ChipFilter: Microfluidic-Based Comprehensive Sample Preparation Methodology for Microbial Consortia.

The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.

Multiomics to Characterize the Molecular Events Underlying Impaired Glucose Tolerance in FXR-Knockout Mice.

The prevalence of different metabolic syndromes has grown globally, and the farnesoid X receptor (FXR), a metabolic homeostat for glucose, lipid, and bile acid metabolisms, may serve an important role in the progression of metabolic disorders. Glucose intolerance by FXR deficiency was previously reported and observed in our study, but the underlying biology remained unclear. To investigate the ambiguity, we collected the nontargeted profiles of the fecal metaproteome, serum metabolome, and liver proteome in Fxr-null (Fxr-/-) and wild-type (WT) mice with LC-HRMS. FXR deficiency showed a global impact on the different molecular levels we monitored, suggesting its serious disruption in the gut microbiota, hepatic metabolism, and circulating biomolecules. The network and enrichment analyses of the dysregulated metabolites and proteins suggested the perturbation of carbohydrate and lipid metabolism by FXR deficiency. Fxr-/- mice presented lower levels of hepatic proteins involved in glycogenesis. The impairment of glycogenesis by an FXR deficiency may leave glucose to accumulate in the circulation, which may deteriorate glucose tolerance. Lipid metabolism was dysregulated by FXR deficiency in a structural-dependent manner. Fatty acid ß-oxidations were alleviated, but cholesterol metabolism was promoted by an FXR deficiency. Together, we explored the molecular events associated with glucose intolerance by impaired FXR with integrated novel multiomic data.

Upgrade from aerated static pile to agitated bed systems promotes lignocellulose degradation in large-scale composting through enhanced microbial functional diversity.

Composting presents a viable management solution for lignocellulose-rich municipal solid waste. However, our understanding about the microbial metabolic mechanisms involved in the biodegradation of lignocellulose, particularly in industrial-scale composting plants, remains limited. This study employed metaproteomics to compare the impact of upgrading from aerated static pile (ASP) to agitated bed (AB) systems on physicochemical parameters, lignocellulose biodegradation, and microbial metabolic pathways during large-scale biowaste composting process, marking the first investigation of its kind. The degradation rates of lignocellulose including cellulose, hemicellulose, and lignin were significantly higher in AB (8.21%-32.54%, 10.21%-39.41%, and 6.21%-26.78%) than those (5.72%-23.15%, 7.01%-33.26%, and 4.79%-19.76%) in ASP at three thermal stages, respectively. The AB system in comparison to ASP increased the carbohydrate-active enzymes (CAZymes) abundance and production of the three essential enzymes required for lignocellulose decomposition involving a mixture of bacteria and fungi (i.e., Actinobacteria, Bacilli, Sordariomycetes and Eurotiomycetes). Conversely, ASP primarily produced exoglucanase and ß-glucosidase via fungi (i.e., Ascomycota). Moreover, AB effectively mitigated microbial stress caused by acetic acid accumulation by regulating the key enzymes involved in acetate conversion, including acetyl-coenzyme A synthetase and acetate kinase. Overall, the AB upgraded from ASP facilitated the lignocellulose degradation and fostered more diverse functional microbial communities in large-scale composting. Our findings offer a valuable scientific basis to guide the engineering feasibility and environmental sustainability for large-scale industrial composting plants for treating lignocellulose-rich waste. These findings have important implications for establishing green sustainable development models (e.g., a circular economy based on material recovery) and for achieving sustainable development goals.

Evaluation of protein extraction methods to improve meta-proteomics analysis of treated wastewater biofilms.

