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The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has spurred an extraordinary scientific effort to better understand the disease's pathophysiology and develop diagnostic and prognostic tools to guide more precise and effective clinical management. Among the biological samples analyzed for biomarker identification, urine stands out due to its low risk of infection, non-invasive collection, and suitability for frequent, large-volume sampling. Integrating data from omics studies with standard biochemical analyses offers a deeper and more comprehensive understanding of COVID-19. This review aims to provide a detailed summary of studies published to date that have applied omics and clinical analyses on urine samples to identify potential biomarkers for COVID-19. In July 2024, an advanced search was conducted in Web of Science using the query: "covid* (Topic) AND urine (Topic) AND metabol* (Topic)". The search included results published up to 14 October 2024. The studies retrieved from this digital search were evaluated through a two-step screening process: first by reviewing titles and abstracts for eligibility, and then by retrieving and assessing the full texts of articles that met the specific criteria. The initial search retrieved 913 studies, of which 45 articles were ultimately included in this review. The most robust biomarkers identified include kynurenine, neopterin, total proteins, red blood cells, ACE2, citric acid, ketone bodies, hypoxanthine, amino acids, and glucose. The biological causes underlying these alterations reflect the multisystemic impact of COVID-19, highlighting key processes such as systemic inflammation, renal dysfunction, critical hypoxia, and metabolic stress.

Alterations in metabolism caused by SARS-CoV-2 infection have been highlighted in various investigations and have been used to search for biomarkers in different biological matrices. However, the selected biomarkers vary greatly across studies. Our objective is to provide a robust selection of biomarkers, including results from different sample treatments in the analysis of volatile organic compounds (VOCs) present in urine samples from patients with COVID-19. Between September 2021 and May 2022, urine samples were collected from 35 hospitalized COVID-19 patients and 32 healthy controls. The samples were analyzed by headspace (HS) solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). Analyses were conducted on untreated urine samples and on samples that underwent specific pretreatments: lyophilization and treatment with sulfuric acid. Partial Least Squares Linear Discriminant Analysis (PLS-LDA) and Subwindow Permutation Analysis (SPA) models were established to distinguish patterns between COVID-19 patients and healthy controls. The results identify compounds that are present in different proportions in urine samples from COVID-19 patients compared to those from healthy individuals. Analysis of urine samples using HS-SPME-GC-MS reveals differences between COVID-19 patients and healthy individuals. These differences are more pronounced when methods that enhance VOC formation are used. However, these pretreatments can cause reactions between sample components, creating additional products or removing compounds, so biomarker selection could be altered. Therefore, using a combination of methods may be more informative when evaluating metabolic alterations caused by viral infections and would allow for a better selection of biomarkers.

In this study, the biodegradation properties of leather treated with various finishing chemicals were evaluated in order to enhance the sustainability of leather processing. We applied advanced analytical techniques, including FT-IR, thermogravimetric analysis (TGA), and solid-state NMR spectroscopy. Leather samples treated with different polymers, resins, bio-based materials, and traditional finishing agents were subjected to a composting process under controlled conditions to measure their biodegradability. The findings revealed that bio-based polyurethane finishes and acrylic wax exhibited biodegradability, while traditional chemical finishes like isocyanate and nitrocellulose lacquer showed moderate biodegradation levels. The results indicated significant differences in the biodegradation rates and the impact on plant germination and growth. Some materials, such as black pigment, nitrocellulose lacquer and wax, were beneficial for plant growth, while others, such as polyurethane materials, had adverse effects. These results support the use of eco-friendly finishes to reduce the environmental footprint of leather production. Overall, this study underscores the importance of selecting sustainable finishing chemicals to promote eco-friendly leather-manufacturing practices.

During adverse atmospheric events, enormous damage can occur at marine aquaculture facilities, as was the case during Storm Gloria in the southeastern Spanish Mediterranean in January 2020, with massive fish escapes. Fishes that escape were caught by professional fishermen. The objective of this study was to identify biomarkers in fish that enable differentiation among wild fish, escaped farm-raised fish, and farm-raised fish kept in aquaculture facilities until their slaughter. We focused on gilthead sea bream ( Sparus aurata ). We used nuclear magnetic resonance to search for possible biomarkers. We found that wild gilthead sea bream showed higher levels of taurine and trimethylamine-N-oxide (TMAO) in their muscle and higher levels of ω-3 fatty acids, whereas farm-escaped and farmed gilthead sea bream raised until slaughter exhibit higher levels of ω-6 fatty acids. From choline, carnitine, creatinine, betaine, or lecithin, trimethylamine (TMA) is synthesized in the intestine by the action of bacterial microflora. In the liver, TMA is oxidized to TMAO and transported to muscle cells. The identified biomarkers will improve the traceability of gilthead sea bream by distinguishing wild specimens from those raised in aquaculture., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

