The fundamental role of pH in CH4 bioconversion into polyhydroxybutyrate in mixed methanotrophic cultures.

Climate change and plastic pollution are likely the most relevant challenges for the environment in the 21st century. Developing cost-effective technologies for the bioconversion of methane (CH4) into polyhydroxyalkanoates (PHAs) could simultaneously mitigate CH4 emissions and boost the commercialization of biodegradable polymers. Despite the fact that the role of temperature, nitrogen deprivation, CH4:O2 ratio or micronutrients availability on the PHA accumulation capacity of methanotrophs has been carefully explored, there is still a need for optimization of the CH4-to-PHA bioconversion process prior to becoming a feasible platform in future biorefineries. In this study, the influence of different cultivation broth pH values (5.5, 7, 8.5 and 10) on bacterial biomass growth, CH4 bioconversion rate, PHA accumulation capacity and bacterial community structure was investigated in a stirred tank bioreactor under nitrogen deprivation conditions. Higher CH4 elimination rates were obtained at increasing pH, with a maximum value of 50.4 ± 2.7 g CH4·m-3·h-1 observed at pH 8.5. This was likely mediated by an increased ionic strength in the mineral medium, which enhanced the gas-liquid mass transfer. Interestingly, higher PHB accumulations were observed at decreasing pH, with the highest PHB contents recorded at a pH 5.5 (43.7 ± 3.4 %w·w-1). The strong selective pressure of low pH towards the growth of Type II methanotrophic bacteria could explain this finding. The genus Methylocystis increased its abundance from 34 % up to 85 and 90 % at pH 5.5 and 7, respectively. On the contrary, Methylocystis was less abundant in the community enriched at pH 8.5 (14 %). The accumulation of intracellular PHB as energy and carbon storage material allowed the maintenance of high CH4 biodegradation rates during 48 h after complete nitrogen deprivation. The results here obtained demonstrated for the first time a crucial and multifactorial role of pH on the bioconversion performance of CH4 into PHA.

Extracellular ATP hydrolysis in Caco-2 human intestinal cell line.

Extracellular nucleotides and nucleosides activate signaling pathways that play major roles in the physiology and pathophysiology of the gastrointestinal tract. Ectonucleotidases hydrolyze extracellular nucleotides and thus regulate ligand exposure to purinergic receptors. In this study, we investigated the expression, localization and activities of ectonucleotidases using Caco-2 cells, a model of human intestinal epithelial cells. In addition, by studying ATP release and the rates of extracellular ATP (eATP) hydrolysis, we analyzed the contribution of these processes to the regulation of eATP in these cells. Results show that Caco-2 cells regulate the metabolism of eATP and by-products by ecto-nucleoside triphosphate diphosphohydrolase-1 and -2, a neutral ecto-phosphatase and ecto-5'-nucleotidase. All these ectoenzymes were kinetically characterized using intact cells, and their presence confirmed by denatured and native gels, western blot and cytoimmunofluorescence techniques. In addition, regulation of eATP was studied by monitoring the dynamic balance between intracellular ATP release and ectoATPase activity. Following mechanical and hypotonic stimuli, Caco-2 cells triggered a strong but transient release of intracellular ATP, with almost no energy cost, leading to a steep increase of eATP concentration, which was later reduced by ectoATPase activity. A data-driven algorithm allowed quantifying and predicting the rates of ATP release and ATP consumption contributing to the dynamic accumulation of ATP at the cell surface.

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis.

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.

Multi-production of high added market value metabolites from diluted methane emissions via methanotrophic extremophiles.

This study constitutes the first-proof-of-concept of a methane biorefinery based on the multi-production of high profit margin substances (ectoine, hydroxyectoine, polyhydroxyalkanoates (PHAs) and exopolysaccharides (EPS)) using methane as the sole carbon and energy source. Two bubble column bioreactors were operated under different magnesium concentrations (0.2, 0.02 and 0.002 g L-1) to validate and optimize this innovative strategy for valorization of CH4 emissions. High Mg2+ concentrations promoted the accumulation of ectoine (79.7-94.2 mg g biomass-1), together with high hydroxyectoine yields (up to 13 mg g biomass-1) and EPS concentrations (up to 2.6 g L culture broth-1). Unfortunately, PHA synthesis was almost negligible (14.3 mg L-1) and only found at the lowest Mg2+ concentration tested. Halomonas, Marinobacter, Methylophaga and Methylomicrobium, previously described as ectoine producers, were dominant in both bioreactors, Methylomicrobium being the only described methanotroph. This study encourages further research on CH4 biorefineries capable of creating value out of GHG mitigation.

Anaerobic Digestion of Sugarcane Vinasse Through a Methanogenic UASB Reactor Followed by a Packed Bed Reactor.

