Abstract
Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea are essential for methane production within AD microbial communities. Methanogens are a phylogenetically diverse group that can be classified into three metabolically distinct lineages based on the substrates they use to produce methane. While process optimization based on physicochemical parameters is well established in AD, measurements that could allow manipulation of the underlying microbial community are seldom used as they tend to be non-specific, expensive, or time-consuming, or a combination of all three. Loop-mediated isothermal amplification (LAMP) assays combine a simple, rapid, low-cost detection technique with high sensitivity and specificity. Here, we describe the optimization of LAMP assays for the detection of four different genera of hydrogenotrophic methanogens: Methanoculleus, Methanothermobacter, Methanococcus, and Methanobrevibacter spp. By targeting archaeal elongation factor 2 (aEF2), these LAMP assays provide a rapid, low-cost, presence/absence indication of hydrogenotrophic methanogens that could be used as a real-time measure of process conditions. The assays were shown to be sensitive to 1 pg of DNA from most tested methanogen species, providing a route to a quantitative measure through simple serial dilution of samples. The LAMP assays described here offer a simple, fast, and affordable method for the specific detection of four different genera of hydrogenotrophic methanogens. Our results indicate that this approach could be developed into a quantitative measure that could provide rapid, low-cost insight into the functioning and optimization of AD and related systems.
Highlights
In addition to its vital and well-established role in stabilization of wastewater biosolids, anaerobic digestion (AD) is increasingly used for resource recovery from a wide variety of agri- and food-processing wastes and industrial effluents
We confirmed that the archaeal elongation factor 2 (aEF-2) gene was a good Loop-mediated isothermal amplification (LAMP) assay candidate by comparing these results to similar searches that we performed for other potential gene targets including the following genes involved in methanogenesis pathways: methyl-CoA reductase, methylene tetrahydromethanopterin reductase, energy-converting hydrogenase A, the 30S ribosomal protein S28e, and 50S ribosomal protein L44e (Table 2)
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Summary
In addition to its vital and well-established role in stabilization of wastewater biosolids, anaerobic digestion (AD) is increasingly used for resource recovery from a wide variety of agri- and food-processing wastes and industrial effluents. Hydrolysis of polymers leads, via a cascade of metabolic pathways, to the production of small molecules and to carbon dioxide and methane, the main energy-carrying component of biogas, and a nutrient-rich digestate residue [1] This process is mediated by hundreds of different interacting microbial species. This is compounded by our inability to grow many species in isolation [10] Molecular methods such as PCR-based techniques (including qPCR) are sensitive but require expensive equipment and reagents and take a number of hours. Approaches such as 16S rRNA amplicon or whole metagenome high-throughput sequencing provide excellent specificity, but are expensive, slow. Conserved regions of genes were identified by visual inspection of the alignment and selected based on a threshold of >80% identity per nucleotide position to establish nucleotide consensus sequences (Figure S1)
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