328 The Potential Role of Two Red Seaweeds That Promote Anti-methanogenic Activity and Rumen Fermentation Profiles Under Laboratory Conditions

Ruminal methane (CH4) production results from carbohydrate fermentation by ruminal microbiota (methanogens) to produce CH4, volatile fatty acids (VFA), carbon dioxide (CO2), nitrous oxide (N2O), and hydrogen (H2) in a reduction pathway. The aims of this study were to assess the effects of two red seaweed (RSW) species (Asparagopsis taxiformis and A. armata; collected from Messina, Italy). The two RSW were identified by DNA barcoding and genetic data were deposited in BOLD Systems (REAPP006-21, REAPP004-21, respectively). The two RSW were used at dietary inclusion levels (0, 2, and 4% as-fed basis) in an anaerobic in vitro study (39°C for 48-h) to examine greenhouse gas (GHG) production and VFA profiles. Gases were collected using an ANKOM Gas Production system and analyzed for CH4 and N2O by gas chromatography. Asparagopsis taxiformis contained higher levels of bromoform (201 vs. 7.0 mg/kg DM), iodine (4820 vs. 3260 ppm), and crude protein (16 vs. 15.6% DM), than A. armata, which contained higher levels of acid detergent fiber (ADF; 7.7 vs. 19.0%) and neutral detergent fiber (NDF; 13.2 vs. 19.2%), respectively. RSW supplementation increased total gas, butyrate and valerate production (P < 0.01), while production of CH4 (mg/g DM), acetate (A), propionate (P), A/P ratios and in vitro dry matter digestibility (IVDMD; % DM) were reduced (P < 0.01) as RSW supplementation increased. In the presence of A. taxiformis, production of N2O (µg/g DM), tended to be less (P = 0.1) at 2% DM, but increased (P < 0.01) N2O production with A. armata at the 2 and 4% DM. Therefore, it may be possible to suppress methanogenesis both directly and indirectly by addition of RSW. To efficiently use seaweeds as feed ingredients with nutritional and environmental benefits, more research is required to determine the mechanisms underlying seaweed and dietary substrate interactions.

Anaerobic Biofilm Enriched With an Ammonia Tolerant Methanogenic Consortium to Improve Wastewater Treatment in The Fishing Industry

The digestion efficiency of liquid industrial wastes increases when using bioreactors colonized by microbial biofilms. High concentrations of proteins derived from the fish processing industry lead to the production of ammonia, which inhibits methane production. Two bioreactors were constructed to compare methanogenic activity: one enriched with mMPA consortia (control bioreactor), and the second with NH3 tolerant consortia (treatment bioreactor). Ammonia tolerant activity was assessed by applying an ammonia shock (755 mg NH3/L). Methane production, consumption of total organic carbon (TOC) and the taxonomic composition of bacteria and archaea was evaluated using 16S rDNA in the acclimatization, ammonia shock, and recovery phases. The ammonia shock significantly affected both methane production and the consumption of TOC in the control reactor (p<0.05) and taxonomical composition of the microbial consortia (OTU). These values remained constant in the treatment reactor. The analysis of biofilm composition showed a predominance of Methanosarcinaceae (Methanomethylovorans sp., and probably two different species of Methanosarcina sp.) in bioreactors. These results demonstrate that using acclimated biofilms enriched with ammonia tolerant methanogens control the inhibitory effect of ammonia on methanogenesis.

Microbial Involvement in Carbon Transformation via CH4 and CO2 in Saline Sedimentary Pool

Methane and carbon dioxide are one of the most important greenhouse gases and significant components of the carbon cycle. Biogeochemical methane transformation may occur even in the extreme conditions of deep subsurface ecosystems. This study presents methane-related biological processes in saline sediments of the Miocene Wieliczka Formation, Poland. Rock samples (W2, W3, and W4) differed in lithology (clayey salt with veins of fibrous salt and lenses of gypsum and anhydrite; siltstone and sandstone; siltstone with veins of fibrous salt and lenses of anhydrite) and the accompanying salt type (spiza salts or green salt). Microbial communities present in the Miocene strata were studied using activity measurements and high throughput sequencing. Biological activity (i.e., carbon dioxide and methane production or methane oxidation) occurred in all of the studied clayey salt and siltstone samples but mainly under water-saturated conditions. Microcosm studies performed at elevated moisture created more convenient conditions for the activity of both methanogenic and methanotrophic microorganisms than the intact sediments. This points to the fact that water activity is an important factor regulating microbial activity in saline subsurface sediments. Generally, respiration was higher in anaerobic conditions and ranged from 36 ± 2 (W2200%t.w.c) to 48 ± 4 (W3200%t.w.c) nmol CO2 gdw−1 day−1. Methanogenic activity was the highest in siltstone and sandstone (W3, 0.025 ± 0.018 nmol CH4 gdw−1 day−1), while aerobic methanotrophic activity was the highest in siltstone with salt and anhydrite (W4, 220 ± 66 nmol CH4 gdw−1 day−1). The relative abundance of CH4-utilizing microorganisms (Methylomicrobium, Methylomonas, Methylocystis) constituted 0.7–3.6% of all taxa. Methanogens were represented by Methanobacterium (0.01–0.5%). The methane-related microbes were accompanied by a significant number of unclassified microorganisms (3–64%) and those of the Bacillus genus (4.5–91%). The stable isotope composition of the CO2 and CH4 trapped in the sediments suggests that methane oxidation could have influenced δ13CCH4, especially in W3 and W4.

