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. ... Read more

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. ... Read more

One of the main challenges society currently deals with is the depletion of fossil fuels. To navigate this issue, we must embrace the concept of circularity and turn waste into a resource. Waste streams are omnifarious and their conversion into new chemical building blocks is not always trivial. Luckily, we can take a look at nature’s problem solving skills to help us out. Because nature, in due time, always finds a solution and there is a (micro)organism for everything.

But.. we can also give nature a hand by simplifying the problem. The diversity and complexity of waste streams can be reduced by using gasification, where the waste is combusted at a high temperature with small amounts of oxygen. This yields syngas, a mixture consisting of mainly carbon monoxide, carbon dioxide and hydrogen gas. Syngas can be converted chemically into i.e. ethanol, but the success of this process highly depends on the ratios of CO, CO₂ and H₂ and the absence of impurities in the gas. Microorganisms can deal with much more variability, making them a promising biocatalyst for the conversion of syngas to chemical building blocks. Yet, we have to understand the microorganisms to be able to work together with them in combatting climate change. The work in this thesis is aimed at increasing our understanding of two specific types of microorganisms that can help us to turn waste into new chemicals: syngas fermenting bacteria and chain elongating bacteria. Together, they can form a team that turns a C1 molecule (carbon monoxide) all the way into a C6 molecule (hexanoate). To make the team as effective as possible, we studied both team members in detail. The syngas fermenting bacterium we studied goes by the name Clostridium autoethanogenum, and is already being used at industrial scale by the company LanzaTech. For its chain-elongating counterpart, however, we used a mixed community of microorganisms that was specifically selected to perform chain elongation. We used this mixed community because the single, optimal partner for C. autoethanogenum has yet to be found.

It has been established previously, by other researchers, that producing a lot of hexanoate is easiest when you feed chain elongating organisms a substrate with a high ethanol-to-acetate ratio. C. autoethanogenum naturally produces ethanol and acetate, but usually in a low ethanol-to-acetate ratio. In Chapter 2 we use a theoretical framework based on thermodynamics, as well as data from literature to understand what triggers C. autoethanogenum to make ethanol. We found that acetate conversion into ethanol is a stress response used to deal with a (too) high load of CO, which can be classified as overflow metabolism. We show that this behavior not only takes place when feeding CO alone, but also in the presence of both CO and H₂, underlining its relevance in syngas fermentation processes. The stress response can be induced by tuning the operational parameters of the bioreactor, such as the CO supply rate or the growth rate.

In Chapter 3 we quantify this effect in the laboratory ourselves. We use a steady-state culture of C. autoethanogenum in a chemostat bioreactor and repeatedly disturb it for periods of one hour with increasing amounts of CO in the inlet gas, up to a CO partial pressure of 1.2 atm. We see that ethanol production increases with increasing CO partial pressures, and at a pCO of 0.6 atm or higher external acetate is even consumed to sustain higher ethanol production rates. This proves that the product spectrum of syngas fermentation can be directed by changing the operational conditions. Furthemore, the experimental method that we used allowed for the identification of the CO uptake rate at each CO partial pressure, directly via the off-gas measurements. We observed biomass-specific CO uptake rates of up to –119 ± 1 mmol·g_x⁻¹·h⁻¹, which is much higher than has previously been reported for this organism. The biomass-specific uptake rate is instrumental for obtaining an accurate mathematical description (or: kinetic model) of this microorganism, which in turn allows for more accurate bioprocess design.

Chapter 4 focusses on the chain-elongating counterpart of our syngas fermenter. C. autoethanogenum prefers to grow at a pH of 5 –5.5, and most chain elongators that have been described in literature rather grow at neutral pH (± 7.0). This chapter revolves around this discrepancy. By using enrichment cultures in a sequencing batch bioreactor, we select for chain elongating microorganisms both at pH 7.0 and pH 5.5. In doing so, we establish that chain elongators can live at pH 5.5 and that a very comparable microbial community (on genus-level) develops at both pH. However, the behavior in the bioreactors was not the same. At lower pH, a significantly smaller fraction of the supplied ethanol was converted to hexanoate. Instead, more of the C₄ molecule butyrate was produced, likely because it is less toxic to the microorganisms than hexanoate. This means that pH is an important parameter to control the product spectrum of chain elongation and that establishing an effective microbial team for C₁-to-C₆ conversion likely requires more than finding microbes with the same preferred pH.

In Chapter 5 we delve into the biochemistry of chain elongating microbes. They are known to be very flexible in their metabolism, and they can deal with a wide range of ethanol-to-acetate ratios. Theoretically, this ratio could even be infinite (i.e. feeding only ethanol), which would lead to the production of only hexanoate and no butyrate. We call this ethanol-only chain elongation. This is interesting from a fundamental as well as a process design perspective. Therefore, we test whether it is also possible in practice by using well-monitored batch experiments in bioreactors. We use different initial conditions: only ethanol, ethanol and a small amount of acetate and ethanol and a small amount of butyrate. We observe in the bioreactors that ethanol-only chain elongation is possible, but that it proceeds very slowly. Beside that, the microorganisms prefer the presence of either acetate or butyrate so much that they eventually start producing these compounds from ethanol themselves when they are not available. This behavior has never been observed before, nor was it regarded as possible.

