Microplate-Based Cell Viability Assay as a Cost-Effective Alternative to Flow Cytometry for Microalgae Analysis.

Cell viability is a critical indicator for assessing culture quality in microalgae cultivation for biorefinery and bioremediation. Fluorescent dyes that distinguish viable from nonviable cells can enable viability quantification based on the percentage of live cells. However, fluorescence analysis using the typical flow cytometry method is costly and impractical for industrial applications. To address this, we developed new microplate assays utilizing fluorescein diacetate as a live cell stain and erythrosine B as a dead cell stain. These assays provide a low-cost, simple, and reliable method of assessing cell viability. The proposed microplate assays were successfully applied to monitor the viability of the microalgae Dunaliella viridis under carbon and nitrogen limitation stresses and demonstrated good agreement with flow cytometry measurements. We conducted a systematic investigation of the effects of dye concentration, incubation time, and background fluorescence on the microplate assays' performance. Further, we provide a comprehensive review of commonly used fluorescent dyes for microalgae staining, discuss strategies to enhance assay performance, and offer recommendations for dye selection and protocol development. This study presents a comprehensive new method for microplate-based viability analysis, providing valuable insights for future microalgae viability assessments and applications.

Effects of UV-C Disinfection on N95 and KN95 Filtering Facepiece Respirator Reuse.

The objective of this study was to evaluate the effectiveness of UV technology for virus disinfection to allow FFR reuse. UV is a proven decontamination tool for microbial pathogens, including the SARS-CoV-2 virus. Research findings suggest that the impacts of UV-C treatment on FFR material degradation should be confirmed using microbial surrogates in addition to the commonly performed abiotic particle testing. This study used the surrogates, E. coli and MS-2 bacteriophage, as they bracket the UV response of SARS-CoV-2. Lower log inactivation was observed on FFRs than predicted by aqueous-based UV dose-response data for MS-2 bacteriophage and E. coli. In addition, the dose-response curves did not follow the trends commonly observed with aqueous data for E. coli and MS-2. The dose-response curves for the respirators in this study had a semicircle shape, where the inactivation reached a peak and then decreased. This decrease in UV inactivation is thought to be due to the degradation of the fibers of the FFR and allows for more viral and bacterial cells to wash through the layers of the respirator. This degradation phenomenon was observed at UV doses at and above 2,000 mJ/cm2. Results have demonstrated that FFR materials yield various results in terms of effective disinfection in experiments conducted on KN95 and N95 face respirators. The highest inactivation for both surrogates was observed with the KN95 respirator made by Purism, yielding 3 and 2.75 log inactivation for E. coli and MS-2 at UV doses of 1,500 mJ/cm2. The KN95 made by Anboruo yielded the lowest inactivation for MS-2 at 0.75 log when exposed to 1,000 mJ/cm2. To further test the degradation theory, experiments used a collimated beam device to test the hypothesis further that degradation is occurring at and above UV doses of 1,500 mJ/cm2. The experiment aimed to determine the effect of "predosing" a respirator with UV before inoculating the respirator with MS-2. In this test, quantification of the penetrated irradiance value and the ability of each layer to retain MS-2 were quantified. The results of the experiments varied from the intact FFR degradation experiments but displayed some data to support the degradation theory. IMPORTANCE Research suggests degradation of FFR materials at high UV doses is important. There appears to be a peak inactivation dose at approximately 1,500 mJ/cm2. The subsequent dose increases appear to have the reverse effect on inactivation values; these trends have shown true with both the N95 and KN95-Purism respirators.

Dynamic Modeling of Microalgae Growth and Lipid Production under Transient Light and Nitrogen Conditions.

We developed a new dynamic model to characterize how light and nitrogen regulate the cellular processes of photosynthetic microalgae leading to transient changes in the production of neutral lipids, carbohydrates, and biomass. Our model recapitulated the versatile neutral lipid synthesis pathways via (i) carbon reuse from carbohydrate metabolism under nitrogen sufficiency and (ii) fixed carbon redirection under nitrogen depletion. We also characterized the effects of light adaptation, light inhibition hysteresis, and nitrogen limitation on photosynthetic carbon fixation. The formulated model was calibrated and validated with experimental data of Dunaliella viridis cultivated in a lab-scale photobioreactor (PBR) under various light (low/moderate/high) and nitrogen (sufficient/limited) conditions. We conducted the identifiability, uncertainty, and sensitivity analyses to verify the model reliability using the profile likelihood method, the Markov chain Monte Carlo (MCMC) technique, and the extended Fourier Amplitude Sensitivity Test (eFAST). Our model predictions agreed well with experimental observations and suggested potential model improvement by incorporating a lipid degradation mechanism. The insights from our model-driven analysis helped improve the mechanistic understanding of transient algae growth and bioproducts formation under environmental variations and could be applied to optimize biofuel and biomass production.

