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.

Meeting the Water and Sanitation Challenges of Underbounded Communities in the U.S.

Water and sanitation (wastewater) infrastructure in the United States is aging and deteriorating, with massive underinvestment over the past several decades. For many years, lack of attention to water and sanitation infrastructure has combined with racial segregation and discrimination to produce uneven access to water and wastewater services resulting in growing threats to human and environmental health. In many metropolitan areas in the U.S., those that often suffer disproportionately are residents of low-income, minority communities located in urban disadvantaged unincorporated areas on the margins of major cities. Through the process of underbounding (the selective expansion of city boundaries to exclude certain neighborhoods often based on racial demographics or economics), residents of these communities are disallowed municipal citizenship and live without piped water, sewage lines, and adequate drainage or flood control. This Perspective identifies the range of water and sanitation challenges faced by residents in these communities. We argue that future investment in water and sanitation should prioritize these communities and that interventions need to be culturally context sensitive. As such, approaches to address these problems must not only be technical but also social and give attention to the unique geographic and political setting of local infrastructures.

SARS-CoV-2 Wastewater Surveillance for Public Health Action.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has garnered extensive public attention during the coronavirus disease pandemic as a proposed complement to existing disease surveillance systems. Over the past year, methods for detection and quantification of SARS-CoV-2 viral RNA in untreated sewage have advanced, and concentrations in wastewater have been shown to correlate with trends in reported cases. Despite the promise of wastewater surveillance, for these measurements to translate into useful public health tools, bridging the communication and knowledge gaps between researchers and public health responders is needed. We describe the key uses, barriers, and applicability of SARS-CoV-2 wastewater surveillance for supporting public health decisions and actions, including establishing ethics consideration for monitoring. Although wastewater surveillance to assess community infections is not a new idea, the coronavirus disease pandemic might be the initiating event to make this emerging public health tool a sustainable nationwide surveillance system, provided that these barriers are addressed.

Global Water, Sanitation, and Hygiene Approaches: Anthropological Contributions and Future Directions for Engineering.

Anthropologists contribute key insights toward a comprehensive understanding of water, sanitation, and hygiene (WASH) as a multidimensional, multiscalar, and culturally embedded phenomenon. Yet, these insights have yet to be sufficiently operationalized and implemented in WASH development and wider WASH access-related paradigms. Ensuring WASH security requires a comprehensive approach to identifying both human health risk and environmental impact of WASH-related programs and strategies. It requires an understanding of how sanitation is integrated into households and communities and how individuals within particular cultural contexts practice sanitation and hygiene. This work facilitates that goal by outlining the major contributions of anthropology and allied social sciences to WASH, as well as outlining key considerations for future work and collaboration. We identify six major themes that, if applied in future engineering approaches, will more equitably integrate stakeholders and multiple vantage points in the successful implementation of WASH projects for marginalized and diverse groups. These include a critical understanding of previous approaches, culturally aware interventions, capacity building that considers (un)intended impact, co-created technology, collaboration between fields such as anthropology and engineering, and challenge-ready initiatives that respond to historic and emergent social and environmental inequity.

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.

Microbial ecological succession during municipal solid waste decomposition.

The decomposition of landfilled refuse proceeds through distinct phases, each defined by varying environmental factors such as volatile fatty acid concentration, pH, and substrate quality. The succession of microbial communities in response to these changing conditions was monitored in a laboratory-scale simulated landfill to minimize measurement difficulties experienced at field scale. 16S rRNA gene sequences retrieved at separate stages of decomposition showed significant succession in both Bacteria and methanogenic Archaea. A majority of Bacteria sequences in landfilled refuse belong to members of the phylum Firmicutes, while Proteobacteria levels fluctuated and Bacteroidetes levels increased as decomposition proceeded. Roughly 44% of archaeal sequences retrieved under conditions of low pH and high acetate were strictly hydrogenotrophic (Methanomicrobiales, Methanobacteriales). Methanosarcina was present at all stages of decomposition. Correspondence analysis showed bacterial population shifts were attributed to carboxylic acid concentration and solids hydrolysis, while archaeal populations were affected to a higher degree by pH. T-RFLP analysis showed specific taxonomic groups responded differently and exhibited unique responses during decomposition, suggesting that species composition and abundance within Bacteria and Archaea are highly dynamic. This study shows landfill microbial demographics are highly variable across both spatial and temporal transects.

Shifts in microbial communities in bioaugmented grease interceptors removing fat, oil, and grease (FOG).

