Thermodynamic Analysis of Intermediary Metabolic Steps and Nitrous Oxide Production by Ammonium-Oxidizing Bacteria.

Nitrous oxide (N2O) is a greenhouse gas emitted from wastewater treatment, soils, and agriculture largely by ammonium-oxidizing bacteria (AOB). While AOB are characterized by being aerobes that oxidize ammonium (NH4+) to nitrite (NO2-), fundamental studies in microbiology are revealing the importance of metabolic intermediates and reactions that can lead to the production of N2O. These findings about the metabolic pathways for AOB were integrated with thermodynamic electron-equivalents modeling (TEEM) to estimate kinetic and stoichiometric parameters for each of the AOB's nitrogen (N)-oxidation and -reduction reactions. The TEEM analysis shows that hydroxylamine (NH2OH) oxidation to nitroxyl (HNO) is the most energetically efficient means for the AOB to provide electrons for ammonium monooxygenation, while oxidations of HNO to nitric oxide (NO) and NO to NO2- are energetically favorable for respiration and biomass synthesis. The respiratory electron acceptor can be O2 or NO, and both have similar energetics. The TEEM-predicted value for biomass yield, maximum-specific rate of NH4+ utilization, and maximum specific growth rate are consistent with empirical observations. NO reduction to N2O is thermodynamically favorable for respiration and biomass synthesis, but the need for O2 as a reactant in ammonium monooxygenation likely precludes NO reduction to N2O from becoming the major pathway for respiration.

Chemical Oxygen Demand Can Be Converted to Gross Energy for Food Items Using a Linear Regression Model.

BACKGROUND: Human and microbial metabolism are distinct disciplines. Terminology, metrics, and methodologies have been developed separately. Therefore, combining the 2 fields to study energetic processes simultaneously is difficult. OBJECTIVES: When developing a mechanistic framework describing gut microbiome and human metabolism interactions, energy values of food and digestive materials that use consistent and compatible metrics are required. As an initial step toward this goal, we developed and validated a model to convert between chemical oxygen demand (COD) and gross energy (${E_g}$) for >100 food items and ingredients. METHODS: We developed linear regression models to relate (and be able to convert between) theoretical gross energy (${E_g}^{\prime}$) and chemical oxygen demand (COD'); the latter is a measure of electron equivalents in the food's carbon. We developed an overall regression model for the food items as a whole and separate regression models for the carbohydrate, protein, and fat components. The models were validated using a sample set of computed ${E_g}^{\prime}$ and COD' values, an experimental sample set using measured ${E_g}$ and COD values, and robust statistical methods. RESULTS: The overall linear regression model and the carbohydrate, protein, and fat regression models accurately converted between COD and ${E_g}$, and the component models had smaller error. Because the ratios of COD per gram dry weight were greatest for fats and smallest for carbohydrates, foods with a high fat content also had higher ${E_g}$ values in terms of kcal · g dry weight-1. CONCLUSION: Our models make it possible to analyze human and microbial energetic processes in concert using a single unit of measure, which fills an important need in the food-nutrition-metabolism-microbiome field. In addition, measuring COD and using the regressions to calculate ${E_g}$ can be used instead of measuring ${E_g}$ directly using bomb calorimetry, which saves time and money.

The complex puzzle of dietary silver nanoparticles, mucus and microbiota in the gut.

Hundreds of consumer and commercial products containing silver nanoparticles (AgNPs) are currently used in food, personal-care products, pharmaceutical, and many other applications. Human exposure to AgNPs includes oral intake, inhalation, and dermal contact. The aim of this review was to focus on oral intake, intentional and incidental of AgNPs where well-known antimicrobial characteristics that might affect the microbiome and mucus in the gastrointestinal tract (GIT). This critical review summarizes what is known regarding the impacts of AgNPs on gut homeostasis. It is fundamental to understand the forms of AgNPs and their physicochemical characterization before and during digestion. For example, lab-synthesized AgNPs differ from "real" ingestable AgNPs used as food additives and dietary supplements. Similarly, the gut environment alters the chemical and physical state of Ag that is ingested as AgNPs. Emerging research on in vitro and in vivo rodent and human indicated complex multi-directional relationships among AgNPs, the intestinal microbiota, and the epithelial mucus. It may be necessary to go beyond today's descriptive approach to a modeling-based ecosystem approach that might quantitatively integrate spatio-temporal interactions among microbial groups, host factors (e.g., mucus), and environmental factors, including lifestyle-based stressors. It is suggested that future research (1) utilize more representative AgNPs, focus on microbe/mucus interactions, (2) assess the effects of environmental stressors for longer and longitudinal conditions, and (3) be integrated using quantitative modeling.

