Co-Culture of Halotolerant Bacteria to Produce Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Using Sewage Wastewater Substrate.

The focus of the current study was the use of sewage wastewater to obtain PHA from a co-culture to produce a sustainable polymer. Two halotolerant bacteria, Bacillus halotolerans 14SM (MZ801771) and Bacillus aryabhattai WK31 (MT453992), were grown in a consortium to produce PHA. Sewage wastewater (SWW) was used to produce PHA, and glucose was used as a reference substrate to compare the growth and PHA production parameters. Both bacterial strains produced PHA in monoculture, but a copolymer was obtained when the co-cultures were used. The co-culture accumulated a maximum of 54% after 24 h of incubation in 10% SWW. The intracellular granules indicated the presence of nucleation sites for granule initiation. The average granule size was recorded to be 231 nm; micrographs also indicated the presence of extracellular polymers and granule-associated proteins. Fourier transform infrared spectroscopy (FTIR) analysis of the polymer produced by the consortium showed a significant peak at 1731 cm-1, representing the C=O group. FTIR also presented peaks in the region of 2800 cm-1 to 2900 cm-1, indicating C-C stretching. Proton nuclear magnetic resonance (1HNMR) of the pure polymer indicated chemical shifts resulting from the proton of hydroxy valerate and hydroxybutyrate, confirming the production of poly(3-hydroxybutyrate-co-3-hydroxy valerate) (P3HBV). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that the copolymer was biocompatible, even at a high concentration of 5000 µg mL-1. The results of this study show that bacterial strains WK31 and 14SM can be used to synthesize a copolymer of butyrate and valerate using the volatile fatty acids present in the SWW, such as propionic acid or pentanoic acid. P3HBV can also be used to provide an extracellular matrix for cell-line growth without causing any cytotoxic effects.

PFOS Mass Flux Reduction/Mass Removal: Impacts of a Lower-Permeability Sand Lens within Otherwise Homogeneous Systems.

Perfluorooctane sulfonic acid (PFOS) is one of the most common per- and polyfluoroalkyl substances (PFAS) and is a significant risk driver for these emerging contaminants of concern. A series of two-dimensional flow cell experiments was conducted to investigate the impact of flow field heterogeneity on the transport, attenuation, and mass removal of PFOS. A simplified model heterogeneous system was employed consisting of a lower-permeability fine sand lens placed within a higher-permeability coarse sand matrix. Three nonreactive tracers with different aqueous diffusion coefficients, sodium chloride, pentafluorobenzoic acid, and ß-cyclodextrin, were used to characterize the influence of diffusive mass transfer on transport and for comparison to PFOS results. The results confirm that the attenuation and subsequent mass removal of the nonreactive tracers and PFOS were influenced by mass transfer between the hydraulically less accessible zone and the coarser matrix (i.e., back diffusion). A mathematical model was used to simulate flow and transport, with the values for all input parameters determined independently. The model predictions provided good matches to the measured breakthrough curves, as well as to plots of reductions in mass flux as a function of mass removed. These results reveal the importance of molecular diffusion and pore water velocity variability even for systems with relatively minor hydraulic conductivity heterogeneity. The impacts of the diffusive mass transfer limitation were quantified using an empirical function relating reductions in contaminant mass flux (MFR) to mass removal (MR). Multi-step regression was used to quantify the nonlinear, multi-stage MFR/MR behavior observed for the heterogeneous experiments. The MFR/MR function adequately reproduced the measured data, which suggests that the MFR/MR approach can be used to evaluate PFOS removal from heterogeneous media.

The Co-Transport of PFAS and Cr(VI) in porous media.

