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Enginneered nanoparticles and microbial activity- Dinesh et al (2012)


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This presentation is based on our review paper ‘Engineered nanoparticles in the soil and their potential implications to microbial activity’, Geoderma, 2012, 173-174, 19-27 (

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Enginneered nanoparticles and microbial activity- Dinesh et al (2012)

  1. 1. Engineered nanoparticles in the soil and their potential implications to microbial activity R. Dinesh M. Anandaraj V. Srinivasan S. Hamza Indian Institute of Spices Research (Indian Council of Agricultural Research) Marikunnu PO., Calicut-673012 Kerala State, IndiaThis presentation is based on our review paper ‘Engineered nanoparticles in the soil and their potential implications tomicrobial activity’, Geoderma, 2012, 173-174, 19-27 (
  2. 2. NanotechnologyThe U.S. National Nanotechnology Initiative (NNI) has defined nanotechnology asthescience, engineering, and technology conducted at thenanoscale, which is about 1 to 100 nanometers (nm).Nanoscience and nanotechnology are the study and application of extremely smallthings and can be used across all the other science fields, such as chemistry,biology, physics, materials science, and engineering. Nanotechnology is not just anew field of science and engineering, but a new way of looking at and studying atthe nanoscale where unique phenomena enable novel applications(; accessed on 16 February 2012).A joint report by the British Royal Society and the Royal Academy of Engineeringsimilarly defined nanotechnology as “the design, characterization,production, and application of structures, devices and systemsby controlling shape and size at nanometer scale.
  3. 3. The schematic figure(a) depicts a logarithmic-length scaleshowing the size of a classicalnanomaterial (C60 fullerene) comparedwith various biological components(adapted from 17). Particles of varioussizes are drawn to scale.(b) Rat macrophage cells withinternalized rope-like bundles ofsingle-walled carbon nanotubes(SWCNT). For comparison,mitochondria are marked with arrows.Human macrophages are up to twotimes larger than their ratcounterparts.(c) Human lung carcinoma cells withevidence of internalization of ironoxide nanoparticles of ∼20 nm indiameter.Source: Shvedova et al. (2010) 3
  4. 4. [Source:]4
  5. 5. Types of NPsNanoparticle: A sub-classification of ultrafine particles with lengths intwo or three dimensions greater than 1 nanometer (nm) and smaller than about100 nm, and which may or may not exhibit size-related intensive properties.Natural nanoparticles: Particles with one or more dimensions at thenanoscale originating from natural processes, e.g. soil colloids.Incidental nanoparticles: Nanoparticles formed as a by-product of man-made or natural processes, e.g. welding, milling, grinding or combustion.Engineered nanoparticles (less frequently also “manufacturednanoparticles”): Nanoparticles manufactured to have specific properties or aspecific composition.
  6. 6. Types of ENPs • Fullerenes (grouping Buckminster fullerenes, CNTs, nanocones etc.) • Metal ENPs (e.g. elemental Ag, Au, Fe) • Oxides (or binary compounds when including carbides, nitrides etc.). E.g. TiO2, Fe oxides.[Source: Norwegian Pollution Control Authority. 2008] • Complex compounds (alloys, composites, nanofluids etc., consisting of two or more elements) e.g. Cobalt-zinc iron oxide. • Quantum dots (or q-dots) are binary or complex compounds often coated with a polymer. They are usually regarded apart due to unique use and composition. Q-dots are ENPs that exhibit size-dependent electronic or optical properties due to quantum confinement. E.g. cadmium-selenide (CdSe) which has light emission peaks that varies according to particle size; green for 3 nm diameter particles, red for 5 nm, etc. Used in electronics/experimental biology/medicine. • Organic polymers (dendrimers, polystyrene, etc.) Animations courtesy Dr S. Dr. Maruyama;
