Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach

Anaerobic digestion is an important biotechnology treatment process for conversion of waste to energy. In this study, a comparative core microbiome approach, i.e., determining taxa that are shared in functioning digesters but not shared in non-functioning digesters, was used to determine microbial taxa that could play key roles for effective anaerobic digestion. Anaerobic digester functions were impaired by adding the broad-spectrum antimicrobial triclosan (TCS) or triclocarban (TCC) at different concentrations, and the core microbiomes in both functioning and non-functioning anaerobic digesters were compared. Digesters treated with high (2500 mg/kg) or medium (450 mg/kg) TCS and high (850 mg/kg) TCC concentrations lost their function, i.e., methane production decreased, effluent volatile fatty acid concentrations increased, and pH decreased. Changes in microbial community diversity and compositions were assessed using 16S rRNA gene amplicon sequencing. Microbial richness decreased significantly in non-functioning digesters (p < 0.001). Microbial community compositions in non-functioning digesters significantly differed from those in functioning digesters (p = 0.001, ANOSIM). Microbes identified as potentially key taxa included previously known fatty acid-degrading syntrophs and amino acid-degrading syntrophs. A diverse group of syntrophs detected in this study had low relative abundance in functioning digesters, suggesting the importance of rare microbes in anaerobic digester operation. The comparative microbiome approach used in this study can be applied to other microbial systems where a community-driven biological phenomena can be observed directly.

Fujimoto, M., Carey, D. E., Zitomer, D. H., & McNamara, P. J. (2019). Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-019-09862-4

16s sequencing is one of the go to sequencing methods to complete microbiome studies. 16s sequencing involves sequencing of the 16s rRNA gene found in all bacteria and archaea. The fact that the 16s rRNA gene can be found in all prokaryotes make the 16s gene the ideal candidate to characterize the microbiome of numerous environments. Another key factor that makes the 16s gene an ideal candidate for microbiome characterization is the fact that there are both highly conserved regions and highly variable regions within the gene. The highly conserved regions within the 16s rRNA gene make great targets for PCR primers bind to and replicate thereby producing millions of copies of the targeted hypervariable region that is flanked by the highly conserved regions. The 16s rRNA gene contains 9 hypervariable regions that can be targeted in order to gain insight into the diversity amongst prokaryotes.

16s rRNA sequencing has grown in popularity over the last decade due in large part to next-generation sequencing. NGS, also known as high-throughput sequencing, has drastically reduced the cost of DNA sequencing due to its capability to sequence hundreds of samples at a time. In the past, capillary electrophoresis was able to produce ~1Mb per run, and today, with instruments such as the Illumina HiSeq, we are able to produce ~500 Gb per run. Until recently, one metric that Sanger sequencing had as an advantage over NGS instruments such as the Illumina MiSeq and Ion Torrent was read length. On average, capillary electrophoresis was able to achieve read lengths up to 1,000bp in comparison to the 400-600bp read lengths provided by Illumina and Ion Torrent. However, the PacBio Sequel now allows researchers the ability to achieve read lengths ranging from 10-30Kb. What does this mean for 16s rRNA sequencing? We can now sequence the entire 16s rRNA gene, which is approximately 1.5Kb in length. Whether your goal is to target one specific hypervarible region or the entire 16s rRNA gene, there is a NGS platform for you.

Metagenome sequencing is a term that can cause a lot of confusion, as well as bring so much joy to microbiologists around the world.  Metagenome sequencing is a sequencing method that investigates the DNA extracted from an environmental sample as a whole. Whereas certain DNA sequencing techniques will target a specific organism from a specific environment, metagenome sequencing targets all microbial organisms found in a certain environment. There are predominantly two methods used to completely metagenomics studies, 16s rRNA sequencing and Shotgun metagenome sequencing. The difference between the two sequencing methods can be found in their names. 16s rRNA sequencing, as you would expect, targets only the 16s rRNA gene, while Shotgun metagenome sequencing targets all genes present in your sample. How is this accomplished?

Shotgun metagenome sequencing involves the random shearing of all DNA present in a particular sample. These smaller fragments are sequenced on NGS platforms such the Illumina MiSeq and then reassembled. Because shotgun metagenomics is non-discriminatory, not only are you able to gain taxonomic information, similar to 16s sequencing, but you are also able to gain insight to the presence of functional genes as well. Overall, shotgun metagenome sequencing provides a more complete picture of your environmental sample. NGS platforms such as the Illumina HiSeq allow researchers to ability to sequence their metagenome samples to a much greater depth. Why is sequencing depth important? Naturally, the most abundant organisms in your sample will receive the most amounts of data, and because you are not specifically targeting certain organisms, without sufficient sequencing coverage, you run the risk of not identifying those organisms that may be underrepresented. From the human microbiome, to pond water, and to the rumen of cattle, shotgun metagenome sequencing paired with NGS platforms has the ability to take your ecological study to the next level.