This work evaluates the effect of agricultural plastic waste (APW) in two particle sizes, microplastic and film debris, and subjected to a pre-treatment by exposure to UV-C, in the development of the vermicomposting process. Eisenia fetida health status and metabolic response and the vermicompost quality and enzymatic activity were determined. The environmental significant of this study is mainly related to how can affect plastic presence (depending on plastic type, size and/or if it is partially degraded) not only to this biological process of organic waste degradation, but also to the vermicompost characteristics, since these organic materials will be reintroduced in the environment as organic amendments and/or fertilizers in agriculture. The plastic presence induced a significant negative effect in survival and body weight of E. fetida with an average decrease of 10% and 15%, respectively, and differences on the characteristics of the vermicomposts obtained, mainly related with NPK content. Although the plastic proportion tested (1.25% f. w.) did not induce acute toxicity in worms, effects of oxidative stress were found. Thus, the exposure of E. fetida to AWP with smaller size or pre-treated with UV seemed to induce a biochemical response, but the mechanism of oxidative stress response did not seem to be dependent on the size or shape of plastic fragments or pre-treated plastic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Since the state of alarm was declared due to the COVID-19 pandemic, hospitals have been the main ones in charge of registering the therapeutic follow-up of affected people. The analysis of these data has allowed those different biochemical markers have been identified as predictors of the severity of the disease, but most of the published studies tend to be eminently descriptive and do not propose a biochemical hypothesis to explain the alteration of the results they are showing. Our objective is to recognize the main metabolic processes that are occurring in COVID-19 patients, as well as the identification of clinical parameters that are decisive to predict the severity of the disease., Methods: A multivariate analysis was carried out from the clinical parameters collected in the database of the HM hospitals in Madrid, to determine the most relevant variables to predict the severity of the disease. Chemometric methods allow these variables to be obtained by applying a classification strategy with PLS-LDA., Findings and Interpretation: The variables that most contribute to separation are age in men and, in both sexes, the concentration of lactate dehydrogenase, urea and C-reactive protein.Oxygen deficiency in the tissues, due to the loss of functionality of the lungs, could be affecting the muscle tissue with special severity. Inflammation and tissue damage is related to increased LDH and CRP. The loss of muscle mass and the increase in the concentration of urea and LDH is explained by the adaptation of muscle metabolism to this oxygen deficiency., Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profits sectors., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Analysis of urine samples from COVID-19 patients by 1 H NMR reveals important metabolic alterations due to SAR-CoV-2 infection. Previous studies have identified biomarkers in urine that reflect metabolic alterations in COVID-19 patients. We have used 1 H NMR to better define these metabolic alterations since this technique allows us to obtain a broad profile of the metabolites present in urine. This technique offers the advantage that sample preparation is very simple and gives us very complete information on the metabolites present. To detect these alterations, we have compared urine samples from COVID-19 patients (n = 35) with healthy people (n = 18). We used unsupervised (Robust PCA) and supervised (PLS-LDA) multivariate analysis methods to evaluate the differences between the two groups: COVID-19 and healthy controls. The differences focus on a group of metabolites related to energy metabolism (glucose, ketone bodies, glycine, creatinine, and citrate) and other processes related to bacterial flora (TMAO and formic acid) and detoxification (hippuric acid). The alterations in the urinary metabolome shown in this work indicate that SARS-CoV-2 causes a metabolic change from a normal situation of glucose consumption towards a gluconeogenic situation and possible insulin resistance., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The global consumption of plastic is growing year by year, producing small plastic pieces known as microplastics (MPs) that adversely affect ecosystems. The use of organic amendments (compost and manure) polluted with MPs affects the quality of agricultural soils, and these MPs can be incorporated into the food chain and negatively impact human health. Current European legislation only considers large plastic particles in organic amendments. There is no information regarding MP pollution. Thus, the development of a methodology to support future legislation ensuring the quality of agricultural soils and food safety is necessary. This proposed methodology is based on thermogravimetry coupled with mass spectrometry to quantify polyethylene and polystyrene (PE and PS) MPs through their mass spectrometry signal intensity of characteristic PE (m/z 41, 43 and 56) and PS (m/z 78 and 104) ions. This method has been validated with several organic amendments where the MP content ranged from 52.6 to 4365.7 mg kg -1 for PE-MPs and from 1.1 to 64.3 mg kg -1 for PS-MPs. The proposed methodology is a quick and robust analytical method to quantify MPs in organic amendments that could support new legislation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The entomopathogenic fungus Beauveria bassiana (Bb) is used to control the red palm weevil (RPW) Rhyncophorus ferrugineus (Oliver). Beuveria bassiana can infect and kill all developmental stages of RPW. We found that a solid formulate of B. bassiana isolate 203 (Bb203; CBS 121097), obtained from naturally infected RPW adults, repels RPW females. Fungi, and entomopathogens in particular, can produce volatile organic compounds (VOCs). VOCs from Bb203 were analyzed using gas chromatography-mass spectrometry (GC-MS). GC-MS identified more than 15 VOCs in B. bassiana not present in uninoculated (control) formulate. Both ethenyl benzene and benzothiazole B. bassiana VOCs can repel RPW females. Our findings suggest that B. bassiana and its VOCs can be used for sustainable management of RPW. They could act complementarily to avoid RPW infestation in palms.

