Anaerobic co-digestion (AcoD) is a promising strategy to increase the methane production of anaerobic digestion plants treating wastewater sludge (WAS). In this work the degradability of six different mixtures of WAS with fish waste (FW) or garden-grass (GG) was evaluated and compared to the three mono-digestions. Degradation performances and methanogenic 2 pathways, determined with the isotopic signatures of biogas, were compared across time. Fish and grass mono-digestion provided a higher final methane production than WAS monodigestion. In co-digestion the addition of 25 % of fish was enough to increase the final methane production from WAS while 50 % of grass was necessary. To determine the optimal blend of WAS co-digestion two indicators were specifically designed, representing the maximum potential production (ODI) and the expected production in mono-digestion conditions (MDI). The comparison between these indicators and the experimental results showed that the most productive blend was composed of 75% of co-substrate, fish or grass, with WAS. Indeed, the final methane production was increased by 1.9 times with fish and by 1.7 times with grass associated to an increase of the methane production rate by 1.5 times.Even if the same succession of methanogenic pathways across time was observed for the different mixtures, their relative proportions were different. Sewage sludge degradation was mostly achieved through hydrogenotrophic pathway as confirmed by the archaeal analysis while acetoclastic archaea were identified for fish and grass degradation.

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Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two continuous stirred tank reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After 3 months of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein-inferred and 16S ribosomal DNA tags based taxonomic community profiles were not consistent. PCA however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow-derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4 × 10(6) reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (11 vs. 4% of spectral identification rate for the combined samples). Surprisingly, a high proportion of proteins from members of the "Candidatus Competibacter" group, a key microbial player usually found in activated sludge plants, was retrieved in our anaerobic digester samples. Data are available via ProteomeXchange with identifier PXD002420 (http://proteomecentral.proteomexchange.org/dataset/PXD002420).