Stable isotope probing: Technical considerations when resolving 15N-labeled RNA in gradients

Addison, Sarah; McDonald, Ian R.; Lloyd‐Jones, Gareth

doi:10.1016/j.mimet.2009.11.002

Cited by 18 publications

(10 citation statements)

References 29 publications

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“…Conceptually, a dual 13 C and 15 N SIP experiment might be the ideal way to address the known interactions between C and N cycles (Hessen et al, 2004) at the individual taxon level. While 13 C SIP has become part of the standard microbial ecology toolkit, 15 N SIP has been less widely applied (Wawrik et al, 2009(Wawrik et al, , 2012Gallagher et al, 2010;Andeer et al, 2012) and is a more challenging procedure requiring at least 30 atm% 15 N enrichment for DNA or more for 15 N RNA SIP (Buckley et al, 2007;Addison et al, 2010). A new approach to SIP, Chip-SIP, circumvents some of the difficulties of separating 15 N DNA and RNA in a density gradient by separating microbial community RNA on a phylogenetic microarray for subsequent isotopic analysis with a Cameca NanoSIMS 50 (Cameca, Gennevilliers, France), an imaging secondary ion mass spectrometer (Mayali et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Taxon-specific C/N relative use efficiency for amino acids in an estuarine community

Mayali

Weber

Pett‐Ridge

2012

FEMS Microbiol Ecol

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Microbial activity plays a critical role in determining the nutrient status of an ecosystem (i.e. N or C limitation). While the balance of C/N assimilation has been measured at the whole community scale, quantitative detection of N and C assimilation from a single substrate at the scale of individual taxa has not been carried out. We recently developed Chip-SIP, a microarray and NanoSIMS-based method for linking microbial phylogeny and function that allows simultaneous measurement of (15)N and (13)C incorporation. Here, we measured the relative incorporation of C and N from dual-labeled substrates by individual microbial taxa in bottle incubations of samples collected from an estuary. Incubation times < 24 h were sufficient to successfully detect active microbes incorporating (15)N ammonium. In subsequent experiments, we used the incorporation of labeled amino acids (AAs) as a proxy for heterotrophic activity and showed different levels of incorporation among different taxonomic groups. Taxon-specific differences in the net incorporation of AA-derived C and N indicate that the C/N relative use efficiency ranged from 0.8 to 1.4, where 1 reflects stoichiometric incorporation of C and N. Our results revealed that microbial organic matter processing is affected by taxon-specific physiological diversity, both in terms of general activity levels and in the ratio of assimilated C/N.

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Section: Introductionmentioning

confidence: 99%

Taxon-specific C/N relative use efficiency for amino acids in an estuarine community

Mayali

Weber

Pett‐Ridge

2012

FEMS Microbiol Ecol

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“…Since the content of nitrogen in RNA is approximately 2.5 times less than carbon, it presents the difficulty of a lower density gain after a 15 N pulse. Addison et al (2010) observed that 15 Nlabelled RNA increased in buoyant density compared to unlabelled RNA when centrifuged individually, but not when centrifuged together. Similarly, labelled RNA extracted from a paper mill effluent microcosm after a 15 N 2 pulse, showed a limited separation by density centrifugation in CsTFA.…”

Section: Rna-stable Isotope Probing (Rna-sip)mentioning

confidence: 90%

An appraisal of methods for linking environmental processes to specific microbial taxa

Gutierrez-Zamora

Manefield

2010

Rev Environ Sci Biotechnol

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The last decade has witnessed a revolution in the development of methods and technology available to investigate the ecological roles of microorganisms in the environment. As a consequence, microbial ecologists have gained a better understanding of the functional aspects of microorganisms in marine, groundwater and freshwater systems, soils, sediments, hot springs, wastewater treatment plants, landfills, the rhizosphere and the animal gut. This review provides a compilation and critical comparison of the currently available methods linking microbial function with phylogeny, including a description and advantages and limitations of each method. Examples are also provided to illustrate their application. The ongoing improvements of these 'function-identity' methods points to a bright future in our understanding of complex ecological processes and to improved management of microbe dependent ecosystem services.

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“…Nitrogen-fixing activity has also been reported in a number of pulp and paper aerated lagoon systems (Gauthier et al 2000;Yu & Mohn 2001;Smith et al 2003), including the two New Zealand kraft mills (Clark et al 1997;Addison et al 2007Addison et al , 2009Bowers et al 2008). Another useful ''rule-ofthumb'' would be the BOD:N ratio at which a wastewater could be considered suitable for nitrogen-fixing treatment.…”

Section: Introductionmentioning

confidence: 98%

The relationship between BOD:N ratio and wastewater treatability in a nitrogen-fixing wastewater treatment system

Slade

Thorn

Dennis

2011

Water Science and Technology

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A BOD:N:P ratio of 100:5:1 is often used as a benchmark for nutrient addition in nutrient limited wastewaters. The impact of varying nitrogen levels, whilst maintaining phosphorus constant, was studied in a simulated aerated lagoon (BOD:N of 100:0; 100:1.3; 100:1.8; 100:2.7 and 100:4.9). A synthetic wastewater was prepared using methanol, glucose and acetate as the combined carbon source, ammonium chloride as the nitrogen source and dipotassium hydrogen phosphate as the phosphorus source. Nitrogen levels did not impact organic carbon removal, but did strongly influence floc structure. With no supplemental nitrogen, growth was dispersed. Increasing the nitrogen level increased filamentous growth, with a marked change in filamentous species occurring between a BOD:N ratio of 100:1.8 and 100:2.7. Nitrogen fixation occurred at a BOD:N ratio of 100:0; 100:1.3 and 100:1.8, with nitrogen loss at BOD:N ratios of 100:2.7 and 100:4.9. At a BOD:N ratio of 100:4.9, ammonium discharge was significantly greater (1.8 mg/L) than at the lower nitrogen levels (0.04 - 0.18 mg/L). Phosphorus behaviour was more variable, however significantly more phosphorus was discharged at the lowest nitrogen level than at the highest (p<0.05). Based on readily available nitrogen, the BOD:N ratio at which nitrogen fixation no longer occurred was around 100:1.9.

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Stable isotope probing: Technical considerations when resolving 15N-labeled RNA in gradients

Cited by 18 publications

References 29 publications

Taxon-specific C/N relative use efficiency for amino acids in an estuarine community

Taxon-specific C/N relative use efficiency for amino acids in an estuarine community

An appraisal of methods for linking environmental processes to specific microbial taxa

The relationship between BOD:N ratio and wastewater treatability in a nitrogen-fixing wastewater treatment system

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