Effect of model methanogens on the electrochemical activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes

Abstract

Combining anaerobic digestion (AD) and microbial electrochemical technologies (MET) in AD-MET holds great potential. Methanogens have been identified as one cause of decreased electrochemical activity and deterioration of Geobacter spp. biofilm anodes. A better understanding of the different interactions between methanogenic genera/species and Geobacter spp. biofilms is needed to shed light on the observed reduction in electrochemical activity and stability of Geobacter spp. dominated biofilms as well as observed changes in microbial communities of AD-MET. Here, we have analyzed electrochemical parameters and changes in the microbial community of Geobacter spp. biofilm anodes when exposed to three representative methanogens with different metabolic pathways, i.e., Methanosarcina barkeri, Methanobacterium formicicum, and Methanothrix soehngenii. M. barkeri negatively affected the performance and stability of Geobacter spp. biofilm anodes only in the initial batches. In contrast, M. formicicum did not affect the stability of Geobacter spp. biofilm anodes but caused a decrease in maximum current density of ~37%. M. soehngenii induced a coloration change of Geobacter spp. biofilm anodes and a decrease in the total transferred charge by ~40%. Characterization of biofilm samples after each experiment by 16S rRNA metabarcoding, whole metagenome nanopore sequencing, and shotgun sequencing showed a higher relative abundance of Geobacter spp. after exposure to M. barkeri as opposed to M. formicicum or M. soehngenii, despite the massive biofilm dispersal observed during initial exposure to M. barkeri.

Fig. 1. Geobacter spp. biofilms exposed to varied media conditions.

Transferred charge ($\mathrm{Q}$), current density (j_max) and chemical oxygen demand removal (ΔCOD) upon exposure of Geobacter spp. biofilms to a 50:50, v/v mixture of acetate-based medium and: (a) abiotic BFS01 medium, (b) M. barkeri cultures in BFS01, (c) M. formicicum cultures in BFS01, (d) M. soehngenii cultures in BFS01. C1-C3: successive control batches with only acetate-based medium, B1*-B4*: four successive batch cycles with abiotic BFS01 medium, B1-B4: four successive batch cycles with the methanogenic cultures grown in the BFS01 medium (see Fig. 4), n ≥ 3, error bars indicate confidence interval CI.

Fig. 2. Exploring the effect of M. barkeri age on Geobacter spp. biofilm.

Transferred charge ($\mathrm{Q}$), current density (j_max) and chemical oxygen demand removal (ΔCOD) at the end of each batch cycle upon exposure of Geobacter spp. biofilm to a 50:50, v/v mixture of acetate-based medium and M. barkeri cultures in BFS01 medium. C1-C3: control batches with only acetate-based medium, B1-B4: four successive batch cycles with Geobacter spp. biofilms exposed to M. barkeri cultures aged 3–6 weeks, respectively, n = 4, error bars indicate confidence interval CI.

Fig. 3. Characterisation of Geobacter spp. biofilm using varied profiling techniques.

a Metabarcoding abundance profiling targeting the 16S rRNA V3-V4 region, (b) whole metagenome shotgun read percentages, and (c) whole metagenome nanopore read percentages of biofilm samples at the end of the experiment upon exposure of Geobacter spp. biofilm to the abiotic BFS01 medium and each methanogenic culture in BFS01 medium, respectively. [CX1-CX3], [GB1-GB3], [GF1-GF3], and [GS1-GS3] indicate triplicate biological biofilm samples after each electrochemical experiment, i.e., CX1-CX3: after biofilm exposure to abiotic BFS01 medium, GB1-GB3: after biofilm exposure to M. barkeri, GF1-GF3: after biofilm exposure to M. formicicum, and GS1-GS3: after biofilm exposure to M. soehngenii, respectively. (Other genera < 2%).

Fig. 4. Overview of electrochemical experiments.

Each batch cycle always lasts one week. “C1 to C3” indicate the three successive control batch cycles during the pre-growth of Geobacter spp. biofilms with 100% acetate-based medium, “Exposure batch cycles” indicate the successive batch cycles during the electrochemical control experiment without methanogens, i.e., B1*-B4* with the 50:50, v/v mixture of acetate-based medium and abiotic BFS01 medium (top) as well as the electrochemical experiment with methanogens, i.e., B1-B4 with the 50:50, v/v mixture of acetate-based medium and each methanogenic culture (bottom).