Heterogeneity of atmosfpheric ammonia at the landscape scale and consequences for environmental impact assessment.

We examined the consequences of the spatial heterogeneity of atmospheric ammonia (NH3) by measuring and modelling NH3 concentrations and deposition at 25 m grid resolution for a rural landscape containing intensive poultry farming, agricultural grassland, woodland and moorland. The emission pattern gave rise to a high spatial variability of modelled mean annual NH3 concentrations and dry deposition. Largest impacts were predicted for woodland patches located within the agricultural area, while larger moorland areas were at low risk, due to atmospheric dispersion, prevailing wind direction and low NH3 background. These high resolution spatial details are lost in national scale estimates at 1 km resolution due to less detailed emission input maps. The results demonstrate how the spatial arrangement of sources and sinks is critical to defining the NH3 risk to semi-natural ecosystems. These spatial relationships provide the foundation for local spatial planning approaches to reduce environmental impacts of atmospheric NH3.

The application of inverse-dispersion and gradient methods to estimate ammonia emissions from a penguin colony.

Penguin colonies represent some of the most concentrated sources of ammonia emissions to the atmosphere in the world. The ammonia emitted into the atmosphere can have a large influence on the nitrogen cycling of ecosystems near the colonies. However, despite the ecological importance of the emissions, no measurements of ammonia emissions from penguin colonies have been made. The objective of this work was to determine the ammonia emission rate of a penguin colony using inverse-dispersion modelling and gradient methods. We measured meteorological variables and mean atmospheric concentrations of ammonia at seven locations near a colony of Adélie penguins in Antarctica to provide input data for inverse-dispersion modelling. Three different atmospheric dispersion models (ADMS, LADD and a Lagrangian stochastic model) were used to provide a robust emission estimate. The Lagrangian stochastic model was applied both in ‘forwards’ and ‘backwards’ mode to compare the difference between the two approaches. In addition, the aerodynamic gradient method was applied using vertical profiles of mean ammonia concentrations measured near the centre of the colony. The emission estimates derived from the simulations of the three dispersion models and the aerodynamic gradient method agreed quite well, giving a mean emission of 1.1 g ammonia per breeding pair per day (95% confidence interval: 0.4–2.5 g ammonia per breeding pair per day). This emission rate represents a volatilisation of 1.9% of the estimated nitrogen excretion of the penguins, which agrees well with that estimated from a temperature-dependent bioenergetics model. We found that, in this study, the Lagrangian stochastic model seemed to give more reliable emission estimates in ‘forwards’ mode than in ‘backwards’ mode due to the assumptions made.

Toward a climate-dependent paradigm of ammonia emission & deposition.

Existing descriptions of bi-directional ammonia (NH3) land–atmosphere exchange incorporate temperature and moisture controls, and are beginning to be used in regional chemical transport models. However, such models have typically applied simpler emission factors to upscale the main NH3 emission terms. While this approach has successfully simulated the main spatial patterns on local to global scales, it fails to address the environment- and climate-dependence of emissions. To handle these issues, we outline the basis for a new modelling paradigm where both NH3 emissions and deposition are calculated online according to diurnal, seasonal and spatial differences in meteorology. We show how measurements reveal a strong, but complex pattern of climatic dependence, which is increasingly being characterized using ground-based NH3 monitoring and satellite observations, while advances in process-based modelling are illustrated for agricultural and natural sources, including a global application for seabird colonies. A future architecture for NH3 emission–deposition modelling is proposed that integrates the spatio-temporal interactions, and provides the necessary foundation to assess the consequences of climate change. Based on available measurements, a first empirical estimate suggests that 5°C warming would increase emissions by 42 per cent (28–67%). Together with increased anthropogenic activity, global NH3 emissions may increase from 65 (45–85) Tg N in 2008 to reach 132 (89–179) Tg by 2100.

Improving the low-wind performance of the AERMOD atmospheric dispersion model for predicting short-range impacts of livestock ammonia emissions.

