Quickly, the show is about the start. Date: 3.5 thousand million years ago, location: planet Earth, event: life. Naturally, life is starting small, even microscopically tiny. Life in the form of microorganisms endures eons of time in which the world changes. They survived, failed, adapted, thrived, and they actually changed the world. They have seen humankind step into the light of day, and they will be there when we see no more. Microorganisms are the link between the inanimate, mineral planet and the living world. They facilitate the natural cycle of the elements. The CO2 we breathe out is transformed by phototropic algae to oxygen. The nitrogen in proteins that we eat finds its way to the nitrogen gas in the air, and back into the roots of plants through countless microorganisms. And central to our life: carbon, it is the food that we eat, the oil that we burn, and the plastics that will immortalize humans’ existence. We are life, we flourish, and like all living things, we are greedy. So greedy that we disrupted the circularity of nature. We are far from the first, nor the most successful, organism to change the face of the earth. Algae made the world aerobic, and the first trees covered the world in meters of indigestible wood for millions of years. And while nature seems to have found a new balance, those algae and trees now form the oil and coal that drive our manic existence. What differentiates us from those earlier life forms is that we can appreciate that we are running on borrowed time, as we can see the world changing, fast. Over the past century, it has become clear that we are shaping a linear society, predominantly driven by fossil fuels. If we, by contrast, could manage to convert our waste streams back into resources at the same rate that we produce them, that would chime in a new era. And even more profound is that we are living in a world shaped and dominated by microorganisms. We need to start cooperating with them for our health and prosperity, which requires a better understanding of the microbial world. And although we are making significant progress; time is ticking and we could use all the help there is. This thesis is on how we can explore and utilize 3.5 billion years of help. In the first chapter the vastness, complexity and wealth of the microbial world are introduced. It focusses on a fraction of that wealth, the specific topic of interest, which is the production of biopolymers by microbial communities. These biopolymers are important building blocks for a circular society, as they can serve as precursor to oil, plastics, food, and specialty materials. Of the many biopolymers in nature, the predominant one within this thesis are polyhydroxyalkanoates (PHA), which are produced by microorganisms as their equivalent to human fat, and can be used by us to produce bioplastics. In the second chapter our key contribution to the scientific field of microbial community research is made. A key aspect that is holding back research on microbial communities is the lack of experimental freedom to bring nature to the lab. In this work, we attempt to bring cultivation research into the 21st century with a more flexible biodiscovery cultivation platform. This chapter describes a part of the hardware and software that was developed to significantly assist parallel enrichment research in dynamic conditions, it elaborates on the bioreactor setups of 8 systems, the automatization, on-line data processing, and process modelling. We demonstrate a generalized respiration rate reconstruction tool for dynamic operated bioreactors. The setup and tools described here have facilitated over twenty research topics that were conducted during and alongside this Doctoral research. The third chapter demonstrates how the setup can be used to increase the research intensity of enrichment studies. We investigated the influence of temperature on the enrichment of PHA accumulating microbial communities, which yielded several noteworthy findings. Besides an explanation for the global temperature optimum of 30°C, we identified other competitive strategies in feast-famine enrichment systems, that of fast-growth and decay, and subsequent growth on cell lysis. Furthermore, we were able to align shifts in microbial function with microbial community shifts, and addressed important issues of reproducibility in microbial community enrichments. The results demonstrate that a rigorous experimental approach involving parallel cultivation allows for unambiguous identification of competitive strategies in microbial communities. And a major improvement with this approach is that we can pinpoint where our knowledge is lacking. The fourth chapter follows a systematic investigation of a specific surprising observation that was made possible by the close monitoring of the enrichment systems. During a study investigating the influence of pH on the enrichment of PHA accumulating microbial communities (analogous to the temperature study), we noticed markedly different microbial community structure and behavior between enrichments, that seemed solely based on the type of acid used for pH control. We demonstrated that the observed changes were 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. In chapter five, we investigate a novel enrichment process for PHA accumulating microbial communities. The strict uncoupling in time of nutrient supply of two growth nutrients is investigated. The setup was used to optimize the process by investigating the influence of (i) nitrogen or phosphorous uncoupling from carbon, (ii) increased carbon to nutrient ratios, and (iii) increased exchange ratios. The uncoupling strategy resulted in stable enrichments, that achieved 89 wt% (gPHA/gDW) in eight hours, every operational cycle, making this the most PHA rich production system to date. The proposed strict uncoupling strategy yields stable microbial communities with an unprecedented combination of PHA storing capacity, productivity, product yield, and general applicability for feed streams without nitrogen or phosphate. Chapter six looks forward on the future of microbial community research, it explores the collaborative efforts between Wageningen University and Delft University in the 24 million euro UNLOCK project, for which the work in this thesis laid a principal foundation.@en

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.@en

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.@en

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.@en

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.@en

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.@en

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.@en

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.@en

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.”@en

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.@en

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.@en

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.@en

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.@en

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.@en

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.@en