Earthworms drastically change fungal and bacterial communities during vermicomposting of sewage sludge

The composition of bacterial and fungal microbiotas changes during vermicomposting of sewage sludge

The bacterial community of the raw sewage sludge included 19 phyla and was mainly comprised of Bacteroidota, Bdellovibrionota, Campilobacterota, Firmicutes and Proteobacteria (Fig. 1). Bacterial communities of fresh earthworm casts were dominated by the phyla Bacteroidota, Proteobacteria and Verrucomicrobiota (Fig. 1). Large changes in bacterial community composition were found after transit of the sewage sludge through the gut of the earthworms (GAP), with significant decreases in the abundance of Campilobacterota, Firmicutes and Bacteroidota, and significant increases in the abundance of Verrucomicrobiota, Proteobacteria and Bacteroidota (Supplementary Table S1). At the genus level, transit through the gut significantly reduced the abundance of bacterial genera Terrimonas, Acetoanaerobium, Bacteroides, Cloacibacterium, Proteocatella and Macellibacteroides among others (Fig. 1, Supplementary Table S2), and increased significantly the abundance of Dyadobacter, Aeromonas, Luteolibacter, Edaphobaculum, Cellvibrio, Pedobacter, Sphingomonas, Devosia, Cetobacterium and Rhodanobacter among others (Fig. 1, Supplementary Table S2). At ASV level, transit through the earthworm gut significantly reduced the relative abundance of 49 bacterial ASVs and increased the relative abundance of 54 bacterial ASVs (Supplementary Table S3).

The bacterial community of the vermicompost was dominated by the phyla Actinobacteria, Bacteroidota, Gemmatimonadota, Proteobacteria, Sumerlaeota and Verrucomicrobiota (Fig. 1). The cast-associated processes (CAP) of the vermicomposting significantly increased the abundance of Actinobacteriota, Gemmatimonadota and Sumerlaeota, and reduced the abundance of Verrucomicrobiota, Bacteroidota and Proteiobacteria (Fig. 1, Supplementary Table S1). At the genus level, CAP processes of vermicomposting resulted in the increase of the abundance of the bacterial genera Alcanivorax, Sumerlaea, Arenibacter, Nitrosomonas among others, and significantly reduced the abundance of the bacterial genera Dyadobacter, Aeromonas, Brevundimonas, Chryseobacterium, Sphingomonas, Acinetobacter and Devosia among others (Fig. 1, Supplementary Table S2). At ASV level, CAP processes of vermicomposting significantly increased the relative abundance of 40 ASVs and reduced the relative abundance of 15 ASVs (Supplementary Table S3).

The fungal community of the sewage sludge was almost exclusively dominated by the phylum Basidiomycota (Fig. 2). Large changes in fungal community composition were found after transit of the sewage sludge through the gut of the earthworms (GAP), with significant decreases in the abundance of Basidiomycota and significant increases in the abundance of Ascomycota and Mortierellomycota (Fig. 2, Supplementary Table S1).

At the genus level, transit through the gut significantly decreased the abundance of Apiotrichum, Candida, Kazachstania and Trichosporon (Fig. 2, Supplementary Table S2) and increased significantly the abundance of the fungal genera Clonostachys, Fusarium, Coprinus, Humicola, Paracremonium and Mortierella among others (Fig. 2, Supplementary Table S2). At ASV level, transit through the gut significantly reduced the relative abundance of 25 fungal ASVs and increased the relative abundance of 105 bacterial ASVs (Supplementary Table S3).

The fungal community of the vermicompost was composed of the same phyla as the fresh casts. CAP processes of vermicomposting significantly increased the abundance of the phyla Blastocladiomycota and Mortierellomycota (Fig. 2, Supplementary Table S1). At the genus level, CAP processes of vermicomposting resulted in the increase of the abundance of Debaryomyces, Mortierella, Cephaliophora, Scedosporium and Trichosporon, and significantly reduced the abundance of Scutellinia, Apiotrichum, Paracremonium and Boubovia (Fig. 2, Supplementary Table S2). At ASV level, CAP processes of vermicomposting significantly increased the relative abundance of 74 fungal ASVs and reduced the relative abundance of 91 fungal ASVs (Supplementary Table S3).

Our results partially agree with our previous findings about the vermicomposting of green wastes, where the bacterial composition of the starting materials changed into a vermicompost dominated mainly by Proteobacteria, Bacteroidota, Actinobacteriota and Verrucomicrobiota^12,18,19,20. Regarding fungi, few studies have characterized fungal biodiversity in compost and vermicompost; the majority of the most abundant fungal genera found in this study were not described in previous studies^21,22. As with bacteria, fungal composition of cast and vermicompost was radically different from those of sewage sludge.

These results highlight how deeply vermicomposting modifies bacterial and fungal microbiotas of sewage sludge and demonstrates the critical effect of earthworm gut associated processes. These changes of the microbiota produced by earthworm activity were postulated as the main reasons for the mitigation of antibiotic resistance genes (ARGs) during vermicomposting of sewage sludge^23,24,25.

Bacterial and fungal α- and β-diversity change during vermicomposting of sewage sludge

Bacterial α-diversity decreased moderately in sewage sludge after transit through the earthworm gut (GAP), with significant decreases in ASV richness, Faith phylogenetic diversity (Fig. 3) and Chao1 richness (Supplementary Fig. S1). The cast-associated processes (CAP) of vermicomposting slightly increased bacterial α-diversity, with significant increases in Faith phylogenetic diversity (Fig. 3).

Fungal α-diversity in the sewage sludge increased greatly after transit through the earthworm gut (GAP), with significant increases in fungal ASV richness, inverse Simpson diversity (Fig. 3) and Chao1 richness (Supplementary Fig. S1). The cast-associated processes (CAP) of vermicomposting decreased fungal α-diversity, with significant decreases in richness and diversity (Fig. 3).

Our results disagree with previous findings about the vermicomposting of green wastes, including Scotch broom and different types of grape marc, where bacterial α-diversity increased with vermicomposting^{12,18,19,20,26}. In those studies, the starting material had not previously been processed by an animal gut, and bacterial diversity was low. Here, vermicomposting of sewage sludge, a material already processed by the human gut and therefore more microbially rich, reduced its bacterial diversity due to the earthworm gut-associated processes. These findings indicate that microbial succession during vermicomposting is strongly influenced by the starting substrate. On this regard, and since sewage sludge is highly variable due to its heterogeneous nature and the different methodologies applied in wastewater treatment plants, it would be necessary to verify the performance and magnitude of the vermicomposting process on different types of sludge and or biosolids.

Changes in bacterial and fungal α-diversity of sewage sludge during vermicomposting were accompanied by drastic changes in bacterial and fungal β-diversity during both GAP and CAP associated processes (Fig. 4). Thus, bacterial and fungal communities of sewage sludge, fresh earthworm casts and vermicompost (3 months old) were all significantly different in PCoA 1 and PCoA 2 for Bray–Curtis, Jaccard (Fig. 4) and weighted and unweighted UniFrac distance matrices (Supplementary Fig. S2).

Only 6 bacterial and fungal ASVs were shared or present in sewage sludge, fresh earthworm casts and vermicompost (Fig. 5a,c, Supplementary Tables S4–S5). This suggests that vermicomposting eliminates 96% of the initial bacterial ASVs and 91% of the initial fungal ASVs as sludge passes through the earthworm gut, the GAP processes of the vermicomposting process (Fig. 5b,d). The CAP processes increased the diversity of the bacterial community and decreased the diversity of the fungal community of the vermicompost derived from sewage sludge (Fig. 5a,c, Supplementary Tables S4, S5). According with the observed differences in β-diversity, bacterial and fungal communities of sewage sludge, fresh earthworm casts and vermicompost were largely composed by their own or exclusive ASVs (Fig. 5, Supplementary Tables S4, S5).

Several studies have found important levels of reduction of microbial human pathogens during vermicomposting of sewage sludge^27,28,29 and animal manures^30,31. We have found that earthworm activity, mainly during the gut-associated processes, is a critical factor leading to the rapid reduction of pathogens during vermicomposting.

The mechanisms involved in the reduction or elimination of the bacterial and fungal taxa in the sewage sludge may include direct effects of physical disruption during grinding in the earthworm gizzard, microbial inhibition by antimicrobial substances or microbial antagonists produced by the earthworms themselves, and destruction of microorganisms by enzymatic digestion and assimilation¹⁵.

This underscores the critical importance of maintaining vermicomposting reactors at the highest possible stocking densities or at maximum charge capacity, so optimal operation or performance of the vermicomposting process is ensured.

This study describes how vermicomposting drastically modifies bacterial and fungal communities of sewage sludge and stresses the critical effect of earthworms during that process. Bacterial and fungal composition and structure changes significantly during gut-associated processes (GAP) and cast-associated processes (CAP). Most of the microbial taxa present in the sewage sludge were eliminated during vermicomposting, mainly in the GAP. Given that earthworms change drastically microbial communities of the organic wastes during vermicomposting and vermicompost microbiome resembles the microbial communities of the earthworm gut, studying the effect of the starting material in the configuration of the earthworm gut microbiome is paramount.

Source: Ecology - nature.com