Metaproteomics can be used to study functionally active biofilm-based bacterial populations in reclaimed water distribution systems, which in turn result in bacterial regrowth that impacts the water quality. However, existing protein extraction methods have differences in their protein recovery and have not been evaluated for their efficacies in reclaimed water biofilm samples. In this study, we first evaluated six different protein extraction methods with diverse chemical and physical properties on a mixture of bacterial cell culture. Based on a weighting scores-based evaluation, the extraction protocols in order of decreasing performance are listed as B-PER > RIPA > PreOmics > SDS > AllPrep > Urea. The highest four optimal methods on cell culture were further tested against treated wastewater non-chlorinated and chlorinated effluent biofilms. In terms of protein yield, our findings showed that RIPA performed the best; however, the highest number of proteins were extracted from SDS and PreOmics. Furthermore, SDS and PreOmics worked best to rupture gram-positive and gram-negative bacterial cell walls. Considering the five evaluation factors, PreOmics obtained highest weighted score, indicating its potential effectiveness in extracting proteins from biofilms. This study provides the first insight into evaluating protein extraction methods to facilitate metaproteomics for complex reclaimed water matrices.

MetaFS: Performance assessment of biomarker discovery in metaproteomics.

Metaproteomics suffers from the issues of dimensionality and sparsity. Data reduction methods can maximally identify the relevant subset of significant differential features and reduce data redundancy. Feature selection (FS) methods were applied to obtain the significant differential subset. So far, a variety of feature selection methods have been developed for metaproteomic study. However, due to FS's performance depended heavily on the data characteristics of a given research, the well-suitable feature selection method must be carefully selected to obtain the reproducible differential proteins. Moreover, it is critical to evaluate the performance of each FS method according to comprehensive criteria, because the single criterion is not sufficient to reflect the overall performance of the FS method. Therefore, we developed an online tool named MetaFS, which provided 13 types of FS methods and conducted the comprehensive evaluation on the complex FS methods using four widely accepted and independent criteria. Furthermore, the function and reliability of MetaFS were systematically tested and validated via two case studies. In sum, MetaFS could be a distinguished tool for discovering the overall well-performed FS method for selecting the potential biomarkers in microbiome studies. The online tool is freely available at https://idrblab.org/metafs/.

Multi-omics analysis of fecal samples in colorectal cancer Egyptians patients: a pilot study.

BACKGROUND: Colorectal cancer (CRC) is a public health concern and the second most common disease worldwide. This is due to genetic coding and is influenced by environmental aspects, in which the gut microbiota plays a significant role. The purpose of this study was to compare the microbiota makeup of CRC patients with that of healthy control and to identify upregulated and downregulated proteins and metabolites in CRC patients. Using a next-generation sequencing approach, fecal samples of five females (4 CRC patients and one healthy control) were analyzed by BGI DNBSEQ-T7, Hong Kong, China. Furthermore, proteomics and metabolomics analysis were performed using LC-MS/MS technique. RESULTS: Dysbiosis of gut microbiota has been observed in patients with CRC, with an increase in microbiota diversity at all taxonomic levels relative to healthy control. Where, at the functional level the bacterial species participate in many different pathways among them de novo nucleotide synthesis and amino acids pathways were aberrantly upregulated in CRC patients. Proteomics and metabolomics profiles of CRC patients showed different proteins and metabolites, a total of 360 and 158 proteins and metabolites, respectively were highly expressed compared to healthy control with fold change ≥ 1.2. Among the highly expressed proteins were transketolase, sushi domain-containing protein, sulfide quinone oxidoreductase protein, AAA family ATPase protein, carbonic anhydrase, IgG Fc-binding protein, nucleoside diphosphate kinase protein, arylsulfatase, alkaline phosphatase protein, phosphoglycerate kinase, protein kinase domain-containing protein, non-specific serine/threonine protein kinase, Acyl-CoA synthetase and EF-hand domain-containing protein. Some of the differential metabolites, Taurine, Taurocholic acid, 7-ketodeoxycholic acid, Glycochenodeoxycholic acid, Glycocholic acid, and Taurochenodeoxycholic acid that belong to bile acids metabolites. CONCLUSIONS: Some bacterial species, proteins, and metabolites could be used as diagnostic biomarkers for CRC. Our study paves an insight into using multi-omics technology to address the relationship between gut microbiota and CRC.

