Size Shapes the Active Microbiome of Methanogenic Granules, Corroborating a Bioﬁlm Life Cycle

Methanogenic archaea are key players in cycling organic matter in nature but also in engineered waste treatment systems, where they generate methane, which can be used as a renewable energy source. In such systems in the built environment, complex methanogenic consortia are known to aggregate into highly organized, spheri-cal granular bioﬁlms comprising the interdependent microbial trophic groups mediating the successive stages of the anaerobic digestion (AD) process. This study separated methanogenic granules into a range of discrete size fractions, hypothesizing different bioﬁlm growth stages, and separately supplied each with speciﬁc substrates to stimulate the activity of key AD trophic groups, including syntrophic acid oxidizers and methanogens. Rates of speciﬁc methanogenic activity were measured, and amplicon sequencing of 16S rRNA gene transcripts was used to resolve phylotranscriptomes across the series of size fractions. Increased rates of methane production were observed in each of the size fractions when hydrogen was supplied as the substrate compared with those of volatile fatty acids (acetate, propionate, and butyrate). This was connected to a shift toward hydrogenotrophic methanogenesis dominated by Methanobacterium and Methanolinea . Interestingly, the speciﬁc active microbiomes measured in this way indicated that size was signiﬁcantly more important than substrate in driving the structure of the active community in granules. Multivariate integration studywise discriminant analysis identiﬁed 56 genera shaping changes in the active community across both substrate and size. Half of those were found to be upregulated in the medium-sized granules, which were also the most active and potentially of the most important size, or life stage, for preci-sion management of AD systems. IMPORTANCE Biological wastewater conversion processes collectively constitute one of the single biggest worldwide applications of microbial communities. There is an obvious requirement, therefore, to study the microbial systems central to the suc-cess of such technologies. Methanogenic granules, in particular, are architecturally fascinating bioﬁlms that facilitate highly organized cooperation within the metabolic network of the anaerobic digestion (AD) process and, thus, are especially intriguing model systems for microbial ecology. This study, in a way not previously reported, provoked syntrophic and methanogenic activity and the structure of the microbial community, using speciﬁc substrates targeting the key trophic groups in AD. Unex-pectedly, granule size more strongly than substrate shaped the active portion of the microbial community. Importantly, the ﬁndings suggest the size, or age, of granules inherently shapes the active microbiome linked to a life cycle. This provides exciting insights into the function of, and the potential for additional modeling of bioﬁlm development in, methanogenic granules. active microbiome assays indicate that size, more than substrate, shapes the microbial community of methanogenic granules, corroborating a bioﬁlm life-cycle in anaerobic digesters SMA and biomass sampling. SMA assays were set up, in triplicate, to determine the maximum rate of methane production in biomass from each of the nine size fractions, A to I, against each of acetate, propionate, butyrate, and H 2 /CO 2 and including no-added-substrate (or N 2 /CO 2 in the case of negative controls for H 2 /CO 2 ) controls ( n (cid:2) 162) by following previously described protocols (20, 21). Biogas production was monitored daily using a handheld pressure transducer (Centrepoint Electronics, Inc., Galway, Ireland). Separately, and once the SMA assays were concluded, each different incubation condition was set up again, but this time with a no-added-substrate control incubation for each condition for each of the size fractions ( n (cid:2) 72) to facilitate sacriﬁcial biomass sampling at one time point during the exponential phase of biogas production. Biomass was immediately ﬂash-frozen in liquid N 2 and stored at –80°C, while the liquid samples were stored for volatile fatty acid (VFA) analysis. VFA analysis. VFA concentration proﬁles were determined for acetic, propionic, butyric, isobutyric, valeric, isovaleric, caproic, and isocaproic acids in the samples using a gas chromatograph equipped with a Combi PAL autosampler system (Varian, Inc., Walnut Creek, CA) and a 25-m-long CP-WAX 58 (FFAP) capillary column (SGE Analytical Sciences) with an internal diameter of 0.32 mm and 0.2- (cid:2) m ﬁlm thickness. Liquid samples in 1-ml aliquots were injected into the front injector (port type 1177) at 250°C. ﬂow of 1 ml min under a constant ﬂow and pressure 0.1 lb/in . The oven temperature 60°C ﬁrst 0.1 min, 100°C 1.17 min, and 200°C 12.97 VFA a ﬂame ionization (FID) 300°C and identiﬁed and