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In the tropics, frequent nitrogen (N) fertilization of grazing areas can potentially increase nitrous oxide (N2O) emissions. The application of nitrification inhibitors has been reported as an effective management practice for potentially reducing N loss from the soil-plant system and improving N use efficiency (NUE). The aim of this study was to determine the effect of the co-application of nitrapyrin (a nitrification inhibitor, NI) and urea in a tropical Andosol on the behavior of N and the emissions of N2O from autotrophic and heterotrophic nitrification. A greenhouse experiment was performed using a soil (pH 5.9, organic matter content 78 g kg–1, and N 5.6 g kg–1) sown with Cynodon nlemfuensis at 60% water-filled pore space to quantify total N2O emissions, N2O derived from fertilizer, soil ammonium (NH4+) and nitrate (NO3–), and NUE. The study included treatments that received deionized water only (control, CK) and two doses of 15N-enriched urea (65 (UR) and 129 mg N kg–1 (UD)) without or with 350 g nitrapyrin for each 100 kg N (UR + NI and UD + NI). No significant differences were observed in soil NH+ content between the UR and UR + NI treatments, probably because of soil mineralization and immobilization (influenced by high soil organic matter content). Nitrapyrin application failed to maintain a stable pool of labeled NO3− due to the additional NO− produced by heterotrophic nitrification, which is not effectively inhibited by nitrapyrin. After 56 d, N2O emissions in UR (0.51 ± 0.12 mg N2O-N kg–1) and UR + NI (0.45 ± 0.13 mg N2O-N kg–1) were not significantly different; by contrast, emissions were 36.3% lower in UD + NI than in UD. It was concluded that the soil organic N mineralization and heterotrophic nitrification are the main processes of NH4+ and NO3– production. Additionally, it was found that N2O emissions were partially a consequence of the direct oxidation of the soil's organic N via heterotrophic nitrification coupled to denitrification. Finally, the results suggest that nitrapyrin would likely exert significant mitigation on N2O emissions only if a substantial N surplus exists in soils with high organic matter content. Universidad de Costa Rica/[802-B7-505]/UCR/Costa Rica Organismo Internacional de Energía Atómica/[No. CRP DI.50.16]/IAEA/Estados Unidos UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

High hydrostatic pressure (HHP) processing is a non-thermal technology reported to increase desirable metabolites in plant foods. This work evaluated changes in carotenoid accumulation in fresh-cut papaya fruit as affected by HHP treatment (50–400 MPa for 3–60 min) and during subsequent storage at 4 °C; simultaneously, transcriptional activities of carotenoid biosynthetic genes and oxidative stress markers were evaluated. LC–MS analyses revealed that HHP treatment increased carotenoid precursors and carotenes contents following processing and storage: lycopene levels increased up to 11-fold compared to the non-treated samples, and H2O2 and lipid peroxidation were concomitantly increased. qRT-PCR of intact RNA showed that the amount of phytoene desaturase transcripts increased after HHP treatment, and that they were correlated with carotene accumulation. This is the first study to show that HHP treatment triggers de novo carotenoid biosynthesis, which is regulated at the transcriptional level, possibly by inducing oxidative stress signaling in fruit tissue.

Environmental pollution is a matter of increasing concern worldwide, for which new and innovative solutions are constantly required. Bioremediation, the transformation of contaminants into less toxic substances using microorganisms, can provide an economically viable and ecologically friendly method of removing these pollutants. The identification of the microorganisms capable of degradation and their degradative genes is typically the first and most important step, when bioremediation of a new compound is devised. Once known, the gene encoding the degradative enzyme can be used to confer the degradative ability to either the indigenous microbial population or to ‘non-native’ genetically modified microorganisms. Furthermore, the identification of the genes can also allow for the presence and activity of degradative organisms to be monitored in order to adequately follow and predict the outcome of a bioremediation process. Traditional microbiological methods have been used for this purpose for several decades. Nonetheless, recent advances in molecular biology are allowing increasingly accurate and informative techniques to be applied. This review provides an overview of the genetic methods used to identify degradative genes, with an emphasis on pesticides, and their application in relation to gene monitoring in a bioremediation setting. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

A monitoring network was established in streams within a catchment near the Costa Rican Pacific coast (2008–2011) to estimate the impact of pesticides in surface water (84 samples) and sediments (84 samples) in areas under the influence of melon and watermelon production. A total of 66 (water) and 47 (sediment) pesticides were analyzed, and an environmental risk assessment (ERA) was performed for four taxa (algae, Daphnia magna, fish and Chironomus riparius). One fungicide and seven insecticides were detected in water and/or sediment; the fungicide azoxystrobin (water) and the insecticide cypermethrin (sediments) were the most frequently detected pesticides. The insecticides endosulfan (5.76 μg/L) and cypermethrin (301 μg/kg) presented the highest concentrations in water and sediment, respectively. The ERA revealed acute risk in half of the sampling points of the melon-influenced area and in every sampling point from the watermelon-influenced area. Safety levels were exceeded within and around the crop fields, suggesting that agrochemical contamination was distributed along the catchment, with potential influence of nearby crops. Acute risk was caused by the insecticides chlorpyrifos, cypermethrin and endosulfan to D. magna, fish and C. riparius; the latter was the organism with the overall highest/continuous risk. High chronic risk was determined in all but one sampling point, and revealed a higher number of pesticides of concern. Cypermethrin was the only pesticide to pose chronic risk for all benchmark organisms. The results provide new information on the risk that tropical crops pose to aquatic ecosystems, and highlight the importance of including the analysis of sediment concentrations and chronic exposure in ERA. Universidad de Costa Rica/[802-A8-016]/UCR/Costa Rica Universidad de Costa Rica/[802-B7-508]/UCR/Costa Rica Organismo Internacional de Energía Atómica/[RLA5/050]/IAEA/Austria Organismo Internacional de Energía Atómica/[RLA5/053]/IAEA/Austria Organismo Internacional de Energía Atómica/[RLA5/061]/IAEA/Austria Unión Europea/[690918 H2020-MSCA-RISE-2015]/UE/Paises Bajos UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

SummaryOlive (Olea europaea) is an important crop in Europe, with high cultural, economic, and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized.We discovered two novel, bi-functional cytochrome P450 enzymes, catalysing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus Secologanin Synthase (CrSLS), the substrate and product profiles differ.Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity.Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin independent. Notably, sequence data of cultivated and wild olives, points to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs.