Integrating the serum proteomic and fecal metaproteomic to analyze the impacts of overweight/obesity on IBD: a pilot investigation.

BACKGROUND: Inflammatory bowel disease (IBD) encompasses a group of chronic relapsing disorders which include ulcerative colitis (UC) and Crohn's disease (CD). The incidences of IBD and overweight/obesity are increasing in parallel. Here, we investigated alterations in proteomic in serum and metaproteomic in feces of IBD patients with overweight/obesity and aimed to explore the effect of overweight/ obesity on IBD and the underlying mechanism. METHODS: This prospective observational study (n = 64) comprised 26 health control subjects (HC, 13 with overweight/obesity) and 38 IBD patients (19 with overweight/obesity) at a tertiary hospital. Overweight/obesity was evaluated by body mass index (BMI) and defined as a BMI greater than 24 kg/m2. The comprehensive serum proteomic and fecal metaproteomic analyses were conducted by ultra-performance liquid chromatography-Orbitrap Exploris 480 mass spectrometry. RESULTS: UC and CD presented similar serum molecular profiles but distinct gut microbiota. UC and CD serum exhibited higher levels of cytoskeleton organization- associated and inflammatory response-related proteins than the HC serum. Compared the serum proteome of UC and CD without overweight/obesity, inflammatory response-associated proteins were dramatically decreased in UC and CD with overweight/obesity. Fecal metaproteome identified 66 species in the feces. Among them, Parasutterella excrementihominis was increased in CD compared with that in HC. UC group had a significant enrichment of Moniliophthora roreri, but had dramatically decreased abundances of Alistipes indistinctus, Clostridium methylpentosum, Bacteroides vulgatus, and Schizochytrium aggregatum. In addition, overweight/obesity could improve the microbial diversity of UC. Specifically, the UC patients with overweight/obesity had increased abundance of some probiotics in contrast to those without overweight/obesity, including Parabacteroides distasonis, Alistipes indistincus, and Ruminococcus bromii. CONCLUSION: This study provided high-quality multi-omics data of IBD serum and fecal samples, which enabled deciphering the molecular bases of clinical phenotypes of IBD, revealing the impacts of microbiota on IBD, and emphasizing the important role of overweight/obesity in IBD.

The active synergetic microbiota with Aspergillus as the core dominates the metabolic network of ester synthesis in medium-high temperature Daqu.

The active ester-synthesis microorganisms in medium-high temperature Daqu (MHT-Daqu) largely impact the strong-flavor Baijiu quality, while their actual composition and metabolic mechanism remain unclear. Here, to explore how the active microbiota contributes to MHT-Daqu ester biosynthesis, metatranscriptomic and metaproteomic analyses coupled with experimental verification were performed. The results showed that the MHT-Daqu microbiota with the higher ester-forming ability exhibited a more active dynamic alteration from transcription to translation. The genera Aspergillus, Bacillus, Leuconostoc, and Pediococcus could transcribe and translate obviously more ester-forming enzymes. In the ester-synthesis metabolic network, the synergetic microbiota confirmed by interaction analysis, containing Eurotiales, Bacillales, and Saccharomycetales, played an essential role, in which the Eurotiales and its representative genus Aspergillus contributed the highest transcript and protein abundance in almost every metabolic process, respectively. The recombined fermentation verified that their corresponding genera could produce the ester and precursor profiles very close to that of the original MHT-Daqu active microbiota, while the microbiota without Aspergillus caused a polar separation. These results indicated that the synergetic microbiota with Aspergillus as the core dominated the metabolic network of ester synthesis in MHT-Daqu. Our study provides a detailed framework of the association between the active synergetic microbiota and ester synthesis in MHT-Daqu.

