Abstract
BackgroundAnaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage two-phase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures. To follow the adaptive response of the biomass-attached cellulolytic/hydrolytic biofilms at increasing ammonium/ammonia contents, genome-centered metagenomics and transcriptional profiling based on metagenome assembled genomes (MAGs) were conducted.ResultsIn total, 78 bacterial and archaeal MAGs representing the most abundant members of the communities, and featuring defined quality criteria were selected and characterized in detail. Determination of MAG abundances under the tested conditions by mapping of the obtained metagenome sequence reads to the MAGs revealed that MAG abundance profiles were mainly shaped by the temperature but also by the OLR. However, the OLR effect was more pronounced for the mesophilic systems as compared to the thermophilic ones. In contrast, metatranscriptome mapping to MAGs subsequently normalized to MAG abundances showed that under thermophilic conditions, MAGs respond to increased OLRs by shifting their transcriptional activities mainly without adjusting their proliferation rates. This is a clear difference compared to the behavior of the microbiome under mesophilic conditions. Here, the response to increased OLRs involved adjusting of proliferation rates and corresponding transcriptional activities. The analysis led to the identification of MAGs positively responding to increased OLRs. The most outstanding MAGs in this regard, obviously well adapted to higher OLRs and/or associated conditions, were assigned to the order Clostridiales (Acetivibrio sp.) for the mesophilic biofilm and the orders Bacteroidales (Prevotella sp. and an unknown species), Lachnospirales (Herbinix sp. and Kineothrix sp.) and Clostridiales (Clostridium sp.) for the thermophilic biofilm. Genome-based metabolic reconstruction and transcriptional profiling revealed that positively responding MAGs mainly are involved in hydrolysis of grass silage, acidogenesis and / or acetogenesis.ConclusionsAn integrated -omics approach enabled the identification of new AD biofilm keystone species featuring outstanding performance under stress conditions such as increased OLRs. Genome-based knowledge on the metabolic potential and transcriptional activity of responsive microbiome members will contribute to the development of improved microbiological AD management strategies for biomethanation of renewable biomass.
Highlights
Anaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage twophase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures
An integrated -omics approach enabled the identification of new AD biofilm keystone species featuring outstanding performance under stress conditions such as increased OLRs
The summarized average biogas yields from hydrolytic reactors (HR) and anaerobic filters (AF) were constantly 601 ± 18 l normalized for standard temperature and pressure (LN) per kg volatile substances (VS) (M1) and 599 ± 26 Liter normalized (LN) kilogram volatile substances (kgVS)− 1 (M2) for the reactor systems operated under mesophilic temperature regime with an average methane content of 56 ± 2% (v/v) (Fig. 2)
Summary
Anaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage twophase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures. To digest crop biomass as sole substrate and at high organic loading rates (OLRs), specially designed biogas reactors are advantageous such as staged degradation step (‘phase’) separated reactor systems consisting of a cellulolysis / hydrolysis fermenter and a downstream methanogenesis reactor. Crop biomass is colonized by a cellulolytic biofilm which composition varies with the abiotic environmental conditions, as example, the process temperature, and relies on the physico-chemical characteristics of crop material and process liquids [7]. Once the biofilm is established, it functions as a cooperative consortium leading to enhanced biomass degradation and, in consequence, to biogas production [8, 9]
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