDjA/e4BPi8GNvDRpHh4Jw5mz89KNeRjSjDccYDM3wKtFIscMpIVxw20Qg+Fw4oYDPGwS+B0G0WK/4eYZkJb/hLUw3IBoSdxwgwek5QBhLQZn3hhbzuGRSJ7Zk5cmOccg2XjmYTYz/A5rzzG88banzraf/ewxiTcVdrJ9x5uf4XcYAwOLBGMP3FIgZiagHqTkA8MPwqpGwSgYBaNgBAMASL8+4UzsI08AAAAASUVORK5CYII=","orcid":"","institution":"Beijing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yujiao","middleName":"","lastName":"Sun","suffix":""},{"id":481492126,"identity":"e1304214-ebc4-489e-a24c-e83c0ae647ae","order_by":2,"name":"Xueqian Ren","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xueqian","middleName":"","lastName":"Ren","suffix":""},{"id":481492127,"identity":"1f03c30c-b0c3-4959-a8c8-f3e5448e71c1","order_by":3,"name":"Yujie Sun","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Sun","suffix":""},{"id":481492128,"identity":"d762ea57-0aed-438a-a7d6-90960d783488","order_by":4,"name":"Meijun Liu","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Meijun","middleName":"","lastName":"Liu","suffix":""},{"id":481492129,"identity":"be3b963c-f937-4e88-9b83-d6f1575b86f5","order_by":5,"name":"Guomin Bai","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Guomin","middleName":"","lastName":"Bai","suffix":""}],"badges":[],"createdAt":"2025-06-06 10:18:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6836071/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6836071/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86237619,"identity":"ac7129f9-b5a3-4853-80a7-dc949f205590","added_by":"auto","created_at":"2025-07-08 09:56:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28294,"visible":true,"origin":"","legend":"\u003cp\u003eRank-Abundance curves during the single carbon source acclimation process. W5, W10, W20, and W35 represent samples from days 5, 10, 20, and 35 of the single carbon source acclimation process, respectively.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/43a8542043ca1d00ca2f5b35.png"},{"id":86237901,"identity":"86c2f391-f8e4-45ce-ac80-2c2afd86c310","added_by":"auto","created_at":"2025-07-08 10:04:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28414,"visible":true,"origin":"","legend":"\u003cp\u003eRank-Abundance curves during the complex carbon source acclimation process. LB5, LB10, LB20, and LB35 represent samples from days 5, 10, 20, and 35 of the complex carbon source acclimation process, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/75d35853c2da767c657ef7c9.png"},{"id":86238787,"identity":"6d34a7c2-bf91-40d2-8d1b-3d04db86dc37","added_by":"auto","created_at":"2025-07-08 10:12:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35903,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) plot of samples. Points labeled with W represent samples acclimated with single carbon source, points labeled with LB represent samples acclimated with complex carbon source, and samples labeled with 0 represent the original samples from day 0. The arrow indicates the direction of the environmental factor 1,2-DCP concentration. PC1 is the principal component with the maximum variance among samples, explaining 37.45% of the variance; PC2 is the principal component with the second largest variance among samples, explaining 18.3% of the variance.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/3218aa68febcd353c3d5eb08.png"},{"id":86237620,"identity":"922b72fd-9bc8-4ea6-be88-178230ff9f32","added_by":"auto","created_at":"2025-07-08 09:56:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40237,"visible":true,"origin":"","legend":"\u003cp\u003eBar chart of community structure at the phylum level for all samples. Sample information are detailed in Fig. 1 and 2\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/8944803ee042e8288e050a2b.png"},{"id":86237902,"identity":"04a7e77a-35c5-4fa1-ac2f-1b0a4e3e1dc8","added_by":"auto","created_at":"2025-07-08 10:04:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54377,"visible":true,"origin":"","legend":"\u003cp\u003eBar chart of community structure at the genus level for all samples. Sample information are detailed in Fig. 1 and 2\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/bd064fdfd78b616d968a97c9.png"},{"id":86237632,"identity":"917bfb96-17f7-4607-8924-3b03cd37ec7d","added_by":"auto","created_at":"2025-07-08 09:56:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":327966,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of microbial communities at the genus level for all samples. Sample information are detailed in Fig. 1 and 2\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/df2b960aafd5c1ce887af856.png"},{"id":86237906,"identity":"03661313-7c0f-4454-acc4-10ebeabc35f2","added_by":"auto","created_at":"2025-07-08 10:04:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":117117,"visible":true,"origin":"","legend":"\u003cp\u003eBar chart of COG functional statistics\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/a1d3824bb24b581974701ef6.png"},{"id":86239068,"identity":"40aecfbe-e7fb-4586-9e0d-a19d21488a37","added_by":"auto","created_at":"2025-07-08 10:20:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":84964,"visible":true,"origin":"","legend":"\u003cp\u003eBar chart of COG functional statistics for sample W35\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/ba05b689007159a417680d14.png"},{"id":86237911,"identity":"309777af-5829-48b6-a4fc-7c799c7b51ba","added_by":"auto","created_at":"2025-07-08 10:04:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":87286,"visible":true,"origin":"","legend":"\u003cp\u003eBar chart of COG functional statistics for sample LB35\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/4316abf14ef0f4246d11e0fb.png"},{"id":88205369,"identity":"34d1481c-ed7b-4f16-818a-38d7cb607893","added_by":"auto","created_at":"2025-08-04 02:41:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1570185,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6836071/v1/a13aba33-da84-4eeb-a33b-dda1a1624571.pdf"}],"financialInterests":"","formattedTitle":"Analysis and optimization of single and composite carbon source substrates for acclimation and screening of 1,2-Dichloropropane degrading bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003e1,2-Dichloropropane (1,2-DCP) represents a versatile yet highly toxic chlorinated hydrocarbon contaminant that has been extensively utilized as a solvent, pesticide, and intermediate in organic synthesis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to its volatility, high stability, and environmental persistence, 1,2-DCP has been demonstrated to pose significant ecological risks to soil and groundwater ecosystems, while concurrently presenting substantial threats to human health. Prolonged exposure to 1,2-DCP has been associated with deleterious effects on the central nervous system and visceral organs, and exhibits potential carcinogenicity according to epidemiological and toxicological evidence[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrent remediation technologies for 1,2-DCP contamination primarily encompass physical, chemical, and biological approaches. Physical remediation techniques, such as adsorption and isolation, can rapidly control contaminant migration to a certain extent, but cannot completely remove contaminants[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Chemical remediation strategies facilitate rapid degradation of contaminants through oxidation-reduction reactions, but may induce secondary contamination and adverse effects on soil ecosystems[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast, bioremediation technologies utilize the metabolic degradation capabilities of microorganisms to reduce 1,2-DCP toxicity, offering significant advantages including cost-effectiveness and environmental friendliness, which has established this approach as a prominent research focus in chlorinated hydrocarbon contamination remediation[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Loffler et al.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] conducted enrichment cultures under anaerobic conditions and observed that following a 4-week lag period, 1,2-DCP was transformed into 1-chloropropane and 2-chloropropane, and further converted to propene, indicating that halogenated propanes can be completely dechlorinated by anaerobic bacteria. Maness et al.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] demonstrated that \u003cem\u003eDehalogenimonas alkenigignens\u003c/em\u003e and \u003cem\u003eDehalogenimonas lykanthroporepellens\u003c/em\u003e possess the capacity to reductively dechlorinate elevated concentrations of 1,2-dichloroethane, 1,2-DCP, and 1,1,2-trichloroethane.\u003c/p\u003e\u003cp\u003eIn investigations of microbial degradation of 1,2-DCP, carbon source selection strategies exert substantial influence on degradation efficiency and microbial community acclimation outcomes. In the sole carbon source approach, 1,2-DCP functions as the single carbon and energy source for microbial proliferation, thereby facilitating the screening of functional bacterial strains possessing specialized degradation capabilities. Schmidt et al.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] demonstrated aerobic degradation utilizing cis-1,2-dichloroethene as the sole carbon and energy source, establishing a more promising engineered bioremediation approach for cis-dichloroethylene (cDCE) contamination compared to reductive dechlorination or cometabolic degradation pathways. Pan et al.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] isolated, purified, and identified a bacterial strain DDT-1 from soil contaminated with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) that could utilize DDT as the sole source of carbon and energy. Compared to single carbon sources, composite carbon source strategies enhance microbial cometabolic degradation of 1,2-DCP through supplementation with readily biodegradable carbon sources including glucose, organic acids, fatty acids, etc., resulting in superior degradation efficiency and accelerated microbial community acclimation kinetics[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Yang et al.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] supplemented atrazine-contaminated soil with varying concentrations of biodegradable composite carbon sources; microbial community analyses revealed that the application of composite substrates significantly reduced microbial community richness and diversity, while concurrently enhancing nitrogen metabolism and atrazine degradation efficiency. Dolinov et al.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] demonstrated that under aerobic conditions, diverse methanotrophic bacteria, toluene-degrading bacteria, and phenol-degrading bacteria possess the capability to degrade chlorinated ethenes, including perchloroethylene (PCE), trichloroethylene (TCE), cDCE, and vinyl chloride (VC) through cometabolic pathways.\u003c/p\u003e\u003cp\u003eDespite the considerable potential of bioremediation, microbial degradation research targeting 1,2-DCP continues to encounter numerous challenges. Primarily, the elevated toxicity and environmental persistence of chlorinated hydrocarbons significantly constrain the degradation efficiency of indigenous microbial communities[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, the dynamic succession patterns of microbial communities during 1,2-DCP degradation under diverse cultivation strategies and their ecological mechanisms remain insufficiently elucidated, introducing significant uncertainties for the optimization and practical implementation of microbial remediation approaches[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, the mechanistic influence of various carbon substrates on the expression and stability of microbial degradative functional genes remains poorly understood, thereby constraining the identification and application of highly efficient degradative strains.\u003c/p\u003e\u003cp\u003eThis study aims to address the aforementioned challenges by investigating 1,2-DCP-contaminated soils through acclimatization and enrichment cultivation using both 1,2-DCP as a sole carbon source and combined 1,2-DCP with supplementary organic carbon substrates, thereby evaluating the differential impacts of carbon source compositions on functional microbial consortia development. Integrating high-throughput sequencing and functional analyses, this study comparatively analyzes the successional patterns of microbial community structures, functional characteristics, and the screening advantages of potential remediation strains during the adaptation process to elucidate the influence of medium composition on microbial adaptability. Concurrently, for the highly efficient 1,2-DCP degrading microbial strains screened, their degradation functions and remediation potential will be predicted. This research provides both theoretical foundations and microbial resources for rapid and efficient remediation strategies targeting 1,2-DCP and related halogenated organic contaminants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample Source\u003c/h2\u003e\u003cp\u003eSoil samples were collected from a 1,2-DCP contaminated site, at depths of 0.5 m and 1.5 m. The collected samples were processed to remove large particles and sieved through a 2 mm mesh, then sealed in zip-lock bags and stored in a refrigerator at 2℃ until use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCulture Media\u003c/h3\u003e\n\u003cp\u003eComplex carbon source medium: NaCl 10 g/L, tryptone 10 g/L, yeast extract 5 g/L. Single carbon source inorganic salt medium: Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 2440.0 mg/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1520.0 mg/L, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 500.0 mg/L, MgSO\u003csub\u003e4\u003c/sub\u003e 98.0 mg/L, CaCl\u003csub\u003e2\u003c/sub\u003e 37.8 mg/L, etc. For solid culture media preparation, 15 g/L of agar was added to the medium.\u003c/p\u003e\n\u003ch3\u003eAcclimation and Enrichment of 1,2-DCP Degrading Bacteria\u003c/h3\u003e\n\u003cp\u003eSingle carbon source acclimation: 20 g of processed soil samples were added to 600 mL of single carbon source medium, with 1,2-DCP (initial concentration of 10 mg/L) as the sole carbon source for acclimation. The mixture was sealed and incubated in a constant temperature incubator at 30℃. Every 5 days, the supernatant was collected and sealed for storage in a refrigerator at 2℃ for future use. Fresh single carbon source medium was replenished to the conical flask, and the concentration of 1,2-DCP was gradually increased (10 mg/L, 20 mg/L, 40 mg/L, and 80 mg/L) to screen for dominant bacterial communities capable of tolerating high pollution concentrations.