Copper nanoparticles (NCu) have been proposed as an antimicrobial agent in agriculture. Therefore, NCu may interact with numerous pollutants including pesticides. Little is known about the combined effects of NCu and pesticides in soil. This study aimed at assessing the impact of NCu combined with the herbicide atrazine (ATZ) on soil. We focused on assessing the adsorption and dissipation of ATZ in the presence of NCu and the changes in microbial community profiles. First, ATZ adsorption isotherms (described using the Freundlich equation) were evaluated. After that, soil samples were spiked with NCu (40–60 nm) at 0.05 and 0.15% w/w and ATZ (3 mg a.i kg−1) and incubated for 30 days. The results showed that ATZ adsorption is favored by the presence of NCu. On the other hand, NCu at 0.15% w/w caused a significant decrease in ATZ dissipation, increasing its half-life from 6 to 37 days. Microbial community profiles (bacteria, fungi and nitrifying bacteria) remained relatively stable throughout the evaluated period. Therefore, our findings suggest that NCu can increase the persistence of ATZ in soil, which may be mostly associated to physical-chemical interaction with soil particles more than a microbial impact. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

A pesticide monitoring study including 80 and 60 active ingredients (in surface waters and sediments, respectively) was carried out in a river basin in Costa Rica during 2007–2012. A special emphasis was given on the exceptional ecological conditions of the tropical agro-ecosystem and the pesticide application strategies in order to establish a reliable monitoring network. A total of 135 water samples and 129 sediment samples were collected and analyzed. Long-term aquatic ecotoxicological risk assessment based on risk quotient in three trophic levels was conducted. Short-term risk assessment was used to calculate the toxic unit and prioritization of sampling sites was conducted by the sum of toxic units in both aquatic and sediment compartments. Dimethoate (61.2 μg/L), propanil (30.6 μg/L), diuron (22.8 μg/L) and terbutryn (4.8 μg/L) were detected at the highest concentrations in water samples. Carbendazim and endosulfan were the most frequently detected pesticides in water and sediment samples, respectively. Triazophos (491 μg/kg), cypermethrin (71.5 μg/kg), permethrin (47.8 μg/kg), terbutryn (38.7 μg/kg), chlorpyrifos (18.2 μg/kg) and diuron (11.75 μg/kg) were detected at the highest concentrations in sediment samples. The pesticides carbendazim, diuron, endosulfan, epoxyconazole, propanil, triazophos and terbutryn showed non-acceptable risk even when a conservative scenario was considered. Sum TUsite higher than 1 was found for one and two sampling sites in water and sediment compartments, respectively, suggesting high acute toxicity for the ecosystem. Main finding of the work Exceptional ecological conditions of the tropical agro-ecosystem affect the fate of pesticides in water and sediment environment differently than the temperate one.

The evolution of chemical complexity has been a major driver of plant diversification, with novel compounds serving as key innovations. The species-rich mint family (Lamiaceae) produces an enormous variety of compounds that act as attractants and defense molecules in nature and are used widely by humans as flavor additives, fragrances, and anti-herbivory agents. To elucidate the mechanisms by which such diversity evolved, we combined leaf transcriptome data from 48 Lamiaceae species and four outgroups with a robust phylogeny and chemical analyses of three terpenoid classes (monoterpenes, sesquiterpenes, and iridoids) that share and compete for precursors. Our integrated chemical–genomic–phylogenetic approach revealed that: (1) gene family expansion rather than increased enzyme promiscuity of terpene synthases is correlated with mono- and sesquiterpene diversity; (2) differential expression of core genes within the iridoid biosynthetic pathway is associated with iridoid presence/absence; (3) generally, production of iridoids and canonical monoterpenes appears to be inversely correlated; and (4) iridoid biosynthesis is significantly associated with expression of geraniol synthase, which diverts metabolic flux away from canonical monoterpenes, suggesting that competition for common precursors can be a central control point in specialized metabolism. These results suggest that multiple mechanisms contributed to the evolution of chemodiversity in this economically important family.

Constructed wetlands (CWs) and biopurification systems (BPS) present two low-cost approaches for the removal of pesticides from waters of agricultural origin. Both strategies were tested in the treatment of the herbicide terbuthylazine, a triazine of worldwide use. Three horizontal subsurface flow (HSF) CW systems were operated continuously for one year; the planted CWs (containing either Phragmites australis or Typha latifolia) were able to remove up to 73.7% and 58.4% of the pesticide, respectively, and exhibited a markedly superior performance compared to the unplanted CW. However, by the end of the treatment period, some symptoms of phytotoxicity were observed in the plants of the CWs, which are related to high terbuthylazine concentrations in plant material. A coconut fiber-containing biomixture was used in BPS which was able to rapidly remove terbuthylazine, with an estimated half-life of 8.1 d, the fastest so far reported in these systems. However, the biomixture failed to detoxify the matrix, according to ecotoxicological tests of seed germination. The current data suggests that coupled/hybrid configurations comprising CWs and BPS in series could provide increased effectiveness and a low-cost technology to remove terbuthylazine from highly contaminated water.

An experimental study evaluating the effect of bioaugmentation and antibiotic (oxytetracycline) application on pesticide degradation and microbial community structure of a biomixture used in a biopurification system (BPR) was conducted. The bioaugmentation employed a carbofuran-degrading bacterial consortium. The non-bioaugmented biomixture showed excellent performance for removal of atrazine (t1/2: 9.9 days), carbendazim (t1/2: 3.0 days), carbofuran (t1/2: 2.8 days), and metalaxyl (t1/2: 2.7 days). Neither the addition of oxytetracycline nor bioaugmentation affected the efficiency of pesticide removal or microbial community (bacterial and fungal) structure, as determined by DGGE analysis. Instead, biomixture aging was mainly responsible for microbial population shifts. Even though the bioaugmentation did not enhance the biomixtures′ performance, this matrix showed a high capability to sustain initial stresses related to antibiotic addition; therefore, simultaneous elimination of this particular mixture of pesticides together with oxytetracycline residues is not discouraged.

Antibiotic-containing wastewaters produced in agricultural activities may depress the pesticidedegrading capacity of biomixtures contained in biopurification systems. This work aimed to assay the effect of oxytetracycline (OTC) on the removal of carbofuran (CFN) in an optimized biomixture, and to determine the capacity of the system to dissipate OTC. During co-application of CFN + OTC, CFN removal and its accelerated degradation were not negatively affected. Similarly, different doses of OTC (10–500 mg kg−1) did not significantly affect CFN mineralization, and the process even exhibited a hormetic-like effect. Moreover, the biomixture was able to remove OTC with a half-life of 34.0 d. DGGEcluster analyses indicated that fungal and bacterial communities remained relatively stable during OTC application and CFN + OTC co-application, with similarities of over 70% (bacteria) and 80% (fungi). Overall, these findings support the potential use of this matrix to discard OTC-containing wastewater in this system originally intended for CFN removal. Universidad de Costa Rica/[802-B4-503]/UCR/Costa Rica Universidad de Costa Rica/[802-B6-137]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-093-13]/MICITT/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)

Bioaugmentation with ligninolytic fungi represents a potential way to improve the performance of biomixtures used in biopurification systems for the treatment of pesticide-containing agricultural wastewater. The fungus Trametes versicolor was employed in the bioaugmentation of a biomixture to be used in the simultaneous removal of seven fungicides. Liquid cultures of the fungus were able to remove tebuconazole, while no evidence of carbendazim, metalaxyl and triadimenol depletion was found. When applied in the biomixture, the bioaugmented matrix failed to remove all the triazole fungicides (including tebuconazole) under the assayed conditions, but was efficient to eliminate carbendazim, edifenphos and metalaxyl (the latter only after a second pesticide application). The re-addition of pesticides markedly increased the elimination of carbendazim and metalaxyl; nonetheless, no clear enhancement of the biomixture performance could be ascribed to fungal bioaugmentation, not even after the re-inoculation of fungal biomass. Detoxification efficiently took place in the biomixture (9 d after pesticide applications) according to acute tests on Daphnia magna. DGGE-analysis revealed only moderate time-divergence in bacterial and fungal communities, and a weak establishment of T. versicolor in the matrix. Data suggest that the non-bioaugmented biomixture is useful for the treatment of fungicides other than triazoles.

