Induction of Microbial Oxidative Stress as a New Strategy to Enhance the Enzymatic Degradation of Organic Micropollutants in Synthetic Wastewater.

Organic micropollutants (OMPs) are pervasive anthropogenic contaminants of receiving waters where they can induce various adverse effects to aquatic life. Their ubiquitous environmental occurrence is primarily attributed to discharge from wastewater treatment plants due to incomplete removal by common biological wastewater treatment processes. Here, we assess a new strategy for promoting the degradation of six representative OMPs (i.e., sulfamethoxazole, carbamazepine, tylosin, atrazine, naproxen, and ibuprofen) by intentionally stimulating the production of microbial oxidoreductases to counter oxidative stress caused by oxygen perturbations. Mixed microbial cultures from a dairy farm wastewater were subjected to cyclic perturbations of dissolved oxygen (DO). A distance-based redundancy analysis was used to show that DO perturbations correlate with the abundance of Pseudomonadaceae and Rhodocyclaceae families, activities of peroxidases and cytochromes, and the degradation of OMPs. DO perturbation of 0.25 and 0.5 cycles/h led to most abundance of Pseudomonadaceae and Rhodocyclaceae families, showed higher activity of peroxidase and cytochrome, and gave largest removal of OMPs (removal of 92 ± 3% for sulfamethoxazole, 84 ± 3% for naproxen, 82 ± 3% for ibuprofen, 66 ± 2% for carbamazepine, 57 ± 15% for tylosin, and 88 ± 1% for atrazine).

Modulation of Nitrous Oxide (N2O) Accumulation by Primary Metabolites in Denitrifying Cultures Adapting to Changes in Environmental C and N.

Metabolomics provides insights into the actual physiology of cells rather than their mere "potential", as provided by genomic and transcriptomic analysis. We investigate the modulation of nitrous oxide (N2O) accumulation by intracellular metabolites in denitrifying bacteria using metabolomics and genome-based metabolic network modeling. Profiles of metabolites and their rates of production/consumption were obtained for denitrifying batch cultures under four conditions: initial COD:N ratios of 11:1 and 4:1 with and without nitrite spiking (28 mg-N L-1). Only the nitrite-spiked cultures accumulated N2O. The NO2- spiked cultures with an initial COD:N = 11:1 accumulated 3.3 ± 0.57% of the total nitrogen added as N2O and large pools of tricarboxylic acid cycle intermediates and amino acids. In comparison, the NO2- spiked cultures with COD:N = 4:1 showed significantly higher (p = 0.028) N2O accumulation (8.5.3 ± 0.9% of the total nitrogen added), which was linked to the depletion of C11-C20 fatty acids. Metabolic modeling analysis shows that at COD:N of 4:1 the denitrifying cells slowly generate electron equivalents as FADH2 through ß-oxidation of saturated fatty acids, while COD:N of 11:1 do it through the TCA cycle. When combined with NO2- shock, this prolonged the duration over which insufficient electron equivalents were available to completely reduce NOx to N2, resulting in increased N2O accumulation. Results extend the understanding of how organic carbon and nitrite loads modulate N2O accumulation in denitrification, which may contribute to further design strategies to control greenhouse gas emissions from agricultural soils or wastewater treatment systems.

Assessment of nitric oxide (NO) redox reactions contribution to nitrous oxide (N2 O) formation during nitrification using a multispecies metabolic network model.

Over the coming decades nitrous oxide (N2O) is expected to become a dominant greenhouse gas and atmospheric ozone depleting substance. In wastewater treatment systems, N2O is majorly produced by nitrifying microbes through biochemical reduction of nitrite (NO2(-)) and nitric oxide (NO). However it is unknown if the amount of N2O formed is affected by alternative NO redox reactions catalyzed by oxidative nitrite oxidoreductase (NirK), cytochromes (i.e., P460 [CytP460] and 554 [Cyt554 ]) and flavohemoglobins (Hmp) in ammonia- and nitrite-oxidizing bacteria (AOB and NOB, respectively). In this study, a mathematical model is developed to assess how N2O formation is affected by such alternative nitrogen redox transformations. The developed multispecies metabolic network model captures the nitrogen respiratory pathways inferred from genomes of eight AOB and NOB species. The performance of model variants, obtained as different combinations of active NO redox reactions, was assessed against nine experimental datasets for nitrifying cultures producing N2O at different concentration of electron donor and acceptor. Model predicted metabolic fluxes show that only variants that included NO oxidation to NO2(-) by CytP460 and Hmp in AOB gave statistically similar estimates to observed production rates of N2O, NO, NO2(-) and nitrate (NO3(-)), together with fractions of AOB and NOB species in biomass. Simulations showed that NO oxidation to NO2(-) decreased N2O formation by 60% without changing culture's NO2(-) production rate. Model variants including NO reduction to N2O by Cyt554 and cNor in NOB did not improve the accuracy of experimental datasets estimates, suggesting null N2O production by NOB during nitrification. Finally, the analysis shows that in nitrifying cultures transitioning from dissolved oxygen levels above 3.8 ± 0.38 to <1.5 ± 0.8 mg/L, NOB cells can oxidize the NO produced by AOB through reactions catalyzed by oxidative NirK.

