Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Study on Burkholderia sp: Arsenic Resistant Bacteria Isolated from Contaminated Soil

Aritri Laha*, Somnath Bhattacharyya, Sudip Sengupta, Kallol Bhattacharyya, Sanjoy GuhaRoy

Department of Botany, West Bengal State University, Barasat, Kolkata 700126, West Bengal, India

Abstract: The threat of arsenic (As) pollution being severe warrants opting for low cost microbial remediation strategies. Our present study of identifying suitable bacterial strain led to isolation of As-tolerant strain from the As contaminated soil of West Bengal, India. The isolated bacterial strain LAR-2 had a high MIC (minimum inhibitory concentration) towards As(V) (300.67 mM) and As(III) (31.3 mM) and transformed 78.4% of arsenite to arsenate under laboratory condition. Based on 16S rRNA homology the LAR-2 was identified as Burkholderia sp (MK634685) and emerged as the most potent strain for As decontamination.

Keywords: Arsenic (As), Burkholderia sp, minimum inhibitory concentration, phylogenetic tree

Received November 21, 2020; Revised December 25, 2020; Accepted January 08, 2021

Citation: Aritri Laha, Somnath Bhattacharyya, Sudip Sengupta, Kallol Bhattacharyya, Sanjoy GuhaRoy, Study on Burkholderia sp: Arsenic Resistant Bacteria Isolated from Contaminated Soil. Applied Ecology and Environmental Sciences, 9 (2). 144-148. January 2021. doi:10.12691/aees-9-2-4

1. Introduction

The eastern India (West Bengal mainly) and Bangladesh are severe sufferers of arsenic contamination owing to exposure of living system via water and food [1,2]. The use of arsenic or heavy metal enriched water in agricultural fields is the reason of its significant build-up in soil [3,4] and subsequent accumulation in standing crop [5]. As residues are generally found in the top layer of soil or surface soil and enter the plants easily because of its low volatility and low solubility [6]. As has both organic and inorganic forms [7] and in environment found as an oxyanion [8]. Several soil detoxification methods such as stabilization, containment, and solidification etc. have been offered for the in-situ remediation of contaminated soils, but the high-risk involvement and high cost of remediation [9] emphasizes the scope of bioremediation through novel As-tolerant microbes [10]. Bioremediation through novel As-tolerant microbes may be an easy, eco-friendly, low cost and emerging approach to mitigate arsenic contamination.

Soil microorganisms play a crucial role in arsenic geocycling [11]. As(V) is taken up by the chromosomal and plasmid ars genetic system into the cell and it gets reduced to As(III). These enzymatic systems influence speciation and mobility of As affecting its bioavailability [12]. These microbes can either oxidize arsenite to arsenate or reduce arsenate to arsenite. A group of soil microorganisms can volatilize arsenic from the soil [13,14]. Alcaligenes faecalis, Bordetella sp. and Rhodobacter sphaeroides are three most important arsenic volatilizing bacteria also previously reported.

In this backdrop, we have categorized the study and identified efficient As-resistant bacterium from contaminated soil, and accrue steps to contribute an accelerated remediation of arsenic polluted soils.

2. Materials and Methods

2.1. Collection and Analysis of Soil Samples

Soil samples (2 cm diameter, 10 cm depth) were collected aseptically from arsenic (As) contaminated rhizospheric zone of lentil in Chakdaha, West Bengal (23°05' N latitude and 88°54' E longitude), India, noted for As concentrations in the ground water above World Health Organization (WHO)-defined safe limit [15]. Atomic Absorption Spectrophotometer (AAS) was used to measure the total [16] and available As [17] of the soil samples.

2.2. Isolation of As-tolerant Bacteria

2 g of soil (taken from lentil rhizosphere separately) was suspended in 2 mL sterile distilled water, 1mL of each was re-suspended in Yeast Extract Mannitol (YEM) liquid medium separately in two conical flasks, spiked with 1 mM As(III) and 1 mM As(V). The total experimental set up was incubated for 48 hours at 30°C [18]. After incubation 2 ml culture was further inoculated in a YEM liquid medium. Procedure was repeated twice. Around 0.1 mL of As spiked culture was spread on a YEMA (Yeast Extract Mannitol Agar) solid medium plate to isolate the As tolerant bacteria.

