ABSTRACT
The preservation of nucleic acids for high-throughput sequencing is an ongoing challenge for field scientists. In particular, samples that are low biomass, or that have to be collected and preserved in logistically challenging environments (such as remote sites or during long sampling campaigns) can pose exceptional difficulties. With this work, we compare and assess the effectiveness of three preservation methods for DNA and RNA extracted from microbial communities of glacial snow and ice samples. Snow and ice samples were melted and filtered upon collection in Iceland, and filters were preserved using: (i) liquid nitrogen flash freezing, (ii) storage in RNAlater, or (iii) storage in Zymo DNA/RNA Shield. Comparative statistics covering nucleic acid recovery, sequencing library preparation, genome assembly, and taxonomic diversity were used to determine best practices for the preservation of DNA and RNA samples from these environments. Our results reveal that microbial community composition based on DNA was comparable at the class level across preservation types. Based on extracted RNA, the taxonomic composition of the active community was primarily driven by the filtered sample volume (i.e., biomass content). In low biomass samples (where <200 ml of sample volume was filtered) the taxonomic and functional signatures trend toward the composition of the control samples, while in samples where a larger volume (more biomass) was filtered our data showed comparable results independent of preservation type. Based on all comparisons our data suggests that flash freezing of filters containing low biomass is the preferred method for preserving DNA and RNA (notwithstanding the difficulties of accessing liquid nitrogen in remote glacial field sites). Generally, RNAlater and Zymo DNA/RNA Shield solutions work comparably well, especially for DNA from high biomass samples, but Zymo DNA/RNA Shield is favored due to its higher yield of preserved RNA. Biomass quantity from snow and ice samples appears to be the most important factor in regards to the collection and preservation of samples from glacial environments.
ABSTRACT
Anaerobic digestion can produce biogas as an eco-friendly energy source, driven by a microbial community-dependent process and, as such, suffer influences from many biotic and abiotic factors. Understanding the players and how they interact, the mechanisms involved, what the factors are, and how they influence the biogas process and production is an important way to better control it and make it more efficient. Metagenomic approach is a powerful tool to assess microbial diversity and further, allow correlating changes in microbial communities with multiple factors in virtually all environments. In the present study, we used metagenomic approach to assess microbial community structure changes in two biodigesters, differing in their biogas production capacity, architecture, and feed. A total of 1,440,096 reads of the 16S rRNA gene V4 region were obtained and analyzed. The main bacterial phyla were Firmicutes and Bacteroidetes in both biodigesters, but the biodiversity was greater in the Upflow Anaerobic Sludge Blanket (UASB) reactor fed with bovine manure than in the Continuous Stirred Tank Reactor (CSTR) fed with swine manure, which also correlated with an increase in biogas or methane production. Microbial community structure associated with biodigesters changed seasonally and depended on animal growth stage. Random forest algorithm analysis revealed key microbial taxa for each biodigester. Candidatus Cloacomonas, Methanospirillum, and Methanosphaera were the marker taxa for UASB and the archaea groups Methanobrevibacter and Candidatus Methanoplasma were the marker taxa for CSTR. A high abundance of Candidatus Methanoplasma and Marinimicrobia SAR406 clade suggested lower increments in methane production. Network analysis pointed to negative and positive associations and specific key groups, essential in maintaining the anaerobic digestion (AD) process, as being uncultured Parcubacteria bacteria, Candidatus Cloacomonas, and Candidatus Methanoplasma groups, whose functions in AD require investigation.
