University of Innsbruck
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 Austrian Science Fund project Phenylodigest

Occurrence and fate of phenyl-acids PAA, PPA, and PBA and
their impact on the microbiology of anaerobic digestion processes


2016 - 2020

Principal Investigator

Andreas O. Wagner

Concept and goals

For purpose of anaerobic digestion, in past years substrate spectra have been broadened as well as the application of pre-treatment strategies progressively were extended to better exploit the energy present in the substrate. An improved application and disintegration of substrates, however, also entails risks in view of undesired by-product formation including aromatic compounds like phenylacetic-, -propionic-, and -butyric acid, which are known to possibly accumulate under unfavourable digestion conditions and to exert a negative effect on the anaerobic digestion processes.

The goals of the project therefore comprised the investigation of

the formation of phenylacetic-, -propionic-, and -butyric acid (PAA, PPA, PBA) during mesophilic and thermophilic anaerobic digestion (AD) from defined precursor substrates (protein rich, lignin derivatives),

the impact of PAA, PPA, PBA on the anaerobic digestion process and its microbial consortium under mesophilic and thermophilic conditions,

the ability of microbial consortia to adapt to different phenyl acid concentrations, and

the identification of organisms/communities capable of handling phenyl acid while still maintaining high methane productivity.

Most important results and a brief description of their significance

Results of this project were published in several papers in SCI indexed, peer reviewed scientific journals. Due to space restrictions, the most important findings only are presented here in the way the results were published. For details and a deeper insight, please refer to the original publications.

Formation of phenylacetic acid and phenyl propionic acid under different overload conditions during mesophilic and thermophilic anaerobic digestion, Biotechnology for Biofuels, 2019, DOI: 10.1186/s13068-019-1370-6

This paper, constituting a large data set derived from WP1, deals with the formation of phenyl acids from protein-rich and amino acid precursor substrates under different overload conditions. Applying both, mesophilic and thermophilic mixed microbial communities it could be shown, that the phenyl acids PAA and PPA accumulated up to concentrations of > 20 mM. During thermophilic AD, the presence of amino acids tended to result in PPA, whereas complex, protein-rich substrates promoted the accumulation of PAA. The effect of phenyl acid formation was mainly substrate load dependent, whereas it was less inoculum and temperature driven. Once formed, the formation of phenyl acids constituted a reversible process during mesophilic AD, while during thermophilic incubation phenyl acids tended to accumulate without further degradation.

The hypothesis that phenyl acids formed during overload conditions in anaerobic digestion reactors would generally inhibit the methanation process had to be rejected. However, it could be demonstrated that phenyl acids seem to play an important role in the microbial response to overloaded biogas systems.

Microbial and Phenyl Acid Dynamics during the Start-up Phase of Anaerobic Straw Degradation in Meso- and Thermophilic Batch Reactors, Microorganisms, 2019, DOI: 10.3390/microorganisms7120657

In this study, during straw degradation a cascade-like PAA-PPA-PBA pattern could be observed for mesophilic medium - and for thermophilic high overload samples. PAA was probably derived from easily available substrates and was an early indicator for overload conditions. An increase in PBA indicated the end of the start-up and the beginning of the degradation of more complex materials. Methanosarcina spp. dominated in those samples, and thus confirmed its essential role for stabilising overloaded reactors. Although the role of phenyl acids during anaerobic digestion processes remains to be further elucidated, these dynamics during the start-up phase might be relevant for monitoring the process stability and the start of degradation of more recalcitrant waste portions in biogas plants.

Microbial community dynamics in mesophilic and thermophilic batch reactors under methanogenic, phenyl acid-forming conditions, Biotechnology for Biofuels, 2020, DOI: 10.1186/s13068-020-01721-z

This publication can be assigned to WP 1 and refers to aim 4. Here, various microbiological aspects regarding the effect of phenyl acid on the anaerobic digestion process under overload conditions using amino acid and protein-rich co-substrates could be covered.

For amino acid and protein-rich substrate fed biogas reactors under both, meso- and thermophilic conditions, Sedimentibacter spp., Tepidanaerobacter spp., Acetomicrobium spp., and Sporanaerobacter spp. were shown to be significant biomarkers for high phenyl acid concentrations during. Members of the genus Syntrophus took part in the anaerobic benzene ring cleavage in mesophilic samples at low overload conditions. They might be important players in preventing phenyl acid accumulation and reactor performance deterioration. Acetoclastic methanogenesis dominated over all mesophilic samples, whereas a shift from acetoclastic to SAO-induced hydrogenotrophic methanogenesis took place in thermophilic samples. This methanogenic pathway seemed to be quite robust when proteinaceous materials/precursors were degraded in high loads. However, interactions between microbes involved in the formation/degradation dynamics of aromatic compounds were highly complex and further studies on phenyl acid formation dynamics are still needed, especially when considering the influence of further factors like temperature, substrate, and substrate load.

Lignin intermediates lead to phenyl acid formation and microbial community shifts in meso- and thermophilic batch reactors, Biotechnology for Biofuels, 2021, DOI: 10.1186/s13068-020-01855-0

Here, various lignin intermediates were tested in different concentrations on their impact on the overall, meso- and thermophilic digestion process and the involved microbial community:

Even though coming from a low carbon load system, the mesophilic sludge community could cope far better with lignin intermediates than the thermophilic one. Acetoclastic methanogens were shown to be especially susceptible to aromatic compound additions. High concentrations of lignin derivatives led to a complete inhibition of methanogenesis. It could be shown, that lignin intermediates also led to an inhibition of acetogenic microorganisms. Not only the load and the temperature regime, but also the chemical structure (like the length of the aliphatic side chain, methoxy-, hydroxy-groups) had an influence on the overall digestion performance. The phenyl acids PAA, PPA, and PBA accumulated when lignin intermediates were metabolically utilised, but stuck in degradation phases prior to methanogenesis. Associations of genera like Fastidiosipila, Syntrophomonas (mesophilic), or Bacillus, Lactobacillus, Geobacillus, and Tissierella (thermophilic) with anaerobic phenyl acid formation were reproduced; hence, these genera can be seen as bioindicators for phenyl acid formation and/or process failures. Although the impact of various lignin intermediates on the generation of phenyl acids could be established, the exact degradation pathways remain elusive.

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