Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs.

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.

Looking for lipases and lipolytic organisms in low-temperature anaerobic reactors treating domestic wastewater.

Poor lipid degradation limits low-temperature anaerobic treatment of domestic wastewater even when psychrophiles are used. We combined metagenomics and metaproteomics to find lipolytic bacteria and their potential, and actual, cold-adapted extracellular lipases in anaerobic membrane bioreactors treating domestic wastewater at 4 and 15 °C. Of the 40 recovered putative lipolytic metagenome-assembled genomes (MAGs), only three (Chlorobium, Desulfobacter, and Mycolicibacterium) were common and abundant (relative abundance ≥ 1%) in all reactors. Notably, some MAGs that represented aerobic autotrophs contained lipases. Therefore, we hypothesised that the lipases we found are not always associated with exogenous lipid degradation and can have other roles such as polyhydroxyalkanoates (PHA) accumulation/degradation and interference with the outer membranes of other bacteria. Metaproteomics did not provide sufficient proteome coverage for relatively lower abundant proteins such as lipases though the expression of fadL genes, long-chain fatty acid transporters, was confirmed for four genera (Dechloromonas, Azoarcus, Aeromonas and Sulfurimonas), none of which were recovered as putative lipolytic MAGs. Metaproteomics also confirmed the presence of 15 relatively abundant (≥ 1%) genera in all reactors, of which at least 6 can potentially accumulate lipid/polyhydroxyalkanoates. For most putative lipolytic MAGs, there was no statistically significant correlation between the read abundance and reactor conditions such as temperature, phase (biofilm and bulk liquid), and feed type (treated by ultraviolet light or not). Results obtained by metagenomics and metaproteomics did not confirm each other and extracellular lipases and lipolytic bacteria were not easily identifiable in the anaerobic membrane reactors used in this study. Further work is required to identify the true lipid degraders in these systems.

Temperature and immigration effects on quorum sensing in the biofilms of anaerobic membrane bioreactors.

Quorum sensing (QS), a microbial communication mechanism modulated by acyl homoserine lactone (AHL) molecules impacts biofilm formation in bioreactors. This study investigated the effects of temperature and immigration on AHL levels and biofouling in anaerobic membrane bioreactors. The hypothesis was that the immigrant microbial community would increase the AHL-mediated QS, thus stimulating biofouling and that low temperatures would exacerbate this. We observed that presence of immigrants, especially when exposed to low temperatures indeed increased AHL concentrations and fouling in the biofilms on the membranes. At low temperature, the concentrations of the main AHLs observed, N-dodecanoyl-L-homoserine lactone and N-decanoyl-L-homoserine lactone, were significantly higher in the biofilms than in the sludge and correlated significantly with the abundance of immigrant bacteria. Apparently low temperature, immigration and denser community structure in the biofilm stressed the communities, triggering AHL production and excretion. These insights into the social behaviour of reactor communities responding to low temperature and influx of immigrants have implications for biofouling control in bioreactors.

Mitigation of membrane biofouling in membrane bioreactor treating sewage by novel quorum quenching strain of Acinetobacter originating from a full-scale membrane bioreactor.

A novel quorum quenching (QQ) strain, Acinetobacter guillouiae ST01, was isolated from a full-scale membrane bioreactor (MBR) and characterized for its QQ activities. Batch reactor studies at lab-scale showed that A. guillouiae ST01 exhibited higher QQ activity against acyl homoserine lactones (AHLs) with an oxo group compared to those without an oxo group. The organism was then inoculated (10%) in an MBR (Q-MBR) treating sewage over 48 days and was found to reduce quorum sensing (QS) activity by reducing AHL concentrations in the sludge and the biofilm of the Q-MBR. The concentration of polysaccharides was reduced up to 30% in both the biofilm and sludge relative to the control, whereas protein concentrations were reduced by 40% and 47% in the sludge and biofilm, respectively. The Q-MBR fouling rates were halved. These results indicate that A. guillouiae ST01 is a promising strain for biofouling reduction in MBR treating real wastewater.

Integrated air cathode microbial fuel cell-aerobic bioreactor set-up for enhanced bioelectrodegradation of azo dye Acid Blue 29.

