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.

Biofilm-based simultaneous nitrification, denitrification, and phosphorous uptake in wastewater by Neurospora discreta.

Biological removal of nitrogen and phosphorous from wastewater conventionally involves multiple processing steps to satisfy the differing oxygen requirements of the microbial species involved. In this work, simultaneous nitrification, denitrification, and phosphorous removal from synthetic wastewater were achieved by the fungus Neurospora discreta in a single-step, biofilm-based, aerobic process. The concentrations of carbon, nitrogen, and phosphorous in the synthetic wastewater were systematically varied to investigate their effects on nutrient removal rates and biofilm properties. Biofilm growth was significantly (p < 0.05) affected by carbon and nitrogen, but not by phosphorous concentration. Scanning electron microscopy revealed the effects of nutrients on biofilm microstructure, which in turn correlated with nutrient removal efficiencies. The carbohydrate and protein content in the biofilm matrix decreased with increasing carbon and nitrogen concentrations but increased with increasing phosphorous concentration in the wastewater. High removal efficiencies of carbon (96%), ammonium (86%), nitrate (100%), and phosphorus (82%) were achieved under varying nutrient conditions. Interestingly, decreasing the phosphorus concentration increased the nitrification and denitrification rates, and decreasing the nitrogen concentration increased the phosphorus removal rates significantly (p < 0.05). Correlations between biofilm properties and nutrient removal rates were also evaluated in this study.

Targeting Microbial Biofouling by Controlling Biofilm Formation and Dispersal Using Rhamnolipids on RO Membrane.

Finding new biological ways to control biofouling of the membrane in reverse osmosis (RO) is an important substitute for synthetic chemicals in the water industry. Here, the study was focused on the antimicrobial, biofilm formation, and biofilm dispersal potential of rhamnolipids (RLs) (biosurfactants). The MTT assay was also carried out to evaluate the effect of RLs on biofilm viability. Biofilm was qualitatively and quantitatively assessed by crystal violet assay, light microscopy, fluorescence microscopy (bacterial biomass (µm2), surface coverage (%)), and extracellular polymeric substances (EPSs). It was exhibited that RLs can reduce bacterial growth. The higher concentrations (≥100 mg/L) markedly reduced bacterial growth and biofilm formation, while RLs exhibited substantial dispersal effects (89.10% reduction) on preformed biofilms. Further, RLs exhibited 79.24% biomass reduction while polysaccharide was reduced to 60.55 µg/mL (p < 0.05) and protein to 4.67 µg/mL (p < 0.05). Light microscopy revealed biofilm reduction, which was confirmed using fluorescence microscopy. Microscopic images were processed with BioImageL software. It was revealed that biomass surface coverage was reduced to 1.1% at 1000 mg/L of RLs and that 43,245 µm2 of biomass was present for control, while biomass was reduced to 493 µm2 at 1000 mg/L of RLs. Thus, these data suggest that RLs have antimicrobial, biofilm control, and dispersal potential against membrane biofouling.

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.

The experimental determination of reliable biodegradation rates for mono-aromatics towards evaluating QSBR models.

Quantitative Structure Biodegradation Relationships (QSBRs) are a tool to predict the biodegradability of chemicals. The objective of this work was to generate reliable biodegradation data for mono-aromatic chemicals in order to evaluate and verify previously developed QSBRs models. A robust biodegradation test method was developed to estimate specific substrate utilization rates, which were used as a proxy for biodegradation rates of chemicals in pure culture. Five representative mono-aromatic chemicals were selected that spanned a wide range of biodegradability. Aerobic biodegradation experiments were performed for each chemical in batch reactors seeded with known degraders. Chemical removal, degrader growth and CO2 production were monitored over time. Experimental data were interpreted using a full carbon mass balance model, and Monod kinetic parameters (Y, Ks, qmax and µmax) for each chemical were determined. In addition, stoichiometric equations for aerobic mineralization of the test chemicals were developed. The theoretically estimated biomass and CO2 yields were similar to those experimentally observed; 35% (s.d ±â€¯8%) of the recovered substrate carbon was converted to biomass, and 65% (s.d ±â€¯8%) was mineralised to CO2. Significant correlations were observed between the experimentally determined specific substrate utilization rates, as represented by qmax and qmax/Ks, at high and low substrate concentrations, respectively, and the first order biodegradation rate constants predicted by a previous QSBR study. Similarly, the correlation between qmax and selected molecular descriptors characterizing the chemicals structure in a previous QSBR study was also significant. These results suggest that QSBR models can be reliable and robust in prioritising chemical half-lives for regulatory screening purposes.

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.

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].

Effect of cycle run time of backwash and relaxation on membrane fouling removal in submerged membrane bioreactor treating sewage at higher flux.

Intermittent backwashing and relaxation are mandatory in the membrane bioreactor (MBR) for its effective operation. The objective of the current study was to evaluate the effects of run-relaxation and run-backwash cycle time on fouling rates. Furthermore, comparison of the effects of backwashing and relaxation on the fouling behavior of membrane in high rate submerged MBR. The study was carried out on a laboratory scale MBR at high flux (30 L/m2·h), treating sewage. The MBR was operated at three relaxation operational scenarios by keeping the run time to relaxation time ratio constant. Similarly, the MBR was operated at three backwashing operational scenarios by keeping the run time to backwashing time ratio constant. The results revealed that the provision of relaxation or backwashing at small intervals prolonged the MBR operation by reducing fouling rates. The cake and pores fouling rates in backwashing scenarios were far less as compared to the relaxation scenarios, which proved backwashing a better option as compared to relaxation. The operation time of backwashing scenario (lowest cycle time) was 64.6% and 21.1% more as compared to continuous scenario and relaxation scenario (lowest cycle time), respectively. Increase in cycle time increased removal efficiencies insignificantly, in both scenarios of relaxation and backwashing.

Study of physio-psychological effects on traffic wardens due to traffic noise pollution; exposure-effect relation.

BACKGROUND: Noise pollution has increased to alarming extent in most of the urban areas in Pakistan. It is assumed even more perilous than air and water pollution due to its direct acute and chronic physio-psychological effects. The objective of this study is to analyze and evaluate the psychological and physiological effects caused by traffic noise on traffic wardens and to find relation type between exposure time and effect. METHODS: Three wardens check posts near roads were selected for survey in Taxila and Islamabad cities of Pakistan. Survey conducted included noise measurements at aforementioned check posts for one month and Performa based interviews of traffic wardens. RESULTS AND CONCLUSIONS: Analysis of results showed that noise levels varied between 85-106 dB hence violating OSHA regulations. Major psychological effects found in wardens were aggravated depression 58%, stress 65%, public conflict 71%, irritation and annoyance 54%, behavioral affects 59% and speech interference 56%. Physiological effects found were hypertension 87%, muscle tension 64%, exhaustion 48%, low performance levels 55%, concentration loss 93%, hearing impairment 69%, headache 74% and cardiovascular issue 71%. Relation between exposure time and effects were evaluated by using simple regression test in excel. Percentage of psychological and physiological effects in wardens varied with the exposure time; aggravated depression (R(2) = 0.946, P = 0.133), stress suffering (R(2) = 0.014, P = 0.173), public conflict (R(2) = 0.946, P = 0.133), irritation and annoyance (R(2) = 0.371, P = 0.137), behavioral affects (R(2) = 0.596, P = 0.0616) and speech interference (R(2) = 0.355, P = 0.445), hypertension (R(2) = 0.96, P = 0.00095) and cardiovascular issue (R(2) = 0.775, P = 0.044).