2021, 61(4):iii-iv.
Abstract:
Peng Liang , Bo Liu , Yu Wang , Ying Xue , Bei Li
2021, 61(4):781-792. DOI: 10.13343/j.cnki.wsxb.20200595
Abstract:Most microorganisms are still unculturable in vitro, therefore studies of the “microbial dark matter” have great significance for investigating the evolution of microorganisms and making the most of microbial resources. Research on the isolation and cultivation of uncultivated microorganisms has become a hot research topic in the world. In this paper, we introduce the principle and development of different single cell sorting techniques, such as micromanipulation, fluorescence activated cell sorting, microfluidic cell sorting, optical tweezer, laser-induced forward transfer, to isolate and culture microorganisms. According to the advantages and disadvantages of each sorting technology, we mainly describe the advantages of laser-induced forward transfer and prospect the application of this technology to the research of uncultivated microorganisms.
Chen Yang , Weiyan Zhang , Dawoon Jung , Shan He
2021, 61(4):793-806. DOI: 10.13343/j.cnki.wsxb.20200669
Abstract:Ninty nine percentage of microorganisms in the environment remains uncultured, called “uncultured” microorganisms or microbial “dark matter”. Unraveling the mysteries of these microbial “dark matter” is especially helpful to understand the diversity of microbes and their metabolic characteristics. DNA genome sequences of microbial “dark matter” could be recovered from the environment samples by population binning of metagenomics and single-cell genomics, independently or combined synergistically. However, microbial functions often cannot be deduced from genetic information alone, and the isolation of uncultured microorganisms remains a powerful approach. Many cultivation-based methods for culturing microorganisms have been developed, such as in situ cultivation, co-culture and continuous flow bioreactor. In this mini-review, we briefly introduce the development of new culture-dependent technology of continuous flow bioreactor and indicate the future research opportunities in this area.
Qing Liu , Leilei Yang , Yuguang Zhou , Yuhua Xin , Xiuzhu Dong
2021, 61(4):807-815. DOI: 10.13343/j.cnki.wsxb.20200793
Abstract:Glaciers, as the main cryosphere on earth, are inhabited by abundant cold-adapted microorganisms. In 1976, Inoue & Komagata isolated a psychrophilic bacterium from Antarctica. Until 1997, genus Cryobacterium was validly published with Cryobacterium psychrophilum as type species and this psychrophilic strain as type strain. The species in genus Cryobacterium were mainly distributed in cold environments such as Antarctic, Arctic, permafrost and glacier in Tibetan Plateau. Cryobacterium belongs to rare group, since its abundance is lower than other common groups in glacier environment. At present, there are 15 species in genus Cryobacterium with validly published names, including nine psychrophilic species. Different species show phenotypic divergence of thermotolerance. Therefore, Cryobacterium strains can be excellent materials for studying the evolution and speciation of bacteria in the cold environment. Cryobacterium strains can produce β-carotenoids, cold-adapted enzymes and other bioactive substances. In this paper, the distribution, biological characteristics and application potential of Cryobacterium were reviewed. The diversity of Cryobacterium was discussed by calculation and cluster analysis of the average nucleotide identity (ANI) using the whole genome sequences of Cryobacterium which were downloaded from GenBank database. The application potential of Cryobacterium in food processing, medicine and health field was discussed.
