Presence and role of viruses in anaerobic digestion of food waste under environmental variability.

BACKGROUND: The interaction among microorganisms in the anaerobic digestion of food waste (ADFW) reactors lead to the degradation of organics and the recycling of energy. Viruses are an important component of the microorganisms involved in ADFW, but are rarely investigated. Furthermore, little is known about how viruses affect methanogenesis. RESULTS: Thousands of viral sequences were recovered from five full-scale ADFW reactors. Gene-sharing networks indicated that the ADFW samples contained substantial numbers of unexplored anaerobic-specific viruses. Moreover, the viral communities in five full-scale reactors exhibited both commonalities and heterogeneities. The lab-scale dynamic analysis of typical ADFW scenarios suggested that the viruses had similar kinetic characteristics to their prokaryotic hosts. By associating with putative hosts, a majority of the bacteria and archaea phyla were found to be infected by viruses. Viruses may influence prokaryotic ecological niches, and thus methanogenesis, by infecting key functional microorganisms, such as sulfate-reducing bacteria (SRB), syntrophic acetate-oxidizing bacteria (SAOB), and methanogens. Metabolic predictions for the viruses suggested that they may collaborate with hosts at key steps of sulfur and long-chain fatty acid (LCFA) metabolism and could be involved in typical methanogenesis pathways to participate in methane production. CONCLUSIONS: Our results expanded the diversity of viruses in ADFW systems and suggested two ways that viral manipulated ADFW biochemical processes. Video Abstract.

Leaching risk of antibiotic resistance contamination from organic waste compost in rural areas.

Composting is an important decentralized technology for treating multiple biodegradable organic wastes in rural areas. However, compared to industrial composting (i.e., time and temperature protocols), rural composting is less well-controlled, and the risk of antibiotic resistance genes (ARGs) in these composts needs to be determined. We performed a quantitative determination of ARGs and both prokaryotes and eukaryotes to investigate the liquid-solid leaching ratio and the relationship between ARGs and microbial communities in solid and water extracts of composts collected from rural areas. We observed a high level of sulfonamides resistance genes and tetracyclines resistance genes (10-4-10-2 copies/16S copies). Tet-C and tet-X show the strongest leaching potential in rural organic waste composts with complex hosts in solid and liquid phases. This study showed high ARG abundances in compost solid and water extracts, highlighting the leaching risk of compost ARGs when exposed to runoff or groundwater during open storage and field application.

Metagenomic analysis of microbiological risk in bioaerosols during biowaste valorization using Musca domestica.

Bioconversion using insects has gradually become a promising technology for biowaste management and protein production. However, knowledge about microbiological risk of insect related bioaerosols is sparse and conventional methods failed to provide higher resolved information of environmental microbe. In this study, a metagenomic analysis including microorganisms, antibiotic resistance genes (ARGs), virulence factor genes (VFGs), mobile gene elements (MGEs), and endotoxin distribution in bioaerosols during biowaste conversion via Musca domestica revealed that bioaerosols in Fly rearing room possess the highest ARGs abundances and MGEs diversity. Through a metagenome-assembled genomes (MAGs)-based pipeline, compelling evidence of ARGs/VFGs host assignment and ARG-VFG co-occurrence pattern were provided from metagenomic perspective. Bioaerosols in Bioconversion and Maggot separation zone were identified to own high density of MAGs carrying both ARGs and VFGs. Bacteria in Proteobacteria, Actinobacteriota, and Firmicutes phyla were predominate hosts of ARGs and VFGs. Multidrug-Motility, Multidrug-Adherence, and Beta lactam-Motility pairs were the most common ARG-VFG co-occurrence pattern in this study. Results obtained are of great significance for microbiological risk assessment during housefly biowaste conversion process.

Emission of odor pollutants and variation in microbial community during the initial decomposition stage of municipal biowaste.