\u003c/p\u003e\u003cp\u003eComplex carbon source acclimation: The procedure for complex carbon source acclimation and enrichment culture was consistent with the above-mentioned single carbon source acclimation.\u003c/p\u003e\n\u003ch3\u003eMicrobial Community Diversity Analysis\u003c/h3\u003e\n\u003cp\u003eTo thoroughly investigate the succession changes in microbial communities during the acclimation process, 50 mL samples were collected from the early (0 d), middle (10 d, 20 d), and late (35 d) stages of acclimation. The bacterial cell pellets were collected by centrifugation at 7000 rcf for 10 min at 4℃, and sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for genomic DNA extraction. Subsequently, PCR amplification of the 16S rRNA gene was performed followed by sequencing. The obtained PE (paired-end) reads were assembled based on overlap relationships, while sequence quality control and filtering were conducted. After sample differentiation, OTU (Operational Taxonomic Unit) clustering analysis, taxonomic classification, and community function prediction were carried out.\u003c/p\u003e\n\u003ch3\u003eScreening and Identification of Degrading Bacterial Strains\u003c/h3\u003e\n\u003cp\u003eSamples were taken from the acclimation culture when the concentration reached 80 mg/L. The gradient dilution method (1, 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) was used to dilute the culture, which was then spread on selective media containing 80 mg/L of 1,2-DCP. After 48 hours of constant temperature incubation at 30℃, different single colonies were selected based on colony characteristics and purified through streak plate method to obtain pure isolates. The isolates were simultaneously inoculated into liquid medium for expanded cultivation. The obtained strains were sent to Beijing Ruibiotech Biological Technology Co., Ltd. for genomic DNA extraction. The universal primers 27F and 1492R were used to amplify the 16S rDNA sequences of the bacteria through PCR to obtain gene sequences[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. BLAST tool was used for comparative analysis in the NCBI database to determine the genus and species of the strains.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of Metabolic Models for Degrading Bacterial Strains\u003c/h2\u003e\u003cp\u003eThe genome files of the screened degrading bacterial strains were uploaded to KBase. The RAST database was used to re-annotate the data files and compare them with the annotated microbial genome database. Model SEED was utilized to perform functional analysis on the re-annotated genomic data and construct metabolic models of the strains.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eMicrobial Community\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003eChanges in Alpha Diversity\u003c/h2\u003e\u003cp\u003eWhen acclimating degrading bacteria with a single carbon source, the Rank-Abundance curves (Fig.\u0026nbsp;1) exhibited a steep downward trend, indicating that species with low OTU rankings had higher abundance, while species with high OTU rankings had lower abundance. The four curves covered a wide range on the horizontal axis, suggesting high species richness throughout the acclimation process. Figure\u0026nbsp;2 shows the Rank-Abundance curves for the degrading bacteria cultured with complex carbon sources, which similarly displayed steep downward trends. At days 5 and 10, species with low OTU rankings had relatively high abundance in the community. However, as time progressed, at days 20 and 35, the relative abundance of species with high OTU rankings gradually increased.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe increase in 1,2-DCP concentration during the acclimation process led to varying degrees of changes in microbial diversity indices in different culture media (Table\u0026nbsp;1). In the single carbon source medium, as time progressed, the Chao index showed a gradual upward trend, while the Shannon index first decreased and then increased. In the complex carbon source medium, the Chao index also gradually increased over time, while the Shannon index first decreased and then increased, especially at days 20 and 35, when the Chao index exceeded 300.\u003c/p\u003e\u003cp\u003eThe principal component analysis (PCA) results at the OTU level (Fig.\u0026nbsp;3) indicate that samples from the same acclimation process were relatively closely distributed in the principal component space, suggesting similar microbial community structures. The significant differences between original samples and acclimated samples demonstrate that the acclimation process induced fundamental structural reorganization of the microbial communities. Samples under different culture medium conditions were clearly separated in the PCA plot, indicating that the type of culture medium had a significant impact on microbial community structure. The community succession trajectory of samples using complex carbon source medium was more consistent with the direction of 1,2-DCP concentration changes, suggesting that their community structure changes might respond more directly to the selective pressure of the degradation substrate. Samples acclimated with single carbon source were concentrated within a smaller range and evolved along different directions, indicating that the microbial community structures among these samples were more consistent, and they adopted different adaptation strategies under nutrient-limited conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eComparison of Community Structure\u003c/h2\u003e\u003cp\u003eThe microbial community structure exhibited significant succession characteristics during the acclimation period. Figure\u0026nbsp;4 shows the community structure bar chart of each sample at the phylum level. In the original samples from day 0, the microbial community displayed a well-balanced and nutritious state, with the relative abundance of major bacterial phyla (such as \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eActinobacteriota\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, etc.) distributed in a relatively balanced state. In the early samples acclimated with single carbon source (days 5 and 10), the microbial community was predominantly dominated by \u003cem\u003eProteobacteria\u003c/em\u003e. This indicates that \u003cem\u003eProteobacteria\u003c/em\u003e has an advantage in nutrient-poor single carbon source environments, with the ability to quickly adapt to and utilize limited resources. By day 20, the microbial community structure became more diverse. The abundance of \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e increased significantly, while other phyla such as \u003cem\u003eChloroflexi\u003c/em\u003e began to appear. By day 35, the community composition further diversified, with a significant increase in the proportion of \u003cem\u003eFirmicutes\u003c/em\u003e and a decrease in the proportion of \u003cem\u003eProteobacteria\u003c/em\u003e. In the complex carbon source culture system, the community composition remained relatively simple and stable throughout the cultivation period, with \u003cem\u003eFirmicutes\u003c/em\u003e consistently dominating the samples and other species representing smaller proportions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo gain a deeper understanding of the impact of different culture media on microbial community structure during the acclimation process, a comparative analysis of the microbial community structure at the genus level (Fig.