Chujin Ruan , Xiaowei Zheng , Li Wang , Yi Wang , Yaxin Zhu , Jian Wang , Juanli Yun , Zhiyang Dong , Zujun Lu , Ying Huang , Wenbin Du , Li Huang , Xin Dai
2021, 61(4):816-827. DOI: 10.13343/j.cnki.wsxb.20200459
Abstract:[Objective] To test a single cell cultivation method involving flow cytometry-based high-throughput cell sorting and single cell cultivation in the isolation of microorganisms from deep-sea samples. [Methods] The flow cytometer sorted to microbial cells of interest based on the size and complexity of the particles using forward versus side scatter gating (SSC vs. FSC) without the need for fluorescence labeling. Then, the single cells were cultivated in 96-well plates and further transferred to agar plate for scale-up cultivation and taxonomic identification. The performance of this method to cultivate microorganisms from deep-sea water and sediment samples was evaluated. [Results] An optimal sort region was chosen to sort microbial cells from the deep-sea samples, following by high-throughput single-cell broth cultivation. A total of 61 potential novel microbial strains, which belong to 6 novel genus or species, were obtained from deep-sea samples from the Indian Ocean. The novel strains accounted for 26.29% of the total isolates and shared 89.79%-95.37% similarity at the 16S rRNA gene sequence level. [Conclusion] The FCM-based high-throughput cell sorting and single-cell cultivation method is more suitable for sorting and cultivating of sea-water microorganisms. This methed may help increase the efficiency of identifying novel species from deep seas.
Yu Lin , Ronghua Liu , Shun Zhou , Xiaoyu Zhu , Jinyan Wang , Xiaohua Zhang
2021, 61(4):828-844. DOI: 10.13343/j.cnki.wsxb.20200668
Abstract:[Objective] The Mariana Trench is the deepest trench on earth. It has the characteristics of ultra-high hydrostatic pressure, low temperature and lack of light and contains unique microbial resources. Dimethylsulfoniopropionate (DMSP) is one of the marine's most abundant organosulfur molecules. Marine heterotrophic microorganisms can cleave DMSP and release “cooling gas” dimethyl sulfide (DMS), which plays an important role in driving global sulfur cycling and regulating global climate change. The heterotrophic bacteria from sediments of the Mariana Trench were isolated and identified and their ability to degrade DMSP was studied. Our study provides unique microbial resources for elucidating the life process of abyssal microorganisms. [Methods] Our study collected the sediments from five stations of the Mariana Trench as subject, using three kinds of conventional heterotrophic bacteria culture media (2216E, R2A and TCBS) and two kinds of heterotrophic bacteria enrichment culture media (TCBS broth and alkaline peptone water) for isolation and cultivation of bacteria at 4 ℃, 16 ℃ and 28 ℃, respectively. [Results] A total of 1057 strains were isolated and identified. And these isolates belonged to four phyla, seven classes and 76 genera. Gammaproteobacteria was the most abundant class, accounting for 61.4% of the total isolates. Pseudomonas and Halomonas were the most abundant genera; Proteobacteria was dominated in samples of all layers of sediments. The relative abundance of deep layer of Firmicutes was higher than that of the surface layer; The alkaline peptone water (AP) medium were found to have a better selectivity to Actinobacteria, and the TCBS broth medium were found to have a better selectivity to Bacilli; 101 strains were potential novel taxa of bacteria; In this study, the ability to degrade DMSP of 134 representative heterotrophic bacteria were tested, and 52 strains were found to have the ability to cleave DMSP and release DMS, accounting for 38.8% of the tested strains. [Conclusion] These results indicate that there is a high diversity of culturable bacteria and strains that have the ability to degrade DMSP from sediments of the Mariana Trench. Our research provides valuable microbial resources for further research on the life process of abyssal microorganisms.
