Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRNA analysis were checked giving special attention to the approach used for high throughput sequencing. More specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing and shotgun DNA underestimated the Methanoculleus genus, probably due to the low 16S rRNA gene copy number encoded in this taxon

Performance and genome-centric metagenomics of thermophilic single and two-stage anaerobic digesters treating cheese wastes

The present research is the first comprehensive study regarding the thermophilic anaerobic degradation of cheese wastewater, which combines the evaluation of different reactor configurations (i.e. single and two-stage continuous stirred tank reactors) on the process efficiency and the in-depth characterization of the microbial community structure using genome-centric metagenomics. Both reactor configurations showed acidification problems under the tested organic loading rates (OLRs) of 3.6 and 2.4 g COD/L-reactor day and the hydraulic retention time (HRT) of 15 days. However, the two-stage design reached a methane yield equal to 95% of the theoretical value, in contrast with the single stage configuration, which reached a maximum of 33% of the theoretical methane yield. The metagenomic analysis identified 22 new population genomes and revealed that the microbial compositions between the two configurations were remarkably different, demonstrating a higher methanogenic biodiversity in the two-stage configuration. In fact, the acidogenic reactor of the serial configuration was almost solely composed by the lactose degrader Bifidobacterium crudilactis UC0001. The predictive functional analyses of the main population genomes highlighted specific metabolic pathways responsible for the AD process and the mechanisms of main intermediates production. Particularly, the acetate accumulation experienced by the single stage configuration was mainly correlated to the low abundant syntrophic acetate oxidizer Tepidanaerobacter acetatoxydans UC0018 and to the absence of aceticlastic methanogens

Converting mesophilic upflow sludge blanket (UASB) reactors to thermophilic by applying axenic methanogenic culture bioaugmentation

The application of thermophilic conditions in anaerobic digesters leads to higher methane production rates and better sanitation of the effluents compared to mesophilic operation. However, an increase in operational temperature is challenging due to the tremendous selective pressure imposed on the microbial consortium. The adaptation of microbial community to a new environment or condition can be accelerated by a process known as \u201cbioaugmentation\u201d or \u201cmicrobial community manipulation\u201d, during which exogenous microorganisms harbouring specific metabolic activities are introduced to the reactor. The aim of the current study was to rapidly convert the operational temperature of up-flow anaerobic sludge blanket (UASB) reactors from mesophilic to thermophilic conditions by applying microbial community manipulation techniques. Three different bioaugmentation strategies were compared and it was proven that the injection of axenic methanogenic culture was the most efficient approach leading to improved biomethanation process with 40% higher methane production rate compared to the control reactor. Microbial community analyses revealed that during bioaugmentation, the exogenous hydrogenotrophic methanogen could be encapsulated in granular structures and concomitantly promote the growth of syntrophic fatty acid oxidizing bacteria. The results derived from the current study indicated that microbial community manipulation is an efficient alternative method to speed up transition of UASB reactors from mesophilic to thermophilic conditions