Anaerobic digestion of wastewater from hydrothermal liquefaction of sewage sludge and combined wheat straw-manure.

Hydrothermal liquefaction (HTL) shows promise for converting wet biomass waste into biofuel, but the resulting high-strength process water (PW) requires treatment. This study explored enhancing energy recovery by anaerobic digestion using semi-batch reactors. Co-digesting manure with HTL-PW from wheat straw-manure co-HTL yielded methane (43-49% of the chemical oxygen demand, COD) at concentrations up to 17.8 gCOD·L-1, whereas HTL-PW from sewage sludge yielded methane (43% of the COD) up to only 12.8 gCOD·L-1 and complete inhibition occurred at 17 gCOD·L-1. Microbial community shifts confirmed inhibition of methanogenic archaea, while hydrolytic-fermentative bacteria were resilient. Differences in chemical composition, particularly higher levels of N-containing heterocyclic compounds in PW of sewage sludge, likely caused the microbial inhibition. The considerable potential of combining HTL with anaerobic digestion for enhanced energy recovery from straw-manure in an agricultural context is demonstrated, yet sewage sludge HTL-PW requires more advanced approaches to deal with methanogenesis inhibitors.

Energetically exploiting lignocellulose-rich residues in anaerobic digestion technologies: from bioreactors to proteogenomics.

The biogas produced through anaerobic digestion (AD) of renewable feedstocks is one of the promising alternatives to replace fossil-derived energy. Even though lignocellulosic biomass is the most abundant biomass on earth, only a small fraction is being used towards resources recovery, leaving a great potential unexploited. In this study, the combination of state-of-art genomic techniques and engineered systems were used to further advance the knowledge on biogas production from lignocellulosic-rich residues and the microbiome involved in the anaerobic digestion hereof. A long-term adapted anaerobic microbiome capable of degrading wheat straw as the sole substrate was investigated using protein stable isotope probing (protein-SIP). The results indicated that a diverse microbial community, primarily composed of Firmicutes and Methanogens, played crucial roles in cellulose degradation and methane production. Notably, Defluviitoga tunisiensis, Syntrophothermus lipocalidus, and Pelobacter carbinolicus were identified as direct metabolizers of cellulose, while Dehalobacterium assimilated labelled carbon through cross-feeding. This study provides direct evidence of primary cellulose degraders and sheds light on their genomic composition. By harnessing the potential of lignocellulosic biomass and understanding the microbial communities involved, we can promote sustainable biogas production, contributing to energy security and environmental preservation.

Effect of biofilm thickness on the activity and community composition of phosphorus accumulating bacteria in a moving bed biofilm reactor.

Can biofilms enhance the rates of phosphorus removal in wastewater treatment? In order to narrow the scientific gap on the effect of biofilm thickness on the activity and microbial community of phosphorus-accumulating bacteria, this study investigated biofilms of 30 to 1000 µm thickness in a moving bed biofilm reactor. Measurements on 5 different biofilm carriers showed that biomass-specific phosphorus release and uptake rates increased as a function of biofilm thickness for biofilms thinner than about 110 µm but were lower for thicker biofilms of about 550-1000 µm. The reduced phosphorus uptake and release rates in the thickest biofilms can result from substrate mass transfer limitations whereas the low activity in the thinnest biofilms can be related to a too high turnover rate in the biofilm due to heterotrophic growth. Additionally, the microbial ecology of the different biofilms confirms the observed phosphorus uptake and release rates. The results from the full-length 16S rRNA gene sequencing of the bacterial community showed that the thicker biofilms were characterized by higher relative abundance (40-58%) of potential phosphorus accumulating genera Zoogloea, Acinetobacter, Dechloromonas and Ca. Accumulibacter. In contrast, the thinner biofilms were dominated by the genus Ferribacterium (34-60%), which might be competing with phosphorus-accumulating bacteria as indicated by the relatively high acetate uptake rates in the thinner biofilms. It is concluded that there is an optimal biofilm thickness of 100-500 µm, at which the phosphorus accumulating bacteria have the highest activity.

Pig slurry organic matter transformation and methanogenesis at ambient storage temperatures.

