Abstract:Microbiome engineering is an emerging interdisciplinary field that systematically investigates and applies engineering methods to uncover the functions, structures, and interaction mechanisms of microbial communities with their environments, offering critical insights into global challenges. To showcase the latest advancements and achievements in this field, Chinese Journal of biotechnology has specially organized a special issue, inviting experts and scholars from multiple domestic institutions to elaborate on the practical applications and potential of microbiome engineering in agriculture and industrial production, environmental and ecological restoration, and health and medical treatment, from perspectives of fundamental research, technological innovation, and engineering applications. Additionally, this issue explores future trends in the field, providing valuable references to promote innovation and contribute to the sustainable development of human society.

Abstract:Microbiomes in natural environments have diverse functions and harbor vast exploitable potential of modifying the nature and hosts, being significant resources for development. The inherent high complexity and uncontrollability of natural microbiomes, as well as the selection by the nature and hosts, impose significant constraints on practical applications. Synthetic microbiomes, serving as precisely defined engineered microbiomes, demonstrate enhanced functionality, stability, and controllability compared with natural microbiomes. These engineered microbiomes emerge as a prominent research focus and are potentially having applications across various fields including environmental bioremediation and host health management. Nevertheless, substantial challenges persist in both fundamental research and practical application of synthetic microbiomes. This review systematically summarizes three core design principles for synthetic microbiomes, introduces current construction strategies including top-down, bottom-up, and integrated approaches, and comprehensively lists their applications in environmental remediation, agricultural innovation, industrial biotechnology, and healthcare. Furthermore, it critically examines existing technical and conceptual challenges while proposing strategic recommendations, thereby providing theoretical guidance for future advancements in the design, engineering, and application of synthetic microbiomes.

Abstract:Traditional Chinese medicine (TCM) plays an important role in preventing and treating diseases and improving human health. However, the complex bioactive components and regulation of signaling pathway and network restrict the elucidation of the mechanisms of action of TCM. A human being is regarded as a super “symbiont” composed of body cells and commensal microorganisms. Gut microbiota is the core commensal microorganism system of a human body, being considered as “the second genome” and the new “organ”. Alterations in gut microbiota reflect the state of body health and progression of diseases. Recent investigations have revealed that the TCM rich in polysaccharides and polyphenols can modulate gut microbiota metabolites to rehabilitate gut homeostasis, thus ameliorating diseases via regulating gut-liver axis or gut-brain axis. This review summarizes the causal relationship and mechanisms of action of TCM in the treatment of diseases from the perspective of gut microbiota metabolites. Our findings are expected to provide new insights into the mechanisms of TCM in preventing and treating diseases and guidance for TCM-based drug discovery in the future.

Abstract:Acinetobacter baumannii is a Gram-negative opportunistic pathogen widely distributed in hospital settings. It can survive for a long time and cause a variety of infections, including pneumonia, septicemia, urinary tract infections, and meningitis. The bacterium demonstrates extensive resistance, particularly to critical antibiotics like carbapenems and polymyxins, posing a serious threat to the recovery of severely ill patients. Carbapenem-resistant A.baumannii has been designated as a pathogen of critical priority on the World Health Organization (WHO) Bacterial Pathogen Priority List, requiring urgent development of new therapeutic agents. Phages, as a novel biological control approach, exhibit substantial potential in combating A.baumannii infections due to their specific ability to infect and lyse bacteria. This review highlights the application and potential of phages and phage-derived enzymes against multidrug-resistant A. baumannii, considering the epidemiological trends of A.baumannii in China, with the aim of providing innovative insights and strategies for phage therapy of drug-resistant bacterial infections.

Abstract:The intestine is a complex symbiotic system, and the gut microbiota is closely related to host health. Studies have indicated that the gut microbiota influences physiological functions of the host by producing a variety of metabolites, which act as signaling molecules and substrates for metabolic reactions in the host. Dysbiosis of the gut microbiota affects the abundance of gut microbiota-derived metabolites, thereby influencing host health by disrupting signal transduction in multiple organs. Additionally, dietary compounds can shape the gut microbiota, affecting gut microbiota-derived metabolite levels and regulating host metabolism. This article introduces the methods for mining gut microbiota-derived metabolites, reviews the roles of these metabolites in metabolic diseases and related dietary interventions. Which provides a perspective on the prevention and treatment of metabolic diseases by targeting these metabolites, enriching the knowledge on the role of gut microbiota in the regulation of host metabolism.