Role of hydrogen (H2) mass transfer in microbiological H2-threshold studies.

Gas-to-liquid mass transfer of hydrogen (H2) was investigated in a gas-liquid reactor with a continuous gas phase, a batch liquid phase, and liquid mixing regimes relevant to assessing kinetics of microbial H2 consumption. H2 transfer was quantified in real-time with a H2 microsensor for no mixing, moderate mixing [100 rotations per minute (rpm)], and rapid mixing (200 rpm). The experimental results were simulated by mathematical models to find best-fit values of volumetric mass transfer coefficients-kLa-for H2, which were 1.6/day for no mixing, 7/day for 100 rpm, and 30/day for 200 rpm. Microbiological H2-consumption experiments were conducted with Methanobacterium bryantii M.o.H. to assess effects of H2 mass transfer on microbiological H2-threshold studies. The results illustrate that slow mixing reduced the gas-to-liquid H2 transfer rate, which fell behind the rate of microbiological H2 consumption in the liquid phase. As a result, the liquid-phase H2 concentration remained much lower than the liquid-phase H2 concentration that would be in equilibrium with the gas-phase H2 concentration. Direct measurements of the liquid-phase H2 concentration by an in situ probe demonstrated the problems associated with slow H2 transfer in past H2 threshold studies. The findings indicate that some of the previously reported H2-thresholds most likely were over-estimates due to slow gas-to-liquid H2 transfer. Essential requirements to conduct microbiological H2 threshold experiments are to have vigorous mixing, large gas-to-liquid volume, large interfacial area, and low initial biomass concentration.

pH Dependency in Anode Biofilms of Thermincola ferriacetica Suggests a Proton-Dependent Electrochemical Response.

Monitoring the electrochemical response of anode respiring bacteria (ARB) helps elucidate the fundamental processes of anode respiration and their rate limitations. Understanding these limitations provides insights on how ARB create the complex interfacing of biochemical metabolic processes with insoluble electron acceptors and electronics. In this study, anode biofilms of the thermophilic (60 °C) Gram-positive ARB Thermincola ferriacetica were studied to determine the presence of a proton-dependent electron transfer response. The effects of pH, the presence of an electron donor (acetate), and biofilm growth were varied to determine their influence on the electrochemical midpoint potential ( EKA) and formal redox potential ( E°') under nonturnover conditions. The EKA and E°' are associated with an enzymatic process within ARB's metabolism that controls the rate and energetic state of their respiration. Results for all conditions indicate that pH was the major contributor to altering the energetics of T. ferriacetica anode biofilms. Electrochemical responses measured in the absence of an electron donor and with a minimal proton gradient within the anode biofilms resulted in a 48 ± 7 mV/pH unit shift in the E°', suggesting a proton-dependent rate-limiting process. Given the limited energy available for anode respiration (<200 mV when using acetate as electron donor), our results provide a new perspective in understanding proton-transport limitations in ARB biofilms, one in which ARB are thermodynamically limited by pH gradients. Since the anode biofilms of all ARB that perform direct extracellular electron transfer (EET) investigated thus far exhibit an n = 1 Nernstian behavior, and because this behavior is affected by changes in pH, we hypothesize that the Nernstian response is associated with membrane proteins responsible for proton translocation. Finally, this study shows that the EKA and E°' are a function of pH within the physiological range of ARB, and thus, given the significant effect pH has on this parameter, we recommend reporting the EKA and E°' of ARB biofilms at a specific bulk pH.

pH-dependent speciation and hydrogen (H2 ) control U(VI) respiration by Desulfovibrio vulgaris.

In situ bioreduction of soluble hexavalent uranium U(VI) to insoluble U(IV) (as UO2 ) has been proposed as a means of preventing U migration in the groundwater. This work focuses on the bioreduction of U(VI) and precipitation of U(IV). It uses anaerobic batch reactors with Desulfovibrio vulgaris, a well-known sulfate, iron, and U(VI) reducer, growing on lactate as the electron donor, in the absence of sulfate, and with a 30-mM bicarbonate buffering. In the absence of sulfate, D. vulgaris reduced >90% of the total soluble U(VI) (1 mM) to form U(IV) solids that were characterized by X-ray diffraction and confirmed to be nano-crystalline uraninite with crystallite size 2.8 ± 0.2 nm. pH values between 6 and 10 had minimal impact on bacterial growth and end-product distribution, supporting that the mono-nuclear, and poly-nuclear forms of U(VI) were equally bioavailable as electron acceptors. Electron balances support that H2 transiently accumulated, but was ultimately oxidized via U(VI) respiration. Thus, D. vulgaris utilized H2 as the electron carrier to drive respiration of U(VI). Rapid lactate utilization and biomass growth occurred only when U(VI) respiration began to draw down the sink of H2 and relieve thermodynamic inhibition of fermentation.

