A highly pure biomethane stream (≈97% CH₄) was produced continuously under halo-alkaline conditions (pH > 9, 0.6 M Na⁺) from complex alkaline organic waste residue originating from biopolymer extraction from sewage sludge. During the proof-of-concept operation, the substrate was degraded with similar efficiency (40% of the volatile solids, VS) compared to neutral conditions (36% of the VS). Operational data was utilised in a technical evaluation to identify bottlenecks for full-scale implementation at an early stage of process development and for comparison to conventional biogas upgrading using pressure swing and membranes. Initially identified bottlenecks for alkaline fermentation were related to overcautious assumptions, while others could be technically solved. Alkaline fermentation offers an attractive method for supplying increasingly needed high-purity biomethane using various recalcitrant substrates that have undergone alkaline pre-treatment. This is more feasible than the conventional ex-situ biogas upgrading. Next, upscaling steps for alkaline fermentation should be pursued. Strategies for integrated CO₂ sequestration and nutrient recovery are outlined, which will offer additional benefits in the future.

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.

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.

Anaerobic microbial communities can produce carboxylic acids of medium chain length (e.g., caproate, caprylate) by elongating short chain fatty acids through reversed β-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.

Volatile fatty acids (VFA) may serve as building blocks for the production of chemicals and polymers. A technology enabling high-rate VFA production from carbohydrate-rich wastewater is the anaerobic granular sludge process. In this study, the characteristics of an anaerobic granular sludge process fermenting glucose was evaluated at different solid retention times (SRT). A lab-scale anaerobic sequencing batch reactor fed with 6 g·L-1 glucose was operated at a pH of 5.5 and at SRT values of 1-2, 10-20, and 40-50 days and operated in total for 215 days. A low sludge volume index (SVI) of 1,144 mL·gTSS-1 allowed for the high SRT and high volatile suspended solid (VSS) concentration that reached 59 gVSS·L-1. This high VSS concentration enabled a glucose consumption rate of 1,100 gCOD·L-1·day-1 at an SRT of 40-50 days. Two product spectra were obtained: (1) a propionate:acetate mixture with a ratio of 2.05∶1 (molpropionate:molacetate) produced at an SRT of 40-50 days; and (2) an acetate dominated product spectrum was obtained at 1-2 days and 10-20 days SRT (0.71-0.75 molacetate·molVFA-1). Overall, a high VFA yield between 0.77 and 0.79 was obtained throughout all enrichments. This work demonstrates that high-rate VFA production combining high yields and low solid concentrations in the effluent technologically can be achieved. This work contributes to the implementation of waste-based production of VFA using anaerobic granular sludge.

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.”

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.

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.

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 (K_S) 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.