Short-range impacts to sensitive ecosystems as a result of ammonia emitted by livestock farms are often assessed using atmospheric dispersion modelling systems such as AERMOD. These assessments evaluate mean annual atmospheric concentrations of ammonia and nitrogen deposition rates at the ecosystem location for comparison with ecosystem damage thresholds. However, predictions of mean annual atmospheric concentrations can be dominated by periods of stable night-time conditions, which can contribute significantly to mean concentrations. AERMOD has been demonstrated to overestimate concentrations in certain stable low-wind conditions and so the model could potentially overestimate the short-range impacts of livestock ammonia emissions. This paper tests several modifications to the parameterisation of AERMOD (v12345) that aim to improve model predictions in low-wind conditions. The modifications are first described and then are applied to three pig farm case studies in the USA, Denmark and Spain to assess whether the modifications improve long-term mean ammonia concentration predictions through improved model performance. For these three case studies, most of the modifications tested improved model performance as a result of reducing the long-term mean concentration predictions, with the largest effect for low- or ground-level sources (e.g. slurry lagoons or naturally ventilated housing).

Modelling agro-forestry scenarios for ammonia abatement in the landscape

Ammonia emissions from livestock production can have negative impacts on nearby protected sites and ecosystems that are sensitive to eutrophication and acidification. Trees are effective scavengers of both gaseous and particulate pollutants from the atmosphere making tree belts potentially effective landscape features to support strategies aiming to reduce ammonia impacts. This research used the MODDAS-THETIS a coupled turbulence and deposition turbulence model, to examine the relationships between tree canopy structure and ammonia capture for three source types?animal housing, slurry lagoon, and livestock under a tree canopy. By altering the canopy length, leaf area index, leaf area density, and height of the canopy in the model the capture efficiencies varied substantially. A maximum of 27% of the emitted ammonia was captured by tree canopy for the animal housing source, for the slurry lagoon the maximum was 19%, while the livestock under trees attained a maximum of 60% recapture. Using agro-forestry systems of differing tree structures near ?hot spots? of ammonia in the landscape could provide an effective abatement option for the livestock industry that complements existing source reduction measures.

Cost-effective mitigation of ammonia emissions from fertilizer use in Spanish agroecosystems

Mitigación GEI sistemas agrícolas

Ammonia and Greenhouse Gases Emission from Slurry Storage with impermeable Cover and Landspreading of Cattle Slurry

Intensive farm systems handle large volume of livestock wastes, resulting in adverse environmental effects, such as gaseous losses into the atmosphere in form of ammonia (NH3) and greenhouse gases (GHG), i.e. methane (CH4), carbon dioxide (CO2) and nitrous oxide (N2O). In this study, the manure management continuum of slurry storage with impermeable cover and following cattle slurry band spreading and incorporation to soil was assessed for NH3 and GHG emissions. The experiment was conducted in an outdoor covered storage (flexible bag system) (study I), which collected the slurry produced in 7 dairy cattle farms (2,000 m3 slurry) during 12 days in the northern Spain.

Comparison of micrometeorological techinques for estimating ammonia emission from covered slurry storage and land spreading of cattle slurry

The micrometeorological mass-balance integrated horizontal flux (IHF) technique has been commonly employed for measuring ammonia (NH3) emissions inon-field experiments. However, the inverse-dispersion modeling technique, such as the backward Lagrangian stochastic (bLS) modeling approach, is currently highlighted as offering flexibility in plot design and requiring a minimum number of samplers (Ro et al., 2013). The objective of this study was to make a comparison between the bLS technique with the IHF technique for estimating NH3 emission from flexible bag storage and following landspreading of dairy cattle slurry. Moreover, considering that NH3 emission in storage could have been non uniform, the effect on bLS estimates of a single point and multiple downwind concentration measurements was tested, as proposed by Sanz et al. (2010).

