# A novel approach to enhance methane production in anaerobic digestion of waste activated sludge via pre-enrichment by a microbial consortium of degrading fungi

## TL;DR

A microbial consortium of fungi improves methane production from wastewater sludge by breaking down fungal cell walls and releasing organic matter.

## Contribution

The study introduces a novel microbial consortium of degrading fungi that enhances anaerobic digestion efficiency and methane yield from waste-activated sludge.

## Key findings

- MCDF enrichment increased methane production by 35% in waste-activated sludge.
- MCDF degraded fungal cell wall polysaccharides and released intracellular organics from four fungal species.
- Key hydrolase activities in MCDF were significantly higher than in untreated sludge.

## Abstract

Both fungi and bacteria contribute to the efficiency of activated sludge processes. However, fungal degradability has historically been overlooked in the anaerobic fermentation of waste-activated sludge (WAS). This study investigated the role of an enriched microbial consortium of degrading fungi (MCDF) in WAS fermentation. MCDF was initially enriched using a mixture of WAS and two mesophilic anaerobic sludges with chitin as the substrate. Fungal diversity and key cell wall polysaccharides, including chitin, mannan, and glucans, were identified in WAS. The addition of enriched MCDF to WAS significantly increased methane production by 35% and disrupted the floc structure through degradation of native fungi. The enriched MCDF also utilized fungal polysaccharides from four fungal species (Candida albicans, Trichosporon asahii, Geotrichum sp., and Magnusiomyces capitatus) and facilitated the release of intracellular organics. Proteinophilum and Petrimonas were identified as the main producers of chitinase (EC 3.2.1.14). Additional microbial producers were identified for mannanase (EC 3.2.1.78), β−1,4-glucanase (EC 3.2.1.4), and β−1,3-glucanase (EC 3.2.1.6). The relative activities of these four hydrolases in WAS were only 0.01%–12.3% of those in enriched MCDF. These results highlight the multifunctional potential of MCDF enrichment in WAS digestion, including degradation of fungal cell wall polysaccharides, release of intracellular fungal organics, and hydrolysis of bacterial extracellular organics. Overall, this study demonstrates that the destruction of native fungi in WAS provides a promising approach to enhance methane production and anaerobic digestion efficiency in wastewater treatment plants.

Both fungi and bacteria contribute to maintaining the floc structure of waste-activated sludge (WAS). This study highlights that MCDF enrichment significantly enhances methane production and disrupts the floc structure through the degradation of native fungi in WAS. Fungal diversity and key fungal cell wall polysaccharides—chitin, mannan, and glycans—were identified in WAS. The MCDF consortium was shown to utilize extracellular organics from four fungal species (Candida albicans, Trichosporon asahii, Geotrichum sp., and Magnusiomyces capitatus) and promote the release of intracellular organics. Multi-omics analysis revealed Proteinophilum and Petrimonas as the main chitinase producers in MCDF. Additional hydrolytic activities were observed: Fermentimonas and Petrimonas for mannanase, Mesotoga and Proteinophilum for β−1,4-glucanase, and Syntrophorhabdus for β−1,3-glucanase. Notably, the activities of these four hydrolases were significantly higher in MCDF than in WAS, indicating that MCDF is the main source of key functional enzymes. These findings highlight the multifunctional potential of MCDF enrichment in enhancing methane production during WAS digestion.