Impact of Aggregate-scale Heterogeneity of Transport and Biogeochemical Processes on Selenium Mobility in Soil

Transcription

1 Mission Kearney Foundation of Soil Science: Understanding and Managing Soil-Ecosystem Functions Across Spatial and Temporal Scales Final Report: , 1/1/ /31/2009 Impact of Aggregate-scale Heterogeneity of Transport and Biogeochemical Processes on Selenium Mobility in Soil Matteo Kausch, Juyoung Ha and Céline Pallud * Project Objectives Selenium (Se) plays a role of two-fold importance in the environment: Trace amounts are essential to animal and microorganism health, while toxicity rapidly ensues at elevated concentrations. Bioavailable Se oxyanions can be immobilized in nature by microbial reduction to solid, elemental Se. In structured soils, which are characterized by strong systematic heterogeneity at the mm-cm scale, mass transfer limitations arising from the existence of predominantly diffusive transport domains within soil aggregates are likely to affect Se reduction rates. Working with simplified experimental systems in conjunction with reactive transport modeling, we investigated the heterogeneity in microbial Se reduction within artificial soil aggregates and elucidated their driving factors. The overarching objective of this project was to determine how the combined effects of local variation in mass transfer and biogeochemical processes that operate from micron to cm scales govern the fate and transport of selenium in soils. The specific objectives that have been addressed so far are: i) to determine the aggregatescale physical and biogeochemical processes controlling the fate and transport of Se within soils; ii) to identify diffusional constraints on biogeochemical reactions of Se and associated mobility within soils; iii) to assess the differential impact of this aggregate scale heterogeneity on two model Se-reducing organisms, Enterobacter cloacae SLD1a-1 and Thauera selenatis; and iv) to develop a mechanistic reactive transport model that describes the spatial variation of microbial iron-reduction and secondary mineral formation observed in artificial aggregate systems analogous to those employed in this study (PALLUD et al., 2010b) and commence the adaptation of this model to the biogeochemistry of Se described here. Approach and procedures Variable factors investigated for their impact on Se-reduction Experiments were performed using (i) two Se-reducing bacteria (Enterobacter cloacae SLD1a-1 and Thauera selenatis) that have been extensively characterized in the literature and are known to differ with respect to their sensitivity to oxygen (LOSI and FRANKENBERGER, 1998; YEE and KOBAYASHI, 2008) and their enzymatic Se-reduction kinetics (RIDLEY et al., 2006; SCHRÖDER et al., 1997), (ii) under aerobic and anaerobic conditions to constrain the influence of oxygen on Se reduction, (iii) using different concentrations of organic carbon sources and dissolved selenate in the input solution, since they are limiting factors of Se reduction by these microbes (MA et al., 2007; RIDLEY et al., 2006), and (iv) in artificial aggregates composed of either pure sand (IOTA quartz sand, grain size of μm) or ferrihydrite-coated sand, since iron-oxides are 1 Environmental Science, Policy and Management Department, UC Berkeley *Principal Investigator For more information contact Dr. Céline Pallud (cpallud@berkeley.edu)

2 known to significantly impact the transport behavior of selenium oxyanions via sorption (BALISTRIERI and CHAO, 1990; DUC et al., 2003; SU and SUAREZ, 2000). Artificial aggregates and flow-through reactors Flow-through experiments were performed using newly developed systems consisting of an artificial spherical aggregate (2.5 cm ID) contained in a cylindrical reactor cell (5.1 cm ID, 3.7 cm long) and surrounded by free fluid flowing upwards through the reactor (Figure 1; PALLUD et al., 2010b). Flow rate and composition of the input solution were controlled and effluent solution composition was monitored over time. Aggregates were synthesized in the laboratory using the protocol of PALLUD et al. (2010b). Aggregates were composed of either pure quartz sand or ferrihydrite-coated sand and inoculated with either E. cloacae or T. selenatis. Before synthesizing the aggregates, the sand or ferrihydrite-coated sand was homogenously inoculated with one bacterial strain to a density of approximately 10 8 cells/g dry sand. Cell densities were measured at the start of experiments by performing dilution series and colony forming unit (CFU) plate counts. No carbon source was present in the aggregate at the time of synthesis. The inoculated sand or ferrihydrite-coated sand was molded using a spherical press (2.5 cm ID) and hydrogel agarose was used as a surrogate for natural polymers, which promote soil particle aggregation. A total of 150 g of 0.5 wt % agarose and 30 ml of bacterial suspension were homogenized with 300 g of sand. The constructed aggregates have an average pore diameter of approximately 39 m, an apparent dry bulk density of 1.19 g cm -3, a particle density of 2.83 g cm -3 and a porosity of 0.58 (PALLUD et al., 2010b). output channel L=3.7 cm =5.1 cm Aggregate =2.5 cm input channel Figure 1: Schematic drawing of the artificial aggregate-reactor system set-up. The input solution consisted of artificial groundwater medium (prepared in 10 mm PIPES with (mg/l): NaCl, 30; NH 4 Cl, 0.95; KCl, 5; MgSO 4, 50; KH 2 PO 4, and 1 ml/l mineral Wolfe s solution. ph was adjusted to 7.2. Pyruvate or acetate was added as the electron donor, for E. cloacae and T. selenatis, respectively, to a final concentration of 0.3 or 1.2 mm from a 2