In this study, an azo dye (Acid Blue 29 or AB29) was efficiently degraded with acetate as co-substrate into less contaminated biodegraded products using an integrated single chamber microbial fuel cell (SMFC)-aerobic bioreactor set-up. The decolorization efficiencies were varied from 91 ± 2% to 94 ± 1.9% and more than 85% of chemical oxygen demand (COD) removal was achieved for all dye concentrations after different operating time. The highest coulombic efficiency (CE) and cell potential were 3.18 ± 0.45% and 287.2 mV, respectively, for SMFC treating 100 mg L-1 of AB29. Electrochemical impedance spectroscopy (EIS) revealed that the anode resistance was 0.3 Ω representing an entirely grown biofilm on the anode surface resulted in higher electron transfer rate. Gas chromatography coupled mass spectrometry (GC-MS) investigation demonstrated that initially biodegradation of AB29 started with the cleavage of the azo bond (-N=N-), resulted the biotransformation into aromatic amines. In successive aerobic treatment stage, these amines were biodegraded into lower molecular weight compounds. The 16S rRNA microbial community analysis indicated that at phylum level, both inoculum and dye acclimated cultures were mainly consisting of Proteobacteria which was 27.9, 53.6 and 68.9% in inoculum, suspension and anodic biofilm, respectively. At genus level, both suspension and biofilm contained decolorization as well as electrochemically active bacteria. The outcomes exhibited that the AB29 decolorization would contest with electrogenic bacteria for electrons.

Diversity of Acyl Homoserine Lactone Molecules in Anaerobic Membrane Bioreactors Treating Sewage at Psychrophilic Temperatures.

This study explores the types of acyl homoserine lactone (AHL) and their concentrations in different compartments of different conventional anaerobic bioreactors: (i) an upflow anaerobic membrane bioreactor (UAnMBR, biofilm/mixed liquor (sludge)); (ii) an anaerobic membrane bioreactor (AnMBR, biofilm/mixed liquor (sludge)); and (iii) an upflow sludge blanket (UASB, sludge only), all operating at 15 °C. Ten types of the AHL, namely C4-HSL, 3-oxo-C4-HSL, C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-HSL, and 3-oxo-C12-HSL, which were investigated in this study, were found in UAnMBR and UASB, whilst only six of them (C4-HSL, 3-oxo-C4-HSL, C8-HSL, C10-HSL, 3-oxo-C10-HSL, and C12-HSL) were found in AnMBR. Concentrations of total AHL were generally higher in the biofilm than the sludge for both membrane bioreactors trialed. C10-HSL was the predominant AHL found in all reactors (biofilm and sludge) followed by C4-HSL and C8-HSL. Overall, the UAnMBR biofilm and sludge had 10-fold higher concentrations of AHL compared to the AnMBR. C10-HSL was only correlated with bacteria (p < 0.05), whilst other types of AHL were correlated with both bacteria and archaea. This study improves our understanding of AHL-mediated Quorum Sensing (QS) in the biofilms/sludge of UAnMBR and AnMBR, and provides new information that could contribute to the development of quorum quenching anti-fouling strategies in such systems.

Low-Temperature Pretreatment of Organic Feedstocks with Selected Mineral Wastes Sustains Anaerobic Digestion Stability through Trace Metal Release.

A low-cost approach for enhancing mesophilic (37 °C) anaerobic digestion (AD) of organic waste using a low-temperature (37 °C) pretreatment with different mineral wastes (MW) was investigated. A higher and stable methane production rate, in comparison to MW-free controls, was achieved for 80 days at organic loading rates of 1-2 g VS/L·d, using a feed substrate pretreated with incinerator bottom ash (IBA). The boiler ash and cement-based waste pretreatments also produced high methane production rates but with some process instability. In contrast, an incinerator fly ash pretreatment showed a progressive decrease in methane production rates and poor process stability, leading to reactor failure after 40 days. To avoid process instability and/or reactor failure, two metrics had to be met: (a) a methanogenesis to fermentation ratio higher than 0.6 and (b) a cell-specific methanogenic activity to cell-specific fermentation activity ratio of >1000. The prevalence of Methanofastidiosum together with a mixed community of acetoclastic (Methanosaeta) and hydrogenotrophic (Methanobacterium) methanogens in the stable IBA treatment indicated the importance of Methanofastidiosum as a potential indicator of a healthy and stable reactor.

Improving the methane productivity of anaerobic digestion using aqueous extracts from municipal solid waste incinerator ash.

