Extraction of microorganisms from soil: evaluation of the efficiency by counting methods and activity measurements

Riis, V.; Lorbeer, Helmut; Babel, Wolfgang

doi:10.1016/s0038-0717(97)00232-0

Cited by 68 publications

(31 citation statements)

References 25 publications

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“…The soil composition was as follows: total salt content, 6.43% (wt/wt); pH 7.5; original water content, 6.44% (wt/wt); hydrocarbon content, 41 Isolation and propagation of the bacterial consortium. The microorganisms were extracted after mixing and sonicating the soil in 0.2% Na 4 P 2 O 7 solution (adjusted to pH 8.5) as described previously (58), except that 7.5% NaCl was added. The consortium was enriched in shaken flasks containing 250 ml mineral medium amended with diesel fuel (800 l liter Ϫ1 ), paraffin (400 l liter Ϫ1 ), and yeast extract (120 mg liter Ϫ1 ).…”

Section: Methodsmentioning

confidence: 99%

Population Dynamics within a Microbial Consortium during Growth on Diesel Fuel in Saline Environments

Kleinsteuber

Riis

Fetzer

et al. 2006

Appl Environ Microbiol

134

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The diversity and dynamics of a bacterial community extracted from an exploited oil field with high natural soil salinity near Comodoro Rivadavia in Patagonia (Argentina) were investigated. Community shifts during long-term incubation with diesel fuel at four salinities between 0 and 20% NaCl were monitored by singlestrand conformation polymorphism community fingerprinting of the PCR-amplified V4-V5 region of the 16S rRNA genes. Information obtained by this qualitative approach was extended by flow cytometric analysis to follow quantitatively the dynamics of community structures at different salinities. Dominant and newly developing clusters of individuals visualized via their DNA patterns versus cell sizes were used to identify the subcommunities primarily involved in the degradation process. To determine the most active species, subcommunities were separated physically by high-resolution cell sorting and subsequent phylogenetic identification by 16S rRNA gene sequencing. Reduced salinity favored the dominance of Sphingomonas spp., whereas at elevated salinities, Ralstonia spp. and a number of halophilic genera, including Halomonas, Dietzia, and Alcanivorax, were identified. The combination of cytometric sorting with molecular characterization allowed us to monitor community adaptation and to identify active and proliferating subcommunities.Many oil fields are located in semiarid regions characterized by soils of high natural salinity. Careless operation during oil extraction and processing contaminates the soil, for instance, via the generation of large volumes of oily and saline wastewater. Strategies are thus needed to effectively treat oil pollution of highly saline waters and soils.The degradation of hydrocarbons as major components of mineral oil is generally brought about by microorganisms. At the salinity of seawater (ϳ3% total salts, mainly NaCl) or below, most low-molecular-weight components of mineral oil are easily degraded by a variety of microorganisms. However, the range of degrading organisms decreases with increasing salinity. There are only a few reports on the degradation of hydrocarbons in hypersaline environments. Enrichment cultures from the Great Solar Lake grew on mineral oil at salinities up to 17.2% (70), and a bacterial consortium and an extremely halophilic archaeon grew on hexadecane and crude oil at salinities of 15% and 32%, respectively (5). Degradation of hydrocarbons (mainly n-alkanes) by archaea (Halobacteria) at 15 to 32% NaCl was also reported (40). An extremely halophilic archaeon from a salt marsh degraded various saturated and aromatic hydrocarbons at salinities of up to 31% and grew optimally at 22% NaCl (10). In our own laboratory, microbial communities from saline soils in Patagonia degraded diesel fuel up to a salinity of 17.5% (59). Although some members of the communities were identified, their contributions to the diesel fuel biodegradation remained obscure.Information on the phylogenetic diversity of microbial communities can be obtained by molecular methods, like...

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Section: Methodsmentioning

confidence: 99%

Population Dynamics within a Microbial Consortium during Growth on Diesel Fuel in Saline Environments

Kleinsteuber

Riis

Fetzer

et al. 2006

Appl Environ Microbiol

134

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“…The second dilution (50 ml; 10 ±4 concentration) was then centrifuged at 100 g for 10 min. This low-speed centrifugation was recommended by Riis et al (1998), who compared different methods of extracting micro-organisms from soil. The supernatant¯uid (150 ll) was added to each well of a Biolog GN and MT plate.…”

Section: Inoculating Platesmentioning

confidence: 99%

Microbial communities in different soil types do not converge after diesel contamination

2002

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“…Epifluorescence microscopy (EFM) was developed as a standardised method allowing direct microscopic counts of microbial cells stained with fluorescence dyes. This method was successfully employed to count bacteria in freshwater (Jones, 1979), marine water (Daley, 1979), soil (Barra Caracciolo et al, 2005;Elazhari-Ali et al, 2013;Riis et al, 1998) and sediments samples (Amalfitano and Fazi, 2008). The main factors hampering the wider application of these methods for biomass quantification include the laborious and time-consuming nature of the analytical procedure, especially when working with solid matrices.…”

Section: Introductionmentioning

confidence: 99%

Flow-cytometric quantification of microbial cells on sand from water biofilters

et al. 2018

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a b s t r a c tRapid quantification of absolute microbial cell abundances is important for a comprehensive interpretation of microbiome surveys and crucial to support theoretical modelling and the design of engineered systems. In this paper, we propose a protocol specifically optimised for the quantification of microbial abundances in water biofilters using flow cytometry (FCM). We optimised cell detachment from sand biofilter particles for FCM quantification through the evaluation of five chemical dispersants (NaCl, Triton-X100, CaCl 2 , sodium pyrophosphate (PP), Tween 80 combined with PP), different mechanical pretreatments (low and high energy sonication and shaking) and two fixation methods (glutaraldehyde and ethanol). The developed protocol was cross-compared using other established and commonly employed methods for biomass quantification in water filter samples (adenosine triphosphate (ATP) quantification, real-time quantitative PCR (qPCR) and volatile solids (VS)). The highest microbial count was obtained by detaching the biofilm from biofilter grains and dispersing clusters into singles cells using Tween 80 and sodium pyrophosphate combined with four steps of high energy sonication (27W, for 80 s each step); glutaraldehyde was shown to be the best fixative solution. The developed protocol was reliable and highly reproducible and produced results that are comparable to data from alternative quantification methods. Indeed, high correlations were found with trends obtained through ATP and qPCR (r ¼ 0.98 and r ¼ 0.91) measurements. The VS content was confirmed as an inaccurate method to express biomass in sand samples since it correlated poorly with all the other three methods (r ¼ 0.005 with FCM, 0.002 with ATP and 0.177 with qPCR). FCM and ATP showed the strongest agreement between absolute counts with a slope of the correlation equal to 0.7, while qPCR seemed to overestimate cell counts by a factor of ten. The rapidity and reproducibility of the method developed make its application ideal for routine quantification of microbial cell abundances on sand from water biofilters and thus useful in revealing the ecological patterns and quantifying the metabolic kinetics involved in such systems.

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Extraction of microorganisms from soil: evaluation of the efficiency by counting methods and activity measurements

Cited by 68 publications

References 25 publications

Population Dynamics within a Microbial Consortium during Growth on Diesel Fuel in Saline Environments

Population Dynamics within a Microbial Consortium during Growth on Diesel Fuel in Saline Environments

Microbial communities in different soil types do not converge after diesel contamination

Flow-cytometric quantification of microbial cells on sand from water biofilters

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