There is a growing need of substrate flexibility for biobased production of energy and value-added products that allows the application of variable biodegradable residues within a circular economy. It can be used to balance fluctuating energy provision of other renewable sources. Hydrolysis presents one of the biggest limitations during anaerobic digestion. Methods to improve it will result in broader process applicability and improved integration into regional material cycles. Recently, one focus of anaerobic digestion research has been directed to systems with a separate hydrolysis–acidogenesis stage as it might be promised to improve process performance. Conditions can be adjusted to each class of microorganisms individually without harming methanogenic microorganisms. Extensive research of separate biomass pretreatment via biological, chemical, physical or mixed methods has been conducted. Nevertheless, several methods lack economic efficiency, have a high environmental impact or focus on specific substrates. Pretreatment via a separate hydrolysis stage as cell-driven biotransformation in a suspension might be an alternative that enables high yields, flexible feeding and production, and a better process control. In this review, we summarize existing technologies for microbial hydrolytic biotransformation in a separate reactor stage and the impacts of substrate, operational parameters, combined methods and process design as well as remaining challenges.

In this work, the effect of bioaugmentation on the hydrolysis and acidogenesis efficiency of bedding straw mixed with maize silage is examined. A plug-flow bioreactor was operated for 70 weeks with maize silage as a reference feedstock and subsequently with an increasing straw content of 30% and 66% (w/w). Bioaugmentation with two Paenibacillus species was conducted at each process condition to investigate the impact on hydrolysis of the recalcitrant lignocellulosic feedstock. A stable acidogenic digestion of the substrates was achieved, during which acetic and butyric acid were accumulated as main byproducts. Specific hydrolysis rates between 258 and 264 gO₂ kg⁻¹_VS were determined for pure maize silage and maize silage mixed with 30% of straw, while the specific hydrolysis rate decreased to 195 gO₂ kg⁻¹_VS when a mixture with 66% of straw was applied. Bioaugmentation with Paenibacillus spp. increased the specific hydrolysis rate by up to 41–63% for pure maize silage and the mixture with 30% of straw, while no increase was observed with a mixture of 66% of straw. Acid production, however, was enhanced by 21 to 42% following bioaugmentation for all substrate mixtures. A positive effect on the physiological state of cultures, as recorded with frequency-dispersed polarizability, was seen after bioaugmentation, which remained for two retention times during the continuous fermentation mode. Recirculation of the thin sludge further prolonged the positive effects of bioaugmentation. The results of this work provide a basis to optimize the amount of the bioaugmented microorganisms and hydrolysis of biogenic material with respect to sustainable effects on process performance and costs.

Background: Two parallel plug-flow reactors were successfully applied as a hydrolysis stage for the anaerobic pre-digestion of maize silage and recalcitrant bedding straw (30% and 66% w/w) under variations of the hydraulic retention time (HRT) and thin-sludge recirculation. Results: The study proved that the hydrolysis rate profits from shorter HRTs while the hydrolysis yield remained similar and was limited by a low pH-value with values of 264–310 and 180–200 g_O2 kg_VS⁻¹ for 30% and 66% of bedding straw correspondingly. Longer HRT led to metabolite accumulation, significantly increased gas production, a higher acid production rate and a 10–18% higher acid yield of 78 g_SCCA kg_VS⁻¹ for 66% of straw. Thin-sludge recirculation increased the acid yield and stabilized the process, especially at a short HRT. Hydrolysis efficiency can thus be improved by shorter HRT, whereas the acidogenic process performance is increased by longer HRT and thin-sludge recirculation. Two main fermentation patterns of the acidogenic community were found: above a pH-value of 3.8, butyric and acetic acid were the main products, while below a pH-value of 3.5, lactic, acetic and succinic acid were mainly accumulating. During plug-flow digestion with recirculation, at low pH-values, butyric acid remained high compared to all other acids. Both fermentation patterns had virtually equal yields of hydrolysis and acidogenesis and showed good reproducibility among the parallel reactor operation. Conclusions: The suitable combination of HRT and thin-sludge recirculation proved to be useful in a plug-flow hydrolysis as primary stage in biorefinery systems with the benefits of a wider feedstock spectrum including feedstock with cellulolytic components at an increased process robustness against changes in the feedstock composition. Graphical

Hydrolysis at changing hydraulic retention time, recirculation, bedding straw content in the feed, bioaugmentation and the impact of those changes on gradient formation in the liquid phase in plug-flow reactors (PFRs) was examined. The pH-value, conductivity and oxidation–reduction potential (ORP) were monitored at three spots along the PFRs to study potential correlations to process performance during a total process time of 123 weeks. The on-line monitoring showed good correlations to acidogenesis: namely, the pH and ORP to the acidification, to butyric (and lactic) acid concentration and to the acid yield. The ORP (measured at the inlet) showed the most stable correlation to acidogenesis under dynamic operation, while the conductivity (at the outlet) correlated to the acid concentration in dependence on the feedstock. Multiple measurement spots as used in this study allow to gain more information about acidogenic fermentation than a single spot, simplifying process control and automation attempts with recalcitrant feedstock. Graphical Abstract