2.3. MIC (Minimum Inhibitory Concentration) Value of the Bacteria

The MIC is the lowest concentration of As(V) or As(III) which entirely inhibits microbial growth and activity [19,20]. 1.0 mL of overnight bacterial culture was inoculated in two conical flasks containing 99.0 mL of YEM liquid medium, containing either As(III) (NaAsO₂; 1-50 mM) or As(V) (Na₂HAsO₄·7H₂O; 1-500 mM) separately and incubated at 30ºC with 48 hrs of shaking. The OD (optical density, measurement of microbial growth) of the bacterial cultures was detected using a UV-Vis spectrophotometer at 600 nm wavelength.

2.4. Oxidation and Reduction of Arsenic by the Isolates

Standard silver nitrate (AgNO₃) method was employed to screen As-oxidizing bacterial isolates [21]. The Arsenic-oxidizing ability of the bacterial strains was further confirmed using a modified micro plate technique [22].

2.5. Arsenite Oxidase Assay

The Arsenic tolerant bacterial isolates were grown in CDM medium spiked with 30 mM of arsenite. After centrifugation at 10000 rpm for 2 minutes late log-phase cells were collected. Then cells were washed by using 50 mM Tris-HCl buffer (pH 8.0) and suspended in 2 mL buffer having 0.5 mM phenyl methyl sulfonyl fluoride (PMSF) and lysozyme. Cell suspensions were chrono-sequentially incubated for 2 h and sonicated for disrupting the cellular membrane. Then after centrifugation at 10,000 rpm for 30 min cell debris was removed. Finally, the protein concentrations were measured by using bovine serum albumin (Sigma) as a standard [23]. The arsenite oxidase assay was performed using a method previously described [24].

2.6. Identification of the As Tolerant Bacteria

The As tolerant bacterial genomic DNA was isolated and PCR amplification for molecular identification by 16S rRNA gene sequence analysis was done [25]. The bacterial isolate was studied for Gram reaction, colony morphology and characterized for catalase, urease, and oxidase activities by standard protocols [26].

2.7. Scanning Electron Microscopic (SEM) Study

For the SEM study the bacterial cells were harvested first and then washed with sodium phosphate buffer (pH 7.4). A thin bacterial smear was prepared on a cover glass, heat fixed over a flame for 1-2 sec followed by fixation with 2.5% glutaraldehyde for 45 min [27]. The slides were then dehydrated with 50-90% of alcohol solutions and finally through absolute alcohol for 5 min each and observed under a 15 kV scanning electron microscope (HITACHI, S-530, SEM and ELKO Engineering).

2.8. Statistical Analysis

Statistical computations were performed using Microsoft Excel 2016 and SPSS version 23.0.

3. Results

3.1. Characterization of Experimental Site

The total As concentration and available As concentration of experimental rhizospheric soil of lentil were measured. Results revealed a considerable load of the total and available arsenic i.e. 17.2 ± 1.72 and 1.50 ± 0.27 mg kg⁻¹ respectively (presented as mean of three observations ± SD).

3.2. Isolation and Identification of the As Tolerant Bacteria

A few distinct colonies were found in the YEMA medium (Figure 1). From these a distinct colony was picked and allowed for further study.

Figure 1. Burkholderia sp. in congo red YEMA medium

The isolated strain of Burkholderia was found to be gram negative and rod shaped. This isolated strain was studied for phenotypic and biochemical studies and have been presented in Table 1. The bacterial isolate was tested for catalase, urease, citrate, oxidase, indole, MR, VP.

Table 1. Morphological and biochemical properties of the bacteria

Further employing 16S rRNA gene sequencing a phylogenetic tree was prepared (Figure 2). These identified bacterial isolates thus assumed to be Burkholderia sp (LAR-2, accession number MK634685).

Figure 2. Phylogenetic tree based on partial 16S rRNA gene sequences of arsenic and heavy metal tolerant bacterial isolates from arsenic contaminated soil and other arsenic oxidising bacterial isolates from the database with accession numbers

Figure 3. SEM image of the LAR-2

Further to confirm the results, the SEM study of the bacterial isolates (Figure 3) was also categorized.