3 filtered-sterile stock solution. Selenate was added to a final concentration of 0.25 or 0.50 mm. Input solution composition remained constant throughout the experiments. Constant flow rates between 0.6 and 1.4 ml/h were imposed at the reactor s lower boundary using a peristaltic pump and effluent samples were collected for 180 hours using a fraction collector for quantification of total dissolved Se and selenite concentration. At the conclusion of experiments aggregates were shaved into three concentric sections (core: radius 0-5 mm, mid-section: radius 5-9 mm, and exterior: radius mm, corresponding to relative volumes of 6.4%, 30.9% and 62.7%, respectively) for solid phase Se extractions. Solid phase Se-extraction and determination of Se concentrations and speciation In order to obtain information about the Se concentrations and speciation associated with the solid phase it was necessary to develop an extraction method that could yield quantitative recoveries from our matrices (agarose/sand and agarose/ferrihydrite-coated sand) while preserving Se speciation. A number of protocols from the literature (TOKUNAGA et al., 1991; ZHANG and FRANKENBERGER, 2003) and using various concentrations of HNO 3, extraction times, and temperatures were tested on quality control samples consisting of the same ferrihydritecoated sand/agarose mixture used to construct aggregates, but spiked through the addition of known concentrations of selenate and/or selenite which were added dissolved in di-h 2 O. Quality control samples were fully air-dried prior to extraction. Optimal results were obtained for 0.5 g of sample extracted in sealed Teflon vessels with 5 ml of 16 M HNO 3 on a rotary shaker at room temperature for h. The average recoveries obtained with this method for quality control samples ranged from 96% to 105% and are reported in Table 1. Method blanks were below the detection limit for both Se(IV) and total Se. Solid phase samples from the aerobic T. selenatis flow-through experiments were analyzed with the developed method. For the exterior and mid section of each aggregate triplicate extractions were performed on 0.5 g subsamples, after thorough mixing of the sample. A single extraction on a 0.5 g subsample was performed on core sections due to limited sample amount. Extracted samples were diluted 5-fold for analysis of total Se and selenite concentrations. Table 1: Recoveries for HNO 3 extraction of spiked quality control (QC) samples. Se concentration of spike in samples Average recovery standard deviation # of samples mol/g Se(IV) 96.7% 15.8% mol/g Se(IV) 104.9% 12.4% 2 5 mol/g Se(IV) 104.0% 12.8% 4 1 mol/g Se(total) 96.3% 7.2% 3 10 mol/g Se(total) 97.2% 4.8% 14 Effluent samples and solid phase extraction products from flow-though experiments were analyzed for total Se and selenite concentrations using a Perkin Elmer 5300 DV optical emission ICP. Total Se was determined using the ICP-OES operating in regular mode and selenite concentrations were measured via ICP-OES coupled to a hydride generation set-up developed for this project based on the phase separator set-up described by BOSNAK and DAVIDOWSKI (2004) and with flow rates as used by Brooks (BROOKS, 1991) in manifold 2. Due to a significant 3