Development of Photochemical Microsensors for Evaluating Photosynthetic Light Dose Distributions in Microalgal Photobioreactors.

We describe the development and testing of a Lagrangian method for quantifying light dose distributions within photobioreactors (PBRs) using novel photochemical microsensors. These microsensors were developed using 3-µm microspheres coated with a fluorescent dye that responds to wavelengths of visible light that are critical for photosynthesis. The dose-response kinetics of the microsensors was established by varying known doses of collimated light and quantifying the fluorescence responses of individual particles using flow cytometry. A deconvolution scheme was used to determine the light dose distribution from the fluorescence distribution of the microsensors. As proof-of-concept, the microsensors were used to quantify the photosynthetic light dose distributions within a gently mixed, 3 L flat-plate, batch PBR with and without algae and no gas bubbling and without algae but with gas bubbling. The microsensor approach not only provided information about the photosynthetic light distributions within the PBRs but also predicted the average light attenuation due to algal cells within 1% of estimates made with an in situ light sensor. The results showed that bubbles, under the conditions tested, increased the overall light irradiance by 18%; a result not captured by static measurements. The Lagrangian microsensors provide a novel approach for quantifying light within a photobioreactor.

Complete proteomic-based enzyme reaction and inhibition kinetics reveal how monolignol biosynthetic enzyme families affect metabolic flux and lignin in Populus trichocarpa.

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.

Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid:coenzyme A ligase protein complex formation, regulation, and numerical modeling.

As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.

Heat Generation and Accumulation in Municipal Solid Waste Landfills.

There have been reports of North American landfills that are experiencing temperatures in excess of 80-100 °C. However, the processes causing elevated temperatures are not well understood. The objectives of this study were to develop a model to describe the generation, consumption and release of heat from landfills, to predict landfill temperatures, and to understand the relative importance of factors that contribute to heat generation and accumulation. Modeled heat sources include energy from aerobic and anaerobic biodegradation, anaerobic metal corrosion, ash hydration and carbonation, and acid-base neutralization. Heat removal processes include landfill gas convection, infiltration, leachate collection, and evaporation. The landfill was treated as a perfectly mixed batch reactor. Model predictions indicate that both anaerobic metal corrosion and ash hydration/carbonation contribute to landfill temperatures above those estimated from biological reactions alone. Exothermic pyrolysis of refuse, which is hypothesized to be initiated due to a local accumulation of heat, was modeled empirically to illustrate its potential impact on heat generation.

Good modelling practice in applying computational fluid dynamics for WWTP modelling.

Computational fluid dynamics (CFD) modelling in the wastewater treatment (WWT) field is continuing to grow and be used to solve increasingly complex problems. However, the future of CFD models and their value to the wastewater field are a function of their proper application and knowledge of their limits. As has been established for other types of wastewater modelling (i.e. biokinetic models), it is timely to define a good modelling practice (GMP) for wastewater CFD applications. An International Water Association (IWA) working group has been formed to investigate a variety of issues and challenges related to CFD modelling in water and WWT. This paper summarizes the recommendations for GMP of the IWA working group on CFD. The paper provides an overview of GMP and, though it is written for the wastewater application, is based on general CFD procedures. A forthcoming companion paper to provide specific details on modelling of individual wastewater components forms the next step of the working group.

Population balance models: a useful complementary modelling framework for future WWTP modelling.