Chuang Sun , Jinyan Wang , Yulin Zhang , Yunhui Zhang , Xiaoyu Zhu , Zhaohui Chen , Xiaohua Zhang
2021, 61(4):845-861. DOI: 10.13343/j.cnki.wsxb.20200687
Abstract:[Objective] The complex marine environment of the Western Pacific has unique ecosystems and biological communities, and contains abundant marine microbial resources. The community structure of bacteria in different water depths in the Western Pacific was studied based on isolation culture technique. We tried to improve the capturability of marine bacteria by modifying the culture medium. [Methods] We used five different culture media in this study: improved 2216E agar medium (IMA), R2A agar medium (R2A), MBM agar medium (MBM), thiosulfate citrate bile salts sucrose agar medium (TCBS) and improved 2216E liquid enriched medium (IMB). Through strain isolation and 16S rRNA gene sequence identification, the community structure and diversity of culturable bacteria from the surface layer to 6000 meters water depth of the Western Pacific were analyzed. We compared the similarities and differences of bacterial diversity in different culture media. [Results] In this study, we isolated and identified 1293 strains in total, which affiliated to four phyla, seven classes, 14 orders, 26 families, 52 genera and 119 species. Proteobacteria were the dominant group recovered from the seawaters. At the class level, γ-Proteobacteria, α-Proteobacteria, and Actinobacteria_c were the dominant microflora. The most dominant phyla obtained from the five cultures were all Proteobacteria, and the most dominant class was γ-Proteobacteria. Except for the TCBS in which the dominant order was Vibrionales, the most dominant order in other four media was Alteromonadales. In addition, the five media exhibited different selectivity at each taxonomic level. The diversity of the cultivable bacteria in the five culture media from high to low was R2A, IMA, MBM, TCBS and IMB. The most unique genera (up to 10) were isolated from R2A. With the increase of water depth, the number of cultivable heterotrophic microorganisms showed a decreasing trend. A total of 68 strains were potential new bacteria among all the isolates, and the rates of new bacteria were relatively high among IMA, R2A and MBM. [Conclusion] In this study, cultivable bacteria from the seawater of the Western Pacific were studied by using five different culture media, showing a high diversity and revealing the selectivity of different media for cultivable marine bacteria. This research has provided valuable microbial resources for other ecological and molecular studies, and it has also brought more inspiration for using improved culture medium to isolate uncultured marine microorganisms in the future.
Jinyou Liang , Junliang Zhang , Xiaoli Zheng , Shuotian Chen , Peng Yang , Mengbin Liao , Ying Xu
2021, 61(4):862-874. DOI: 10.13343/j.cnki.wsxb.20200771
Abstract:[Objective] The present work aims to study the amounts and species of cultivable microbes and bioactive metabolite producing bacteria from a marine invertebrate collected in Shenzhen. [Methods] We dissected the invertebrate into five parts to isolated bacteria by R2A plate and identified them through the 16S rRNA sequence. Then use the culture broth and supernatant to screen the activity of degrading quorum-sensing signal molecules and anti-biofilm activity. [Results] A total of 216 strains of bacteria were isolated from the invertebrate, belonging to 87 species and 54 genera, and 16 strains were suspected to be new bacteria. Distributed in the phylum Proteobacteria (126 strains), Bacteroidetes (44 strains), Firmicutes (34 strains), Actinobacteria (10 strains), and Planctomycetes (1 strain). The number and species of bacteria in the eggs of the invertebrate were the most diverse. Twenty-eight strains were screened to degrade quorum-sensing signal molecules, and eight strains showed anti-biofilm activity. [Conclusion] It is the first report that the marine invertebrate Homoiodoris japonica-associated bacteria contains were abundant and diverse, including potential new species of bacteria and natural product resources, which may provide a research basis for future research on the Homoiodoris japonica-associated microbes.
Mengxuan Du , Minzhi Jiang , Chang Liu , Shuangjiang Liu
2021, 61(4):875-890. DOI: 10.13343/j.cnki.wsxb.20200737
Abstract:The gut microbiome (GM) is extremely complicated in composition and function. Many culture-independent studies have demonstrated that the GM plays a pivotal role in host health and its imbalance has close relevance with various diseases. Meanwhile, increasing more researchers have realized that the culturable gut microbial biobanks are the resource foundations to facilitate the in-depth studies of GM by transition from meta-data based on relevance analysis to experimental validation of host-microbe interactions and GM-disease causality and further application in human health management. In this review, we summarize the recent progresses on large-scale gut microbial isolation and cultivation from different hosts, and provide a few examples for application of cultured microbial resources in high throughput sequencing data analyzing and in host-microbe interaction studies.