Abstract:Uric acid (UA) is the final metabolite of purines in the human body. An imbalance in UA production and excretion that disrupts homeostasis leads to elevated blood UA levels and the development of hyperuricemia (HUA). Approximately one-third of UA is excreted through the intestinal tract. As a crucial component of the intestinal microenvironment, the gut microbiota plays a pivotal role in regulating blood UA levels. Alterations or imbalances in gut microbiota composition are linked to the onset of HUA, which implies the potential of gut microbiota as a novel target for the prevention and treatment of HUA. This review introduces the occurrence mechanism and damage of hyperuricemia, examines the association between HUA and the gut microbiota and their metabolites, and explores the molecular mechanisms underlying gut microbiota-targeted therapies for HUA. Furthermore, it discusses the potential applications of probiotics, prebiotics, and traditional Chinese medicine (including both single herbs and compound formulas) with UA-lowering effects, along with cutting-edge technologies such as fecal microbiota transplantation and machine learning in HUA treatment. This review provides valuable perspectives and strategies for improving the prevention and treatment of HUA.

Abstract:Phages, as obligate bacterial and archaeal parasites, constitute a virus group of paramount ecological significance due to their exceptional abundance and genetic diversity. These biological entities serve as critical regulators in Earth’s ecosystems, driving biogeochemical cycles, energy fluxes, and ecosystem services across terrestrial and marine environments. Within soil microbiomes, phages function as microbial “dark matter,” maintaining the soil-plant system balance through precise modulation of the microbial community structure and functional dynamics. Despite the growing research interests in soil phages in recent years, the proportion of such studies in environmental virology remains disproportionately low, which is primarily attributed to researchers’ limited familiarity with the research methodologies for phage microecology, incomplete technical frameworks, and inherent challenges posed by soil environmental complexity. To address these challenges, this review synthesizes cutting-edge methodologies for soil phage investigation from four aspects: (1) tangential flow filtration (TFF)-based phage enrichment strategies; (2) integrated quantification approaches combining double-layer agar plating, epifluorescence microscopy, and flow cytometry; (3) multi-omics analytical pipelines leveraging metagenomics and viromics datasets; and (4) computational frameworks merging machine learning algorithms with eco-evolutionary theory for deciphering phage-host interaction networks. Through comparative analysis of methodological principles, technical merits, and application scopes, we establish a comprehensive workflow for soil phage research. Future research in this field should prioritize: (1) construction of soil phage resource libraries, (2) exploration of RNA phages based on transcriptomes, (3) functional characterization of unknown genes, and (4) deep integration and interaction validation of multi-omics data. This systematic methodological synthesis provides critical technical references for addressing fundamental challenges in characterizing soil phages regarding the community structure, functional potential, and interaction mechanisms with hosts.

Abstract:Microbial denitrification is a major pathway for nitrogen removal from water bodies. However, denitrification is often difficult to continue when there is a lack of microbially available organic matter in the water body to serve as electron donors. In recent years, studies have shown that some denitrifying bacteria can directly utilize photoelectrons generated by sunlight-excited semiconductor minerals or natural organic matter for denitrification without the need for bioavailable organic matter as electron donors. This process is defined as microbial photoelectrotrophic denitrification. The discovery of microbial photoelectrotrophic denitrification phenomenon reshapes the previous knowledge about the chemoheterotrophic mode of denitrifying bacteria and broadens the pathway of nitrogen removal by the new photoelectrotrophic metabolism, which is of great significance to our understanding and exploration of sunlight-driven nitrogen cycling process. In this paper, we comprehensively sort out the existing research reports in the field of microbial photoelectrotrophic denitrification, systematically summarize the principle and the current research progress of microbial photoelectrotrophic denitrification, deeply analyze the problems and challenges faced by this technology, and make an outlook on the future research directions and application prospects of this technology, providing a reference for the further research and application of this technology.