PFAS and Cr are present at some sites as co-contaminants. The objective of this research was to investigate the co-transport behavior of per- and polyfluoroalkyl substances (PFAS) and hexavalent chromium (Cr(VI)) in porous media. Miscible-displacement experiments were conducted using two soils and an aquifer sediment with different geochemical properties. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) were employed as model PFAS. The retardation of PFOS was decreased in the presence of Cr(VI). Conversely, the transport and retardation of PFOA was not affected by the presence of Cr(VI). The reduction of PFOS retardation caused by Cr(VI) is likely due to sorption competition for both organic-carbon and inorganic (metal-oxides and clay minerals) domains. The relative contributions of the three soil constituents to PFOS sorption and the potential for competition between PFOS and Cr(VI) is a function of the geochemical composition of the porous media (i.e., organic carbon, metal-oxides and clay minerals). The PFAS had minimal impact on the retention and transport of Cr(VI). To our knowledge, the results presented herein represent the first reported data for PFOS and Cr(VI) co-transport in porous media. The results of this study indicate that the presence of Cr(VI) has the potential to increase the migration potential of PFOS in soil and groundwater, which should be considered when characterizing electroplating facilities, leather tanning facilities, and other co-contaminated sites.

Identification and Computational Analysis of Novel Pathogenic Variants in Pakistani Families with Diverse Epidermolysis Bullosa Phenotypes.

Epidermolysis bullosa (EB) includes a group of rare gesnodermatoses that result in blistering and erosions of the skin and mucous membranes. Genetically, pathogenic variants in around 20 genes are known to alter the structural and functional integrity of intraepidermal adhesion and dermo-epidermal anchorage, leading to four different types of EB. Here we report the underlying genetic causes of EB phenotypes segregating in seven large consanguineous families, recruited from different regions of Pakistan. Whole exome sequencing, followed by segregation analysis of candidate variants through Sanger sequencing, identified eight pathogenic variants, including three novel (ITGB4: c.1285G>T, and c.3373G>A; PLEC: c.1828A>G) and five previously reported variants (COL7A1: c.6209G>A, and c.1573C>T; FERMT1: c.676insC; LAMA3: c.151insG; LAMB3: c.1705C>T). All identified variants were either absent or had very low frequencies in the control databases. Our in-silico analyses and 3-dimensional (3D) molecular modeling support the deleterious impact of these variants on the encoded proteins. Intriguingly, we report the first case of a recessively inherited form of rare EBS-Ogna associated with a homozygous variant in the PLEC gene. Our study highlights the clinical and genetic diversity of EB in the Pakistani population and expands the mutation spectrum of EB; it could also be useful for prenatal diagnosis and genetic counseling of the affected families.

Transport of PFOS in aquifer sediment: Transport behavior and a distributed-sorption model.

The objectives of this research were to examine the transport of perfluorooctane sulfonic acid (PFOS) in aquifer sediment comprising different geochemical properties, and to compare the behavior to that observed for PFOS transport in soil and sand. PFOS retardation was relatively low for transport in all aquifer media. The PFOS breakthrough curves were asymmetrical and exhibited extensive concentration tailing, indicating that sorption/desorption was significantly nonideal. The results of model simulations indicated that rate-limited sorption/desorption was the primary cause of the nonideal PFOS transport. Comparison of PFOS transport in aquifer media to data reported for PFOS transport in two soils and a quartz sand showed that PFOS exhibited more extensive elution tailing for the soils, likely reflecting differences in the relative contributions of various media constituents to sorption. A three-component distributed-sorption model was developed that accounted for contributions from soil organic carbon, metal oxides, and silt + clay fraction. The model produced very good predictions of Kd for the five media with lower soil organic­carbon contents (≤0.1%). Soil organic carbon was estimated to contribute 19-42% of the total sorption for all media except the sand, to which it contributed ~100%. The contribution of silt + clay ranged from 51 to 80% for all media except the sand. The only medium for which the contribution of metal-oxides was significant is Hanford, with an estimated contribution of 15%. Overall, the results of the study indicate that sorption of PFOS by these aquifer media comprised contributions from multiple soil constituents.

Column versus batch methods for measuring PFOS and PFOA sorption to geomedia.

