Perturbations to common gardens of anaerobic co-digesters reveal relationships between functional resilience and microbial community composition.

We report the relationship between enrichment of adapted populations and enhancement of community functional resilience in methanogenic bioreactors. Although previous studies have shown the positive effects of acclimation, this work directly investigated the relationships between microbiome dynamics and performance of anaerobic co-digesting reactors in response to different levels of an environmental perturbation (loading of grease interceptor waste [GIW]). Using the methanogenic microbiome from a full-scale digester, we developed eight sets of microbial communities in triplicate using different feed sources. These substrate-specific microbiomes were then exposed to three independent disturbance events of low-, mid- and high-GIW loading rates. This approach allowed us to directly attribute differences in community responses to differences in community composition. Despite identical inocula, environment (digester operation, substrate loading rate, and feeding patterns) and general whole-community function (methane production and effluent quality) during the cultivation period, different substrates led to different microbial community assemblies. Lipid pre-acclimation led to enrichment of a pool of specialized populations, along with thriving of sub-dominant communities. The enrichment of these populations improved functional resilience and process performance when exposed to a low level of lipid-rich perturbation compared with less-acclimated communities. At higher levels of perturbation, the communities were not able to recover methanogenesis, indicating a loading limit to the resilience response. This study extends our current understanding of environmental perturbations, feed-specific adaptation, and functional resilience in methanogenic bioreactors.IMPORTANCEThis study demonstrates, for the first time for GIW co-digestion, how applying similar perturbations to different microbial communities was used to directly identify the causal relationships between microbial community, function, and environment in triplicate anaerobic microbiomes. We evaluated the impact of feed-specific adaptation on methanogenic microbiomes and demonstrated how microbiomes can be influenced to improve their functional (methanogenic) resilience to GIW inhibition. These findings demonstrate how an ecological framework can help improve a biological engineering application, and more specifically, increase the potential of anaerobic co-digestion for converting wastes to energy.

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.

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.

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.

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.

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.

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.

Reducing fat, oil, and grease (FOG) deposits formation and adhesion on sewer collection system structures through the use of fly ash replaced cement-based materials.

The accumulation of fat, oil, and grease (FOG) deposits in sewer pipes reduces their conveyance and results in Sanitary Sewer Overflows (SSOs). Previous research has shown that concrete used in sewer lines is a significant source for calcium ion, which participates in the FOG deposit formation mechanism. However, no research has been conducted to understand the effect of calcium leaching from cement on FOG deposits formation and adhesion. This study quantifies the reduction in FOG deposit formation when Fly Ash (FA), a Supplementary Cementitious Material (SCM), is used to replace cement in the production of High Volume Fly Ash (HVFA) concrete materials. Results show that after 90 days of leaching test under controlled pH conditions, 75% and 86% reduction in calcium release were achieved from 50% and 75% FA replacement, respectively. After 30 days of FOG deposits formation tests on HVFA samples, 58% and 81% reduction in FOG deposit formation was found for 50% and 75% FA replacement, respectively. FTIR analyses of FOG deposits formed on concrete samples without FA replacement exhibited high calcium soap content (48%), while, FOG deposit formed on HVFA concrete materials showed low calcium soap percentage (22~29%). Furthermore, FTIR analyses report the first spatial variation found in FOG deposits that includes a surface layer of hard FOG deposits with high calcium soap absorbance and an outer layer of soft FOG deposits consisting of a low calcium absorbance. FTIR analyses revealed that the FOG deposit formation mechanism is affected by the availability of calcium and pH near the concrete surface. Finally, HVFA concrete materials were tested for compressive strength and durability against microbially induced concrete corrosion (MICC). After 180 days of sealed curing, HVFA concrete exhibited adequate compressive strength necessary for the sewer line construction and 50% FA replacement revealed satisfactory durability against MICC.

Modeling cell aggregate morphology during aerobic granulation in activated sludge processes reveals the combined effect of substrate and shear.

Past research on AGS (aerobic granular sludge technology) has mainly focused on macro-environment factors, such as settling time, feeding pattern, OLR (organic loading rate), SRT (sludge retention time), among others, and their effects on the granulation process. The biomass granulation process, however, is significantly affected by the micro-environment surrounding these biomass aggregates. In this research, an in silico computational approach was adopted to study the impact of the micro-environment on the biomass granulation process. A 2-D biofilm model based on the cellular automata algorithm and computational fluid dynamics was used to simulate the development of an individual biomass aggregate under specific hydrodynamic and substrate availability conditions. The simulation results indicated that shear and bulk substrate concentration combined to create the optimal conditions for aerobic granule formation. This process can be characterized by the RT (reversed Thiele) modulus value, which is the ratio of the maximum substrate transport over the maximum substrate reaction rate and an indicator of substrate availability. For AGS formation, the RT value should be greater than 0.1. Many common strategies, such as the application of batch reactors, selection for slow-growing microorganism, F/M (food/mass) ratio adjustment, feast and famine condition, and short settling time, for biomass granulation production can be explained by the RT value. The results suggest that rethinking unit process configurations in wastewater treatment facilities will be required to achieve reliable AGS formation.

Increased loading stress leads to convergence of microbial communities and high methane yields in adapted anaerobic co-digesters.

