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Design and application of a novel culturing chip (cChip) for culturing the uncultured aquatic microorganisms

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Abstract

Culturing uncultured microorganisms is an important aspect of microbiology. Once cultured, these microorganisms can be a source of useful antibiotics, enzymes etc. In this study, we have designed a novel culturing chip (cChip) to facilitate the growth of uncultured aquatic bacterial community by concentrating the samples. cChip was optimized for microbial growth using known bacteria in the laboratory as a pre-experiment. Then microorganisms from a freshwater lake were concentrated and inoculated, before putting the inoculated cChip in a simulated lake environment and further sub-culturing on laboratory media. High-throughput sequencing and traditional culturing were also performed for comparison. These three methods were able to detect 265 genera of microorganisms in the sample, of which 78.87% were detected by high-throughput sequencing, 30.94% by cChip, while only 6.42% were obtained by traditional culture. Moreover, all microorganisms obtained by traditional culture were also cultured using the cChip. A total of 45 new strains were isolated from the cChip, and their 16S rRNA gene sequences were 91.35% to 98.7% similar to their closest relatives according to NCBI GenBank database. We conclude that the design and simple operation of cChip can improve the culture efficiency of traditional culture by almost 5 times. To the best of our knowledge, this is the first report comparing a novel culturing method with high-throughput sequencing data and traditional culturing of the same samples.

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Data availability

Data have been submitted to the NCBI GenBank database under the accession numbers mentioned in Table 1, and the high-throughput sequencing section (lines 311–312) of this manuscript.

References

  • Alain K, Querellou J (2009) Cultivating the uncultured: limits, advances and future challenges. Extremophiles 13:583–594

    Article  PubMed  Google Scholar 

  • Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berdy B, Spoering AL, Ling LL, Epstein SS (2017) In situ cultivation of previously uncultivable microorganisms using the ichip. Nat Protoc 12:2232

    Article  PubMed  Google Scholar 

  • Bokulich NA, Subramanian S, Faith JJ et al (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10:57–59

    Article  CAS  PubMed  Google Scholar 

  • Bollmann A, Lewis K, Epstein SS (2007) Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol 73:6386–6390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bollmann A, Palumbo AV, Lewis K, Epstein SS (2010) Isolation and physiology of bacteria from contaminated subsurface sediments. Appl Environ Microbiol 76:7413–7419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Caporaso JG, Kuczynski J, Stombaugh J et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chaudhary DK, Khulan A, Kim J (2019) Development of a novel cultivation technique for uncultured soil bacteria. Sci Rep 9:6666

    Article  PubMed  PubMed Central  Google Scholar 

  • Colwell RR, Grimes DJ et al (2000) Nonculturable microorganisms in the environment. ASM press

    Book  Google Scholar 

  • Cui Z, Luan X, Li S et al (2022) Occurrence and distribution of cyclic-alkane-consuming psychrophilic bacteria in the Yellow Sea and East China Sea. J Hazard Mater 427:128129

    Article  CAS  PubMed  Google Scholar 

  • Davies J (2007) Small molecules: the lexicon of biodiversity. J Biotechnol 129:3–5

    Article  CAS  PubMed  Google Scholar 

  • Demain AL, Sanchez S (2009) Microbial drug discovery: 80 years of progress. J Antibiot (tokyo) 62:5

    Article  CAS  PubMed  Google Scholar 

  • Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996

    Article  CAS  PubMed  Google Scholar 

  • Edgar RC, Haas BJ, Clemente JC et al (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Farooq Lodhi A, Zhang Y, Adil M, Deng Y (2018) Antibiotic discovery: combining isolation chip (iChip) technology and co-culture technique. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-018-9193-0

    Article  Google Scholar 

  • Ferrari BC, Binnerup SJ, Gillings M (2005) Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria. Appl Environ Microbiol 71:8714–8720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferrari BC, Winsley T, Gillings M, Binnerup S (2008) Cultivating previously uncultured soil bacteria using a soil substrate membrane system. Nat Protoc 3:1261

