Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are two microbial processes that compete for oxidized nitrogen compounds in the environment. The objective of this work was to determine the role of nitrite versus nitrate as terminal electron acceptor on the competition between DNRA and denitrification. Initially, a mixed culture chemostat was operated under nitrate limitation and performed DNRA. Stepwise, the influent nitrate was replaced with nitrite until nitrite was the sole electron acceptor and N-source present. Despite changing the electron acceptor from nitrate to nitrite, the dominant process remained DNRA and the same dominant organism closely related to Geobacter lovleyi was identified. Contrary to previous studies conducted with a complex substrate in marine microbial communities, the conclusion of this work is that nitrate versus nitrite as electron acceptor does not generally control the competition between DNRA and denitrification. Our results show that the effect of this ratio must be interpreted in combination with other environmental factors, such as the type and complexity of the electron donor, pH, or sulfide concentrations.@en

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.@en

Microbial fermentations are a key process in naturally and man-made ecosystems. Microbial fermentations play a key role in creating and digesting our food and they are useful in designing bioprocesses that can produce biogas, biofuels, bioplastics, and many other functional molecules (Chapter 1). Furthermore, studying the competition and cooperation in microbial fermentative ecosystems can help to solve the question how microbial diversity is shaped. Glucose is a molecule central to most forms of life, therefore glucose was chosen as a model substrate to perform fermentative enrichment studies. Xylose is an important monomer in many types of hemicellulose and was therefore chosen as second model substrate. Glucose and xylose can be fermented to volatile fatty acids, alcohols or lactic acid. The biomass specific uptake and production rates at which microbial fermentations are performed are high compared to other biological anaerobic carbon conversions. This rate difference is useful when studying fermentation using an enrichment culture approach. Such fermentative enrichment cultures can be used to develop mixed culture fermentation technologies, which offer alternative technological possibilities for processing feedstocks and residual streams containing carbohydrates (Chapter 1). Biogas production is a relatively well-established industry, but remains to be economically outcompeted by natural gas. The market for (bio)hydrogen production is relatively big, as the hydrogen economy stood for 130 billion USD in 2017. Actual large-scale hydrogen production and capture using biological systems has yet to prove itself. Lactate and ethanol can both be produced using mixed culture fermentation, where ethanol production remains to be a challenging business case due to small profit margins. Medium chain fatty acids are also a potential product. These molecules are expected to have many applications, with a likely higher value than biogas or biofuel, thus promising a healthy business case. Producing polyhydroxyalkanoates from volatile fatty acids produced by mixed culture fermentation promises a healthy industrial feasibility. When assuming solely competition on substrates to occur, limiting a single substrate in a microbial ecosystem is expected to result in one dominant species. The results of Chapter 2 confirm this hypothesis, to the extent of >85% of the observed cell surface belonging to a single species for three out of the four enrichment cultures. A population of Enterobacter cloacae and Citrobacter freundii dominated the glucose and xylose limited sequencing batch cultures respectively. Continuous glucose limitation showed the dominance of Clostridium intestinale. A xylose limited continuous enrichment culture resulted in the coexistence of Citrobacter freundii, and a Lachnospiraceae and Muricomes population. Chapter 3 aims to answer the question how dual substrate limitation influences a fermentative microbial community. Dual xylose and glucose limitation led to a generalist population of Clostridium intestinale in continuous feeding, and a generalist population of Citrobacter freundii in sequencing batch culturing. No apparent carbon catabolite repression was observed when analysing a batch cycle or when performing a batch experiment in the continuous dual limited enrichment culture. This response is of value when designing large scale fermentative bioprocesses, as in industry, typically microorganisms are used which show carbon catabolite repression in mixtures of glucose and xylose. The kinetic, stoichiometric and bioenergetic analysis of enrichment cultures in continuously limited or sequencing batch environments showed that sequencing batch enrichments select for rate, while continuous limited enrichments select for efficiency (Chapter 2). Rate is considered as the biomass-specific substrate uptake rate (qsmax) and efficiency is considered as yield of biomass on ATP