Manure management is a significant source of global methane emissions, and there is an increased interest in understanding and predicting emissions. The hydrolysis rate of manure organic matter is critical for understanding and predicting methane emissions. We estimated hydrolysis rate constants of crude protein, fibers, and lipids and used the Arrhenius equation to describe its dependency on temperature. Simultaneously, measurements of methane emission, 13/12 C isotope ratios, and methanogen community were conducted. This was achieved by incubating fresh pig manure without inoculum at 10°C, 15°C, 20°C, and 25°C for 85 days in a lab-scale setup. Hydrolysis of hemicellulose and cellulose increased more with temperature than crude protein, but still, hydrolysis rate of crude protein was highest at all temperatures. Results suggested that crude protein consisted of multiple substrate groups displaying large differences in degradability. Lipids and lignin were not hydrolyzed during incubations. Cumulative methane emissions were 7.13 ± 2.69, 24.6 ± 8.00, 66.7 ± 4.8, and 105.7 ± 7.14 gCH4 kgVS -1 at 10°C, 15°C, 20°C, and 25°C, respectively, and methanogenic community shifted from Methanosphaera toward Methanocorpusculum over time and more quickly at higher temperatures. This study provides important parameter estimates and dependencies on temperature, which is important in mechanistic methane emission models. Further work should focus on characterizing quickly degradable substrate pools in the manure organic matter as they might be the main carbon source of methane emission from manure management.

Proteomic Changes in Methicillin-Resistant Staphylococcus aureus Exposed to Cannabinoids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to ß-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

Assessing labelled carbon assimilation from poly butylene adipate-co-terephthalate (PBAT) monomers during thermophilic anaerobic digestion.

PBAT (poly butylene adipate-co-terephthalate) is a widely used biodegradable plastic, but the knowledge about its metabolization in anaerobic environments is very limited. In this study, the anaerobic digester sludge from a municipal wastewater treatment plant was used as inoculum to investigate the biodegradability of PBAT monomers in thermophilic conditions. The research employs a combination of 13C-labelled monomers and proteogenomics to track the labelled carbon and identify the microorganisms involved. A total of 122 labelled peptides of interest were identified for adipic acid (AA) and 1,4-butanedio (BD). Through the time-dependent isotopic enrichment and isotopic profile distributions, Bacteroides, Ichthyobacterium, and Methanosarcina were proven to be directly involved in the metabolization of at least one monomer. This study provides a first insight into the identity and genomic potential of microorganisms responsible for biodegradability of PBAT monomers during anaerobic digestion under thermophilic conditions.

Proteomic characterisation of polyethylene terephthalate and monomer degradation by Ideonella sakaiensis.

Synthetic plastics, like polyethylene terephthalate (PET), have become an essential part of modern life. Many of these products are remarkably persistent in the environment, and the accumulation in the environment is recognised as a major threat. Therefore, an increasing interest has been focusing on the screening for organisms able to degrade and assimilate the plastic. Ideonella sakaiensis originally isolated from a plastisphere has been reported as a bacterium that was solely thriving on the degradation on PET films. The processes affected by the presence of PET and its monomeric substances terephthalic acid, ethylene glycol, ethyl glycolate, and sodium glyoxylate monohydrate were elucidated by analysis of differential protein expression. The exposure of PET and its monomers induced the MHETase and affect two major pathways: the TCA cycle and the ß-oxidation pathway. The increased expression of proteins directly or indirectly involved in these pathways suggests their underlying importance in the degradation of PET by I. sakaiensis since these proteins are mechanistically supporting the enzymes involved in the degradation of PET and its monomers.

Protein fractionation and shotgun proteomics analysis of enriched bacterial cultures shed new light on the enzymatically catalyzed degradation of acesulfame.

The removal of organic micropollutants in municipal wastewater treatment is an extensively studied field of research, but the underlying enzymatic processes have only been elucidated to a small extent so far. In order to shed more light on the enzymatic degradation of the artificial sweetener acesulfame (ACE) in this context, we enriched two bacterial taxa which were not yet described to be involved in the degradation of ACE, an unknown Chelatococcus species and Ensifer adhaerens, by incubating activated sludge in chemically defined media containing ACE as sole carbon source. Cell-free lysates were extracted, spiked with ACE and analyzed via target LC-MS/MS, demonstrating for the first time enzymatically catalyzed ACE degradation outside of living cells. Fractionation of the lysate via two-dimensional fast protein liquid chromatography (FPLC) succeeded in a partial separation of the enzymes catalyzing the initial transformation reaction of ACE from those catalyzing the further transformation pathway. Thereby, an accumulation of the intermediate transformation product acetoacetamide-n-sulfonic acid (ANSA) in the ACE-degrading fractions was achieved, providing first quantitative evidence that the cleavage of the sulfuric ester moiety of ACE is the initial transformation step. The metaproteome of the enrichments was analyzed in the FPLC fractions and in the unfractionated lysate, using shotgun proteomics via UHPLC-HRMS/MS and label-free quantification. The comparison of protein abundances in the FPLC fractions to the corresponding ACE degradation rates revealed a metallo-ß-lactamase fold metallo-hydrolase as most probable candidate for the enzyme catalyzing the initial transformation from ACE to ANSA. This enzyme was by far the most abundant of all detected proteins and amounted to a relative protein abundance of 91% in the most active fraction after the second fractionation step. Moreover, the analysis of the unfractionated lysate resulted in a list of further proteins possibly involved in the transformation of ACE, most striking a highly abundant amidase likely catalyzing the further transformation of ANSA, and an ABC transporter substrate-binding protein that may be involved in the uptake of ACE into the cell.

