Post-treatment of dewatered digested sewage sludge by thermophilic high-solid digestion for pasteurization with positive energy output.

This study investigated the possibility to use thermophilic anaerobic high solid digestion of dewatered digested sewage sludge (DDS) at a wastewater treatment plant (WWTP) as a measure to increase total methane yield, achieve pasteurization and reduce risk for methane emissions during storage of the digestate. A pilot-scale plug-flow reactor was used to mimic thermophilic post-treatment of DDS from a WWTP in Linköping, Sweden. Process operation was evaluated with respect to biogas process performance, using both chemical and microbiological parameters. Initially, the process showed disturbance, with low methane yields and high volatile fatty acid (VFA) accumulation. However, after initiation of digestate recirculation performance improved and the specific methane production reached 46 mL CH4/g VS. Plug flow conditions were assessed with lithium chloride and the hydraulic retention time (HRT) was determined to be 19-29 days, sufficient to reach successful pasteurization. Degradation rate of raw protein was high and resulted in ammonia-nitrogen levels of up to 2.0 g/L and a 30% lower protein content in the digestate as compared to DDS. Microbial analysis suggested a shift in the methane producing pathway, with dominance of syntrophic acetate oxidation and the candidate methanogen family WSA2 by the end of the experiment. Energy balance calculations based on annual DDS production of 10000 ton/year showed that introduction of high-solid digestion as a post-treatment and pasteurization method would result in a positive energy output of 340 MWh/year. Post-digestion of DDS also decreased residual methane potential (RMP) by>96% compared with fresh DDS.

Comparative study of industrial-scale high-solid biogas production from food waste: Process operation and microbiology.

Anaerobic high-solid treatment (HST) for processing food waste and biogas production is a viable technology with considerable commercial potential. In this study, we examined and compared mesophilic and thermophilic industrial-scale plug-flow digesters. The HSTs demonstrated reasonable biogas yields from food waste (0.4-0.6 Nm3 CH4/kg volatile solids). However, during operation at thermophilic conditions ammonia inhibition (~2 g NH3-N/L) and acid accumulation (6-14 g/L) caused severe process disturbance. Microbial community structures diverged between the processes, with temperature appearing to be a strong driver. A unique feature of the thermophilic HSTs was high abundance of the uncultivated Clostridia group MBA03 and temperature fluctuations in one mesophilic HST were linked to drastically decreased abundance of methanogens and relative abundance of Cloacimonetes. The process data obtained in this study clearly demonstrate both potential and challenges in HST of food waste but also possibilities for management approaches to tackle process imbalance and restore process function.

Effects of thermal hydrolytic pre-treatment on biogas process efficiency and microbial community structure in industrial- and laboratory-scale digesters.

This study examined the impact of thermal hydrolysis process (THP) pre-treatment on anaerobic co-digestion of wastewater sludge and household waste and assessed whether THP was vital to achieve higher process capacity. Performance data were collected for both industrial- and laboratory-scale digesters and response in microbial community structure was evaluated by Illumina sequencing. Implementation of THP at the industrial-scale plant increased methane yield by 15% and enhanced substrate degradability. Possibility to extend the sludge retention time due to a higher solid content of the substrate, sanitisation of the digestate and improved fertiliser quality of the digestate were other industrial-scale benefits of THP installation. Continuously-fed laboratory-scale digesters were fed THP-treated or untreated substrate at an organic loading rate (OLR) of 5 g volatile solid (VS)/L/day, a feeding rate necessary at the corresponding industrial-scale plant to meet the estimated population increase within the municipality. The results indicated that the plant could have increased the capacity with unimpaired stability independently of THP installation, even though the retention time was significantly shortened during operation with untreated substrate. Microbial community analyses revealed increased contribution of the Clostridia class after THP installation in industrial-scale digesters and positive correlation between Firmicutes:Bacteriodetes and methane yield in all digesters. Differentiated profiles in laboratory-scale digesters indicated that a temperature increase from 37 to 42 °C in association with THP installation and altered substrate composition were strong determining factors shaping the microbial community. Overall, these findings can assist industrial-scale plants in choosing management strategies aimed at improving the efficiency of anaerobic digestion processes.

Novel Syntrophic Populations Dominate an Ammonia-Tolerant Methanogenic Microbiome.