\u0026nbsp;5) was conducted. The results showed that the main dominant genera in the original samples were \u003cem\u003eAcinetobacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e, accounting for 23.59% and 11.07%, respectively. Compared with the original samples, the composition of the microbial community acclimated with single carbon source underwent significant changes. The abundance of \u003cem\u003eDelftia\u003c/em\u003e and \u003cem\u003eRalstonia\u003c/em\u003e showed an increasing trend in the early acclimation period (W5, W10, and W20), while \u003cem\u003eCurvibacter\u003c/em\u003e, which dominated in the initial acclimation stage (W5), gradually decreased in abundance during the acclimation process. In the late acclimation period (W35), as the 1,2-DCP concentration increased, the community structure changed significantly. The abundance of the previously dominant genera rapidly decreased, and \u003cem\u003eArthrobacter\u003c/em\u003e became the most dominant genus, accounting for approximately 19.57%. In the early stages of complex carbon source acclimation (LB5 and LB10), \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eParaclostridium\u003c/em\u003e, which had relatively low abundance in the original samples, increased in abundance and became the absolutely dominant genera. In the late acclimation period (LB20 and LB35), \u003cem\u003eTissierella\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e increased significantly and became dominant. As shown in the clustering tree (Fig.\u0026nbsp;6), samples acclimated with complex carbon source and samples acclimated with single carbon source formed two distinct clusters in terms of microbial community structure, with significant differences in the abundance of species (such as \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003eParaclostridium\u003c/em\u003e) between different culture media.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFunctional Prediction of Bacterial Communities\u003c/h2\u003e\u003cp\u003eThe results of COG functional analysis (Fig.\u0026nbsp;7) indicate that in the single carbon source culture system, the functional abundance of Category P (inorganic ion transport and metabolism) and Category J (translation and ribosomal structure) slightly increased in the late acclimation period (W35), possibly related to the processing of chloride ions during the dehalogenation process, and increased demand for protein synthesis-related functions[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the complex carbon source culture system, the functional abundance of Category J (translation and ribosomal structure) and Category K (transcription) significantly increased in the late acclimation period (LB20 and LB35), indicating more active gene expression. The functional abundance of Category L (replication, recombination, and repair) showed a notable increasing trend during the acclimation process, suggesting an increased demand for DNA damage repair[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Meanwhile, Category M (cell wall/membrane/envelope biogenesis) showed an upward trend during the acclimation process, possibly related to cellular defense against the toxicity of 1,2-DCP[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA comparative analysis of community functional distribution was conducted on samples from day 35 of acclimation with complex carbon source and single carbon source (LB35 and W35) (Figs.\u0026nbsp;8 and 9). The results showed that, except for Category D (cell cycle control, cell division, chromosome partitioning) and Category N (cell motility), the gene abundance of functional categories in the single carbon source acclimated sample was higher than that in the complex carbon source acclimated sample. Among them, the sample acclimated with single carbon source had higher functional abundance in Category C (energy production and conversion) and Category P (inorganic ion transport and metabolism). The sample acclimated with complex carbon source had higher proportions in Category J (translation and ribosomal structure) and Category K (transcription) functions, but relatively lower abundance in Category Q (secondary metabolite biosynthesis) functions,\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of Degrading Bacteria\u003c/h2\u003e\u003cp\u003eThrough acclimation and enrichment of bacterial communities in chlorinated hydrocarbon contaminated soil, 7 strains were successfully isolated and screened (Table\u0026nbsp;2), including \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eMicrobacterium proteolyticum\u003c/em\u003e, \u003cem\u003eStutzerimonas stutzeri\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eMethylorubrum lusitanum\u003c/em\u003e strain, and \u003cem\u003eSphingomonas\u003c/em\u003e sp. Among them, \u003cem\u003eMethylorubrum lusitanum\u003c/em\u003e strain and \u003cem\u003eSphingomonas\u003c/em\u003e sp. only appeared in the early stages of acclimation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eFunctional Prediction\u003c/h2\u003e\u003cp\u003eThe microbial degradation of 1,2-DCP involves the synergistic action of multiple enzyme systems, forming a complete metabolic network. Through analysis of the screened strains' species information and corresponding genomic data, their potential degradation mechanisms were revealed (Table\u0026nbsp;3), including monooxygenases, hydrolases, oxidoreductases, and glutathione-related enzymes.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eComparison of Microbial Community Composition\u003c/h2\u003e\u003cp\u003eSignificant differences in microbial community structures under different cultivation conditions. In the single carbon source medium, the initial community was dominated by \u003cem\u003eProteobacteria\u003c/em\u003e, but over time, \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e increased significantly, resulting in a more diverse community structure. This succession process reflects how different bacterial species gradually formed a relatively stable diverse structure through competitive and symbiotic relationships. In the complex carbon source medium, \u003cem\u003eFirmicutes\u003c/em\u003e consistently dominated, and the community structure remained simple and stable, indicating that nutritional conditions are key factors in shaping microbial community structure. More degradation-functional bacteria were enriched in the single carbon source medium because under low-nutrient conditions, bacteria need to efficiently utilize the target pollutant as a carbon source to maintain growth, thus making it easier to screen out specialized degrading bacteria. Microorganisms in the complex carbon source medium possessed broader metabolic capabilities but were not necessarily highly specialized, and might tend to enrich species that adapt to complex nutritional environments but have relatively weak pollutant degradation abilities.