scholarly journals Identification of Ciliate Grazers of Autotrophic Bacteria in Ammonia-Oxidizing Activated Sludge by RNA Stable Isotope Probing

2010 ◽  
Vol 76 (7) ◽  
pp. 2203-2211 ◽  
Author(s):  
Ana Maria Moreno ◽  
Carsten Matz ◽  
Staffan Kjelleberg ◽  
Mike Manefield
Keyword(s):  
Escherichia Coli ◽  
Stable Isotope ◽  
Nutrient Cycling ◽  
Activated Sludge ◽  
Functional Groups ◽  
Clone Library ◽  
Bacterial Biomass ◽  
Stable Isotope Probing ◽  
Autotrophic Bacteria

ABSTRACT It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that 13C-labeled protozoan RNA can be retrieved from activated sludge amended with 13C-labeled protozoa or 13C-labeled Escherichia coli cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to CO2 under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.

scholarly journals Subgroup level differences of physiological activities in marine Lokiarchaeota

2020 ◽  
Author(s):  
Xiuran Yin ◽  
Mingwei Cai ◽  
Yang Liu ◽  
Guowei Zhou ◽  
Tim Richter-Heitmann ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Marine Sediments ◽  
Inorganic Carbon ◽  
Bacterial Biomass ◽  
Stable Isotope Probing ◽  
Aromatic Compound Degradation ◽  
Lactate Degradation ◽  
Globally Distributed ◽  
Mud Area

Abstract Asgard is a recently discovered archaeal superphylum, closely linked to the emergence of eukaryotes. Among Asgard archaea, Lokiarchaeota are abundant in marine sediments, but their in situ activities are largely unknown except for Candidatus ‘Prometheoarchaeum syntrophicum’. Here, we tracked the activity of Lokiarchaeota in incubations with Helgoland mud area sediments (North Sea) by stable isotope probing (SIP) with organic polymers, 13C-labelled inorganic carbon, fermentation intermediates and proteins. Within the active archaea, we detected members of the Lokiarchaeota class Loki-3, which appeared to mixotrophically participate in the degradation of lignin and humic acids while assimilating CO2, or heterotrophically used lactate. In contrast, members of the Lokiarchaeota class Loki-2 utilized protein and inorganic carbon, and degraded bacterial biomass formed in incubations. Metagenomic analysis revealed pathways for lactate degradation, and involvement in aromatic compound degradation in Loki-3, while the less globally distributed Loki-2 instead rely on protein degradation. We conclude that Lokiarchaeotal subgroups vary in their metabolic capabilities despite overlaps in their genomic equipment, and suggest that these subgroups occupy different ecologic niches in marine sediments.

scholarly journals Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography

2005 ◽  
Vol 71 (12) ◽  
pp. 8683-8691 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Batch Reactor ◽  
External Source ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
The Impact

ABSTRACT The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

scholarly journals Shotgun Isotope Array for Rapid, Substrate-Specific Detection of Microorganisms in a Microbial Community

2011 ◽  
Vol 77 (20) ◽  
pp. 7430-7432 ◽  
Author(s):  
Tomohiro Tobino ◽  
Futoshi Kurisu ◽  
Ikuro Kasuga ◽  
Hiroaki Furumai
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Prior Knowledge ◽  
Community Composition ◽  
Stable Isotope Probing ◽  
Proof Of Concept ◽  
Specific Detection

ABSTRACTThe shotgun isotope array method has been proposed to be an effective new tool for use in substrate-specific microbe exploration without any prior knowledge of the community composition. Proof of concept was demonstrated by detection of acetate-degrading microorganisms in activated sludge and further verified by independent stable isotope probing (SIP).

Towards safer stable isotope probing — effect of formamide on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation

2020 ◽  
Vol 66 (8) ◽  
pp. 491-494
Author(s):  
Severin Weis ◽  
Sylvia Schnell ◽  
Markus Egert
Keyword(s):  
Escherichia Coli ◽  
Stable Isotope ◽  
Microbial Ecology ◽  
Distribution Pattern ◽  
Density Gradient ◽  
Stable Isotope Probing ◽  
Density Gradient Ultracentrifugation ◽  
Molecular Microbial Ecology ◽  
Environmental Mixtures ◽  
Labeled Rna

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.

scholarly journals Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

2004 ◽  
Vol 70 (1) ◽  
pp. 588-596 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Philip Hugenholtz ◽  
Holger Daims ◽  
Michael Wagner ◽  
Jürg Keller ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Microbial Community ◽  
Stable Isotope ◽  
Fluorescence In Situ Hybridization ◽  
Clone Library ◽  
Stable Isotope Probing ◽  
Denitrification Rate ◽  
Biological Nutrient Removal ◽  
Group A

ABSTRACT A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3 −-N mg of mixed-liquor volatile suspended solids (MLVSS)−1 h−1 to a steady-state value of 0.06 mg of NO3 −-N mg of MLVSS−1 h−1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

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