Diluted Luria-Bertani medium vs. sewage sludge as growth media: comparison of community structure and diversity in the culturable bacteria

Abstract

Because colony formation is essential to seek bacterial functions by the direct observation of phenotype, the diversification of colony formation for culturable bacteria is a big challenge in the research field of Environmental Biotechnology. In this study, the biodiversity of cultivable bacteria (colony or liquid culture) was compared by using Luria-Bertani (LB) medium and waste sewage sludge (WSS) under different dilutions and temperatures. When WSS was used as a bacterial source, whereas the highest number of colonies was found at the concentration of WSS (5%), a particular concentration of LB (10%) or WSS (1%) as a growth medium showed the best number of the operational taxonomic units (OTUs) of colonies. The results of bacterial community structure indicated that there are 1, 8, and 12 bacterial genera found uniquely in the agar plates of LB, 10% LB, and 5% WSS. By contrast, when palm oil mill effluent sludge was used as a bacterial source, the effect of dilution was different with WSS. When comparing the biodiversity between colonies and liquid culture, a high OTU value was observed in the colonies on the plate. In addition, 30°C showed the highest number of colonies in LB, 10% LB, and 5% WSS whereas the best OTUs were observed at 37°C for LB and 10% LB, and at 25°C for 5% WSS. This study demonstrates the diversification of cultivable bacteria through the number of OTUs in diluted LB medium and WSS, which is beneficial to isolate a unique bacterial strain.

Key points

Impacts of diluted LB medium and WSS for colony formation were determined.

Difference of concentration of LB and WSS made different effects on colony formation.

Temperature change affected on diluted LB and WSS as media.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Barba M, Czosnek H, Hadidi A (2014) Historical perspective, development and applications of next-generation sequencing in plant virology. Viruses 6:106–136. https://doi.org/10.3390/v6010106

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Bertani G (1951) Studies on lysogenesis. J Bacteriol 62:293–300. https://doi.org/10.1128/JB.62.3.293-300.1951

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Birošová L, Mackuľak T, Bodík I, Ryba J, Škubák J, Grabic R (2014) Pilot study of seasonal occurrence and distribution of antibiotics and drug resistant bacteria in wastewater treatment plants in Slovakia. Sci Total Environ 490:440–444. https://doi.org/10.1016/j.scitotenv.2014.05.030

    CAS  Article  PubMed  Google Scholar 

  4. 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. https://doi.org/10.1128/AEM.01309-07

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Chaudhary DK, Khulan A, Kim J (2019) Development of a novel cultivation technique for uncultured soil bacteria. Sci Rep 9:1–11. https://doi.org/10.1038/s41598-019-43182-x

    CAS  Article  Google Scholar 

  6. Davis KER, Joseph SJ, Janssen PH (2005) Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl Environ Microbiol 71:826–834. https://doi.org/10.1128/AEM.71.2.826-834.2005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. George IF, Hartmann M, Liles MR, Agathos SN (2011) Recovery of as-yet-uncultured soil Acidobacteria on dilute solid media. Appl Environ Microbiol 77:8184–8188. https://doi.org/10.1128/AEM.05956-11

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Hahn MW, Stadler P, Wu QL, Pöckl M (2004) The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria. J Microbiol Methods 57:379–390. https://doi.org/10.1016/j.mimet.2004.02.004

    CAS  Article  PubMed  Google Scholar 

  9. Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68:669–685. https://doi.org/10.1128/mmbr.68.4.669-685.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Hara S, Isoda R, Tahvanainen T, Hashidoko Y (2012) Trace amounts of furan-2-carboxylic acids determine the quality of solid agar plates for bacterial culture. PLoS One 7:e41142. https://doi.org/10.1371/journal.pone.0041142

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Hou D, Huang Z, Zeng S, Liu J, Wei D, Deng X, Weng S, He Z, He J (2017) Environmental factors shape water microbial community structure and function in shrimp cultural enclosure ecosystems. Front Microbiol 8:2359. https://doi.org/10.3389/fmicb.2017.02359

    Article  PubMed  PubMed Central  Google Scholar 

  12. Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M (2002) Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ Microbiol 68:2391–2396. https://doi.org/10.1128/AEM.68.5.2391-2396.2002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Jung D, Aoi Y, Epstein S (2016) In situ cultivation allows for recovery of bacterial types competitive in their natural environment. Microbes Environ 31:456–459. https://doi.org/10.1264/jsme2.ME16079

    Article  PubMed  PubMed Central  Google Scholar 

  14. Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296(80):1127–1129. https://doi.org/10.1126/science.1070633

    CAS  Article  PubMed  Google Scholar 

  15. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1. https://doi.org/10.1093/nar/gks808

