A UPLC-MS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

Arinole, a novel auxin-stimulating benzoxazole, affects root growth and promotes adventitious root formation.

The triple response phenotype is characteristic for seedlings treated with the phytohormone ethylene or its direct precursor 1-aminocyclopropane-carboxylic acid and is often employed to find novel chemical tools to probe ethylene responses. We identified a benzoxazole-urea derivative (B2) partially mimicking ethylene effects in a triple response bioassay. A thorough phenotypic analysis demonstrated that B2 and its closest analogue arinole (ARI) induced phenotypic responses reminiscent of seedlings with elevated levels of auxin, including impaired hook development and inhibition of seedling growth. Specifically, ARI reduced longitudinal cell elongation in roots, while promoting cell division. In contrast to other natural or synthetic auxins, ARI mostly acts as an inducer of adventitious root development, with only limited effects on lateral root development. Quantification of free auxins and auxin biosynthetic precursors as well as auxin-related gene expression demonstrated that ARI boosts global auxin levels. In addition, analyses of auxin reporter lines and mutants, besides pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment resulted in AR formation in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, a desirable trait in both agriculture and horticulture.

Optimization of methane gas-liquid mass transfer during biogas-based ectoine production in bubble column bioreactors.

Nowadays, the utilization of biogas for energy generation is hindered by the declining production costs of solar and wind power. A shift towards the valorization of biogas into ectoine, a highly valuable bioproduct priced at 1000 €â¸±kg-1, offers a novel approach to fostering a more competitive biogas market while contributing to carbon neutrality. This study evaluated the optimization of CH4 gas-liquid mass transfer in 10 L bubble column bioreactors for CH4 conversion into ectoine and hydroxyectoine using a mixed methanotrophic culture. The influence of the empty bed residence time (EBRTs of 27, 54, and 104 min) at different membrane diffuser pore sizes (0.3 and 0.6 mm) was investigated. Despite achieving CH4 elimination capacities (CH4-ECs) of 10-12 g⸱m-3⸱h-1, an EBRT of 104 min mediated CH4 limitation within the cultivation broth, resulting in a negligible biomass growth. Reducing the EBRT to 54 min entailed CH4-ECs of 21-24 g⸱m-3⸱h-1, concomitant to a significant increase in biomass growth (up to 0.17 g⸱L⸱d-1) and reaching maximum ectoine and hydroxyectoine accumulation of 79 and 13 mg⸱gVSS-1, respectively. Conversely, process operation at an EBRT of 27 min lead to microbial inhibition, resulting in a reduced biomass growth of 0.09 g⸱L⸱d-1 and an ectoine content of 47 mg⸱gVSS-1. While the influence of diffuser pore size was less pronounced compared to EBRT, the optimal process performance was observed with a diffuser pore size of 0.6 mm.

Genome sequence analysis and characterization of Bacillus altitudinis B12, a polylactic acid- and keratin-degrading bacterium.

Keratin-rich wastes, mainly in the form of feathers, are recalcitrant residues generated in high amounts as by-products in chicken farms and food industry. Polylactic acid (PLA) is the second most common biodegradable polymer found in commercial plastics, which is not easily degraded by microbial activity. This work reports the 3.8-Mb genome of Bacillus altitudinis B12, a highly efficient PLA- and keratin-degrading bacterium, with potential for environmental friendly biotechnological applications in the feed, fertilizer, detergent, leather, and pharmaceutical industries. The whole genome sequence of B. altitudinis B12 revealed that this strain (which had been previously misclassified as Bacillus pumilus B12) is closely related to the B. altitudinis strains ER5, W3, and GR-8. A total of 4056 coding sequences were annotated using the RAST server, of which 2484 are core genes of the pan genome of B. altitudinis and 171 are unique to this strain. According to the sequence analysis, B. pumilus B12 has a predicted secretome of 353 proteins, among which a keratinase and a PLA depolymerase were identified by sequence analysis. The presence of these two enzymes could explain the characterized PLA and keratin biodegradation capability of the strain.

Continuous polyhydroxybutyrate production from biogas in an innovative two-stage bioreactor configuration.