Hydrocarbon Toxicity towards Hydrogenotrophic Methanogens in Oily Waste Streams

Hydrocarbon-containing wastes and wastewaters are produced worldwide by the activities of the oil and gas industry. Anaerobic digestion has the potential to treat these waste streams, while recovering part of its energy potential as biogas. However, hydrocarbons are toxic compounds that may inhibit the microbial processes, and particularly the methanogens. In this work, the toxicity of hexadecane (0–30 mM) towards pure cultures of hydrogenotrophic methanogens (Methanobacterium formicicum and Methanospirillum hungatei) was assessed. Significantly lower (p < 0.05) methane production rates were only verified in the incubations with more than 15 mM hexadecane and represented up to 52% and 27% inhibition for M. formicicum and M. hungatei, respectively. The results obtained point out that 50% inhibition of the methanogenic activity would likely occur at hexadecane concentrations between 5–15 mM and >30 mM for M. formicicum and M. hungatei, respectively, suggesting that toxic effects from aliphatic hydrocarbons towards hydrogenotrophic methanogens may not occur during anaerobic treatment. Hydrocarbon toxicity towards hydrogenotrophic methanogens was further assessed by incubating an anaerobic sludge with H2/CO2 in the presence of a complex mixture of hydrocarbons (provided by the addition of an oily sludge from a groundwater treatment system). Specific methanogenic activity from H2/CO2 decreased 1.2 times in the presence of the hydrocarbons, but a relatively high methane production (~30 mM) was still obtained in the assays containing the inoculum and the oily sludge (without H2/CO2), reinforcing the potential of anaerobic treatment systems for methane production from oily waste/wastewater.

Impact of Alum Water Treatment Residues on the Methanogenic Activity in the Digestion of Primary Domestic Wastewater Sludge

The effect of adding alum water treatment residues (WTR) on the methanogenic activity in the digestion of primary domestic wastewater sludge was evaluated through laboratory experiments in sedimentation columns, using total suspended solids (TSS) concentrations from 0.37 to 1.23 g/L. The addition of WTR to primary clarifiers can benefit its effluent water quality in terms of colour, turbidity, chemical oxygen demand (COD), and TSS. However, the presence of WTR can negatively influence the production of methane gas during organic sludge digestion in primary clarifiers, for concentrations of TSS between 14.43 and 25.23 g/L and of VSS between 10.2 and 11.85 g/L. The activity of the Methanothrix sp., curved bacilli, methanococci, and Methanosarcina sp. decreases considerably after 16 days of anaerobic digestion, and methane production seems to only be associated with fluorescent methanogenic bacilli.

Enhancement of Methanogenic Activity in Volumetrically Undersized Reactor by Mesophilic Co-Digestion of Sewage Sludge and Aqueous Residue

To date, energy recovery from biological sewage sludge (BSS) by anaerobic digestion has been very popular. However, it can often happen that anaerobic reactors are volumetrically undersized, thus reducing performance in terms of biogas production. A continuous-flow pilot-scale plant was used to investigate, for the first time, the effects of mesophilic anaerobic co-digestion (MACoD) of sewage sludge and aqueous residue (AR) from a biosolids treatment plant (BTP) on methanogenic activity under low hydraulic retention time (HRT) conditions (to simulate the undersizing of the reactor). The results showed that the digestate is always more rapidly biodegradable than the matrices fed, while particulate COD hydrolyzed (12 ± 1.3%) is independent of the quantity of AR dosed. Feeding over 35% of soluble OLR, the total VFAs in the system strongly decreased, despite the low HRT. In correspondence with higher dosages of AR, the percentage of CH4 increased up to 77–78% and the CO2 CH4−1 ratio decreased to 0.25 ± 0.2. Specific methane production increased from 0.09 ± 0.01 m3CH4 kgCODremoved−1 with BSS alone to 0.28 ± 0.01 m3CH4CH4 kgCODremoved−1 in the case of BSS co-digested with AR. Moreover, co-digestion with AR from a BTP allowed continuous specific methanogenic activity to be enhanced from 1.76 ± 0.02 m3CH4 tVSS−1 d−1 to 6.48 ± 0.88 m3CH4 tVSS−1 d−1. Therefore, the MACoD of BSS and AR from a BTP could be a good solution to enhance methanogenic activity in a volumetrically undersized anaerobic digester with reduced HRT.