Deciphering different effects of ZVI and NaOH on metabolic characteristics in the process of methanogenesis recovery from VFA suppression.

Dosing zero valent iron (ZVI) or sodium hydroxide (NaOH) is the common method of addressing acidification in anaerobic digestion (AD) systems; however, few studies have discussed and compared their effects on microbial metabolism. In the present study, microbial syntrophy and metabolic pathways under ZVI and NaOH regulation are comparatively analyzed through microbial network analysis and metagenomic/metaproteomic analyses. CH4 yield in the ZVI reactor was 414 mL/gVS, an increase of 23% when compared with that in the reactor with NaOH dosing (336 mL/gVS). The methanogenesis recovery period in the ZVI reactor (37 days) was shorter than that in the NaOH reactor (48 days). Co-occurrence networks indicated that ZVI promoted Methanoculleus and Methanosarcina to establish a complex syntrophic association with SAO bacteria (Syntrophaceticus and Aminobacterium) and syntrophic acetogens (Syntrophomonas), strengthening SAO-hydrogenotrophic methanogenesis (HM) and acetoclastic methanogenesis (AM) pathways simultaneously. Metagenomic analysis showed that the relative abundance of mcrA and fwdB in the ZVI reactor was higher 27% than that in the NaOH reactor. Furthermore, through metaproteomics analysis, much more enzymes related to glucose degradation, bioconversion of butyric acid and pyruvate, conversion of formate and acetate to CO2, and production of CH4 from acetate and CO2 were significantly upregulated under ZVI regulation than under NaOH regulation (fold change relative to control [FC] > 1.5, p < 0.05). The results of the present study enhance our understanding of methanogenic mechanisms under the regulation of ZVI, providing a theoretical basis for its practical application in AD systems experiencing VFA suppression.

Gut microbiome alterations and gut barrier dysfunction are associated with host immune homeostasis in COVID-19 patients.

BACKGROUND: COVID-19 is an infectious disease characterized by multiple respiratory and extrapulmonary manifestations, including gastrointestinal symptoms. Although recent studies have linked gut microbiota to infectious diseases such as influenza, little is known about the role of the gut microbiota in COVID-19 pathophysiology. METHODS: To better understand the host-gut microbiota interactions in COVID-19, we characterized the gut microbial community and gut barrier function using metagenomic and metaproteomic approaches in 63 COVID-19 patients and 8 non-infected controls. Both immunohematological parameters and transcriptional profiles were measured to reflect the immune response in COVID-19 patients. RESULTS: Altered gut microbial composition was observed in COVID-19 patients, which was characterized by decreased commensal species and increased opportunistic pathogenic species. Severe illness was associated with higher abundance of four microbial species (i.e., Burkholderia contaminans, Bacteroides nordii, Bifidobacterium longum, and Blautia sp. CAG 257), six microbial pathways (e.g., glycolysis and fermentation), and 10 virulence genes. These severity-related microbial features were further associated with host immune response. For example, the abundance of Bu. contaminans was associated with higher levels of inflammation biomarkers and lower levels of immune cells. Furthermore, human-origin proteins identified from both blood and fecal samples suggested gut barrier dysfunction in COVID-19 patients. The circulating levels of lipopolysaccharide-binding protein increased in patients with severe illness and were associated with circulating inflammation biomarkers and immune cells. Besides, proteins of disease-related bacteria (e.g., B. longum) were detectable in blood samples from patients. CONCLUSIONS: Our results suggest that the dysbiosis of the gut microbiome and the dysfunction of the gut barrier might play a role in the pathophysiology of COVID-19 by affecting host immune homeostasis.

Lead removal in flue gas from sludge incineration by denitrification: Insights from metagenomics and metaproteomics.