Abstract:Diabetic chronic wounds are characterized by difficult healing, recurrent progression, and high rates of disability and mortality, which make their clinical treatment a medical challenge urgent to be addressed. However, the complex local microenvironment conditions of chronic wounds, such as high protease activity and persistent inflammatory responses, result in low bioavailability of exogenous cytokines (e.g., chemokine CXCL12) at the wound site, limiting their clinical application. In this study, we utilized Lactobacillus plantarum WCFS1 as the chassis to develop an efficient CXCL12 delivery system based on synthetic biology. Subsequently, we evaluated the role of the engineered probiotic strain in promoting the chronic wound healing in diabetic mice. Firstly, we fused the endogenous secretion signal peptide lp_3050 (SP_{lp_3050}) of L.plantarumWCFS1 and the commonly used secretion signal peptide usp45 (SP_usp45) of lactic acid bacteria with the reporter gene gusA and inserted them into the pTRK892-P_32(pgm) plasmid by molecular cloning. Then, we prepared the engineered strains and characterized the efficacy of the two signal peptides in driving the secretion of GusA. The results showed that SP_{lp_3050} efficiently drove the secretion of GusA inL.plantarum WCFS1, increasing the activity of GusA in the culture supernatant by nearly five times compared with that of SP_{lp_3050}. Further, we fused SP_{lp_3050} and codon-optimized CXCL12 gene to construct an engineered probiotic strain Lpw-CXCL12 for CXCL12 delivery. The results demonstrated that the content of CXCL12 in the culture supernatant reached (13.40±0.20) μg/mL. Finally, we found that the engineered probiotic strain Lpw-CXCL12 accelerated chronic wound healing in a diabetic mouse model. In conclusion, these results support an engineered probiotic strain in promoting diabetic chronic wound healing, providing a new strategy and technological foundation for the management of diabetic chronic wounds in the future.

Abstract:N-dodecanoyl-l-homoserine lactone (C12-HSL) is a signaling molecule that mediates bacterial quorum sensing, regulating bacterial population behaviors. This study investigated the effects of C12-HSL on the isolation and cultivation of gut microbiota, with the goal of enriching the diversity and number of cultivable bacterial strains from the mouse gut microbiota. Using a culture medium supplemented with C12-HSL, we isolated and cultivated bacterial strains from mouse intestinal contents, obtaining a total of 235 isolates. Preliminary identification based on the 16S rRNA gene revealed 54 bacterial species, including 4 potential new species, 4 potential new genera and 1 potential new family. Compared with the previously established mouse gut microbial biobank (mGMB), this study newly identified 42 bacterial species, enhancing the diversity of the strain library. Statistical analysis showed that the proportion of Gram-negative bacteria, particularly those belonging to Proteobacteria, isolated by this method was significantly higher than that obtained by conventional isolation and cultivation methods without the addition of C12-HSL. Subsequent cultivation experiments with one of the newly discovered bacterial species indicated that exogenous C12-HSL at 20-200 μmol/L significantly promoted the growth of this species, while higher concentrations of C12-HSL significantly reduced the cell density of this bacterium. This work confirms that quorum sensing molecules, such as C12-HSL, can enhance the growth, isolation, and cultivation of Gram-negative bacteria in the gut within a specific concentration range. Although the mechanism by which C12-HSL promotes the growth of gut bacterial strains requires further investigation, the findings of this study provide new insights into the targeted isolation, cultivation, and regulation of gut microbiota using bacterial quorum sensing signal molecules.

Abstract:Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B.fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B.fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.

