Exploring the Specificity of Extracellular Wastewater Peptidases to Improve the Design of Sustainable Peptide-Based Antibiotics.

New antimicrobial peptides are emerging as promising alternatives to conventional antibiotics because of their specificity for target pathogens and their potential to be rapidly hydrolyzed (i.e., inactivated) by extracellular peptidases during biological wastewater treatment, thereby limiting the emergence and propagation of antibiotic resistance in the environment. However, little is known about the specificity of extracellular peptidases derived from wastewater microbial communities, which is a major impediment for the design of sustainable peptide-based antibiotics that can be hydrolyzed by wastewater peptidases. We used a set of natural peptides to explore the specificity of dissolved extracellular wastewater peptidases. We found that enzyme-catalyzed hydrolysis occurred at specific sites and that a subset of these hydrolyses was conserved across enzyme pools derived from three independent wastewater microbial communities. An analysis of the amino-acid residues flanking the hydrolyzed bonds revealed a set of residue motifs that were linked to enzyme-catalyzed hydrolysis and are therefore candidates for incorporation into new and sustainable peptide-based antibiotics.

Singlet Oxygen Phosphorescence as a Probe for Triplet-State Dissolved Organic Matter Reactivity.

Triplet-state chromophoric dissolved organic matter (3CDOM*) plays an important role in aquatic photochemistry, yet much remains unknown about the reactivity of these intermediates. To better understand the kinetic behavior and reactivity of 3CDOM*, we have developed an indirect observation method based on monitoring time-resolved singlet oxygen (1O2) phosphorescence kinetics. The underpinning principle of our approach relies on the fact that O2 quenches almost all triplets with near diffusion limited rate constants, resulting in the formation of 1O2, which is kinetically linked to the precursors. A kinetic model relating 1O2 phosphorescence kinetics to triplet excited states produced from isolated humic substances and in whole natural-water samples (hereafter referred to as 3CDOM*) was developed and used to determine rate constants governing 3CDOM* natural lifetimes and quenching by oxygen and 2,4,6-trimethylphenol (TMP), a common triplet probe molecule. 3CDOM* was found to exhibit smaller O2 and TMP quenching rate constants, ∼9 × 108 and ∼8 × 108 M-1 s-1, respectively, compared with model sensitizers, such as aromatic ketones. Findings from this report shed light on the fundamental photochemical properties of CDOM in organic matter isolates and whole waters and will help refine photochemical models to more accurately predict pollutant fate in the environment.

Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin.

The antibiotic monensin is fed to dairy cows to increase milk production efficiency. A fraction of this monensin is excreted into the cow manure. Previous studies have found that cow manure containing monensin can negatively impact the performance of anaerobic digesters, especially upon first introduction. Few studies have examined whether the anaerobic digester microbiome can adapt to monensin during the operating time. Here, we conducted a long-term time series study of four lab-scale anaerobic digesters fed with cow manure. We examined changes in both the microbiome composition and function of the anaerobic digesters when subjected to the dairy antibiotic monensin. In our digesters, monensin was not rapidly degraded under anaerobic conditions. The two anaerobic digesters that were subjected to manure from monensin feed-dosed cows exhibited relatively small changes in microbiome composition and function due to relatively low monensin concentrations. At higher concentrations of monensin, which we dosed directly to control manure (from dairy cows without monensin), we observed major changes in the microbiome composition and function of two anaerobic digesters. A rapid introduction of monensin to one of these anaerobic digesters led to the impairment of methane production. Conversely, more gradual additions of the same concentrations of monensin to the other anaerobic digester led to the adaptation of the anaerobic digester microbiomes to the relatively high monensin concentrations. A member of the candidate OP11 (Microgenomates) phylum arose in this anaerobic digester and appeared to be redundant with certain Bacteroidetes phylum members, which previously were dominating.IMPORTANCE Monensin is a common antibiotic given to dairy cows in the United States and is partly excreted with dairy manure. An improved understanding of how monensin affects the anaerobic digester microbiome composition and function is important to prevent process failure for farm-based anaerobic digesters. This time series study demonstrates how anaerobic digester microbiomes are inert to low monensin concentrations and can adapt to relatively high monensin concentrations by redundancy in an already existing population. Therefore, our work provides further insight into the importance of microbiome redundancy in maintaining the stability of anaerobic digesters.