The agri-food industry is at the centre of the circular economy, since the co-composting of its residual flows allows their management and adds value producing fertilisers. In this work, six composting piles were prepared combining agri-food sludge (AS), different fresh vegetable wastes (pepper waste (P), tomato waste (T), and leek waste (L), and, as bulking agents, vine shoot pruning (VS), garlic stalks (GS) and avocado leaves (AL)). Classical physico-chemical and chemical determinations and advanced instrumental methods (excitation-emission fluorescence (EEM) and gravimetric (TG, DTG and DTA) techniques) were used and compared to assess organic matter evolution and evaluate the quality of the composts obtained. The thermal profiles of the composting processes were viable to show the stabilization of the agri-food sludge with the different materials tested in the mixtures, reaching adequate levels of stabilization of organic matter. Preferential degradation of peptides and proteins was observed by fluorescence. This seemed to induce a limitation in the biodegradation of the remaining organic matter, indicating that these biomolecules are key in composting dynamics, acting as limiting components during the process. The results from thermogravimetric analysis indicated the degradation of labile compounds (e.g., carbohydrates and proteins), the most recalcitrant material becoming predominant at the maturity stage of the composting process. The rise in the thermogravimetric parameter R2 was associated with the increase in the concentration of more refracting compounds, which need more energy for their decomposition., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this work, we use electrophysiological and metabolomic tools to determine the role of chitosan as plant defense elicitor in soil for preventing or manage root pests and diseases sustainably. Root exudates include a wide variety of molecules that plants and root microbiota use to communicate in the rhizosphere. Tomato plants were treated with chitosan. Root exudates from tomato plants were analyzed at 3, 10, 20, and 30 days after planting (dap). We found, using high performance liquid chromatography (HPLC) and excitation emission matrix (EEM) fluorescence, that chitosan induces plant hormones, lipid signaling and defense compounds in tomato root exudates, including phenolics. High doses of chitosan induce membrane depolarization and affect membrane integrity. 1 H-NMR showed the dynamic of exudation, detecting the largest number of signals in 20 dap root exudates. Root exudates from plants irrigated with chitosan inhibit ca. twofold growth kinetics of the tomato root parasitic fungus Fusarium oxysporum f. sp. radicis-lycopersici. and reduced ca. 1.5-fold egg hatching of the root-knot nematode Meloidogyne javanica ., (Copyright © 2020 Suarez-Fernandez, Marhuenda-Egea, Lopez-Moya, Arnao, Cabrera-Escribano, Nueda, Gunsé and Lopez-Llorca.)

