Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria.

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification.

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named "Candidatus Nitrospira kreftii", and performed a detailed genomic and physiological characterization. The complete genome of "Ca. N. kreftii" allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (Km(app)_NO2- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of "Ca. N. kreftii" indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.

The draft genome sequence of "Nitrospira lenta" strain BS10, a nitrite oxidizing bacterium isolated from activated sludge.

The genus Nitrospira is considered to be the most widespread and abundant group of nitrite-oxidizing bacteria in many natural and man-made ecosystems. However, the ecophysiological versatility within this phylogenetic group remains highly understudied, mainly due to the lack of pure cultures and genomic data. To further expand our understanding of this biotechnologically important genus, we analyzed the high quality draft genome of "Nitrospira lenta" strain BS10, a sublineage II Nitrospira that was isolated from a municipal wastewater treatment plant in Hamburg, Germany. The genome of "N. lenta" has a size of 3,756,190 bp and contains 3968 genomic objects, of which 3907 are predicted protein-coding sequences. Thorough genome annotation allowed the reconstruction of the "N. lenta" core metabolism for energy conservation and carbon fixation. Comparative analyses indicated that most metabolic features are shared with N. moscoviensis and "N. defluvii", despite their ecological niche differentiation and phylogenetic distance. In conclusion, the genome of "N. lenta" provides important insights into the genomic diversity of the genus Nitrospira and provides a foundation for future comparative genomic studies that will generate a better understanding of the nitrification process.

Evaluation of a novel quorum quenching strain for MBR biofouling mitigation.

Membrane biofouling, due to Soluble Microbial Products (SMP) and Extracellular Polymeric Substances (EPS) deposition, results in reduction of the performance of Membrane Bioreactors (MBRs). However, recently, a new method of biofouling control has been developed, utilizing the interference of the bacterial inter- and intra-species' communication. Bacteria use Quorum Sensing (QS) to regulate the production of SMP and EPS. Therefore, disruption of Quorum Sensing (Quorum Quenching: QQ), by enzymes or microorganisms, may be a simple mean to control membrane biofouling. In the present study, a novel QQ-bacterium, namely Lactobacillus sp. SBR04MA, was isolated from municipal wastewater sludge and its ability to mitigate biofouling was evaluated by monitoring the changes in critical flux and transmembrane pressure, along with the production of EPS and SMP, in a lab-scale MBR system treating synthetic wastewater. Lactobacillus sp. SBR04MA showed great potential for biofouling control, which was evidenced by the ∼3-fold increase in critical flux (8.3 → 24.25 L/m2/h), as well as by reduction of the SMP and EPS production, which was lower during the QQ-period when compared against the control period. Furthermore, the addition of the QQ-strain did not affect the COD removal rate. Results suggested that Lactobacillus sp. SBR04MA represents a novel and promising strain for biofouling mitigation and enhancement of MBRs performance.