    Article  CAS  PubMed  Google Scholar 

  • Filippidou S, Junier T, Wunderlin T et al (2015) Under-detection of endospore-forming Firmicutes in metagenomic data. Comput Struct Biotechnol J 13:299–306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Franklin RB, Garland JL, Bolster CH, Mills AL (2001) Impact of dilution on microbial community structure and functional potential: comparison of numerical simulations and batch culture experiments. Appl Environ Microbiol 67:702–712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Giovannoni SJ, Rappe M (2000) Revolution, diversity and molecular ecology of marine prokaryotes. In D. L. Kirchman (ed.), Microb Ecol Ocean, 1st edn. WileyLiss. pp. 47–84

  • Goers L, Freemont P, Polizzi KM (2014) Co-culture systems and technologies: taking synthetic biology to the next level. J R Soc Interface 11:20140065

    Article  PubMed  PubMed Central  Google Scholar 

  • Haas BJ, Gevers D, Earl AM et al (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21:494–504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Heberle H, Meirelles GV, da Silva FR et al (2015) InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinform 16:169

    Article  Google Scholar 

  • Imazaki I, Kobori Y (2010) Improving the culturability of freshwater bacteria using FW70, a low-nutrient solid medium amended with sodium pyruvate. Can J Microbiol 56:333–341

    Article  CAS  PubMed  Google Scholar 

  • Jannasch HW, Jones GE (1959) Bacterial populations in sea water as determined by different methods of enumeration. Limnol Oceanogr 4:128–139

    Article  Google Scholar 

  • Jung D, Seo E-Y, Epstein SS et al (2014) Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia. FEMS Microb Ecol 90(2):417–23

  • Kaeberlein T, Lewis K, Epstein SS (2002) Isolating" uncultivable" microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129

    Article  CAS  PubMed  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Li M et al (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lewis K, Epstein S, D’onofrio A, Ling LL (2010) Uncultured microorganisms as a source of secondary metabolites. J Antibiot (tokyo) 63:468

    Article  CAS  PubMed  Google Scholar 

  • Livermore DM, for Antimicrobial Chemotherapy Working Party on The Urgent Need: Regenerating Antibacterial Drug Discovery BS, Development et al (2011) Discovery research: the scientific challenge of finding new antibiotics. J Antimicrob Chemother 66:1941–1944

    Article  CAS  PubMed  Google Scholar 

  • Martiny JBH, Bohannan BJM, Brown JH et al (2006) Microbial biogeography: putting microorganisms on the map. Nat Rev Microbiol 4:102–112

    Article  CAS  PubMed  Google Scholar 

  • Marx CJ (2009) Getting in touch with your friends. Science 324:1150–1151

    Article  CAS  PubMed  Google Scholar 

  • Meyer-Reil L-A (1978) Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural waters. Appl Environ Microbiol 36:506–512

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nadell CD, Xavier JB, Foster KR (2008) The sociobiology of biofilms. FEMS Microbiol Rev 33:206–224

    Article  PubMed  Google Scholar 

  • Newton RJ, Jones SE, Eiler A et al (2011) A guide to the natural history of freshwater lake bacteria. Microbiol Mol Biol Rev 75:14–49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nichols D, Cahoon N, Trakhtenberg EM et al (2010) Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol 76:2445–2450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piddock LJV (2015) Teixobactin, the first of a new class of antibiotics discovered by iChip technology? J Antimicrob Chemother 70:2679–2680

    Article  CAS  PubMed  Google Scholar 

  • Pulschen AA, Bendia AG, Fricker AD et al (2017) Isolation of uncultured bacteria from antarctica using long incubation periods and low nutritional media. Front Microbiol 8:1346