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eComparison of microbial community functions\u003c/h2\u003e\u003cp\u003eDespite the two acclimation strategies leading to distinctly different microbial community structures, they maintained a considerable degree of stability at the functional level, suggesting that microbial community adaptation to 1,2-DCP might be achieved more through adjusting the expression intensity of specific functional genes rather than changing the overall functional composition[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The bacterial community acclimated to the single carbon source exhibited more prominent functions in energy production and conversion and inorganic ion transport and metabolism, indicating that this microbial community could efficiently utilize 1,2-DCP as the sole carbon source and energy source, while developing good ion balance regulation mechanisms to ensure that chloride ions released during the dehalogenation process could be effectively transported out of the cells, thereby avoiding toxic accumulation and maintaining cellular environmental homeostasis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].The sample acclimated with complex carbon source had higher proportions in translation and ribosomal structure and transcription functions, but relatively lower abundance in secondary metabolite biosynthesis functions, indicating that this community utilized rich organic substances in the culture medium as carbon and energy sources, maintaining an active basal metabolic state[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of bacterial degradation function\u003c/h2\u003e\u003cp\u003eThis study successfully isolated and identified four strains with high efficiency in degrading 1,2-DCP. There were significant differences in the dominant degrading bacterial communities screened under different culture medium conditions. The dominant degrading bacteria in the single carbon source medium were mainly \u003cem\u003eStutzerimonas stutzeri\u003c/em\u003e, \u003cem\u003eMicrobacterium proteolyticum\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, while in the complex carbon source medium, the dominant degrading bacteria were primarily \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e was enriched in both single carbon source and complex carbon source media, possibly because of its ability to utilize various organic carbon sources and inorganic compounds as nutritional sources, showing strong adaptability and surviving under different culture conditions[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It may play an auxiliary role in the degradation process, for example, by enhancing the degradation rate of certain organic pollutants through co-metabolism.\u003c/p\u003e\u003cp\u003ePrevious studies have reported various efficient 1,2-DCP degrading bacteria, including \u003cem\u003eMicrobacterium proteolyticum\u003c/em\u003e, \u003cem\u003eStutzerimonas stutzeri\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e isolated in this study. Although \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eMethylorubrum lusitanum\u003c/em\u003e strain, and \u003cem\u003eSphingomonas\u003c/em\u003e sp. were detected in this study, it is speculated that these strains may not possess the capability to directly degrade chlorinated hydrocarbons, and their presence may be related to symbiotic relationships or the utilization of secondary metabolites.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMicrobacterium proteolyticum\u003c/em\u003e is a facultatively anaerobic or aerobic Gram-positive rod-shaped bacterium that exhibits strong tolerance to various environmental conditions. Previous studies have shown that \u003cem\u003eMicrobacterium\u003c/em\u003e strains can degrade BTEX compounds (benzene, toluene, ethylbenzene, and xylene) and naphthalene, demonstrating high degradation capacity for pollutants in groundwater and soil environments[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Rybkina et al.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] isolated \u003cem\u003eMicrobacterium\u003c/em\u003e sp. B51 from soil contaminated with chemical industrial waste, which was capable of oxidizing the ortho-chlorinated rings of 2,2'-DCB and 2,4'-DCB, as well as the para-chlorinated ring of 4,4'-DCB. This strain possesses highly active HOPDA hydrolase (BphD), which exhibits dioxygenase activity toward the carbon atoms at positions 2 and 3 of the ortho-chlorinated ring, cleaving the chlorine atoms.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStutzerimonas stutzeri\u003c/em\u003e is an obligate aerobic Gram-negative rod, non-spore-forming, using oxygen as the final electron acceptor. Dijk et al.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] studied that \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e JJ could initially degrade 2-chloroethanol to acetaldehyde under denitrifying conditions, further oxidize it to glycolic acid, then metabolize it to glyoxylic acid, and finally enter the tricarboxylic acid (TCA) cycle, achieving complete mineralization. Ryoo et al.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] reported that the bacterium \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e OX1 aerobically degraded 0.56 \u0026micro;mol of 2.0 \u0026micro;mol PCE within 21 hours through the expression of toluene-o-xylene monooxygenase (ToMO).\u003c/p\u003e\u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is a Gram-negative bacterium, non-spore-forming and non-flagellated, with a relatively thick capsule, widely distributed in various environments and possessing strong adaptability. Li et al.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] isolated \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e from underground sediments that could dechlorinate carbon tetrachloride to chloroform. Setlhare et al.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] enriched and isolated \u003cem\u003eKlebsiella\u003c/em\u003e KZNSA from activated sludge samples, which could utilize 2,4-DCP as the sole carbon and energy source. It possesses catabolic genes encoding enzymes involved in the degradation of 2,4-DCP via the ortho pathway.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is an obligate aerobic Gram-negative bacterium, non-spore forming, capable of forming capsules. Verce et al.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] found that \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e MF1, cultivated with vinyl chloride and dichloroethylene as the main substrates, demonstrated strong co-metabolic capability and could degrade vinyl chloride and dichloroethylene. Stancu[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] discovered that the genome of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e IBBCt8 contains alkB (870 bp) and rhlAB (216 bp) genes, exhibiting high tolerance and degradation efficiency for hexadecane, decane, and cyclohexane. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e possesses enzymes involved in the oxidation of alkanes to fatty acids, including alkane hydroxylase, alcohol dehydrogenase, and aldehyde dehydrogenase, which can hydroxylate the C-H bonds in chlorinated hydrocarbons and gradually oxidize them[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e can produce abundant rhamnolipid biosurfactants, increasing the water solubility of chlorinated hydrocarbons, making them more accessible for microbial contact and degradation[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003ePrediction of bacterial strain function\u003c/h2\u003e\u003cp\u003eBased on genomic functional prediction of the isolated strains, their degradation mechanisms mainly involve monooxygenases, oxidoreductases, and glutathione-related enzyme systems. The FMNH\u003csub\u003e2\u003c/sub\u003e-dependent monooxygenase likely plays a key role in the initial transformation of 1,2-dichloropropane, utilizing FMNH\u003csub\u003e2\u003c/sub\u003e as a cofactor to introduce oxygen atoms into organic molecules, promoting dechlorination reactions. Rudolph et al.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] found that flavin-dependent monooxygenases can hydroxylate natural phenols at carbon atoms with hydrogen substituents, transforming pentachlorophenol into the highly active product tetrachlorobenzoquinone. Iwakiri et al.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] genetically engineered a \u003cem\u003eRhodococcus\u003c/em\u003e strain KF711, which expresses modified camphor monooxygenase and hybrid dioxygenase, capable of completely degrading pentachloroethane. Oxidoreductases such as F\u003csup\u003e2+\u003c/sup\u003e:NAD\u003csup\u003e+\u003c/sup\u003e oxidoreductase likely provide essential electron transfer support during the degradation process, facilitating the removal of chlorine atoms and subsequent oxidation reactions[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Glutathione (GSH) is an important antioxidant in cells, playing a crucial role in maintaining redox homeostasis and preventing oxidative stress[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The degradation of 1,2-dichloropropane may produce cytotoxic intermediates or reactive oxygen species, and glutathione-related enzyme systems such as glutathione:hydrogen-peroxide oxidoreductase may play a protective role during the degradation process, participating in detoxification mechanisms with GSH as a co-substrate, and regulating cellular redox homeostasis through maintaining thiol/disulfide balance[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis investigation elucidated the influence patterns of carbon source complexity on the succession and functional expression of 1,2-DCP degrading bacterial communities. The single carbon source acclimation strategy is more conducive to screening efficient and specific degrading functional bacteria, suitable for isolating specialized strains targeting specific pollutants. In contrast, the complex carbon source acclimation strategy is more favorable for enriching microbial communities with broad metabolic capabilities, appropriate for bioremediation of complex contaminated environments. This research provides theoretical foundation and bacterial resources for optimizing bioremediation technologies for organic halogenated hydrocarbon pollutants such as 1,2-DCP.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (51978056).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Fan Jiang. The first draft of the manuscript was written by Fan Jiang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eField, J.A., Sierra-Alvarez, R. (2004). Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds. Reviews in environmental Science and Bio/technology, 3, 185-254.\u003c/li\u003e\n\u003cli\u003eYamada, K., Kumagai, S., Nagoya, T., Endo, G. (2014). Chemical exposure levels in printing workers with cholangiocarcinoma. journal of Occupational Health, 56, 332-338.\u003c/li\u003e\n\u003cli\u003eMackenzie, K., Battke, J., Koehler, R., Kopinke, F. (2005). Catalytic effects of activated carbon on hydrolysis reactions of chlorinated organic compounds: Part 2. 1, 1, 2, 2-Tetrachloroethane. Applied Catalysis B: Environmental, 59, 171-179.\u003c/li\u003e\n\u003cli\u003eTseng, H., Su, J., Liang, C. (2011). Synthesis of granular activated carbon/zero valent iron composites for simultaneous adsorption/dechlorination of trichloroethylene. Journal of hazardous materials, 192, 500-506.\u003c/li\u003e\n\u003cli\u003eWang, C., Zhang, W. (1997). Synthesizing nanoscale iron particles for rapid and complete dechlorination of TCE and PCBs. Environmental science \\\u0026amp; technology, 31, 2154-2156.\u003c/li\u003e\n\u003cli\u003eDe Wildeman, S., Verstraete, W. (2003). The quest for microbial reductive dechlorination of C 2 to C 4 chloroalkanes is warranted. Applied microbiology and biotechnology, 61, 94-102.\u003c/li\u003e\n\u003cli\u003eLoffler, F.E., Champine, J.E., Ritalahti, K.M., Sprague, S.J., Tiedje, J.M. (1997). Complete reductive dechlorination of 1, 2-dichloropropane by anaerobic bacteria. Applied and environmental microbiology, 63, 2870-2875.\u003c/li\u003e\n\u003cli\u003eManess, A.D., Bowman, K.S., Yan, J., Rainey, F.A., Moe, W.M. (2012). Dehalogenimonas spp. can reductively dehalogenate high concentrations of 1, 2-dichloroethane, 1, 2-dichloropropane, and 1, 1, 2-trichloroethane. Amb Express, 2, 1-7.\u003c/li\u003e\n\u003cli\u003eSchmidt, K.R., Augenstein, T., Heidinger, M., Ertl, S., Tiehm, A. (2010). Aerobic biodegradation of cis-1,2-dichloroethene as sole carbon source: Stable carbon isotope fractionation and growth characteristics. Chemosphere, 78, 527-532.\u003c/li\u003e\n\u003cli\u003ePan, X., Lin, D., Zheng, Y., Zhang, Q., Yin, Y., Cai, L., Fang, H., Yu, Y. (2016). Biodegradation of DDT by Stenotrophomonas sp. DDT-1: characterization and genome functional analysis. Scientific reports, 6, 21332.\u003c/li\u003e\n\u003cli\u003eMa, Q., Yi, J., Tang, Y., Geng, Z., Zhang, C., Sun, W., Liu, Z., Xiong, W., Wu, H., Xie, X. (2024). Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals. Biotechnology Advances, 73, 108380.\u003c/li\u003e\n\u003cli\u003eYang, Z., Lou, Y., Pan, H., Wang, H., Yang, Q., Sun, Y., Zhuge, Y. (2023). Reinforced Bioremediation of Excessive Nitrate in Atrazine-Contaminated Soil by Biodegradable Composite Carbon Source. Polymers, 15, 2765.\u003c/li\u003e\n\u003cli\u003eDolinov A, I., V S Trojsov A, M., V C Ern I K, M., N V E Me V C Ek, J., Mach A V C Kov A, J.V.R.I., V S Evc Uu, A. (2017). Microbial degradation of chloroethenes: a review. Environmental Science and Pollution Research, 24, 13262-13283.\u003c/li\u003e\n\u003cli\u003eJuwarkar, A.A., Singh, S.K., Mudhoo, A. (2010). A comprehensive overview of elements in bioremediation. Reviews in Environmental Science and bio/technology, 9, 215-288.\u003c/li\u003e\n\u003cli\u003eZhang, M., Yoshikawa, M. (2016). An overview of remediation technologies for sites contaminated with volatile organic compounds. Geo-Chicago 2016, 295-301.\u003c/li\u003e\n\u003cli\u003eYin, H., Meng, L., Li, L., Xiao, J., Liang, L., Huang, N., Shi, Y., Zhao, A., Hou, J. (2025). Polydopamine-modified biochar supported polylactic acid and zero-valent iron affects the functional microbial community structure for 1, 1, 1-trichloroethane removal in simulated groundwater. Chinese Chemical Letters, 36, 110313.