Comparing the inhibitory thresholds of dairy manure co-digesters after prolonged acclimation periods: Part 2--correlations between microbiomes and environment.

Here, we studied the microbiome succession and time-scale variability of four mesophilic anaerobic reactors in a co-digestion study with the objective to find links between changing environmental conditions and the microbiome composition. The changing environmental conditions were ensured by gradual increases in loading rates and mixing ratios of three co-substrates with a constant manure-feeding scheme during an operating period longer than 900 days. Each co-substrate (i.e., alkaline hydrolysate, food waste, and glycerol) was co-digested separately. High throughput 16S rRNA gene sequencing was used to examine the microbiome succession. The alkaline hydrolysate reactor microbiome shifted and adapted to high concentrations of free ammonia, total volatile fatty acids, and potassium to maintain its function. The addition of food waste and glycerol as co-substrates also led to microbiome changes, but to a lesser extent, especially in the case of the glycerol reactor microbiome. The divergence of the food waste reactor microbiome was primarily linked to increasing free ammonia levels in the reactor; though, these levels remained below previously reported inhibitory levels for acclimated biomass. The glycerol reactor microbiome succession included an increase in Syntrophomonadaceae family members, which have previously been linked to long-chain fatty acid degradation. The glycerol reactor exhibited rapid failure and limited adaptation at the end of the study.

Methane Emission in a Specific Riparian-Zone Sediment Decreased with Bioelectrochemical Manipulation and Corresponded to the Microbial Community Dynamics.

Dissimilatory metal-reducing bacteria are widespread in terrestrial ecosystems, especially in anaerobic soils and sediments. Thermodynamically, dissimilatory metal reduction is more favorable than sulfate reduction and methanogenesis but less favorable than denitrification and aerobic respiration. It is critical to understand the complex relationships, including the absence or presence of terminal electron acceptors, that govern microbial competition and coexistence in anaerobic soils and sediments, because subsurface microbial processes can effect greenhouse gas emissions from soils, possibly resulting in impacts at the global scale. Here, we elucidated the effect of an inexhaustible, ferrous-iron and humic-substance mimicking terminal electron acceptor by deploying potentiostatically poised electrodes in the sediment of a very specific stream riparian zone in Upstate New York state. At two sites within the same stream riparian zone during the course of 6 weeks in the spring of 2013, we measured CH4 and N2/N2O emissions from soil chambers containing either poised or unpoised electrodes, and we harvested biofilms from the electrodes to quantify microbial community dynamics. At the upstream site, which had a lower vegetation cover and highest soil temperatures, the poised electrodes inhibited CH4 emissions by ∼45% (when normalized to remove temporal effects). CH4 emissions were not significantly impacted at the downstream site. N2/N2O emissions were generally low at both sites and were not impacted by poised electrodes. We did not find a direct link between bioelectrochemical treatment and microbial community membership; however, we did find a correspondence between environment/function and microbial community dynamics.

Correlation between microbial community and granule conductivity in anaerobic bioreactors for brewery wastewater treatment.

Prior investigation of an upflow anaerobic sludge blanket (UASB) reactor treating brewery wastes suggested that direct interspecies electron transfer (DIET) significantly contributed to interspecies electron transfer to methanogens. To investigate DIET in granules further, the electrical conductivity and bacterial community composition of granules in fourteen samples from four different UASB reactors treating brewery wastes were investigated. All of the UASB granules were electrically conductive whereas control granules from ANAMMOX (ANaerobic AMMonium OXidation) reactors and microbial granules from an aerobic bioreactor designed for phosphate removal were not. There was a moderate correlation (r=0.67) between the abundance of Geobacter species in the UASB granules and granule conductivity, suggesting that Geobacter contributed to granule conductivity. These results, coupled with previous studies, which have demonstrated that Geobacter species can donate electrons to methanogens that are typically predominant in anaerobic digesters, suggest that DIET may be a widespread phenomenon in UASB reactors treating brewery wastes.