Enhancing biogas production, while avoiding inhibition of methanogenesis during co-digestion of grease interceptor waste (GIW), can help water resource recovery facilities reduce their carbon footprint. Here we used pre-adapted and non-adapted digesters to link microbial community structure to digester function. Before disturbance, the pre-adapted and non-adapted digesters showed similar methane production and microbial community diversity but dissimilar community composition. When exposed to an identical disturbance, the pre-adapted digester achieved better performance, while the non-adapted digester was inhibited. When re-exposed to disturbance after recovery, communities and performance of both digesters converged, regardless of the temporal variations. Co-digestion of up to 75% GIW added on a volatile solids (VS) basis was achieved, increasing methane yield by 336% from 0.180 to 0.785 l-methane/g-VS-added, the highest methane yield reported to date for lipid-rich waste. Progressive perturbation substantially enriched fatty acid-degrading Syntrophomonas from less than 1% to 24.6% of total 16S rRNA gene sequences, acetoclastic Methanosaeta from 2.3% to 11.9%, and hydrogenotrophic Methanospirillum from less than 1% to 6.6% in the pre-adapted digester. Specific hydrolytic and fermentative populations also increased. These ecological insights demonstrated how progressive perturbation can be strategically used to influence methanogenic microbiomes and improve co-digestion of GIW.

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline.

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier. Particle size and shape are both misleadingly 'simple' measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation. Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs. We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

Flux modeling for monolignol biosynthesis.

The pathway of monolignol biosynthesis involves many components interacting in a metabolic grid to regulate the supply and ratios of monolignols for lignification. The complexity of the pathway challenges any intuitive prediction of the output without mathematical modeling. Several models have been presented to quantify the metabolic flux for monolignol biosynthesis and the regulation of lignin content, composition, and structure in plant cell walls. Constraint-based models using data from transgenic plants were formulated to describe steady-state flux distribution in the pathway. Kinetic-based models using enzyme reaction and inhibition constants were developed to predict flux dynamics for monolignol biosynthesis in wood-forming cells. This review summarizes the recent progress in flux modeling and its application to lignin engineering for improved plant development and utilization.

Properties influencing fat, oil, and grease deposit formation.

Fat, oil, and grease (FOG) deposits are the reported cause of 50 to 75% of sanitary sewer overflows in the United States, resulting in 1.8 X 10(6) m3 (500 mil. gal) of raw wastewater released into the environment annually. The objective of this research was to characterize the chemical and physical properties of FOG deposits. Twenty-three cities from around the United States contributed FOG samples for the study. The FOG deposits showed a wide range in yield strength (4 to 34 kPa), porosity (10 to 24%), and moisture content (10 to 60%), suggesting uncontrolled formation processes. A majority of these deposits display hard, sandstonelike texture, with distinct layering effects, suggesting a discontinuous formation process. The results found that 84% of FOG deposits contained high concentrations of saturated fatty acids and calcium, suggesting preferential accumulation.

Controlling aerobic biological floc size using Couette-Taylor Bioreactors.

Biological floc size is an important reactor microenvironment parameter that is often not experimentally controlled due to a lack of suitable methods. Here, we introduce the Couette-Taylor bioreactor (CTB) as an improved tool for controlling biological floc size, specifically as compared with bubble-column sequencing batch reactors (SBRs). A CTB consists of two concentric walls, either of which may be rotated to induce fluid motion. The induced flow produces hydrodynamic shear which is more uniform than that produced through aeration in SBRs. Because hydrodynamic shear is a major parameter controlling floc size, we hypothesized the ability to better control shear rates within a CTB would enable better-controlled floc sizes. To test this hypothesis, we measured the particle size distributions of activated sludge flocs from CTBs with either inner (iCTB) or outer (oCTB) rotating walls as well as SBRs with varying height to diameter ratios (0.5, 1.1, and 9.4). The rotation speed of the CTBs and aeration rate of the SBRs were varied to produce predicted mean shear rates from 25 to 250 s-1. Further, the shear rate distributions for each experiment were estimated using computational fluid dynamics (CFD). In all SBR experiments, the floc distributions did not significantly vary with shear rate or geometry, likely because shear rates (estimated by CFD) differed much less than originally predicted by theory. In the CTB experiments, the mean particle size decreased proportionally with increased hydrodynamic shear, and iCTBs produced particle size distributions with smaller coefficients of variation than oCTBs (0.3 vs. 0.5-0.7, respectively).

Assessing the impact of the 4CL enzyme complex on the robustness of monolignol biosynthesis using metabolic pathway analysis.

Lignin is a polymer present in the secondary cell walls of all vascular plants. It is a known barrier to pulping and the extraction of high-energy sugars from cellulosic biomass. The challenge faced with predicting outcomes of transgenic plants with reduced lignin is due in part to the presence of unique protein-protein interactions that influence the regulation and metabolic flux in the pathway. Yet, it is unclear why certain plants have evolved to create these protein complexes. In this study, we use mathematical models to investigate the role that the protein complex, formed specifically between Ptr4CL3 and Ptr4CL5 enzymes, have on the monolignol biosynthesis pathway. The role of this Ptr4CL3-Ptr4CL5 enzyme complex on the steady state flux distribution was quantified by performing Monte Carlo simulations. The effect of this complex on the robustness and the homeostatic properties of the pathway were identified by performing sensitivity and stability analyses, respectively. Results from these robustness and stability analyses suggest that the monolignol biosynthetic pathway is resilient to mild perturbations in the presence of the Ptr4CL3-Ptr4CL5 complex. Specifically, the presence of Ptr4CL3-Ptr4CL5 complex increased the stability of the pathway by 22%. The robustness in the pathway is maintained due to the presence of multiple enzyme isoforms as well as the presence of alternative pathways resulting from the presence of the Ptr4CL3-Ptr4CL5 complex.