The study of viruses of Methanosaeta, a key acetoclastic methanogen for digestion process stability

[[abstract]]Stable digestion processes is important for wastewater treatment plants to treat organic solid waste produced from primary and secondary sludge. The stability of the digestion process has greatly improved but process failure still occurs. Toxicants and inadequate operation have been demonstrated to cause failure in some cases. However, in some other cases, there is no obvious explanation for observed upsets. Digestion failure is usually indicated by a decreased rate of methane production, accumulation of acetic acid, and decreased pH, which suggest that the primary consumer of acetate in most mesophilic anaerobic digesters, Methanosaeta, has been lost. In natural ecosystems, viruses can target the most rapid growing populations ("killing the winner" theory) and influence the composition of microbial communities. The most dominant methanogenic Archaea, Methanosaeta in mesophilic digesters, appear to be a favorable target for virus attack. This virus-host pair may provide a mechanism for many observed digestion process upsets. No virus of Methanosaeta has been reported previously and the cultivation of Methanosaeta is challenging due to the requirement of strict anaerobic conditions and their slow growth rate. The growth of Methanosaeta concilii was improved using modified DSM 334 solid media. Methanosaeta concilii DSM 6752 colonies appeared on the surface of media (0.5% of gellan) after one month of incubation in serum bottles at 35oC using spread plating. By pour plating (0.5% of gellan), growth of Methanosaeta was found after about one week incubation at 35oC. In addition, ammonia toxicity to Methanosaeta concilii DSM 6752 in liquid media was also investigated. No inhibition of the methane production when the concentration of ammonia-nitrogen was below 16.8 mg/L. The suppression of methane yield (day 4.8) was 15.6%, 39.6%, and 51.3% at free ammonia-nitrogen of 28.7 mg/L, 47.9 mg/L, and 71.2 mg/L, respectively. Viruses in two lab-scale reactors (the daily-fed and hourly-fed reactors) dominated by Methanosaeta were investigated because Methanosaeta viruses were possibly present. All of the observed VLPs were of head-tailed morphology but virus populations between the two reactors were distinct. VBR values were low in both the daily-fed reactor (0.123) and the hourly-fed reactor (0.093) suggesting that lysogenic or pseudolysogenic life cycles could be the favorable strategy for viruses targeting slow-growing hosts in the methanogenic environment. The presence of many VLPs with 60 nm capsid and 120 nm tail or viruses having the same genome sizes (30 kbp) in both reactors may suggest they target a common host such as Methanosaeta, Proteobacteria or Firmicutes. The connection between viruses and Methanosaeta was studied using a genomic approach based on clustered regularly interspaced short palindromic repeats (CRISPRs). Methanosaeta spacers matched viral contigs were found in the two reactors (daily-fed and hourly-fed reactors) and two digesters (West Point and South Plant), suggesting that the Methanosaeta viruses might still active in these systems. Presence of various phage proteins in contigs targeted by Methanosaeta spacers suggests these sequences are originated from Methanosaeta viruses. The connection between viruses and Methanosaeta is further confirmed by finding conserved PAMs on viral contigs targeted by both CRISPR/Cas systems.[[notice]]補正完