Aaron Marc Saunders

Archaea Dominate the Ammonia-Oxidizing Community in the Rhizosphere of the Freshwater Macrophyte Littorella uniflora

ABSTRACT Archaeal and bacterial ammonia monooxygenase genes (amoA) had similar low relative abundances in freshwater sediment. In the rhizosphere of the submersed macrophyte Littorella uniflora, archaeal amoA was 500- to >8,000-fold enriched compared to bacterial amoA, suggesting that the enhanced nitrification activity observed in the rhizosphere was due to ammonia-oxidizing Archaea.

A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants

The microbial populations in 25 full-scale activated sludge wastewater treatment plants with enhanced biological phosphorus removal (EBPR plants) have been intensively studied over several years. Most of the important bacterial groups involved in nitrification, denitrification, biological P removal, fermentation, and hydrolysis have been identified and quantified using quantitative culture-independent molecular methods. Surprisingly, a limited number of core species was present in all plants, constituting on average approx. 80% of the entire communities in the plants, showing that the microbial populations in EBPR plants are rather similar and not very diverse, as sometimes suggested. By focusing on these organisms it is possible to make a comprehensive ecosystem model, where many important aspects in relation to microbial ecosystems and wastewater treatment can be investigated. We have reviewed the current knowledge about these microorganisms with focus on key ecophysiological factors and combined this into a conceptual ecosystem model for EBPR plants. It includes the major pathways of carbon flow with specific organic substances, the dominant populations involved in the transformations, interspecies interactions, and the key factors controlling their presence and activity. We believe that the EBPR process is a perfect model system for studies of microbial ecology in water engineering systems and that this conceptual model can be used for proposing and testing theories based on microbial ecosystem theories, for the development of new and improved quantitative ecosystem models and is beneficial for future design and management of wastewater treatment systems.

A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) is widely used for removal of phosphorus from wastewater. In this study, a metagenome (18.2 Gb) was generated using Illumina sequencing from a full-scale EBPR plant to study the community structure and genetic potential. Quantitative fluorescence in situ hybridization (qFISH) was applied as an independent method to evaluate the community structure. The results were in qualitative agreement, but a DNA extraction bias against gram positive bacteria using standard extraction protocols was identified, which would not have been identified without the use of qFISH. The genetic potential for community function showed enrichment of genes involved in phosphate metabolism and biofilm formation, reflecting the selective pressure of the EBPR process. Most contigs in the assembled metagenome had low similarity to genes from currently sequenced genomes, underlining the need for more reference genomes of key EBPR species. Only the genome of ‘Candidatus Accumulibacter’, a genus of phosphorus-removing organisms, was closely enough related to the species present in the metagenome to allow for detailed investigations. Accumulibacter accounted for only 4.8% of all bacteria by qFISH, but the depth of sequencing enabled detailed insight into their microdiversity in the full-scale plant. Only 15% of the reads matching Accumulibacter had a high similarity (>95%) to the sequenced Accumulibacter clade IIA strain UW-1 genome, indicating the presence of some microdiversity. The differences in gene complement between the Accumulibacter clades were limited to genes for extracellular polymeric substances and phage-related genes, suggesting a selective pressure from phages on the Accumulibacter diversity.

Effect of Lake Trophic Status and Rooted Macrophytes on Community Composition and Abundance of Ammonia-Oxidizing Prokaryotes in Freshwater Sediments

ABSTRACT Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species Littorella uniflora, Juncus bulbosus, and Myriophyllum alterniflorum were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (amoA) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting amoA, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with M. alterniflorum (0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of L. uniflora and J. bulbosus. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like L. uniflora and J. bulbosus, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked.

Influence of starvation on potential ammonia-oxidizing Activity and amoA mRNA levels of Nitrosospira briensis

ABSTRACT The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NOx biosensor. The apparent half-saturation constant [Km(app)] value of the NH3 oxidation of N. briensis was 3 μM NH3 for cultures grown both in continuous and batch cultures as determined by a NOx biosensor. Cells grown on the wall of the vessel had a lower Km(app) value of 1.8 μM NH3. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 μM N h−1, and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH4+, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH4+ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH4+ availability.

The activated sludge ecosystem contains a core community of abundant organisms.