harvested in catabolism (Yx,ATP). These findings fit within the r- and K-selection theory. Furthermore, it was found that butyrate production is linked to a lower uptake rate than combined acetate and ethanol production. Potentially, more energy is harvested in butyrate production than in combined acetate and ethanol production, through electron bifurcation. More microbial diversity (i.e. more than one species) was observed than what was expected from a competitive point of view in all six enrichments performed in Chapter 2 and 3. Therefore, in Chapter 5 a complementary approach of metabolomics, metagenomics and isolation studies where performed to generate an evidence based hypothesis on how the Enterobacteriaceae and Clostridiales populations in the continuous xylose limited enrichment culture interacted. The metagenomic evaluation resulted in three dominant bins, one for Citrobacter freundii, one for “Ca. Galacturonibacter soehngenii” and one for a Ruminococcus sp. The interaction between Citrobacter freundii and “Ca. Galacturonibacter soehngenii” is proposed to be a sharing of biotin, pyridoxine and alanine by Citrobacter freundii with “Ca. Galacturonibacter soehngenii”. A differential enrichment study showed that indeed the fraction of “Ca. Galacturonibacter soehngenii” increased and Enterobacteriaceae decreased, when these three metabolites were directly supplemented to the enrichment culture. Thus, commensalism and competition were likely to driving microbial diversity in this culture. Chapter 4 aimed to study the ecology of lactic acid bacteria. Bacteria can produce lactic acid from glucose, which is a different metabolism than producing acetate and butyrate. Sequencing batch reactors were used to enrich, comparing a mineral and complex medium. The media were identical, except for the addition of peptides and 9 B vitamins in the complex medium. Glucose was fermented to a mixture of lactic acid and ethanol when using the complex medium, thereby a heterofermentation. Using the mineral medium, glucose was fermented to a mixture of acetate, butyrate and hydrogen, with smaller amounts of lactic acid and ethanol. A population of Lactobacillus, Lactococcus and Megasphaera was enriched on complex medium. On mineral medium, a population of Ethanoligenens dominated the enrichment with a small fraction of Clostridium. Lactic acid producing bacteria are hypothesised to have taken over the fermentation, due to a 94% increase in biomass-specific substrate uptake rate, leading to a higher growth rate. The increase in growth rate is argued to be caused due to resource allocation, whereby lactic acid bacteria optimise their enzyme levels in anabolism and catabolism, attaining a higher growth rate than mineral-type fermenters such as Ethanoligenens. Chapter 6 aims to direct further research, which lies in studying the effect of different parameters on fermentative ecosystems. These parameters are concentrations of: gaseous compounds (I), cations used to neutralise (II), nutrients, such as B vitamins (III). Also, very low pH environments (pH<3.5) are considered an opportunity (IV). Finally, analysing the composition of “real” fermentable streams and their effect on the arising product spectra is of interest (V). Kinetics and bioenergetics are discussed using enzymatic Michaelis-Menten kinetics and the concept of resource allocation. In this way, efforts can be directed into the ability to predict product formation a priori in fermentative ecosystems. Future experimentation is guided to take place on four distinct levels, and useful experiments to verify concepts in this thesis are outlined. Finally, commensalism and/or mutualism might both be relevant in open microbial ecosystems which remains to be settled by future work. @en

Efficient industrial fermentation of lignocellulosic waste containing a large part of glucose and xylose is desirable to implement a circular economy. Mixed culture biotechnologies can aid in realizing this goal. The effect of feeding equivalent substrates to a microbial community, such a xylose and glucose, is not well understood in terms of the number of dominant species and how these species compete for the substrates. We compared the metabolism and microbial community structure in a continuous-flow stirred tank reactor (CSTR) and a sequencing batch reactor (SBR) fed with a mixture of xylose and glucose, inoculated with bovine rumen at pH 8, 30°C and a hydraulic retention time of 8 h. We hypothesised that a CSTR will select for generalist species, taking up both substrates. We used 16S rRNA gene sequencing and fluorescent in situ hybridisation to accurately determine the microbial community structures. Both enrichments were stoichiometrically and kinetically characterised. The CSTR enrichment culture was dominated by Clostridium intestinale (91% ± 2%). The SBR showed an abundance of Enterobacteriaceae (75% ± 8%), dominated by Citrobacter freundii and a minor fraction of Raoultella ornithinolytica. C. freundii ferments xylose and glucose in a non-diauxic fashion. Clearly, a non-diauxic generalist outcompetes specialists and diauxic generalists in SBR environments.@en

