Holger Daims

The Domain-specific Probe EUB338 is Insufficient for the Detection of all Bacteria: Development and Evaluation of a more Comprehensive Probe Set

In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.

Deciphering the evolution and metabolism of an anammox bacterium from a community genome

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycles major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics—the reconstruction of genomic data directly from the environment—to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organisms special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants.

ABSTRACT Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of theNitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genusNitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates ofNitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospiramicrocolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources byNitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, theNitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO3− or as CO2) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by theNitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring

The recent discovery of ammonia-oxidizing archaea (AOA) dramatically changed our perception of the diversity and evolutionary history of microbes involved in nitrification. In this study, a moderately thermophilic (46°C) ammonia-oxidizing enrichment culture, which had been seeded with biomass from a hot spring, was screened for ammonia oxidizers. Although gene sequences for crenarchaeotal 16S rRNA and two subunits of the ammonia monooxygenase (amoA and amoB) were detected via PCR, no hints for known ammonia-oxidizing bacteria were obtained. Comparative sequence analyses of these gene fragments demonstrated the presence of a single operational taxonomic unit and thus enabled the assignment of the amoA and amoB sequences to the respective 16S rRNA phylotype, which belongs to the widely distributed group I.1b (soil group) of the Crenarchaeota. Catalyzed reporter deposition (CARD)–FISH combined with microautoradiography (MAR) demonstrated metabolic activity of this archaeon in the presence of ammonium. This finding was corroborated by the detection of amoA gene transcripts in the enrichment. CARD-FISH/MAR showed that the moderately thermophilic AOA is highly active at 0.14 and 0.79 mM ammonium and is partially inhibited by a concentration of 3.08 mM. The enriched AOA, which is provisionally classified as “Candidatus Nitrososphaera gargensis,” is the first described thermophilic ammonia oxidizer and the first member of the crenarchaeotal group I.1b for which ammonium oxidation has been verified on a cellular level. Its preference for thermophilic conditions reinvigorates the debate on the thermophilic ancestry of AOA.

Complete nitrification by Nitrospira bacteria

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Microbial community composition and function in wastewater treatment plants.

Biological wastewater treatment has been applied for more than a century to ameliorate anthropogenic damage to the environment. But only during the last decade the use of molecular tools allowed to accurately determine the composition, and dynamics of activated sludge and biofilm microbial communities. Novel, in many cases yet not cultured bacteria were identified to be responsible for filamentous bulking and foaming as well as phosphorus and nitrogen removal in these systems. Now, methods are developed to infer the in situ physiology of these bacteria. Here we provide an overview of what is currently known about the identity and physiology of some of the microbial key players in activated sludge and biofilm systems.

Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes

Fluorescence in situ hybridisation with rRNA-targeted nucleic acid probes can be used to directly identify microorganisms within complex samples in a few hours and therefore has widespread application in environmental and medical microbiology. The past year has seen significant methodological improvements in fluorescence in situ hybridisation, as well as in the combination of this method with other techniques for inferring functional traits of microorganisms within their environment.

Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers

Nitrification is a core process in the global nitrogen cycle that is essential for the functioning of many ecosystems. The discovery of autotrophic ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota has changed our perception of the microbiology of nitrification, in particular since their numerical dominance over ammonia-oxidizing bacteria (AOB) in many environments has been revealed. These and other data have led to a widely held assumption that all amoA-encoding members of the Thaumarchaeota (AEA) are autotrophic nitrifiers. In this study, 52 municipal and industrial wastewater treatment plants were screened for the presence of AEA and AOB. Thaumarchaeota carrying amoA were detected in high abundance only in four industrial plants. In one plant, thaumarchaeotes closely related to soil group I.1b outnumbered AOB up to 10,000-fold, and their numbers, which can only be explained by active growth in this continuous culture system, were two to three orders of magnitude higher than could be sustained by autotrophic ammonia oxidation. Consistently, 14CO2 fixation could only be detected in AOB but not in AEA in actively nitrifying sludge from this plant via FISH combined with microautoradiography. Furthermore, in situ transcription of archaeal amoA, and very weak in situ labeling of crenarchaeol after addition of 13CO2, was independent of the addition of ammonium. These data demonstrate that some amoA-carrying group I.1b Thaumarchaeota are not obligate chemolithoautotrophs.

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

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.

Microbial landscapes: new paths to biofilm research.

It is the best of times for biofilm research. Systems biology approaches are providing new insights into the genetic regulation of microbial functions, and sophisticated modelling techniques are enabling the prediction of microbial community structures. Yet it is also clear that there is a need for ecological theory to contribute to our understanding of biofilms. Here, we suggest a concept for biofilm research that is spatially explicit and solidly rooted in ecological theory, which might serve as a universal approach to the study of the numerous facets of biofilms.