Exploring Microbial Diversity: Extending the boundaries of biopolymer production using parallel cultivation

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.