Flue gas lead emission during sludge incineration damages to human health and ecological environment seriously. Therefore, a denitrifying bio-trickling filter (DNBTF) for lead removal in flue gas from sludge incineration was investigated. Lead removal efficiency was up to 90.7% in 60 days' operation. Lead speciation in biofilms of DNBTF consists of 84.27% residue lead, 15.18% organic bound lead, and less than 1% exchangeable and reducible lead. Lead resistant bacteria and lead resistant-denitrifying bacteria accounted for 85.04% and 58.25%, respectively. Lead resistant microorganisms(Pseudomonas, Azoarcus, Stappia, Pararhodobacter, Paracoccus, Azospirillum, Hyphomonas, Rhodobacter, Polymorphum, Brevunimonas, Stenotrophomonas) could resist the toxicity of Pb2+ in flue gas by transport protein and binding protein, and detoxify Pb2+ in flue gas by extracellular polymeric substances (EPS) adsorption, protein binding and precipitation under the action of resistance genes, such as pbrAB, golT, troABCD, znuABC, czcABCD, pcoB, copA, as shown by integrated metagenomic and metaproteomic analyses. The biofilm was characterized by FTIR, XRD, 3D-EEM, and SEM-EDS. XRD and SEM-EDS spectra indicated the formation of pyromorphite from bioconversion of lead in flue gas. Lead-containing flue gas was bio-stabilized in the form of pyromorphite and HA-Pb via complexation of humic acids in extracellular polymeric substances (EPS), biosorption and biodeposition. This provides a new way of sludge incineration flue gas lead removal using a denitrifying biotricking filter.

Integrated Metagenomic and Metaproteomic Analyses Unravel Ammonia Toxicity to Active Methanogens and Syntrophs, Enzyme Synthesis, and Key Enzymes in Anaerobic Digestion.

During anaerobic digestion, the active microbiome synthesizes enzymes by transcription and translation, and then enzymes catalyze multistep bioconversions of substrates before methane being produced. However, little information is available on how ammonia affects truly active microbes containing the expressed enzymes, enzyme synthesis, and key enzymes. In this study, an integrated metagenomic and metaproteomic investigation showed that ammonia suppressed not only the obligate acetotrophic methanogens but also the syntrophic propionate and butyrate oxidation taxa and their assistant bacteria (genus Desulfovibrio), which declined the biotransformations of propionate and butyrate → acetate → methane. Although the total population of the hydrolyzing and acidifying bacteria was not affected by ammonia, the bacteria with ammonia resistance increased. Our study also revealed that ammonia restrained the enzyme synthesis process by inhibiting the RNA polymerase (subunits A' and D) during transcription and the ribosome (large (L3, L12, L13, L22, and L25) and small (S3, S3Ae, and S7) ribosomal subunits) and aminoacyl-tRNA synthesis (aspartate-tRNA synthetase) in translation. Further investigation suggested that methylmalonyl-CoA mutase, acetyl-CoA C-acetyltransferase, and CH3-CoM reductase, which regulate propionate and butyrate oxidation and acetoclastic methanation, were significantly downregulated by ammonia. This study provides intrinsic insights into the fundamental mechanisms of how ammonia inhibits anaerobic digestion.

Meta-omics reveal microbial assortments and key enzymes in bean sauce mash, a traditional fermented soybean product.

BACKGROUND: Dajiang is fermented based on the metabolism of microbial communities in bean sauce mash, a traditional fermented soybean product in China. The current study first investigated the metaproteome of bean sauce mash. This was followed by an analysis of its biological functions and its microbial community to reveal information about strains and about the expressed proteins to better understand the roles of the microbiota in bean sauce mash. RESULTS: The metaproteomic results demonstrated that a total of 1415 microbial protein clusters were expressed mainly by members of the Penicillium and Rhizopus genera and were classified into 100 cellular components, 238 biological processes, and 220 molecular function categories by gene ontology (GO) annotation. Enzymes associated with glycolysis metabolic pathways were also identified. These can provide the energy required for microbial fermentation. Illumina MiSeq sequencing technology results showed that the microorganism communities of bean sauce mash exhibited a high level of diversity. Microbiological analysis demonstrated that the Penicillium, Mucor, Fusarium, Aspergillus, and Rhizopus fungi, and Lactobacillus, Enterococcus, Fructobacillus, Staphylococcus, Carnobacterium genera were predominant 22 samples. CONCLUSION: The profiles and insights in the current study are important for research on bean sauce mash and related products in terms of their food microbial ecology. The information obtained from this study will help the development of stable sufu starter cultures with unique sensory qualities. © 2019 Society of Chemical Industry.