    CAS  Article  PubMed  Google Scholar 

  16. Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis ER (2013) The next-generation sequencing revolution and its impact on genomics. Cell 155:27

    CAS  Article  Google Scholar 

  17. Kogure K, Simidu U, Taga N (1979) A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 25:415–420. https://doi.org/10.1139/m79-063

    CAS  Article  PubMed  Google Scholar 

  18. Kudla G, Murray AW, Tollervey D, Plotkin JB (2009) Coding-sequence determinants of gene expression in Escherichia coli. Science 324(80):255–258. https://doi.org/10.1126/science.1170160

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Lloret E, Pascual JA, Brodie EL, Bouskill NJ, Insam H, Juárez MFD, Goberna M (2016) Sewage sludge addition modifies soil microbial communities and plant performance depending on the sludge stabilization process. Appl Soil Ecol 101:37–46. https://doi.org/10.1016/j.apsoil.2016.01.002

    Article  Google Scholar 

  20. Locey KJ, Lennon JT (2016) Scaling laws predict global microbial diversity. Proc Natl Acad Sci 113:5970–5975. https://doi.org/10.1073/PNAS.1521291113

    CAS  Article  PubMed  Google Scholar 

  21. Luo G, De Francisci D, Kougias PG, Laura T, Zhu X, Angelidaki I (2015) New steady-state microbial community compositions and process performances in biogas reactors induced by temperature disturbances. Biotechnol Biofuels 8:3. https://doi.org/10.1186/s13068-014-0182-y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Ma Z, Wen X, Zhao F, Xia Y, Huang X, Waite D, Guan J (2013) Effect of temperature variation on membrane fouling and microbial community structure in membrane bioreactor. Bioresour Technol 133:462–468. https://doi.org/10.1016/j.biortech.2013.01.023

    CAS  Article  PubMed  Google Scholar 

  23. Maeda T, Yoshimura T, Shimazu T, Shirai Y, Ogawa HI (2009) Enhanced production of lactic acid with reducing excess sludge by lactate fermentation. J Hazard Mater 168:656–663. https://doi.org/10.1016/j.jhazmat.2009.02.067

    CAS  Article  PubMed  Google Scholar 

  24. Maeda T, Yoshimura T, García-Contreras R, Ogawa HI (2011) Purification and characterization of a serine protease secreted by Brevibacillus sp. KH3 for reducing waste activated sludge and biofilm formation. Bioresour Technol 102:10650–10656. https://doi.org/10.1016/j.biortech.2011.08.098

    CAS  Article  PubMed  Google Scholar 

  25. Martín HG, Ivanova N, Kunin V, Warnecke F, Barry KW, McHardy AC, Yeates C, He S, Salamov AA, Szeto E, Dalin E, Putnam NH, Shapiro HJ, Pangilinan JL, Rigoutsos I, Kyrpides NC, Blackall LL, McMahon KD, Hugenholtz P (2006) Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities. Nat Biotechnol 24:1263–1269. https://doi.org/10.1038/nbt1247

    CAS  Article  Google Scholar 

  26. Mello BL, Alessi AM, McQueen-Mason S, Bruce NC, Polikarpov I (2016) Nutrient availability shapes the microbial community structure in sugarcane bagasse compost-derived consortia. Sci Rep 6:1–8. https://doi.org/10.1038/srep38781

    CAS  Article  Google Scholar 

  27. Mohd-Nor D, Ramli N, Sharuddin SS, Hassan MA, Mustapha NA, Ariffin H, Sakai K, Tashiro Y, Shirai Y, Maeda T (2019) Dynamics of microbial populations responsible for biodegradation during the full-scale treatment of palm oil mill effluent. Microbes Environ 34:121–128. https://doi.org/10.1264/jsme2.ME18104

    Article  PubMed  PubMed Central  Google Scholar 

  28. Mustapha NA, Hu A, Yu CP, Sharuddin SS, Ramli N, Shirai Y, Maeda T (2018) Seeking key microorganisms for enhancing methane production in anaerobic digestion of waste sewage sludge. Appl Microbiol Biotechnol 102:5323–5334. https://doi.org/10.1007/s00253-018-9003-8

    CAS  Article  PubMed  Google Scholar 

  29. Nguyen TM, Seo C, Ji M, Paik MJ, Myung SW, Kim J (2018) Effective soil extraction method for cultivating previously uncultured soil bacteria. Appl Environ Microbiol 84:e01145–18. https://doi.org/10.1128/AEM.01145-18

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Nguyen PDT, Mustapha NA, Kadokami K, Garcia-Contreras R, Wood TK, Maeda T (2019) Quorum sensing between Gram-negative bacteria responsible for methane production in a complex waste sewage sludge consortium. Appl Microbiol Biotechnol 103:1485–1495. https://doi.org/10.1007/s00253-018-9553-9

