Nanoscale zerovalent iron (nZVI) impact on soil microbial communities

Lead Research Organisation: University of Reading
Department Name: Geography and Environmental Sciences


The release of particles of nanometre scale to the environment for the clean up of pollution is an emerging technology. At the forefront is the development of nanoscale zerovalent iron (nZVI) to clean up environments that have become polluted with chlorinated organic compounds. The nZVI works by removal of chlorine atoms from the pollutant, usually resulting in the production of a less toxic and less persistent compound. nZVI has been most commonly used for clean up of ground water, however, application to contaminated soil is now showing promise with the development of ways to deploy nZVI into the soil matrix without it sticking to the soil, or itself. Because of their dimensions, nZVI particles have the potential to interact at a scale relevant to microbes living in the soil, either directly through contact with microbial cells, or, indirectly through altering the chemical environment in which soil microbes live. It is important to assess whether nZVI has detrimental impacts on soil microbes because of the important functions the microbes carry out; these include biogeochemical cycling with resulting provision of nutrients to plants and the breakdown of organic pollutants. Thus, this proposal will test the impacts of nZVI on two important microbial groups; one involved in the decomposition of a major class of pollutants, the polychlorinated biphenyls (PCBs), and the other responsible for symbiotic improvement of plant growth and thus for enhancing and stabilising soils. The context of the study will be a two-step soil clean up scenario. The first step involves the use of nZVI to reduce the number of chlorines in the PCBs, which reduces their toxicity. The second, biological, step involves use of native soil microbes to decompose the nZVI-produced mono or dechlorinated products with subsequent planting of the soil to stabilise the site and stimulate beneficial pollutant decomposing microbes. For laboratory experiments, we will use commercially produced nZVI of different sizes and formulation. In consequence, we will address nZVI commercial applications whilst defining the environmental impact for a range of nZVI reactivities. In the course of our research we will answer the following questions: 1. Does nZVI impact on the numbers, activity and diversity of soil microbial communities responsible for the breakdown of pollutants? 2. Does nZVI impact on plant-microbe symbioses? Specifically: (i) legume symbiosis with symbiotic rhizobia bacteria responsible for formation of nitrogen-fixing nodules on plant roots; and, (ii) symbiotic arbuscular mycorrhizal fungi which form a 'fungus root' and benefit the plant host in many ways, including improvement of phosphorus nutrition. 3. Does the nZVI impact on soil microbial communities depend on the reactivity of nZVI and its environmental behaviour? We will assess the diversity of microbial communities using a nucleic-acid based fingerprinting method and measure their activity by quantifying the rate at which they decompose an added pollutant chemical. The impact on symbiosis will be determined by: (i) counting numbers of root nodules and the rate at which they fix nitrogen; and, (ii) the extent of root colonisation by arbuscular mycorrhizal fungi and plant phosphorus levels. We will manipulate nZVI reactivity by varying the size of the particles (bigger surface area = higher reactivity) and formulation (addition of a surfactant to stop them from agglomerating). We will see how the nZVI particles behave in soil by using an electron microscope. Also, we will apply to use the synchrotron 'super-microscope', a new facility in Oxfordshire, to assess its potential to look at nZVI agglomeration in an undisturbed soil situation.
Description 1. Overview of the project aim and experiments conducted
The overall aim of the proposed research was to advance the understanding of nanoscale zerovalent iron (nZVI) impacts on microbial communities important for bioremediation of soils contaminated with chlorinated aromatic compounds. In order to achieve this aim, we carried out two experiments. The first examined the short-term (over 28 days) effects of nZVI (mean particle size = 12.5 ± 0.3 nm by TEM analysis) on soil chemistry, the number, activity and diversity of soil microorganisms involved in the decomposition of organic matter and specifically the biodegradation of chloroaromatic compounds. In this experiment, we made comparisons to ZVI in microscale (mZVI; particle size = 1500 nm, TEM). In the second experiment, we focussed on the effects of palladized (Pd, 5%) nZVI (particle size = 13.9 ± 6 nm) aged in soil for 28 days on chloroaromatic degradative microbes and the growth and symbioses of Trifolium pratense and Lolium perenne. In both experiments, soil was artificially contaminated with a polychlorinated biphenyl mix (aroclor 1242), the particles were delivered in a polyacrylic acid (PAA) dispersant and we characterized particle slurries using electron microscopy. Additionally we assessed the potential of Small Angle Neutron Scattering (SANS) to examine nZVI agglomeration in an undisturbed soil situation.

