García Martínez, Sandra

Universitat Autònoma de Barcelona. Facultat de Biociències

García-Martínez, Sandra

Diposit Digital de Documents de la UAB

Bioremediation of irrigation water contaminated witharsenical pesticidesIntroductionNowadays pollution by heavy metals of soils and water continues being a serious problem for both environment and human health due to itspotential inclusion into food networks and its bioaccumulation [1].The arsenic is a metalloid and in nature we can find different arsenic species (As0, As-3, As+3 i As+5). Arsenic forms have different toxicities [2][Fig. 1].Currently arsenic used in pesticide fabrication and arsenic consumption derives to population intoxications through the irrigation of contaminatedwater [3] .Initial hypotesisMaterials and methodsSample collection:The samples will be taken from the delta of the River Ganges in India [Fig. 3] where an excess level ofarsenic pollution higher than what FAO/OMS allows was detected (10µg/L).Samples of river water will be put into sterilized bottles which shall be immersed 20 cm deep and thenfilled. Next the bottles will be labeled.Fig. 1: Some arsenic species that it isarranged in descending order oftoxicity: 1.Di-methyl arsenate (DMAV);2.Monomethyl arsenate (MMAV);3.Arsenate (AsV); 4.Di-methyl arsenite(DMAIII); 5.Monomethylarsenite(MMAIII); 6. Arsenite (AsIII) [2].Fig.2. Diagram of the different microbial processes involved in arsenicbiochemistry in the environment [4].Bioremediation of arsenic can be conducted by different processes depending on the microorganism. The most used methods are: [Fig.2][4]:1. Arsenic unspecific absortion: Arsenic enters the cells through the phosphate transporters (arsenate) or the aqua-glyceroporins (arsenite).2. Arsenic resistance/tolerance: Once inside the cells, arsenate is reduced to arsenite by ArsC, which further extrude out of the cell by the specificpump ArsB.3. Arsenic accumulation: Arsenite can also be detoxified by complexation with Cys-rich peptides.4. Arsenite oxidation: Arsenite can serve as electron donor by oxidation to arsenate.5. Arsenate reduction: Arsenate can be used as the ultimate electron acceptor during respiration.6. Arsenic methylation: Inorganic arsenic can also be transformed into organic species in a methylation cascade.Sandra García Martínez, Grau de Microbiologia, UAB, Tutor: Antoni SoléObjectivesThe project aims are:- Define resistance/tolerance to arsenic of different strains.- Check if the chosen strains actually retain the arsenic ions in pollutedwater contaminated by the environment.- Check efficiency for a posterior use in bioremediation.In the Banerjee, S. et al [5] article resolves that some microorganismschange the status of oxidation of the arsenate/arsenitet which does not .In this research we shall take some strains described in article byBanerjee, S. et al [5] and they will use for establishing targets.Measuring physiochemical parametersThe pH will be measured with pH meter (Cyberscan PC510, Eutech Instrumens; Netherlands) andarsenic concentration with an analytic test (Merck, Spectroquant 1.01747.0001).Processing of the water:Water will be filtered for removing particles in suspension, which could later interfere. Afterwards thewater will be sterilized in an autoclave (ST200, Yamato Scientific Co.) at 121ºC during 20 minutes [7].Fig. 3 Arsenic levels in delta Ganges river [6]. Fig. 4. (a) A. lwoffii in absence of arsenic, (b) A. lwoffii in presenceof arsenic, (c)  Transmission electron micrograph of A. lwoffii in presence of arsenic [5]Research of resistant strains:The strains that will be used in the study are [Fig. 4]:- Pseudomonas aeruginosa- Bacillus circulants- Acinetobacter lwoffiiEvaluation of resistance to arsenic:To evaluate arsenic resistance strains will be grown in an enrichment medium (LB) with differentconcentrations of arsenate (0-150mM), arsenite (0-50mM) and without the ion (control) during 18 hours.The growth will be measured using optic density at 600nm with spectrophotometer (UNICO, S-1200-E)[8].To evaluate the maximum resistance levels of strains, the concentration of arsenate or arsenite necessary forinhibit the growth of any that is isolated will be analysed. This arsenite or arsenate concentration will bemeasured with a spectrophotometer (UNICO, S-1200-E)..Defining bacterial activity:The ability of oxiding arsenite and reducing arsenate of each strain will be tested with the use of silverPhysiologic characterization:Temperaute optimum of growth will be achieved by incubing different cultures into different temperatures for 24 hours.pH optimum will be measured by incubing the cultures into pH between 2 and 13 for 24 hours.The growth is established by measuring the optical density of the culture in the spectrophotometer (UNICO, S-1200-E) at 600nm.Expected outcomesIt is expected that the strains used in this project must have a hightolerance/resistance to arsenic and a high efficiency when retainingarsenate or arsenite which has oxidized or reduced, respectively, in thecontaminated environment. They would be chosen to measure theconcentration of arsenite and arsenate [Fig. 7].Also the efficiency of each isolated bioremediate shall be comparedseparately or as a consortium [Fig.8]. Depending on the result they willbe used in one way or another.Fig. 7. Example of removal and uptake capacity A. ludwffii in presence of arsenate and arseniteions [5]. a b cReferences:1. Smedley, P. and Kinniburgh, D. A review of the source, behaviour and distribution of arsenic in natural waters, Applied Geochemistry. 