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

Foaming in manure based digesters: Causes and solutions

Anaerobic digestion foaming is one of the major problems that occasionally occurred in the Danish full-scale biogas plants, affecting negatively the overall digestion process. The foam is typically formatted in the main biogas reactor or in the pre-storage tank and the entrapped solids in the foam cause severe operational problems, such as blockage of mixing devices, and collapse of pumps. Furthermore, the foaming problem is linked with economic consequences for biogas plants, due to income losses derived from the reduced biogas production, extra labour work and additional maintenance costs. Moreover, foaming presents adverse environmental impacts owing to the overflowing of the pre-storage or digester tanks. So far, there has never been thoroughly investigation of foaming problem in manure-based digester, which is the main anaerobic digestion applied in Denmark. The purpose of the present study was to identify potential causes of foaming in manure based digesters. Moreover, it was also an aim to investigate possible solutions to counteract foam formation with the use of antifoam agents. Thus, the impact of organic loading rate and content of feeding substrate on anaerobic digestion foaming was studied in continuous mode experiments. Two sets of treatments were examined in duplicate using 5 continuous stirred tank reactors (working volume 1.5L), operating in thermophilic conditions. Two replicate reactors were fed with cattle manure and gelatine, as a representative of proteins, while the other two replicates were fed with cattle manure and Na-oleate, as a representative of lipids. One reactor was kept as a control and was fed only with cattle manure. The experiment was divided in 5 periods. During the 1st, 3rd and 5th period the organic loading rate of all reactors was increased by the addition of glucose in the feeding substrate. During the 2nd and 4th period the organic loading rate was maintained constant, but instead of glucose, higher concentration of Na-oleate or gelatine was added in the feeding substrate. The results obtained from the above experiment showed that the organic loading rate has a significant impact on foam formation, lowering the methane yield of the reactor. Moreover, it was found that an increase in gelatine concentration does not promote foam, while an increase in Na-oleate concentration enhances stable foam. Based on the above results, a new experiment was designed, where the antifoam efficiency of different commercial and non-commercial compounds, was investigated. The antifoam potential of the compounds was determined by aeration method. The apparatus comprised of a glass cylinder with a diffuser placed at the bottom. A 50 mL sample, derived from a foaming reactor, was aerated in the cylinder with an air flow rate of 60 mL/min for 10 minutes. After that, the aeration was repeated adding different concentrations of antifoam solutions in the sample. The foam height in the cylinder was measured as soon as the aeration was stopped and again 1 hour later. The antifoam potential was defined using two parameters: foaming tendency and foam stability. Foaming tendency (mL-foam/(mL-air·min)) was calculated from the volume of foam (mL) right after aeration divided by air flow rate (mL/min). Foam stability was determined as percentage of foam remaining in the cylinder 1 h after aeration compared to the volume of foam right after aeration

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