The aim of this study was to evaluate the effect of considering different composting times on compost quality before the end of the bio-oxidative phase and after the maturation and storage phases. This study may provide useful information to optimize the composting process and increase the economic feasibility of the technology and its adoption without decreasing the quality of the end-product. In this study, three composts were prepared using chicken meat processing wastes mixed with urban tree trimmings, serving as a bulking agent, to evaluate the effect of reducing the durations of the bio-oxidative phase and the maturity stage on the different physico-chemical properties. Specifically, we evaluate water-soluble organic matter transformations by using excitation-emission matrix (EEM) fluorescence spectroscopy. The obtained results have shown that a composting time of 35 days combined with eight turnings was efficient for achieving the standard process control parameters for agro-industrial waste composting in the studied conditions and allowed for the production of stable and mature compost that is suitable for agricultural use., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the Mediterranean countries, there is an increasing production of date palm wastes (Phoenix dactylifera L.), not only due to the raising production of date palm fruits, but also derived from the maintenance of urban and peri-urban green areas, especially in those affected by red palm weevil (Rhynchophorus ferrugineus). The management of this increasing volume of green wastes usually concludes with a controlled disposal that implies an important loss of resources, in terms of organic matter, nutrients and energy. In addition, the rise of wastewater generation and the incentive of the wastewater treatment processes have derived in an increase of the amount of the sludge produced, which makes difficult its management. This work studies the feasibility of co-composting palm wastes with sludge from the urban and agri-food sectors as alternative treatment to manage these organic waste streams and to obtain added-value compost. For this, four mixtures (P1, P2, P3 and P4) were prepared using as main component palm leave waste (PL) mixed with different types of sludge. In the piles P1, P2 and P3, sewage sludge (SS) was used as co-composting agent, while agri-food sludge (AS) was used in P4. Throughout composting, the thermal profile of the composting piles was assessed, as were physical, chemical, physico-chemical and maturity parameters. In addition, the changes in water-soluble organic matter were assessed using chemical analytical methods and the excitation-emission matrix (EEM) fluorescence spectroscopy. The results obtained showed the viability of the co-composting process to obtain end-products with adequate maturity degree and physical characteristics for their potential use as substrates, except for the salt contents that can limit their use in some agricultural sectors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This work evaluates several co-composting scenarios based on the use of Arundo donax biomass (AD) as bulking agent for the co-composting of sewage sludge (MS) and agri-food sludge (AS), to manage these organic wastes and to produce balanced organic fertilizers by optimizing the process. For this, six piles were prepared in commercial composting conditions, using AD in a range of 40%-80% (on a dry weight basis). Physico-chemical and chemical parameters and the thermal behaviour were evaluated during the process, as were the physical and chemical parameters of the final composts. The proportion of AD in the mixtures has a significant effect on the development of the thermophilic stage of composting, showing the piles with higher proportion of AD a quicker organic matter degradation. In addition, the evolution of the thermal indices R1 and R2 was different depending on the origin of the sludge used, indicating an increase in the relative concentration of more recalcitrant materials in the piles prepared with AS. The estimation of the global warming potential showed that the use of higher proportion of AD in the composting mixture may be a strategy to mitigate the emission of greenhouse gases during the composting process. Moreover, the end-products obtained had an additional marketable value, with a balanced nutrient content and a good degree of maturity, which indicates the viability of the composting process as a method for the stabilization of these organic wastes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Currently, the management of urban waste streams in developing countries is not optimized yet, and in many cases these wastes are disposed untreated in open dumps. This fact causes serious environmental and health problems due to the presence of contaminants and pathogens. Frequently, the use of specific low-cost strategies reduces the total amount of wastes. These strategies are mainly associated to the identification, separate collection and composting of specific organic waste streams, such as vegetable and fruit refuses from food markets and urban gardening activities. Concretely, in the Chimborazo Region (Ecuador), more than 80% of municipal solid waste is dumped into environment due to the lack of an efficient waste management strategy. Therefore, the aim of this study was to develop a demonstration project at field scale in this region to evaluate the feasibility of implanting the composting technology not only for the management of the organic waste fluxes from food market and gardening activities to be scaled-up in other developing regions, but also to obtain an end-product with a commercial value as organic fertilizer. Three co-composting mixtures were prepared using market wastes mixed with pruning of trees and ornamental palms as bulking agents. Two piles were created using different proportions of market waste and prunings of trees and ornamental palms: pile 1 (50:33:17) with a C/N ratio 25; pile 2: (60:30:10) with C/N ratio 24 and pile 3 (75:0:25) with C/N ratio 33), prepared with market waste and prunings of ornamental palm. Throughout the process, the temperature of the mixtures was monitored and organic matter evolution was determined using thermogravimetric and chemical techniques. Additionally, physico-chemical, chemical and agronomic parameters were determined to evaluate compost quality. The results obtained indicated that all the piles showed a suitable development of the composting process, with a significant organic matter decomposition, reached in a shorter period of time in pile 3. At the end of the process, all the composts showed absence of phytotoxicity and suitable agronomic properties for their use as organic fertilizers. This reflects the viability of the proposed alternative to be scaled-up in developing areas, not only to manage and recycle urban waste fluxes, but also to obtain organic fertilizers, including added value in economic terms related to nutrient contents.

