Net growth rate of continuum heterogeneous biofilms with inhibition kinetics

Biofilm systems can be modeled using a variety of analytical and numerical approaches, usually by making simplifying assumptions regarding biofilm heterogeneity and activity as well as effective diffusivity. Inhibition kinetics, albeit common in experimental systems, are rarely considered and analytical approaches are either lacking or consider effective diffusivity of the substrate and the biofilm density to remain constant. To address this obvious knowledge gap an analytical procedure to estimate the effectiveness factor (dimensionless substrate mass flux at the biofilm-fluid interface) was developed for a continuum heterogeneous biofilm with multiple limiting-substrate Monod kinetics to different types of inhibition kinetics. The simple perturbation technique, previously validated to quantify biofilm activity, was applied to systems where either the substrate or the inhibitor is the limiting component, and cases where the inhibitor is a reaction product or the substrate also acts as the inhibitor. Explicit analytical equations are presented for the effectiveness factor estimation and, therefore, the calculation of biomass growth rate or limiting substrate/inhibitor consumption rate, for a given biofilm thickness. The robustness of the new biofilm model was tested using kinetic parameters experimentally determined for the growth of Pseudomonas putida CCRC 14365 on phenol. Several additional cases have been analyzed, including examples where the effectiveness factor can reach values greater than unity, characteristic of systems with inhibition kinetics. Criteria to establish when the effectiveness factor can reach values greater than unity in each of the cases studied are also presented.Fil: Gonzo, Elio Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; ArgentinaFil: Wuertz, Stefan. Nanyang Technological University; SingapurFil: Rajal, Verónica Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; Singapu

Three-dimensional distribution of GFP-tagged Pseudomonas putida during biofilm formation on solid PAHs assessed by confocal laser scanning microscopy

Confocal laser scanning microscopy was used to monitor the colonization pattern of the gfp-labelled derivative strain of Pseudomonas putida ATCC 17514 on fluorene and phenanthrene crystals. The in situ experiments showed that P. putida tends to grow directly on phenanthrene, forming a biofilm on accessible crystalline surfaces. On the other hand, no significant biofilm formation was observed in the presence of fluorene. The higher biopolymers production in the presence of phenanthrene rather than fluorene may be a strategy to increase substrate accessibility.Fundação para a Ciência e a Tecnologia (FCT), Instituto de Biotecnologia e Química Fina (IBQF)

Three-dimensional distribution of GFP-labeled Pseudomonas putida during biofilm formation on solid PAHs assessed by confocal laser scanning microscopy

Confocal laser scanning microscopy was used to monitor the colonization pattern of the gfplabeled derivative strain of Pseudomonas putida ATCC 17514 on fluorene and phenanthrene crystals. The in situ experiments showed that P. putida tends to grow directly on phenanthrene, forming a biofilm on accessible crystalline surfaces. On the other hand, no significant biofilm formation was observed in the presence of fluorene. The results obtained showed that substrate properties affected bacterial strategy regarding uptake.Fundação para s Ciência e a Teconologia (FCT) - PRAXISXXI/BD/15944/98. Instituto de Biotecnologia e Química Fina (IBQF). EC Biotech (contrato Bio4-ct97-2015)

Variably improved microbial source tracking with digital droplet PCR

This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74–100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.Fil: Nshimyimana, Jean Pierre. Michigan State University; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos. Nanyang Technological University; SingapurFil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; SingapurFil: Wuertz, Stefan. Nanyang Technological University; SingapurFil: Thompson, Janelle R.. Massachusetts Institute of Technology; Estados Unido

Microbial abundance and community composition in biofilms on in-pipe sensors in a drinking water distribution system