Population balance models (PBMs) represent a powerful modelling framework for the description of the dynamics of properties that are characterised by distributions. This distribution of properties under transient conditions has been demonstrated in many chemical engineering applications. Modelling efforts of several current and future unit processes in wastewater treatment plants could potentially benefit from this framework, especially when distributed dynamics have a significant impact on the overall unit process performance. In these cases, current models that rely on average properties cannot sufficiently capture the true behaviour and even lead to completely wrong conclusions. Examples of distributed properties are bubble size, floc size, crystal size or granule size. In these cases, PBMs can be used to develop new knowledge that can be embedded in our current models to improve their predictive capability. Hence, PBMs should be regarded as a complementary modelling framework to biokinetic models. This paper provides an overview of current applications, future potential and limitations of PBMs in the field of wastewater treatment modelling, thereby looking over the fence to other scientific disciplines.

Monolignol pathway 4-coumaric acid:coenzyme A ligases in Populus trichocarpa: novel specificity, metabolic regulation, and simulation of coenzyme A ligation fluxes.

4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stem-differentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis.

Microbial community assembly in engineered bioreactors.

Microbial community assembly (MCA) processes that shape microbial communities in environments are being used to analyze engineered bioreactors such as activated sludge systems and anaerobic digesters. The goal of studying MCA is to be able to understand and predict the effect of design and operation procedures on bioreactor microbial composition and function. Ultimately, this can lead to bioreactors that are more efficient, resilient, or resistant to perturbations. This review summarizes the ecological theories underpinning MCA, evaluates MCA analysis methods, analyzes how these MCA-based methods are applied to engineered bioreactors, and extracts lessons from case studies. Furthermore, we suggest future directions in MCA research in engineered bioreactor systems. The review aims to provide insights and guidance to the growing number of environmental engineers who wish to design and understand bioreactors through the lens of MCA.

Understanding the spatial formation and accumulation of fats, oils and grease deposits in the sewer collection system.

Sanitary sewer overflows are caused by the accumulation of insoluble calcium salts of fatty acids, which are formed by the reaction between fats, oils and grease (FOG) and calcium found in wastewaters. Different sewer structural configurations (i.e., manholes, pipes, wet wells), which vary spatially, along with other obstructions (roots intrusion) and pipe deformations (pipe sags), may influence the detrimental buildup of FOG deposits. The purpose of this study was to quantify the spatial variation in FOG deposit formation and accumulation in a pilot-scale sewer collection system. The pilot system contained straight pipes, manholes, roots intrusion, and a pipe sag. Calcium and oil were injected into the system and operated at alkaline (pH = 10) and neutral (pH = 7) pH conditions. Results showed that solid accumulations were slightly higher at neutral pH. Fourier transform infrared (FTIR) analysis on the solids samples confirmed that the solids were indeed calcium-based fatty acid salts. However, the fatty acid profiles of the solids deviated from the profile found from FOG deposits in sewer systems, which were primarily saturated fatty acids. These results confirm the work done previously by researchers and suggest an alternative fate of unsaturated fatty acids that does not lead to their incorporation in FOG deposits in full-scale sewer systems.

Growth of Dunaliella viridis in multiple cycles of reclaimed media after repeated high pH-induced flocculation and harvesting.

Minimizing the use of water for growing microalgae is crucial for lowering the energy and costs of animal feed, food, and biofuel production from microalgae. Dunaliella spp., a haloterant species that can accumulate high intracellular levels of lipids, carotenoids, or glycerol can be harvested effectively using low-cost and scalable high pH-induced flocculation. However, the growth of Dunaliella spp. in reclaimed media after flocculation and the impact of recycling on the flocculation efficiency have not been explored. In this study, repeated cycles of growth of Dunaliella viridis in repeatedly reclaimed media from high pH-induced flocculation were studied by evaluating cell concentrations, cellular components, dissolved organic matter (DOM), and bacterial community shifts in the reclaimed media. In reclaimed media, D. viridis grew to the same concentrations of cells and intracellular components as fresh media-107 cells/mL with cellular composition of 3 % lipids, 40 % proteins, and 15 % carbohydrates-even though DOM accumulated and the dominant bacterial populations changed. There was a decrease in the maximum specific growth rate and flocculation efficiency from 0.72 d-1 to 0.45 d-1 and from 60 % to 48 %, respectively. This study shows the potential of repeated (at least five times) flocculation and reuse of media as a possible way of reducing the costs of water and nutrients with some tradeoffs in growth rate and flocculation efficiency.

Biotransformation of micropollutants in moving bed biofilm reactors under heterotrophic and autotrophic conditions.