Odor pollution often occurs in the initial decomposition stage of municipal biowaste, including throwing/collection and transportation. However, this aspect of odor impact from municipal biowaste has not been well studied. In this study, a practical dustbin (120 L) equipped with flux chamber and filled with three types of municipal biowaste was used to simulate garbage storage conditions. The result indicated that the emission rate of odor pollutants for uncooked food waste (UFW) represented a nearly linear growth trend, reaching the maximum (3963 ± 149 µg kg-1 DM h-1) at 72 h. Cooked food waste (CFW) increased rapidly from 8 h to 24 h, and then remain fluctuated, reached the maximum (2026 ± 77 µg kg-1 DM h-1) at 72 h. Comparatively, household kitchen waste (HKW) reached the maximum emission rate (10,396 ± 363 µg kg-1 DM h-1) at 16 h. Sulfide and aldehydes ketones were identified as dominant odor contributor to UFW and CFW, respectively. While aldehydes ketones and sulfides were both dominant odor contributor to HKW. Moreover, the microbial diversity analysis suggests that Acinetobacter was the dominant genus in UFW, and Lactobacillus was the dominant genus in CFW and HKW. In addition, it was evident that each odorous pollutant was significantly associated with two or more bacterial genera, and most bacterial genera such as Acinetobacter, were also significantly associated with multiple odorous pollutants. The variation of odorants composition kept consistent with microbial composition. The present study could provide essential evidence for a comprehensive understanding of odorant generation in the initial decomposition stage of municipal biowaste. It could contribute to setting out strategies for odor control and abatement in municipal biowaste management systems.

Syntrophic Acetate-Oxidizing Microbial Consortia Enriched from Full-Scale Mesophilic Food Waste Anaerobic Digesters Showing High Biodiversity and Functional Redundancy.

Syntrophic acetate oxidation (SAO) coupled with hydrogenotrophic methanogenesis (HM) plays a vital role in the anaerobic digestion of protein-rich feedstocks such as food wastes. However, current knowledge of the biodiversity and genetic potential of the involved microbial participants, especially syntrophic acetate-oxidizing bacteria (SAOB), is limited due to the low abundance of these microorganisms and challenges in their isolation. The intent of this study was to enrich and identify potential SAOB. Therefore, we conducted continuous acetate feeding under high ammonia concentrations using two separate inoculum consortia of microorganisms that originated from full-scale mesophilic food waste digesters, which lasted for more than 200 days. Using 16S rRNA gene amplicon and metagenomic analyses, we observed a convergence of the experimental microbial communities during the enrichment regarding taxonomic composition and metabolic functional composition. Stable carbon isotope analyses of biogas indicated that SAO-HM was the dominant methanogenic pathway during the enrichment process. The hydrogenotrophic methanogen Methanoculleus dominated the archaeal community. The enriched SAO community featured high biodiversity and metabolic functional redundancy. By analyzing the metagenome-assembled genomes, the known SAOB Syntrophaceticus schinkii and six uncultured populations were identified to have the genetic potential to perform SAO through the conventional reversed Wood-Ljungdahl pathway, while another six bacteria were found to encode the reversed Wood-Ljungdahl pathway combined with a glycine cleavage system as novel SAOB candidates. These results showed that the food waste anaerobic digesters harbor diverse SAOB and highlighted the importance of the glycine cleavage system for acetate oxidation. IMPORTANCE Syntrophic acetate oxidation to CO2 and H2, together with hydrogenotrophic methanogenesis, contributes to much of the carbon flux in the anaerobic digestion of organic wastes, especially at high ammonia concentrations. A deep understanding of the biodiversity, metabolic genetic potential, and ecology of the SAO community can help to improve biomethane production from wastes for clean energy production. Here, we enriched the SAO-HM functional guild obtained from full-scale food waste anaerobic digesters and recorded dynamic changes in community taxonomic composition and functional profiles. By reconstructing the metabolic pathways, diverse known and novel bacterial members were found to have SAO potential via the reversed Wood-Ljungdahl (WL) pathway alone, or via the reversed WL pathway with a glycine cleavage system (WLP-GCS), and those catalyzing WLP-GCS showed higher microbial abundance. This study revealed the biodiversity and metabolic functional redundancy of SAOB in full-scale anaerobic digester systems and provided inspiration for further genome-centric studies.