Biosynthetic potential of uncultured anammox community bacteria revealed through multi-omics analysis.

Microbial secondary metabolites (SMs) and their derivatives have been widely used in medicine, agriculture, and energy. Growing needs for renewable energy and the challenges posed by antibiotic resistance, cancer, and pesticides emphasize the crucial hunt for new SMs. Anaerobic ammonium-oxidation (anammox) systems harbor many uncultured or underexplored bacteria, representing potential resources for discovering novel SMs. Leveraging HiFi long-read metagenomic sequencing, 1,040 biosynthetic gene clusters (BGCs) were unearthed from the anammox microbiome with 58% being complete and showcasing rich diversity. Most of them showed distant relations to known BGCs, implying novelty. Members of the underexplored lineages (Chloroflexota and Planctomycetota) and Proteobacteria contained lots of BGCs, showcasing substantial biosynthetic potential. Metaproteomic results indicated that Planctomycetota members harbored the most active BGCs, particularly those involved in producing potential biofuel-ladderane. Overall, these findings underscore that anammox microbiomes could serve as valuable resources for mining novel BGCs and discovering new SMs for practical application.

Metaproteomic investigation of enzyme profile in daqu used for the production of Nongxiangxing baijiu.

Enzymes and microbiota in daqu are essential for the brewing of Nongxiangxing baijiu. Uncover the key enzymes and functional strains in daqu is beneficial to improve the flavor and quality of Nongxiangxing baijiu. In this study, metaproteome technology was employed to determine the enzyme profiles in Nongxiangxing daqu, and strains with high saccharification activity were screened and identified. 933 proteins were identified in daqu, of which 463 belonged to enzymes, including 140 oxidoreductases, 98 transferases, 91 hydrolases, 49 ligases, 41 lyases and 27 isomerases, and hydrolase is the enzyme with the highest abundance in baijiu brewing. Among hydrolases, a total of 36 carbohydrate metabolism-related enzymes (CMEs) were identified, and 12 of them were key enzymes related to glycoside hydrolysis. Four major glycoside hydrolysis enzymes glucoamylase (EC 3.2.1.3), glucan 1,4-alpha-glucosidase (EC 3.2.1.3), glucanase (EC 3.2.1.-) and ß-glucosidase (EC 3.2.1.21) were revealed, and their sources were Byssochlamys spectabilis, Lichtheimia ramosa and Thermoascus aurantiacus, respectively. Then, strains Aspergillus A2, A3, A7, Lichtheimia L1, L4, L5, and Saccharomycopsis S2, S4, S6 with high saccharifying enzyme-producing capacity were screened through culture-dependent approach. Resents presented in this study can further reveal the enzyme profiles and identify the main functional strains in daqu, which can provide theoretical support for the brewing of Nongxiangxing baijiu.

Metagenomics and proteomics profiling of extracellular polymeric substances from municipal waste sludge and their application for soil and water bioremediation.