Microbial Communities and Interactions of Nitrogen Oxides With Methanogenesis in Diverse Peatlands of the Amazon Basin

Tropical peatlands are hotspots of methane (CH4) production but present high variation and emission uncertainties in the Amazon region. This is because the controlling factors of methane production in tropical peats are not yet well documented. Although inhibitory effects of nitrogen oxides (NOx) on methanogenic activity are known from pure culture studies, the role of NOx in the methane cycling of peatlands remains unexplored. Here, we investigated the CH4 content, soil geochemistry and microbial communities along 1-m-soil profiles and assessed the effects of soil NOx and nitrous oxide (N2O) on methanogenic abundance and activity in three peatlands of the Pastaza-Marañón foreland basin. The peatlands were distinct in pH, DOC, nitrate pore water concentrations, C/N ratios of shallow soils, redox potential, and 13C enrichment in dissolved inorganic carbon and CH4 pools, which are primarily contingent on H2-dependent methanogenesis. Molecular 16S rRNA and mcrA gene data revealed diverse and novel methanogens varying across sites. Importantly, we also observed a strong stratification in relative abundances of microbial groups involved in NOx cycling, along with a concordant stratification of methanogens. The higher relative abundance of ammonia-oxidizing archaea (Thaumarchaeota) in acidic oligotrophic peat than ammonia-oxidizing bacteria (Nitrospira) is noteworthy as putative sources of NOx. Experiments testing the interaction of NOx species and methanogenesis found that the latter showed differential sensitivity to nitrite (up to 85% reduction) and N2O (complete inhibition), which would act as an unaccounted CH4 control in these ecosystems. Overall, we present evidence of diverse peatlands likely differently affected by inhibitory effects of nitrogen species on methanogens as another contributor to variable CH4 fluxes.

Methanogenesis at High Temperature, High Ionic Strength and Low pH in the Volcanic Area of Dallol, Ethiopia

The Dallol geothermal area originated as a result of seismic activity and the presence of a shallow underground volcano, both due to the divergence of two tectonic plates. In its ascent, hot water dissolves and drags away the subsurface salts. The temperature of the water that comes out of the chimneys is higher than 100 °C, with a pH close to zero and high mineral concentration. These factors make Dallol a polyextreme environment. So far, nanohaloarchaeas, present in the salts that form the walls of the chimneys, have been the only living beings reported in this extreme environment. Through the use of complementary techniques: culture in microcosms, methane stable isotope signature and hybridization with specific probes, the methanogenic activity in the Dallol area has been assessed. Methane production in microcosms, positive hybridization with the Methanosarcinales probe and the δ13CCH4-values measured, show the existence of extensive methanogenic activity in the hydrogeothermic Dallol system. A methylotrophic pathway, carried out by Methanohalobium and Methanosarcina-like genera, could be the dominant pathway for methane production in this environment.

Anaerobic Co-Digestion of Tannery and Slaughterhouse Wastewater for Solids Reduction and Resource Recovery: Effect of Sulfate Concentration and Inoculum to Substrate Ratio

Anaerobic digestion is considered unsuitable for the bioremediation of tannery effluent due to process inhibition, mainly due to high concentrations of sulfur species, and the accumulation of H2S and/or NH3. This study using the standardized biochemical methane potential protocol showed that efficient processing is possible with slaughterhouse wastewater, provided sufficient functional biomass is present at the start of the process and the SO42− concentration is below inhibition threshold. Methanogenic activity (K = 13.4–17.5 and µm = 0.15–0.27) and CH4 yields were high when reactors were operated ISR ≥ 3 and/or lower SO42− ≤ 710 mg/L while high SO42− ≥ 1960 mg/L and ISR < 3.0 caused almost complete inhibition regardless of corresponding ISR and SO42−. The theoretical optimum operating conditions (922 mg/L SO42−, ISR = 3.72) are expected to generate 361 mL biogas/gVS, 235 mL CH4/gVS with reduction efficiencies of 27.5% VS, 27.4% TS, 75.1% TOC, 75.6% SO42−, and 41.1% COD. This implies that tannery sludge will be reduced by about 27% (dry mass) and SO42− by 76%, with a fraction of it recovered as S0. The models displayed a perfect fit to the cumulative CH4 yields with high precision in the order Logistic > Cone > modified Gompertz > first order.