A steady-state biofilm model for simultaneous reduction of nitrate and perchlorate, part 2: parameter optimization and results and discussion.

Part 1 of this work developed a steady-state, multispecies biofilm model for simultaneous reduction of nitrate and perchlorate in the H(2)-based membrane biofilm reactor (MBfR) and presented a novel method to solve it. In Part 2, the half-maximum-rate concentrations and inhibition coefficients of nitrate and perchlorate are optimized by fitting data from experiments with different combinations of influent nitrate and perchlorate concentrations. The model with optimized parameters is used to quantitatively and systematically explain how three important operating conditions (nitrate loading, perchlorate loading, and H(2) pressure) affect nitrate and perchlorate reduction and biomass distribution in these reducing biofilms. Perchlorate reduction and accumulation of perchlorate-reducing bacteria (PRB) in the biofilm are affected by four promotion or inhibition mechanisms: simultaneous use of nitrate and perchlorate by PRB and competition for H(2), the same resources in PRB, and space in a biofilm. For the hydrogen pressure evaluated experimentally, a low nitrate loading (<0.1 g N/m(2)-d) slightly promotes perchlorate removal, because of the beneficial effect from PRB using both acceptors. However, a nitrate loading of >0.6 g N/m(2)-d begins to inhibit perchlorate removal, as the competition effects become dominant.

A steady-state biofilm model for simultaneous reduction of nitrate and perchlorate, part 1: model development and numerical solution.

A multispecies biofilm model is developed for simultaneous reduction of nitrate and perchlorate in the H(2)-based membrane biofilm reactor. The one-dimension model includes dual-substrate Monod kinetics for a steady-state biofilm with five solid and five dissolved components. The solid components are autotrophic denitrifying bacteria, autotrophic perchlorate-reducing bacteria, heterotrophic bacteria, inert biomass, and extracellular polymeric substances (EPS). The dissolved components are nitrate, perchlorate, hydrogen (H(2)), substrate-utilization-associated products, and biomass-associated products (BAP). The model explicitly considers four mechanisms involved in how three important operating conditions (H(2) pressure, nitrate loading, and perchlorate loading) affect nitrate and perchlorate removals: (1) competition for H(2), (2) promotion of PRB growth due to having two electron acceptors (nitrate and perchlorate), (3) competition between nitrate and perchlorate reduction for the same resources in the PRB: electrons and possibly reductase enzymes, and (4) competition for space in the biofilm. Two other special features are having H(2) delivered from the membrane substratum and solving directly for steady state using a novel three-step approach: finite-difference for approximating partial differential and/or integral equations, Newton-Raphson for solving nonlinear equations, and an iterative scheme to obtain the steady-state biofilm thickness. An example result illustrates the model's features.

Advanced control for photoautotrophic growth and CO2-utilization efficiency using a membrane carbonation photobioreactor (MCPBR).

A membrane carbonation (MC) module uses bubbleless gas-transfer membranes to supply inorganic carbon (C(i)) for photoautotrophic cyanobacterial growth in a photobioreactor (PBR); this creates the novel MCPBR system, which allows precise control of the CO(2)-delivery rate and minimal loss of CO(2) to the atmosphere. Experiments controlled the supply rate of C(i) to the main PBR by regulating the recirculation rate (Q(R)) between the module of MC chamber and the main PBR. The experiments evaluated how Q(R) controls the CO(2) mass transport in MC chamber and how it connects with the biomass production rate, C(i) concentration, pH in the PBR, and CO(2)-utilization efficiency. The biomass production rate and C(i) concentration increased in response to the C(i) supply rate (controlled by Q(R)), but not in linear proportion. The biomass production rate increased less than C(i) due to increased light limitation. Except for the highest Q(R), when the higher C(i) concentration caused the pH to decrease, CO(2) loss to gas ventilation was negligible. The results demonstrate that this MCPBR offers independent control over the growth of photoautotrophic biomass, pH control, and minimal loss of CO(2) to the atmosphere.

Integrative and quantitative bioenergetics: Design of a study to assess the impact of the gut microbiome on host energy balance.

