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.