Biogas biorefineries have opened up new horizons beyond heat and electricity production in the anaerobic digestion sector. Added-value products such as polyhydroxyalkanoates (PHAs), which are environmentally benign and potential candidates to replace conventional plastics, can be generated from biogas. This work investigated the potential of an innovative two-stage growth-accumulation system for the continuous production of biogas-based polyhydroxybutyrate (PHB) using Methylocystis hirsuta CSC1 as cell factory. The system comprised two turbulent bioreactors in series to enhance methane and oxygen mass transfer: a continuous stirred tank reactor (CSTR) and a bubble column bioreactor (BCB) with internal gas recirculation. The CSTR was devoted to methanotrophic growth under nitrogen balanced growth conditions and the BCB targeted PHB production under nitrogen limiting conditions. Two different operational approaches under different nitrogen loading rates and dilution rates were investigated. A balanced nitrogen loading rate along with a dilution rate (D) of 0.3 day-1 resulted in the most stable operating conditions and a PHB productivity of ~53 g PHB m-3 day-1 . However, higher PHB productivities (~127 g PHB m-3 day-1 ) were achieved using nitrogen excess at a D = 0.2 day-1 . Overall, the high PHB contents (up to 48% w/w) obtained in the CSTR under theoretically nutrient balanced conditions and the poor process stability challenged the hypothetical advantages conferred by multistage vs single-stage process configurations for long-term PHB production.

Resilience and robustness of alphaproteobacterial methanotrophs upon methane feast-famine scenarios.

Most of methane (CH4) emissions contain low CH4 concentrations and typically occur at irregular intervals, which hinders the implementation and performance of methane abatement processes. This study aimed at understanding the metabolic mechanisms that allow methane oxidizing bacteria (MOB) to survive for long periods of time under methane starvation. To this aim, we used an omics-approach and studied the diversity and metabolism of MOB and non-MOB in bioreactors exposed to low CH4 concentrations under feast-famine cycles of 5 days and supplied with nutrient-rich broth. The 16S rRNA and the pmoA transcripts revealed that the most abundant and active MOB during feast and famine conditions belonged to the alphaproteobacterial genus Methylocystis (91-65%). The closest Methylocystis species were M. parvus and M. echinoides. Nitrifiers and denitrifiers were the most representative non-MOB communities, which likely acted as detoxifiers of the system. During starvation periods, the induced activity of CH4 oxidation was not lost, with the particulate methane monooxygenase of alphaproteobacterial MOB playing a key role in energy production. The polyhydroxyalkanoate and nitrification metabolisms of MOB had also an important role during feast-famine cycles, maintaining cell viability when CH4 concentrations were negligible. This research shows that there is an emergence and resilience of conventional alphaproteobacterial MOB, being the genus Methylocystis a centrepiece in environments exposed to dilute and intermittent methane emissions. This knowledge can be applied to the operation of bioreactors subjected to the treatment of dilute and discontinuous emissions via controlled bioaugmentation.

Standards-Compliant Multi-Protocol On-Board Unit for the Evaluation of Connected and Automated Mobility Services in Multi-Vendor Environments.

Vehicle-to-everything (V2X) communications enable real-time information exchange between vehicles and infrastructure, which extends the perception range of vehicles beyond the limits of on-board sensors and, thus, facilitating the realisation of cooperative, connected, and automated mobility (CCAM) services that will improve road safety and traffic efficiency. In the context of CCAM, the successful deployments of cooperative intelligent transport system (C-ITS) use cases, with the integration of advanced wireless communication technologies, are effectively leading to make transport safer and more efficient. However, the evaluation of multi-vendor and multi-protocol based CCAM service architectures can become challenging and complex. Additionally, conducting on-demand field trials of such architectures with real vehicles involved is prohibitively expensive and time-consuming. In order to overcome these obstacles, in this paper, we present the development of a standards-compliant experimental vehicular on-board unit (OBU) that supports the integration of multiple V2X protocols from different vendors to communicate with heterogeneous cloud-based services that are offered by several original equipment manufacturers (OEMs). We experimentally demonstrate the functionalities of the OBU in a real-world deployment of a cooperative collision avoidance service infrastructure that is based on edge and cloud servers. In addition, we measure end-to-end application-level latencies of multi-protocol supported V2X information flows to show the effectiveness of interoperability in V2X communications between different vehicle OEMs.

Elucidating the key environmental parameters during the production of ectoines from biogas by mixed methanotrophic consortia.

Anaerobic digestion (AD) is a robust biotechnology for the valorisation of organic waste into biogas. However, the rapid decrease in renewable electricity prices requires alternative uses of biogas. In this context, the engineering of innovative platforms for the bio-production of chemicals from CH4 has recently emerged. The extremolyte and osmoprotectant ectoine, with a market price of ~1000€/Kg, is the industrial flagship of CH4-based bio-chemicals. This work aimed at optimizing the accumulation of ectoines using mixed microbial consortia enriched from saline environments (a salt lagoon and a salt river) and activated sludge, and biogas as feedstock. The influence of NaCl (0, 3, 6, 9 and 12 %) and Na2WO4 (0, 35 and 70 µg L-1) concentrations and incubation temperature (15, 25 and 35 °C) on the stoichiometry and kinetics of the methanotrophic consortia was investigated. Consortia enriched from activated sludge at 15 °C accumulated the highest yields of ectoine and hydroxyectoine at 6 % NaCl (105.0 ± 27.2 and 24.2 ± 5.4 mgextremolyte gbiomass-1, respectively). The consortia enriched from the salt lagoon accumulated the highest yield of ectoine and hydroxyectoine at 9 % NaCl (56.6 ± 2.5 and 51.0 ± 2.0 mgextremolyte gbiomass-1, respectively) at 25 °C. The supplementation of tungsten to the cultivation medium did not impact on the accumulation of ectoines in any of the consortia. A molecular characterization of the enrichments revealed a relative abundance of ectoine-accumulating methanotrophs of 7-16 %, with Methylomicrobium buryatense and Methylomicrobium japanense as the main players in the bioconversion of methane into ectoine.

Halotolerance mechanisms of the methanotroph Methylomicrobium alcaliphilum.

Methylomicrobium alcaliphilum is an alkaliphilic and halotolerant methanotroph. The physiological responses of M. alcaliphilum to high NaCl concentrations, were studied using RNA sequencing and metabolic modeling. This study revealed that M. alcaliphilum possesses an unusual respiratory chain, in which complex I is replaced by a Na+ extruding NQR complex (highly upregulated under high salinity conditions) and a Na+ driven adenosine triphosphate (ATP) synthase coexists with a conventional H+ driven ATP synthase. A thermodynamic and metabolic model showing the interplay between these different components is presented. Ectoine is the main osmoprotector used by the cells. Ectoine synthesis is activated by the transcription of an ect operon that contains five genes, including the ectoine hydroxylase coding ectD gene. Enzymatic tests revealed that the product of ectD does not have catalytic activity. A new Genome Scale Metabolic Model for M. alcaliphilum revealed a higher flux in the oxidative branch of the pentose phosphate pathway leading to NADPH production and contributing to resistance to oxidative stress.

Pre-Surgical Nutrition Support Reduces the Incidence of Surgical Wound Complications in Oncological Patients.

Patients who suffer from cancer are at a higher risk of complications when they experience malnutrition. Evidence shows that oral nutritional supplements favor the healing process. The main objective of this study was to evaluate the efficacy of preoperative oral nutritional intervention in oncological patients undergoing surgery. This study assessed retrospectively 55 cancer patients who previously had undergone abdominal surgery and did not have receive pre-surgical nutritional support (control group), and prospectively 30 oncological patients undergoing gastrointestinal surgery and received pre-surgical high-protein nutritional support (experimental group). All patients had to have a NRS 2002 score ≥ 3. Analytical and clinical parameters were analyzed and the NRS 2002 screening test was performed. Post-operative assessments of surgical wound complications were also carried out to determine the impact of nutrition support. Pre-surgical nutritional interventions reduced the incidence and severity of wound complications as well as the length of hospital stays. Only 26.7% of patients in the experimental group had complications compared to 60% of the control group (P = 0.003). We conclude that pre-surgical nutritional interventions of patients undergoing surgery can improve post-surgical patient outcomes of malnourished patients.