Using carbon substrate as a selection pressure to enhance the potential of aerobic granular sludge microbial communities for removing contaminants of emerging concern.

The ability of aerobic granular sludge (AGS) technology to biotransform contaminants of emerging concern (CECs) is largely unknown. AGS supplemented with either acetate, 2-propanol, glycerol, or a 1:1:1 mixture of all three, were cultivated to investigate the link between carbon supplements and biotransformation of six CECs. Carbon substrate had a significant effect on the microbial community composition, as assessed by 16S rRNA gene sequence analyses. Substrate degradation requiring a larger number of catabolic reactions (i.e., glycerol and the mix) was associated with greater microbial richness. The biotransformation of CECs was 45.9% greater in communities supplemented with glycerol (60.3 ±â€¯30.2 µg L-1 VSS-1) compared to acetate (20.9 ±â€¯29.7 µg L-1 VSS-1). Database surveys of metabolic reactions indicate that microbial communities supplemented with glycerol have the greatest capacity for the degradation of aromatic compounds, while those supplemented with acetate community have the lowest.

Nitrosomonas europaea adaptation to anoxic-oxic cycling: Insights from transcription analysis, proteomics and metabolic network modeling.

In suboxic or anoxic environments, nitrous oxide (N2O) can be produced by ammonia oxidizing bacteria (AOB) as a potent greenhouse gas. Although N2O producing inventory and pathways have been well-characterized using archetypal AOB, there is little known about their adaptive responses to oxic-anoxic cycling, which is a prevalent condition in soil, sediment, and wastewater treatment bioreactors. In this study, cellular responses of Nitrosomonas europaea 19718 to sustained anoxic-oxic cycling in a chemostat bioreactor were evaluated at transcriptomic, proteomic, and fluxomic levels. During a single oxic-anoxic transition, the accumulations of major intermediates were found at the beginning of anoxia (nitric oxide, NO) and post anoxia (hydroxylamine, NH2OH, and N2O). Anoxic-oxic cycling over thirteen days led to significantly reduced accumulations of NH2OH, NO and N2O. Distinct from short-term responses, which were mostly regulated at the mRNA level, adapted cells seemed to sustain energy generation under repeated anoxia by partially sacrificing the NO detoxification capacities, and such adaptation was mainly regulated at the protein level. The proteomic data also suggested the potential contributions of the newly discovered cytochrome P460-mediated NH2OH oxidation pathway to N2O productions. Flux balance analysis was performed based on a metabolic network model consisting of 49 biochemical reactions involved in nitrogen respiration, and changes in metabolic fluxes after the anoxic-oxic cycling were found to be better correlated with intracellular protein concentrations rather than mRNA levels. Previous studies focusing on single anoxic-oxic transition might have overlooked the adaptive responses of nitrifiers to anoxic-oxic cycling, and thus overestimated NO and N2O emission levels from natural and engineered nitrification systems.

Metabolic Network Modeling of Microbial Interactions in Natural and Engineered Environmental Systems.

We review approaches to characterize metabolic interactions within microbial communities using Stoichiometric Metabolic Network (SMN) models for applications in environmental and industrial biotechnology. SMN models are computational tools used to evaluate the metabolic engineering potential of various organisms. They have successfully been applied to design and optimize the microbial production of antibiotics, alcohols and amino acids by single strains. To date however, such models have been rarely applied to analyze and control the metabolism of more complex microbial communities. This is largely attributed to the diversity of microbial community functions, metabolisms, and interactions. Here, we firstly review different types of microbial interaction and describe their relevance for natural and engineered environmental processes. Next, we provide a general description of the essential methods of the SMN modeling workflow including the steps of network reconstruction, simulation through Flux Balance Analysis (FBA), experimental data gathering, and model calibration. Then we broadly describe and compare four approaches to model microbial interactions using metabolic networks, i.e., (i) lumped networks, (ii) compartment per guild networks, (iii) bi-level optimization simulations, and (iv) dynamic-SMN methods. These approaches can be used to integrate and analyze diverse microbial physiology, ecology and molecular community data. All of them (except the lumped approach) are suitable for incorporating species abundance data but so far they have been used only to model simple communities of two to eight different species. Interactions based on substrate exchange and competition can be directly modeled using the above approaches. However, interactions based on metabolic feedbacks, such as product inhibition and synthropy require extensions to current models, incorporating gene regulation and compounding accumulation mechanisms. SMN models of microbial interactions can be used to analyze complex "omics" data and to infer and optimize metabolic processes. Thereby, SMN models are suitable to capitalize on advances in high-throughput molecular and metabolic data generation. SMN models are starting to be applied to describe microbial interactions during wastewater treatment, in-situ bioremediation, microalgae blooms methanogenic fermentation, and bioplastic production. Despite their current challenges, we envisage that SMN models have future potential for the design and development of novel growth media, biochemical pathways and synthetic microbial associations.