Biopurification systems constitute a biological approach for the treatment of pesticide-containing wastewaters produced in agricultural activities, and contain an active core called biomixture. This work evaluated the performance of a biomixture to remove and detoxify a combination of three triazine herbicides (atrazine/terbuthylazine/terbutryn) and one insecticide (chlorpyrifos), and this efficiency was compared with dissipation in soil alone. The potential enhancement of the process was also assayed by bioaugmentation with the ligninolytic fungi Trametes versicolor. Globally, the non-bioaugmented biomixture exhibited faster pesticide removal than soil, but only in the first stages of the treatment. After 20 d, the largest pesticide removal was achieved in the biomixture, while significant removal was detected only for chlorpyrifos in soil. However, after 60 d the removal values in soil matched those achieved in the biomixture for all the pesticides. The bioaugmentation failed to enhance, and even significantly decreased the biomixture removal capacity. Final removal values were 82.8% (non-bioaugmented biomixture), 43.8% (fungal bioaugmented biomixture), and 84.7% (soil). The ecotoxicological analysis revealed rapid detoxification (from 100 to 170 TU to

The biopurification systems (BPS) used for the treatment of pesticide-containing wastewater must present a versatile degrading ability, in order to remove different active ingredients according to the crop protection programs. This work aimed to assay the simultaneous removal of several pesticides (combinations of herbicides/insecticides/fungicides, or insecticides/fungicides) in a biomixture used in a BPS over a period of 115 d, and in the presence of oxytetracycline (OTC), an antibiotic of agricultural use that could be present in wastewater from agricultural pesticide application practices. The biomixture was able to mostly remove the herbicides during the treatment (removal rates: atrazine ≈ linuron > ametryn), and suffered no inhibition by OTC (only slightly for ametryn). Two fungicides (carbendazim and metalaxyl) were removed, nonetheless, in the systems containing only fungicides and insecticides, a clear increase in their half-lives was obtained in the treatments containing OTC. The neonicotinoid insecticides (imidacloprid and thiamethoxam) and the triazole fungicides (tebuconazole and triadimenol) were not significantly eliminated in the biomixture. Globally, the total removal of active ingredients ranged from 40.9% to 61.2% depending on the system, following the pattern: herbicides > fungicides > insecticides. The ecotoxicological analysis of the process revealed no detoxification towards the microcrustacean Daphnia magna, but a significant decay in the phytotoxicity towards Lactuca sativa in some cases, according to seed germination tests; in this case, OTC proved to be partially responsible for the phytotoxicity. The patterns of pesticide removal and detoxification provide inputs for the improvement of BPS use and their relevance as devices for wastewater treatment according to specific pesticide application programs.

The aim of this work was to determine the efficiency of a straw/compost/soil biomixture for pesticide depuration during its aging and continuous use, for a period of over a year, based on its capacity to remove carbofuran (CFN), while simultaneously monitoring the variations in microbial community structure. Successive CFN spikings were applied in the biomixture at 6-week intervals, and the removal efficiency was determined 48 h post-application. Initially, only a discrete degradation performance was observed (9.9%), but one CFN application was sufficient to induce efficient elimination (>88.5%) of the pesticide at subsequent influxes for a period of over 6 months. A statistically significant reduction on CFN removal efficiency after this time was detected, reaching levels similar to the fresh-prepared biomixture (14.8%) at the end of the experiment. Simultaneous DGGE analyses showed only modest changes on microbial community patterns through time for both, bacteria and fungi. The clustering of genetic fingerprints in chronological groups corresponding to significantly different CFN degradation efficiencies indicates that biomixture aging changes not only the composition of microbial communities, but also their suitability to engage in pesticide degradation. Periodic substitution of straw/compost/soil biomixture in biopurification systems or regular provision of easily-degradable organic substrates should be considered to maintain an adequate depuration capacity on this system.

Biopurification systems are useful in the management of pesticide residues and provide an option to dispose wastewaters of agricultural origin derived from pesticide application practices. The analysis of pesticide residues in the biopurification system biomixture is necessary to determine whether the removal of the target compounds occurs with reliable results. In this study, the pesticide extraction methodology was optimized and validated in a biomixture composed of coconut fiber, compost and soil, to determine a total of 43 molecules, distributed among triazines (10), triazoles (13) and organophosphates (20) using liquid chromatography coupled to a triple quadrupole mass spectrometer. For the validation, the parameters of linearity, matrix effect, limit of determination (LOD), specificity, selectivity, precision, trueness and robustness in the proposed biomixture were evaluated. The analyses of those parameters revealed satisfactory results of the method for most of the compounds, with the exception of diclorvos and ciromazine, for which the development of an alternative method is recommended. Once the extraction methodology was validated, the removal of eight molecules was assayed in a biopurification system used for the simultaneous treatment of a mixture of pesticide commercial formulations. Although most of the compounds were at least partially removed, none of them was eliminated at levels below the LOD. The removal pattern of ametryn, atrazine, chlorpyrifos, malathion and terbutryn was comparable to those obtained in other efficient biomixtures, and the highly recalcitrant triadimenol was eliminated; nonetheless, tebuconazole and diazinon were not significantly removed. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

Biopurification systems (BPS) or biobeds have been developed to attenuate point-source contamination due to inappropriate pesticide handling or disposal of agricultural wastewaters. The biomixture used for this strategy should be able to remove different active ingredients but its efficiency can vary due to the constant load of pesticides from crop application programs. For that reason, the performance of biomixtures in conditions that mimic the real pesticide treatment before their implementation in field settings should be assayed. This study aimed to evaluate the removal and detoxifying capacity of a previously formulated biomixture (coconut fiber, 50% v/v; compost, 25%; and soil pre-exposed to pesticides, 25%) during a simulated cycle of pesticide application (93 days) for potato production. The scheme included a first application of linuron followed by a weekly alternated treatment of the mixtures chlorpyrifos/metalaxyl and malathion/dimethomorph, and antibiotics at day 72. The biomixture showed efficient removal of linuron (half-life

The presence of oxyhaemoglobin and biliverdin interferes with the method recommended by the UK NEQAS Specialist Advisory group for EQA of CSF Proteins and Biochemistry for estimating of the net bilirubin absorbance in CSF. This is easily demonstrated by using solutions with different concentrations of these three substances.The two secondary peaks of the oxyhaemoglobin spectrum at 540 nm and 577 nm are used as reference to minimize these interferences. Those peaks have the same absorbance as at 456 nm in the oxyhaemoglobin spectrum, independent of its concentration. This wavelength is very close to the maximum absorption of bilirubin and, therefore, is suitable for estimating the net bilirubin absorbance.A preliminary study with 48 spectrophotometric analyses of CSF from patients who were suspected of having subarachnoid haemorrhage were used to compare both net bilirubin absorbance estimation methods.The new method is practically free of oxyhaemoglobin and biliverdin interference. This allows for higher sensitivity and a more realistic estimation of the bilirubin concentration in a sample.A better estimation of the bilirubin concentration can have special relevance for diminishing the amount of equivocal or inconclusive cases and also to improve the prematurity of the diagnosis.