Metabolic Regulation of Mesophilic Methanosarcina barkeri to Ammonium Inhibition.

Undesirable ammonium concentrations can lead to unstable anaerobic digestion processes, and Methanosarcina spp. are the representative methanogens under inhibition. However, no known work seems to exist for directly exploring the detailed metabolic regulation of pure cultured representative Methanosarcina spp. to ammonium inhibition. We used transcriptomics and proteomics to profile the metabolic regulation of Methanosarcina barkeri to 1, 4, and 7 g N/L of total ammoniacal nitrogen (TAN), where free ammonia concentrations were between 1.5 and 36.1 mg N/L. At the initial stages of ammonium inhibition, the genes participating in the acquisition and assimilation of reduced nitrogen sources showed significant upregulation where the minimal fold change of gene transcription was about 2. Apart from nitrogen metabolism, the transcription of some genes in methanogenesis also significantly increased at the initial stages. For example, the genes encoding alternative heterodisulfide reductase subunits (HdrAB), energy-converting hydrogenase subunit (EchC), and methanophenazine-dependent hydrogenase subunits (VhtAC) were significantly upregulated by at least 2.05 times. For the element translocation at the initial stages, the genes participating in the uptake of ferrous iron, potassium ion, and molybdate were significantly upregulated with a minimal fold change of 2.10. As the cultivation proceeded, the gene encoding the cell division protein subunit (FtsH) was significantly upregulated by 13.0 times at 7 g N/L of TAN; meanwhile, an increment in OD600 was observed at the terminal sampling point of 7 g N/L of TAN. The present study explored the metabolic regulation of M. barkeri in stress response, protein synthesis, signal transduction, nitrogen metabolism, methanogenesis, and element translocation. The results would contribute to the understanding of the metabolic effects of ammonium inhibition on methanogens and have significant practical implication in inhibited anaerobic digestion.

Monitor process state of batch anaerobic digestion in reliance on volatile and semi-volatile metabolome.

It has been a challenge to recognize appropriate compounds as indicators for monitoring and early-warning of the anaerobic digestion process. A strategy was initiated to explore the evolution of the panorama profile of volatile and semi-volatile metabolites. Non-target analysis using high-resolution gas chromatography coupled with Orbitrap mass spectrometry was applied to construct a time-series molecular fingerprint of 218 metabolites classified in 14 categories. Alkanes accounted for the main part in early and late stages of methanization and aromatic compounds were the major in middle stage. Spearman correlation analysis and partial least squares analysis unwind that Trichococcus (1.49%-83.96%) was positively related to most of metabolites at early and middle stages, while Brevundimonas (0%-24.04%) was positively related to acylamide at late stage. This indicated that microbial volatile organic compounds were possible to serve as biochemical indicators for anaerobic digestion performance and to build nexus of "what" (metabolites), "who" (microorganism), and "how" (kinetics).

Functional genome-centric view of the CO-driven anaerobic microbiome.