The anaerobic treatment of raw vinasse in a combined system consisting in two methanogenic reactors, up-flow anaerobic sludge blanket (UASB) + anaerobic packed bed reactors (APBR), was evaluated. The organic loading rate (OLR) was varied, and the best condition for the combined system was 12.5 kg COD m-3day-1 with averages of 0.289 m3 CH4 kg COD r-1for the UASB reactor and 4.4 kg COD m-3day-1 with 0.207 m3 CH4 kg COD r-1 for APBR. The OLR played a major role in the emission of H2S conducting to relatively stable quality of biogas emitted from the APBR, with H2S concentrations <10 mg L-1. The importance of the sulphate to COD ratio was demonstrated as a result of the low biogas quality recorded at the lowest ratio. It was possible to develop a proper anaerobic digestion of raw vinasse through the combined system with COD removal efficiency of 86.7% and higher CH4 and a lower H2S content in biogas.

Integral approaches to wastewater treatment plant upgrading for odor prevention: Activated Sludge and Oxidized Ammonium Recycling.

Traditional physical/chemical end-of-the-pipe technologies for odor abatement are relatively expensive and present high environmental impacts. On the other hand, biotechnologies have recently emerged as cost-effective and environmentally friendly alternatives but are still limited by their investment costs and land requirements. A more desirable approach to odor control is the prevention of odorant formation before being released to the atmosphere, but limited information is available beyond good design and operational practices of the wastewater treatment process. The present paper reviews two widely applicable and economic alternatives for odor control, Activated Sludge Recycling (ASR) and Oxidized Ammonium Recycling (OAR), by discussing their fundamentals, key operating parameters and experience from the available pilot and field studies. Both technologies present high application potential using readily available plant by-products with a minimum plant upgrading, and low investment and operating costs, contributing to the sustainability and economic efficiency of odor control at wastewater treatment facilities.

Evaluating odour control technologies using reliability and sustainability criteria--a case study for water treatment plants.

Technologies for odour control have been widely reviewed and their optimal range of application and performance has been clearly established. Selection criteria, mainly driven by process economics, are usually based on the air flow volume, the inlet concentrations and the required removal efficiency. However, these criteria are shifting with social and environmental issues becoming as important as process economics. A methodology is illustrated to quantify sustainability and robustness of odour control technology in the context of odour control at wastewater treatment or water recycling plants. The most commonly used odour abatement techniques (biofiltration, biotrickling filtration, activated carbon adsorption, chemical scrubbing, activated sludge diffusion and biotrickling filtration coupled with activated carbon adsorption) are evaluated in terms of: (1) sustainability, with quantification of process economics, environmental performance and social impact using the sustainability metrics of the Institution of Chemical Engineers; (2) sensitivity towards design and operating parameters like utility prices (energy and labour), inlet odour concentration (H2S) and design safety (gas contact time); (3) robustness, quantifications of operating reliability, with recommendations to improve reliability during their lifespan of operations. The results show that the odour treatment technologies with the highest investments presented the lowest operating costs, which means that the net present value (NPV) should be used as a selection criterion rather than investment costs. Economies of scale are more important in biotechniques (biofiltration and biotrickling filtration) as, at increased airflows, their reduction in overall costs over 20 years (NPV20) is more extreme when compared to the physical/chemical technologies (chemical scrubbing and activated carbon filtration). Due to their low NPV and their low environmental impact, activated sludge diffusion and biotrickling filtration are in general the most cost-effective, and probably the technologies to be considered first for odour treatment in a wastewater treatment or water recycling plant. When, in an economical and risk evaluation, the reliability is counted to be as relevant as the overall costs, a hybrid technology (biotrickling filtration with activated carbon polishing) would be comparable to biotrickling filtration and activated sludge diffusion as the most preferred technologies, when all technologies are designed to have a 99% reduction of H2S and a 95% reduction of the odour concentration.

A membrane bioreactor for the simultaneous treatment of acetone, toluene, limonene and hexane at trace level concentrations.

The performance of a flat-membrane biofilm reactor (MBR) for the removal of acetone, toluene, limonene and hexane at concentrations ranging from 1.3 to 3.2 mg m(-3) was investigated at different gas residence times (GRT): 60, 30, 15 and 7 s. A preliminary abiotic test was conducted to assess the mass transport of the selected volatile organic compounds (VOCs) through the membrane. A reduced transport of limonene and hexane was observed with water present over the dense side of the membrane. The presence of a biofilm attached on the dense side of the membrane following bioreactor inoculation significantly increased VOC transport. High acetone and toluene removals (>93%) were recorded in the MBR regardless of the GRT. To remediate the low hexane removal performance (RE < 24%) recorded at the initial stages of the process, a re-inoculation of the membrane with a hexane-degrading consortium embedded in silicon oil was performed. Although hexane removal did not exceed 27%, this re-inoculation increased limonene removals up to 90% at a GRT of 7 s. The absence of inhibition of hexane biodegradation by substrate competition confirmed that hexane removal in the MBR was indeed limited by the mass transfer through the membrane. Despite the low carbon source spectrum and load, the microbiological analysis of the communities present in the MBR showed high species richness (Shannon-Wiener indices of 3.2-3.5) and a high pair-wise similarity (84-97%) between the suspended and the attached biomass.