\u003c/li\u003e\n\u003cli\u003eSun, Y., Sun, Y., Ren, X., Xuan, Y., Liu, M., Bai, G., Jiang, F. (2025). Enhancement of volatile fatty acids to extremely high content in fermentation of food waste: Optimization of conditions, microbial functional genes, and mechanisms. Bioresource Technology, 416, 131735.\u003c/li\u003e\n\u003cli\u003eThomas, P.C. (1973). Microbial protein synthesis. Proceedings of the Nutrition Society, 32, 85-91.\u003c/li\u003e\n\u003cli\u003eSirbu, B.M., Cortez, D. (2013). DNA damage response: three levels of DNA repair regulation. Cold Spring Harbor perspectives in biology, 5, a12724.\u003c/li\u003e\n\u003cli\u003eArjoon, A., Olaniran, A.O., Pillay, B. (2013). Co-contamination of water with chlorinated hydrocarbons and heavy metals: challenges and current bioremediation strategies. International Journal of Environmental Science and Technology, 10, 395-412.\u003c/li\u003e\n\u003cli\u003eChen, Y., Geng, N., Hu, T., Baeyens, J., Wang, S., Su, H. (2022). Adaptive regulation of activated sludge\u0026apos;s core functional flora based on granular internal spatial microenvironment. Journal of Environmental Management, 319, 115714.\u003c/li\u003e\n\u003cli\u003eFeng, G., Xie, T., Wang, X., Bai, J., Tang, L., Zhao, H., Wei, W., Wang, M., Zhao, Y. (2018). Metagenomic analysis of microbial community and function involved in cd-contaminated soil. BMC microbiology, 18, 1-13.\u003c/li\u003e\n\u003cli\u003ePu, D., Zhao, J., Chang, K., Zhuo, X., Cao, B. (2023). \u0026ldquo;Superbugs\u0026rdquo; with hypervirulence and carbapenem resistance in Klebsiella pneumoniae: the rise of such emerging nosocomial pathogens in China. Science Bulletin, 68, 2658-2670.\u003c/li\u003e\n\u003cli\u003eHao, P., Wu, S., Zhang, X., Gou, C., Wang, Y., Wang, L., Zhu, Y., Basang, W., Gao, Y. (2022). Characterization and Degradation Pathways of Microbacterium resistens MZT7, A Novel 17\u0026beta;-Estradiol-Degrading Bacterium. International Journal of Environmental Research and Public Health, 19, 11097.\u003c/li\u003e\n\u003cli\u003eRybkina, D.O., Plotnikova, E.G., Dorofeeva, L.V., Mironenko, Y.L., Demakov, V.A. (2003). A new aerobic gram-positive bacterium with a unique ability to degrade ortho-and para-chlorinated biphenyls. Microbiology, 72, 672-677.\u003c/li\u003e\n\u003cli\u003eDijk, J.A., Gerritse, J., Schraa, G., Stams, A.J.M. (2004). Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions. Archives of Microbiology, 182, 514-519.\u003c/li\u003e\n\u003cli\u003eRyoo, D., Shim, H., Canada, K., Barbieri, P., Wood, T.K. (2000). Aerobic degradation of tetrachloroethylene by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1. Nature Biotechnology, 18, 775-778.\u003c/li\u003e\n\u003cli\u003eLi, X.M., Zhou, S.G., Li, F.B., Wu, C.Y., Zhuang, L., Xu, W., Liu, L. (2009). Fe(III) oxide reduction and carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17. Journal of Applied Microbiology, 106, 130-139.\u003c/li\u003e\n\u003cli\u003eSetlhare, B., Kumar, A., Aregbesola, O.A., Mokoena, M.P., Olaniran, A.O. (2021). 2, 4-dichlorophenol Degradation by Indigenous Pseudomonas sp. P KZNSA and Klebsiella pneumoniae Kp KZNSA: Kinetics, Enzyme Activity and Catabolic Gene Detection. Applied Biochemistry and Microbiology, 57, 656-665.\u003c/li\u003e\n\u003cli\u003eVerce, M.F., Gunsch, C.K., Danko, A.S., Freedman, D.L. (2002). Cometabolism of cis-1,2-Dichloroethene by Aerobic Cultures Grown on Vinyl Chloride as the Primary Substrate. Environmental Science \u0026amp; Technology, 36, 2171-2177.\u003c/li\u003e\n\u003cli\u003eStancu, M.M. (2022). ALIPHATIC HYDROCARBONS BIODEGRADATION BY A Pseudomonas STRAIN. AgroLife Scientific Journal, 11,\u003c/li\u003e\n\u003cli\u003eVandecasteele, J.P., Blanchet, D., Tassin, J.P., Bonamy, A.M., Guerrillot, L. (1983). Enzymology of alkane degradation in Pseudomonas aeruginosa. Acta biotechnologica, 3, 339-344.\u003c/li\u003e\n\u003cli\u003eReis, R.S., Pereira, A.G., Neves, B.C., Freire, D.M. (2011). Gene regulation of rhamnolipid production in Pseudomonas aeruginosa--a review. Bioresource technology, 102, 6377-6384.\u003c/li\u003e\n\u003cli\u003eRudolph, J., Erbse, A.H., Behlen, L.S., Copley, S.D. (2014). A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum. Biochemistry, 53, 6539-6549.\u003c/li\u003e\n\u003cli\u003eIwakiri, R., Yoshihira, K., Futagami, T., Goto, M., Furukawa, K., Others (2004). Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase. Bioscience, biotechnology, and biochemistry, 68, 1353-1356.\u003c/li\u003e\n\u003cli\u003eYakovlev, G., Reda, T., Hirst, J. (2007). Reevaluating the relationship between EPR spectra and enzyme structure for the iron--sulfur clusters in NADH: quinone oxidoreductase. Proceedings of the National Academy of Sciences, 104, 12720-12725.\u003c/li\u003e\n\u003cli\u003eCouto, N., Wood, J., Barber, J. (2016). The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radical Biology and Medicine, 95, 27-42.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Alpha diversity indices\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"529\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOTUs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChao\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eShannon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCoverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eW5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e237.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e3.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eW10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e264.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eW20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e256.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eW35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e291.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e3.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eLB5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e295\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e157.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eLB10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e217.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e2.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eLB20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e3.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eLB35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e295\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e302.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Identification of 1,2-DCP degrading bacteria\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eStrain No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClosest Related Strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNCBI\u003c/p\u003e\n \u003cp\u003eReference Sequence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSequence Similarity（%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCarbon Source Substrate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus faecalis\u0026nbsp;\u003c/em\u003estrain SR5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePP218401.