    Article  PubMed  PubMed Central  Google Scholar 

  • Pushpakumara BLDU, Tandon K, Willis A, Verbruggen H (2023) Unravelling microalgal-bacterial interactions in aquatic ecosystems through 16S rRNA gene-based co-occurrence networks. Sci Rep 13:2743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qu Y, Zhang X, Shen W et al (2016) Illumina MiSeq sequencing reveals long-term impacts of single-walled carbon nanotubes on microbial communities of wastewater treatment systems. Bioresour Technol 211:209–215

    Article  CAS  PubMed  Google Scholar 

  • Reasoner DJ, Geldreich E (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49:1–7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roszak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment. Microbiol Rev 51:365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Shimoyama T, Kato S, Ishii S, Watanabe K (2009) Flagellum mediates symbiosis. Science 323(80):1574

    Article  CAS  PubMed  Google Scholar 

  • Silver LL (2011) Challenges of antibacterial discovery. Clin Microbiol Rev 24:71–109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Smrhova T, Jani K, Pajer P et al (2022) Prokaryotes of renowned Karlovy Vary (Carlsbad) thermal springs: phylogenetic and cultivation analysis. Environ Microbiome 17:1–17

    Article  Google Scholar 

  • Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346

    Article  CAS  PubMed  Google Scholar 

  • Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194:4151–4160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taguchi Y-H, Oono Y (2005) Relational patterns of gene expression via non-metric multidimensional scaling analysis. Bioinformatics 21:730–740

    Article  CAS  PubMed  Google Scholar 

  • Van Dorst JM, Hince G, Snape I, Ferrari BC (2016) Novel culturing techniques select for heterotrophs and hydrocarbon degraders in a subantarctic soil. Sci Rep 6:1–13

    Google Scholar 

  • Vartoukian SR, Palmer RM, Wade WG (2010) Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiol Lett 309:1–7

    CAS  PubMed  Google Scholar 

  • Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yarza P, Yilmaz P, Pruesse E et al (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12:635–645

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Deng C-P, Shen B et al (2016) Syntrophic interactions within a butane-oxidizing bacterial consortium isolated from Puguang gas field in China. Microb Ecol 72:538–548

    Article  CAS  PubMed  Google Scholar 

  • Zhao J, Shakir Y, Deng Y, Zhang Y (2023) Use of modified ichip for the cultivation of thermo-tolerant microorganisms from the hot spring. BMC Microbiol 23:56

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors are thankful to Talal Jamil Qazi, Mohibullah Kakar, Muhammad Akram, Salabat Khan Wazir, Iqbal Ahmed Alvi, other friends, students of Deng’s family and Ying’s lab for providing logistic, technical, and moral support during the research work. The authors are thankful to Research Square for adding this study as a preprint under the https://doi.org/10.21203/rs.3.rs-91678/v1.

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Author notes

  1. Adil Farooq Lodhi and Ying Zhang have contributed equally to this work.

Authors and Affiliations

  1. Beijing Key Laboratory for Separation and Analysis in Biomedicine and Pharmaceutical, School of Life Science, Beijing Institute of Technology, Beijing, 100081, China

    Adil Farooq Lodhi, Ying Zhang & Yulin Deng

  2. Department of Microbiology, Faculty of Biological and Health Sciences, Hazara University, Mansehra, Pakistan

    Adil Farooq Lodhi & Maria Adil

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  1. Adil Farooq Lodhi
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Contributions

AFL and YZ: discussed the idea and designed the study under the supervision of YD. AFL: performed all the experimental work including sampling and solved experimental hurdles in consultation with YZ, MA, and YD. AFL, and YZ: wrote the manuscript, MA helped in corrections. All the facilities for the study were provided by YD, and progress was regularly monitored.

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Correspondence to Yulin Deng.

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Communicated by Ran Wang.

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Lodhi, A.F., Zhang, Y., Adil, M. et al. Design and application of a novel culturing chip (cChip) for culturing the uncultured aquatic microorganisms. Arch Microbiol 205, 285 (2023). https://doi.org/10.1007/s00203-023-03613-w

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  • DOI: https://doi.org/10.1007/s00203-023-03613-w

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