The objective of this study is to compare the consistency between column and batch experiment methods for measuring solid-phase sorption coefficients and isotherms for per and polyfluoroalkyl substances (PFAS). Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are used as representative PFAS, and experiments are conducted with three natural porous media with differing geochemical properties. Column-derived sorption isotherms are generated by conducting multiple experiments with different input concentrations (multi-C0 method) or employing elution-front integration wherein the entire isotherm is determined from a single breakthrough curve (BTC) elution front. The isotherms generated with the multi-C0 column method compared remarkably well to the batch isotherms over an aqueous concentration range of 3-4 orders of magnitude. Specifically, the 95% confidence intervals for the individual isotherm variables overlapped, producing statistically identical regressions. The elution-front integration isotherms generally agreed with the batch isotherms, but exhibited noise and systematic deviation at lower concentrations in some cases. All data sets were well described by the Freundlich isotherm model. Freundlich N values ranged from 0.75 to 0.81 for PFOS and was 0.87 for PFOA and are consistent with values reported in the literature for different geomedia. The results of this study indicate that column and batch experiments can measure consistent sorption isotherms and sorption coefficients for PFOS and PFOA when robust experimental setup and data analysis are implemented.

Natural attenuation method for contaminant remediation reagent delivery assessment for in situ chemical oxidation using aqueous ozone.

A Monitored Natural Attenuation (MNA) assessment approach typically used for contaminant remediation feasibility assessment was developed here for remediation-reagent delivery assessment. Subsurface delivery of oxidants, such as aqueous ozone (O3) for in situ chemical oxidation (ISCO) of groundwater contaminants, is naturally attenuated by oxidant demand and reactivity. We compared mixed reactor kinetic experiments, sand column tracer transport experiments, and reactive transport modeling and assessment methods to quantify natural attenuation kinetics, aqueous O3 solute transport, oxidant demand kinetics, and ISCO reagent delivery limitations. Sorption of aqueous O3 to quartz sand was observed during transport of O3 through water-saturated porous media. Pseudo 1st order decomposition rate constants of O3 bulk attenuation with transport were comparable to mixed reactor experiments without transport, and reactive transport modeling of miscible-displacement column experiments was used to quantify each attenuation process. Aqueous ionic strength was correlated with O3 decomposition rate constants, which was the dominant reagent delivery attenuation process. These results suggest that aqueous O3 decomposition and oxidant delivery attenuation can be predictable upon characterization of the sediment oxidant demand and dispersion, and increasing groundwater velocity during aqueous O3 injection can maximize transport distance for reagent delivery.

Molecular Diagnosis and Identification of Genetic Variants Underlying Distal Renal Tubular Acidosis in Pakistani Patients Using Whole Exome Sequencing.

Introduction: Primary distal renal tubular acidosis (dRTA) is a rare genetic disorder characterized by an impaired urinary acidification process in distal nephrons that results in the production of alkaline urine. Loss of function variants in any of the three genes, ATP6V0A4, ATP6V1B1, or SLC4A1, which all play a role in normal acidification of urine by kidneys, may lead to dRTA. Objective: This study was designed to identify genetic variants underlying dRTA in Pakistani patients using whole exome sequencing, followed by confirmatory Sanger sequencing. Materials and Methods: Patients were identified following presentation with characteristic clinical features of dRTA including vomiting, dehydration, and highly alkaline urine with metabolic acidosis during the first few days of life. Whole exome sequencing and Sanger sequencing were employed for genetic analyses of the patients. In silico analyses of the identified variants were performed using web-based bioinfomatics programs. Results: Through whole exome sequencing, we identified two splice site variants (c.2257 + 1G>A and c.722 + 5G>A) in the ATP6V0A4 gene that likely underly the disease phenotype in the two families. Multiple in silico tools predicted these variants to affect the respective splice sites supporting their likely role in pathogenesis. Conclusion: The study extends the spectrum of ATP6V0A4 variants associated with dRTA and should benefit the genetic counseling and prenatal diagnosis of the affected families.

Nonideal Transport and Extended Elution Tailing of PFOS in Soil.