Ecotoxicological evaluations require the use of assays with several bioindicators from different trophic levels. Only a few ecotoxicological tests using fungi have been developed, reason why, detection of adverse effects from compounds that exert fungicide action may be overlooked. This work developed a toxicity test based on the inhibition of laccase enzymatic activity in the fungus Trametes versicolor. The test was applied to several fungicides and succeeded to determine inhibition values (half maximum effective concentration, EC50) for most of them (flusilazole, imazalil, pyrimethanil, tetraconazole), though a clear dose-response was not evident for others (thiabendazole, metalaxyl). The application on atrazine (herbicide), imidacloprid (insecticide) and oxytetracycline (antibiotic), proved the proposed test is suitable towards other agrochemicals. The test was also used to estimate the detoxification resulting from two different approaches employed in the removal of agrochemicals. (a) First, in the liquid-phase elimination by fungal biomass simultaneously removing atrazine, imazalil, tebuconazole and triadimenol, the test showed a significant decrease in toxicity by biodegradation (adsorption contribution to detoxification was negligible). (b) Second, a solid-phase biomixture (used for pesticide degradation from agricultural wastewater) partially removed atrazine, imazalil, metalaxyl and pyrimethanil after 33 d; nonetheless, this system could not reduce the toxicity of the matrix, and higher laccase inhibition was detected after the treatment. The design test increases the battery of available bioassays to determine the toxicity of agrochemicals, and provides an interesting tool to monitor biodegradation processes. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Complementary clues from enzyme and genome evolution reveal how a mint turned into catmint., Catnip or catmint (Nepeta spp.) is a flowering plant in the mint family (Lamiaceae) famed for its ability to attract cats. This phenomenon is caused by the compound nepetalactone, a volatile iridoid that also repels insects. Iridoids are present in many Lamiaceae species but were lost in the ancestor of the Nepetoideae, the subfamily containing Nepeta. Using comparative genomics, ancestral sequence reconstructions, and phylogenetic analyses, we probed the re-emergence of iridoid biosynthesis in Nepeta. The results of these investigations revealed mechanisms for the loss and subsequent re-evolution of iridoid biosynthesis in the Nepeta lineage. We present evidence for a chronology of events that led to the formation of nepetalactone biosynthesis and its metabolic gene cluster. This study provides insights into the interplay between enzyme and genome evolution in the origins, loss, and re-emergence of plant chemical diversity.

Biopurification systems (BPS) are employed for the treatment of pesticide-containing wastewaters. In this work, a biomixture (active core of BPS) complemented by the addition of the fungus Trametes versicolor was evaluated for the elimination of a mixture of pesticides under different treatment conditions. The biomixture achieved high removal of all the pesticides assayed after 16 d: atrazine (68.4%, t1/2: 9.6 d), carbendazim (96.7%, t1/2: 3.6 d), carbofuran (98.7%, t1/2: 3.1 d) and metalaxyl (96.7%, t1/2: 3.8 d). Variations in the treatment conditions including addition of the antibiotic oxytetracycline and co-bioaugmentation with a bacterial consortium did not significantly affect the removal performance of the biomixture. Bacterial and fungal community profiles determined by DGGE analyses revealed changes that responded to biomixture aging, and not to antibiotic or pesticide addition. The proposed biomixture exhibits very efficient elimination during simultaneous pesticide application; moreover, the matrix is highly stable during stressful conditions such as the co-application of antibiotics of agricultural use. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

Las biomezclas constituyen la parte biológicamente activa de los sistemas de biopurificación (BPS), que se utilizan para tratar las aguas residuales que contienen pesticidas. El objetivo de este trabajo fue determinar si la co-aplicación de clorpirifos (CLP) afecta la eliminación de carbofurano (CFN) (ambos insecticidas / nematicidas) en una mezcla de fibra de coco y suelo biológico (biomezcla FCS), después del envejecimiento o exposición previa a CFN. La eliminación de CFN y dos de sus productos de transformación (3-hidroxicarbofurano y 3-cetocarbofurano) se mejoró en las biomexias preexpuestas en comparación con las biomezclas envejecidas. La aplicación conjunta de CLP no afectó la eliminación de CFN, lo que sugiere que CLP no inhibe las poblaciones microbianas a cargo de la transformación de CFN. Al contrario del comportamiento de eliminación, la mineralización de 14 radiomarcadoLos pesticidas C mostraron tasas de mineralización más altas de CFN en biomezclos envejecidos (con respecto a las biomezclas recién preparadas o expuestas previamente). En el caso de CLP, se favoreció la mineralización en biomezclas recién preparadas, lo que podría atribuirse a una alta sorción durante el envejecimiento y a la inhibición microbiana por CFN en la preexposición. Independientemente de los resultados de remoción y mineralización, los ensayos toxicológicos revelaron una fuerte disminución en la toxicidad aguda de la matriz en el microcrustáceo Daphnia magna (más del 97%) después de 8 días de tratamiento de pesticidas individuales o la mezcla CFN / CLP. Los resultados sugieren que las biomezclas FCS son adecuadas para ser usadas en BPS para el tratamiento de aguas residuales en campos donde se emplean ambos pesticidas. Biomixtures constitute the biologically active part of biopurification systems (BPS), which are used to treat pesticide-containing wastewater. The aim of this work was to determine whether co-application of chlorpyrifos (CLP) affects the removal of carbofuran (CFN) (both insecticide/nematicides) in a coconut fiber–compost–soil biomixture (FCS biomixture), after aging or previous exposure to CFN. Removal of CFN and two of its transformation products (3-hydroxycarbofuran and 3-ketocarbofuran) was enhanced in pre-exposed biomixtures in comparison to aged biomixtures. The co-application of CLP did not affect CFN removal, which suggests that CLP does not inhibit microbial populations in charge of CFN transformation. Contrary to the removal behavior, mineralization of radiolabeled 14C-pesticides showed higher mineralization rates of CFN in aged biomixtures (with respect to freshly prepared or pre-exposed biomixtures). In the case of CLP, mineralization was favored in freshly prepared biomixtures, which could be ascribed to high sorption during aging and microbial inhibition by CFN in pre-exposure. Regardless of removal and mineralization results, toxicological assays revealed a steep decrease in the acute toxicity of the matrix on the microcrustacean Daphnia magna (over 97%) after 8 days of treatment of individual pesticides or the mixture CFN/CLP. Results suggest that FCS biomixtures are suitable to be used in BPS for the treatment of wastewater in fields where both pesticides are employed. Universidad de Costa Rica/[802-B2-046]/UCR/Costa Rica Universidad de Costa Rica/[802-B4-503]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Comunicaciones/[FI-093-13]/MICITT/Costa Rica Food and Agriculture Organization of the United Nations/[TC COS5/029]/FAO/Italia International Atomic Energy Agency/[TC COS5/029]/IAEA/Austria UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

This work presents experimental results on the catalytic effect of volcanic sand on the simultaneous removal of naproxen, ketoprofen, and cimetidine by ozonation. Catalyst characterization included XRD, EDX, XRF, SEM and physisorption analysis. Removal assays at laboratory scale in a semi-batch reactor showed enhanced removal by the presence of the catalyst at pH 4, but no difference was found at higher pH values. Furthermore, the immobilization of different amounts of TiO2 (2.5 % and 5.0 %) on the sand surface revealed that TiO2 exerted an adverse effect on the catalytic activity at pH 4, lowering ketoprofen removal by up to 41.3 %. Likewise, the highest and fastest removal at pH 4 was obtained using a catalyst dose of 1 g/L and dosing ozone at 15 mg/L (72.5 % ketoprofen; 90.7 % total pharmaceuticals), reaching more than 80 % total elimination after only one minute of treatment. Catalyst reusability evaluated over 5 cycles demonstrated efficient elimination of the pharmaceuticals, with only slight decrease in ketoprofen elimination. Finally, heterogeneous catalytic ozonation of ketoprofen was applied in a real wastewater matrix, obtaining a removal of 75.9 % after 40 min of reaction; this represents a similar efficiency to that achieved in synthetic wastewater. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

Nepenthes is a genus of carnivorous plants that evolved a pitfall trap, the pitcher, to catch and digest insect prey to obtain additional nutrients. Each pitcher is part of the whole leaf, together with a leaf blade. These two completely different parts of the same organ were studied separately in a non-targeted metabolomics approach in Nepenthes x ventrata, a robust natural hybrid. The first aim was the analysis and profiling of small (50&ndash, 1000 m/z) polar and non-polar molecules to find a characteristic metabolite pattern for the particular tissues. Second, the impact of insect feeding on the metabolome of the pitcher and leaf blade was studied. Using UPLC-ESI-qTOF and cheminformatics, about 2000 features (MS/MS events) were detected in the two tissues. They showed a huge chemical diversity, harboring classes of chemical substances that significantly discriminate these tissues. Among the common constituents of N. x ventrata are phenolics, flavonoids and naphthoquinones, namely plumbagin, a characteristic compound for carnivorous Nepenthales, and many yet-unknown compounds. Upon insect feeding, only in pitchers in the polar compounds fraction, small but significant differences could be detected. By further integrating information with cheminformatics approaches, we provide and discuss evidence that the metabolite composition of the tissues can point to their function.