17:517-56, 20022. Routt, J. and Roberts, J. Reconocimiento y manejo de los envenenamientos por pesticidas, Capitulo IV, 140-151, 19993. Consumer reports.org. Arsenic in your food, Consumer Reports magazine, 20124. Páez-Espino, D., Tamames, J., De Lorenzo, V., Cánovas, D.; Microbial responses to environmental arsenic, Biometals, 22:117–130, 20095. Banerjee, S., Datta, S., Chattyopadhyay, D., Sarkar, P.; Arsenic accumulating and transforming bacteria isolated from contaminated soil for potential use in bioremediation, Journal of Environmental Science and Health Part A, 46:1736–1747, 20116. Kinniburgh, D .i Smedley, P,, Arsenic contamination of groundwater in Bangladesh, British Geological Survey, Report WC/00/19., 20017. Sierra-Alvarez R., Yenal, U. Field J.A., Kopplin M., Gandolfi J.A., Garbarino J.R.; Anaerobic biotransformation of organoarsenical pesticides monomethylarsonic acid and dimethylarsinic acid, Journal Agricultural And Food Chemistry, 54:3959-3966, 20068. Mateos, L., Ordoñez, E., Letek, M., Gil, J.A.; Corynebacterium glutamicum as a model bacterium for the bioremediation of arsenic, International microbiology, 9:207-215, 20069. Rehman A., Butt S.A, Hasnain S. Isolation and characterization of arsenite oxidizing Pseudomonas lubricans and its potential use in bioremediation of wastewater, African Journal of Biotechnology, 9:1493:1498, 201010. Simeonova D., Lievremon D., Lagarde F., Muller D., Groudeva V., Lett M.C.; Microplate screening assay for detection of arseniteoxidizing and arsenate-reducing bacteria, FEMS Microbiology Letters, 237:249–253, 200411. Pelczar M. i Reid R. Pure cultures and growth characteristics, Microbiology, McGraw-Hill Book Company, 76–84, 195812. Héry, M., Gault, G., Rowland, A.L., Lear, G., Polya, D.A., Lloyd, J.R.; Molecular and cultivation-dependent analysis of metal-reducing bacteria implicated in arsenic mobilisation in south-east asian aquifers, Applied Geochemistry, 23:3215–3223, 200813. Gong, Z., Lu, X., Cullen, W.R., Le, X.C.; Unstable trivalent arsenic metabolites, monomethylarsonous acid and dimethylarsinous acid, Journal of Analytical Atomic Spectrometry, 16:1409-1413, 200114. Frankenberger W.T. , M.E. Losi, H.D. Skipper i R.F. Turco, Applications of Bioremediation in the Cleanup of Heavy Metals and Metalloids, 1995Fig. 5. Appearance of brownish precipitateafter the application of AgNO3 in agarplate supplemented with NaH2AsO3 inwhich P. lubricans was growing [9].nitrate (AgNO3). This test will be made in a YEM Agar (Yeast Agar Manitol). To this medium 1mM of arsenitefor the determination of the oxidation of arsenite and the same agar will be added, but adding 5mm ofarsenate, for fixing the reduction of arsenate [Fig. 5].The cultures will be incubated in 30ºC with 0,1M d’AgNO3 for 7 days and the reaction, that is a chromogenicprecipitate, will be measured in visu. The brown precipitate means that Ag3AsO4 is present in the medium,and therefore the strain has oxidase arsenite. In the same medium (without the arsenite), 5mm of arsenatewill summed for fixing the reduction of arsenate [10].Fig. 6. Microbial interactions with As and impact on As(III) release in the groundwater [12]. Evaluation of bioremediation:Bioremediative potential will be determined by measuring absence/presence of arsenite or arsenatein water samples that have been taken and treated before.300 ml will be taken from the different axenic cultures, and inoculated separately into 1L of differentsamples of environmental water; this culture will be maintained in an optimum pH and temperatureand in a constant agitation [11].After measuring separately the oxidative or reductive activity of the strains, this activity will beanalysed as a consortia [Fig. 6]. To analyse the mentioned activity, a 100 mL of the axenic culture shallbe taken and inoculated to a sole sample of environmental water. This culture will be maintained inthe before mentioned pH and temperature parameters and in a constant agitation.The initial and final concentration of arsenite/arsenate in the medium will be measured with a HPLC-ICP-MS (7500 Series, Agilent Technologies) [11] and the bacterial growth will be measured with theoptical density by spectrophotometer (UNICO, S-1200-E) in 600nm. The duplication time and theconstant rating of growth (k) during the logarithmic phase (of the growth curve) will be established byrepresenting graphically [13].Fig. 8. Example of a bioreactor for arsenicelimination [14]. Dissemination planThe dissemination plan would follow these processes: first carry out aprocess of publication in various journals of the bioremediation field ofmicrobes which have a high impact index such as EnviromentalMicrobiology, FEMS Microbiology reviews, Advances in AppliedMicrobiology or Journal of Hazardous Materials.In addition the project will be presented at international conferences suchas FEMS (Federation of European Microbiological Societies) or national ofthe SEM ((Sociedad Española de Microbiología)

Bioremediation of irrigation water contaminated with arsenical pesticides

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Bioremediation of irrigation water contaminated with arsenical pesticides

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