Proteogenomics identification of TBBPA degraders in anaerobic bioreactor.

Tetrabromobisphenol A (TBBPA) is the most used flame retardant worldwide and has become a threat to aquatic ecosystems. Previous research into the degradation of this micropollutant in anaerobic bioreactors has suggested several identities of putative TBBPA degraders. However, the organisms actively degrading TBBPA under in situ conditions have so far not been identified. Protein-stable isotope probing (protein-SIP) has become a cutting-edge technique in microbial ecology for enabling the link between identity and function under in situ conditions. Therefore, it was hypothesized that combining protein-based stable isotope probing with metagenomics could be used to identify and provide genomic insight into the TBBPA-degrading organisms. The identified 13C-labelled peptides were found to belong to organisms affiliated to Phytobacter, Clostridium, Sporolactobacillus, and Klebsilla genera. The functional classification of identified labelled peptides revealed that TBBPA is not only transformed by cometabolic reactions, but also assimilated into the biomass. By application of the proteogenomics with labelled micropollutants (protein-SIP) and metagenome-assembled genomes, it was possible to extend the current perspective of the diversity of TBBPA degraders in wastewater and predict putative TBBPA degradation pathways. The study provides a link to the active TBBPA degraders and which organisms to favor for optimized biodegradation.

Unravelling gradient layers of microbial communities, proteins, and chemical structure in aerobic granules.

Most bacteria live in microbial assemblages like biofilms and granules, and each layer of these assemblages provides a niche for certain bacteria with specific metabolic functions. In this study, a gentle (non-destructive) extraction approach based on a cation exchange resin and defined shear was employed to gradually disintegrate biomass and collect single layers of aerobic granules from a full-scale municipal wastewater treatment plant. The microbial community composition of granule layers was characterized using next-generation sequencing (NGS) targeting the 16S rRNA gene, and protein composition was investigated using metaproteomics. The chemical composition of eroded layers was explored using Fourier Transformed Infrared Spectroscopy. On the surface of the granules, the microbial structure (flocculation-supporting Nannocystis sp.) as well as composition of extracellular polymers (extracellular DNA) and proteome (chaperonins and binding proteins) favored microbial aggregation. Extracellular polymeric substances in the granules were composed of mostly proteins and EPS-producers, such as Tetrasphaera sp. and Zoogloea sp., were evenly distributed throughout the granule structure. The interior of the granules harbored several denitrifiers (e.g., Thauera sp.), phosphate-accumulating denitrifiers (Candidatus Accumulibacter, Dechloromonas sp.) and nitrifiers (Candidatus Nitrotoga). Proteins associated with glycolytic activity were identified in the outer and middle granule layers, and proteins associated with phosphorus conversions, in the deeper layers. In conclusion, the use of an existing cation-exchange resin for gradual biomass disintegration, combined with NGS and metaproteomic analysis was demonstrated as a promising approach for simultaneously investigating the identity and functions of microbes in multilayered biofilm structures.

Tetrabromobisphenol A (TBBPA) biodegradation in acidogenic systems: One step further on where and who.

