Barth F. Smets

We demonstrate the first viscoelastic-based flow-integrated Raman spectroscopy (FIRS) platform using spontaneous Raman spectroscopy with a simplified fluidic design that precisely controls particle transit speed and predicts arrival time to the Raman interrogation region, together with time synchronized triggered acquisition. Using viscoelastic flow focusing and optical detection of velocity for hardware triggering, the setup enables high-throughput, real-time Raman analysis of particles in continuous flow. The system operates with a single pressure pump, two sets of fiber-coupled lasers and detectors, and a microcapillary precisely aligned in a projection micro-stereolithography 3D-printed mount, and ensures detection stability across various flow rates. The demonstrated platform is modular and scalable. Detection rates of up to 80 cells per minute were achieved and dinoflagellate Alexandrium ostenfeldii cultures were analyzed. The integrated optical trigger mechanism supports future extensions such as cell sorting or additional analyses. A user-friendly graphical interface provides full control of fluidics, spectroscopy, and real-time monitoring, making this compact, low-cost FIRS platform accessible across a wide range of laboratory environments.

Hydroxylamine, nitrous acid, and nitric oxide are obligate intermediates or side metabolites in different nitrogen-converting microorganisms. These compounds are unstable and susceptible to the formation of highly reactive nitrogen species, including nitrogen dioxide, dinitrogen trioxide, nitroxyl, and peroxynitrite. Due to the high reactivity and cytotoxicity, the buildup of reactive nitrogen can affect the interplay of microorganisms/microbial processes, stimulate the reactions with organic compounds like organic micropollutants (OMP) and act as the precursors of nitrous oxide (N₂O). However, there is little understanding of the occurrence and significance of reactive nitrogen during biological nitrogen conversions in engineered water systems. In this review, we evaluate the formation and fate of reactive nitrogen produced by microorganisms involved in biological nitrogen removal (BNR) processes, i.e., nitritation/nitrification, denitratation/denitrification, anammox, and the combined processes. While the formation of reactive nitrogen intermediates is entirely controlled by microbial activities, the consumption can be either biological or purely chemical. Changes in environmental conditions, such as redox transition, pH, and substrate availability, can imbalance the production and consumption of these reactive intermediates, thus leading to the transient accumulation of species. Based on previous experimental evidence, environmental relevance of reactive nitrogen in BNR systems, particularly related to abiotic N₂O production and OMP transformation, is demonstrated.

Mating assays are common laboratory experiments for measuring the conjugation frequency, i.e. efficiency at which a plasmid transfers from a population of donor cells to a population of recipient cells. Selective plating remains a widely used quantification method to enumerate transconjugants at the end of such assays. However, conjugation frequencies may be inaccurately estimated because plasmid transfer can occur on transconjugant-selective plates rather than only during the intended mating duration. We investigated the influence of cell density on this phenomenon. We conducted mating experiments with IncPα plasmid RP4 harbored in Escherichia coli at a fixed cell density and mating conditions, inoculated a serial dilution of the mating mixture on transconjugant-selective plates or in transconjugant-selective broth, and compared the results to a model of cell-to-cell distance distribution. Our findings suggest that irrespective of the mating mode (liquid vs solid), the enumeration of transconjugants becomes significantly biased if the plated cell density exceeds 28 Colony Forming Unit (CFU)/mm² (or 1.68•10⁵ CFU/standard 9 cm Petri dish). This threshold is determined with a 95% confidence interval of ± 4 CFU/mm² (± 2.46•10⁴ CFU/standard 9 cm Petri dish). Liquid mating assays were more sensitive to this bias because the conjugation frequency of RP4 is several orders of magnitude lower in suspension compared to surface mating. Therefore, if selective plating is used, we recommend to plate at this density threshold and that negative controls are performed where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating assay. As an alternative, a liquid enumeration method can be utilized to increase the signal-to-noise ratio and allow for more accurate enumeration of transconjugants.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments, and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske‐type dioxygenases and flavin‐dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth‐supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4‐hydroxybenzoic acid, 4‐hydroxybenzaldehyde; Group 2: p ‐coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre‐growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor ( Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost‐efficient approach that reveals mechanistic insights into OMP‐DOM biodegradation.