To understand the effect of daily bioaugmentation in full-scale grease interceptors (GIs), we compared the microbial communities occurring in two full-scale GIs during bioaugmented and non-bioaugmented cycles. The changes in microbial communities were determined using terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene clone library construction. Differences in the microbial community structure between control and bioaugmented cycles were observed in all cases, although the dominant terminal restriction fragments in the biological product were not detected. The addition of bioaugmentation products and changes in the GI microbial ecology were related to differences in GI performance. Understanding the shifts due to bioaugmentation will result in more informed assessments of the benefits of bioadditives on FOG removal in GIs as well as the effects on downstream sewer lines.

Structural integrity affects nitrogen removal activity of granules in semi-continuous reactors.

Granules were observed after more than two years of operation in two semi-continuously fed intermittently aerated reactors treating swine wastewater with aerobic:anoxic cycles of 1:1 h and 1:4 h. Subsequently, the granules and flocs were compared with respect to physical characteristics, activity, and microbial community structure. Granules exhibited higher specific nitrification and denitrification rates than flocs. However, once granule structural integrity was disrupted, the rates decreased to levels similar to those of flocs. Membrane hybridizations using 16S rRNA-targeted probes showed that ammonia oxidizing bacteria populations in flocs and granules were dominated by Nitrosomonas and Nitrosococcus mobilis. Granules provided better conditions for Nitrospira compared to flocs. The diversities of the dominant bacterial populations in granules and flocs were not significantly different. Our findings highlight the importance of structural integrity of granules to their nitrogen removing activity.

Linking microbial population dynamics in anaerobic bioreactors to food waste type and decomposition stage.

A key question in anaerobic microbial ecology is how microbial communities develop over different stages of waste decomposition and whether these changes are specific to waste types. We destructively sampled over time 26 replicate bioreactors cultivated on fruit/vegetable waste (FVW) and meat waste (MW) based on pre-defined waste components and composition. To characterize community shifts, we examined 16S rRNA genes from both the leachate and solid fractions of the waste. Waste decomposition occurred faster in FVW than MW, as accumulation of ammonia in MW reactors led to inhibition of methanogenesis. We identified population succession during different stages of waste decomposition and linked specific populations to different waste types. Community analyses revealed underrepresentation of methanogens in the leachate fractions, emphasizing the importance of consistent and representative sampling when characterizing microbial communities in solid waste.

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.

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.

Identification of nitrite-reducing bacteria using sequential mRNA fluorescence in situ hybridization and fluorescence-assisted cell sorting.

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.

Physico-chemical characterization of grease interceptors with and without biological product addition.

Hardened and insoluble fat, oil, and grease (FOG) deposits are the primary cause of sewer line blockages leading to sanitary sewer overflows (SSOs). However, there have been very few long-term assessments of the physico-chemical characteristics of full-scale grease interceptors (GIs), the first "line of defense" against FOG buildup in sewer lines. In this study, we assessed the physico-chemical characteristics of two full-scale GIs (at a restaurant and a retirement community kitchen) over a one-year period. Statistically significant differences between bioaugmented and untreated cycles were detected for several chemical and physical properties. The treated cycles had lower BOD and COD at the grease interceptor outlet. While the combined data for all treated cycles did not show lower FOG concentrations in the GI outlet compared to the combined data for all untreated cycles, comparison of specific individual treated and untreated cycles show a positive effect due to the addition of product.

Multifaceted Assessment of Wastewater-Based Epidemiology for SARS-CoV-2 in Selected Urban Communities in Davao City, Philippines: A Pilot Study.

Over 60 countries have integrated wastewater-based epidemiology (WBE) in their COVID-19 surveillance programs, focusing on wastewater treatment plants (WWTP). In this paper, we piloted the assessment of SARS-CoV-2 WBE as a complementary public health surveillance method in susceptible communities in a highly urbanized city without WWTP in the Philippines by exploring the extraction and detection methods, evaluating the contribution of physico-chemical-anthropogenic factors, and attempting whole-genome sequencing (WGS). Weekly wastewater samples were collected from sewer pipes or creeks in six communities with moderate-to-high risk of COVID-19 transmission, as categorized by the City Government of Davao from November to December 2020. Physico-chemical properties of the wastewater and anthropogenic conditions of the sites were noted. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. A subset of nine samples were subjected to WGS using the Minion sequencing platform. SARS-CoV-2 RNA was detected in twenty-two samples (91.7%) regardless of the presence of new cases. Cycle threshold values correlated with RNA concentration and attack rate. The lack of a sewershed map in the sampled areas highlights the need to integrate this in the WBE planning. A combined analysis of wastewater physico-chemical parameters such as flow rate, surface water temperature, salinity, dissolved oxygen, and total dissolved solids provided insights on the ideal sampling location, time, and method for WBE, and their impact on RNA recovery. The contribution of fecal matter in the wastewater may also be assessed through the coliform count and in the context of anthropogenic conditions in the area. Finally, our attempt on WGS detected single-nucleotide polymorphisms (SNPs) in wastewater which included clinically reported and newly identified mutations in the Philippines. This exploratory report provides a contextualized framework for applying WBE surveillance in low-sanitation areas.