This study assessed the components of anaerobically digested sludge, activated sludge, and microbial and extracellular polymeric substance (EPS) enzymes to identify the mechanisms underlying nitrogen removal and soil regeneration. 16S rRNA gene amplicon-based sequencing was used to determine the microbial community composition and the related National Center for Biotechnology Information (NCBI) protein database was used to construct a conventional library from the observed community. EPS components were identified using gel-free proteomic (Liquid Chromatography with tandem mass spectrometry-LC/MS/MS) methods. Alginate-like EPS from aerobically activated sludge have strong potential for soil aggregation and water-holding capacity, whereas total EPS from anaerobic sludge have significant potential for ammonia removal under salt stress. Fourier transform infrared spectroscopy (FTIR) revealed that both EPS may contain proteins, carbohydrates, humic compounds, uronic acid, and DNA and determined the presence of O-H, N-H, C-N, CO, and C-H functional groups. These results demonstrate that the overall enzyme activity may be inactivated at 30 g L-1 of salinity. An annotation found in Kyoto Encyclopedia of Genes and Genomes (KEGG)- KEGG Automatic Annotation Server (KAAS) revealed that the top two metabolic activities in the EPS generated from the anaerobic sludge were methane and nitrogen metabolism. Therefore, we focused on the nitrogen metabolism reference map 00910. EPS from the anaerobically digested sludge exhibited nitrate reductase, nitrite reductase, and dehydrogenase activities. Assimilatory nitrate reduction, denitrification, nitrification, and anammox removed ammonia biochemically. The influence of microbial extracellular metabolites on water-holding capacity and soil aggregation was also investigated. The KAAS-KEGG annotation server was used to identify the main enzymes in the activated sludge-derived alginate-like extracellular EPS (ALE-EPS) samples. These include hydrolases, oxidoreductases, lyases, ligases, and transporters, which contribute to soil fertility and stability. This study improves our understanding of the overall microbial community structure and the associated biochemical processes, which are related to distinct functional genes or enzymes involved in nitrogen removal and soil aggregation. In contrast to conventional methods, microbial association with proteomics can be used to investigate ecological relationships, establishments, key player species, and microbial responses to environmental changes. Linking the metagenome to off-gel proteomics and bioinformatics solves the problem of analyzing metabolic pathways in complex environmental samples in a cost-effective manner.

Nonmonotonic effect of CuO nanoparticles on medium-chain carboxylates production from waste activated sludge.

The growing applications of CuO nanoparticles (NPs) in industrial and agriculture has increased their concentrations in wastewater and subsequently accumulated in waste activated sludge (WAS), raising concerns about their impact on reutilization of WAS, especially on the medium-chain carboxylates (MCCs) production from anaerobic fermentation of WAS. Here we showed that CuO NPs at 10-50 mg/g-TS can significantly inhibit MCCs production, and reactive oxygen species generation was revealed to be the key factor linked to the phenomena. At lower CuO NPs concentrations (0.5-2.5 mg/g-TS), however, MCCs production was enhanced, with a maximum level of 37% compared to the control. The combination of molecular approaches and metaproteomic analysis revealed that although low dosage CuO NPs (2.5 mg/g-TS) weakly inhibited chain elongation process, they displayed contributive characteristics both in WAS solubilization and transport/metabolism of carbohydrate. These results demonstrated that the complex microbial processes for MCCs production in the anaerobic fermentation of WAS can be affected by CuO NPs in a dosage-dependent manner via regulating microbial protein expression level. Our findings can provide new insights into the influence of CuO NPs on anaerobic fermentation process and shed light on the treatment option for the resource utilization of CuO NPs polluted WAS.

The differences in carbohydrate utilization ability between six rounds of Sauce-flavor Daqu.

