Propositions 1. Actinomyces succiniciruminis has potential for industrial succinate production from starchy waste (this thesis) 2. Knowing the relative abundance of each microbial species in a mixed rumen inoculum does not help to predict the fermentation products (this thesis) 3. Phytoplanktons are the real-world savers for the global warming crisis as they are world’s biggest oxygen producers and carbon sequesters (Witman, S. (2017) World’s biggest oxygen producers living in swirling ocean waters. Eos, 98) 4. The best way to protect endangered floras is to bring them into a commercial breeding system 5. Studying abroad elevates cooking skills to the master level 6. There is no real waste in our world Propositions belonging to this thesis entitled: “Organic acid production from starchy waste by gut derived microorganisms” Susakul Palakawong Na Ayudthaya Wageningen, 7 September 2018 Organic acid production from starchy waste by gut derived microorganisms Susakul Palakawong Na Ayudthaya Thesis committee Promotors Prof. Dr Alfons J.M. Stams Personal chair at the Laboratory of Microbiology Wageningen University & Research Prof. Dr Willem M. de Vos Professor of Microbiology Wageningen University & Research Co-promotor Dr Caroline M. Plugge Associate professor, Laboratory of Microbiology Wageningen University & Research Other members Prof. Dr Grietje Zeeman, Wageningen University & Research Dr Bundit Fungsin, Thailand Institute of Scientific and Technological Research, Pathum Thani, Thailand Prof. Dr Gert-Jan W. Euverink, University of Groningen, The Netherlands Dr Marieke E. Bruins, Wageningen University & Research This research was conducted under the auspices of the Graduate School for Socio-Economic and Natural Sciences of the Environment (SENSE). Organic acid production from starchy waste by gut derived microorganisms Susakul Palakawong Na Ayudthaya Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 7 September 2018 at 11 a.m. in the Aula. Susakul Palakawong Na Ayudthaya Organic acid production from starchy waste by gut derived microorganisms, 220 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018) With references, with summary in English ISBN: 978-94-6343-788-2 DOI: https://doi.org/10.18174/451805 Table of Contents Chapter 1 General introduction and thesis outline 7 Chapter 2 Organic acid production from potato starch waste fermentation 27 by rumen microbial communities from Dutch and Thai dairy cows Chapter 3 Actinomyces succiniciruminis sp. nov. and Actinomyces 69 glycerinitolerans sp. nov., two novel organic acid-producing bacteria isolated from rumen Chapter 4 Optimization of succinate production from potato starch waste 97 by Actinomyces succiniciruminis strain Am4T Chapter 5 Microbial diversity and organic acid production of guinea pig 123 fecal samples Chapter 6 Streptococcus caviae sp. nov., isolated from guinea pig fecal 153 samples Chapter 7 General discussion 173 Summary 187 References 191 List of publications 211 Co-author affiliations 212 About the author 213 Acknowledgements 215 SENSE Diploma 218 CHAPTER 1 General introduction and thesis outline Chapter 1 Global energy consumption and waste streams Global energy consumption is steadily increasing. The transportation, chemical and many other industry sectors are largely relying on petroleum and fossil fuels as a primary source of energy. In 2015, fossil fuels comprised of oil (31.7%), coal (28.1%) and natural gas (21.6%) contributed for more than 80% of the total energy supply (13,647 Mt) (IEA, 2017). The rate at which worldwide fossil fuels are consumed is increasing due to world's population increase and the rise in living standards in parts of the world that had consumed very little energy in the past. Fossil fuel reserves are limited, and exploitation will become expensive. Besides, consumption of fossil fuels results in pollution and carbon dioxide formation, which contributes to global warming. Therefore, the search for other energy sources such as natural gas, wind, nuclear and renewable organics is essential to guarantee energy supply. Current bio-based economy prospective Fossil reserves are also important as chemicals precursors, including building blocks for the chemical industry. Hence, chemical technologies based on fossil (petroleum) resources are currently being replaced by green technologies that make use of alternative biobased resources, in the so called biorefinery concept. This is often relying on microbial fermentation and when compared to chemical refinery processes, it is more environmental friendly and more sustainable (less pollution and no net CO2 emission to the atmosphere). In Table 1 the chemical and the bio-fermentation routes are compared. One of the guideline principles of green chemistry defined by the US Environmental Protection Agency (EPA) is promoting the use of renewable feedstocks (http://www.epa.gov/) (Fiorentino and Ripa, 2017). 