Abstract:The gut microbiota is closely related to human health, and various gut microbiota health indices have been developed to assist in evaluating the health of the gut microbiota and even the overall health of the human body. Diets are one of the main factors that regulate the gut microbiota, while there is still no good method for evaluating the regulatory effects of dietary factors. To assess the regulatory effects of dietary factors on the gut microbiota of overweight individuals, we conducted an in vitro fermentation experiment based on 17 dietary factors, and developed an evaluation method for the regulatory effects of dietary factors based on the health index with principal component analysis (hiPCA). The results showed that most dietary factors had positive regulatory effects on the gut microbiota of overweight individuals. Galactooligosaccharides (GOS) and puerarin were the most significant dietary factors in regulating the gut microbiota of overweight individuals. The analysis of the contribution of species to the hiPCA indicated that GOS and puerarin might inhibit the activities of bacteria associated with overweight by regulating Eubacterium dolichum, Lactobacillus salivarius, Clostridium clostridioforme, Clostridium citroniae, and Lachnospiraceae bacterium 9_1_43BFAA. In addition, GOS may further enhance the inhibition of these activities by regulating Lachnospiraceae bacterium 6_1_63FAA, thereby reducing the gut health risks in overweight individuals. In summary, this study evaluated the health effects of dietary factors based on the hiPCA and specifically analyzed the role of different dietary factors in regulating the gut microbiota of overweight individuals. This provides new ideas and methods for improving gut microbiota health and has potential applications in the field of precision nutrition.

Abstract:The enterotoxigenic Escherichia coli (ETEC) infection is a major factor restricting the development of animal husbandry. However, the abuse of antibiotics will lead to the antibiotic residues and emergence of antibiotic-resistant bacteria. The existing vaccines face challenges in stimulating intestinal immunity, demonstrating limited prevention effects. Therefore, it is indispensable to develop a new vaccine that is safe and suitable as a feed additive to activate intestinal immunity. This study constructed yeast-cell microcapsules (YCM) carrying the DNA vaccine against ETEC by genetic engineering. Furthermore, animal experiments were carried out to explore the regulatory effects of feeding YCM on the intestinal immune system and intestinal microbiota. Saccharomyces cerevisiae was selected as the oral delivery vehicle (microcapsules) of the DNA vaccine. The codon-optimized nucleic acid sequence of K88, the main antigen of mammal-derived ETEC, was synthesized, and the yeast shuttle vector containing the corresponding DNA vaccine expression cassette was constructed by DNA recombination. The recombinant strain of YCM was prepared by transforming JMY1. Additionally, the characteristics of the YCM strain and its feasibility as an oral vaccine were comprehensively evaluated by the fluorescence reporter assay, gastrointestinal fluid tolerance assay, intestinal epithelial cell adhesion assay, intestinal retention assessment, antiserum detection, and intestinal microbiota detection. The experimental results showed that the DNA vaccine expression cassette was expressed in mammals, and the recombinant strain of YCM could tolerate up to 8 hours of gastrointestinal fluid digestion and had good adhesion to intestinal epithelial cells. The results of mouse feeding experiments indicated that the recombinant strain of YCM could stay in the intestinal tract for at least two weeks, and the DNA vaccine expression cassette carried by YCM entered the intestinal immune system and triggered an immune response to induce the production of specific antibodies. Moreover, feeding YCM recombinant bacteria also improved the abundance of gut microbiota in mice, demonstrating a positive effect in regulating intestinal flora. In summary, we prepared the recombinant strain of YCM carrying the DNA vaccine against ETEC and comprehensively evaluated its characteristics and feasibility as an oral vaccine. Feeding the recombinant YCM could induce specific immune responses and regulate intestinal microbiota. The findings provide a reference for the immunoprevention of ETEC-related animal diseases.

Abstract:Polyethylene (PE) is widely used due to its excellent properties. However, the improper disposal of PE waste has led to serious environmental pollution. Microbial degradation of PE is a low-carbon, environmentally friendly, and highly efficient method of homogeneous recycling. The use of microbial degradation technology to treat polyethylene waste has become one of the current research hotspots. As a result, employing microbial degradation technology to address polyethylene waste has become a key focus of current research. A PE-degrading strain ETX1 was screened from waste plastics in a landfill by the enrichment culture method. The strain was identified as Lysinibacillus sp.. After incubating PE powder with the strain for 20 days, a weight loss of 29.41% was observed. Fourier transform infrared spectroscopy (FTIR) showed that special absorption peaks such as carbonyl and hydroxyl groups appeared, proving that ETX1 had the effect of degrading PE. The degradation effect of this strain was characterized by the weight loss of PE film, FTIR, scanning electron microscopy, and contact angle. The results showed that ETX1 reduced the PE film weight by up to 5.23% within 120 days. The film structure was damaged, with holes formed by erosion on the film surface, and the hydrophilicity was enhanced. Additionally, a stronger carbonyl absorption peak appeared. The discovery of the PE-degrading strain ETX1 not only enriches the resources of PE plastic-degrading strains but also lays a foundation for mining efficient PE-degrading elements, obtaining degrading enzymes, and deciphering related degradation pathways.