Ammonia and methane gas emissions from pig slurry: variability induced through feeding strategies

Esta Tesis doctoral fue desarrollada para estudiar las emisiones de amoniaco (NH3) y metano (CH4) en purines de cerdos, y los efectos ocasionados por cambios en la formulación de la dieta. Con este propósito, fueron llevados a cabo tres estudios. El experimento 1 fue realizado con el objetivo de analizar los factores de variación de la composición de purines y establecer ecuaciones de predicción para emisiones potenciales de NH3 y CH4. Fueron recogidas setenta y nueve muestras de piensos y purines durante dos estaciones del año (verano y invierno) de granjas comerciales situadas en dos regiones de España (Centro y Mediterráneo). Se muestrearon granjas de gestación, maternidad, lactación y cebo. Se determinó la composición de piensos y purines, y la emisión potencial de NH3 y CH4. El contenido de nutrientes de los piensos fue usado como covariable en el análisis. La espectroscopia de reflectancia del infrarrojo cercano (NIRS) se evaluó como herramienta de predicción de la composición y potencial emisión de gases del purín. Se encontró una amplia variabilidad en la composición de piensos y purines. Las granjas del Mediterráneo tenían mayor pH (P<0,001) y concentración de cenizas (P =0,02) en el purín que las del Centro. El tipo de granja también afectó al contenido de extracto etéreo (EE) del purín (P =0,02), observando los valores más elevados en las instalaciones de animales jóvenes. Los resultados sugieren un efecto tampón de la fibra de la dieta en el pH del purín y una relación directa (P<0,05) con el contenido de fibra fecal. El contenido de proteína del pienso no afectó al contenido de nitrógeno del purín, pero disminuyó (P=0,003) la concentración de sólidos totales (ST) y de sólidos volátiles (SV). Se obtuvieron modelos de predicción de la emisión potencial de NH3 (R2=0,89) y CH4 (R2=0,61) partir de la composición del purín. Los espectros NIRS mostraron una buena precisión para la estimación de la mayor parte de los constituyentes, con coeficientes de determinación de validación cruzada (R2cv) superiores a 0,90, así como para la predicción del potencial de emisiones de NH3 y CH4 (R2cv=0,84 y 0,68, respectivamente). El experimento 2 fue realizado para investigar los efectos del nivel de inclusión de dos fuentes de sub-productos fibrosos: pulpa de naranja (PN) y pulpa de algarroba (PA), en dietas iso-fibrosas de cerdos de cebo, sobre la composición del purín y las emisiones potenciales de NH3 y CH4. Treinta cerdos (85,4±12,3 kg) fueron alimentados con cinco dietas iso-nutritivas: control comercial trigo/cebada (C) y cuatro dietas experimentales incluyendo las dos fuentes de sub-productos a dos niveles (75 y 150 g/kg) en una estructura 2 × 2 factorial. Después de 14 días de periodo de adaptación, heces y orina fueron recogidas separadamente durante 7 días para medir la digestibilidad de los nutrientes y el nitrógeno (N) excretado (6 réplicas por dieta) en cerdos alojados individualmente en jaulas metabólicas. Las emisiones de NH3 y CH4 fueron medidas después de la recogida de los purínes durante 11 y 100 días respectivamente. La fuente y el nivel de subproductos fibrosos afectó a la eficiencia digestiva de diferentes formas, ya que los coeficientes de digestibilidad total aparente (CDTA) para la materia seca (MS), materia orgánica (MO), fracciones fibrosas y energía bruta (EB) aumentaron con la PN pero disminuyeron con la inclusión de PA (P<0,05). El CDTA de proteína bruta (PB) disminuyó con la inclusión de las dos fuentes de fibra, siendo más bajo al mayor nivel de inclusión. La concentración fecal de fracciones fibrosas aumentó (P<0,05) con el nivel de inclusión de PA pero disminuyó con el de PN (P<0,01). El nivel más alto de las dos fuentes de fibra en el pienso aumentó (P<0,02) el contenido de PB fecal pero disminuyó el contenido de N de la orina (de 205 para 168 g/kg MS, P<0,05) en todas las dietas suplementadas comparadas con la dieta C. Adicionalmente, las proporciones de nitrógeno indigerido, nitrógeno soluble en agua, nitrógeno bacteriano y endógeno excretado en heces no fueron afectados por los tratamientos. Las características iniciales del purín no difirieron entre las diferentes fuentes y niveles de fibra, excepto para el pH que disminuyó con la inclusión de altos niveles de sub-productos. La emisión de NH3 por kg de purín fue más baja en todas las dietas suplementadas con fibras que en la dieta C (2,44 vs.1,81g de promedio, P<0,05). Además, purines de dietas suplementadas con alto nivel de sub-productos tendieron (P<0,06) a emitir menos NH3 por kg de nitrógeno total y mostraron un potencial más bajo para emitir CH4, independientemente de la fuente de fibra. El experimento 3 investigó los efectos de la fuente de proteína en dietas prácticas. Tres piensos experimentales fueron diseñados para sustituir una mescla de harina y cascarilla de soja (SOJ) por harina de girasol (GIR) o por DDGS del trigo (DDGST). La proporción de otros ingredientes fue modificada para mantener los