"Candidatus Siderophilus nitratireducens": a putative nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales.

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens," carries genes central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). Using thermodynamics, we demonstrate that iron oxidation coupled to nap based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe3+/Fe2+ and NO3-/NO2- concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.

Product Inhibition and pH Affect Stoichiometry and Kinetics of Chain Elongating Microbial Communities in Sequencing Batch Bioreactors.

Anaerobic microbial communities can produce carboxylic acids of medium chain length (e.g., caproate, caprylate) by elongating short chain fatty acids through reversed ß-oxidation. Ethanol is a common electron donor for this process. The influence of environmental conditions on the stoichiometry and kinetics of ethanol-based chain elongation remains elusive. Here, a sequencing batch bioreactor setup with high-resolution off-gas measurements was used to identify the physiological characteristics of chain elongating microbial communities enriched on acetate and ethanol at pH 7.0 ± 0.2 and 5.5 ± 0.2. Operation at both pH-values led to the development of communities that were highly enriched (>50%, based on 16S rRNA gene amplicon sequencing) in Clostridium kluyveri related species. At both pH-values, stably performing cultures were characterized by incomplete substrate conversion and decreasing biomass-specific hydrogen production rates during an operational cycle. The process stoichiometries obtained at both pH-values were different: at pH 7.0, 71 ± 6% of the consumed electrons were converted to caproate, compared to only 30 ± 5% at pH 5.5. Operating at pH 5.5 led to a decrease in the biomass yield, but a significant increase in the biomass-specific substrate uptake rate, suggesting that the organisms employ catabolic overcapacity to deal with energy losses associated to product inhibition. These results highlight that chain elongating conversions rely on a delicate balance between substrate uptake- and product inhibition kinetics.

Experimental infrastructure requirements for quantitative research on microbial communities.

Natural microbial communities are composed of a large diversity of interacting microorganisms, each with a specific role in the functional properties of the ecosystem. The objectives in microbial ecology research are related to identifying, understanding and exploring the role of these different microorganisms. Because of the rapidly increasing power of DNA sequencing and the rapid increase of genomic data, main attention of microbial ecology research shifted from cultivation-oriented studies towards metagenomic studies. Despite these efforts, the direct link between the molecular properties and the measurable changes in the functional performance of the ecosystem is often poorly documented. A quantitative understanding of functional properties in relation to the molecular changes requires effective integration, standardization, and parallelization of experiments. High-resolution functional characterization is a prerequisite for interpretation of changes in metagenomic properties, and will improve our understanding of microbial communities and facilitate their exploration for health and circular economy related objectives.

Seemingly trivial secondary factors may determine microbial competition: a cautionary tale on the impact of iron supplementation through corrosion.

Microbial community engineering aims for enrichment of a specific microbial trait by imposing specific cultivation conditions. This work demonstrates that things may be more complicated than typically presumed and that microbial competition can be affected by seemingly insignificant variables, like in this case the type of acid used for pH control. Aerobic bioreactors pulse fed with acetate operated with hydrochloric acid resulted in the enrichment of Plasticicumulans acidivorans, and changing the pH controlling agent to sulfuric acid shifted the community towards Zoogloea sp. Further research demonstrated that the change in community structure was not directly caused by the change in acid used for pH control, but resulted from the difference in corrosive strength of both acids and the related iron leaching from the bioreactor piping. Neither system was iron deficient, suggesting that the biological availability of iron is affected by the leaching process. Our results demonstrate that microbial competition and process development can be affected dramatically by secondary factors related to nutrient supply and bioavailability, and is way more complex than generally assumed in a single carbon substrate limited process.

"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of "Candidatus Galacturonibacter soehngenii," from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of "Ca. G. soehngenii" was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the "Ca. G. soehngenii" MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of "Ca. G. soehngenii" enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of "Ca. G. soehngenii."

Simultaneous growth and poly(3-hydroxybutyrate) (PHB) accumulation in a Plasticicumulans acidivorans dominated enrichment culture.

The wide variety of organic carbon to nitrogen and phosphorous ratios that are encountered in different wastewaters has a major impact on the poly(3-hydroxybutyrate) (PHB) accumulation potential of microbial communities. In this study we investigated the influence of the substrate composition in terms of the carbon to nitrogen (C/N) or phosphorus (C/P) ratio on the PHB accumulation performance. A multi-reactor set-up was used, enabling parallel experiments using identical inoculum of an enrichment culture dominated by Plasticicumulans acidivorans. In all experiments simultaneous PHB production and growth was observed. Generally, when trace amounts of growth nutrients were present the PHB production yield on substrate remained high for at least 12 h. Interestingly, from the carbon to nutrient ratio in the substrate, the PHB wt% could be accurately predicted in the accumulations. This study demonstrates that strict uncoupling of microbial growth and PHA accumulation is not required for achieving high cellular PHA-contents. Herewith the range of wastewaters that enable a cellular PHA content of 80 % or higher for at least 12 h is expanded to C:N and C:P-ratios exceeding COD:N of 26 gCOD:gNH4-N and COD:P of 511 gCOD:gPO4-P respectively.

Temperature as competitive strategy determining factor in pulse-fed aerobic bioreactors.

Exposing a microbial community to alternating absence and presence of carbon substrate in aerobic conditions is an effective strategy for enrichment of storage polymers (polyhydroxybutyrate, PHB) producing microorganisms. In this work we investigate to which extent intermediate storage polymer production is a temperature independent microbial competition determining factor. Eight parallel bioreactors were operated in the temperature range of 20-40 °C, but intermediate storage polymer production was only obtained at 25-35 °C. Besides PHB production and consumption, cell decay and subsequent cryptic growth on lysis products was found to determine process properties and the microbial community structure at all operational temperatures. At 40 °C decay processes cannot be overcome with additional energy from storage polymers, and fast-growing microorganisms dominate the system. At 20 °C, highly competitive communities with ambiguous storage properties were enriched. The results described here demonstrate that a rigorous experimental approach could aid in the understanding of competitive strategies in microbial communities.

O2 versus N2O respiration in a continuous microbial enrichment.

Despite its ecological importance, essential aspects of microbial N2O reduction-such as the effect of O2 availability on the N2O sink capacity of a community-remain unclear. We studied N2O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N2O and O2 can occur, (ii) the mechanism governing the competition for N2O and O2, and (iii) how the N2O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N2O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O2. When supplied simultaneously, N2O reduction to N2 was only detected when the O2 concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N2O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture's affinity constant (KS) for O2 was found to be two orders of magnitude lower than the value for N2O, explaining the preferential use of O2 over N2O under most environmentally relevant conditions.

Ecology-based selective environments as solution to contamination in microalgal cultivation.

Large-scale production of energetic storage compounds by microalgae is hampered by competition and evolution. Both phenomena result in contamination and arise due to a mismatch between the desired productive microalgal strain and the constructed environment. The prevailing approach to solve this issue involves increasing the survival potential of the desired strain, for example by working in closed systems or at extreme conditions. We advocate adjusting the environment in such a way that lipid production, or any other desired characteristic, gives a competitive advantage. Competition and evolution become a value rather than a threat to processes in which the desired characteristic is ensured by a selective environment. Research and cultivation efforts will benefit from this approach as it harnesses the microalgal diversity in nature.