None of the health claims about pomegranate juices has been approved yet by European Food Safety Authority (EFSA). There is a general perception among consumers that organic foods are healthier, tastier, and more nutritive than the conventional products. The aim of this research was to study the differences in the biological activity between ready-for-consumption juices obtained from pomegranates fruits grown under conventional and organic agricultural practices. Antioxidant activity has been evaluated by three methods (DPPH • , ABTS + , and FRAP), together with the total contents of phenolics and punicalagin (HPLC-DAD); besides, the Ames test was used to evaluate the antimutagenic potential of the juices. Pomegranate juice, either from conventionally or organically grown fruits, was antimutagenic (mean of 51 and 90 % for Salmonella typhimurium TA100 and TA98, respectively) and it was capable of protecting DNA from both, base-pair or frame-shift type of mutations. In fact, the antimutagenicity of conventional pomegranate juice was higher than that achieved by the organic sample; this finding was linked to a higher punicalagin content (201 and 104 mg L -1 for conventional and organic juices, respectively).

In this work, different analytical techniques (thermal analysis, (13)C cross-polarization magic angle spinning (CPMAS) NMR and Fourier transform infrared (FT-IR) spectroscopy) have been used to study the organic matter changes during the co-composting of pig slurry with cotton gin waste. To ensure the validity of the findings, the composting process was developed in different scenarios: under experimental pilot plant conditions, using the static pile system, and under real conditions on a pig farm, using the turning pile system. Also, the thermal stability index (R1) was determined before and after an extraction with water, to evaluate the effect of eliminating water-soluble inorganic salts on the thermal analysis. The results of the thermal methods showed the degradation of the most labile organic matter during composting; R1 increased during composting in all piles, without any influence of the presence of water-soluble inorganic ions in the sample. The NMR showed a decrease in the abundance of the carbohydrate molecules and an increase in the aliphatic materials during composting, due to a concentration effect. Also, FT-IR spectroscopy was a useful technique to study the trends of polysaccharides and nitrate, as indicators of organic matter transformations during composting.

The objective of this work was to study the co-composting process of wastes from the winery and distillery industry with animal manures, using the classical chemical methods traditionally used in composting studies together with advanced instrumental methods (thermal analysis, FT-IR and CPMAS 13C NMR techniques), to evaluate the development of the process and the quality of the end-products obtained. For this, three piles were elaborated by the turning composting system, using as raw materials winery-distillery wastes (grape marc and exhausted grape marc) and animal manures (cattle manure and poultry manure). The classical analytical methods showed a suitable development of the process in all the piles, but these techniques were ineffective to study the humification process during the composting of this type of materials. However, their combination with the advanced instrumental techniques clearly provided more information regarding the turnover of the organic matter pools during the composting process of these materials. Thermal analysis allowed to estimate the degradability of the remaining material and to assess qualitatively the rate of OM stabilization and recalcitrant C in the compost samples, based on the energy required to achieve the same mass losses. FT-IR spectra mainly showed variations between piles and time of sampling in the bands associated to complex organic compounds (mainly at 1420 and 1540 cm-1) and to nitrate and inorganic components (at 875 and 1384 cm-1, respectively), indicating composted material stability and maturity; while CPMAS 13C NMR provided semi-quantitatively partition of C compounds and structures during the process, being especially interesting their variation to evaluate the biotransformation of each C pool, especially in the comparison of recalcitrant C vs labile C pools, such as Alkyl /O-Alkyl ratio.

Sewage sludge application to agricultural soils is a common practice in several countries in the European Union. Nevertheless, the application dose constitutes an essential aspect that must be taken into account in order to minimize environmental impacts. In this study, near infrared reflectance spectroscopy (NIRS) was used to estimate in sewage sludge samples several parameters related to agronomic and environmental issues, such as the contents in organic matter, nitrogen and other nutrients, metals and carbon fractions, among others. In our study (using 380 biosolid samples), two regression models were fitted: the common partial least square regression (PLSR) and the penalized signal regression (PSR). Using PLSR, NIRS became a feasible tool to estimate several parameters with good goodness of fit, such as total organic matter, total organic carbon, total nitrogen, water-soluble carbon, extractable organic carbon, fulvic acid-like carbon, electrical conductivity, Mg, Fe and Cr, among other parameters, in sewage sludge samples. For parameters such as C/N ratio, humic acid-like carbon, humification index, the percentage of humic acid-like carbon, the polymerization ratio, P, K, Cu, Pb, Zn, Ni and Hg, the performance of NIRS calibrations developed with PLSR was not sufficiently good. Nevertheless, the use of PSR provided successful calibrations for all parameters., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We present a new methodology to predict embryo viability in assisted reproductive technology (ART) treatments by determining the relative amino acid concentrations in human embryo culture medium on day 3, using high-performance liquid chromatography with mass spectroscopy analysis without derivatization. The model was performed with soft independent modeling of class analogy for the samples from nonpregnancy and pregnancy cases., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The content and chemical forms of P in compost are essential variables for its proper management with an agricultural purpose, especially considering the increasing P over-fertilization in agrosystems. In this study, the estimation of P content and dynamics in different composting scenarios was developed using near infrared reflectance spectroscopy (NIRS) coupled with a statistical tool for calibration, a penalized signal regression. Samples were analyzed on total P and partitioned using NaOH-solution (31)P NMR spectroscopy quantifying pyrophosphate, orthophosphate, orthophosphate diesters, phospholipids, and orthophosphate monoesters pools. According to the results obtained, total P content (r(2)=0.99 and root mean square error of cross-validation=0.53) and P forms can be estimated during composting using NIRS, as well as in the mature product, orthophosphate and orthophosphate monoesters being the most abundant P forms throughout the experiment. Penalized signal regression allows detecting the significant wavenumbers in each composting period, and also with the different P pools in the composting pile depending on time.