In Chapter 6 we present a dataset of well-controlled bioreactor experiments in 9 different initial conditions, including the experiments described in the previous chapter. This dataset can be used to refine the current mathematical description of chain-elongating microbes. We describe the initial analysis of this dataset and how we assure its quality and usability for kinetic modelling using data reconciliation. With this reconciled dataset we test the accuracy of the currently available kinetic model. From this overall analysis we set out the next steps for the formulation of a more accurate kinetic model of chain elongating microbes in the future.

Chapter 7 recapitulates the significant findings from this thesis, but more importantly provides a list of questions that still remain to be answered. These questions are grouped around three different themes to provide some structure: the inner world of microorganisms, the interactions between (communities of different) microorganisms and the design of efficient (new) bioprocesses for a more sustainable world. To conclude, I reflect on the societal role of a scientist.
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Medium chain length polyhydroxyalkanoate (mcl-PHA) is a form of polyhydroxyalkanoate (PHA) which has properties similar to rubber. However, most of the research on mcl-PHA has been focused on pure cultures with artificial substrates, both of which increase the cost of the production to economically unviable levels. This is especially the case since the biological and the chemical production are very cheap. To reduce the costs, a mixed culture could be used. By investigating which conditions lead to high mcl-PHA production with a mixed culture, a step can be taken towards an economically viable biodegradable rubber replacement. To develop a culture which was able to produce mcl-PHA, an enrichment was first performed in a sequential batch reactor with a feast-famine regime to select for PHA producers. Additional selection pressures like substrate selection, pH, oxygen flow rate and time between carbon source addition and nitrogen source addition were imposed on the culture. After the culture reached pseudo-steady state, an accumulation was done. The substrate was one of the most important selection pressures. Using octanoate as sole carbon and energy source, the microbial community was successfully enriched in a feast-famine regime with the capacity to produce mcl-PHA. The use of octanoate with a pH of 7 and no reduced oxygen flow resulted in mcl-PHA weight percentages of 27 wt% polyhydroxyoctanoate (PHO) and 5 wt% polyhydroxyhexanoate (PHH) during the cycle. This was raised to 30 wt% PHO and 10 wt% PHH during the accumulation. During the second enrichment, the carbon source, and the oxygen flow of the first enrichment were maintained, but the pH was increased to 8. This led to an increase in mcl-PHA production, to 33 wt% PHO and 7 wt% PHH during the cycle and 44 wt% PHO and 9 wt% PHH during the accumulation. Two other condition changes were also investigated: lowered oxygen flow and uncoupling the system. For the lowered oxygen flow, the oxygen flow rate was reduced to 5% of the original flow rate and the increased pH of 8 of the second enrichment was kept. This resulted in similar values as the enrichment with only increased pH. It is therefore unclear whether the lowered oxygen flow rate could be attributed to the high mcl-PHA production or that the culture performed well despite the lowered oxygen flow. The uncoupled system was a system where the nitrogen source was added 2 hours after addition of the carbon source. Based on the oxygen profile and preliminary results of the enrichment, the culture mainly grew after the addition of carbon and little mcl-PHA was produced. However, scl-PHA production was quite high, with a total of 51 wt% PHB. This work proves that high levels of mcl-PHA could be produced from mixed cultures. This is a good step towards producing an economically viable, biodegradable rubber replacement. ... Read more

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. ... Read more

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. ... Read more

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. ... Read more

The anthropogenic nitrogen inputs in the environment exceed the input by natural processes and impact the global nitrogen cycle considerably . Human meddling in the N-cycle occurs mainly in agricultural ecosystems. Loss of nitrogen from the agricultural soils, other than crop harvest, can have polluting effects on other environments. The three main processes through which the losses occur are ammonia volatilization, the production of gaseous nitrogen compounds and leaching of nitrate , contributing to acid rain, ozone depletion and eutrophication respectively. To reduce N-pollution and improve mitigation strategies, we need to expand our understanding of the metabolic and environmental controls of the nitrogen cycle processes. This thesis focuses on the microbial competition for nitrate between two dissimilatory nitrate reduction processes in the nitrogen cycle, as the different end-products entail important biogeochemical consequences for nitrogen retention in aquatic ecosystems such as wastewater treatment plants, as well as the successful operation of wastewater treatment systems. Nitrate can be reduced to nitrogen gas in the denitrification process, removing the nitrogen from the environment, which is desired for alleviation of eutrophication or treatment of waste water. Alternatively, in the process of dissimilatory nitrate reduction to ammonium (DNRA), ammonium is the end product, and the nitrogen is conserved in the environment, which can be beneficial in fertilizer management. ... Read more