Understanding the microbial ecology of a system requires that the observed population dynamics can be linked to their metabolic functions. However, functional characterization is laborious and the choice of organisms should be prioritized to those that are frequently abundant (core) or transiently abundant, which are therefore putatively make the greatest contribution to carbon turnover in the system. We analyzed the microbial communities in 13 Danish wastewater treatment plants with nutrient removal in consecutive years and a single plant periodically over 6 years, using Illumina sequencing of 16S ribosomal RNA amplicons of the V4 region. The plants contained a core community of 63 abundant genus-level operational taxonomic units (OTUs) that made up 68% of the total reads. A core community consisting of abundant OTUs was also observed within the incoming wastewater to three plants. The net growth rate for individual OTUs was quantified using mass balance, and it was found that 10% of the total reads in the activated sludge were from slow or non-growing OTUs, and that their measured abundance was primarily because of immigration with the wastewater. Transiently abundant organisms were also identified. Among them the genus Nitrotoga (class Betaproteobacteria) was the most abundant putative nitrite oxidizer in a number of activated sludge plants, which challenges previous assumptions that Nitrospira (phylum Nitrospirae) are the primary nitrite-oxidizers in activated sludge systems with nutrient removal.

MiDAS: the field guide to the microbes of activated sludge.

The Microbial Database for Activated Sludge (MiDAS) field guide is a freely available online resource linking the identity of abundant and process critical microorganisms in activated sludge wastewater treatment systems to available data related to their functional importance. Phenotypic properties of some of these genera are described, but most are known only from sequence data. The MiDAS taxonomy is a manual curation of the SILVA taxonomy that proposes a name for all genus-level taxa observed to be abundant by large-scale 16 S rRNA gene amplicon sequencing of full-scale activated sludge communities. The taxonomy can be used to classify unknown sequences, and the online MiDAS field guide links the identity to the available information about their morphology, diversity, physiology and distribution. The use of a common taxonomy across the field will provide a solid foundation for the study of microbial ecology of the activated sludge process and related treatment processes. The online MiDAS field guide is a collaborative workspace intended to facilitate a better understanding of the ecology of activated sludge and related treatment processes—knowledge that will be an invaluable resource for the optimal design and operation of these systems. Database URL: http://www.midasfieldguide.org

Microbial communities involved in enhanced biological phosphorus removal from wastewater: A model system in environmental biotechnology

Enhanced biological phosphorus removal (EBPR) is one of the most advanced and complicated wastewater treatment processes applied today, and it is becoming increasingly popular worldwide as a sustainable way to remove and potentially reuse P. It is carried out by complex microbial communities consisting primarily of uncultured microorganisms. The EBPR process is a well-studied system with clearly defined boundaries which makes it very suitable as a model ecosystem in microbial ecology. Of particular importance are the transformations of C, N, and P, the solid-liquid separation properties and the functional and structural stability. A range of modern molecular methods has been used to study these communities in great detail including single cell microbiology, various -omics methods, flux analyses, and modeling making this one of the best studied microbial ecosystems so far. Recently, an EBPR core microbiome has been described and we present in this article some highlights and show how this complex microbial community can be used as model ecosystem in environmental biotechnology.

The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods.

The bacterial microbiota plays an important role in the prolonged healing of chronic venous leg ulcers. The present study compared the bacterial diversity within ulcer material from 14 skin graft operations of chronic venous leg ulcers using culture‐based methods and molecular biological methods, such as 16S rRNA gene sequencing, fingerprinting, quantitative polymerase chain reaction, and fluorescence in situ hybridization. Each wound contained an average of 5.4 species but the actual species varied between wounds. The diversity determined by culture‐based methods and the molecular biological methods was different. All the wounds contained Staphylococcus aureus, whereas Pseudomonas aeruginosa was in six out of 14 wounds. Molecular methods detected anaerobic pathogens in four ulcers that were not detected with anaerobic culture methods. Quantitative polymerase chain reaction was used to compare the abundance of S. aureus and P. aeruginosa at different locations in the ulcers and their numbers varied greatly between samples taken at different locations in the same ulcer. This should be considered when ulcers are investigated in routine clinical care. The differences between the results obtained with culture‐based and molecular‐based approaches demonstrate that the use of one approach alone is not able to identify all of the bacteria present in the wounds.