The literature is replete with clinical studies that characterize the structure, diversity, and function of the gut microbiome and correlate the results to different disease states, including obesity. Whether the microbiome has a direct impact on obesity has not been established. To address this gap, we asked whether the gut microbiome and its bioenergetics quantitatively change host energy balance. This paper describes the design of a randomized crossover clinical trial that combines outpatient feeding with precisely controlled metabolic phenotyping in an inpatient metabolic ward. The target population was healthy, weight-stable individuals, age 18-45 and with a body mass index ≤30 kg/m2. Our primary objective was to determine within-participant differences in energy balance after consuming a control Western Diet versus a Microbiome Enhancer Diet intervention specifically designed to optimize the gut microbiome for positive impacts on host energy balance. We assessed the complete energy-balance equation via whole-room calorimetry, quantified energy intake, fecal energy losses, and methane production. We implemented conditions of tight weight stability and balance between metabolizable energy intake and predicted energy expenditure. We explored key factors that modulate the balance between host and microbial nutrient accessibility by measuring enteroendocrine hormone profiles, appetite/satiety, gut transit and gastric emptying. By integrating these clinical measurements with future bioreactor experiments, gut microbial ecology analysis, and mathematical modeling, our goal is to describe initial cause-and-effect mechanisms of gut microbiome metabolism on host energy balance. Our innovative methods will enable subsequent studies on the interacting roles of diet, the gut microbiome, and human physiology. CLINICALTRIALSGOV IDENTIFIER: NCT02939703. The present study reference can be found here: https://clinicaltrials.gov/ct2/show/NCT02939703.

Uptake of phosphate by Synechocystis sp. PCC 6803 in dark conditions: Removal driving force and modeling.

Rapid uptake of inorganic phosphate (Pi) by microalgae should occur through two processes operating in parallel: onto extracellular polymeric substances (EPS) and intracellular polymeric substances (IPS). Most previous studies focused only on overall Pi uptake and ignored the roles of EPS. We investigated the two-step removal of Pi by Synechocystis sp. PCC 6803 in dark conditions (i.e., without incorporation of Pi into newly synthesized biomass). We also developed a model to simulate both steps. Experimental results with Synechocystis confirmed that Pi in the bulk solution was removed by the two uptake mechanisms operating in parallel, but with different kinetics. All uptake rates decreased with time, and the Pi uptake rate by IPS was much higher than that by EPS at all times, but EPS had a larger maximum Pi-storage capacity -- 33-48 mgP/gCODEPS versus 15-17 mgP/gCODIPS. Synechocystis had a maximum Pi-storage capacity in the range of 22-28 mgP/g dry biomass. Protein in EPS and IPS played the key role for binding Pi, and biomass with higher protein content had greater Pi-storage capacity. Furthermore, biomass with low initial stored Pi had faster Pi-uptake kinetics, leading to more Pi removed from the bulk solution. This work lays the foundation for using microalgae as a means to remove Pi from polluted water and for understanding competition for Pi in microbial communities.

pH-Mediated Microbial and Metabolic Interactions in Fecal Enrichment Cultures.

pH and fermentable substrates impose selective pressures on gut microbial communities and their metabolisms. We evaluated the relative contributions of pH, alkalinity, and substrate on microbial community structure, metabolism, and functional interactions using triplicate batch cultures started from fecal slurry and incubated with an initial pH of 6.0, 6.5, or 6.9 and 10 mM glucose, fructose, or cellobiose as the carbon substrate. We analyzed 16S rRNA gene sequences and fermentation products. Microbial diversity was driven by both pH and substrate type. Due to insufficient alkalinity, a drop in pH from 6.0 to ~4.5 clustered pH 6.0 cultures together and distant from pH 6.5 and 6.9 cultures, which experienced only small pH drops. Cellobiose yielded more acidity than alkalinity due to the amount of fermentable carbon, which moved cellobiose pH 6.5 cultures away from other pH 6.5 cultures. The impact of pH on microbial community structure was reflected by fermentative metabolism. Lactate accumulation occurred in pH 6.0 cultures, whereas propionate and acetate accumulations were observed in pH 6.5 and 6.9 cultures and independently from the type of substrate provided. Finally, pH had an impact on the interactions between lactate-producing and -consuming communities. Lactate-producing Streptococcus dominated pH 6.0 cultures, and acetate- and propionate-producing Veillonella, Bacteroides, and Escherichia dominated the cultures started at pH 6.5 and 6.9. Acid inhibition on lactate-consuming species led to lactate accumulation. Our results provide insights into pH-derived changes in fermenting microbiota and metabolisms in the human gut. IMPORTANCE The human gut is a dynamic environment in which microorganisms consistently interact with the host via their metabolic products. Some of the most important microbial metabolic products are fermentation products such as short-chain fatty acids. Production of these fermentation products and the prevalence of fermenting microbiota depend on pH, alkalinity, and available dietary sugars, but details about their metabolic interactions are unknown. Here, we show that, for in vitro conditions, pH was the strongest driver of microbial community structure and function and microbial and metabolic interactions among pH-sensitive fermentative species. The balance between bicarbonate alkalinity and formation of fatty acids by fermentation determined the pH, which controlled microbial community structure. Our results underscore the influence of pH balance on microbial function in diverse microbial ecosystems such as the human gut.