Lingxu Fang , Long Li , Zhongyi Lu , Meng Li
2021, 61(4):891-902. DOI: 10.13343/j.cnki.wsxb.20200613
Abstract:Representing a fundamental part of the human microbiota, the oral microbial community is characterized by its diverse and unique composition. Oral diseases like dental caries and periodontitis are directly associated with oral microorganisms. Therefore, it is crucial to deepen our understanding of oral microbiota. Advances in high-throughput sequencing technology provide extensive information of the diversity of oral uncultivated microorganisms, prompting an increasing need for microbial isolation and cultivation techniques. This review presents recent research progress on uncultivated oral microorganisms and lists factors that possibly hinder isolation attempts. In addition, advances in methodologies and techniques used for culturing previously uncultured microorganisms and their applications in oral microbiology studies are summarized, giving valuable insights into various aspects of uncultivated oral microorganisms.
2021, 61(4):903-922. DOI: 10.13343/j.cnki.wsxb.20200258
Abstract:[Objective] To clarify the source references of the paradigm that 99% of microorganisms are noncuturable; and to quantitatively evaluate the proportion of culturable diazotrophs in an agricultural soil. [Methods] Total microbial DNA was directly extracted from soil. Classic cultivation methods were used to obtain diazotrophic colonies from the first and second generation of solid and liquid culture medium for DNA extration. The abundance and composition of microbiome were analyzed by high-throughput sequencing of nifH and 16S rRNA genes. Community composition and proportions of culturable diazotrophs were then identified through phylogenetic classification.[Results] At the genus level, phylogenetic analysis of nifH gene showed the proportion of culturable diazotrophs in the soil was (22.4±4.5)%-(28.4±6.3)%, and result of 16S rRNA genes was (31.6±3.4)%-(41.4±13.1)%. Phylogeny of nifH genes revealed a total of 67 genera in soil; 39 genera could grow in liquid or solid medium, but only 4 genera were significantly enriched. The solid meidum led to significant enrichment of Azotobacter of Proteobacteria as the dominant nitrogen-fixing bacteria with a relative abundance of (98.2±0.94)%. Meanwhile, liquid medium enriched members within two genera of Paenibacillus and Clostridium of Firmicutes significantly, and the relative abundance of the first generation was as high as (76.7±3.9)% and (21.9±4.0)%, respectively. As for 16S rRNA gene-based result, a total of 255 genera within 14 phyla were obtained in soil; 248 genera could grow in liquid or solid medium, but the sequences of up to 226 genera and 5 phya showed high similariry to bacterium in which N2-fixing physiology has not been demonstrated. Members within 6 genera of Proteobacteria were significantly enriched in solid medium, while liquid medium encirhed significantly 5 genera of Firmicutes. [Conclusion] Literature survey reveals that 99% unculturability paradigm is not yet experimentally verified; and it is more a synonym for "the plate count anomaly", depsite it is widely accpeted in past 30 years. It indicates that direct microscopic counts of total microorganisms in environment is often much more than dilution plate counts of culturable microorganisms. Using N2-fixer as an emxple, we show that up to 58.2% (nifH gene) and 97.3% (16S rRNA gene) of diazotrophs in soil can form colony in solid or liquid medium; but 91.1% of these colony have not been isolated yet with known physiology of N2-fixing activity. Meanwhile, the majority of diazotrophs are rare taxa (with relative abundance<0.1%) at genus level on the basis of nifH and 16S rRNA genes, representing 69.5% and 78.5% of the total diazotrophs in soil, respectively. Desite of the clony forming in medium, these numerically less dominant taxa could thus liekly escaped isolation, due to the continous enrichment and long-term subcutlure strategies, and the members within the phyla of Proteobacteria and Firmicutes were particularly enriched. Our study thus highlight that the term “unculturable” should be avoided, and the difficult-to-cultivated term would be more appropriate because >90% of diazotrophs could form colony in medium, although they are not isolated yet. Cultivation effort is warranted for future studies with focused target of these rare taxa.
Shuoxing Yi , Yang Zhou , Xianjiao Zhang , Qing Yao , Huaping Li , Honghui Zhu
2021, 61(4):923-934. DOI: 10.13343/j.cnki.wsxb.20200340
Abstract:[Objective] We modified myxobacterial isolation methods based on the in-situ simulation, the nutritional requirements of cultured myxobacteria and myxobacterial interactions with bacteria inferred from co-occurrence network, to obtain more cultured myxobacteria. [Methods] We set experiment by adding soil extract into isolation medium, using Gram-positive bacteria as bait and changing the inoculation method of bait, with traditional methods as control. [Results] More types of myxobacteria fruiting bodies were induced by the modified methods than traditional methods. A total of 40 myxobacteria strains were isolated from 9 soil samples including subtropical forest soil, saline-alkaline soil, and wetland sediment. The isolated myxobacteria were from genera of Archangium, Chondromyces, Corallococcus, Myxococcus, Nannocystis, Polyangium and Pyxidicoccus. [Conclusion] Compared with traditional methods, the addition of soil extract and bait inoculated by spotting can obviously increase the types of fruiting bodies. Although the strains of bait showed little effect on the fruiting bodies, Gram-positive bacteria could induce specific fruiting bodies. In this study, we modified isolation methods of myxobacteria and greatly increased the types of fruiting bodies, but the number of purified myxobacteria were far less than that of fruiting bodies observed, indicating that the improvement of purification is required to obtain more cultured myxobacteria.
Xiaodi Liu , Zengwei Feng , Honghui Zhu , Qing Yao
2021, 61(4):935-945. DOI: 10.13343/j.cnki.wsxb.20200653
Abstract:[Objective] To reveal the effects of abscisic acid (ABA) on the colonization and sporulation of arbuscular mycorrhiza (AM) fungi, and to establish a highly efficient propagation methods by promoting spore production with exogenous ABA. [Methods] We established the dual culture system with tomato hairy roots and AM fungus Rhizophagus irregularis DAOM 197198, and the exogenous ABA and gibberellin (GA) were applied, or the deficient mutants of ABA and GA were employed. The mycorrhizal colonization was observed after staining, the expression of genes involved in the development of arbuscules and the synthesis and transfer of lipids were measured with qRCR, and the numbers of arbuscules and spores were counted, in order to reveal the effects of ABA on the colonization and sporulation of AM fungus. [Results] In the ABA-deficient mutant not, F% (mycorrhizal frequency), a% (arbuscular abundance), number of arbuscules, and the expression of arbuscule development-specific gene EXO70A1-like (LOC101253481) and the lipid synthesis and transfer related genes RAM2 and STR2 were significantly lower than those in the wild-type MT. Exogenous application of ABA significantly promoted F%, M% (mycorrhizal intensity), number of arbuscules, spore production, and the expression of RAM2 and STR2. The spore production applied with exogenous ABA was approximately 4.5 times of that without application. Exogenous GA significantly inhibited all parameters of mycorrhizal colonization and spore production. The spore production of GA-deficient mutant gib3 was significantly higher than that of the wild-type MM, although there was no significant difference in mycorrhizal colonization between gib3 and MM. [Conclusion] By promoting the lipid synthesis and transfer, ABA increases the colonization and arbuscular formation of AM fungi, and further enhances AM fungal sporulation.
Lei Cheng , Shichun Ma , Kejia Wu , Hui Zhang , Yu Deng
2021, 61(4):946-968. DOI: 10.13343/j.cnki.wsxb.20200448
Abstract:Anaerobes are the largest number of microorganisms on earth but rarely reported from the point view of taxonomy. Owing to oxygen-sensitive and slow-growth, cultivation and isolation of fastidious anaerobe faces a great challenge. In this review, we introduce the research history of anaerobes, and analyze the factors affecting the isolating efficiency of anaerobes. Furthermore, we discuss the current status of isolation and cultivation methods and strategies, and summarize the research progress on systematic taxonomy of anaerobes. In the end, we give an outlook for isolating and cultivating anaerobes.