Neonicotinoid insecticides show high persistence in the environment, and standard biological approaches such as biopurification systems have shown mostly inefficient removal of such compounds. In this work, soil pre-exposed to imidacloprid was used to obtain presumptive imidacloprid-degrading consortia. Cometabolic enrichment yielded a microbial consortium composed of eight bacterial and one yeast strains, capable of degrading not only this compound, but also thiamethoxam and acetamiprid, as demonstrated in cross-degradation assays. The biological removal process was scaled-up to batch stirred tank bioreactors (STBR); this configuration was able to simultaneously remove mixtures of imidacloprid + thiamethoxam or imidacloprid + thiamethoxam + acetamiprid, reaching elimination of 95.8% and 94.4% of total neonicotinoids, respectively. Removal rates in the bioreactors followed the pattern imidacloprid > acetamiprid > thiamethoxam, including >99% elimination of imidacloprid in 6 d and 17 d (binary and ternary mixtures, respectively). A comprehensive evaluation of the detoxification in the STBR was performed using different biomarkers: seed germination (Lactuca sativa), bioluminescence inhibition (Vibrio fischeri), and acute oral tests in honeybees. Overall, ecotoxicological tests revealed partial detoxification of the matrix, with clearer detoxification patterns in the binary mixture. This biological approach represents a promising option for the removal of neonicotinoids from agricultural wastewater; however, optimization of the process should be performed before application in farms. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

Pesticide biopurification systems contain a biologically active matrix (biomixture) responsible for the accelerated elimination of pesticides in wastewaters derived from pest control in crop fields. Biomixtures have been typically prepared using the volumetric composition 50:25:25 (lignocellulosic substrate/humic component/soil); nonetheless, formal composition optimization has not been performed so far. Carbofuran is an insecticide/nematicide of high toxicity widely employed in developing countries. Therefore, the composition of a highly efficient biomixture (composed of coconut fiber, compost, and soil, FCS) for the removal of carbofuran was optimized by means of a central composite design and response surface methodology. The volumetric content of soil and the ratio coconut fiber/compost were used as the design variables. The performance of the biomixture was assayed by considering the elimination of carbofuran, the mineralization of 14C-carbofuran, and the residual toxicity of the matrix, as response variables. Based on the models, the optimal volumetric composition of the FCS biomixture consists of 45:13:42 (coconut fiber/compost/soil), which resulted in minimal residual toxicity and ∼99 % carbofuran elimination after 3 days. This optimized biomixture considerably differs from the standard 50:25:25 composition, which remarks the importance of assessing the performance of newly developed biomixtures during the design of biopurification systems. Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-093-13/802-B4-503]/MICITT/ Costa Rica Food and Agriculture Organization/[TC COS5/029]/FAO/Costa Rica Universidad de Costa Rica/[802-B2-046]/UCR/Costa Rica Universidad de Costa Rica/[802-B4-503]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

Environmental contamination with neonicotinoid insecticides represents an issue of wide concern due to their negative effects on pollinators. The goal of this work was to evaluate the potential use of biomixtures employed in biopurification systems (BPS) to remove two neonicotinoid pesticides, imidacloprid and thiamethoxam, from wastewater of agricultural origin. The removal was assayed by quantification of the parent compounds and the detection of putative transformation products of imidacloprid by means of LC-MS/MS, and mineralization of radiolabeled imidacloprid. Two biomixtures (B1, B2) were prepared using coconut fiber, compost and two soils pre-exposed to imidacloprid (volumetric composition 50:25:25). After spiking of neonicotinoids and 228 days of treatment, the removal ranged from 22.3%–30.3% and 38.6%–43.7% for imidacloprid and thiamethoxam, respectively. Transformation products imidacloprid-urea, desnitro-imidacloprid and desnitro-olefin-imidacloprid were detected in both biomixtures. The mineralization of 14C-imidacloprid revealed DT50 (mineralization half-lives) values of 3466 and 7702 days in the biomixtures B1 and B2, respectively, markedly lower than those in the soil used in their preparation (8667 and 9902 days, respectively). As demonstrated by these findings, the high persistence of these compounds in the BPS suggests that additional biological (or physicochemical) approaches should be explored in order to decrease the impact of neonicotinoid-containing wastewater of agricultural origin.

Lauraceous acetogenins are fatty acid derivatives with an odd-carbon aliphatic chain found in avocado (Persea americana Mill.). These compounds display a wide range of bioactivities that makes them candidates for use as antimicrobial and proapoptotic agents in the food industry and against cancer cells respectively. Existing knowledge about its metabolism in planta is scarce. This work quantifies eight different acetogenins accumulated in fruit tissues (peel, seed, and pulp) from 22 avocado cultivars to sample the existing variation using a targeted metabolomics approach. Multivariate analyses uncovered correlations among acetogenins present in fruit tissues and their chemical backbone that allowed a proposal for classifying them in three families (Avocatins, Pahuatins and Persenins). The seed acetogenin profile differed from that of the pulp and peel, which while different in concentration (peel accumulated low acetogenin amounts), had the same profile. Acetogenins from samples of known origin were also separated by variety using descriptive Linear Discriminant Analysis (LDA), and a chemotaxonomic model was generated via predictive LDA and was tested on samples from unknown origin. This work effectively sampled acetogenin contents and profile variability in seed (1.09–8.33 mg per g FW), peel (0.22–12.5 mg per g FW), and pulp (0.49–9.58 mg per g FW) from avocado fruit, as well as provides a putative classification to seven avocado cultivars. Results from this work show that the eight acetogenins followed are produced in all 22 avocado cultivars, which points to conserved metabolism among avocado plants.

Background Avocado fruit contains aliphatic acetogenins (oft-acetylated, odd-chain fatty alcohols) with promising bioactivities for both medical and food industries. However, we have scarce knowledge about their metabolism. The present work aimed to study changes in acetogenin profiles from mesocarp, lipid-containing idioblasts, and seeds from ‘Hass’ cultivar during fruit development, germination, and three harvesting years. An untargeted LC-MS based lipidomic analysis was also conducted to profile the lipidome of avocado fruit in each tissue. Results The targeted analysis showed that acetogenin profiles and contents remained unchanged in avocado mesocarp during maturation and postharvest ripening, germination, and different harvesting years. However, a shift in the acetogenin profile distribution, accompanied with a sharp increase in concentration, was observed in seed during early maturation. Untargeted lipidomics showed that this shift was accompanied with remodeling of glycerolipids: TAGs and DAGs decreased during fruit growing in seed. Remarkably, the majority of the lipidome in mature seed was composed by acetogenins; we suggest that this tissue is able to synthesize them independently from mesocarp. On the other hand, lipid-containing idioblasts accumulated almost the entire acetogenin pool measured in the whole mesocarp, while only having 4% of the total fatty acids. The lipidome of this cell type changed the most when the fruit was ripening after harvesting, TAGs decreased while odd-chain DAGs increased. Notably, idioblast lipidome was more diverse than that from mesocarp. Conclusions Evidence shown here suggests that idioblasts are the main site of acetogenin biosynthesis in avocado mesocarp. This work unveiled the prevalence of aliphatic acetogenins in the avocado fruit lipidome and evidenced TAGs as initial donors of the acetogenin backbones in its biosynthesis. It also sets evidence for acetogenins being included in future works aimed at characterizing the avocado seed, as they are a main component of their lipidome. Electronic supplementary material The online version of this article (10.1186/s12870-017-1103-6) contains supplementary material, which is available to authorized users.

Biopurification systems for the treatment of pesticide-containing wastewaters contain a biomixture constituted by low-cost materials (soil, lignocellulosic substrates and humic components). In particular, the use of bioaugmented lignocellulosic materials as part of the biomixture components has been scarcely studied. The use of the fungus Trametes versicolor grown in rice husk (RH) was evaluated as an option of lignocellulosic bioaugmented substrate of potential application in biomixtures for carbofuran (CFN) elimination. RH supported growth and activity of the fungus, as evidenced visually and by laccase activity. The system was able to degrade 55.1% CFN in 34 d with a half-life of 29.9 d, and produced 3-hydroxycarbofuran (but not 3-ketocarbofuran) as a transformation product. This metabolite was successfully eliminated during the process. Moreover, a small fraction of CFN was mineralized (7.4%) within 64 d. Toxicological evaluation revealed a marked detoxification during the process, achieving an 89% decrease in the toxicity after 34 d. Overall performance of the bioaugmented substrate indicates that RH/T. versicolor is an interesting option to take into account in the design of biomixtures for pesticide degradation.

Degradation and mineralization behavior of selected nematicides was studied in soil samples from fields cultivated with banana, potato, and coffee. Degradation assays in most of the studied soils revealed shorter half-lives for carbofuran (CBF) and ethoprophos (ETP) in samples with a history of treatment with these compounds, which may have been caused by enhanced biodegradation. A short half-life value for CBF degradation was also observed in a banana field with no previous exposure to this pesticide, but with a recent application of the carbamate insecticide oxamyl, which supports the hypothesis that preexposure to oxamyl may cause microbial adaptation towards degradation of CBF, an observation of a phenomenon not yet tested according to the literature reviewed. Mineralization assays for CBF and terbufos (TBF) revealed that history of treatment with these nematicides did not cause higher mineralization rates in preexposed soils when compared to unexposed ones, except in the case of soils from coffee fields. Mineralization half-lives for soils unexposed to these pesticides were significantly shorter than most reports in the literature in the same conditions. Mineralization rates for soils with a previous exposure to these pesticides were also obtained, adding to the very few reports found. This paper contributes valuable data to the low number of reports dealing with pesticide fate in soils from tropical origin.

An on-farm biopurification system (BPS) biomixture, with proven capacity to degrade carbofuran (CBF) was used as inoculum for a selective-enrichment process to obtain a bacterial consortium capable of using CBF as a sole source of carbon. Bacterial strains that comprise the consortium were identified as members of the genera Cupriavidus, Achromobacter and Pseudomonas. The consortium was tested in batch bioreactors, with initial CBF concentrations ranging from 50 to 200 mg L−1, in which complete degradation was attained in 16–17 h. Continuous operation of the bioreactor was optimized to achieve conditions of complete removal of up to 100 mg L−1 of CBF at a HRT of 23 h by the microbial consortium. Cross-degradation assays showed that the consortium was also capable of degrading high concentrations of other carbamates such as aldicarb, methiocarb and methomyl in batch bioreactors. The use of this consortium for the treatment of CBF and other carbamates at reactor scale represents a potential approach for the removal of these pesticides from agricultural wastewater, as an alternative to BPS. © 2016 Elsevier Ltd, This work was supported by the Costa Rican Ministry of Science, Technology and Telecommunications MICITT (project FI-093-13), Vicerrectoría de Investigación, UCR (project 802-B4-503). The Department of Chemical Engineering (UAB), is the Unit of Biochemical Engineering of the Xarxa de Referència en Biotecnologia de la Generalitat de Catalunya. C.E. Rodríguez-Rodríguez acknowledges UCR for a postdoctoral scholarship.

The use of antibiotics in agriculture produces residues in wastewaters. The disposal of such wastewaters in biopurification systems (BPS) employed for the treatment of pesticides could result in the inhibition of the degrading capacity of the biomixtures used in the BPS. We assayed the effect of two commercial formulations of antibiotics used in agriculture, one containing kasugamycin (KSG) and the other oxytetracycline plus gentamicin (OTC+GTM), on the biomixture performance. Doses from 0.1mgkg-1 to 1000mgkg-1 of KSG increased the respiration of the biomixture, and low doses enhanced the mineralization rate of the insecticide 14C-chlorpyrifos. On the contrary, OTC+GTM depressed the respiration of the biomixture and the initial mineralization rate of 14C-chlorpyrifos; nonetheless, the antibiotics did not decrease overall mineralization values. The application of both formulations in the biomixture at a relevant concentration did not harm the removal of the fungicides carbendazim and metalaxyl, or their enhanced degradation; on the other hand, the biomixture was unable to dissipate tebuconazol or triadimenol, a result that was unchanged during the addition of the antibiotic formulations. These findings reveal that wastewater containing these antibiotics do not affect the performance of BPS. However, such a response may vary depending on the type of pesticide and microbial consortium in the biomixture.

Biopurification systems (BPS) are design to remove pesticides from agricultural wastewater. This work assays for the first time the potential effect of an antibiotic of agricultural use (oxytetracycline, OTC) on the performance of a biomixture (biologically active core of BPS), considering that antibiotic-containing wastewaters are also produced in agricultural labors. The respiration of the biomixture was stimulated in the presence of increasing doses of OTC (≥100 mg kg −1 ), and only slightly increased with lower doses (≤10 mg kg −1 ). When co-applied during the removal of chlorpyrifos, OTC increased chlorpyrifos mineralization rates at low doses, resembling a hormetic effect. The biomixture was also able to remove three herbicides (atrazine, ametryn and linuron) with half-lives of 24.3 d, 43.9 d and 30.7 d; during co-application of OTC at a biomixture-relevant concentration, only the removal of ametryn was significantly inhibited, increasing its half-life to 92.4 d. Ecotoxicological assays revealed that detoxification takes place in the biomixture during the removal of herbicides in the presence of OTC. Overall results suggest that co-application of OTC in a biomixture does not negatively affect the performance of the matrix in every case; moreover, the co-application of this antibiotic could improve the mineralization of some pesticides.