  7. 7. Properties of ENPs• ENPs have different optical, electrical, magnetic, chemical and mechanical properties from their bulk counterparts are that in this size-range (between 1-100 nm) quantum effects start to predominate and the surface-area-to-volume ratio (sa/vol) becomes very large.• The sa/vol of most materials increases gradually as their particles become smaller, which results in increased adsorption of the surrounding atoms and changes their properties and behavior.• Materials reduced to the nano-scale can suddenly show very different properties, compared to what they exhibit on the macro-scale, which enables unique applications.• For example, opaque substances become transparent (copper); stable materials become combustible (aluminum); inert materials become catalysts (platinum); insulators become conductors (silicon); solids turn into liquids at room temperature (gold) Source: Hristozov and Malsch (2009)
  8. 8. Source:
  9. 9. Research in nanotechnology has resulted in applications across a wide range ofareas like medical and pharmaceutical sectors, the development of new materials,personal care products, to applications in agriculture and food. Today, nanoscalematerials find use in a variety of different areas.According to the Project on Emerging Nanotechnologies (PEN) over 1,300manufacturer-identified, nanotechnology-enabled products have enteredthe commercial marketplace around the world and If the current trendcontinues, the number of products could reach 3,400 by 2020.(; accessed on 30 January2012)Because of the potential of this technology there has been a worldwide increase ininvestment in nanotechnology research and development (Guzman et al., 2006).The production of engineered nanoparticles (ENPs) was estimated to be2000 tons in 2004 and is expected to increase to 58,000 tons in 2011-2020 (Maynard, 2006). 9
  10. 10. ENPs and microorganismsThey may have an impact on soil microorganisms via(1) a direct effect (toxicity)(2) changes in the bioavailability of toxins or nutrients(3) indirect effects resulting from their interaction with natural organic compounds and(4) interaction with toxic organic compounds which would amplify or alleviate their toxicityWhile toxicity mechanisms have not yet been completely elucidated for most ENPs, possiblemechanisms include:• disruption of membranes or membrane potential• oxidation of proteins• genotoxicity• interruption of energy transduction• formation of reactive oxygen species (ROS) and• release of toxic constituents [Source: Simonet and Valcárcel (2009); Klaine et al. (2008)] This is a magnification of E. coli exposed to a low concentration (10 mg/L) of titanium dioxide nanoparticles. Cells with compromised membranes are stained red. [ rse.environmental.effects]
  11. 11. Fig. Possible mechanisms of nanomaterials toxicity to bacteriaSource: Klaine et al. (2008) 11
  12. 12. Source: Lellouche et al. (2009) Scanning electron microscope analysis of normal and MgF2·Nps treated cells. (a) E. coli and (b) S.aureus untreated cells after overnight growth; (c) E. coli and (d) S. aureus treated with MgF2·Nps (1 mg/ml).Transmitted electron microscopy of MgF2·Nps treated and untreatedcells. TEM micrographs of E. coli and S. aureus thin sections:Untreated E. coli (a,b) and S. aureus (c,d); MgF2·Nps (1 mg/ml)treated E. coli (e,f) and S. aureus (g,h). Arrows indicate MgF2 12nanoparticles.
  13. 13. Antimicrobial activity of carbon based ENPs• C60 was harmful or has neutral biological consequences.• C60 aggregates inhibited Escherichia coli and Bacillus subtilis.• Fullerene water suspensions (FWS) exhibited relatively strong antibacterial activity and were more toxic to B. subtilis.• FWS exerts ROS-independent oxidative stress in bacteria, with evidence of protein oxidation, changes in cell membrane potential, and interruption of cellular respiration.• Fullerenols (C60 (OH) 12, C60 (OH) 36.8H2O, and C60 (OH) 44.8H2O) have been found to be toxic to six kinds of bacteria and two kinds of fungi.• Carbon based ENPs like CNTs have been found to inactivate E. coli, Staphylococcus epidermis, beneficial soil microbes like P. aeruginosa, B. subtilis as well as diverse microbial communities of river and waste water effluent.The toxicity of C60 has been attributed to its ability to bind and deform the DNA stands,thereby interfering with DNA repair mechanisms. Source: Lyon et al. (2005) , Jia et al. (2005); Fortner et al. (2005); Lyon et al. (2008); Aoshima et al. (2009); Kang et al. (2009); Zhou et al. (2005)