We investigated the transformation of four pharmaceuticals (Diclofenac, Naproxen, Ibuprofen and Carbamazepine) in a moving bed biofilm reactor subjected to different COD/N ratios in four experimental phases. The shift from medium to high range COD/N ratio (i.e., 5:1 to 100:1) intensified the competition between heterotrophs and nitrifying communities, leading to a transition from co-existence of heterotrophic and autotrophic conditions with high COD removal and nitrification rate in phase I to dominant heterotrophic conditions in phase II. At lower range COD/N ratios (i.e., 1:2 and 1:8) in phase III and IV, autotrophic conditions prevailed, resulting in increased nitrification rates and high abundance of amoA gene in the biofilm. Such shifts in the operating condition were accompanied by notable changes in the biofilm concentrations, composition and abundance of microbial populations as well as biodiversity in the biofilms, which collectively affected the degradation rates of the pharmaceuticals. We observed higher kinetic rates per unit of biofilm concentration under autotrophic conditions compared to heterotrophic conditions for all compounds except Naproxen, indicating the importance of nitrification in the transformation of such compounds. The results also revealed a positive relationship between biodiversity and biomass-normalized kinetic rates of most compounds.

Field characterization of external grease abatement devices.

This study characterized some of the physical and chemical features of large outside field grease abatement devices (GADs). 24-hour measurements of several food service establishments' (FSEs') influent GAD flowrates indicated highly intermittent conditions with hydraulic retention times (HRTs) that exceeded the common recommendation (30 minutes) by two to five times. Investigation into the chemical characteristics of GADs indicated highly variable influent and effluent fat, oil, and grease (FOG) concentrations. Low pH and dissolved oxygen values were measured throughout the GAD, indicating the likely occurrence of anaerobic microbial processes. Detailed spatial and temporal observations of the accumulation of FOG and food solids were also discussed. Though the FOG layer remained relatively constant for all GAD configurations investigated, results indicated that commonly-used GAD configurations with a straight submerged inlet tee or no-inlet tee configuration may result in the transport of food solids into the second compartment. The present research showed increased accumulation of food solids in the first compartment with a retro-fit flow distributive inlet. This retro-fit displays promise for potentially improving the separation characteristics of existing GADs.

Evidence for fat, oil, and grease (FOG) deposit formation mechanisms in sewer lines.

The presence of hardened and insoluble fats, oil, and grease (FOG) deposits in sewer lines is a major cause of line blockages leading to sanitary sewer overflows (SSOs). Despite the central role that FOG deposits play in SSOs, little is known about the mechanisms of FOG deposit formation in sanitary sewers. In this study, FOG deposits were formed under laboratory conditions from the reaction between free fatty acids and calcium chloride. The calcium and fatty acid profile analysis showed that the laboratory-produced FOG deposit displayed similar characteristics to FOG deposits collected from sanitary sewer lines. Results of FTIR analysis showed that the FOG deposits are metallic salts of fatty acid as revealed by comparisons with FOG deposits collected from sewer lines and pure calcium soaps. Based on the data, we propose that the formation of FOG deposits occurs from the aggregation of excess calcium compressing the double layer of free fatty acid micelles and a saponification reaction between aggregated calcium and free fatty acids.

Assessment of internal and external grease interceptor performance for removal of food-based fats, oil, and grease from food service establishments.

A research study was performed to determine the fats, oil, and grease (FOG) removal efficiency of internal flow-based grease interceptors (FGI). A passive-flow and a mechanical-flow FGI unit were tested and their performance was compared to an external retention-based grease interceptor (RGI). Experiments involved multiple parameters including three oil/water emulsion strengths, two influent liquid temperatures, and two flow rates. Overall, the RGI achieved approximately 80% FOG removal and the FGIs removed less than 50% under the tested conditions. One exception occurred during the passive FGI testing during which removal reached approximately 80%. This increased efficiency was likely due to the relatively weak emulsion exhibiting significantly large FOG globules. The FOG removal efficiency decreased with increased temperature because of increased breakage of FOG globules at the elevated temperature. These results suggest that emulsion strength significantly affects FOG removal efficiency of FGIs and should be considered in future manufacturer testing protocol.