Biogas reactors operating with protein-rich substrates have high methane potential and industrial value; however, they are highly susceptible to process failure because of the accumulation of ammonia. High ammonia levels cause a decline in acetate-utilizing methanogens and instead promote the conversion of acetate via a two-step mechanism involving syntrophic acetate oxidation (SAO) to H2 and CO2, followed by hydrogenotrophic methanogenesis. Despite the key role of syntrophic acetate-oxidizing bacteria (SAOB), only a few culturable representatives have been characterized. Here we show that the microbiome of a commercial, ammonia-tolerant biogas reactor harbors a deeply branched, uncultured phylotype (unFirm_1) accounting for approximately 5% of the 16S rRNA gene inventory and sharing 88% 16S rRNA gene identity with its closest characterized relative. Reconstructed genome and quantitative metaproteomic analyses imply unFirm_1's metabolic dominance and SAO capabilities, whereby the key enzymes required for acetate oxidation are among the most highly detected in the reactor microbiome. While culturable SAOB were identified in genomic analyses of the reactor, their limited proteomic representation suggests that unFirm_1 plays an important role in channeling acetate toward methane. Notably, unFirm_1-like populations were found in other high-ammonia biogas installations, conjecturing a broader importance for this novel clade of SAOB in anaerobic fermentations. IMPORTANCE The microbial production of methane or "biogas" is an attractive renewable energy technology that can recycle organic waste into biofuel. Biogas reactors operating with protein-rich substrates such as household municipal or agricultural wastes have significant industrial and societal value; however, they are highly unstable and frequently collapse due to the accumulation of ammonia. We report the discovery of a novel uncultured phylotype (unFirm_1) that is highly detectable in metaproteomic data generated from an ammonia-tolerant commercial reactor. Importantly, unFirm_1 is proposed to perform a key metabolic step in biogas microbiomes, whereby it syntrophically oxidizes acetate to hydrogen and carbon dioxide, which methanogens then covert to methane. Only very few culturable syntrophic acetate-oxidizing bacteria have been described, and all were detected at low in situ levels compared to unFirm_1. Broader comparisons produced the hypothesis that unFirm_1 is a key mediator toward the successful long-term stable operation of biogas production using protein-rich substrates.

Expression of barley SUSIBA2 transcription factor yields high-starch low-methane rice.

Atmospheric methane is the second most important greenhouse gas after carbon dioxide, and is responsible for about 20% of the global warming effect since pre-industrial times. Rice paddies are the largest anthropogenic methane source and produce 7-17% of atmospheric methane. Warm waterlogged soil and exuded nutrients from rice roots provide ideal conditions for methanogenesis in paddies with annual methane emissions of 25-100-million tonnes. This scenario will be exacerbated by an expansion in rice cultivation needed to meet the escalating demand for food in the coming decades. There is an urgent need to establish sustainable technologies for increasing rice production while reducing methane fluxes from rice paddies. However, ongoing efforts for methane mitigation in rice paddies are mainly based on farming practices and measures that are difficult to implement. Despite proposed strategies to increase rice productivity and reduce methane emissions, no high-starch low-methane-emission rice has been developed. Here we show that the addition of a single transcription factor gene, barley SUSIBA2 (refs 7, 8), conferred a shift of carbon flux to SUSIBA2 rice, favouring the allocation of photosynthates to aboveground biomass over allocation to roots. The altered allocation resulted in an increased biomass and starch content in the seeds and stems, and suppressed methanogenesis, possibly through a reduction in root exudates. Three-year field trials in China demonstrated that the cultivation of SUSIBA2 rice was associated with a significant reduction in methane emissions and a decrease in rhizospheric methanogen levels. SUSIBA2 rice offers a sustainable means of providing increased starch content for food production while reducing greenhouse gas emissions from rice cultivation. Approaches to increase rice productivity and reduce methane emissions as seen in SUSIBA2 rice may be particularly beneficial in a future climate with rising temperatures resulting in increased methane emissions from paddies.

Methanogenic population and CH4 production in swedish dairy cows fed different levels of forage.

Methanogenic community structure, methane production (CH(4)), and volatile fatty acid (VFA) profiles were investigated in Swedish dairy cows fed a diet with a forage/concentrate ratio of 500/500 or 900/100 g/kg of dry matter (DM) of total DM intake (DMI). The rumen methanogenic population was evaluated using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR (qRT-PCR). Mean CH(4) yields did not differ (P > 0.05) between diets, being 16.9 and 20.2 g/kg DMI for the 500/500 and 900/100 diets, respectively. The T-RFLP analysis revealed that populations differed between individual cows and that each individual population responded differently to the diets. The 16S rRNA gene libraries revealed that Methanobrevibacter spp. dominated for both diets. CH(4) production was positively correlated with a dominance of sequences representing T-RFs related to Methanobrevibacter thaueri, Methanobrevibacter millerae, and Methanobrevibacter smithii relative to Methanobrevibacter ruminantium and Methanobrevibacter olleyae. Total numbers of methanogens and total numbers of Methanobacteriales were significantly higher with the 500/500 diet (P < 0.0004 and P < 0.002, respectively). However, no relationship was found between CH(4) production and total number of methanogens. No differences were seen in total VFA, propionic acid, or acetic acid contents, but the molar proportion of butyric acid in the rumen was higher for the 500/500 diet than for the 900/100 diet (P < 0.05). Interestingly, the results also revealed that a division of the identified methanogenic species into two groups, suggested in the work of King et al. (E. E. King, R. P. Smith, B. St-Pierre, and A. D. G. Wright, Appl. Environ. Microbiol. 77:5682-5687, 2011), increased the understanding of the variation in CH(4) production between different cows.