Lan Liu , Yuzhen Ming , Aiping Lv , Jianyu Jiao , Wenjun Li
2021, 61(4):969-986. DOI: 10.13343/j.cnki.wsxb.20200755
Abstract:The anaerobic ammonia oxidation (anammox) reaction with nitrite as an electron acceptor and nitrogen as a product is mediated by bacteria which belong to Planctomycetales. AnAOB are widely present in marine, freshwater and terrestrial ecosystem, it also can be found in some other extreme environments. In this study, we reviewed the most new research advance on taxonomy, characteristics, metabolism, distribution, biotechniques used for the analysis of AnAOB and application. Finally, we discussed some questions exiting in this field and prospected future research of anaerobic ammonium oxidation microorganisms.
Chao Sun , Xiang Zeng , Guangyu Li , Yaping Du , Zhaoshou Wang , Zongze Shao
2021, 61(4):987-1001. DOI: 10.13343/j.cnki.wsxb.20200715
Abstract:[Objective] Organic matters are rich in the mangrove sediments. This research was aimed to understand the community structure and degradation process of microorganisms involved in natural organic polymers and their environmental roles. [Methods] Using anaerobic microbial culture technology to enrich, isolate and analyze the microbial diversity of anaerobic bacteria that involved in degrading cellulose, chitin and lignin in the mangrove sediments of the Jiulong River Estuary in Zhangzhou. [Results] A total of 202 anaerobes (82 obligate anaerobes and 120 facultative anaerobes) were isolated. Among those strains, four candidate novel genera (named Lachnotalea sp. MCCC 1A16036, Varunaivibrio sp. MCCC 1A15903, Clostridium sp. MCCC 1A15884 and Caminicella sp. MCCC 1A17445, respectively) and four candidate novel species (named Sunxiuqinia sp. MCCC 1A15904, Pseudodesulfovibrio sp. MCCC 1A16040, Pseudodesulfovibrio sp. MCCC 1A16038 and Mangrovibacterium lignilyticum MCCC 1A15882, respectively) were isolated. The dominant cultivable bacteria isolated from different substrate enrichments mainly belong to phyla Proteobacteria, Bacteroides and Firmicutes, but the population is slightly different. The species Prolixibacter bellariivorans and Mangrovibacterium lignilyticum, which belong to the phylum Bacteroides, and the species Desulfovibrio salexigenes and Vibrio alginolyticus, which belong to the phylum Proteobacteria, are most dominant in cellulose and chitin-enriched microbial communities. The strains Demequina salsinemoris MCCC 1A15890 and Brevibacterium celere MCCC 1A17451 (in the phylum Actinobacteria) showed the highest degradation activity to cellulose, while the strains Propionigenium maris MCCC 1A15874 and Ilyobacter polytropus MCCC 1A15889 (in the phylum Fusobacteria) showed the highest degradation activity to chitin. Among the lignin enriched bacteria, Mangrovebacterium lignlyticum (in the phylum Bacteroides) and Clostridium amygdalinum (in the phylum Firmicutes) had high relative abundance. The strains Desulfomicrobium apsheronum MCCC 1A15932 (in the phylum Proteobacteria) and Mangrovibacterium lignilyticum MCCC 1A15882 (in the phylum Bacteroides) have significant efficiency on lignin degradation. [Conclusion] There are diverse, novel and difficult-to-cultivable anaerobic bacteria in mangrove sediments, and most of them have the ability to degrade cellulose, chitin or lignin in anaerobic condition. The results will provide relevant theoretical basis and bacterial resources to explore the biogeochemical cycle of organic polymer carbon in environment of mangrove sediments in situ.