The objective of this research was to examine the influence of nonideal sorption/desorption on the transport of polyfluorinated alkyl substances (PFASs) in soil, with a specific focus on characterizing and quantifying potential extended, mass-transfer-limited elution behavior. Perfluorooctane sulfonic acid (PFOS) was used as a representative PFAS, and miscible-displacement experiments were conducted with two soils comprising contrasting geochemical properties. The influence of nonlinear, rate-limited, hysteretic, and irreversible sorption/desorption on transport was investigated through experiments and model simulations. The breakthrough curves measured for PFOS transport in the two soils were asymmetrical and exhibited extensive elution tailing, indicating that sorption/desorption was significantly nonideal. The widely used two-domain sorption kinetics model could not fully simulate the observed transport behavior, whereas a multirate model employing a continuous distribution of sorption domains was successful. The overall results indicated that sorption/desorption was significantly rate-limited and that nonlinear, hysteretic, and irreversible sorption/desorption had minimal impact on PFOS transport. Comparison of PFOS transport data to data reported for two hydrophobic organic contaminants (HOCs) showed that the HOCs exhibited much more extensive elution tailing, likely reflecting differences in sorption/desorption mechanisms. The projected influence of rate-limited sorption/desorption on PFOS transport at the field scale was investigated through simulation. The results of the study suggest that rate-limited sorption/desorption may affect the field-scale transport of PFOS and other PFAS for systems influenced by transient or short-residence-time conditions and in some cases could possibly increase the amount of flushing required to reduce PFOS concentrations to levels below those associated with human-health concerns.

Cyclodextrin-enhanced 1,4-dioxane treatment kinetics with TCE and 1,1,1-TCA using aqueous ozone.

Enhanced reactivity of aqueous ozone (O3) with hydroxypropyl-ß-cyclodextrin (HPßCD) and its impact on relative reactivity of O3 with contaminants were evaluated herein. Oxidation kinetics of 1,4-dioxane, trichloroethylene (TCE), and 1,1,1-trichloroethane (TCA) using O3 in single and multiple contaminant systems, with and without HPßCD, were quantified. 1,4-Dioxane decay rate constants for O3 in the presence of HPßCD increased compared to those without HPßCD. Density functional theory molecular modeling confirmed that formation of ternary complexes with HPßCD, O3, and contaminant increased reactivity by increasing reactant proximity and through additional reactivity within the HPßCD cavity. In the presence of chlorinated co-contaminants, the oxidation rate constant of 1,4-dioxane was enhanced. Use of HPßCD enabled O3 reactivity within the HPßCD cavity and enhanced 1,4-dioxane treatment rates without inhibition in the presence of TCE, TCA, and radical scavengers including NaCl and bicarbonate. Micro-environmental chemistry within HPßCD inclusion cavities mediated contaminant oxidation reactions with increased reaction specificity.

Molecular Study of Nephronophthisis in 7 Unrelated Pakistani Families.

Nephronophthisis is an autosomal recessive cystic kidney disease characterized by tubular interstitial infiltration, periglomerular fibrosis, and cysts, and is the most frequent genetic cause of end-stage renal disease in children. Nephronophthisis is pleiotropic as almost all the causative genes are involved in primary cilium and centrosome function which are found in almost all human cells. Genetic heterogeneity in nephronophthisis makes the molecular and genetic diagnosis somewhat difficult. Homozygous deletions in the nephronophthisis 1 (NPHP1) gene are the major contributor of nephronophthisis cases, while other genes accounts for less than 3% each. Nephronophthisis-related ciliopathy is a term used for extrarenal symptoms in addition to nephronophthisis. Herein, we are reporting the molecular study of 7 children from independent families fulfilling the criteria of nephronophthisis. A deletion analysis of the NPHP1 gene was performed in each case, and NPHP5 mutation screening was performed in the absence of such deletion in patients with Senior Loken syndrome.

Spectroscopic methods for aqueous cyclodextrin inclusion complex binding measurement for 1,4-dioxane, chlorinated co-contaminants, and ozone.