Microbial community dynamics and stability during an ammonia-induced shift to syntrophic acetate oxidation.

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.

Metabolite transfer with the fermentation product 2,3-butanediol enhances virulence by Pseudomonas aeruginosa.

The respiratory tract of cystic fibrosis (CF) patients harbor persistent microbial communities (CF airway microbiome) with Pseudomonas aeruginosa emerging as a dominant pathogen. Within a polymicrobial infection, interactions between co-habitant microbes can be important for pathogenesis, but even when considered, these interactions are not well understood. Here, we show with in vitro experiments that, compared with glucose, common fermentation products from co-habitant bacteria significantly increase virulence factor production, antimicrobial activity and biofilm formation of P. aeruginosa. The maximum stimulating effect was produced with the fermentation product 2,3-butanediol, which is a substrate for P. aeruginosa, resulting in a metabolic relationship between fermenters and this pathogen. The global transcription regulator LasI LasR, which controls quorum sensing, was upregulated threefold with 2,3-butanediol, resulting in higher phenazine and exotoxin concentrations and improved biofilm formation. This indicates that the success of P. aeruginosa in CF airway microbiomes could be governed by the location within the food web with fermenting bacteria. Our findings suggest that interbacterial metabolite transfer in polymicrobial infections stimulates virulence of P. aeruginosa and could have a considerable impact on disease progression.

Substrate type drives variation in reactor microbiomes of anaerobic digesters.

The goal of this study was to obtain causative information about beta-diversity (differentiation between microbiomes) by comparing sequencing information between studies rather than just knowledge about alpha-diversity (microbiome richness). Here, published sequencing data were merged representing 78 anaerobic digester samples originating from 28 different studies for an overall comparison of beta-diversity (measured using unweighted UniFrac). It was found that digester microbiomes based on bacterial sequences clustered by substrate type, independent of the study of origin, and that this clustering could be attributed to distinct bacterial lineages.

Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut.

Gut mucosal barrier breakdown and inflammation have been associated with high levels of flagellin, the principal bacterial flagellar protein. Although several gut commensals can produce flagella, flagellin levels are low in the healthy gut, suggesting the existence of control mechanisms. We find that mice lacking the flagellin receptor Toll-like receptor 5 (TLR5) exhibit a profound loss of flagellin-specific immunoglobulins (Igs) despite higher total Ig levels in the gut. Ribotyping of IgA-coated cecal microbiota showed Proteobacteria evading antibody coating in the TLR5(-/-) gut. A diversity of microbiome members overexpressed flagellar genes in the TLR5(-/-) host. Proteobacteria and Firmicutes penetrated small intestinal villi, and flagellated bacteria breached the colonic mucosal barrier. In vitro, flagellin-specific Ig inhibited bacterial motility and downregulated flagellar gene expression. Thus, innate-immunity-directed development of flagellin-specific adaptive immune responses can modulate the microbiome's production of flagella in a three-way interaction that helps to maintain mucosal barrier integrity and homeostasis.

Host remodeling of the gut microbiome and metabolic changes during pregnancy.

Many of the immune and metabolic changes occurring during normal pregnancy also describe metabolic syndrome. Gut microbiota can cause symptoms of metabolic syndrome in nonpregnant hosts. Here, to explore their role in pregnancy, we characterized fecal bacteria of 91 pregnant women of varying prepregnancy BMIs and gestational diabetes status and their infants. Similarities between infant-mother microbiotas increased with children's age, and the infant microbiota was unaffected by mother's health status. Gut microbiota changed dramatically from first (T1) to third (T3) trimesters, with vast expansion of diversity between mothers, an overall increase in Proteobacteria and Actinobacteria, and reduced richness. T3 stool showed strongest signs of inflammation and energy loss; however, microbiome gene repertoires were constant between trimesters. When transferred to germ-free mice, T3 microbiota induced greater adiposity and insulin insensitivity compared to T1. Our findings indicate that host-microbial interactions that impact host metabolism can occur and may be beneficial in pregnancy.