Junwei Liu , Xuan Zhang , Yixuan Bao , Jianyi Xu , Jiguo Qiu , Jian He
2021, 61(4):1002-1015. DOI: 10.13343/j.cnki.wsxb.20200645
Abstract:[Objective] The aim of this study was to isolate anaerobic bacteria capable of degrading butachlor. [Methods] By enrichment and acclimation method using butachlor as carbon source for enrichment, we screened an anaerobic butachlor-degrading bacterium from paddy soil. The isolated strain was preliminarily identified based on morphological and biochemical characteristics as well as 16S rRNA phylogenetic analysis, and the metabolites of butachlor degradation were identified by liquid chromatography-time of flight mass spectrometry. [Results] An anaerobic bacterium designated as BAD-20 was screened and identified as Proteiniphilum. The optimum conditions for butachlor degradation by strain BAD-20 were 30-35 ℃, pH 7.5-8.0 and 0-0.5% NaCl. Under the optimal conditions, 90% butachlor was degraded within 10 days by Proteiniphilum sp. BAD-20. Under aerobic conditions, the stain lost the ability to degrade butachlor. Proteiniphilum sp. BAD-20 could also degrade alachlor, acetochlor and propionate with the degradation efficiency following the order: alachlor > acetochlor > propranolol > butachlor. The degradation kinetics to these chloroacetamide herbicides fit to a first-order kinetic equation. Two metabolites, N-(2,6-diethylphenyl)-N-(butoxymethyl)acetanilide and N-(2,6-diethylphenyl)acetamide, were identified, indicating that the initial two steps of butachlor degradation are dechlorination and dealkylation. [Conclusion] An anaerobic butachlor-degrading bacterium BAD-20 was enriched and isolated from paddy soil and identified as Proteiniphilum. This study provides a basis for further study on the anaerobic catabolism of butachlor and the development of anaerobic biological treatment technology for butachlor-containing wastewater.
Yan Lü , Xiuying Li , Jingjing Wang , Huijuan Jin , Yiru Cui , Yi Yang , Jun Yan
2021, 61(4):1016-1029. DOI: 10.13343/j.cnki.wsxb.20200353
Abstract:[Objective] To isolate and characterize a novel organohalide-respiring bacterium and expand the diversity of dehalogenating microorganisms. [Methods] Based on the specialized energy metabolism and antibiotic resistance of certain dehalogenating microorganisms, we adapted the dilution-to-extinction strategy in defined mineral salts medium for the isolation of an anaerobic bacterium from a dehalogenating enrichment culture. Culture purity was investigated by yeast extract addition to growth medium and PCR-RFLP analysis on 16S rRNA gene amplicon. The basic characteristics of this novel microorganism were described in terms of cell morphology, 16S rRNA gene phylogeny and the ability to utilize chlorinated compounds as electron acceptors. [Results] A novel organohalide-respiring bacterial isolate, strain GP, belonging to Dehalogenimonas genus, was obtained. Strain GP is able to dechlorinate 1,1-dichloroethene (1,1-DCE) and grow at the presence of 1 g/L ampicillin and 0.1 g/L vancomycin. Cell morphology of strain GP is irregular disc-shaped coccus with a 0.4-0.8 μm diameter. Strain GP couples growth with reductive dechlorination of 1,1-DCE and vinyl chloride to nontoxic ethene using acetate as carbon source and hydrogen as electron donor. Phylogenetic analysis indicated that the 16S rRNA gene of strain GP shares 99.5% sequence similarity to that of Dehalogenimonas formicexedens NSZ-14, which grows with different types of chlorinated compounds. [Conclusion] A novel organohalide-respiring bacterium strain GP was isolated from a dehalogenating enrichment culture. This work expands the Dehalogenimonas pangenome and provides new biomaterial for detailed biochemical and physiological characterizations of Dehalogenimonas microorganisms.
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