  14. 14. Antimicrobial activity of metal and metal oxide ENPs• Microbial toxicity has been reported for metal NPs, like elemental Ag, Au, Fe; oxides like TiO2, Fe-oxides, Co-Zn-Fe oxide etc. These NPs raise serious environmental concerns because of their unique dissolution properties and electronic charges, in addition to their small sizes and large surface-to-mass ratios (Wang et al., 2010).• Ag NP is toxic to E. coli and Staphylococcus aureus and B. subtilis.• Even Ag NP biosynthesized by fungi showed potent activity against fungal and bacterial strains like Aspergillus niger, Staphylococcus sp., Bacillus sp. and E. coli.• E. coli and S. aureus and Pseudomonas putida were inhibited by ENPs of Ag, CuO and ZnO. [Source: Rai et al. (2009); Suresh et al. (2010); Jaidev and Narasimha (2010); Jones et al. (2008); Gajjar et al. (2009) ] Zn NP ZnO NP
  15. 15. • Even water suspensions of nanosized titanium dioxide (TiO2), silicon dioxide (SiO2), and zinc oxide (ZnO) were found to be harmful to varying degrees, with antibacterial activity increasing with particle concentration. Antibacterial activity generally increased from SiO2 to TiO2 to ZnO, and B. subtilis was most susceptible to their effects.• Electrospraying of NPs of NiO, CuO, or ZnO (20 nm, 20 μg, in 10 min) reduced the total number of living E. coli by more than 88%, 77% and 71%, respectively.• ZnO, Al2O3 and TiO2 NPs were toxic to the nematode Caenorhabditis elegans inhibiting growth especially the reproductive capability.• Likewise, oxides of Zn, Cu and Ti NPs have been reported to be toxic to the microalgae Pseudokirchneriella subcapitata.• Exposure of earthworm (Eisenia fetida) to ZnO NPs enhanced mortality with increasing concentrations of NPs (Li et al., 2011)Source: Adams et al. (2006); Aruoja et al. (2009); Wang et. al. (2010) 15
  16. 16. Jason Unrine’s UK research team mixed earthworms into artificial soil tainted with goldnanoparticles. After 28 days, Unrine’s team detected gold nanoparticles throughout theearthworm’s bodies, with the highest concentrations in their gut. Some of the exposedworms produced 90 percent fewer offspring. This study can serve as a model for howorganisms take up other kinds of nanoparticles. 16
  17. 17. Mechanisms of toxicity of metal ENPs• Pitting of the cell wall, dissipation of the proton motive force, and finally cell death (Choi et al., 2008).• Ag NP would also bind with bacterial DNA, and this might compromise the DNAs replication fidelity (Rai et al., 2009; Yang et al., 2009).• These metal oxide NPs may act as „Trojan-Horses‟, entering cells and releasing ions intracellularly (Limbach et al., 2007). FE-SEM images of captured E. coli using anti-E. coli antibody functionalized magnetic nanoparticles 4 (a), (b), (c), and (d) four different images at different places of the sample. [Source: Rastogi et al. (2011)] Figure 1. left panel: Silica NPs; right panel: E. coli cells with intrenalized Si NPs [source:]
  18. 18. Schematic diagram of colloidal Ag nanoparticles interaction on captured E. coli cell with NPs 4, over the periodof time and observed biomolecules [Source: Rastogi et al. (2011)] 18
  19. 19. Figure 2: Interaction of amorphous TiO2 nanoparticles with MDCK (Madin Darby canine kidney) culture cells. I h incubation. A – nanoparticles fill folds and invaginations of a cell; B - E – direct contact of the nanoparticles with cell plasma membrane; F – invagination of plasma membrane containing nanoparticles; G, H – nanoparticles in “coated pits”, clathrin particles are visible on the pit cytoplasmic surface; I, J – nanoparticles in endosomes. Arrows show empty “coated pits”. Ultrathin sections. Transmission electron microscopy.Source: Ryabchikova, E; Mazurkova, N; Shikina, N; Ismagilov, Z; “The Crystalline Forms Of Titanium DioxideNanoparticles Affect Their Interactions With Individual Cells”, JMedCBR 8, 27 October 2010[] 19
  20. 20. Effect on soil microorganisms• Plant growth promoting rhizobacteria (PGPR) like P. aeruginosa, P. putida, P. fluorescens, B. subtilis and soil N cycle bacteria viz., nitrifying bacteria and denitrifying bacteria have shown varying degrees of inhibition when exposed to ENPs in pure culture conditions or aqueous suspensions (Mishra and Kumar, 2009).• Metal oxide NPs of Cu (80 to 160 nm) showed antibacterial activity against plant growth promoting Klebsiella pneumoniae, P. aeruginosa, Salmonella paratyphi and Shigella strains (Mahapatra et al., 2008).• Iron and copper based NPs are presumed to react with peroxides present in the environment generating free radicals known to be highly toxic to microorganisms like P. aeruginosa (Saliba et al., 2006).