The aim of the present paper is to evaluate the changes of organic matter during the composting process of fresh winery and distillery residues (WDR) by means of classical and chemometric analysis of (13)C cross-polarization magic angle spinning (CPMAS) NMR and Fourier transform infrared (FT-IR) spectra. (13)C NMR spectroscopy displayed a preferential biodegradation of carbohydrates as well as an accumulation of aliphatic chains (cutin- and suberin-like substances). This preferential biodegradation of the organic fractions reduces the landfill emission potential. Although the composition of the input mixture strongly affects the shape of the infrared (IR) spectra, typical bands of components can be selected and used to follow the composting process; that is, changes in the relative absorbances of the band of nitrate (at 1384 cm(-1)) and in the band of carbohydrates (at 1037 cm(-1)) have been observed. In addition, different chemometric tools, such as partial least-squares (PLS), interval PLS (iPLS), backward iPLS (biPLS), and genetic algorithm (GA), have been used to find the most relevant spectral region during the composting process. Chemometric analysis based on the combined and sequential use of iPLS and GA has been revealed as a very powerful tool for the detection in samples of the most relevant spectral region related to the composting process. From the obtained results, it can be concluded that CPMAS (13)C NMR supported by FT-IR could provide information about the evolution and characteristics of the organic matter during the composting process in order to avoid contamination problems after its use as amendment in agriculture or after landfilling.

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.

The aim of this work was to study the viability of recycling the solid wastes generated by the winery and distillery industry by means of co-composting with animal manures, as well as to evaluate the quality of the composts obtained. Two piles, using exhausted grape marc and cattle manure or poultry manure, respectively (at ratios, on a fresh weight basis, of 70:30), were composted by the Rutgers static pile composting system. Throughout the composting process, a number of parameters were monitored, such as pH, electrical conductivity, organic matter, water-soluble carbon, water-soluble polyphenols, different forms of nitrogen (organic nitrogen, ammonium and nitrate) and humification indices (humification ratio, humification index, percentage of humic acid-like C, polymerisation ratio and cation exchange capacity), as well as the germination index. Organic matter losses followed first-order kinetics equation in both piles, the highest organic matter mineralisation rate being observed with exhausted grape marc and cow manure. On the other hand, the mixture with the lowest C/N ratio, using exhausted grape marc and poultry manure, showed the highest initial ammonium contents, probably due to the higher and more labile N content of poultry manure. The increase in the cation exchange capacity revealed the organic matter humification during composting. In contrast, other humification parameters, such as the humification ratio and the humification index, did not show the expected evolution and, thus, could not be used to assess compost maturity. Composting produced a degradation of the phytotoxic compounds, such as polyphenols, to give composts without a phytotoxic character. Therefore, composting can be considered as an efficient treatment to recycle this type of wastes, due to composts presented a stable and humified organic matter and without phytotoxic effects, which makes them suitable for their agronomic use.

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO (3) (-) , NO (2) (-) or NH (4) (+) ) for growth. Different approaches were considered to study the effect of NH (4) (+) on nitrogen assimilation in Hfx. mediterranei cells grown in KNO(3) medium. The NH (4) (+) addition to KNO(3) medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH (4) (+) media. Both activities increased when NH (4) (+) was removed from culture, showing that the negative effect of NH (4) (+) on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO (3) (-) and NH (4) (+) , NH (4) (+) was preferentially consumed, but NO (3) (-) uptake was not completely inhibited by NH (4) (+) at prolonged time scale. The addition of MSX to NH (4) (+) or NO (3) (-) cultures results in an increase of Nas and NiR activities, suggesting that NH (4) (+) assimilation, rather than NH (4) (+ ) per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei.