3.3. MIC of the Arsenic Resistant Bacterial Isolates

The arsenic tolerant strain was tested for their minimum inhibitory concentration of As. The result showed that LAR-2 (Burkholderia sp.) had a high MIC value (Table 2). Bacterial isolate had emerged with a high MIC towards As(V) (300.67 mM) and As(III) (31.3 mM). Further to establish the As oxidizing potential, silver nitrate test was executed (Figure 4), which also provided the rate of As(III) oxidation by the strain as 988 μM h⁻¹ and capacity of arsenite oxidation to the tune of 78.4%.

Table 2. Minimum Inhibitory Concentration and arsenite oxidase Enzyme activity of the arsenic tolerant bacteria

Figure 4. As oxidizing capability of LAR-2 (silver nitrate test)

4. Discussion

4.1. Identification and Isolation of As-resistant Bacterial Strains from Contaminated Soils

The As pollution in the contaminated experimental zone is geogenic. Soil pH, redox condition (Eh) and organic matter, which also effect on soil microbial populations, governs the degree of arsenic contamination and relative proportions of arsenic species [28]. The arsenic resistant bacteria are diverse and the prolonged contamination induced native microbial communities establish a surviving power to exist under metal stress condition [29]. In our study, the soil microclimate was predominated by heterotrophs [30]. The bulk of the As-resistant microbes were isolated from ground water [31], sediments [32], soil [33] etc. Diverse groups of bacteria including Bordetella [30], Achromobacter [34], Stenotrophomonas [33] etc. have been also found and reported. Arsenite oxidase system was also reported in Proteobacteria [35]. Burkholderia have also been previously identified as arsenite oxidizers [35].

The isolate, in the present investigation, were observed to show resistance to arsenite (up to 31.3 mM) and arsenate (up to 300.67 mM) which were significantly higher than As-oxidising bacteria isolated from ground water [36], soil [37] and estuaries [38].

The isolates under the present investigation, LAR-2 were identified as Burkholderia (based on 16S rRNA gene sequence homology) which can oxidize 988 μM h⁻¹ (78.4 %) presence of an arsenite concentrations (30 mM) within 24 h under laboratory condition. Bacillus megaterium was found to oxidise 1250 μM h⁻¹ of As(III) (100%) in the presence 30 mM As(III) in the culture media under laboratory conditions [25].

5. Conclusion

In pursuit of providing environmental safeguard to restore food safety and sustaining food security to burgeoning population and combat abiotic pollution, a low cost alternative to exorbitant pollution control strategies remained an absolute priority. The outcome of the present investigation revealed the candidate bacterial isolate Burkholderia sp. as a potent, novel bacterial strain for As mitigation and may be useful, through fulfilment of mass production and field validation protocols, in As decontamination.

Acknowledgements

The authors are grateful to the ICAR-Niche area of Excellence-As Research Laboratory, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Kalyani, Nadia as well as Department of Genetics & Plant Breeding and Department of Agronomy, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Mohanpur, Nadia, West Bengal, India for providing technical and all other necessary assistance during the study.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work.