  21. 21. Bright-field micrographs of Anabaena CPB4337 exposed to increasing concentrations of ceria nanoparticles. Rodea-Palomares et al. Toxicol. Sci. 2011;119:135-145(A) Control Anabaena filaments.(B and C) Anabaena filaments exposed to 1 mg/l N10 for 48 h and 80 mg/l N10 for 72 h.(D, E, and F) Anabaena filaments exposed to 0.1 mg/l N25 for 72 h, 1 mg/l N25 for 24 h, and 80 mg/l for 24 h.(G and H) Anabaena filaments exposed to 0.01 mg/l N50 for 48 h and 50 mg/l N50 for 48 h.(I, J, and K) Anabaena filaments exposed to 1 mg/l N60 for 24 h, 50 mg/l N60 for 72 h, and 80 mg/l N60 for 24 h. Bars, 20 μm.P.S. Anabaena is a genus of filamentous cyanobacteria that exists as plankton. It is known for its nitrogen fixing abilities in submerged / paddy soils, and they form symbiotic relationships with certain plants, like Azolla)
  22. 22. Published literature on the effects of ENPs on soil microorganisms ENP Effects Source Carbon containing No inhibition in the activity of dehydrogenase and activities of enzymes Tong et al. fullerenes/ CNTs involved in N (urease), P (acid-phosphatase) and C (β-glucosidase) cycles in (2007) the soil. A slight shift in bacterial DNA, indicating a minor change in the community structure measured using PCR-DGGE. (Incubation study) Number of fast-growing bacteria decreased by three-to four folds Johansen et immediately after incorporation of the C60 and protozoan number decreased al. (2007) only slightly in the beginning of the experiment. A slight shift in bacterial DNA, indicating a minor change in the community structure measured using PCR- DGGE. (Incubation study) No significant effect on the anaerobic community of biosolids from Nyberg et al. anaerobic wastewater treatment sludge over an exposure period of a few (2008) months. No change in methanogenesis and no evidence of substantial microbial community shifts due to treatment with C60. (Microcosm study) Multi-walled CNT significantly inhibited the activities of 1,4-β- Chung et al. glucosidase, cellobiohydrolase, xylosidase, 1,4-β-N-acetylglucosaminidase, (2011) phosphatase and microbial biomass-C and -N in soils. (Incubation study)Source: Dinesh et al. (2012)
  23. 23. Published literature on the effects of ENPs on soil microorganisms ENP Effects Source Metal and Effect of Ag-NP on soil dehydrogenase activity was severe and bacterial Murata et al. metal colony growth was inhibited at levels between 0.1 and 0.5 mg Ag kg-1 soil. (2005) oxide (Incubation study) ENPs Ag-NP inhibited soil denitrifying bacteria when Ag was added to soils in Throbäck et. amounts ranging from 0.003 to 100 mg kg-1 dry weight. (Incubation study) al.(2007) Soil respiration studies show that there were no statistical differences Doshi et al. (2008) between the time and sizes of peaks in CO2 production and the total mineralization of glucose due to addition of nano-Al. (Column study using silica-sand mixture) The influence of Si-, Pd-, Au- and Cu-NPs on microbial communities was Shah & Belozerova insignificant. (Microcosm study) (2009) Ag-NP did not influence microbial biomass-N, enzyme activities, soil pH Hänsch and and organic C. Microbial biomass was significantly decreased while basal Emmerling (2010) respiration and metabolic quotient was increased with increasing Ag-NP application rate. (Incubation study)(Source: Dinesh et al. (2012)
  24. 24. Published literature on the effects of ENPs on soil microorganisms ENP Effects Source Metal and TiO2- and ZnO-NPs reduced both microbial biomass and bacterial Ge et al. (2011) metal oxide diversity and composition indicating that nanoparticulate metal oxides ENPs may measurably and negatively impact soil bacterial communities. (Microcosm study) TiO2- and ZnO-NPs significantly inhibited soil protease, catalase, and Du et al. (2011) peroxidase activities; urease activity was unaffected. (Field study) Zn- and ZnO-NPs inhibited the activities of dehydrogenase, β - Kim et al. (2011) glucosidase and acid phosphatase in soils. (Pot study) Ag-, Cu- and Si-NPs impacted arctic soil bacterial community; Ag-NPs Kumar et al. were highly toxic to a plant beneficial bacterium, Bradyrhizobium (2011) canariense. (Incubation study)(Source: Dinesh et al. (2012)
  25. 25. Fig. Key processes in soil relating to transformation and potential risk frommanufactured nano particles (MNPs; Source: Klaine et al. (2008) 1. Dissolution 2. Sorption/aggregation 3. Plant bioaccumulation 4. Invertebrate accumulation and toxicity 5. Microbial toxicity 6. Direct particle uptake/toxicity 7. Particle migration MNPs 6 2 3 4 1 Dissolved pool 7 5
  26. 26. Conclusions• The anti-microbial activity of metal NPs to soil microbial communities holds great significance.• Little information is available on how metal ENPs act in the soil matrices especially their adsorption to clay minerals, organic fractions, toxic substances, organic pollutants etc.• More information is needed on interaction of ENPs with soil components and more quantitative assessments of aggregation/ dispersion, adsorption/ desorption, precipitation/ dissolution, decomposition, and mobility of ENPs in the soil environment is essential.• Overall, it is apparent from the studies done in vitro that ENPs pose a potential hazard to microorganisms.• Studies done by incubating soils with ENPs, microcosm studies and pot experiments suggests that in most cases ENPs inhibit soil microbial activity.