contenidos de nutrientes similares a través de las dietas. El cambio en la fuente de proteína dio lugar a diferencias en el contenido de fibra neutro detergente ligada a proteína bruta (FNDPB), fibra soluble (FS) y lignina ácido detergente (LAD) en la dieta. Veinticuatro cerdos (ocho por dieta), con 52,3 o 60,8 kg en la primera y segunda tanda respectivamente, fueron alojados individualmente en jaulas metabólicas. Durante un periodo de 7 días fue determinado el balance de MS, el CDTA de los nutrientes y la composición de heces y orina. Se realizó el mismo procedimiento del experimento 2 para medir las emisiones de NH3 y CH4 de los purines de cada animal. Ni la ingestión de MS ni el CDTA de la MS o de la energía fueron diferentes entre las dietas experimentales, pero el tipo de pienso afectó (P<0.001) la digestibilidad de la PB, que fue mayor para GIR (0,846) que para SOJ (0,775), mientras que la dieta DDGST mostró un valor intermedio (0,794). La concentración fecal de PB fue por tanto influenciada (P<0,001) por el tratamiento, observándose la menor concentración de PB en la dieta GIR y la mayor en la dieta SOJ. La proporción de N excretado en orina o heces disminuyó de 1,63 en la dieta GIR hasta 0,650 en la dieta SOJ, como consecuencia de perdidas más bajas en orina y más altas en heces, con todas las fracciones de nitrógeno fecales creciendo en paralelo a la excreción total. Este resultado fue paralelo a una disminución de la emisión potencial de NH3 (g/kg purín) en la dieta SOJ con respecto a la dieta GIR (desde 1,82 a 1,12, P<0,05), dando valores intermedios (1,58) para los purines de la dieta DDGST. Por otro lado, el CDTA de la FS y de la fibra neutro detergente (FND) fueron afectados (P<0,001 y 0,002, respectivamente) por el tipo de dieta, siendo más bajas en la dieta GIR que en la dieta SOJ; además, se observó un contenido más alto de FND (491 vs. 361g/kg) en la MS fecal para la dieta GIR que en la dieta SOJ, presentando la dieta DDGST valores intermedios. El grado de lignificación de la FND (FAD/FND x 100) de las heces disminuyó en el orden GIR>DDGST>SOJ (desde 0,171 hasta 0,109 y 0,086, respectivamente) en paralelo a la disminución del potencial de emisión de CH4 por g de SV del purín (desde 301 a 269 y 256 mL, respectivamente). Todos los purines obtenidos en estos tres experimentos y Antezana et al. (2015) fueron usados para desarrollar nuevas calibraciones con la tecnología NIRS, para predecir la composición del purín y el potencial de las emisiones de gases. Se observó una buena precisión (R2cv superior a 0,92) de las calibraciones cuando muestras de los ensayos controlados (2, 3 y Antezana et al., 2015) fueron añadidas, aumentando el rango de variación. Una menor exactitud fue observada para TAN y emisiones de NH3 y CH4, lo que podría explicarse por una menor homogeneidad en la distribución de las muestras cuando se amplía el rango de variación del estudio. ABSTRACT This PhD thesis was developed to study the emissions of ammonia (NH3) and methane (CH4) from pig slurry and the effects caused by changes on diet formulation. For these proposes three studies were conducted. Experiment 1 aimed to analyse several factors of variation of slurry composition and to establish prediction equations for potential CH4 and NH3 emissions. Seventy-nine feed and slurry samples were collected at two seasons (summer and winter) from commercial pig farms sited at two Spanish regions (Centre and Mediterranean). Nursery, growing-fattening, gestating and lactating facilities were sampled. Feed and slurry composition were determined, and potential CH4 and NH3 emissions measured. Feed nutrient contents were used as covariates in the analysis. Near infrared reflectance spectroscopy (NIRS) was evaluated as a predicting tool for slurry composition and potential gaseous emissions. A wide variability was found both in feed and slurry composition. Mediterranean farms had a higher pH (P<0.001) and ash (P=0.02) concentration than those located at the centre of Spain. Also, type of farm affected ether extract (EE) content of the slurry (P=0.02), with highest values obtained for the youngest animal facilities. Results suggested a buffer effect of dietary fibre on slurry pH and a direct relationship (P<0.05) with fibre constituents of manure. Dietary protein content did not affect slurry nitrogen content (N) but decreased (P=0.003) in total solid (TS) and volatile solids (VS) concentration. Prediction models of potential NH3 emissions (R2=0.89) and biochemical CH4 potential (B0) (R2=0.61) were obtained from slurry composition. Predictions from NIRS showed a high accuracy for most slurry constituents with coefficient of determination of cross validation (R2cv) above 0.90 and a similar accuracy of prediction of potential NH3 and CH4 emissions (R2cv=0.84 and 0.68, respectively) thus models based on slurry composition from commercial farms. Experiment 2 was conducted to investigate the effects of increasing the level of two sources of fibrous by-products, orange pulp (OP) and carob meal (CM), in iso-fibrous diets for