Ammonia, a selective agent for methane production by syntrophic acetate oxidation at mesophilic temperature.

In biogas processes, methane production from acetate proceeds by either aceticlastic methanogenesis or through syntrophic acetate oxidation (SAO). In the present study, the pathway for methane production from acetate was analysed; i) during a gradual increase of the ammonia concentration (final concentration 7 g NH(4)(+) -N/L) in a semi-continuous lab-scale anaerobic digester (4.3 L), operating at mesophilic temperature (37 degrees C) or ii) in diluted enrichment cultures (100 ml) experiencing a gradual increase in ammonia, sodium, potassium and propionic acid. The pathway for methane formation was determined by calculating the (14)CO(2)/(14)CH(4) ratio after incubating samples with (14)C-2-acetate. In the anaerobic digester, as well as in the enrichment cultures, the (14)CO(2)/(14)CH4 ratio clearly increased with increasing ammonium-nitrogen concentration, i.e. as the ammonia concentration increased, a shift from the aceticlastic mechanism to the syntrophic pathway occurred. The shift was very distinct and occurred as the NH(4)(+) -N concentration rose above 3 g/l. No shift in pathway was seen during increasing concentrations of sodium, potassium or propionic acid. The shift to SAO in the biogas digester resulted in a twofold decrease in the specific gas and methane yield.

In situ ammonia production as a sanitation agent during anaerobic digestion at mesophilic temperature.

AIM: To measure the sanitizing effect of mesophilic (37 degrees C) anaerobic digestion in high ammonia concentrations produced in situ. METHODS AND RESULTS: Indicator organisms and salmonella were transferred to small-scale anaerobic batch cultures and D-values were calculated. Batch cultures were started with material from two biogas processes operating at high (46 mmol l(-1)) and low (1.6 mmol l(-1)) ammonia concentration. D-values were shortened from c. 3 days to <1 day for the bacteria. MS2 had the same D-value (1.3 days) independent of ammonia concentration whereas PhiX174 and 28B were faster inactivated in the control (1.1 and 7.9 days) than in the high ammonia (8.9 and 39 days) batch cultures. CONCLUSION: Running biogas processes at high levels of ammonia shortens the time to meet EU regulation concerning reduction of salmonella and enterococci (5 log). Unless a minimum retention time of 2 days, post-treatment digestion is needed to achieve sufficient sanitation in continuous biogas processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Running mesophilic biogas processes at high ammonia level produces residue with a high fertilizer value. With some stipulations concerning management parameters, such processes provide a method of bacterial sanitation without preceding pasteurization of the incoming organic waste.

Fate of Ah-receptor agonists in organic household waste during anaerobic degradation--estimation of levels using EROD induction in organ cultures of chick embryo livers.

The fate of 7-ethoxyresorufin O-deethylase (EROD)-inducing compounds in source-separated organic household waste subject to anaerobic degradation (i.e. mesophilic/thermophilic anaerobic treatment) was investigated using organ cultures of embryonic chicken livers from fertilised hen eggs. This bioassay reflects the combined effect of all EROD-inducing, possibly dioxin-like compounds in a sample, including chemicals that are seldom or never analysed. All samples tested induced EROD in the bioassay, indicating the presence of dioxin-like compounds. In the anaerobic processes, the amounts of acid-resistant EROD-inducing compounds coming out of the reactors were considerably higher than the incoming amounts, especially for the low-temperature (mesophilic) process. This apparent production of EROD-inducing compounds may be due to de novo synthesis or to an increase in the EROD-inducing potency of the compounds in the material.

Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium.

A syntrophic acetate-oxidizing bacterium, strain BST (T = type strain), was isolated from a previously described mesophilic triculture that was able to syntrophically oxidize acetate and form methane in stoichiometric amounts. Strain BST was isolated with substrates typically utilized by homoacetogenic bacteria. Strain BST was a spore-forming, gram-positive, rod-shaped organism which utilized formate, glucose, ethylene glycol, cysteine, betaine, and pyruvate. Acetate and sometimes formate were the main fermentation products. Small amounts of alanine were also produced from glucose, betaine, and cysteine. Strain BST grew optimally at 37 degrees C and pH 7. The G+C content of the DNA of strain BST was 32 mol%. A 16S rRNA sequence analysis revealed that strain BST was a member of a new species of the genus Clostridium. We propose the name Clostridium ultunense for this organism; strain BS is the type strain of C. ultunense.