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSingle Carbon Source, Complex Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eMicrobacterium proteolyticum\u0026nbsp;\u003c/em\u003estrain TC01-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMW928414.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSingle Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eStutzerimonas stutzeri\u003c/em\u003e strain XQ-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMT471993.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSingle Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae subsp. ozaenae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOQ405432.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSingle Carbon Source, Complex Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain XL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePP758286.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eComplex Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eMethylorubrum lusitanum\u0026nbsp;\u003c/em\u003estrain 13635N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEU741086.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSingle Carbon Source, Complex Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eSphingomonas\u003c/em\u003e sp. strain Rb-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOP390782.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSingle Carbon Source, Complex Carbon Source\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Key Enzymes in 1,2-DCP Degradation\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eCategory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eEnzyme Name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003edirection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003edeltag\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 15px;\"\u003e\n \u003cp\u003eMonooxygenases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eFMNH2-dependent monooxygenase (butanesulfonate)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-71.9142\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eFMNH2-dependent monooxygenase (methanesulfonate)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-76.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eFMNH2-dependent monooxygenase (sulfoacetate)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-78.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eFMNH2-dependent monooxygenase (ethanesulfonate)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-80.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eHydrolases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003epropane-1,2-diol hydro-lyase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-9.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15px;\"\u003e\n \u003cp\u003eOxidoreductases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eFe\u0026sup2;⁺:NAD⁺ oxidoreductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. proteolyticum\u003c/em\u003e、\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e50.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003ehydrogen:ferredoxin oxidoreductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. proteolyticum\u003c/em\u003e、\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e22.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eFerredoxin:NADP\u003csup\u003e+\u003c/sup\u003e oxidoreductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. proteolyticum\u003c/em\u003e、\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e14.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15px;\"\u003e\n \u003cp\u003eGlutathione-related\u0026nbsp;\u003cbr\u003e\u0026nbsp;Enzymes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eglutathione:hydrogen-peroxide oxidoreductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. proteolyticum\u003c/em\u003e、\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-74.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eglutathione:NADP+ oxidoreductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003eglutathione gamma-glutamylaminopeptidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. proteolyticum\u003c/em\u003e、\u003cem\u003eS. stutzeri\u003c/em\u003e、\u003cem\u003eK. pneumoniae\u003c/em\u003e、\u003cem\u003eP.aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioremediation, Composite carbon source, 1,2-Dichloropropane, Degradative strain screening, Functional prediction, Sole carbon source","lastPublishedDoi":"10.21203/rs.3.rs-6836071/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6836071/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e1,2-Dichloropropane (1,2-DCP) is a highly toxic and environmentally persistent chlorinated hydrocarbon pollutant that causes serious contamination of soil and groundwater. This study investigated 1,2-DCP contaminated soil using single and complex carbon source cultivation strategies to screen and acclimate bacterial communities. Through high-throughput sequencing, community structure and functional analysis, and pure culture isolation and identification, the study compared the effects of different carbon substrates on the succession patterns, functional characteristics, and screening advantages of degrading bacterial communities. Results showed that communities acclimated with a single carbon source were dominated by \u003cem\u003eProteobacteria\u003c/em\u003eand gradually diversified, while communities acclimated with complex carbon sources were dominated by \u003cem\u003eFirmicutes\u003c/em\u003e and remained relatively stable. Functional prediction revealed that although the two acclimation strategies led to different community structures, functional stability was maintained, with major differences manifested in energy metabolism, ion transport, and DNA repair functions. Four 1,2-DCP degrading strains were isolated and identified, including \u003cem\u003eMicrobacterium proteolyticum\u003c/em\u003e, \u003cem\u003eStutzerimonas stutzeri\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Genomic functional prediction suggested that their degradation mechanisms mainly involve monooxygenases, oxidoreductases, and glutathione-related enzyme systems. The study demonstrated that the single carbon source nutrition with pollution pressure has significant advantages in constructing functional bacterial communities, expressing specific functions, and screening specific strains, while complex carbon source cultivation favors the enrichment of microbial communities with broad metabolic capabilities, providing a theoretical foundation and bacterial resources for 1,2-DCP bioremediation.\u003c/p\u003e","manuscriptTitle":"Analysis and optimization of single and composite carbon source substrates for acclimation and screening of 1,2-Dichloropropane degrading bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-08 09:56:17","doi":"10.21203/rs.3.rs-6836071/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6a0f035-6ecf-4769-8c7a-ed12b4633a21","owner":[],"postedDate":"July 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T02:33:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-08 09:56:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6836071","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6836071","identity":"rs-6836071","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}