growing-finishing pig, slurry composition and potential NH3 and CH4 emissions. Thirty pigs (85.4±12.3 kg) were fed five iso-nutritive diets: a commercial control wheat/barley (C) and four experimental diets including two sources of fibrous by-products OP and CM and two dietary levels (75 and 150 g/kg) in a 2 × 2 factorial arrangement. After a 14-day adaptation period, faeces and urine were collected separately for 7 days to measure nutrient digestibility and the excretory patterns of N from pigs (6 replicates per diet) housed individually in metabolic pens. For each animal, the derived NH3 and CH4 emissions were measured in samples of slurry over an 11 and 100-day storage periods, respectively. Source and level of the fibrous by-products affected digestion efficiency in a different way as the coefficients of total tract apparent digestibility (CTTAD) for dry matter (DM), organic matter (OM), fibre fractions and gross energy (GE) increased with OP but decreased with CM (P<0.05). Crude protein CTTAD decreased with the inclusion of both sources of fibre, being lower at the highest dietary level. Faecal concentration of fibre fractions increased (P<0.05) with the level of inclusion of CM but decreased with that of OP (P<0.01). High dietary level for both sources of fibre increased (P<0.02) CP faecal content but urine N content decreased (from 205 to 168 g/kg DM, P<0.05) in all the fibre-supplemented compared to C diet. Additionally, the proportions of undigested dietary, water soluble, and bacterial and endogenous debris of faecal N excretion were not affected by treatments. The initial slurry characteristics did not differ among different fibre sources and dietary levels, except pH, which decreased at the highest by-product inclusion levels. Ammonia emission per kg of slurry was lower in all the fibre-supplemented diets than in C diet (2.44 vs. 1.81g as average, P<0.05). Additionally, slurries from the highest dietary level of by-products tended (P<0.06) to emit less NH3 per kg of initial total Kjeldahl nitrogen (TKN) and showed a lower biochemical CH4 potential , independently of the fibre source. Experiment 3 investigated the effects of protein source in practical diets. Three experimental feeds were designed to substitute a mixture of soybean meal and soybean hulls (SB diet) with sunflower meal (SFM) or wheat DDGS (WDDGS). The proportion of other ingredients was also modified in order to maintain similar nutrient contents across diets. Changes in protein source led to differences in dietary content of neutral detergent insoluble crude protein (NDICP), soluble fibre (SF) and acid detergent lignin (ADL). Twenty-four pigs (eight per diet), weighing 52.3 or 60.8 kg at the first and second batch respectively, were housed individually in metabolic pens to determine during a 7-day period DM balance, CTTAD of nutrients, and faecal and urine composition. Representative slurry samples from each animal were used to measure NH3 and CH4 emissions over an 11 and or 100-day storage period, respectively. Neither DM intake, nor DM or energy CTTAD differed among experimental diets, but type of feed affected (P<0.001) CP digestibility, which was highest for SFM (0.846) than for SB (0.775) diet, with WDDGS-based diet giving an intermediate value (0.794). Faecal DM composition was influenced (P<0.001) accordingly, with the lowest CP concentration found for diet SFM and the highest for SB. The ratio of N excreted in urine or faeces decreased from SFM (1.63) to SB diet (0.650), as a consequence of both lower urine and higher faecal losses, with all the faecal N fractions increasing in parallel to total excretion. This result was parallel to a decrease of potential NH3 emission (g/kg slurry) in diet SB with respect to diet SFM (from 1.82 to 1.12, P<0.05), giving slurry from WDDGS-based diet an intermediate value (1.58). Otherwise, SF and insoluble neutral detergent fibre (NDF) CTTAD were affected (P<0.001 and P=0.002, respectively) by type of diet, being lower for SFM than in SB-diet; besides, a higher content of NDF (491 vs. 361 g/kg) in faecal DM was observed for SFM with respect to SB based diet, with WDDGS diet being intermediate. Degree of lignification of NDF (ADL/NDF x 100) of faeces decreased in the order SFM>WDDGS>SB (from 0.171 to 0.109 and 0.086, respectively) in parallel to a decrease of biochemical CH4 potential per g of VS of slurry (from 301 to 269 and 256 ml, respectively). All slurry samples obtained from these three experiments and Antezana et al. (2015) were used to develop new calibrations with NIRS technology, to predict the slurry composition and potential gaseous emissions of samples with greater variability in comparison to experiment 1. Better accuracy (R2cv above 0.92) was observed for calibrations when samples from controlled trials experiments (2, 3 and Antezana et al., 2015) were included, increasing the range of variation. A lower accuracy was observed for TAN, NH3 and CH4 gaseous emissions, which might be explained by the less homogeneous distribution with a wider range of data.

