Disruption-induced changes in syntrophic propionate and acetate oxidation: flocculation, cell proximity, and microbial activity

Abstract

Background: Syntrophic propionate- and acetate-oxidising bacteria (SPOB and SAOB) play a crucial role in biogas production, particularly under high ammonia conditions that are common in anaerobic degradation of protein-rich waste streams. These bacteria rely on close interactions with hydrogenotrophic methanogens to facilitate interspecies electron transfer and maintain thermodynamic feasibility. However, the impact of mixing-induced disruption of these essential syntrophic interactions in biogas systems remains largely unexplored. This study investigates how magnetic stirring and orbital shaking influence degradation dynamics, microbial community composition, and gene expression in syntrophic enrichment communities under high-ammonia conditions.

Results: Stirring significantly delayed the initiation of propionate degradation in one culture and completely inhibited it in the other two parallel cultures, whereas acetate degradation was less affected. Computational fluid dynamics modelling revealed that stirring generated higher shear rates (~ 20 s^-1) and uniform cell distribution, while shaking led to lower shear rates and cell accumulation at the bottom of the culture bottle. Visual observations confirmed that stirring inhibited floc formation, while shaking promoted larger flocs compared to the static control condition, which formed smaller flocs and a sheet-like biofilm. Microbial community analysis identified substrate type and degradation progress as primary drivers of community structure, with motion displaying minimal influence. However, metatranscriptomic analysis revealed that motion-induced gene downregulation was associated with motility, surface sensing, and biofilm formation in SAOB and another bacterial species expressing genes for the glycine synthase reductase pathway. Stirring also suppressed oxalate-formate antiporter expression in SPOB, suggesting its dependence on spatial proximity for this energy-conserving mechanism. The strongest gene expression changes of stirring were observed in methanogens, indicating a coupling of the first and last steps of hydrogenotrophic methanogenesis, likely an adaptive strategy for efficient energy conservation. Other downregulated genes included ferrous iron transporters and electron transfer-associated enzymes.

Conclusions: This study highlights that stirring critically disrupts the initial syntrophic connection between SPOB and methanogens, whereas SAOB communities exhibit greater tolerance to shear stress and disruptive conditions that inhibits aggregate formation. These findings emphasize the importance of carefully managing mixing regimes, especially when attempting to reactivate ammonia-tolerant syntrophic propionate degraders in biogas systems experiencing rapid propionate accumulation under high-ammonia conditions.

Keywords: Anaerobic digestion; Computational fluid dynamics; Flocculation; Interspecies electron transfer; Methanogens; Mixing; Syntrophic acetate-oxidizing bacteria; Syntrophic propionate-oxidizing bacteria.

Fig. 1

Degradation dynamics of acetate-fed (top) and propionate-fed (bottom) cultures subjected to different types of agitative motion: no motion (C, left), orbital shaking (Sh, middle), and magnetic stirring (St, right). Solid lines show the acetate concentration, while dashed lines show the propionate concentration (mM) and replicates are shown in different colours. For acetate-fed cultures, additional acetate (up to 50 mM) was added on day 111 and RNA extraction was performed on day 125. In the propionate-fed cultures, propionate was added on day 300 and RNA was extracted on day 338. Note the differing scales on the x-axes for acetate-fed and propionate-fed cultures

Fig. 2

Contour plots depicting shear rates (s⁻¹) for magnetic stirring (top plane: a bottom plane: b and orbital shaking (top plane: c bottom plane: d). Logarithmic scale has been used for both colour legends to highlight the variation in shear rates

Fig. 3

Contour plots corresponding to volume fraction of solid particles, unitless, with an initial value of solid particles 0.1 (1% of total volume) with homogenous distribution for both motions with magnetic stirring to the top and orbital shaking at the bottom. The deviation greater than 0.1 indicates regions with higher concentration of solid particles

Fig. 4

Bubble plot showing the relative abundance of microbial genera over time in acetate-fed (CA, ShA, StA) and propionate-fed (CP, ShP, StP1–3) cultures. All cultures, except stirred propionate-fed, show the relative abundance of merged counts across replicates. Stirred propionate-fed cultures (StP1–3) display individual replicate data instead. This distinction was made, because stirred propionate cultures exhibited substantial differences in ability to degrade propionate. For a detailed view of replicate-specific data for each condition, see Supplementary Figs. S4 and S5. Bubble size represents relative genus abundance, with genera with less than 2% relative abundance grouped as "Minor genera (< 2%)”

Fig. 5

Graphical overview of the highlighted differentially expressed genes in stirred or orbital shaken samples relative to the static control samples. The red and green squares represent differentially downregulated and upregulated genes, respectively. White squares indicate that no differential change in expression was observed