Microwave-assisted organic acids and green hydrogen production during mixed culture fermentation

Abstract

Background: The integration of anaerobic digestion into bio-based industries can create synergies that help render anaerobic digestion self-sustaining. Two-stage digesters with separate acidification stages allow for the production of green hydrogen and short-chain fatty acids, which are promising industrial products. Heat shocks can be used to foster the production of these products, the practical applicability of this treatment is often not addressed sufficiently, and the presented work therefore aims to close this gap.

Methods: Batch experiments were conducted in 5 L double-walled tank reactors incubated at 37 °C. Short microwave heat shocks of 25 min duration and exposure times of 5-10 min at 80 °C were performed and compared to oven heat shocks. Pairwise experimental group differences for gas production and chemical parameters were determined using ANOVA and post-hoc tests. High-throughput 16S rRNA gene amplicon sequencing was performed to analyse taxonomic profiles.

Results: After heat-shocking the entire seed sludge, the highest hydrogen productivity was observed at a substrate load of 50 g/l with 1.09 mol H₂/mol hexose. With 1.01 mol H₂/mol hexose, microwave-assisted treatment was not significantly different from oven-based treatments. This study emphasised the better repeatability of heat shocks with microwave-assisted experiments, revealing low variation coefficients averaging 29%. The pre-treatment with microwaves results in a high predictability and a stronger microbial community shift to Clostridia compared to the treatment with the oven. The pre-treatment of heat shocks supported the formation of butyric acid up to 10.8 g/l on average, with a peak of 24.01 g/l at a butyric/acetic acid ratio of 2.0.

Conclusion: The results support the suitability of using heat shock for the entire seed sludge rather than just a small inoculum, making the process more relevant for industrial applications. The performed microwave-based treatment has proven to be a promising alternative to oven-based treatments, which ultimately may facilitate their implementation into industrial systems. This approach becomes economically sustainable with high-temperature heat pumps with a coefficient of performance (COP) of 4.3.

Keywords: 16-S-rRNA sequencing; Acidification; Dark fermentation; Hydrogen production; Microwave heat shocks; Volatile fatty acids production.

Fig. 1

A Experimental set-up: double-walled reactors (1) with a volume of 5 l each. Each reactor was filled through an input port (2), and acidified hydrolysate was removed through a port at the bottom (3). For heating, a water pipe was connected to the input (9) and output (10) ports of the double-wall clearance. Water was heated using a thermostat (6). The gas produced was measured by a MilliGascounter (5), connected to a tube (4), and stored in a gas bag (7). The time-resolved gas production was recorded with an Arduino (8) connected to the gas counter. B Average incubation time for heat shock up to a minimum of 80 °C for pre-treatment with oven (set to 130 °C) and microwave (set to 1000 W). C Substrate variation tests: Comparison of hydrogen production with sucrose, mixed rye bread, gelatine and coconut oil as feeding substrates and sewage sludge as inoculum. Both control and heat shock (oven and microwave) scenarios were tested

Fig. 2

Application of heat shocks to optimise hydrogen productivity: different concentrations of substrate (sucrose) and different pre-treatments (oven and microwave) were compared. A Maximal gas productivity and B the highest hydrogen production were reached with a substrate concentration of 50 g/l and pretreated with oven heat shock. All fermentations were performed at least 5 times. C The highest hydrogen efficiency was achieved with 50 g/l sucrose in both microwave and oven experiments. D Gas composition depending on the feeding amount and pre-treatment. The median values were linked to a trend line to better visualise the effects of the different substrate concentrations

Fig. 3

A Microbial community on genus level as relative abundances differentiated according to pre-treatment and sucrose concentration. Only the most abundant microorganisms with an abundance > 5% are presented; others with abundances below 5% are summarised as others < 5%. Duplicates were sequenced for each condition. B Alpha Diversity of the microbial community for genus level data of microwave and control samples, substrate concentrations of 50 and 100 g/l of sucrose

Fig. 4

A Graphical representation of statistical significance (significant for p < 0.05) of group comparisons for hydrogen and total gas volumes, hydrogen formation efficiency and hydrogen fraction. The marker in the grey area indicates a significant difference between the respective comparison groups. B Production of volatile fatty acids (median) differentiated by treatment method and feeding amount. Volatile fatty acids were analysed in triplicate in all cases. C Course of gas production over seven days and D first 24 h for untreated (control) and heat-shock-treated (oven and microwave) experiments. Performed with sewage sludge and 75 g/l sucrose as feeding substrate