Collecting biofilm samples from drinking water distribution systems (DWDSs) is challenging due to limited access to the pipes during regular operations. We report here the analysis of microbial communities in biofilm and water samples collected from sensors installed in a DWDS where monochloramine is used as a residual disinfectant. A total of 52 biofilm samples and 14 bulk water samples were collected from 17 pipe sections representing different water ages. Prokaryotic genome copies (bacterial and archaeal 16S rRNA genes, Mycobacterium spp., ammonia-oxidizing bacteria (AOB), and cyanobacteria) were quantified with droplet digital PCR, which revealed the abundance of these genes in both biofilm and water samples. Prokaryotic 16S rRNA gene sequencing analysis was carried out for a subset of the samples (12 samples from four sites). Mycobacterium and AOB species were dominant in the DWDS sections with low water age and sufficient residual monochloramine, whereas Nitrospira species (nitrite-oxidizing bacteria) dominated in the sections with higher water age and depleted monochloramine level, suggesting the occurrence of nitrification in the studied DWDS. The present study provides novel information on the abundance and identity of prokaryotes in biofilms and water in a full-scale operational DWDS.Collecting biofilm samples from drinking water distribution systems (DWDSs) is challenging due to limited access to the pipes during regular operations. We report here the analysis of microbial communities in biofilm and water samples collected from sensors installed in a DWDS where monochloramine is used as a residual disinfectant. A total of 52 biofilm samples and 14 bulk water samples were collected from 17 pipe sections representing different water ages. Prokaryotic genome copies (bacterial and archaeal 16S rRNA genes, Mycobacterium spp., ammonia-oxidizing bacteria (AOB), and cyanobacteria) were quantified with droplet digital PCR, which revealed the abundance of these genes in both biofilm and water samples. Prokaryotic 16S rRNA gene sequencing analysis was carried out for a subset of the samples (12 samples from four sites). Mycobacterium and AOB species were dominant in the DWDS sections with low water age and sufficient residual monochloramine, whereas Nitrospira species (nitrite-oxidizing bacteria) dominated in the sections with higher water age and depleted monochloramine level, suggesting the occurrence of nitrification in the studied DWDS. The present study provides novel information on the abundance and identity of prokaryotes in biofilms and water in a full-scale operational DWDSFil: Kitajima, Masaaki. Hokkaido University; Japón. Singapore-MIT Alliance for Research and Technology; SingapurFil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; SingapurFil: Williams, Rohan Benjamin Hugh. National University of Singapore; SingapurFil: Wuertz, Stefan. National University of Singapore; Singapur. Nanyang Technological University; SingapurFil: Whittle, Andrew J.. Singapore-MIT Alliance for Research and Technology; Singapur. Massachusetts Institute of Technology; Estados Unido

Quantification of viable protozoan parasites on leafy greens using molecular methods

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.Fil: Kim, Minji. University of California at Davis; Estados UnidosFil: Shapiro, Karen. University of California at Davis; Estados UnidosFil: Rajal, Verónica Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; ArgentinaFil: Packham, Andrea. University of California at Davis; Estados UnidosFil: Aguilar, Beatriz. University of California at Davis; Estados UnidosFil: Rueda, Lezlie. University of California at Davis; Estados UnidosFil: Wuertz, Stefan. University of California at Davis; Estados Unido

Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India.

We compared host-associated Bacteroidales qPCR assays developed in the continental United States and Europe for the purpose of measuring the effect of improved sanitation on human fecal exposure in rural Indian communities where both human and animal fecal loading are high. Ten candidate Bacteroidales qPCR assays were tested against fecal samples (human, sewage, cow, buffalo, goat, sheep, dog and chicken) from a test set of 30 individual human, 5 sewage, and 60 pooled animal samples collected in coastal Odisha, India. The two universal/general Bacteroidales assays tested (BacUni, GenBac3) performed equally well, achieving 100% sensitivity on the test set. Across the five human-associated assays tested (HF183 Taqman, BacHum, HumM2, BacH, HF183 SYBR), we found low sensitivity (17 to 49%) except for HF183 SYBR (89%), and moderate to high cross-reactivity with dog (20 to 80%) and chicken fecal samples (60 to 100%). BacHum had the highest accuracy (67%), amplified all sewage samples within the range of quantification (ROQ), and did not cross-react with any fecal samples from cows, the most populous livestock animal in India. Of the ruminant- and cattle-associated assays tested (BacCow, CowM2), BacCow was more sensitive in detecting the full range of common Indian livestock animal fecal sources, while CowM2 only detected cow sources with 50% sensitivity. Neither assay cross-reacted with human sources. BacCan, the dog-associated assay tested, showed no cross-reactivity with human sources, and high sensitivity (90%) for dog fecal samples. Overall, our results indicate BacUni, BacHum, HumM2, BacCan and BacCow would be the most suitable MST assays to distinguish and quantify relative amounts of human-associated and livestock/domestic animal-associated contributions to fecal contamination in Odisha, India

Human and Animal Fecal Contamination of Community Water Sources, Stored Drinking Water and Hands in Rural India Measured with Validated Microbial Source Tracking Assays.