The use of fungal bioaugmentation represents a promising way to improve the performance of biomixtures for the elimination of pesticides. The ligninolyitc fungus Trametes versicolor was employed for the removal of three carbamates (aldicarb, ALD; methomyl, MTM; and methiocarb, MTC) in defined liquid medium; in this matrix ALD and MTM showed similar half-lives (14d), nonetheless MTC exhibited a faster removal, with a half-life of 6.5d. Then the fungus was employed in the bioaugmentation of an optimized biomixture to remove the aforementioned carbamates plus carbofuran (CFN). Bioaugmented and non-bioaugmented systems removed over 99% ALD and MTM after 8d of treatment, nonetheless a slight initial delay in the removal was observed in the bioaugmented biomixtures (removal after 3d: ALD 87%/97%; MTM 86%/99%, in bioaugmented/non-bioaugmented systems). The elimination of the other carbamates was slower, but independent of the presence of the fungus: >98% for MTM after 35d and >99.5% for CFN after 22d. Though the bioaugmentation did not improve the removal capacity of the biomixture, it favored a lower production of transformation products at the first stages of the treatment, and in both cases, a marked decrease in the toxicity of the matrix was swiftly achieved along the process (from 435 to 448 TU to values

Biomixtures are used for the removal of pesticides from agricultural wastewater. As biomixtures employ high content of lignocellulosic substrates, their bioaugmentation with ligninolytic fungi represents a novel approach for their enhancement. Nonetheless, the decrease in the concentration of the pesticide may result in sublethal concentrations that still affect ecosystems. Two matrices, a microcosm of rice husk (lignocellulosic substrate) bioaugmented with the fungus Trametes versicolor and a biomixture that contained fungally colonized rice husk were used in the degradation of the insecticide/nematicide carbofuran (CFN). Elutriates simulating lixiviates from these matrices were used to assay the ecotoxicological effects at sublethal level over Daphnia magna (Straus) and the fish Oreochromis aureus (Steindachner) and Oncorhynchus mykiss (Walbaum). Elutriates obtained after 30 d of treatment in the rice husk microcosms at dilutions over 2.5% increased the offspring of D. magna as a trade-off stress response, and produced mortality of neonates at dilutions over 5%. Elutriates (dilution 1:200) obtained during a 30 d period did not produce alterations on the oxygen consumption and ammonium excretion of O. mykiss, however these physiological parameters were affected in O. aureus at every time point of treatment, irrespective of the decrease in CFN concentration. When the fungally colonized rice husk was used to prepare a biomixture, where more accelerated degradation is expected, similar alterations on the responses by O. aureus were achieved. Results suggest that despite the good removal of the pesticide, it is necessary to optimize biomixtures to minimize their residual toxicity and potential chronic effects on aquatic life. Universidad de Costa Rica/[802-B4-609]/UCR/Costa Rica Universidad de Costa Rica/[802-B2-046]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-093-13]/MICITT/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[802-B4-503]/MICITT/Costa Rica Division of Nuclear Techniques in Food and Agriculture/[TC COS5/029]/FAO/IAEA/Austria UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

The ligninolytic fungus Trametes versicolor was employed in the bioaugmentation of compost- (GCS) and peat-based (GTS) biomixtures for the removal of the insecticide-nematicide carbofuran (CFN). Among several lignocellulosic substrates, fungal colonization was best supported in rice husk, and this pre-colonized substrate was used to prepare the biomixtures. Estimated half-lives for CFN were 3.4 and 8.1 days in the GTS and GCS biomixtures, respectively. The CFN transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at the moment of CFN application, but their concentration continuously decreased to complete removal in both biomixtures. Mineralization of 14Cradiolabeled CFN was faster in GTS (k=0.00248 day−1) than in GCS (k=0.00188 day−1). Complete elimination of the toxicity in the matrices was demonstrated after 48 days. Overall data suggest that the bioaugmentation improved the performance of the GTS rather than the GCS biomixture. Universidad de Costa Rica/[802-B2-046]/UCR/Costa Rica Universidad de Costa Rica/[802-B4-503]/UCR/Costa Rica Universidad de Costa Rica/[802-B4-609]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-093-13]/MICITT/Costa Rica Food and Agriculture Organization/[TC COS5/029]/FAO/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

The ability of Trametes versicolor to degrade soil pollutants has been widely studied. However, the use of such fungus in real soil applications has lead to dissimilar results mainly due to soil colonization limitations. Therefore, it is important to investigate techniques to improve the survival of this white rot fungus in soils. In the present study, several processed and unprocessed low-cost lignocellulosic substrates were employed as inoculum carriers for fungal growth prior to application in soil for bioaugmentation. The fungal growth was determined by means of laccase activity and ergosterol determinations; additionally, the degrading capacity was measured by the naproxen degradation test (ND24). Although T. versicolor was able to colonize all materials, the colonization and enzymatic production was higher on processed agricultural wastes with relative low C/N ratios than in raw lignocellulosic substrates. Soil colonization was successful under both sterile and non-sterile conditions when amended with processed agricultural wastes, yielding even higher laccase production in non-sterile conditions. Moreover, T. versicolor was able to degrade significant amounts of spiked naproxen after 24 h in sterile and non-sterile soil cultures, showing the best results when using a material based on wheat straw as carrier.

For safe biosolid-land-applying, sludge should be contaminant-free. However, it may contain important amounts of micropollutants, not removed in the wastewater-treatment-processes. An alternative treatment with the fungus Trametes versicolor was applied in sterile solid-phase systems consisting of sludge and a lignocellulosic substrate. Fungal colonization and activity were demonstrated during the process, according to monitoring of ergosterol, laccase activity and the naproxen-degradation test (ND24). Fourteen out of 43 analyzed pharmaceuticals were found in the raw sludge. After treatment, phenazone, bezafibrate, fenofibrate, cimetidine, clarithromycin, sulfamethazine and atenolol were completely removed, while removals between 42% and 80% were obtained for the remaining pharmaceuticals. Toxicological analyses (Daphnia magna, Vibrio fischeri and seed germination) showed an important reduction in sludge toxicity after treatment. Results suggest that a solid-phase treatment with T. versicolor may reduce the ecotoxicological impact of micropollutants present in sewage sludge. This is the first report of a fungal-approach for elimination of emerging pollutants from biosolids.

Biomixtures comprise the active part of biopurification systems (BPS) for the removal of pesticide-containing wastewater from agricultural origin. Considering that biomixtures contain an important amount of lignocellulosic substrates, their bioaugmentation with degrading ligninolytic fungi represents a promising way to improve BPS. The fungus Trametes versicolor was employed for the bioaugmentation of rice husk-compost-soil (GCS) biomixtures in order to optimize the removal of the highly toxic insecticide/nematicide carbofuran (CFN). Composition of biomixtures has not been optimized before, and usually, a volumetric composition of 50:25:25 (lignocellulosic substrate:humic component:soil) is employed. Optimization of the biomixture composition was performed with a central composite design, using the volumetric content of rice husk (pre-colonized by the fungus) and the volumetric ratio compost/soil as design variables. Performance of biomixtures was comprehensively assayed considering CFN removal, the production of toxic transformation products (3-hydroxycarbofuran/3-ketocarbofuran), the ability to mineralize [14C]carbofuran, and the residual toxicity in the matrix. According to the models, the optimal volumetric composition of the GCS biomixture is 30:43:27, which maximizes removal and mineralization rate, and minimizes the accumulation of transformation products. Results support the value of assessing new biomixture formulations according to the target pesticide in order to obtain their optimal performance, before their use in BPS. Universidad de Costa Rica/[802-B2-046]/UCR/Costa Rica Universidad de Costa Rica/[802-B4-503]/UCR/Costa Rica Universidad de Costa Rica/[802-B4-609]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-093-13]/MICITT/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[802-B4-503]/MICITT/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