8 General introduction and thesis outline Table 1. A general comparison between chemical and bio-fermentative routes. (modified from Cukalovic and Stevens, 2008; Law and Mohammad, 2017) Parameter Chemical routes Fermentative routes Non-renewable feedstocks – Biobased feedstock – carbohydrates petrochemicals Price considerations Still cheaper than the renewable Feedstock themselves do not contribute to sources. the price as much as downstream processing Availability Availability expected to Abundant and renewable decrease in time Routes Developed routes, established Routes under constant improvement, young technologies technologies Yields and Generally high Sometimes a large number of side products, productivities diluted media, long reaction times Major disadvantages High energy demands (pressure Sensitivity of microorganisms, nutrient and temperature). Catalysts requirements, complicated product disposal issues recovery, large amounts of waste Environment effect Release CO2 that contributes to Consume CO2 via TCA cycle global warming Public awareness Decreasing popularity Increased interest in improving currently applied routes and innovations Current high costs of production prevent bio-based chemicals to be widely used. Thus, production processes from bio-based resources need to be developed and optimized. It is estimated that in 2050 approximately 30% (by weight) of chemicals will be obtained from renewable biomass (http://www.suschem.org) (Fiorentino and Ripa, 2017). Biomass is an abundant carbon-neutral renewable resource that can be used as a carbon source instead of fossil feedstocks (Fiorentino and Ripa, 2017). Production chains resulting from biomass are considered as “short-cycle carbon systems”, which are more sustainable and preferable than those resulting from fossil resources, which are considered as “long-cycle carbon systems” (Kajaste et al., 2014; Fiorentino and Ripa, 2017). The transition from fossil based to bio-based economy needs the development of innovative processes to exploit the potential of biomass (Pleissner et al., 2017). Thus, utilization of food wastes or biomass and their conversion into valuable products require more attention as the implementation of such system is not yet successfully guaranteed. However, many criteria need to be considered for 9 Chapter 1 biomass utilization in the context of efficient strategies, such as the amount of biomass available for energy and material uses and the positive environment from renewable organic materials and economic feasibility (Venus et al., 2018), as well as the factors presented in Table 1. At present, three biorefinery platforms are known; i) the sugar platform: using purified enzymes to convert biomass into five- and six-carbon sugars and then further convert these into fuels and chemicals by fermentation; ii) the syngas platform: using thermochemical systems to convert biomass into synthesis gas (a mixture of CO, H2 and CO2) and then further convert to fuels and chemicals by chemical or biological catalysts, and iii) the carboxylate platform: using hydrolysis and fermentation with often undefined mixed microorganisms to convert organic feedstocks into short chain carboxylates (Agler et al., 2011; Bosma et al., 2013). The differences between these platforms are largely based on the method of biomass conversion and the chemicals resulting from that. Methane is usually the expected product from anaerobic digestion process, however, not only methane is valuable, but the intermediate compounds as organic acids are also important. Abundance of organic waste streams On one hand, the increase of human population and the associated consumption of food and energy results in a steady increase of organic waste. Approximately one third of the global food production is discarded as waste per year according to the Food and Agriculture Organization of the United Nations (FAO). (http://www.unric.org/en/food-waste). Organic waste, such as food and fiber processing of fruit, vegetable waste, garbage, sewage sludge, cattle manure and/or industrial waste are abundant worldwide (Murto et al., 2004). The European Community Landfill Directive (EPA) has led to an increase in the costs of landfill and a reduction of places for landfill in many countries (EPA; http://ec.europa.eu/environment/waste/target_review.htm). Approximately 87.6 million tonnes per year of food waste are produced in Europe