Ammonia acquisition in enteric bacteria: Physiological role of the ammonium/methylammonium transport B (AmtB) protein

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein [called methylammonium/ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya. Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [14C]methylammonium depends on its conversion to γ-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3. Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting—that is, concentrating—the charged species, NH4+.

Identifying potential mechanisms enabling acidophily in the ammonia-oxidizing archaeon "Candidatus Nitrosotalea devanaterra"

FUNDING INFORMATION This work, including the efforts of Luis Sayavedra-Soto, was funded by National Science Foundation (NSF) (0541797). This work, including the efforts of Laura E. Lehtovirta-Morley, James I. Prosser, and Graeme W. Nicol, was funded by Natural Environment Research Council (NERC) (NE/I027835/1). Graeme W. Nicol is also supported by the AXA Research Fund.

Molecular adaptation of Ammonia Monooxygenase during independent pH specialization in Thaumarchaeota

Acknowledgements. This work was funded by Natural Environment Research Council Fellowship NE/J019151/1 and by institutional funding from within the University of Aberdeen.

Isolation of 'Candidatus Nitrosocosmicus franklandus', a novel ureolytic soil archaeal ammonia oxidiser with tolerance to high ammonia concentration

Acknowledgements. The authors would like to thank Mr Kevin Mackenzie and Mrs Gillian Milne (University of Aberdeen) for technical support with scanning electron microscopy, and Dr Robin Walker for access to the Woodlands Field experimental plots at the SRUC,Craibstone Estate, Aberdeen. This work was financially supported by Natural Environmental Research Council (standard grants NE/I027835/1 and NE/L006286/1 and fellowship NE/J019151/1), EC Marie Curie ITN NORA, Grant Agreement No. 316472, the AXA Research Fund and the Centre for Genome Enabled Biology and Medicine, University of Aberdeen.

A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N′-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.