  27. 27. • This underlines the fact that the effects of ENPs on microbial community in soils under field conditions is still in its infancy, the smothering effects of SOM and HA on ENPs are still being speculated and the bacterial self protection-mechanism on encountering ENPs in the soil matrix is yet to be extensively studied.• Considering that attempts are being made to employ some of these ENPs as carrier materials for smart delivery of chemical fertilizers and pesticides to crops, (DeRosa et al., 2010) it is imperative that we set specific standards for the manufacture, use, and disposal of ENPs.• Therefore, conclusive evidences need to be obtained to draw strong conclusions about the potential toxicity of ENPs to microbial activity under field conditions and herein lies one of the main challenges in environmental risk assessment of spreading ENPs.
  28. 28. the risks of NPs forHowever, before these advantages can come into play,the environment and crops have to be defined to ensure theirsustainable and beneficial application.Relevant ecotoxicological information on exposure and effects of NPs as a basisfor a comprehensive risk assessment is needed.(Source: Bucheli TD- Effects of NANOparticles on beneficial soil MIcrobes and CROPS(NANOMICROPS);; accessed on 28/2/20120)
  29. 29. An Engineered Nano Particle Risk Assessment (ENPRA) ApproachENPRA aims at developing and implementing a novel integrated approach for ENP Risk Assessment.This approach is based on the Exposure-Dose-Response Paradigm for ENP (see figure below).• This paradigm states that exposure to ENP of different physico-chemical characteristics via inhalation, ingestion or dermal exposure is likely to lead to their distribution, beyond the portal-of- entry organ to other body systems.• The cumulative dose in a target organ will eventually lead to an adverse response in a dose- response manner. The Exposure-Dose-Response paradigm [Source:] 29
  30. 30. The approach proposed by ENPRA is in line with the grand challenges described by Maynard et al. (2006). The rationale of ENPRA is summarised graphically in the below figure .[Source:; accessed on 29/02/2012] 30
  31. 31. Nanotechnology has not yet been proven to be safe for humans or for the environment.Oxford University and Montreal University linked titanium dioxide and zinc oxide nanoparticles insunscreen to causing free radical and DNA damage in skin. And numerous other studies havefound that nanoparticles are easily absorbed by cells, where they cause other untold harm within thebody.Nanoparticles have been found to cause brain damage in fish and other aquatic speciesexposed to them.ENPs have been found to be toxic to microorganisms.Possible toxicity mechanisms include• disruption of membranes or membrane potential• oxidation of proteins,• genotoxicity,• interruption of energy transduction Quantum dots within D. magnia• formation of reactive oxygen species and [Source:]• release of toxic constituents• structural changes to the microbial cell surface that may eventually lead to cell death.
  32. 32. The key question therefore is how to benefit from nanotechnologies whilelimiting these risks?One suggestion is to ‘take into account the ethical, health, environmental andregulatory considerations linked to nanotechnologies as early on as possible inthe R&D phase, and to encourage dialogue with the public’.The aim is to ‘spark debate early on among all those concerned so as to avoidrepeating the mistakes made with genetically modified organisms (GMOs)where the public were reluctant to accept anything mildly related to GMOs’ (StephenSchaller, EU funded Nanologue project).(Source: ‘Dissecting the pros and cons of nanotechnologies‟ at accessed on 28/2/2012)Further reading : Dinesh, R., Anandaraj, M., Srinivasan, V., Hamza, S., 2012. Engineered nanoparticles in the soil andtheir potential implications to microbial activity. Geoderma 173-174, 19-27.
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