Effect of spatial differences in microbial activity, pH, and substrate levels on methanogenesis initiation in refuse.

The initiation of methanogenesis in refuse occurs under high volatile fatty acid (VFA) concentration and low pH (5.5 to 6.25), which generally are reported to inhibit methanogenic Archaea. One hypothesized mechanism for the initiation of methanogenesis in refuse decomposition is the presence of pH-neutral niches within the refuse that act as methanogenesis initiation centers. To provide experimental support for this mechanism, laboratory-scale landfill reactors were operated and destructively sampled when methanogenesis initiation was observed. The active bacterial and archaeal populations were evaluated using RNA clone libraries, RNA terminal restriction fragment length polymorphism (T-RFLP), and reverse transcription-quantitative PCR (RT-qPCR). Measurements from 81 core samples from vertical and horizontal sections of each reactor showed large spatial differences in refuse pH, moisture content, and VFA concentrations. No pH-neutral niches were observed prior to methanogenesis. RNA clone library results showed that active bacterial populations belonged mostly to Clostridiales, and that methanogenic Archaea activity at low pH was attributable to Methanosarcina barkeri. After methanogenesis began, pH-neutral conditions developed in high-moisture-content areas containing substantial populations of M. barkeri. These areas expanded with increasing methane production, forming a reaction front that advanced to low-pH areas. Despite low-pH conditions in >50% of the samples within the reactors, the leachate pH was neutral, indicating that it is not an accurate indicator of landfill microbial conditions. In the absence of pH-neutral niches, this study suggests that methanogens tolerant to low pH, such as M. barkeri, are required to overcome the low-pH, high-VFA conditions present during the anaerobic acid phase of refuse decomposition.

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.

Critical evaluation of solid waste sample processing for DNA-based microbial community analysis.

Landfills represent a unique microbial ecosystem and play a significant role in global biogeochemical processes. The study of complex ecosystems such as landfills using DNA-based techniques can be advantageous since they allow for analysis of uncultured organisms and offer higher resolution in measuring demographic and metabolic (functional) diversity. However, sample acquisition and processing from refuse is challenging due to material heterogeneity. Decomposed refuse was used to evaluate the effect of seven sample processing methods on Bacteria and Archaea community structure using T-RFLP. Bias was assessed using measured richness and by comparing community structure using multi-dimensional scaling (MDS). Generally, direct methods were found to be most biased while indirect methods (i.e., removal of cellular material from the refuse matrix before DNA extraction) were least biased. An indirect method using PO4 buffer gave consistently high bacterial and archaeal richness and also resulted in 28 and 34% recovery of R. albus and M. formicicum spiked into refuse, respectively. However, the highest recovery of less abundant T-RFs was achieved using multiple processing methods. Results indicate differences in measured T-RF diversity from studies of landfill ecosystems could be caused by methodological (i.e., processing method) variation rather than refuse heterogeneity or true divergence in community structure.

Impact of sampling depth on pathogen detection in pit latrines.

Wastewater based epidemiology (WBE) is increasingly used to provide decision makers with actionable data about community health. WBE efforts to date have primarily focused on sewer-transported wastewater in high-income countries, but at least 1.8 billion people in low- and middle-income countries (LMIC) use onsite sanitation systems such as pit latrines and septic tanks. Like wastewater, fecal sludges from such systems offer similar advantages in community pathogen monitoring and other epidemiological applications. To evaluate the distribution of enteric pathogens inside pit latrines-which could inform sampling methods for WBE in LMIC settings unserved by sewers-we collected fecal sludges from the surface, mid-point, and maximum-depth of 33 pit latrines in urban and peri-urban Malawi and analyzed the 99 samples for 20 common enteric pathogens via multiplex quantitative reverse transcription PCR. Using logistic regression adjusted for household population, latrine sharing, the presence of a concrete floor or slab, water source, and anal cleansing materials, we found no significant difference in the odds of detecting the 20 pathogens from the mid-point (adjusted odds ratio, aOR = 1.1; 95% confidence interval = 0.73, 1.6) and surface samples (aOR = 0.80, 95% CI = 0.54, 1.2) compared with those samples taken from the maximum depth. Our results suggest that, for the purposes of routine pathogen monitoring, pit latrine sampling depth does not strongly influence the odds of detecting enteric pathogens by molecular methods. A single sample from the pit latrines' surface, or a composite of surface samples, may be preferred as the most recent material contributed to the pit and may be easiest to collect.