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.
• 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.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
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
Bertani G (1951) Studies on lysogenesis. J Bacteriol 62:293–300. https://doi.org/10.1128/JB.62.3.293-300.1951
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
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
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
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
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
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
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
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
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
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
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
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
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
Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis ER (2013) The next-generation sequencing revolution and its impact on genomics. Cell 155:27
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Stewart EJ (2012) Growing Unculturable Bacteria. J. Bacteriol. 194:4151–4160
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
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
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
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
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
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
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
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
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
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.
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.
This article does not contain any studies performed with human participants or with animals by any of the authors.
Consent to participate
Consent for publication
Conflict of interest
The authors declare no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
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
- Diverse colony formation
- Diluted media
- Environmental bacterial sources
- Waste sewage sludge