This study investigated the effects of mineral waste extracts (MWE) on laboratory-scale two-stage anaerobic digesters treating synthetic organic waste. MWE was prepared as aqueous extracts from different ash samples (incineration bottom ash (IBA), fly ash (FA) and boiler ash (BA) taken from a municipal solid waste incineration plant. At 20 days hydraulic retention time, all three MWE stimulated hydrogen production in their respective acidogenic reactor by around 35% (c.f. control acidogenic reactor), whilst no difference was seen in the methane productivity of the linked methanogenic reactors (average 527 ± 45 mL CH4/g VS, including control methanogenic reactor). Following a step reduction in hydraulic retention time from 20 to 10 days and a doubling of the organic loading rate from 2.5 g to 5 g VS/L. d, no significant change was seen in hydrogen production (p > 0.05) in the acidogenic reactor amended with MWE from IBA and BA, or the control acidogenic reactor. However, the acidogenic reactor receiving MWE from FA had 45% lower hydrogen productivity. The step change in hydraulic retention time and organic loading rates led to the failure of most methanogenic reactors (≤100 mL CH4/g VS), however, the one receiving feed containing MWE from IBA showed stable performance without signs of failure, and had higher volumetric methane productivity, albeit at lower methane yields (370 ± 20 mL CH4/g VS). 16S rRNA analysis using the Illumina sequencing platform revealed acidogenesis by Lactobacillaceae in the acidogenic reactor and syntrophic acetate oxidation by Synergistaceae linked to enrichment of the candidatus genus Methanofastidiosum, in the stable methanogenic reactor receiving MWE from IBA.

Data of metal and microbial analyses from anaerobic co-digestion of organic and mineral wastes.

High concentrations of minerals, heavy metals are often found in mineral wastes (MWs) originated from municipal solid waste incineration plants, so as construction/demolition sites. Such by-products (minerals) often have buffering capacity. The current work provides analysis of total and soluble (dissolved) metal concentrations released by four different MWs (a. cement-based waste, b. incineration (bottom), c. fly and d. boiler ash) supplemented to anaerobic reactors of organic waste at 37 °C. The reactors (continuous stirred tank reactor (CSTR)) were ran for 75 days at hydrolytic retention time of 20 days. Genomic DNA extraction, and qPCR and Illumina HiSeq (16S V4) analyses were conducted to investigate microbial community population and composition in anaerobic digestate samples collected from these reactors. Output data from Illumina sequencing analysis were FastQ files analysed using the QIIME2 pipeline to produce a feature table listing the frequency of each assigned microbial taxa per samples. Additional study was conducted on the microbial data to visualise variations in microbial communities using the STAMP software and phyloseq R package. Detailed interpretation and discussion of the results can be found in the related research article [1].

Co-digestion of organic and mineral wastes for enhanced biogas production: Reactor performance and evolution of microbial community and function.

Mineral wastes (MWs) from municipal solid waste incineration plants and construction demolition sites are rich in minerals, heavy metals and have acid neutralising capacity. This renders such MWs a promising source of bulk and trace elements to enhance and stabilize biogas production in anaerobic processes. However, finding a MW with typical heavy metal concentrations, which promotes anaerobic digestion (AD) without adverse effects on the microbial community of the reactor is of major importance. To investigate the impact of several MW additives (1. incineration bottom ash; 2. fly ash; 3. boiler ash; 4. cement-based waste) as AD co-substrates, six 5 L single stage mesophilic, continuously stirred tank reactors (CSTR) were setup. Two different feeding regimes were employed including: (a) a liquid-recycled feeding method (LRFM); (b) a draw-and-fill feeding method (DFFM). Under the LRFM regime, one gram MW per gram organic waste enhanced process stability (pH), increased methane production (25-45% increase), and yielded (450-520 mL CH4/g VS); DFFM enhanced digestibility to a lesser degree. Illumina HiSeq 16S rRNA community sequencing of reactors showed that the microbial community compositions were unaffected by the presence of MW additives in comparison to unamended controls, but MW amendment accelerated bacterial growth (determined by qPCR). In contrast, different feeding regimes altered the microbial communities; Methanoculleus (hydrogenotrophic) and Methanosaeta (acetoclastic) were the most abundant methanogenic genera in the LRFM reactors, and the more metabolically versatile Methanosarcina genus dominated under DFFM.