Nitrification is mediated by numerous different microorganisms, but knowledge of their ecophysiologies is insufficient. Leachate in the late stages of landfill operation provides a brackish environment with a high ammonia concentration, and methanol is added as an electron donor for denitrification. Such a unique environment may contain novel nitrifiers. Here, we present metagenomic analysis of the microbiome from a closed landfill leachate treatment facility to investigate the identity and functions of nitrifiers. Using a genome-centric approach with metagenomic analysis, we retrieved draft genomes for a novel complete ammonia-oxidizing (comammox) bacterium Nitrospira LAS72; and canonical Nitrospira LAS18, clustered within a novel sub-lineage VII of Nitrospira ; Candidatus Nitrosocosmicus LAS21 and Nitrosarchaeum LAS73, belonging to the ammonia-oxidizing archaea (AOA). This is the first evidence of comammox Nitrospira in a high-ammonia-containing brackish environment. Canonical ammonia-oxidizing bacteria were not detected. Given the brackish environment and supplementation of methanol used in the facility, we also investigated the methanol metabolism of these nitrifiers and their potential to produce compatible solutes as osmoprotectants. Uniquely among Nitrospira , comammox Nitrospira LAS72 possesses genes associated with formaldehyde reductase and glycine betaine biosynthesis. Thus, Nitrospira LAS72 may proliferate because of the availability of formaldehyde upstream of carbon metabolism and adapt to fluctuating osmotic pressure by producing a variety of compatible solutes. The discovery of this novel comammox Nitrospira, and canonical Nitrospira forming a new sub-lineage VII in an ammonia-concentrated brackish environment broadens our knowledge of the diversity and functions of nitrifying microorganisms.

Microbial communities are found in all habitable environments and often occur in assemblages with self-organized spatial structures developing over time. This complexity can only be understood, predicted, and managed by combining experiments with mathematical modeling. Individual-based models are particularly suited if individual heterogeneity, local interactions, and adaptive behavior are of interest. Here we present the completely overhauled software platform, the i ndividual-based Dyn amics o f Mi crobial C ommunities S imulator, iDynoMiCS 2.0, which enables researchers to specify a range of different models without having to program. Key new features and improvements are: (1) Substantially enhanced ease of use (graphical user interface, editor for model specification, unit conversions, data analysis and visualization and more). (2) Increased performance and scalability enabling simulations of up to 10 million agents in 3D biofilms. (3) Kinetics can be specified with any arithmetic function. (4) Agent properties can be assembled from orthogonal modules for pick and mix flexibility. (5) Force-based mechanical interaction framework enabling attractive forces and non-spherical agent morphologies as an alternative to the shoving algorithm. The new iDynoMiCS 2.0 has undergone intensive testing, from unit tests to a suite of increasingly complex numerical tests and the standard Benchmark 3 based on nitrifying biofilms. A second test case was based on the “biofilms promote altruism” study previously implemented in BacSim because competition outcomes are highly sensitive to the developing spatial structures due to positive feedback between cooperative individuals. We extended this case study by adding morphology to find that (i) filamentous bacteria outcompete spherical bacteria regardless of growth strategy and (ii) non-cooperating filaments outcompete cooperating filaments because filaments can escape the stronger competition between themselves. In conclusion, the new substantially improved iDynoMiCS 2.0 joins a growing number of platforms for individual-based modeling of microbial communities with specific advantages and disadvantages that we discuss, giving users a wider choice. Author summary: Microbes are fascinating in their own right and play a tremendously important role in ecosystems. They often form complex, self-organized communities with spatial heterogeneity that is changing over time. Such complexity is challenging to understand and manage without the help of mathematical models. Individual-based models are one type of mathematical model that is particularly suited if differences between individual microbes, local interactions and adaptive behavior are important. We have developed a completely overhauled version of iDynoMiCS, a software that allows users to develop, run and analyze a wide range of individual-based models without having to program the software themselves. There are several capability enhancements and numerous small improvements, for example the ability to model different shapes of cells combined with physically realistic mechanical interactions between neighboring cells. We showcase this by simulating the competition between filaments, long chains of cells, with single cells and find that filaments outcompete single cells as they can spread quickly to new territory with higher levels of resources. Users now have a wider choice of platforms so we provide guidance on which platform might be most suitable for a given purpose.