A biomixture constitutes the active core of the on-farm biopurification systems, employed for the detoxification of pesticide-containing wastewaters. As biomixtures should be prepared considering the available local materials, the present work aimed to evaluate the performance of ten different biomixtures elaborated with by-products from local farming, in the degradation of the insecticide/nematicide carbofuran (CFN), in order to identify suitable autochthonous biomixtures to be used in the tropics. Five different lignocellulosic materials mixed with either compost or peat and soil were employed in the preparation of the biomixtures. The comprehensive evaluation of the biomixtures included removal of the parent compound, formation of transformation products, mineralization of radiolabeled CFN, and determination of the residual toxicity of the process. Detoxification capacity of the matrices was high, and compost-based biomixtures showed better performance than peat-based biomixtures. CFN removal over 98.5 % was achieved within 16 days (eight out of ten biomixtures), with half-lives below 5 days in most of the cases. 3-Hydroxycarbofuran and 3-ketocarbofuran were found as transformation products at very low concentrations suggesting their further degradation. Mineralization of CFN was also achieved after 64 days (2.9 to 15.1 %); several biomixtures presented higher mineralization than the soil itself. Acute toxicity determinations with Daphnia magna revealed a marked detoxification in the matrices at the end of the process; low residual toxicity was observed only in two of the peat-based biomixtures. Overall best efficiency was achieved with the biomixture composed of coconut fiber-compost-soil; however, results suggest that in the case of unavailability of coconut fiber, other biomixtures may be employed with similar performance. Universidad de Costa Rica/[802-B4-609]/UCR/Costa Rica Universidad de Costa Rica/[802-B2-046]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones de Costa Rica/[FI-093-13]MICITT/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones de Costa Rica/[802-B4-503]MICITT/Costa Rica Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture/[TC COS5/029]IAEA/FAO/Austria Universidad de Costa Rica/[802-B4-609]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)

Altres ajuts: MMAMRM/010/PC08/3-04 Use of agrochemicals is a worldwide practice that exerts an important effect on the environment; therefore the search of approaches for the elimination of such pollutants should be encouraged. The degradation of the insecticides imiprothrin (IP) and cypermethrin (CP), the insecticide/nematicide carbofuran (CBF) and the antibiotic of agricultural use oxytetracycline (OTC) were assayed with the white rot fungus Trametes versicolor. Experiments with fungal pellets demonstrated extensive degradation of the four tested agrochemicals, at rates that followed the pattern IP > OTC > CP > CBF. In vitro assays with laccase-mediator systems showed that this extracellular enzyme participates in the transformation of IP but not in the cases of CBF and OTC. On the other hand, in vivo studies with inhibitors of cytochrome P450 revealed that this intracellular system plays an important role in the degradation of IP, OTC and CBF, but not for CP. The compounds 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA) and 3-phenoxybenzoic acid (PBA) were detected as transformation products of CP, as a result of the breakdown of the molecule. Meanwhile, 3-hydroxycarbofuran was detected as a transformation product of CBF; this metabolite tended to accumulate during the process, nonetheless, the toxicity of the system was effectively reduced. Simultaneous degradation of CBF and OTC showed a reduction in toxicity; similarly, when successive additions of OTC were done during the slower degradation of CBF, the fungal pellets were able to degrade both compounds. The simultaneous degradation of the four compounds successfully took place with minimal inhibition of fungal activity and resulted in the reduction of the global toxicity, thus supporting the potential use of T. versicolor for the treatment of diverse agrochemicals.

Synthesis and characterization of gold nanoclusters (Au NCs) stabilized by a zwitterion ligand (Zw) at different Au : Zw ratios are demonstrated. Au NCs exhibit photoluminescence (PL) emission which is tunable from the near infrared (805 nm) to the red spectral window (640 nm) and strongly influenced by the ligand shell size. Optical and chemical investigations suggest the presence of gold polymeric species and large nanoclusters for a molar ratio of Au : Zw = 1 : 1. For 1 : 5 < Au : Zw < 1 : 1, Zw induces etching of the large clusters and the formation of a monolayer of the bidentate ligands on the Au NCs (cluster size ∼7 to 10 kDa) accompanied by red PL emission at λ = 710 nm. A second organic layer starts to form for larger Zw fractions (Au : Zw < 1 : 5) as a result of electrostatic and covalent interactions of the zwitterion leading to an enhancement and a blue-shift of the PL emission. The effect of temperature and pH on the optical properties of gold clusters is strongly dependent on the ligand shell and demonstrates the importance of defining gold nanoclusters as supramolecular assemblies with a complex environment.

Cassava constitutes an abundant substrate in tropical regions. The production of butanol in ABE fermentation by Clostridium beijerinckii BA101 using cassava flour (CF) was scaled-up to bioreactor level (5 L). Optimized fermentation conditions were applied; that is, 40oC, 60 g/l CF, and enzymatic pretreatment of the substrate. The batch fermentation profile presented an acidogenic phase for the first 24 h and a solventogenic phase afterwards. An average of 37.01 g/l ABE was produced after 83 h, with a productivity of 0.446 g/l/h. Butanol production was 25.71 g/l with a productivity of 0.310 g/l/h, high or similar to analogous batch processes described for other substrates. Solvent separation by different combinations of fractioned and azeotropic distillation and liquid-liquid separation were assessed to evaluate energetic and economic costs in downstream processing. Results suggest that the use of cassava as a substrate in ABE fermentation could be a cost-effective way of producing butanol in tropical regions.

Ultraviolet (UV) filters are xenobiotic compounds that can enter the environment through the liquid effluent of wastewater treatment plants (WWTPs) and through adsorption in the sludge by-product because of their high hydrophobicity, as the sludge is subsequently applied as a fertiliser. A solid-state treatment of WWTP sludge with the white-rot fungus Trametes versicolor is reported in the present work as a feasible method for UV filter degradation, with reductions ranging from 87% in the case of 3-(4'-methylbenzylidene) camphor (4-MBC) to 100% for benzophenone-3 (BP3) and its metabolite 4,4'-dihydroxybenzophenone (4DHB). This study represents a first step in the development of a future fungal treatment for UV filters; thus, it is essential to prove that elimination is due only to the action of the fungus and not that of other microorganisms. To this end, the sludge was sterilised prior to fungal treatment. Biological assays indicate that T. versicolor readily eliminates oestrogenic activity, although it may be inefficient at eliminating other compounds, including some with dioxin-like activity. Degradation studies of 4-MBC in liquid media were also performed, and complete removal was achieved in less than 24 h. The main metabolites were identified, and the first steps of the transformation pathway were elucidated: a mono- or di-hydroxylation by cytochrome P450 and a subsequent conjugation with a pentose. None of 4-MBC transformation products was found to be responsible for increased dioxin-like activity in the sludge.

The white-rot fungus Trametes versicolor has been studied as a potential agent for the removal of environmental pollutants. For long-time solid-phase bioremediation systems a test is required to monitor the metabolic status of T. versicolor and its degradation capability at different stages. A biodegradation test based on the percentage of degradation of a spiked model pharmaceutical (anti-inflammatory naproxen) in 24 h (ND24) is proposed to monitor the removal of pharmaceuticals and personal care products in sewage sludge. ND24 is intended to act as a test complementary to ergosterol quantification as specific fungal biomarker, and laccase activity as extracellular oxidative capacity of T. versicolor . For samples collected over 45 d, ND24 values did not necessarily correlate with ergosterol or laccase amounts but in most cases, they were over 30% degradation, indicating that T. versicolor may be suitable for bioremediation of sewage sludge in the studied period.

Background Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules. Methodology/Principal Findings Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system. Conclusions/Significance Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.