Lactate production in anaerobic carbohydrate fermentations with mixed cultures of microorganisms is generally observed only in very specific conditions: the reactor should be run discontinuously and peptides and B vitamins must be present in the culture medium as lactic acid bacteria (LAB) are typically auxotrophic for amino acids. State-of-the-art anaerobic fermentation models assume that microorganisms optimise the adenosine triphosphate (ATP) yield on substrate and therefore they do not predict the less ATP efficient lactate production, which limits their application for designing lactate production in mixed-culture fermentations. In this study, a metabolic model taking into account cellular resource allocation and limitation is proposed to predict and analyse under which conditions lactate production from glucose can be beneficial for microorganisms. The model uses a flux balances analysis approach incorporating additional constraints from the resource allocation theory and simulates glucose fermentation in a continuous reactor. This approach predicts lactate production is predicted at high dilution rates, provided that amino acids are in the culture medium. In minimal medium and lower dilution rates, mostly butyrate and no lactate is predicted. Auxotrophy for amino acids of LAB is identified to provide a competitive advantage in rich media because less resources need to be allocated for anabolic machinery and higher specific growth rates can be achieved. The Matlab™ codes required for performing the simulations presented in this study are available at https://doi.org/10.5281/zenodo.4031144.@en

Bioethanol production is an established biotechnological process. Margins are low which prevent a larger scale production of bioethanol. As a large part of the production cost is due to the feedstock, the use of low value unsterile feedstocks fermented by microbial communities will enable a more cost-competitive bioethanol production. To select for high yield ethanol producing communities, three selective conditions are proposed: acid washing of the cells after fermentation, a low pH (<5) during the fermentation and microaerobiosis at the start of the fermentation. Ethanol producers, such as Zymomonas species and yeasts, compete for carbohydrates with volatile fatty acid and lactic acid producing bacteria. Creating effective consortia of lactic acid bacteria and homo-ethanol producers at low pH will lead to robust and competitive ethanol yields and titres. A conceptual design of an ecology-based bioethanol production process is proposed using food waste to produce bioethanol, electricity, digestate and heat.@en

A mechanistic understanding of microbial community establishment and product formation in open fermentative systems can aid the development of bioprocesses utilising organic waste. Kinetically, a single rate-limiting substrate is expected to result in one dominant species. Four enrichment cultures were operated to ferment either xylose or glucose in a sequencing batch reactor (SBR) or a continuous-flow stirred tank reactor (CSTR) mode. The combination of 16S rRNA gene-based analysis and fluorescence in situ hybridisation revealed no complete dominance of one species in the community. The glucose-fed and xylose-fed SBR enrichments were dominated >80% by one species. Enterobacteriaceae dominated the SBRs enrichments, with Citrobacter freundii dominant for xylose and Enterobacter cloacae for glucose. Clostridium, Enterobacteriaceae and Lachnospiraceae affiliates dominated the CSTRs enrichments. Independent of substrate, SBR communities displayed 2-3 times higher biomass-specific rate of substrate uptake (qsmax) and 50% lower biomass yield on ATP, to CSTR communities. Butyrate production was linked to dominance of Clostridium and low qsmax (1.06 Cmols Cmolx-1 h-1), while acetate and ethanol production was linked to dominance of Enterobacteriaceae and Lachnospiraceae and high qsmax (1.72 Cmols Cmolx-1 h-1 and higher). Overall, more diversity than expected through competition was observed, indicating mutualistic mechanisms might shape microbial diversity.@en