Shaping reactor microbiomes to produce the fuel precursor n-butyrate from pretreated cellulosic hydrolysates.

To maximize the production of carboxylic acids with open cultures of microbial consortia (reactor microbiomes), we performed experiments to understand which factors affect the community dynamics and performance parameters. We operated six thermophilic (55 °C) bioreactors to test how the factors: (i) biomass pretreatment; (ii) bioreactor operating conditions; and (iii) bioreactor history (after perturbations during the operating period) affected total fermentation product and n-butyrate performance parameters with corn fiber as the cellulosic biomass waste. We observed a maximum total fermentation product yield of 39%, a n-butyrate yield of 23% (both on a COD basis), a maximum total fermentation production rate of 0.74 g COD l(-1) d(-1) and n-butyrate production rate of 0.47 g COD l(-1) d(-1) in bioreactors that were fed with dilute-acid pretreated corn fiber at a pH of 5.5. Pyrosequencing of 16S rRNA genes with constrained ordination and other statistical methods showed that changes in operating conditions to enable dilution of toxic carboxylic acid products, which lead to these maximum performance parameters, also altered the composition of the microbiome, and that the microbiome, in turn, affected the performance. Operating conditions are an important factor (tool for operators) to shape reactor microbiomes, but other factors, such as substrate composition after biomass pretreatment and bioreactor history are also important. Further optimization of operating conditions must relieve the toxicity of carboxylic acids at acidic bioreactor pH levels even more, and this can, for example, be accomplished by extracting the product from the bioreactor solutions.

Relating chloroethene respiration rates in Dehalococcoides to protein and mRNA biomarkers.

Molecular biomarkers could provide critical insight into myriad in situ microbial activities. In this study we explore correlations of both mRNA and protein biomarkers with chloroethene respiration rate in Dehalococcoides. In a series of continuously fed dechlorinating mixed-culture microcosm experiments (n = 26), we varied respiratory substrates, substrate ratios and feeding rates. Transcript levels for most biomarkers were responsive down to 0.01× the culture's maximum respiration rate. The dehalogenase TceA and the Ni-Fe hydrogenase HupL transcripts were positively correlated (Pearson's r of 0.89 and 0.88, respectively) with respiration rates on log-log plots between 1.5 and 280 µeeq/L-hr for mRNA abundances of 10(7) to 10(10) transcripts/mL (0.07-230 transcripts/genome). These trends were independent of the types of chloroethene or electron donors fed. Other mRNA target levels plateaued or declined at respiration rates above 5 µeeq/L-hr. Using both relative and absolute protein quantification methods, we found that per-genome protein abundances of most targeted biomarkers did not statistically change over the experimental time frames. However, quantified enzyme levels allowed us to calculate in vivo enzyme-specific rate constants (k(cat)) for the dehalogenases PceA and TceA: 400 and 22 substrate molecules/enzyme-sec, respectively. Overall, these data support the promise of both mRNA and protein biomarkers for estimating process rates through either empirical (mRNA-based) or kinetic (protein-based) models, but they require follow-up studies in other cultures and at active remediation sites.

Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys.

Taxonomic classification of the thousands-millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases.

Bacterial community structures are unique and resilient in full-scale bioenergy systems.

Anaerobic digestion is the most successful bioenergy technology worldwide with, at its core, undefined microbial communities that have poorly understood dynamics. Here, we investigated the relationships of bacterial community structure (>400,000 16S rRNA gene sequences for 112 samples) with function (i.e., bioreactor performance) and environment (i.e., operating conditions) in a yearlong monthly time series of nine full-scale bioreactor facilities treating brewery wastewater (>20,000 measurements). Each of the nine facilities had a unique community structure with an unprecedented level of stability. Using machine learning, we identified a small subset of operational taxonomic units (OTUs; 145 out of 4,962), which predicted the location of the facility of origin for almost every sample (96.4% accuracy). Of these 145 OTUs, syntrophic bacteria were systematically overrepresented, demonstrating that syntrophs rebounded following disturbances. This indicates that resilience, rather than dynamic competition, played an important role in maintaining the necessary syntrophic populations. In addition, we explained the observed phylogenetic differences between all samples on the basis of a subset of environmental gradients (using constrained ordination) and found stronger relationships between community structure and its function rather than its environment. These relationships were strongest for two performance variables--methanogenic activity and substrate removal efficiency--both of which were also affected by microbial ecology because these variables were correlated with community evenness (at any given time) and variability in phylogenetic structure (over time), respectively. Thus, we quantified relationships between community structure and function, which opens the door to engineer communities with superior functions.