Recalcitrant organic contaminants, such as 1,4-dioxane, typically require advanced oxidation process (AOP) oxidants, such as ozone (O3), for their complete mineralization during water treatment. Unfortunately, the use of AOPs can be limited by these oxidants' relatively high reactivities and short half-lives. These drawbacks can be minimized by partial encapsulation of the oxidants within a cyclodextrin cavity to form inclusion complexes. We determined the inclusion complexes of O3 and three common co-contaminants (trichloroethene, 1,1,1-trichloroethane, and 1,4-dioxane) as guest compounds within hydroxypropyl-ß-cyclodextrin. Both direct (ultraviolet or UV) and competitive (fluorescence changes with 6-p-toluidine-2-naphthalenesulfonic acid as the probe) methods were used, which gave comparable results for the inclusion constants of these species. Impacts of changing pH and NaCl concentrations were also assessed. Binding constants increased with pH and with ionic strength, which was attributed to variations in guest compound solubility. The results illustrate the versatility of cyclodextrins for inclusion complexation with various types of compounds, binding measurement methods are applicable to a wide range of applications, and have implications for both extraction of contaminants and delivery of reagents for treatment of contaminants in wastewater or contaminated groundwater.

Stabilization and prolonged reactivity of aqueous-phase ozone with cyclodextrin.

Recalcitrant organic groundwater contaminants, such as 1,4-dioxane, may require strong oxidants for complete mineralization. However, their efficacy for in-situ chemical oxidation (ISCO) is limited by oxidant decay and reactivity. Hydroxypropyl-ß-cyclodextrin (HPßCD) was examined for its ability to stabilize aqueous-phase ozone (O3) and prolong oxidation potential through inclusion complex formation. Partial transformation of HPßCD by O3 was observed. However, HPßCD proved to be sufficiently recalcitrant, because it was only partially degraded in the presence of O3. The formation of a HPßCD:O3 clathrate complex was observed, which stabilized decay of O3. The presence of HPßCD increased the O3 half-life linearly with increasing HPßCD:O3 molar ratio. The O3 half-life in solutions increased by as much as 40-fold relative to HPßCD-free O3 solutions. Observed O3 release from HPßCD and indigo oxidation confirmed that the formation of the inclusion complex is reversible. This proof-of-concept study demonstrates that HPßCD can complex O3 while preserving its reactivity. These results suggest that the use of clathrate stabilizers, such as HPßCD, can support the development of a facilitated-transport enabled ISCO for the O3 treatment of groundwater contaminated with recalcitrant compounds.

Volatile-organic molecular characterization of shale-oil produced water from the Permian Basin.

Growth in unconventional oil and gas has spurred concerns on environmental impact and interest in beneficial uses of produced water (PW), especially in arid regions such as the Permian Basin, the largest U.S. tight-oil producer. To evaluate environmental impact, treatment, and reuse potential, there is a need to characterize the compositional variability of PW. Although hydraulic fracturing has caused a significant increase in shale-oil production, there are no high-resolution organic composition data for the shale-oil PW from the Permian Basin or other shale-oil plays (Eagle Ford, Bakken, etc.). PW was collected from shale-oil wells in the Midland sub-basin of the Permian Basin. Molecular characterization was conducted using high-resolution solid phase micro extraction gas chromatography time-of-flight mass spectrometry. Approximately 1400 compounds were identified, and 327 compounds had a >70% library match. PW contained alkane, cyclohexane, cyclopentane, BTEX (benzene, toluene, ethylbenzene, and xylene), alkyl benzenes, propyl-benzene, and naphthalene. PW also contained heteroatomic compounds containing nitrogen, oxygen, and sulfur. 3D van Krevelen and double bond equivalence versus carbon number analyses were used to evaluate molecular variability. Source composition, as well as solubility, controlled the distribution of volatile compounds found in shale-oil PW. The salinity also increased with depth, ranging from 105 to 162 g/L total dissolved solids. These data fill a gap for shale-oil PW composition, the associated petroleomics plots provide a fingerprinting framework, and the results for the Permian shale-oil PW suggest that partial treatment of suspended solids and organics would support some beneficial uses such as onsite reuse and bio-energy production.