Monitoring techniques for odour abatement assessment.

Odorous emissions from sewers and wastewater treatment plants are a complex mixture of volatile chemicals that can cause annoyance to local populations, resulting in complaints to wastewater operators. Due to the variability in hedonic tone and chemical character of odorous emissions, no analytical technique can be applied universally for the assessment of odour abatement performance. Recent developments in analytical methodologies, specifically gas chromatography, odour assessment approaches (odour wheels, the odour profile method and dynamic olfactometry), and more recently combined gas chromatography-sensory analysis, have contributed to improvements in our ability to assesses odorous emissions in terms of odorant concentration and composition. This review collates existing knowledge with the aim of providing new insight into the effectiveness of sensorial and characterisation approaches to improve our understanding of the fate of odorous emissions during odour abatement. While research in non-specific sensor array (e-nose) technology has resulted in progress in the field of continuous odour monitoring, more successful long term case-studies are still needed to overcome the early overoptimistic performance expectations. Knowledge gaps still remain with regards to the decomposition of thermally unstable volatile compounds (especially sulfur compounds), the inability to predict synergistic, antagonistic, or additive interactions among odorants in combined chemical/sensorial analysis techniques, and the long term stability of chemical sensors due to sensor drift, aging, temperature/relative humidity effects, and temporal variations. Future odour abatement monitoring will require the identification of key odorants to facilitate improved process selection, design and management.

Effects of extracellular nucleotides and their hydrolysis products on regulatory volume decrease of trout hepatocytes.

In trout hepatocytes, hypotonic swelling is followed by a compensatory shrinkage called regulatory volume decrease (RVD). It has been postulated that extracellular ATP and other nucleotides may interact with type 2 receptors (P(2)) to modulate this response. In addition, specific ectoenzymes hydrolyze ATP sequentially down to adenosine, which may bind to type 1 receptors (P(1)) and also influence RVD. Accordingly, in this study, we assessed the role of extracellular nucleoside 5'-tri- and diphosphates and of adenosine on RVD of trout hepatocytes. The extent of RVD after 40 min of maximum swelling was denoted as RVD(40), whereas the initial rate of RVD was called v(RVD). In the presence of hypotonic medium (60% of isotonic), hepatocytes swelled 1.6 times followed by v(RVD) of 1.7 min(-1) and RVD(40) of 60.2%. ATP, UTP, UDP, or ATPgammaS (P(2) agonists; 5 microM) increased v(RVD) 1.5-2 times, whereas no changes were observed in the values of RVD(40). Addition of 100 microM suramin or cibacron blue (P(2) antagonists) to the hypotonic medium produced no effect on v(RVD) but a 53-58% inhibition of RVD(40). Incubation of hepatocytes in the presence of either 5 microM [gamma-(32)P]ATP or [alpha-(32)P]ATP induced the extracellular release of [gamma-(32)P]P(i) (0.21 nmol.10(-6) cells(-1).min(-1)) and [alpha-(32)P]P(i) ( approximately 8 x 10(-3) nmol.10(-6) cells(-1).min(-1)), suggesting the presence of ectoenzymes capable of fully dephosphorylating ATP. Concerning the effect of P(1) activation on RVD, 5 microM adenosine, both in the presence and absence of 100 microM S-(4-nitrobenzil)-6-tioinosine (a blocker of adenosine uptake), decreased RVD(40) by 37-44%, whereas 8-phenyl theophylline, a P(1) antagonist, increased RVD(40) by 15%. Overall, results indicate that ATP, UTP, and UDP, acting via P(2), are important factors promoting RVD of trout hepatocytes, whereas adenosine binding to P(1) inhibits this process.

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.

An attachment for nondestructive, fast quenching of samples in rapid-mixing experiments.