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration.

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Photolysis of chlortetracycline on a clay surface.

Chlortetracycline, an antibiotic commonly used as a growth promoter in livestock, enters the environment primarily through application of animal waste to open fields. The photochemical loss of chlortetracycline in sunlight-exposed soils is a potentially important process in its environmental fate, especially because it is photochemically labile and sorbs strongly to mineral surfaces. In this study, photolysis on kaolinite clay under simulated sunlight was used as a model system to elucidate the mechanistic kinetics of chlortetracycline photolysis on soil surfaces. The results suggest that photolysis may be an important loss process for chlortetracycline sorbed to sunlight-exposed soils, as well as to suspended clays in surface waters. Under direct irradiation equivalent to noon-time, summer sunlight in the midwestern United States, chlortetracycline at the outer clay surface (before light attenuation) degraded with a rate constant of k(0)(p) = 0.65 +/- 0.30 h(-1). The depth at which photochemical action was reduced by 50% (z(0.5)), one of the parameters of the mechanistic model, was found to be 0.014 +/- 0.004 mm. The quantum yield on the clay surface was estimated to be (1.3 +/- 0.7) x 10(-4), an order of magnitude lower than the quantum yield of the aqueous chlortetracycline zwitterion [(1.3 +/- 0.3) x 10(-3); pH 5], although still significant.

Environmental photochemistry of tylosin: efficient, reversible photoisomerization to a less-active isomer, followed by photolysis.

The environmental photochemical kinetics of tylosin, a common veterinary macrolide antibiotic and growth promoter, were investigated under simulated sunlight. An efficient, reversible photoisomerization was characterized using kinetic, mass spectrometry, and proton nuclear magnetic resonance data. The photoisomerization was confirmed to occur by a rotation about the distal alkene of the ketodiene functionality. Concurrent forward (quantum yield = 0.39 +/- 0.09) and back (quantum yield = 0.32 +/- 0.08) reactions lead to a photochemical equilibrium near a tylosin/photoisomer ratio of 50:50, completed in less than 2 min under a spectrum equivalent to noontime, summer sunlight. The activity of the isomer for the inhibition of Escherichia coli DH5alpha growth was observed to be less than that of tylosin. On a longer time scale than that of isomerization, the isomer mixture undergoes photolysis with a quantum yield of (1.4 +/- 0.3) x 10(-3). The observed quantum yields and UV-vis absorbance data allow for the prediction of the photochemical behavior of tylosin in most environmental systems. Indirect photosensitization was not a significant loss process in solutions of Suwannee River fulvic acid with concentrations from 1 to 20 mg L(-1).

Water hardness as a photochemical parameter: tetracycline photolysis as a function of calcium concentration, magnesium concentration, and pH.

The environmental photochemical kinetics of the antibiotic compound tetracycline were investigated. The aqueous speciation of tetracycline over a range of natural pH and water hardness values is dominated by association with Ca2+ and Mg2+ ions. The association constants necessary to calculate tetracycline aqueous speciation given knowledge of pH, [Ca2+], and [Mg2+] were measured by spectrophotometric titrations and matrix deconvolution of a series of UV-vis absorption spectra into individual component species. A series of photolysis experiments was performed under simulated sunlight, and quantum yields for the solar photolysis of each environmentally relevant species were calculated. The results indicate that the pseudo-first-order rate constant for tetracycline photolysis at varied Mg2+ and Ca2+ concentrations relevant to natural conditions can vary by up to an order of magnitude. A self-sensitization effect was observed and was accounted for by varying the initial tetracycline concentration under each set of photolysis conditions.