CO is a promising substrate for producing biochemicals and biofuels through mixed microbial cultures, where carboxydotrophs play a crucial role. The previous investigations of mixed microbial cultures focused primarily on overall community structures, but under-characterized taxa and intricate microbial interactions have not yet been precisely explicated. Here, we undertook DNA-SIP based metagenomics to profile the anaerobic CO-driven microbiomes under 95 and 35% CO atmospheres. The time-series analysis of the isotope-labeled amplicon sequencing revealed the essential roles of Firmicutes and Proteobacteria under high and low CO pressure, respectively, and Methanobacterium was the predominant archaeal genus. The functional enrichment analysis based on the isotope-labeled metagenomes suggested that the microbial cultures under high CO pressure had greater potential in expressing carboxylate metabolism and citrate cycle pathway. The genome-centric metagenomics reconstructed 24 discovered and 24 under-characterized metagenome-assembled genomes (MAGs), covering more than 94% of the metagenomic reads. The metabolic reconstruction of the MAGs described their potential functions in the CO-driven microbiomes. Some under-characterized taxa might be versatile in multiple processes; for example, under-characterized Rhodoplanes sp. and Desulfitobacterium_A sp. could encode the complete enzymes in CO oxidation and carboxylate production, improving functional redundancy. Finally, we proposed the putative microbial interactions in the conversion of CO to carboxylates and methane.

Biological denitrification potential as an indicator for measuring digestate stability.

Biological stability is an essential parameter for assessing the environmental impact from the land application of digestate as organic amendment. In this paper, a new indicator, biological denitrification potential (BDP), was developed for evaluating the biological stability of digestate. Digestate samples collected along the digestion process from a mesophilic anaerobic batch digester fed with food waste were investigated under different solid retention time. The value of BDP based on nitrate removal ranged from 176.3 to 48.3 mg-N/g-VSdigestate, corresponding well to the digestion time, and strongly correlated with total organic carbon content. Evolution trends similar to respiration index (RI) and biochemical methane potential (BMP) can be also observed for BDP, indicating that values presented of these stability indices decreased with the degree of digestate stabilization. The mass balance of the BDP process indicated that nitrate was mainly converted into N2 gas with mineralizing organic carbon from digestate, implying that biostability evaluated by BDP depends on carbon source and denitrification activity in digestate. The denitrifying bacteria Thiopseudomonas and Pseudomonas accounted for the majority of microorganisms. These findings of this study concluded that BDP can be an efficient indicator to assess the bio-stability of digestate planned for agricultural or land use. Compared with the existing biostability index, BDP has the additional advantage of no exogenous inoculum addition, homogenous test condition and possibility of shortening incubation time.

Responses of Methanosarcina barkeri to acetate stress.

BACKGROUND: Anaerobic digestion of easily degradable biowaste can lead to the accumulation of volatile fatty acids, which will cause environmental stress to the sensitive methanogens consequently. The metabolic characteristics of methanogens under acetate stress can affect the overall performance of mixed consortia. Nevertheless, there exist huge gaps in understanding the responses of the dominant methanogens to the stress, e.g., Methanosarcinaceae. Such methanogens are resistant to environmental deterioration and able to utilize multiple carbon sources. In this study, transcriptomic and proteomic analyses were conducted to explore the responses of Methanosarcina barkeri strain MS at different acetate concentrations of 10, 25, and 50 mM. RESULTS: The trend of OD600 and the regulation of the specific genes in 50 mM acetate, indicated that high concentration of acetate promoted the acclimation of M. barkeri to acetate stress. Acetate stress hindered the regulation of quorum sensing and thereby eliminated the advantages of cell aggregation, which was beneficial to resist stress. Under acetate stress, M. barkeri allocated more resources to enhance the uptake of iron to maintain the integrities of electron-transport chains and other essential biological processes. Comparing with the initial stages of different acetate concentrations, most of the genes participating in acetoclastic methanogenesis did not show significantly different expressions except hdrB1C1, an electron-bifurcating heterodisulfide reductase participating in energy conversion and improving thermodynamic efficiency. Meanwhile, vnfDGHK and nifDHK participating in nitrogen fixation pathway were upregulated. CONCLUSION: In this work, transcriptomic and proteomic analyses are combined to reveal the responses of M. barkeri to acetate stress in terms of central metabolic pathways, which provides basic clues for exploring the responses of other specific methanogens under high organics load. Moreover, the results can also be used to gain insights into the complex interactions and geochemical cycles among natural or engineered populations. Furthermore, these findings also provide the potential for designing effective and robust anaerobic digesters with high organic loads.