A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F46356088%3A_____%2F23%3AN0000009" target="_blank" >RIV/46356088:_____/23:N0000009 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/46747885:24620/23:00010680

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0169131722004045" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0169131722004045</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.clay.2022.106809" target="_blank" >10.1016/j.clay.2022.106809</a>

Jazyk výsledku

angličtina

Název v původním jazyce

A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite

Popis výsledku v původním jazyce

Bentonite is a clay material with a broad range of applications in construction, industry (food, pharmacy), and civil engineering including water treatment and waste disposal. It has also been proposed as a buffer and backfill material in deep geological repositories (DGRs). However, the presence of metabolically active bacteria in bentonite could compromise the long-term safety of such DGRs, highlighting the need for a method for detection of microorganisms in bentonite materials. Here, we propose a novel protocol for the detection of both living (metabolically active and dead indigenous bacterial cells in bentonite. The extraction protocol requires the addition of a dispersant (2.5 mM sodium pyrophosphate (NaPP)/12.5 mM EDTA solution or 1% methanol) to a bentonite sample, followed by a two-step centrifugation over high-density media (i.e., sucrose and histodenz) to separate and concentrate the cells. The extracted bacterial cells can then be examined by epifluorescence microscopy using LIVE/DEAD staining and using molecular biology methods. Overall, the NaPP-based dispersant afforded a better extraction efficiency (20%) than methanol (6%). The light clay fraction acted as a sieve effectively retaining dispersed cells during centrifugation; this light clay fraction with attached cells being later detected in the final extracts. Importantly, the microbial community composition detected in cell extracts by 16S rRNA sequencing corresponded to that in the original suspension. The protocol has been successfully applied on different bentonite samples in different environments, demonstrating the high potential of this approach for evaluation of microbial activity in bentonite.

Název v anglickém jazyce

A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite

Popis výsledku anglicky

Bentonite is a clay material with a broad range of applications in construction, industry (food, pharmacy), and civil engineering including water treatment and waste disposal. It has also been proposed as a buffer and backfill material in deep geological repositories (DGRs). However, the presence of metabolically active bacteria in bentonite could compromise the long-term safety of such DGRs, highlighting the need for a method for detection of microorganisms in bentonite materials. Here, we propose a novel protocol for the detection of both living (metabolically active and dead indigenous bacterial cells in bentonite. The extraction protocol requires the addition of a dispersant (2.5 mM sodium pyrophosphate (NaPP)/12.5 mM EDTA solution or 1% methanol) to a bentonite sample, followed by a two-step centrifugation over high-density media (i.e., sucrose and histodenz) to separate and concentrate the cells. The extracted bacterial cells can then be examined by epifluorescence microscopy using LIVE/DEAD staining and using molecular biology methods. Overall, the NaPP-based dispersant afforded a better extraction efficiency (20%) than methanol (6%). The light clay fraction acted as a sieve effectively retaining dispersed cells during centrifugation; this light clay fraction with attached cells being later detected in the final extracts. Importantly, the microbial community composition detected in cell extracts by 16S rRNA sequencing corresponded to that in the original suspension. The protocol has been successfully applied on different bentonite samples in different environments, demonstrating the high potential of this approach for evaluation of microbial activity in bentonite.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10403 - Physical chemistry

Projekt

<a href="/cs/project/TK02010169" target="_blank" >TK02010169: Limitní faktory pro přežití a proliferaci mikrobiálních společenstev, významných pro korozi bariér hlubinného úložiště radioaktivních odpadů</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Applied Clay Science

ISSN

0169-1317

e-ISSN

1872-9053

Svazek periodika

232

Číslo periodika v rámci svazku

February

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

9

Strana od-do

1-9

Kód UT WoS článku

000917946800001

EID výsledku v databázi Scopus

2-s2.0-85145662746