Measurement of heat release during hydration and carbonation of ash disposed in landfills using an isothermal calorimeter.

Temperatures as high as 100 °C have been reported at a few municipal solid waste (MSW) landfills in the U.S. A recently published model describing landfill heat accumulation identified reactions that contribute significant heat to landfills including the hydration and carbonation of Ca-containing wastes such as ash from MSW and coal combustion. The objective of this study was to develop a method to measure heat release from Ca-containing ash by isothermal calorimetry. The method was confirmed by comparing measured heat release from hydration and carbonation of pure CaO and Ca(OH)2 to the theoretical heat. Theoretical heat release was determined by characterizing test materials before and after experiments using thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Heat recovery efficiencies with both water and synthetic leachate ranged from 79 to 90% for CaO hydration and between 65 and 74% for Ca(OH)2 carbonation, with no effect attributable to leachate. Additionally, simultaneous hydration and carbonation of CaO/Ca(OH)2 mixtures resulted in efficiencies of 65 to 74%. The developed method was applied to eight samples that were excavated from a landfill and known to contain coal ash, and the ratio of measured to theoretical heat was 0.5 to 4. Thus, calculation of theoretical heat release from XRD data was not a good predictor of the experimentally measured heat release. The developed method can be used by landfill operators to evaluate the heat potential of a waste, thereby facilitating decisions on the quantity of a waste that can be buried in consideration of landfill temperatures.

Biodegradation of organophosphorus pesticides in moving bed biofilm reactors: Analysis of microbial community and biodegradation pathways.

We investigated the performance of a lab-scale moving bed biofilm reactor (MBBR) with respect to general bioconversion processes and biotransformation of two commonly used organophosphorus pesticides, Chlorpyrifos (CHL) and Malathion (MAL). The reactor was operated for 300 days under different organic loads by changing hydraulic retention time (HRT). The decrease in organic load resulted in the formation of a thinner biofilm and the growth of more biomass in the bulk, which greatly shifted bioconversion processes. The low organic loading supported more nitrification in the reactor, but an opposite trend was observed for denitrification, which was enhanced at higher organic loading where the formation of anoxic zones in the thick biofilm was favored. 70% and 55% removal corresponding to 210 and 165 µg/m2/d occurred for MAL and CHL, respectively, at an HRT of 3 h and progressively increased with higher HRTs. Phylogenetic analysis revealed a shift in composition and abundance of taxa throughout the reactor operation where lower loading rate supported the growth of a more diverse and evenly distributed community. The analysis also highlighted the dominance of heterotrophic communities such as Flavobacterium and Acinetobacter johnsonii, which could be involved in the biotransformation of CHL and MAL through co-metabolism.

Modeling hydroxyl radical distribution and trialkyl phosphates oxidation in UV-H2O2 photoreactors using computational fluid dynamics.

Advanced Oxidation Processes (AOPs) promoted by ultraviolet light are innovative and potentially cost-effective solutions for treating persistent pollutants recalcitrant to conventional water and wastewater treatment. While several studies have been performed during the past decade to improve the fundamental understanding of the UV-H(2)O(2) AOP and its kinetic modeling, Computational Fluid Dynamics (CFD) has only recently emerged as a powerful tool that allows a deeper understanding of complex photochemical processes in environmental and reactor engineering applications. In this paper, a comprehensive kinetic model of UV-H(2)O(2) AOP was coupled with the Reynolds averaged Navier-Stokes (RANS) equations using CFD to predict the oxidation of tributyl phosphate (TBP) and tri(2-chloroethtyl) phosphate (TCEP) in two different photoreactors: a parallel- and a cross-flow UV device employing a UV lamp emitting primarily 253.7 nm radiation. CFD simulations, obtained for both turbulent and laminar flow regimes and compared with experimental data over a wide range of UV doses, enabled the spatial visualization of hydrogen peroxide and hydroxyl radical distributions in the photoreactor. The annular photoreactor displayed consistently better oxidation performance than the cross-flow system due to the absence of recirculation zones, as confirmed by the hydroxyl radical dose distributions. Notably, such discrepancy was found to be strongly dependent on and directly correlated with the hydroxyl radical rate constant becoming relevant for conditions approaching diffusion-controlled reaction regimes (k(C,OH) > 10(9) M(-1) s(-1)).