Targeting Bacteria and Methanogens To Understand the Role of Residual Slurry as an Inoculant in Stored Liquid Dairy Manure

Abstract

Microbial communities in residual slurry left after removal of stored liquid dairy manure have been presumed to increase methane emission during new storage, but these microbes have not been studied. While actual manure storage tanks are filled gradually, pilot- and farm-scale studies on methane emissions from such systems often use a batch approach. In this study, six pilot-scale outdoor storage tanks with (10% and 20%) and without residual slurry were filled (gradually or in batch) with fresh dairy manure, and methane and methanogenic and bacterial communities were studied during 120 days of storage. Regardless of filling type, increased residual slurry levels resulted in higher abundance of methanogens and bacteria after 65 days of storage. However, stronger correlation between methanogen abundance and methane flux was observed in gradually filled tanks. Despite some variations in the diversity of methanogens or bacteria with the presence of residual slurry, core phylotypes were not impacted. In all samples, the phylum Firmicutes predominated (∼57 to 70%) bacteria: >90% were members of Clostridia . Methanocorpusculum dominated (∼57 to 88%) archaeal phylotypes, while Methanosarcina gradually increased with storage time. During peak flux of methane, Methanosarcina was the major player in methane production. The results suggest that increased levels of residual slurry have little impact on the dominant methanogenic or bacterial phylotypes, but large population sizes of these organisms may result in increased methane flux during the initial phases of storage. IMPORTANCE Methane is the major greenhouse gas emitted from stored liquid dairy manure. Residual slurry left after removal of stored manure from tanks has been implicated in increasing methane emissions in new storages, and well-adapted microbial communities in it are the drivers of the increase. Linking methane flux to the abundance, diversity, and activity of microbial communities in stored slurries with different levels of residual slurry can help to improve the mitigation strategy. Mesoscale and lab-scale studies conducted so far on methane flux from manure storage systems used batch-filled tanks, while the actual condition in many farms involves gradual filling. Hence, this study provides important information toward determining levels of residual slurry that result in significant reduction of well-adapted microbial communities prior to storage, thereby reducing methane emissions from manure storage tanks filled under farm conditions.

Reduction in Methane Emissions From Acidified Dairy Slurry Is Related to Inhibition of Methanosarcina Species

“…Throughout the storage period, CH 4 fluxes from acidified slurries remained low, consistent with levels observed during the lag phase (the first 20 days of storage, Figure 1C ). The lag phase observed in the current study was also observed in our previous studies that did not involve acidification ( Habtewold et al, 2018 ). A previous study from our lab has linked shifts in methanogens to methane emissions ( Habtewold et al, 2018 ), therefore we predicted that the communities would shift after acidification.…”

“…The lag phase observed in the current study was also observed in our previous studies that did not involve acidification ( Habtewold et al, 2018 ). A previous study from our lab has linked shifts in methanogens to methane emissions ( Habtewold et al, 2018 ), therefore we predicted that the communities would shift after acidification. Using qPCR-based quantification and deep sequencing (Illumina MiSeq) of phylogenetic and functional marker genes and transcripts, we demonstrated that slurry acidification (to pH 5.9) did not affect the community structure of most anaerobically-degrading microorganisms except methanogens closely related to the genus Methanosarcina .…”

“…With little impacts of acidification on the abundance and activity of bacteria, accumulated intermediary compounds including propionate, butyrate, and valerate could be converted into acetate by the acetogens ( Demirel and Scherer, 2008 ), making stored liquid dairy manure rich in acetate ( Barret et al, 2013 ; Habtewold et al, 2018 ). Although acetoclastic methanogenesis (using acetate as substrate) is the major contributor of CH 4 produced in many environments ( Conrad, 1999 ), CH 4 production in environments with high concentration of acetate has been found to drastically reduce as pH decline ( Van Kessel and Russell, 1996 ), although the exact mechanism is not yet clear.…”

“…In various manure related environments, culture independent investigations of bacterial and methanogenic communities often involve using phylogenetic and/or functional gene markers (e.g., 16S rRNA and mcrA genes) ( Petersen et al, 2014 ; Pandey et al, 2018 ). However, our previous study indicated that mcrA transcripts were more relevant to methane CH 4 emissions than mcrA genes ( Habtewold et al, 2018 ). Particularly with slurry acidification, where significant number of bacterial and methanogenic communities could be dormant or dead, DNA-based studies of these microbes may not reflect activities.…”

“…The large volumes of manure produced annually from intensive dairy farming operations areusually stored in slurry form ( VanderZaag et al, 2013 ), which create environments conducive to CH 4 production ( Grant et al, 2015 ; Petersen, 2018 ). To reduce CH 4 emissions from such storage systems, strategies such as reduction of aged manure (inoculants), crust development for potential aerobic CH 4 oxidation, and manure acidification using sulfuric acid (H 2 O 4 ) have been reported ( Petersen et al, 2012 ; Sommer et al, 2017 ; Habtewold et al, 2018 ). Sulfuric acid-based acidification of liquid dairy manure has primarily been used to abate ammonia (NH 3 ) emissions, but can also reduce CH 4 emissions ( Ottosen et al, 2009 ; Petersen et al, 2012 ; Fangueiro et al, 2015 ; Sommer et al, 2017 ).…”