Sauce-flavor Daqu is an important source of fermentation power in baijiu brewing. Revealing carbohydrate metabolism will help to explore the underlying reasons for the difference in fermentation performance of Daqu. In this study, metagenomic and metaproteomic technologies were performed to explore the carbohydrate metabolism network and its active functional microorganisms of Sauce-flavor Daqu. The sugar profile was analyzed using LC-MS to confirm the metabolic network. The results showed that 23 fungi and 5 bacteria were involved in carbohydrate metabolism. Starch metabolism, cellulose metabolism, and glucan metabolism were the main metabolic pathways, in which fungi especially Aspergillus were more involved than bacteria. Among these active microorganisms, Saccharomycopsis fibuligera, Aspergillus oryzae, Monascus purpureus, Byssochlamys spectabilis, Lichtheimia ramosa, Thermomyces lanuginosus, and Thermoascus aurantiacus were significant functional microorganisms with the ability to produce multiple enzymes. Lichtheimia ramosa, Lichtheimia corymbifera and Kroppenstedtia eburnea were biomarkers of Daqu in the first round, granting it a better liquefaction ability. ß-amylase derived from wheat also played an important role in starch degradation, and the synergistic effect with α-amylase endowed Daqu with higher liquefaction power in the first two rounds. The results of this study are of great significance for the analysis of the mechanism of Daqu fermentation and provide a reliable theoretical basis for strengthening the fermentation performance of Daqu.

Metaproteomic and Metagenomic-Coupled Approach to Investigate Microbial Response to Electrochemical Conditions in Microbial Fuel Cells.

MFCs represent a promising sustainable biotechnology that enables the direct conversion of organic matter from wastewater into electricity using bacterial biofilms as biocatalysts. A crucial aspect of MFCs is how electroactive bacteria (EAB) behave and their associated mechanisms during extracellular electron transfer to the anode. A critical phase in the MFC start-up process is the initial colonization of the anode by EAB. Two MFCs were operated with an external resistance of 1000 ohms, one with an applied electrical voltage of 500 mV during the initial four days of biofilm formation and the other without any additional applied voltage. After stabilization of electricity production, total DNA and protein were extracted and sequenced from both setups. The combined metaproteomic/metagenomic analysis revealed that the application of voltage during the colonization step predominantly increased direct electron transfer via cytochrome c, mediated primarily by Geobacter sp. Conversely, the absence of applied voltage during colonization resulted in a broader diversity of bacteria, including Pseudomonas and Aeromonas, which participated in electricity production via mediated electron transfer involving flavin family members.

Metagenomic and metaproteomic analyses of microbial amino acid metabolism during Cantonese soy sauce fermentation.

Cantonese soy sauce is an important type of traditional Chinese brewed soy sauce that was developed in southern China, mainly in Guangdong. Due to the long fermentation period and complex microbiota in Cantonese soy sauce, there are few reports on the microbial metaproteomics of Cantonese soy sauce. In this study, integrative metagenomic and metaproteomic analyzes were used to identify the changes in the dominant microbiota and amino acid synthesis-related enzymes and metabolism during Cantonese soy sauce fermentation. Metagenomic analysis revealed that Tetragenococcus halophilus, Weissella confusa, Weissella paramesenteroides, Enterobacter hormaechei, and Aspergillus oryzae were the dominant microbiota. Using the Top 15 dominant microbiota identified by metagenomics as the database, LTQ Orbitrap Velos Pro ETD mass spectrometry was used to obtain metaproteomic information about the microbes in the soy sauce, and the results indicated that the active enzymes involved in the metabolism of amino acids were secreted by microorganisms such as A. oryzae, T. halophilus, and Zygosaccharomyces rouxii. During the Cantonese soy sauce fermentation process. Among them, early fermentation (0-15d) was dominated by A. oryzae and T. halophilus, mid-term fermentation (60-90d) was dominated by Z. rouxii, A. oryzae, and T. halophilus, and late fermentation (90-120d) was dominated by A. oryzae, Z. rouxii, and T. halophilus. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the main enzymes involved in the metabolism of umami amino acids were aspartate aminotransferase, citrate synthase, aconitase, and isocitrate dehydrogenase, which were produced by Z. rouxii and A. oryzae during early fermentation (0-15 d) and the middle fermentation stage (60-90 d). This study constructed a regulatory network of enzymes potentially involved in the metabolism of flavor amino acids, which provided a theoretical basis for studying the amino acid metabolism of Cantonese soy sauce.