The present paper describes a quenching-and-washing chamber (QWC) to be used with a rapid-mixing apparatus (RMA) for the study of processes in the millisecond time scale. The QWC enables fast, nondestructive quenching by cooling and dilution of reactants in particulate systems that can be trapped on a filter. The reaction mixture (e.g., at 25 degrees C) is injected from the RMA into the QWC where it is immediately mixed with a stream of ice-cold solution flowing at a rate of 15-40 ml s-1. Quenching requires that the process studied is slowed considerably by cooling to 0-2 degrees C and/or by removal of reactants by dilution. The equipment was characterized through a study of the tight binding (occlusion) of 86Rb+ to purified, membrane-bound Na+/K+-ATPase. Millipore filters of 0.22-0.80 microm pore size trapped close to 100% of the enzyme protein. Enzyme with occluded 86Rb+ was formed in the RMA under conditions where the rate constant for release of Rb+ at 25 degrees C is up to 25 s-1 and then injected into the QWC. The high off-rate constant is due to the presence of 2.5 mM ATP, which accelerates release of Rb+. The recovery of occluded 86Rb+ on the filter was at least 90%, indicating that both cooling of the reactants and dilution of ATP are fast enough to stop the reaction. The quenching time was 3-4 ms.

[Contraception in adolescents included in a family planning program]. / Anticoncepción en adolescentes incluidas en un programa de planificación familiar.

OBJECTIVE: To describe the motives of consultation, kinds of contraceptive methods and side-effects of oral contraceptives (OCs) in adolescent users of a family planning program. DESIGN: Family planning program at the District Primary Care Unit, Cádiz, Spain. PATIENTS: 283 adolescent females who requested attendance in a family planning program from January 1993 until January 1994. MEASUREMENTS AND MAIN RESULTS: A medical history of every adolescent was carried out and every patient was examined. Those who were recommended the use of OCs passed through 3 controls: at the beginning, after the 6th month and after 1 year. These controls consisted in recording sexual risk behaviours as well as several analytic tests. RESULTS: Most adolescents requested the prescription of contraceptive methods (81.7% of the most prescribed and demanded methods were OCs). We found no serious side-effects after 1 year, so we can conclude that OCs are an usefull contraceptive choice for sexually active adolescents because of its security, acceptance and easy use.

[Anthropometric assessment of the nutritional status of the adolescent population of Cádiz (Spain)]. / Valoración antropométrica del estado nutricional en una población adolescente de Cádiz.

OBJECTIVE: The results of a series of anthropometrical values of nutritional nature, which were used to determine corporal composition, are presented. PATIENTS AND METHODS: The samples were taken from schools in the city of Cádiz. A total of 945 young people between the ages of 10 and 17 years were used. In each case, tricipital skinfold thickness, Quetelet's index, brachial fat area and brachial adipose muscular quotient were determined. RESULTS: A comparison of the values we obtain with those of national studies showed our figures to be higher. CONCLUSIONS: The figures given for our adolescent population are intended to serve as a reference point for use in nutritional studies in this geographical area.

[The USFAM Study: the prevalence of allergic diseases in the school-age population of the province of Cádiz. I. Ubrique, San Fernando, Algeciras and Medina Sidonia]. / Estudio USFAM: prevalencia de las enfermedades alérgicas en la población escolar de la provincia de Cádiz. Parte I.

The purpose of this study was to determine the prevalence of allergic diseases in the school population of the province of Cádiz in order to confirm the hypothesis that these diseases are more frequent there than in the rest of the Spanish provinces. Four cities of our province were chosen (Ubrique, San Fernando, Algeciras and Medina Sidonia, whose initials form the acrostic USFAM) with clear differential characteristics (climatological, orographical, environmental, etc). In each city, schools, both public and private, were chosen at random. Complete classes were used, and the age-limit was that established in each school. A questionnaire was given to all of the parents who, together with the teachers, were given instructions for its completion. Of the 4,000 questionnaires distributed, 2,572 were correctly completed. Of these, 1,988 answered that their child was non-allergic and 584 as allergic (22.7%). The ratio male/female was almost 2/1 (64.4%/35.6%) and the largest prevalence of these allergic diseases was in the group of children between 10 and 13 years of age. We have found that the prevalence of this pathology in the province of Cádiz is higher than the national mean.

[Cardiorespirographic parameters in the first 12 hours of life. Influence of the administration of thiopental and diazepam plus thiopental on labor]. / Parámetros cardiorrespirográficos en las primeras doce horas de vida. Influencia de la administración de tiopental y diacepam más tiopental en el parto.

Cardiorespirographic parameters undergo a series of evolutionary modifications during first hours of life, becoming stable between the sixth and twelfth. The authors analyse how these parameters are influenced by the administration to the mother of thiopental and diazepam plus thiopental during birth. They observed a significant reduction, during the first hour after birth, in the range and frequency of accelerations in the newborn babies whose mothers had been administered one of the drugs. Also, they observed a greater number of cases of decelerations in the newborns who were given drugs. No significant differences were observed in other parameters, and after the twelfth hour no differences were observed at all.