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Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria

Environmental microorganisms are important sources of biotechnology innovations; however, the discovery process is hampered by the inability to culture the overwhelming majority of microbes. To drive the discovery of new biotechnology products from previously unculturable microbes, several methods s...

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Autores principales: Pope, Emily, Cartmell, Christopher, Haltli, Bradley, Ahmadi, Ali, Kerr, Russell G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9441948/
https://www.ncbi.nlm.nih.gov/pubmed/36071955
http://dx.doi.org/10.3389/fmicb.2022.958660
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author Pope, Emily
Cartmell, Christopher
Haltli, Bradley
Ahmadi, Ali
Kerr, Russell G.
author_facet Pope, Emily
Cartmell, Christopher
Haltli, Bradley
Ahmadi, Ali
Kerr, Russell G.
author_sort Pope, Emily
collection PubMed
description Environmental microorganisms are important sources of biotechnology innovations; however, the discovery process is hampered by the inability to culture the overwhelming majority of microbes. To drive the discovery of new biotechnology products from previously unculturable microbes, several methods such as modification of media composition, incubation conditions, single-cell isolation, and in situ incubation, have been employed to improve microbial recovery from environmental samples. To improve microbial recovery, we examined the effect of microencapsulation followed by in situ incubation on the abundance, viability, and diversity of bacteria recovered from marine sediment. Bacteria from marine sediment samples were resuspended or encapsulated in agarose and half of each sample was directly plated on agar and the other half inserted into modified Slyde-A-Lyzer™ dialysis cassettes. The cassettes were incubated in their natural environment (in situ) for a week, after which they were retrieved, and the contents plated. Colony counts indicated that bacterial abundance increased during in situ incubation and that cell density was significantly higher in cassettes containing non-encapsulated sediment bacteria. Assessment of viability indicated that a higher proportion of cells in encapsulated samples were viable at the end of the incubation period, suggesting that agarose encapsulation promoted higher cell viability during in situ incubation. One hundred and 46 isolates were purified from the study (32–38 from each treatment) to assess the effect of the four treatments on cultivable bacterial diversity. In total, 58 operational taxonomic units (OTUs) were identified using a 99% 16S rRNA gene sequence identity threshold. The results indicated that encapsulation recovered greater bacterial diversity from the sediment than simple resuspension (41 vs. 31 OTUs, respectively). While the cultivable bacterial diversity decreased by 43%–48% after in situ incubation, difficult-to-culture (Verrucomicrobia) and obligate marine (Pseudoalteromonas) taxa were only recovered after in situ incubation. These results suggest that agarose encapsulation coupled with in situ incubation in commercially available, low-cost, diffusion chambers facilitates the cultivation and improved recovery of bacteria from marine sediments. This study provides another tool that microbiologists can use to access microbial dark matter for environmental, biotechnology bioprospecting.
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spelling pubmed-94419482022-09-06 Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria Pope, Emily Cartmell, Christopher Haltli, Bradley Ahmadi, Ali Kerr, Russell G. Front Microbiol Microbiology Environmental microorganisms are important sources of biotechnology innovations; however, the discovery process is hampered by the inability to culture the overwhelming majority of microbes. To drive the discovery of new biotechnology products from previously unculturable microbes, several methods such as modification of media composition, incubation conditions, single-cell isolation, and in situ incubation, have been employed to improve microbial recovery from environmental samples. To improve microbial recovery, we examined the effect of microencapsulation followed by in situ incubation on the abundance, viability, and diversity of bacteria recovered from marine sediment. Bacteria from marine sediment samples were resuspended or encapsulated in agarose and half of each sample was directly plated on agar and the other half inserted into modified Slyde-A-Lyzer™ dialysis cassettes. The cassettes were incubated in their natural environment (in situ) for a week, after which they were retrieved, and the contents plated. Colony counts indicated that bacterial abundance increased during in situ incubation and that cell density was significantly higher in cassettes containing non-encapsulated sediment bacteria. Assessment of viability indicated that a higher proportion of cells in encapsulated samples were viable at the end of the incubation period, suggesting that agarose encapsulation promoted higher cell viability during in situ incubation. One hundred and 46 isolates were purified from the study (32–38 from each treatment) to assess the effect of the four treatments on cultivable bacterial diversity. In total, 58 operational taxonomic units (OTUs) were identified using a 99% 16S rRNA gene sequence identity threshold. The results indicated that encapsulation recovered greater bacterial diversity from the sediment than simple resuspension (41 vs. 31 OTUs, respectively). While the cultivable bacterial diversity decreased by 43%–48% after in situ incubation, difficult-to-culture (Verrucomicrobia) and obligate marine (Pseudoalteromonas) taxa were only recovered after in situ incubation. These results suggest that agarose encapsulation coupled with in situ incubation in commercially available, low-cost, diffusion chambers facilitates the cultivation and improved recovery of bacteria from marine sediments. This study provides another tool that microbiologists can use to access microbial dark matter for environmental, biotechnology bioprospecting. Frontiers Media S.A. 2022-08-22 /pmc/articles/PMC9441948/ /pubmed/36071955 http://dx.doi.org/10.3389/fmicb.2022.958660 Text en Copyright © 2022 Pope, Cartmell, Haltli, Ahmadi and Kerr. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Pope, Emily
Cartmell, Christopher
Haltli, Bradley
Ahmadi, Ali
Kerr, Russell G.
Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
title Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
title_full Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
title_fullStr Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
title_full_unstemmed Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
title_short Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
title_sort microencapsulation and in situ incubation methodology for the cultivation of marine bacteria
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9441948/
https://www.ncbi.nlm.nih.gov/pubmed/36071955
http://dx.doi.org/10.3389/fmicb.2022.958660
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