The occurrence of brominated flame retardants such as Tetrabromobisphenol A (TBBPA) in water bodies poses a serious threat to aquatic ecosystems. Degradation of TBBPA in wastewater has successfully been demonstrated to occur through anaerobic digestion (AD), although the involved microorganisms and the conditions favouring the conversion remains unclear. In this study, it was observed that bioconversion of TBBPA did not occur during the hydrolytic stage of the AD, but during the strictly fermentative stage. Bioconversion occurred in hydrolytic-acidogenic as well as in strictly acidogenic continuous bioreactors. This indicates that the microorganisms that degrade TBBPA benefit from the electron flux taking place during glycolysis and further transformations into short-chain fatty acids. The degradation kinetics of TBBPA was inversely proportional to the complexity of the wastewater as the apparent kinetics constants were 2.11, 1.86, and 0.52 h-1·gVSS-1 for glucose, starch, and domestic sewage as carbon source, respectively. Additionally, the micropollutant loading rate relative to the overall organic loading rate is of major importance during the investigation of cometabolic transformations. The long-term exposure to TBBPA at environmentally realistic concentrations did not cause any major changes in the microbiome composition. Multivariate statistical analysis of the evolvement of the microbiome throughout the incubation suggested that Enterobacter spp. and Clostridium spp. are the key players in TBBPA degradation. Finally, a batch enrichment was conducted, which showed that concentrations of 0.5 mg·L-1 or higher are detrimental to Clostridium spp., even though these organisms are putative TBBPA degraders. The Clostridium genus was outcompeted by the Enterobacter and Klebsiella genera, hereby highlighting the effect of unrealistic concentrations frequently used in culture-dependent studies on the microbial community composition.

Wood-Ljungdahl pathway utilisation during in situ H2 biomethanation.

Biogas production from organic waste is a waste-to-energy technology with the potential to contribute significantly to sustainable energy production. Upgrading of biogas using in situ biomethanation with hydrogen has the potential for surplus electricity storage, and delivery of biogas with a methane content of >90%, allowing for easier integration into the natural gas grid, as well as conversion to other products. Microbial communities in biomethanation reactors undergo changes, however, these changes are largely unexplored. In the present study, metagenome-resolved protein stable isotope probing (Protein-SIP) was applied to laboratory scale batch incubations operating under anaerobic digestion, and (pre-adapted) biomethanation conditions, fed with 13C-labelled bicarbonate, in order to gain insight into the microbial activities during CO2-reduction. The strongest and most microbially diverse isotopic incorporation was observed in the pre-adapted biomethanation incubation. Furthermore, divergent incorporation of 13C-labelled bicarbonate was also observed in the Wood-Ljungdahl pathway, with the anaerobic digester incubations primarily showing labelled proteins in the peripheral pathways leading toward production of energy and biomass. The pre-adapted biomethanation incubations consumed H2 and CO2, but did not convert it to CH4, suggesting the production of acetate in these incubations, which was supported by heavy labelling of key enzymes in the Wood-Ljungdahl pathway. Twelve (ten high quality) metagenome-assembled genomes (MAGs) coding for 13C-incorporated proteins were extracted from the metagenome, eight of which contained one or more of the key genes in the Wood-Ljungdahl pathway, one of which was affiliated to Methanosarcina. Together, the findings in the present study deepen our knowledge surrounding microbial communities in biomethanation systems, and contribute to the development of better strategies for implementation of biogas upgrading and microbial management.

Physiological Responses of Aspergillus niger Challenged with Itraconazole.

Aspergillus niger is an opportunistic pathogen commonly found in a variety of indoor and outdoor environments. An environmental isolate of A. niger from a pig farm was resistant to itraconazole, and in-depth investigations were conducted to better understand cellular responses that occur during growth when this pathogen is exposed to an antifungal. Using a combination of cultivation techniques, antibiotic stress testing, and label-free proteomics, this study investigated the physiological and metabolic responses of A. niger to sublethal levels of antifungal stress. Challenging A. niger with itraconazole inhibited growth, and the MIC was estimated to be > 16 mg · liter-1 Through the proteome analysis, 1,305 unique proteins were identified. During growth with 2 and 8 mg · liter-1 itraconazole, a total of 91 and 50 proteins, respectively, were significantly differentially expressed. When challenged with itraconazole, A. niger exhibited decreased expression of peroxidative enzymes, increased expression of an ATP-binding cassette (ABC) transporter most likely involved as an azole efflux pump, and inhibited ergosterol synthesis; however, several ergosterol biosynthesis proteins increased in abundance. Furthermore, reduced expression of proteins involved in the production of ATP and reducing power from both the tricarboxylic acid (TCA) and glyoxylate cycles was observed. The mode of action of triazoles in A. niger therefore appears more complex than previously anticipated, and these observations may help highlight future targets for antifungal treatment.