Clarifying the regulation of NO/N2O production in Nitrosomonas europaea during anoxic-oxic transition via flux balance analysis of a metabolic network model.

The metabolic mechanism regulating the production of nitric and nitrous oxide (NO, N2O) in ammonia oxidizing bacteria (AOB) was characterized by flux balance analysis (FBA) of a stoichiometric metabolic network (SMN) model. The SMN model was created using 51 reactions and 44 metabolites of the energy metabolism in Nitrosomonas europaea, a widely studied AOB. FBA of model simulations provided estimates for reaction rates and yield ratios of intermediate metabolites, substrates, and products. These estimates matched well, deviating on average by 15% from values for 17 M yield ratios reported for non-limiting oxygen and ammonium concentrations. A sensitivity analysis indicated that the reactions catalysed by cytochromes aa3 and P460 principally regulate the pathways of NO and N2O production (hydroxylamine oxidoreductase mediated and nitrifier denitrification). FBA of simulated N. europaea exposure to oxic-anoxic-oxic transition indicated that NO and N2O production primarily resulted from an intracellular imbalance between the production and consumption of electron equivalents during NH3 oxidation, and that NO and N2O are emitted when the sum of their production rates is greater than half the rate of NO oxidation by cytochrome P460.

ORGANIC CARBON SUPPLEMENTATION OF STERILIZED MUNICIPAL WASTEWATER IS ESSENTIAL FOR HETEROTROPHIC GROWTH AND REMOVING AMMONIUM BY THE MICROALGA CHLORELLA VULGARIS(1).

Heterotrophic growth of the microalga Chlorella vulgaris Beij. in synthetic as well as sterilized municipal wastewater of a nonindustrialized city was measured. The city wastewater contained high levels of ammonium and nitrate, medium levels of phosphate, and low levels of nitrite and organic molecules and could not support heterotrophic growth of C. vulgaris. Evaluation of 11 known carbon sources for this microalga that were added to standard synthetic wastewater containing the same levels of nitrogen and phosphorus as the municipal wastewater revealed that the best carbon sources for heterotrophic growth were Na-acetate and d-glucose. These provided the highest growth rates and the largest removal of ammonium. Growth increased with concentration of the supplement to an optimum at 0.12 M Na-acetate. This carbon source was consumed completely within 10 d of incubation. Higher concentrations inhibited the growth of C. vulgaris. The microalgal populations under heterotrophic growth conditions were one level of magnitude higher than that under autotrophic growth conditions that served as a comparison. No growth occurred in the dark in the absence of a carbon source. Na-acetate was superior to d-glucose. In municipal wastewater, when Na-acetate or d-glucose was added, C. vulgaris significantly enhanced ammonium removal under heterotrophic conditions, and its capacity was equal to ammonium removal under autotrophic growth conditions. This study showed that sterilized wastewater can be treated by C. vulgaris under heterotrophic conditions if supplemented with the appropriate organic carbon source for the microalgae.

Heterotrophic cultures of microalgae: metabolism and potential products.

This review analyzes the current state of a specific niche of microalgae cultivation; heterotrophic growth in the dark supported by a carbon source replacing the traditional support of light energy. This unique ability of essentially photosynthetic microorganisms is shared by several species of microalgae. Where possible, heterotrophic growth overcomes major limitations of producing useful products from microalgae: dependency on light which significantly complicates the process, increase costs, and reduced production of potentially useful products. As a general role, and in most cases, heterotrophic cultivation is far cheaper, simpler to construct facilities, and easier than autotrophic cultivation to maintain on a large scale. This capacity allows expansion of useful applications from diverse species that is now very limited as a result of elevated costs of autotrophy; consequently, exploitation of microalgae is restricted to small volume of high-value products. Heterotrophic cultivation may allow large volume applications such as wastewater treatment combined, or separated, with production of biofuels. In this review, we present a general perspective of the field, describing the specific cellular metabolisms involved and the best-known examples from the literature and analyze the prospect of potential products from heterotrophic cultures.