We examined pathways of exposure to fecal contamination of human and animal origin in 24 villages in Odisha, India. In a cross-sectional study during the monsoon season, fecal exposure via community water sources (N = 123) and in the home (N = 137) was assessed using human- and nonhuman-associated Bacteroidales microbial source tracking (MST) markers and fecal coliforms (FCs). Detection rates and marker concentrations were examined to pinpoint pathways of human fecal exposure in the public and domestic domains of disease transmission in study communities. Human fecal markers were detected much more frequently in the domestic domain (45% of households) than in public domain sources (8% of ponds; 4% of groundwater drinking sources). Animal fecal markers were widely detected in both domains (74% of ponds, 96% of households, 10% of groundwater drinking sources), indicating ubiquitous risks of exposure to animal feces and zoonotic pathogens. This study confirms an often suggested contamination link from hands to stored water in the home in developing countries separately for mothers' and children's hands and both human and animal fecal contamination. In contrast to MST markers, FCs provided a poor metric to assess risks of exposure to fecal contamination of human origin in this rural setting

Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone

Polyphosphate-accumulating organisms in full-scale tropical wastewater treatment plants use diverse carbon sources

Enhanced biological phosphorus removal (EBPR) is considered challenging in the tropics, based on a great number of laboratory-based studies showing that the polyphosphate-accumulating organism (PAO) Candidatus Accumulibacter does not compete well with glycogen accumulating organisms (GAOs) at temperatures above 25 °C. Yet limited information is available on the PAO community and the metabolic capabilities in full-scale EBPR systems operating at high temperature. We studied the composition of the key functional PAO communities in three full-scale wastewater treatment plants (WWTPs) with high in-situ EBPR activity in Singapore, their EBPR-associated carbon usage characteristics, and the relationship between carbon usage and community composition. Each plant had a signature community composed of diverse putative PAOs with multiple operational taxonomic units (OTUs) affiliated to Ca. Accumulibacter, Tetrasphaera spp., Dechloromonas and Ca. Obscuribacter. Despite the differences in community composition, ex-situ anaerobic phosphorus (P)-release tests with 24 organic compounds from five categories (including four sugars, three alcohols, three volatile fatty acids (VFAs), eight amino acids and six other carboxylic acids) showed that a wide range of organic compounds could potentially contribute to EBPR. VFAs induced the highest P release (12.0-18.2 mg P/g MLSS for acetate with a P release-to-carbon uptake (P:C) ratio of 0.35-0.66 mol P/mol C, 9.4-18.5 mg P/g MLSS for propionate with a P:C ratio of 0.38-0.60, and 9.5-17.3 mg P/g MLSS for n-butyrate), followed by some carboxylic acids (10.1-18.1 mg P/g MLSS for pyruvate, 4.5-11.7 mg P/g MLSS for lactate and 3.7-12.4 mg P/g MLSS for fumarate) and amino acids (3.66-7.33 mg P/g MLSS for glutamate with a P:C ratio of 0.16-0.43 mol P/mol C, and 4.01-7.37 mg P/g MLSS for aspartate with a P:C ratio of 0.17-0.48 mol P/mol C). P-release profiles (induced by different carbon sources) correlated closely with PAO community composition. High micro-diversity was observed within the Ca. Accumulibacter lineage, which represented the most abundant PAOs. The total population of Ca. Accumulibacter taxa was highly correlated with P-release induced by VFAs, highlighting the latter's importance in tropical EBPR systems. There was a strong link between the relative abundance of individual Ca. Accumulibacter OTUs and the extent of P release induced by distinct carbon sources (e.g., OTU 81 and amino acids, and OTU 246 and ethanol), suggesting niche differentiation among Ca. Accumulibacter taxa. A diverse PAO community and the ability to use numerous organic compounds are considered key factors for stable EBPR in full-scale plants at elevated temperatures.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore