A metatranscriptomic exploration of fungal and bacterial contributions to allochthonous leaf litter decomposition in the streambed

Abstract

The decomposition of organic matter is essential for sustaining the health of freshwater ecosystems by enabling nutrient recycling, sustaining food webs, and shaping habitat conditions, which collectively enhance ecosystem resilience and productivity. Bacteria and fungi play a crucial role in this process by breaking down coarse particulate organic matter (CPOM), such as leaf litter, into nutrients available for other organisms. However, the specific contribution of bacteria and their functional interactions with fungi in freshwater sediments have yet to be thoroughly explored. In the following study, we enriched organic matter through the addition of alder (Alnus glutinosa) leaves into artificial stream channels (AquaFlow mesocosms). We then investigated enzyme expression, metabolic pathways, and community composition of fungi and bacteria involved in the degradation of CPOM through metatranscriptomics and amplicon sequencing. Enzymes involved in the degradation of lignin, cellulose, and hemicellulose were selectively upregulated with increased organic matter. Analysis of ITS and 16S rRNA gene sequences revealed that during decomposition, fungal communities were predominantly composed of Basidiomycota and Ascomycota, while bacterial communities were largely dominated by Pseudomonadota and Bacteroidota. The similar gene expression patterns of CPOM degradation related enzymes observed between bacteria and fungi indicate potential functional interaction between these microbial groups. This correlation in enzyme expression may indicate that bacteria and fungi are jointly involved in the breakdown of coarse particulate organic matter, potentially through mutualistic interaction. This study uncovers the specific enzymatic activities of bacteria and fungi and the importance of microbial interactions in organic matter decomposition, revealing their central role in facilitating nutrient cycling and maintaining the ecological health and stability of freshwater ecosystems.

Keywords: Coarse perticulate organic matter; Enzyme activity; Freshwater ecosystem health; Metatranscriptomics; Microbial interactions; Sequencing.

Figure 1. Alpha diversity of all KO profiles expression over time for (A) bacteria and (B) fungi in sediment samples.

The significance between control (dark green) and treatment (dark raspberry) was tested using ANOVA. Asterisks indicate the significance levels of the ANOVA test with (ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01). The x-axis represents the time period in days, and the y-axis represents the Shannon index.

Figure 2. Temporal patterns for KO expressions of (A) bacteria and (B) fungi from clusters only with ascending expressions in sediment samples.

KO expressions were normalised (rpkm) and used to perform cmeans clustering over time. All KOs without changes were removed using filtering within TCseq. The x-axis shows time points over 10 days and the y-axis represents normalised KO expressions (z-scores). Membership values indicate the degree to which data points belong to a cluster.

Figure 3. Mean expression ratio of CPOM related enzymes such as K01179 (cellulase), K01186 (exo-alpha-sialidase), K15920 (xylan 14-beta-xylosidase), K01728 (pectate lyase) and K12111 (beta-galactosidase) in between control and treatment samples.

The gene expression ratio of listed enzymes was calculated between control and treatment and then the mean expression ratio was plotted for each day.

Figure 4. The MA plot displays the mean expression (A) vs. the log-fold change (M) for significant KEGG Orthologs related to CPOM degradation in (A) bacteria and (B) fungi.

The x-axis represents the log2 mean expression and the y-axis indicates the log2 fold change of KOs. A log-fold change greater than 2 and an adjusted p-value of 0.05 or above are considered upregulated (Up), whereas values below this threshold are deemed non-significant (NS).

Figure 5. Relative abundances of (A) bacterial and (B) fungal phyla for control and treatment samples.

The X-axis represents the percent relative abundance of the top 15 phyla, while the Y-axis indicates the conditions (control and treatment).