2. Significant achievements and outcomes as measured against specific objectives
Objective 1a. In the short term (over 28 days), neither nZVI nor mZVI (both added at a rate of 10 mg kg-1 soil) had a significant (p>0.05) impact on the global hydrolytic activity of soil enzymes (intracellular and extracellular) involved in the depolymerization of organic matter during decomposition (as assessed using a fluorescein diacetate hydrolysis assay). However, nZVI apparently stimulated soil dehydrogenase activity, although we question the validity of this assay for use with nZVI due to the potential alteration of the reactivity of the assay substrate by the nZVI particles. That standard ecotoxicology assays (such as dehydrogenase) may not be suitable for nZVI has implications for the design of a suitable framework for assessing the ecological effects of reactive engineered particles. Addition of nZVI to soil initially (days 0-4) depressed numbers of chloroaromatic degrading microbes but numbers recovered to levels of those in the PAA only control by the end of the 28 experiment. Although degrader numbers appeared to recover with time, their activity was significantly decreased by the particles, implying a direct or indirect effect of nZVI on the expression of the catabolic pathway. This finding has implications for the potential success of combined nZVI/microbial attenuation for site remediation. In contrast, aged Pd/nZVI had no effect on the number or activity of chloroaromatic degradative microbes in the developing Trifolium rhizosphere. The results from the use of DGGE to study impacts on microbial community structure are still under analysis but initial findings do suggest alterations in community composition as a result of nZVI addition.
Objective 1b. Aged Pd/nZVI had no effect (p>0.05) on growth of Trifolium but significantly (p<0.01) enhanced the growth of Lolium at all harvest times (25, 42, 63 & 84 days). We think that the growth response of Lolium was due to a Pd/nZVI impact on microbial soil nitrogen cycling; 28 days after nanoparticle addition nitrate-N concentrations were dramatically increased in the nZVI treatment (implying that nZVI stimulated N mineralization) and this increase in soil available N was reflected in the N content of plant tissues. The lack of a similar growth response in Trifolium may be explained by the greater use of symbiotic N2 fixation by plants in control soil to compensate for lower soil available N. The fact that Trifolium plants in Pd/nZVI soil had fewer nodules and obtained less N by fixation (as determined by 15N dilution) is supportive of this explanation. An interesting finding was that aroclor also significantly inhibited nodulation. Staining of roots for detection of the presence of mycorrhizal fungi revealed a colonization rate too low and uneven for statistical analysis of treatment effects.
Objective 1c. Consistent with the known chemistry of iron corrosion, addition of particles to soil produced an immediate and significant elevation in soil pH of between 1 and 1.5 units, a reduction in redox potential and a reduction in nitrate-N. However, as alluded to above, nitrate-N concentrations increased in the longer term, potentially due to a stimulation of N mineralization from microbial biomass killed by the nZVI treatment.
Objective 1d. PCB dechlorination patterns are currently under analysis.
Objective 2. Our experiences with electron microscopy were that the nZVI readily formed spherical or sub-spherical agglomerates of 1.9 ± 0.06 mm average diameter under sample preparation procedures for SEM, however, through TEM analysis of pipetted slurries we could visualise single particles. SANS analysis suggested that the average diameter of the nZVI within the soil was 36 nm; this is larger than the TEM-observed particle size (see section 1) implying some agglomeration of particles on addition to soil.
Exploitation Route Our findings contribute to the assessment of the environmental safety of nanomaterials when applied for the clean up of contaminated environments.
Sectors Environment