Dating back to 10,000 B.C.E., fermentation has served as a vital tool primarily for food preservation. In the nineteenth century, Louis Pasteur put forth the proposition that the occurrence of fermentation is predominantly facilitated by the presence of microorganisms. Pasteur’s discovery spurred comprehensive studies on microorganisms, revealing their strategic roles in fermentation and microbial ecosystems. However, in striving for stable processes and consistent products, researchers largely focus on simple substrates and pure cultures, yet practical applications of fermentation face obstacles due to substrate complexity and culture maintenance.

This research aims to provide insights into the intricate relationship between the microbial community and the complex substrate. It primarily focuses on the anaerobic acidogenic fermentation of cabbage, with the aim of identifying and understanding the key influences on microbial behavior and fermentation patterns. Cabbage was chosen as the feedstock for this investigation due to its historical and robust use as a substrate, specifically for the generation of lactic acid. This byproduct not only holds economic value but also presents intriguing scientific implications, such as serving as a promising raw material in biodegradable plastic manufacturing. The initial segment of our research endeavors to investigate the fermentation process and characterize the microbial community dynamics observed during the fermentation of chopped cabbage. Interestingly, our analysis of chopped cabbage fermentation revealed a triphasic progression, wherein the Bacilli class—predominant in the initial stages—facilitated lactic acid production, succeeded by Clostridia and Actinobacteria driving butyrate and propionate generation, respectively.

Despite the principles of thermodynamics and ATP yield suggesting that the transformation of sugars into alternative end products, such as short-chain fatty acids, is more favourable, the production of lactic acid is frequently observed, notably in fermentations involving vegetables. State-of-the art flux balance analysis models suggest that these microorganisms achieve dominance when sugar concentration exceeds a certain threshold, leading to enhanced (Lactic Acid Bacteria) LAB growth rates. We hypothesize that by employing a novel water solubilization method to separate the cabbage into soluble and ”insoluble” fractions, it would enable us to manipulate the initial soluble organic load, under the assumption that the substrate composition maintains similarity. This, in turn, could potentially influence the growth rates of LAB. Remarkably, the data revealed Clostridia class dominating the “insoluble” cabbage, while Bacilli class predominated in the soluble cabbage fermentation at the early stages.

The majority of LAB are documented to necessitate specific amino acids for their growth. A crucial aspect of our research was to elucidate whether LAB engaged in cabbage fermentation exhibits a similar dependency on the substrate. To elucidate this, we used the inoculum from cabbage fermentation to ferment solely cabbage sugars in the absence of the cabbage. The objective was to determine if the microbial community could produce similar fermentation patterns without the non-monosaccharide constituents of cabbage, thus investigating their role in LAB growth. Noteworthy, in the absence of cabbage, a divergent pattern with minimal lactic acid and lack of LAB was observed, highlighting their dependence on non-monosaccharide cabbage components.

This research provided critical insights into the interactions between substrates and microbial communities, with potential applications in optimizing fermentation processes in the food industry and biochemical production. It may also lead to a better understanding of microbial behavior in different substrate conditions, providing a foundation for more efficient and sustainable fermentation.

In the treatment of industrial wastewater, the biological removal of aniline & nitrogen often occurs simultaneously within the hyper-saline water matrix. This study focuses on pioneering the research
on combined aniline-nitrogen removal in a hyper-saline environment. The experimental approach assessed the cause of nitrite accumulation phenomena in this specific case study as well as the influence
of oxygen concentration and aniline as a carbon source on nitrification. The hypothesis of increasing aniline biodegradability by making it chemically react with nitrite was experimentally tested. In addition, data analysis of the full scale of this study was performed in order to create analytical models to represent its biological processes. It was found that the correlation between oxygen concentrations and nitrification was non-linear and differs from what is observed in the literature due to the significant influence of different boundary conditions. The unavailability of carbon sources during the nitratation process was the cause of nitrite accumulation, which crucially affects nitrification. The chemical reaction between aniline and nitrite (most likely polymerization) resulted in a compound that did not limit nitrification and had more biodegradable potential. Furthermore, an aerobic model was developed but the promising results were most likely caused by the mathematical optimization, which was done in order to bypass the lack of data. Nevertheless, respirometry was used to build an empirical model to detect nitrite accumulation in the effluent of the full scale, which showed promising results. This study can potentially pave the way for the application of Anammox technology in the combined treatment of aniline & nitrogen, which is discussed in the hypothetical design implementations.

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

Comammox bacteria are capable of catalysing the full nitrification pathway – oxidation of ammonium to nitrate – and have been encountered in many ecosystems recently (Lawson & Lücker, 2018). What the ecological role of comammox bacteria is in hypoxic enrichment cultures remains unclear. Based on the thermodynamics and biochemistry of known nitrogen cycle conversion, we propose that comammox is oxidizing ammonium to nitrite with both oxygen and nitrate as electron acceptor in hypoxic condition. Our hypothesis suggests that when comammox cooperates with anammox, they can harvest most energy per unit of oxygen supplied. We tried to cultivate bacteria toward a community of anammox and comammox consortium by limiting the oxygen and supplying ammonium and nitrate. Although the predicted optimal state has not been achieved during this work, we did observe that the community indeed developed towards higher consumption of ammonium under limited oxygen supply.

A pilot-scale PHA microbial enrichment reactor fed on OFMSW leachate was monitored in order to assess the influence of free ammonia nitrogen (FAN) on its performance. The enrichment reactor consisted of a SBR with a 12-hours cycle which included a feast phase, a settling phase and a famine phase with external nitrogen addition. Based on the microscope pictures and FISH analysis, at least two different PHA producing bacteria were identified: small (< 2.5 μm) and big PHA producing bacteria (< 5 μm). Big PHA bacteria appeared from FAN concentrations higher than 50 mg/L, but concentrations higher than 150 mg/L were highly toxic for the whole PHA microbial enrichment. Finally, it was found that a FAN concentration around 60-70 mg/L might produce a microbial enrichment with good settling properties (SVI30 < 80 mL/g and BLAS < 5%) and maximum PHA yields (around 0.50 g PHA/g sCOD), possibly due to the presence of big PHA producers which are assumed to be more efficient and heavier after feast. However, this FAN concentration may lead to a lower settleable biomass production yield (around 0.30 g VSS/g sCOD). Since these results are not conclusive, it is suggested to test the observations of this pilot study in lab-scale experiments to evaluate the potential of FAN as an additional selective pressure in a PHA microbial enrichment.