Distinctive microbiomes and metabolites linked with weight loss after gastric bypass, but not gastric banding.

Roux-en-Y gastric bypass (RYGB) and laparoscopic adjustable gastric banding (LAGB) are anatomically different bariatric operations. RYGB achieves greater weight loss compared with LAGB. Changes in the gut microbiome have been documented after RYGB, but not LAGB, and the microbial contribution to sustainable surgical weight loss warrants further evaluation. We hypothesized that RYGB imposes greater changes on the microbiota and its metabolism than LAGB, and that the altered microbiota may contribute to greater weight loss. Using multi-omic approaches, we analyzed fecal microbial community structure and metabolites of pre-bariatric surgery morbidly obese (PreB-Ob), normal weight (NW), post-RYGB, and post-LAGB participants. RYGB microbiomes were significantly different from those from NW, LAGB and PreB-Ob. Microbiome differences between RYGB and PreB-Ob populations were mirrored in their metabolomes. Diversity was higher in RYGB compared with LAGB, possibly because of an increase in the abundance of facultative anaerobic, bile-tolerant and acid-sensible microorganisms in the former. Possibly because of lower gastric acid exposure, phylotypes from the oral cavity, such as Escherichia, Veillonella and Streptococcus, were in greater abundance in the RYGB group, and their abundances positively correlated with percent excess weight loss. Many of these post-RYGB microorganisms are capable of amino-acid fermentation. Amino-acid and carbohydrate fermentation products-isovalerate, isobutyrate, butyrate and propionate-were prevalent in RYGB participants, but not in LAGB participants. RYGB resulted in greater alteration of the gut microbiome and metabolome than LAGB, and RYGB group exhibited unique microbiome composed of many amino-acid fermenters, compared with nonsurgical controls.

A Combined Activated Sludge Anaerobic Digestion Model (CASADM) to understand the role of anaerobic sludge recycling in wastewater treatment plant performance.

The Combined Activated Sludge-Anaerobic Digestion Model (CASADM) quantifies the effects of recycling anaerobic-digester (AD) sludge on the performance of a hybrid activated sludge (AS)-AD system. The model includes nitrification, denitrification, hydrolysis, fermentation, methanogenesis, and production/utilization of soluble microbial products and extracellular polymeric substances (EPS). A CASADM example shows that, while effluent COD and N are not changed much by hybrid operation, the hybrid system gives increased methane production in the AD and decreased sludge wasting, both caused mainly by a negative actual solids retention time in the hybrid AD. Increased retention of biomass and EPS allows for more hydrolysis and conversion to methane in the hybrid AD. However, fermenters and methanogens survive in the AS, allowing significant methane production in the settler and thickener of both systems, and AD sludge recycle makes methane formation greater in the hybrid system.

Analysis of a microbial electrochemical cell using the proton condition in biofilm (PCBIOFILM) model.

Common to all microbial electrochemical cells (MXCs) are the anode-respiring bacteria (ARB), which transfer electrons to an anode and release protons that must transport out of the biofilm. Here, we develop a novel modeling platform, Proton Condition in BIOFILM (PCBIOFILM), with a structure geared towards mechanistically explaining: (1) how the ARB half reaction produces enough acid to inhibit the ARB by low pH; (2) how the diffusion of alkalinity carriers (phosphates and carbonates) control the pH gradients in the biofilm anode; (3) how increasing alkalinity attenuates pH gradients and increases current; and (4) why carbonates enable higher current density than phosphates. Analysis of literature data using PCBIOFILM supports the hypothesis that alkalinity limits the maximum current density for MXCs. An alkalinity criterion for eliminating low-pH limitation - 12 mgCaCO(3)/mg BOD--implies that a practical MXC can achieve a maximum current density with an effluent quality comparable to anaerobic digestion.