References

[1]   | Chakraborti, D., Rahman, M.M., Mukherjee, A., Alauddin, M., Hassan, M., Dutta, R.N., Pati, S., Mukherjee, S.C., Roy, S., Quamruzzman, Q. and Rahman, M. 2015. Groundwater arsenic contamination in Bangladesh-21 Years of research. Journal of Trace Elements in Medicine and Biology, 31, 237-248.
[2]   | Bhattacharyya, K. and Sengupta, S. 2020. Arsenic management options in soil-plant-food chain. In: Proceedings of the National Webinar On Arsenic Mitigation: A Nexus Approach; (Prasad Bishun D., Mandal Jajati., Kumar Sunil., Sohane R K, Eds.), pp. 17-23.
[3]   | Abedin, M. J., Cotter-Howells, J., and Meharg, A. A. 2002. Arsenic uptake and accumulation in rice (Oryza sativa L.) irrigated with contaminated water. Plant and soil, 240(2), 311-319.
[4]   | Meharg, A. A., and Rahman, M. M. 2003. Arsenic contamination of Bangladesh paddy field soils: implications for rice contribution to arsenic consumption. Environmental science & technology, 37(2), 229-234.
[5]   | Bogdan, K. and Schenk, M.K. 2009. Evaluation of soil characteristics potentially affecting As concentration in paddy rice (Oryza sativa L.). Environmental Pollution,157, 2617-2621.
[6]   | Das, S., Jean, J. S., Kar, S., and Liu, C. C. 2013. Changes in bacterial community structure and abundance in agricultural soils under varying levels of As contamination. Geomicrobiology Journal, 30(7), 635-644.
[7]   | Matschullat, J. 2000. As in the geosphere - a review. The Science of the Total Environment, 249, 297-312.
[8]   | Frankenberger, W. T. Jr. and Arshad, M. 2002. Volatilization of As. In: Frankenberger, W.T. Jr. (Ed.). Environmental chemistry of As. Marcel Dekker, New York, USA. 363-380.
[9]   | Xiong, X., Liu, X., Iris, K.M., Wang, L., Zhou, J., Sun, X., Rinklebe, J., Shaheen, S.M., Ok, Y.S., Lin, Z. and Tsang, D.C. 2019. Potentially toxic elements in solid waste streams: Fate and management approaches. Environmental Pollution, 253, 680-707.
[10]   | Ghosh, P., Rathinasabapathi, B., and Ma, L. Q. 2011. As-resistant bacteria solubilized As in the growth media and increased growth of As hyperaccumulator Pteris vittataL. Bioresource technology, 102(19), 8756-8761.
[11]   | Mukhopadhyay, R., Rosen, B.P., Phung, L.T., Silver, S., 2002. Microbial arsenic: from geocycles to genes and enzymes. FEMS Microbiol Rev. 26, 311-325.
[12]   | Borch, T., Kretzschmar, R., Kappler, A., Cappellen, P.V., Ginder, V.A., Voegelin, A., Campbell, K., 2010. Biogeochemical redox processes and their impact on contaminant dynamics. Environ. Sci. Technol. 44, 15-23.
[13]   | Turpeinen, R.M., T., 2002. Role of microbes in controlling the speciation of arsenic and production of arsines in contaminated soils. Sci. Total Env. 285, 133-145.
[14]   | Mohapatra, D., Mishra, D., Roy Chaudhury, G., Das, R.P., 2008. Removal of arsenic from arsenic rich sludge by volatilization using anaerobic microorganisms treated with cow dung. Soil Sed. Cont. 17, 301-311.
[15]   | Ma, Y., Prasad, M.N.V., Rajkumar, M. and Freitas, H. 2011. Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnology advances, 29(2), 248-258.
[16]   | Rajkumar, M., Sandhya, S., Prasad, M. N. V., and Freitas, H. 2012. Perspectives of plant-associated microbes in heavy metal phytoremediation. Biotechnology advances, 30(6), 1562-1574.
[17]   | de-Bashan, L.E., Hernandez, J.P. and Bashan, Y. 2012. The potential contribution of plant growth-promoting bacteria to reduce environmental degradation- A comprehensive evaluation. Applied Soil Ecology, 61, 171-189.
[18]   | Ghosh, P.K., Maiti, T.K., Pramanik, K., Ghosh, S.K., Mitra, S. and De, T.K. 2018. The role of As resistant Bacillus aryabhattai MCC3374 in promotion of rice seedlings growth and alleviation of As phytotoxicity. Chemosphere, 211, 407-419.
[19]   | El-Meihy, R.M., Abou-Aly, H.E., Youssef, A.M., Tewfike, T.A., and El-Alkshar, E.A. 2019. Efficiency of heavy metals-tolerant plant growth promoting bacteria for alleviating heavy metals toxicity on sorghum. Environmental and Experimental Botany, 162, 295-301.