Abstract:Small urban wetlands are widely distributed and susceptible to human activities, serving as important sources and sinks of carbon. Microorganisms play a crucial role in carbon cycle, while limited studies have been conducted on the microbial diversity in small urban wetlands and the functions of microbiome in carbon fixation and metabolism. To probe into the microbiome-driven carbon cycling in small urban wetlands and dissect the composition and functional groups of microbiome, we analyzed the relationships between the microbiome structure, element metabolism pathways, and habitat physicochemical properties in sediment samples across three small wetlands in Huzhou City, and compared them with natural wetlands in the Zoige wetland. High-throughput sequencing of 16S rRNA gene amplicons and metagenomics was employed to determine the species and functional groups. Sixty medium to high-quality metagenome-assembled genomes (MAGs) were constructed, including 55 bacterial and 5 archaeal taxa, and their potential in driving elemental cycles were analyzed, with a focus on carbon fixation. Several bacterial species were found to encode a nearly complete carbon fixation pathway, including the Calvin cycle, the reductive tricarboxylic acid cycle, the Wood-Ljungdahl pathway, and the reductive glycine pathway. There were several potential novel carbon-fixing bacterial members, such as those belonging to Syntrophorhabdus (Desulfobacterota) and UBA4417 (Bacteroidetes), which had high relative abundance in the wetland microbiome. Unveiling the genetic potential of these functional groups to facilitate element cycling is of great scientific importance for enhancing the carbon sequestration capacity of small urban wetlands.

Abstract:Three soil conditioners were prepared from granulated food waste and decomposed cattle manure combined with desulfurization gypsum, coal gangue, and maifanite, respectively. Field trials were conducted in the saline field growing Cabernet Sauvignon. The effects of soil conditioners on rhizospheric bacterial communities were studied, with the aim of providing a scientific basis for soil amelioration and restoration. Five treatments were designed, including the control (T1), conventional fertilization (T2), reduced chemical fertilization+organic matter-based soil conditioner with calcium additives (T3), reduced chemical fertilization+organic matter-based soil conditioner with silica additives (T4), and reduced chemical fertilization+organic matter-based soil conditioner with magnesium additives (T5), each with three replications. The results indicated that soil conditioners improved the rhizospheric nutrients, yield, and quality of grape (P<0.05), increased relative abundance of Proteobacteria by 17.32%–23.37%, decreased relative abundance of unidentified_Bacteria and Acidobacteriota by 4.22%–28.42% and 20.88%–35.81%, respectively. The bacterial community composition and diversity were different between treatments. Function analysis showed that the expression levels of the genes involved in chromosome and protein synthesis, mRNA biosynthesis, and glyoxylate and dicarboxylate metabolism were up-regulated in the treatments with soil conditioners. The correlation analysis revealed that multiple environmental factors affected the alpha diversity of rhizospheric bacterial communities, and some bacterial taxa were closely related to the grape yield and quality. It is concluded that soil conditioners can effectively alter rhizosphere nutrient levels and bacterial community structures and functions. T5 treatment outperforms other treatments in improving the physico-chemical and biological characteristics of rhizosphere, and the yield, and quality of grape. It has potential for application, and provides an important basis for development of new-type soil conditioners.