The aim of the present paper is to assess the maturity degree reached by different samples of several mixtures from winery and distillery residues composted using the Rutgers composting system, by means of excitation-emission matrix (EEM) fluorescence spectroscopy. The composts were sampled once a week for about 200d. EEM spectra indicate the presence of different fluorophores. The fluorescence intensities of these peaks show trends related to the maturity of the composting samples selected. The "contour density" of EEM maps is strongly modified through time. We have used the quantitative method of fluorescence regional integration (FRI). The EEMs were delineated into five excitation-emission regions. The degree of compost maturity could be correlated with the percentage of the volumetric integration under the EEM within each region. Further refinement of these techniques should provide a relatively rapid method for assessing the suitability of the compost to soil application.

Sequencing a 6,720-bp segment of the extreme halophilic archaeon Haloferax mediterranei genome has revealed the genomic organization of the putative structural genes for nitrate assimilation. We report a gene operon containing nasABC and nasD gene. nasA encodes an assimilatory nitrate reductase, nasB codes for a membrane protein with similarity to the NarK transporter, nasC encodes a protein with similarity to MobA; and nasD codes for an assimilatory ferredoxin-dependent nitrite reductase. Reverse transcription-PCR and primer extension experiments have demonstrated the existence of one polycistronic messenger nasABC and one monocistronic nasD initiated from a different promoter. The gene order and the grouping in two adjacent transcriptional units constitutes a novel organization of nas genes. The promoter regions harbor direct palindromes reminiscent of target sites for binding of a hypotetical regulatory protein(s). Transcription of the nasABC and nasD regions was found to be repressed by the presence of ammonium as nitrogen source.

The Haloferax mediterranei nar operon has been sequenced and its regulation has been characterized at transcriptional level. The nar operon encodes seven open reading frames(ORFs) (ORF1 narB, narC, ORF4, narG, narH, ORF7 and narJ). ORF1, ORF4 and ORF7 are open reading frames with no assigned function, however the rest of them encoded different proteins. narB codes for a 219-amino-acid-residue iron Rieske protein. narC encodes a protein of 486 amino acid residues identified by databases searches as cytochrome-b (narC). The narG gene encodes a protein with 983 amino acid residues and is identified as a respiratory nitrate reductase catalytic subunit (narG). NarH protein has been identified as an electron transfer respiratory nitrate reductase subunit (narH). The last ORF encodes a chaperonin-like protein (narJ) of 242 amino acid residues. The respiratory nitrate reductase was purified 21-fold from H. mediterranei membranes. Based on SDS-PAGE and gel-filtration chromatography under native conditions, the enzyme complex consists of two subunits of 112 and 61 kDa. The optimum temperature for activity was 70 degrees C at 3.4 M NaCl and the stability did not show a direct dependence on salt concentration. Respiratory nitrate reductase showed maximum activity at pH 7.9 and pH 8.2 when assays were carried out at 40 and 60 degrees C, respectively. The absorption spectrum indicated that Nar contains Fe-S clusters. Reverse transcriptase (RT-PCR) shows that regulation of nar genes occurs at transcriptional level induced by oxygen-limiting conditions and the presence of nitrate.

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe-2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (previously halobium) was solubilized at low salt concentration in reverse micelles of hexadecyltrimethyl-ammoniumbromide in cyclohexane with 1-butanol as co-surfactant. The enzyme maintained its catalytic properties under these conditions. The thermodynamic "solvation-stabilization hypothesis" has been used to explain the bell-shaped dependence of pNPPase activity on the water content of reverse micelles, in terms of protein-solvent interactions. According to this model, the stability of the folded protein depends on a network of hydrated ions associated with acidic residues at the protein surface. At low salt concentration and low water content (the ratio of water concentration to surfactant concentration; w0), the network of hydrated ions within the reverse micelles may involve the cationic heads of the surfactant. The bell-shaped profile of the relationship between enzyme activity and w0 varied depending on the concentrations of NaCl and Mn2+.

The use of halophilic extremozymes in organic media has been limited by the lack of enzymological studies in these media. To explore the behaviour of these extremozymes in organic media, different approaches have been adopted, including the dispersal of the lyophilised enzyme or the use of reverse micelles. The use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could open new fields of application such as the use of these enzymes as biocatalysts in organic media.

Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in catalytic properties and their flexibility in adapting to different environmental conditions. The extremely halophilic alkaline p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum was solubilized in an organic medium consisting of reversed micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as cosurfactant. Hydrolysis of p-nitrophenylphosphate was nonlinear with time when the enzyme was microinjected into reversed micelles that contained substrate. These data are consistent with a kinetic model in which the enzyme is irreversibly converted from an initial form to a final stable form during the first seconds of the encapsulation process. The model features a rate constant (k) for that transition and separate hydrolysis rates, v(1) and v(2), for the two forms of the enzyme. The enzyme conversion may be governed by the encapsulation process., (Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 497-502, 2002.)

A putative partner of the already characterized CopZ from Bacillus subtilis was found, both proteins being encoded by genes located in the same operon. This new protein is highly homologous to eukaryotic and prokaryotic P-type ATPases such as CopA, Ccc2 and Menkes proteins. The N-terminal region of this protein contains two soluble domains constituted by amino acid residues 1 to 72 and 73 to 147, respectively, which were expressed both separately and together. In both cases only the 73-147 domain is folded and is stable both in the copper(I)-free and in the copper(I)-bound forms. The folded and unfolded state is monitored through the chemical shift dispersion of 15N-HSQC spectra. In the absence of any structural characterization of CopA-type proteins, we determined the structure of the 73-147 domain in the 1-151 construct in the apo state through 1H, 15N and 13C NMR spectroscopies. The structure of the Cu(I)-loaded 73-147 domain has been also determined in the construct 73-151. About 1300 meaningful NOEs and 90 dihedral angles were used to obtain structures at high resolution both for the Cu(I)-bound and the Cu(I)-free states (backbone RMSD to the mean 0.35(+/-0.06) A and 0.39(+/-0.07) A, respectively). The structural assessment shows that the structures are accurate. The protein has the typical betaalpha(betabeta)alphabeta folding with a cysteine in the C-terminal part of helix alpha1 and the other cysteine in loop 1. The structures are similar to other proteins involved in copper homeostasis. Particularly, between BsCopA and BsCopZ, only the charges located around loop 1 are reversed for BsCopA and BsCopZ, thus suggesting that the two proteins could interact one with the other. The variability in conformation displayed by the N-terminal cysteine of the CXXC motif in a number of structures of copper transporting proteins suggests that this may be the cysteine which binds first to the copper(I) carried by the partner protein., (Copyright 2002 Elsevier Science Ltd.)

Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl. Consequently, the use of these in organic media seemed to be unlikely. However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration. The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant. The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system. Kinetic parameters were determined in each system. The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only. The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.

Haloferax mediterranei can use nitrate as sole nitrogen source during aerobic growth. We report here the purification and biochemical characterisation of the assimilatory nitrate reductase (EC 1.6.6.2) from H. mediterranei. The enzyme, as isolated, was composed of two subunits (105+/-1.3 kDa and 50+/-1.3 kDa) and behaved as a dimer during gel filtration (132+/-6 kDa). A pH of 9 and elevated temperatures up to 80 degrees C (at 3.1 M NaCl) are necessary for optimum activity. The enzyme stability and activity of the enzyme depend upon the salt concentration. Reduced methyl viologen was as effective as the natural electron donor ferredoxin in the catalytic process. In contrast, NADPH and NADH, which are electron donors in nitrate reductases from different non-photosynthetic bacteria, were ineffective.

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

Reversed micelles were used as a cytoplasmic model to study the effect of the multi-ionic equilibria on kinetics of extreme halophilic enzymes. The enzymatic system used was an alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium). This enzyme was solubilised in reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The p-nitrophenylphosphate phosphatase is a good system to study the regulation of the enzymatic activity, because it utilises manganese, water and potassium or sodium as cofactors and reacts with p-nitrophenylphosphate. Kinetic behaviour was determined by the ratio between [Mn2+] and [Na+] or [K+]. When the [Mn2+] increased and [Na+] or [K+] decreased, the kinetics showed cooperative behaviour. Rabin's model describes the kinetic behaviour of the p-nitrophenylphosphate phosphatase in reversed micelles.

The nitrite reductase from the extreme halophilic archaeon, Haloferax mediterranei, has been purified and characterised. H. mediterranei is capable of growing in a minimal medium (inorganic salts and glucose as a carbon source) with nitrate as the only nitrogen source. The overall purification was 46-fold with about 4% recovery of activity. The enzyme is a monomeric protein of approximately 66 kDa. A pH of 7.5 and high temperatures up to 60 degrees C are necessary for optimum activity. Reduced methyl viologen has been found to be an electron donor as effective as ferredoxin. NADPH and NADH, which are electron donors in nitrite reductases from different non-photosynthetic bacteria, were not effective with nitrite reductase from H. mediterranei.