Towards the integration of oxidative and reductive activities: application to nitrogen removal by co-immobilized microorganisms

BackgroundComplete degradation of many pollutants requires sequenced anaerobic-aerobic biotreatment steps. Many compounds that are difficult to degrade aerobically are readily biotransformed anaerobically. The products of anaerobic biotransformation, however, will frequently resist to further mineralization; yet, they will be good substrates for aerobic biodegradation. Examples of this are the sequential biodegradation of highly chlorinated aromatics and aliphatics, azo-dyes, TNT, inorganic nitrogen compounds (NH 4+ , NO 2- and NO 3- ) and pesticides such as DDT, HCH's or methoxychlor. In waste-and groundwater treatment, these sequenced biotransformations are commonly achieved either by using aerobic and anaerobic (anoxic) reactors in series or by alternating periods of aerobiosis and anaerobiosis in a treatment unit. Ideally, however, these biodegradation processes would take place in a single, compact continuous-reactor system under carefully controlled conditions. In many instances, the benefits of such an integrated system would be clearly greater than the mere sum of the advantages of each individual process.Magic beads: an advanced engineering concept for process integrationThis work addresses the possibilities of integrating oxidative and reductive complementary biodegradation processes in compact systems by using co-immobilized mixed-culture systems. The central idea throughout the book is that aerobic and anaerobic niches will eventually develop and coexist within a single biocatalytic particle so that oxidative and reductive activities (e.g nitrification and denitrification, respectively) can be accomplished simultaneously (Magic-beads). Therefore, multiple-step complementary biodegradation and biotransformation processes could be conducted as single staged (Figure 1). The rationale behind this idea relies on sound experimental evidence (e.g. time-dependent measurements of oxygen gradients across biocatalyst particles or biofilms) that shows that such niches do indeed establish under aerobic process conditions.Figure 1 - Schematic representation of the "Magic-bead Concept".Case study: Integrated nitrification and denitrificationThe potential of the general concept outlined above for combining oxidative and reductive processes with relevance to the biodegadation of recalcitrant compounds is assessed in this work by studying in detail coupled nitrification and denitrification within (double-layered) gel beads for high-rate removal of nitrogen from wastewaters. In such beads, the nitrifying microorganisms (aerobes) immobilized in an outer layer would oxidize ammonium into nitrite that would then diffuse inwards, where immobilized denitrifiers (either facultative heterotrophs or obligate anaerobic ammonium oxidizers) would reduce this nitrite into the harmless gaseous nitrogen. The biocatalyst particle is used optimally because both the external layers and core are active. The beads are placed in a common airlift reactor through which the waste streams can flow at almost any rate, without the need of recirculation to or from any anoxic compartment or reactor.Aims of the dissertation and outlineThis research project aimed at a) the development and characterization of a coupled system for integrated nitrogen removal, b) understanding the mechanisms underlying the processes involved and; c) providing knowledge for the integration of oxidative and reductive activities in a single compact system. With these aims in mind, the stepwise strategy depicted in Figure 2 was developed. Every stage of the project was comprised by a series of self-contained studies addressing different aspects of the proposed system.Figure 2 - Structured contents of this dissertation.In the first stage (chapter 2) the scope of the problem was defined (apparent incompatibility of oxidative and reductive activities of environmental relevance), the needs were addressed (urge to integrate processes and reduce reactor size) and the state-of- the-art of the field (conventional systems and emerging technologies) was presented. In the following phase (chapters 3 to 6), a compact process was proposed and the procedures for biocatalyst production, and its characterization and mechanical stability were assessed. In the next stage (chapter 7 to 9), achievement of in-depth insight into the system's behavior was pursued by means of mathematical modeling and concomitant experimental validation using specific microelectrodes. The knowledge gathered up to this point was subsequently used successfully for the design of a fully autotrophic system for nitrogen removal (chapters 10 and 11). Finally, the possibilities of integrating other oxidative and reductive complementary biodegradation processes in compact systems by using co-immobilized mixed-culture systems were discussed in Chapter 12.</p

SAPP: functional genome annotation and analysis through a semantic framework using FAIR principles

There are currently more than 150.000 sequenced genomes available from which considerable amounts of information can be extracted. However, annotation information is often not interoperable, static, lacks provenance and is quickly outdated. Keeping these datasets up-to-date, and interoperable is a challenging ..

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.<br/

Cofactors revisited - Predicting the impact of flavoprotein-related diseases on a genome scale

Flavin adenine dinucleotide (FAD) and its precursor flavin mononucleotide (FMN) are redox cofactors that are required for the activity of more than hundred human enzymes. Mutations in the genes encoding these proteins cause severe phenotypes, including a lack of energy supply and accumulation of toxic intermediates. Ideally, patients should be diagnosed before they show symptoms so that treatment and/or preventive care can start immediately. This can be achieved by standardized newborn screening tests. However, many of the flavin-related diseases lack appropriate biomarker profiles. Genome-scale metabolic models can aid in biomarker research by predicting altered profiles of potential biomarkers. Unfortunately, current models, including the most recent human metabolic reconstructions Recon and HMR, typically treat enzyme-bound flavins incorrectly as free metabolites. This in turn leads to artificial degrees of freedom in pathways that are strictly coupled. Here, we present a reconstruction of human metabolism with a curated and extended flavoproteome. To illustrate the functional consequences, we show that simulations with the curated model - unlike simulations with earlier Recon versions - correctly predict the metabolic impact of multiple-acyl-CoA-dehydrogenase deficiency as well as of systemic flavin-depletion. Moreover, simulations with the new model allowed us to identify a larger number of biomarkers in flavoproteome-related diseases, without loss of accuracy. We conclude that adequate inclusion of cofactors in constraint-based modelling contributes to higher precision in computational predictions.FWN – Publicaties zonder aanstelling Universiteit Leide

From the Environment to the Host: Re-Wiring of the Transcriptome of Pseudomonas aeruginosa from 22°C to 37°C

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe(2+) extracellular concentration at 37°C and a peak in Fe(3+) extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperatur

Necrotizing soft tissue infections - a multicentre, prospective observational study (INFECT) : Protocol and statistical analysis plan

Background: The INFECT project aims to advance our understanding of the pathophysiological mechanisms in necrotizing soft tissue infections (NSTIs). The INFECT observational study is part of the INFECT project with the aim of studying the clinical profile of patients with NSTIs and correlating these to patient-important outcomes. With this protocol and statistical analysis plan we describe the methods used to obtain data and the details of the planned analyses. Methods: The INFECT study is a multicentre, prospective observational cohort study. Patients with NSTIs are enrolled in five Scandinavian hospitals, which are all referral centres for NSTIs. The primary outcomes are the descriptive variables of the patients. Secondary outcomes include identification of factors associated with 90-day mortality and amputation; associations between affected body part, maximum skin defect and Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score and 90-day mortality; 90-day mortality in patients with and without acute kidney injury (AKI) and LRINEC score of six and above or below six; and association between affected body part at arrival and microbiological findings. Exploratory outcomes include univariate analyses of baseline characteristics associations with 90-day mortality. The statistical analyses will be conducted in accordance with the predefined statistical analysis plan. Conclusion: Necrotizing soft tissue infections result in severe morbidity and mortality. The INFECT study will be the largest prospective study in patients with NSTIs to date and will provide important data for clinicians, researchers and policy makers on the characteristics and outcomes of these patients.</p

Understanding the antimicrobial mechanism of TiO2-based nanocomposite films in a pathogenic bacterium

Titania (TiO2)-based nanocomposites subjected to light excitation are remarkably effective in eliciting microbial death. However, the mechanism by which these materials induce microbial death and the effects that they have on microbes are poorly understood. Here, we assess the low dose radical-mediated TiO2 photocatalytic action of such nanocomposites and evaluate the genome/proteome-wide expression profiles of Pseudomonas aeruginosa PAO1 cells after two minutes of intervention. The results indicate that the impact on the gene-wide flux distribution and metabolism is moderate in the analysed time span. Rather, the photocatalytic action triggers the decreased expression of a large array of genes/proteins specific for regulatory, signalling and growth functions in parallel with subsequent selective effects on ion homeostasis, coenzyme-independent respiration and cell wall structure. The present work provides the first solid foundation for the biocidal action of titania and may have an impact on the design of highly active photobiocidal nanomaterial

Effects of ß-Lactam Antibiotics and Fluoroquinolones on Human Gut Microbiota in Relation to Clostridium difficile Associated Diarrhea

Clostridium difficile infections are an emerging health problem in the modern hospital environment. Severe alterations of the gut microbiome with loss of resistance to colonization against C. difficile are thought to be the major trigger, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. We sequenced 16S rRNA amplicons generated from DNA and RNA/cDNA of fecal samples from three groups of individuals by FLX technology: (i) healthy controls (no antibiotic therapy); (ii) individuals receiving antibiotic therapy (Ampicillin/Sulbactam, cephalosporins, and fluoroquinolones with subsequent development of C. difficile infection or (iii) individuals receiving antibiotic therapy without C. difficile infection. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the effects of alterations of the gut microbiome on C. difficile infection at the DNA (total microbiota) and rRNA (potentially active) levels. A comparison of antibiotic classes showed significant differences at DNA level, but not at RNA level. Among individuals that developed or did not develop a C. difficile infection under antibiotics we found no significant differences. We identified single species that were up- or down regulated in individuals receiving antibiotics who developed the infection compared to non-infected individuals. We found no significant differences in the global composition of the transcriptionally active gut microbiome associated with C. difficile infections. We suggest that up- and down regulation of specific bacterial species may be involved in colonization resistance against C. difficile providing a potential therapeutic approach through specific manipulation of the intestinal microbiome

Distant non-obvious mutations influence the activity of a hyperthermophilic Pyrococcus furiosus phosphoglucose isomerase

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations

Consortia modulation of the stress response: proteomic analysis of single strain versus mixed culture

The high complexity of naturally occurring microbial communities is the major drawback limiting the study of these important biological systems. In this study, a comparison between pure cultures of Pseudomonas reinekei sp. strain MT1 and stable community cultures composed of MT1 plus the addition of Achromobacter xylosoxidans strain MT3 (in a steady-state proportion 9:1) was used as a model system to study bacterial interactions that take place under simultaneous chemical and oxidative stress. Both are members of a real community isolated from a polluted sediment by enrichment in 4-chlorosalicylate (4CS). The analysis of dynamic states was carried out at the proteome, metabolic profile and population dynamic level. Differential protein expression was evaluated under exposure to 4CS and high concentrations of toxic intermediates (4-chlorocatechol and protoanemonin), including proteins from several functional groups and particularly enzymes of aromatic degradation pathways and outer membrane proteins. Remarkably, 4CS addition generated a strong oxidative stress response in pure strain MT1 culture led by alkyl hydroperoxide reductase, while the community showed an enhanced central metabolism response, where A. xylosoxidans MT3 helped to prevent toxic intermediate accumulation. A significant change in the outer membrane composition of P. reinekei MT1 was observed during the chemical stress caused by 4CS and in the presence of A. xylosoxidans MT3, highlighting the expression of the major outer membrane protein OprF, tightly correlated to 4CC concentration profile and its potential detoxification rol