Simultaneous removal of neonicotinoid insecticides by a microbial degrading consortium: Detoxification at reactor scale

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

Crying and Getting a Ph.D. Position

I am not that special. Others dream of becoming scientists and taking the academic path to pursue a doctoral degree. Now, I am a Ph.D. student in beautiful Veneto, Italy, in an entomology group at the University of Padova. I am part of a Crop Science Ph.D. school, where my project focuses on the microbiology of ambrosia beetles. Obtaining the position was not easy. I first attempted to enter graduate school in 2017, and it was not until 2020 that I moved to study in Europe. Experiencing rejection was disappointing, but the application process became more familiar with each attempt. To help reassure others going through this process, I will share my reflections on my setbacks in obtaining a Ph.D. position

Using Molecular Methods to Identify and Monitor Xenobiotic-Degrading Genes for Bioremediation

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 Microbiologí

Selective-enrichment as a Tool to Obtain Microbial Degrading Consortia for the Remediation of Pesticide Residues

Inadequate disposal of pesticides during their extensive use in agricultural practices may result in soil, surface, and groundwater pollution. These agrochemicals affect a wide variety of non-target organisms, and their presence in the environment produces significant toxicological disturbances at acute and chronic levels. The continuous exposure of soils to pesticides strongly selects bacteria with the capacity to tolerate or even degrade these pollutants, an ability that can be exploited for bioremediation purposes. During a selective-enrichment process, an environmental sample, such as soil pre-exposed to pesticides, is repeatedly exposed to a xenobiotic in order to obtain microbial consortia or individual isolates with the potential capacity to remove the target pollutant. This chapter analyzes methodological strategies and problems related to pesticide selective-enrichment processes aimed at biodegradation of agrochemicals. The performances of different approaches employed for the application of the isolated consortia are discussed.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Ecotoxicological test based on inhibition of fungal laccase activity: Application to agrochemicals and the monitoring of pesticide degradation processes

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 Microbiologí

Cimex lectularius Linnaeus, 1758 (Hemiptera: Cimicidae) in Costa Rica: First Case Report Confirmed by Molecular Methods in Central America

Cimex lectularius and Cimex hemipterus are the most common species of bedbugs that infest homes. Although case reports decreased substantially by the end of the 20th century, bed bugs, and especially C. lectularius, are currently suffering a resurgence mostly attributed to insecticide resistance, inadequate pest control, and increased travel. Here, we report, to the best of our knowledge, the first molecular confirmation of C. lectularius in Central America. Specimens were obtained from an apartment located in Heredia, Costa Rica. These specimens were identified morphologically as C. lectularius. The species identification was confirmed by amplifying and sequencing fragments of the cytochrome oxidase subunit I (COI) and the 16S rRNA (16S) genes. The phylogenetic analysis showed that the sequences obtained were more closely related to a C. lectularius mitochondrial complete genome sequence from China, with similarities of 98.84% (686/694) for COI and 98.97% (387/391) for 16S. The finding of C. lectularius in Costa Rica will require further investigation in order to determine the extent of current infestations and the susceptibility to insecticides, especially due to the impact that this species can have in human health, as well as the tourism industry in the region.Universidad de Costa Rica/[430-B5-735]/UCR/Costa RicaUniversidad de Costa Rica/[ED-548]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Impaired pesticide removal and detoxification by biomixtures during the simulated pesticide application cycle of a tropical agricultural system

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 <15 days), and a fluctuating transformation rate for the other compounds. A constant and sustained removal was observed for malathion and methalaxyl. In contrast, lower efficiency and accumulation was described for chlorpyrifos and dimethomorph. Following antibiotic treatment, changes on pesticide removal were observed only in the case of chlorpyrifos, whose removal was slightly enhanced. Furthermore, acute toxicity assays showed limited detoxification of the matrix, especially when compounds began to accumulate. Summarizing, our experiments showed that the proposed biomixture does not support a proper removal of the pesticides during the simulated application cycle of potato production. Further optimization of a biopurification system is required to guarantee the successful elimination of pesticide combinations when applied in field conditions.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Removal of herbicides in a biopurification system is not negatively affected by oxytetracycline or fungally pretreated oxytetracycline

UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro en Investigación en Contaminación Ambiental (CICA

Phylogenetic analyses of antibiotic-producing Streptomyces sp. isolates obtained from the stingless-bee Tetragonisca angustula (Apidae: Meliponini)

Many insects have been associated with actinobacteria in protective symbiosis where antimicrobial metabolites inhibit host pathogens. However, the microbiota of neotropical insects such as the stingless-bee Tetragonisca angustula is poorly explored. T. angustula is a meliponid bee widely distributed in Latin America, its honey is traditionally exploited because of its ethno-pharmacological properties and its antimicrobial activity has been demonstrated. Also, the well-structured nest of this species allows exploration of the microbiota of its different components. Even though Streptomyces spp. have been cultured from stingless-bees, little is known about their role in this insect–microbe relationship. In this study, we examined the association between culturable actinobacteria and T. angustula, and evaluated the isolates’ potential as antimicrobial producers. We isolated 51 actinobacteria from adult bees and different substrates of the hive of T. angustula (pollen and honey storage, garbage pellets and cerumen). We then performed a 16S rRNA phylogenetic analysis that clusters the bacteria to previously described lineages of host-associated Streptomyces. In addition, all the isolates were classified according to their antibacterial activity against human pathogens, measured by a growth inhibition test based on diffusion in agar. More than 50 % of our isolates exhibit antimicrobial activity, mainly to Gram-positive bacteria and fungi and only two against Gram-negative bacteria. Additionally, we obtained electron micrographs of adult bees with what appears to be patches of hyphae with Streptomyces-like cell morphology on their body surface. Our results suggest that T. angustula possibly uptakes and transfers actinobacteria from the environment, acting as vectors for these potentially beneficial organisms. This research provides new insights regarding the microbiota associated with T. angustula and justify future studies exploring the full diversity of the microbial community associated with the hive and the possible exchange of microbes with the crops they pollinate.Universidad de Costa Rica/[810-B3-185]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)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)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de MedicinaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA

Pristine but metal-rich Río Sucio (Dirty River) is dominated by Gallionella and other iron-sulfur oxidizing microbes

Whether the extreme conditions of acidity and heavy metal pollution of streams and rivers originating in pyritic formations are caused primarily by mining activities or by natural activities of metal-oxidizing microbes living within the geological formations is a subject of considerable controversy. Most microbiological studies of such waters have so far focused on acid mine drainage sites, which are heavily human-impacted environments, so it has been problematic to eliminate the human factor in the question of the origin of the key metal compounds. We have studied the physico-chemistry and microbiology of the Río Sucio in the Braulio Carrillo National Park of Costa Rica, 22 km from its volcanic rock origin. Neither the remote origin, nor the length of the river to the sampling site, have experienced human activity and are thus pristine. The river water had a characteristic brownish-yellow color due to high iron-dominated minerals, was slightly acidic, and rich in chemolithoautotrophic iron- and sulfur-oxidizing bacteria, dominated by Gallionella spp. Río Sucio is thus a natural acid-rock drainage system whose metal-containing components are derived primarily from microbial activities.Universidad de Costa Rica/[809-B4-282]/UCR/Costa RicaEuropean Commission-Science and Technology Development for in situ detection and characterization of subsurface life on the Iberian Pyritic Belt/[ERC250350-IPBSL]/ERC IPBSL/Unión EuropeaUSA National Science Foundation/[0959894]//Estados UnidosConsejo Superior de Investigaciones Científicas//CSIC/EspañaEuropean Union FP7 programme /[607346]/EU/UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de QuímicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM