Anaerobic digestion of Green Willow Biomass, a novel lignocellulosic substrate, and the evaluation of H2-induced stress in anaerobic bioreactors by genome-resolved metatranscriptomics

The harmful impact of humanity on the Earth system includes the increasing rate of species extinction, deforestation, climate change, environmental pollution and overpopulation. One central issue is the increasing global energy demand, which is still met with 80% by fossil fuels. The burning of these contributes to many of the beforementioned global issues, as they produce green-house-gases (GHG) and pollutants. Fortunately, reducing their share is the target of several international treaties. Renewable energies provide an alternative to this, but their combined usage is required to provide effective GHG-mitigation. Biomass is a ubiquitous renewable energy source that can be harnessed in many ways. Because its many advantages, one very promising technology for alternative energy generation from biomass is the anaerobic digestion process (AD). In the context of climate change, the generation of biogas as a form of renewable energy has become popular and was intensively examined over the last few decades. In the course of my doctoral work, I aimed to contribute to the following two major challenges that biogas industry faces: 1.) To find novel substrates that have high biogas production potential, yet cheap to produce and do not come into contradiction with the food vs feed debate, as traditional substrates, such as maize do. Willow biomass may be a promising choice, since producing it has many advantages, including low production costs and the possibility of growing it on marginal lands, but the high amounts of lignin in the woody biomass hindered its usage as a substrate for biogas generation until now. To this end, I utilized a novel lignocellulosic biogas substrate: the young shrubs of the short-rotation coppice willow (Salix spp.), which I termed Green Willow Biomass (GWB). 2.) The coupling of other renewable sources, such as photovoltaics and wind energy, with biogas to harness their excess energy produced when the energy consumption rate is lower than its generation. This can be achieved with a process called a power-to-methane (P2bioCH4), wherein the excess energy is used to generate H2 via water electrolysis, which in turn is fed to an anaerobic digester to increase the methane concentration of the produced biogas. To better understand this in situ biological biogas upgrading process (BBU), I investigated the near-immediate effects of hydrogen-addition on the biogas producing microbial community via a metatranscriptomic approach. Within the first part of my research, I analyzed the fermentation efficiency of GWB, based on parameters, such as maximal methane yields (K) and highest methane production rates (µmax). This showed that both the commercial willow genotype (EN) and the novel tetraploid variant (PP) can be viable alternative AD feedstocks. Perhaps even more so when considering CSTR fermentations and industrial-scales operations because the GWB’s higher µmax values, compared to woody willow (WWB). In addition to the laboratory experiments, I estimated the biomass yields (t/ha) based on a small-scale field experiment to compare the energy potential of GWB with other feedstocks. When considering biomass yields, GWB can be an even more promising choice, as the extractable energy potential (GJ/ha) in the August harvests of GWB almost reached and in an estimated September harvest, it can potentially surpass the corresponding yields of either maize silage or combusted woody willow. Many of these advantages come from the presence (or rather, high amounts) of leaf biomass. Therefore, my thesis provides substantial support to promote and recommend green willow biomass as valuable feedstock for biomethanation. Within the course of the second part of my thesis, I examined the early response of the biogas-producing microbial community to the presence of saturating amount of H2. I used a combined metagenomic and metatranscriptomic analysis to determine the changes of the expression levels of the various genes of the metagenome-assembled genomes (MAGs), with a focus to those related to methanogenesis. My results showed that the microbial community (both the Archaea and many Bacteria) responded practically instantaneously (2 hours after the induced H2) to the changed environment. The activity of pluripotential methanogens, mainly Methanosarcina MAGs, reduced significantly, while the hydrogenotrophic Methanobacter MAGs increased. Many MAGs were unique to my samples, but these were not always just side-players, rather several of them are abundant, main-players of the community. Thus, it seems that specific microbes proliferate in the samples, which is likely the case in ultimately any AD environment. This thorough analysis of the differences between the H2-treated metatranscriptomes and corresponding controls identified the early events in the microbial communities brought about by the H2 addition, contributing to our understanding of the BBU mechanism in a P2bioCH4 system