Role of Bioadditions in Sustainable Energy Production

Abstract

This PhD research investigated the effects of yeast bioaddition on anaerobic digestion (AD) of lignin-rich agricultural waste, specifically rye and corn silage, with a focus on understanding the underlying mechanisms of yeast action on the lignocellulosic component of lignin, biogas production and microbial communities. While previous studies have explored bioadditives in lignocellulosic biomass degradation, the detailed mechanisms of these bioadditives, particularly yeast, in enhancing anaerobic digestion remain poorly understood. This study addresses key knowledge gaps by evaluating the impact of yeast addition on process parameters, microbial dynamics, and biogas production in batch and semi-continuous AD systems. The research was divided into four objectives. Firstly, the study assessed the effect of yeast on lignin-rich feedstock, hypothesizing that yeast enhances the degradation of lignin and improves biogas yields. Results revealed that yeast contains enzymes such as polyphenol oxidase (PPO) and phenol oxidizing enzymes (POE), which are able to react with G-units and S-units phenolic compounds following lignin degradation. These compounds, in particular H-units, can have inhibitory effect on methanogens. Some of these PPOs have also been reported to be able to demethylase these phenolics. This is the case in this work as yeast addition to rye, richer in Sunits, results in higher biogas production in the early stages of the reaction, linked to easily digestible methyl-groups. The result also confirms that not all the phenolic compounds following lignin degradation have an inhibitory effect. For example, syringic acid (S-unit) was metabolized and produced increased biogas yields compared to control and yeast addition significantly increased these yields. On the other hand, p-hydroxybenzoic acid, inhibited biogas production, with and without yeast addition. In addition, yeast can provide metals and micronutrients to the process for micronutrients-poor feedstocks such as food waste. This was not the case in this study as both rye and corn silage contained all the required micronutrients. The second objective involved the development of a novel method for full-length 16S rRNA archaeal extraction and sequencing using Oxford Nanopore® Technology (ONT). By combining newly designed primers targeting a broader range of archaeal groups with ONT’s long-read 14 chemistry kit, this method offers a novel alternative to traditional short-read technologies such as Illumina MiSeq. It provides greater taxonomic resolution, enabling comprehensive and real-time detection of methanogens and broader archaeal diversity in iv anaerobic digestion systems, thereby enhancing the detailed identification of critical microbial players for improved biogas production performance. For the third objective, the study explored the short-term and long-term effects of yeast bioadditions on microbial community composition and process performance in pilot-scale reactors digesting rye silage. Yeast addition was found to enhance soluble COD removal efficiency and mitigate ammonia inhibition. Despite comparable soluble COD levels in both the treated (DRY) and untreated (DR) reactors, the DRY reactor demonstrated more stable biogas production, suggesting that yeast bioaddition shifted the COD profile towards more readily biodegradable compounds, sustaining microbial activity. The DRY reactor also exhibited higher concentrations of hydrogenotrophic methanogens, suggesting that yeast supported the growth and activity of these critical microbes, even under elevated NH₃ concentrations. The final objective focused on long-term yeast effects under variable conditions. The study showed that yeast contributed to a more stable syntrophic acetate oxidation (SAO) process, which was linked to the increased presence of SAO bacteria such as Tepidanaerobacter acetatoxydans. This stabilization of the microbial community allowed the treated reactor to maintain high biogas yields, despite high ammonia and volatile fatty acid concentrations. In conclusion, yeast bioaddition positively influenced the degradation of lignocellulose in AD systems, improved microbial resilience, and enhanced biogas production. The findings suggest that yeast can play a critical role in optimizing AD processes, particularly in systems dealing with high-lignin feedstocks. Future research should focus on further understanding the molecular interactions between yeast and the very strong syntrophic association established between Tepidanaerobacter acetatoxydans and Methanoculleus bourgensis that ensured process stability in this study. Also, exploring the scalability of yeast bioadditives for industrial applications is equally recommended