[20]   | Sarkar, S., Basu, B., Kundu, C.K. and Patra, P.K. 2012. Deficit irrigation: An option to mitigate As load of rice grain in West Bengal, India. Agriculture, ecosystems & environment, 146(1), 147-152.
[21]   | Lett, M.C., Paknikar, K., Liиvremont, D., Ciminelli, V.S.T., Garcia, O., 2011. Biohydrometallurgy - fundamentals, technology and sustainable development, part B, Elsevier Science, New York.
[22]   | Simeonova, D.D., , D., , F., , D.A.E., , V.I., , M.C., 2004. Microplate screening assay for the detection of arsenite-oxidizing and arsenate-reducing bacteria. FEMS Microbiol.Letters 237, 249-253.
[23]   | Bradford, M.A., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
[24]   | Anderson, G.L., Williams, J., Hille, R., 1992. The purification and characterization of arsenite oxidase from Alicaligenes faecalis, a molybdenum-containing hydroxylase. J. Biol. Chem. 267, 23674-23682.
[25]   | Majumder, A., Bhattacharyya, K., Bhattacharyya, S. and Kole, S.C. 2013. As-tolerant, As(III)-oxidising bacterial strains in the contaminated soils of West Bengal, India. Science of the total environment, 463, 1006-1014.
[26]   | Holtz, J.D. 1993. Bergey's manual of determinative bacteriology. 9th ed. Baltimore: Williams and Wilkins.
[27]   | Dey, U., Chatterjee, S., and Mondal, N. K. 2016. Isolation and characterization of As-resistant bacteria and possible application in bioremediation. Biotechnology reports, 10, 1-7.
[28]   | Lebrun, E., Brugna, M., Baymann, F., Muller, D., Li ́evremont, D., Lett, M.C., Nitschke, W., 2003. As(III) oxidase, an ancient bioenergetic enzyme. Mol. Biol. Evol. 20, 686-693.
[29]   | Oliveira, A., Pampulha, M.E., Neto, M.M., Almeida, A.C., 2009. Enumeration and characterization arsenic-tolerant diazotrophic bacteria in a long-term heavy-metal-contaminated soil. Water Air Soil Pollut. 200, of 237-243.
[30]   | Bachate, S.P., Cavalca, V., Andreoni, V., 2009. Arsenic-resistant bacteria isolated from agricultural soils of Bangladesh and characterization of arsenate-reducing strains. J. Appl. Microbiol. 107, 145-156.
[31]   | Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y., Wang, G., 2008. Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, Northwestern China. J. Appl. Microbiol. 105, 529-539.
[32]   | Valenzuela, C., Campos, V.L., Yanez, J., Zaror, C.A., Mondaca, M.A., 2009. Isolation of arsenite-oxidizing bacteria from arsenic-enriched sediments from Camarones River, Northern Chile. Bull. Environ. Contam. Toxicol. 82, 593-596.
[33]   | Bahar, M.M., Megharaj, M., Naidu, R., 2012. Arsenic bioremediation potential of a new arsenite-oxidizing bacterium Stenotrophomonas sp. MM-7 isolated from soil. Biodegradation 23, 1-10.
[34]   | Cai, L., Rensing, C., Li, X., Wang, G., 2009. Novel gene clusters involved in As(III) oxidation and resistancein two As(III) oxidizers: Achromobacter sp. SY8 and Pseudomonas sp. TS44. Appl. Microbiol. Biotechnol. 83, 715-725.
[35]   | Quéméneur, M., Heinrich-Salmeron, A., Muller, D., Lièvremont, D., Jauzein, M., Bertin, P.N., Garrido, F., Joulian, C., 2008. Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria. Appl. Environ. Microbiol. 74, 4567-4573.
[36]   | Liao, V.H., Chu, Y.J., Su, Y.C., Hsiao, S.Y., Wei, C.C., Liu, C.W., Liao, C.M., Shen, W.C., Chang, F.J., 2011. Arsenite-oxidizing and arsenate-reducing bacteria associated with arsenic-rich groundwater in Taiwan. J. Contam. Hydrol. 123, 20-29.
[37]   | Srivastava, P.K., Shenoy, B.D., Gupta, M., Vaish, A., Mannan, S., Singh, N., Tewari, S.K., Tripathi, R.D., 2012. Stimulatory effects of arsenic-tolerant soil fungi on plant growth promotion and soil properties. Microbes. Environ. 27, 477-482.
[38]   | Jackson, C.R., Harrison, K.G., Dugas, S.L., 2005. Enumeration and characterization of culturable arsenate resistant bacteria in a large estuary. Syst. Appl. Microbiol. 28, 727-734.