Genome Scale Metabolic Model of the versatile methanotroph Methylocella silvestris.

BACKGROUND: Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. RESULTS: The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. CONCLUSIONS: The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.

Mechanistic Description of Convective Gas-Liquid Mass Transfer in Biotrickling Filters Using CFD Modeling.

The gas-liquid mass transfer coefficient is a key parameter to the design and operation of biotrickling filters that governs the transport rate of contaminants and oxygen from the gas phase to the liquid phase, where pollutant biodegradation occurs. Mass transfer coefficients are typically estimated via experimental procedures to produce empirical correlations, which are only valid for the bioreactor configuration and range of operational conditions under investigation. In this work, a new method for the estimation of the gas-liquid mass transfer coefficient in biotrickling filters is presented. This novel methodology couples a realistic description of the packing media (polyurethane foam without a biofilm) obtained using microtomography with computational fluid dynamics. The two-dimensional analysis reported in this study allowed capturing the mechanisms of the complex processes involved in the creeping porous air and water flow in the presence of capillary effects in biotrickling filters. Model predictions matched the experimental mass transfer coefficients (±30%) under a wide range of operational conditions.

Genome scale metabolic modeling reveals the metabolic potential of three Type II methanotrophs of the genus Methylocystis.

Genome Scale Metabolic Models (GSMMs) of the recently sequenced Methylocystis hirsuta and two other methanotrophs from the genus Methylocystis have been reconstructed. These organisms are Type II methanotrophs with the ability of accumulating Polyhydroxyalkanoates under nutrient limiting conditions. For the first time, GSMMs have been reconstructed for Type II methanotrophs. These models, combined with experimental biomass and PHB yields of Methylocystis hirsuta, allowed elucidating the methane oxidation mechanism by the enzyme pMMO (particulate methane monooxygenase) in these organisms. In contrast to Type I methanotrophs, which use the "direct coupling mechanism", Type II methanotrophs appear to use the so called "redox arm mechanism". The utilization of the "redox arm mechanism", which involves the coupling between methane oxidation and complex I of the respiratory chain, was confirmed by inhibition of complex I with catechol. Utilization of the "redox arm" mechanism leads to lower biomass yields on methane compared to Type I methanotrophs. However, the ability of Type II methanotrophs to redirect high metabolic carbon fluxes towards acetoacetyl-CoA under nitrogen limiting conditions makes these organisms promising platforms for metabolic engineering.

Reconstruction of a Genome Scale Metabolic Model of the polyhydroxybutyrate producing methanotroph Methylocystis parvus OBBP.

BACKGROUND: Methylocystis parvus is a type II methanotroph characterized by its high specific methane degradation rate (compared to other methanotrophs of the same family) and its ability to accumulate up to 50% of its biomass in the form of poly-3-hydroxybutyrate (PHB) under nitrogen limiting conditions. This makes it a very promising cell factory. RESULTS: This article reports the first Genome Scale Metabolic Model of M. parvus OBBP. The model is compared to Genome Scale Metabolic Models of the closely related methanotrophs Methylocystis hirsuta and Methylocystis sp. SC2. Using the reconstructed model, it was possible to predict the biomass yield of M. parvus on methane. The prediction was consistent with the observed experimental yield, under the assumption of the so called "redox arm mechanism" for methane oxidation. The co-consumption of stored PHB and methane was also modeled, leading to accurate predictions of biomass yields and oxygen consumption rates and revealing an anaplerotic role of PHB degradation. Finally, the model revealed that anoxic PHB consumption has to be coupled to denitrification, as no fermentation of PHB is allowed by the reconstructed metabolic model. CONCLUSIONS: The "redox arm" mechanism appears to be a general characteristic of type II methanotrophs, versus type I methanotrophs that use the "direct coupling" mechanism. The co-consumption of stored PHB and methane was predicted to play an anaplerotic role replenishing the serine cycle with glyoxylate and the TCA cycle with succinyl-CoA, which allows the withdrawal of metabolic precursors for biosynthesis. The stored PHB can be also used as an energy source under anoxic conditions when coupled to denitrification.