Abstract:The regulation of rhizosphere bacterial community structure and metabolism by plants in municipal solid waste landfills is a key to enhancing the biodegradation of chlorobenzene (CB). In this study, we employed biodiversity and metabolomics methods to systematically analyze the mechanisms of different plant species in regulating the rhizosphere bacterial community structure and metabolic features and then improved the methane (CH₄) oxidation and CB degradation capacity. The results showed that the rhizosphere soil of Rumex acetosa exhibited the highest CH₄ oxidation and CB degradation capacity of 0.08 g/(kg·h) and 1.72×10^-6 g/(L·h), respectively, followed by the rhizosphere soil of Amaranthus spinosus L., with the rhizosphere soil of Broussonetia papyrifera showing the weakest activity. Rumex acetosa promoted the colonization of Methylocaldum in the rhizosphere, and the small-molecule organic amine, such as triethylamine and N-methyl-aniline, secreted from the roots of this plant enhanced the tricarboxylic acid cycle and nicotinamide metabolism, thereby increasing microbial activity and improving CH₄ and CB degradation efficiency. Conversely, cinnamic acid and its derivatives secreted by Broussonetia papyrifera acted as autotoxins, inhibiting microbial activity and exacerbating the negative effects of salt stress on key microbes such as methanotrophs. This study probed into the mechanisms of typical plants growing in landfill cover soil in regulating bacterial ecological functions, offering theoretical support and practical guidance for the plant-microbe joint control of landfill gas pollution.

Abstract:The biological nitrogen removal technology utilizing heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria has shown effectiveness in wastewater treatment. However, the nitrogen removal efficiency of HN-AD bacteria significantly decreases as the salinity increases. To tackle the challenge of treating high-salt and high-nitrogen wastewater, we isolated a moderately halophilic HN-AD strain 5505 from a salt lake in Xinjiang. The strain was identified based on morphological, physiological, and biochemical characteristics and the 16S rRNA gene sequence. Single-factor experiments were carried out with NH₄⁺-N, NO₃^--N, and NO₂^--N as sole or mixed nitrogen sources to study the nitrifying effect, denitrifying effect, and nitrogen metabolism pathway of the strain. The strain was identified as Halomonas sp.. It can grow in the presence of 1%-25% (W/V) NaCl and exhibited efficient nitrogen removal ability in the presence of 3%-8% NaCl. At the optimal NaCl concentration (8%), the strain showed the NH₄⁺-N, NO₃^--N and NO₂^--N removal rates of 100.0%, 94.11% and 74.43%, respectively. Strain 5505 removed inorganic nitrogen mainly by assimilation, which accounted for over 62.68% of total nitrogen removal. In the presence of mixed nitrogen sources, strain 5505 showed a preference for utilizing ammonia, with a potential HN-AD pathway of NH₄⁺→NH₂OH→NO₂^-→NO₃^-→NO₂^-→NO/N₂O/N₂. The findings provide efficient salt-tolerant bacterial resources, enhance our understanding of biological nitrogen removal, and contribute to the nitrogen removal efficiency improvement in the treatment of high-salt and high-nitrogen wastewater.

Abstract:Chlorobenzene contaminants (CBs) pose a threat to the eco-environment, and functional strains hold considerable potential for the remediation of CB-contaminated sites. To deeply explore the application potential of functional bacteria in the in-situ bioremediation of CBs, this study focused on the biodegradation characteristics and degradation kinetics of CB and 1,2-dichlorobenzene (1,2-DCB) in soil by the isolated strain Serratia marcescens TF-1. Additionally, an in-situ remediation trial was conducted with this strain at a chemical industrial site. Batch serum bottle experiments showed that the degradation rate of CB at the concentrations ranging from 20 to 200 mg/L by TF-1 was 0.22-0.66 mol/(g_cell·h), following the Haldane model, with the optimal concentration at 23.12 mg/L. The results from simulated soil degradation experiments indicated that the combined use of TF-1 and sodium succinate (SS) significantly enhanced the degradation of CBs, with the maximum degradation rate of CB reaching 0.104 d^-1 and a half-life of 6.66 d. For 1,2-DCB, the maximum degradation rate constant was 0.068 7 d^-1, with a half-life of 10.087 d. The in-situ remediation results at the chemically contaminated site demonstrated that the introduction of bacterial inoculant and SS significantly improved the removal of CBs, achieving the removal rates of 84.2%-100% after 10 d. CB, 1,4-dichlorobenzene (1,4-DCB), and benzo[a]pyrene were completely removed. Microbial diversity analysis revealed that the in-situ remediation facilitated the colonization of TF-1 and the enrichment of indigenous nitrogen-fixing Azoarcus, which may have played a key role in the degradation process. This study provides a theoretical basis and practical experience for the in situ bioremediation of CBs-contaminated sites.