    CAS  Article  PubMed  Google Scholar 

  31. Pulschen AA, Bendia AG, Fricker AD, Pellizari VH, Galante D, Rodrigues F (2017) Isolation of uncultured bacteria from antarctica using long incubation periods and low nutritional media. Front Microbiol 8:1346. https://doi.org/10.3389/fmicb.2017.01346

    Article  PubMed  PubMed Central  Google Scholar 

  32. Singh RP, Agrawal M (2008) Potential benefits and risks of land application of sewage sludge. Waste Manag 28:347–358. https://doi.org/10.1016/j.wasman.2006.12.010

    CAS  Article  PubMed  Google Scholar 

  33. Stewart EJ (2012) Growing Unculturable Bacteria. J. Bacteriol. 194:4151–4160

    CAS  Article  Google Scholar 

  34. Sun J, Guo J, Yang Q, Huang J (2019) Diluted conventional media improve the microbial cultivability from aquarium seawater. J Microbiol 57:759–768. https://doi.org/10.1007/s12275-019-9175-7

    CAS  Article  PubMed  Google Scholar 

  35. Tamaki H, Hanada S, Sekiguchi Y, Tanaka Y, Kamagata Y (2009) Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment. Environ Microbiol 11:1827–1834. https://doi.org/10.1111/j.1462-2920.2009.01907.x

    CAS  Article  PubMed  Google Scholar 

  36. Tanaka Y, Hanada S, Manome A, Tsuchida T, Kurane R, Nakamura K, Kamagata Y (2004) Catellibacterium nectariphilum gen. nov., sp. nov., which requires a diffusible compound from a strain related to the genus Sphingomonas for vigorous growth. Int J Syst Evol Microbiol 54:955–959. https://doi.org/10.1099/ijs.0.02750-0

    CAS  Article  PubMed  Google Scholar 

  37. Thompson JR, Randa MA, Marcelino LA, Tomita-Mitchell A, Lim E, Polz MF (2004) Diversity and dynamics of a north Atlantic coastal Vibrio community. Appl Environ Microbiol 70:4103–4110. https://doi.org/10.1128/AEM.70.7.4103-4110.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Vesty A, Biswas K, Taylor MW, Gear K, Douglas RG (2017) Evaluating the impact of DNA extraction method on the representation of human oral bacterial and fungal communities. PLoS One 12:e0169877. https://doi.org/10.1371/journal.pone.0169877

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Wang X, Wang X, Zhang W, Shao Y, Zou X, Liu T, Zhou L, Wan S, Rao X, Li Z, Fu S (2016) Invariant community structure of soil bacteria in subtropical coniferous and broadleaved forests. Sci Rep 6:1–11. https://doi.org/10.1038/srep19071

    CAS  Article  Google Scholar 

  40. Wooley JC, Ye Y (2010) Metagenomics: facts and artifacts, and computational challenges. J Comput Sci Technol 25:71–81. https://doi.org/10.1007/s11390-010-9306-4

    Article  Google Scholar 

  41. Xing MN, Zhang XZ, Huang H (2012) Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis. Biotechnol. Adv. 30:920–929

    CAS  Article  Google Scholar 

  42. Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwelp RR (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb Ecol. 8:313–323

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The study was supported by a grant (No. JPMJAS2004) of aXis program (Accelerating Social Implementation for SDGs Achievement) of the Japan Science and Technology Agency. The authors wish to thank Dr. Mohd Zulkhairi MOHD YUSOFF, a senior lecturer in Universiti Putra Malaysia for providing POME sludge.

Availability of data and material

Available upon the request.

Code availability

Not applicable.

Funding

The study was supported by a grant (No. JPMJAS2004) of aXis program (Accelerating Social Implementation for SDGs Achievement) (of the Japan Science and Technology Agency.

Author information

Affiliations

Authors

Contributions

KY, SS, and TM contributed to the study conception and experimental design. Material preparation, data collection, and analysis were performed by KY, YH, and ST. The draft of the manuscript was initially written by KY and edited by TM. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Toshinari Maeda.

Ethics declarations

Ethics approval

This article does not contain any studies performed with human participants or with animals by any of the authors.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(PDF 307 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yamamoto, K., Toya, S., Sabidi, S. et al. Diluted Luria-Bertani medium vs. sewage sludge as growth media: comparison of community structure and diversity in the culturable bacteria. Appl Microbiol Biotechnol 105, 3787–3798 (2021). https://doi.org/10.1007/s00253-021-11248-4

Download citation

Keywords

  • Diverse colony formation
  • Diluted media
  • Environmental bacterial sources
  • Waste sewage sludge