The microbial diversity in anaerobic digestion (AD) is important because it affects process robustness. High-throughput sequencing offers high-resolution data regarding the microbial diversity and robustness of biological systems including AD; however, to understand the dynamics of microbial processes, knowing the microbial diversity is not adequate alone. Advanced meta-omic techniques have been established to determine the activity and interactions among organisms in biological processes like AD. Results of these methods can be used to identify biomarkers for AD states. This can aid a better understanding of system dynamics and be applied to producing comprehensive models for AD. The paper provides valuable knowledge regarding the possibility of integration of molecular methods in AD. Although meta-genomic methods are not suitable for on-line use due to long operating time and high costs, they provide extensive insight into the microbial phylogeny in AD. Meta-proteomics can also be explored in the demonstration projects for failure prediction. However, for these methods to be fully realised in AD, a biomarker database needs to be developed.

Delignification of steam-exploded birch wood (SEBW) was stimulated using a pretreatment method including Fenton reaction (FR) and fungi. SEBW was employed as a substrate to optimize the Fe(III) and Fe(II) dosage in FR. Maximum iron-binding to SEBW was obtained at pH 3.5. FR pretreatment increased biological methane yields from 257 mL/g vS in control to 383 and 352 mL/ g vS in samples with 0.5 mM Fe(II) and 1.0 mM Fe(III), respectively. Further enzymatic pretreatment using a commercial cellulase cocktail clearly improved methane production rate but only increased the final methane yields by 2-9 %. Finally, pretreatments with the fungi Pleurotus ostreatus (PO) and Lentinula edodes (LE), alone or in combination with FR, were carried out. SEBW pretreated with only LE and samples pretreated with PO and1 mM Fe(III) + H₂O₂ increased the methane production yield to 420 and 419 mL/g vS respectively. These pretreatments delignified SEBW up to 25 %.

Birch wood is a potential feedstock for biogas production in Northern Europe; however, the lignocellulosic matrix is recalcitrant preventing efficient conversion to methane. To improve digestibility, birch wood was thermally pre-treated using steam explosion at 220 °C for 10 min. The steam-exploded birch wood (SEBW) was co-digested with cow manure for a period of 120 days in continuously fed CSTRs where the microbial community adapted to the SEBW feedstock. Changes in the microbial community were tracked by stable carbon isotopes- and 16S r RNA analyses. The results showed that the adapted microbial culture could increase methane production up to 365 mL/g VS day, which is higher than previously reported methane production from pre-treated SEBW. This study also revealed that the microbial adaptation significantly increased the tolerance of the microbial community against the inhibitors furfural and HMF which were formed during pre-treatment of birch. The results of the microbial analysis indicated that the relative amount of cellulosic hydrolytic microorganisms (e.g. Actinobacteriota and Fibrobacterota) increased and replaced syntrophic acetate bacteria (e.g. Cloacimonadota, Dethiobacteraceae, and Syntrophomonadaceae) as a function of time. Moreover, the stable carbon isotope analysis indicated that the acetoclastic pathway became the main route for methane production after long-term adaptation. The shift in methane production pathway and change in microbial community shows that for anaerobic digestion of SEBW, the hydrolysis step is important. Although acetoclastic methanogens became dominant after 120 days, a potential route for methane production could also be a direct electron transfer among Sedimentibacter and methanogen archaea.