4 memory effect a sample injection time of 120 seconds prior to optical emission reading and a rinse time of at least 30 seconds with an additional 100 seconds following samples of concentration 150 ppb or higher were used. Standards were prepared from certified reference stock in a matrix similar to that of samples (i.e. aqueous for flow-through samples and 3.2 M HNO 3 for extraction samples). Reactive transport modelling of aggregate-reactor system In order to interpret the temporally resolved solution data from outflow samples and discrete spatial data obtained through solid phase extraction at a single time point (experiment conclusion) within the context of heterogeneous concentration and reaction rate fields evolving through space and time, reactive transport modelling is necessary. Given the extensive data set available to our group on the microbial iron-reduction and secondary mineral formation observed in an analogous systems (PALLUD et al., 2010b), we developed a reactive transport model describing ironreduction in the artificial aggregate-reactor system (PALLUD et al., 2010a) while conducting the experimental work on Se-reduction here described in parallel. Based on this model we have developed a preliminary reactive transport model describing the biogeochemistry of Se in our system. Reactor geometry and fluid flow were modeled as described in Pallud et al. (2010a), the current model furthermore includes advection and diffusion for pyruvate, selenate, selenite and Se(0). Se reduction is modelled as a two-step process with selenate being reduced to selenite and selenite being reduced to Se(0), both steps requiring the concomitant oxidation of pyruvate (mol/mol). The cell specific reaction rates were determined based on local concentrations via Monod kinetics with maximal reduction rates and half-saturation constants for E. cloacae obtained from the literature (MA et al., 2007). Cell density was treated as a constant, adjustable parameter within the uncertainty range of CFU plate count values obtained at the beginning of the experiment. Results Impact of variable factors on average selenite export rates Given that Se in the input solution was in the form of selenate, selenite concentrations measured in samples collected at reactor outflow provide a proxy for bulk Se reduction rates. Average selenite export rates for each reactor were computed by averaging the concentrations in outflow samples across the time points at which systems had attained quasi-steady state (refer to Figure 5 for examples of characteristic shape of break-through curves) multiplying by the flow-rate at the time of sampling and normalizing by the aggregate dry mass. Concentrations were averaged across a time-span of at least 64 h, or eight consecutive 8 h samples to compute each average rate and its standard deviation. 4

5 average selenite export rates [nmol/h/g_aggregate] Impact of Aggregate-scale Heterogeneity of Transport and Biogeochemical Processes on SeO 4 2- [mm] C-source [mm] anaerobic aerobic anaerobic aerobic Enterobacter cloacae Thauera selenatis Figure 2: Average selenite export rates for flow-through experiments using aggregates consisting of pure quartz sand as a function of bacterial strain, anaerobic versus aerobic solution surrounding aggregates, and input concentrations of selenate and organic carbon source (pyruvate for E. cloacae and acetate for T. selenatis). Error bars represent ±1 standard deviation from the mean. No detectable selenite export was measured for experiments involving ferrihydrite-coated sand aggregates. The average selenite export for flow-through experiments using aggregates consisting of pure quartz sand are shown in Figure 2. Selenite export rates ranged between 0.01 and 3.4 nmol/h/g sand. Lower selenite export rates were consistently observed in response to lower input selenate or carbon concentrations. Rates were lower by a factor of approximately 5 and 25, for E. cloacae and T. selenatis respectively, under aerobic conditions than under anaerobic conditions. Selenite export rates were consistently observed under aerobic conditions, indicating that Se reduction is occurring, with higher rates for T. selenatis than for E. cloacae. Aggregate solid phase Se concentrations and speciation 5

6 Solid phase concentration [umol/g] Impact of Aggregate-scale Heterogeneity of Transport and Biogeochemical Processes on Currently solid phase data are available only for aerobic T. selenatis flow-through experiments (with both pure sand and ferrihydrite-coated sand). Total solid phase Se concentrations within the aggregate sections ranged between 0.1 and 0.6 μmol/g solid phase, selenite concentrations varied between zero and 0.4 μmol/g. Both measurements displayed large variability within aggregate sections as signified by the large standard deviations ( μmol/g) obtained from the triplicate extractions than both the measured values for solid phase total Se and selenite (Figure 3). Solid phase selenate concentrations were computed as the difference between total Se and selenite. These computed values had a much smaller variability (standard deviation between 0.01 and 0.2 μmol/g) (Figure 3) Sand: Lo selenate, Lo acetate Sand: Hi selenate, Lo acetate Sand: Lo selenate, Hi acetate Sand: Hi selenate, Hi acetate Ferri: Hi selenate, Lo acetate Ferri: Lo selenate, Hi acetate Ferri: Hi selenate, Hi acetate E: total Se E: Se(IV) E: total Se-Se(IV) Figure 3: Solid phase total Se concentration, Se(IV) concentrations and their difference in the exterior sections ( mm radius) of aggregates from aerobic T. selenatis flow-through experiments. Error bars represent ±1 standard deviation from the mean (extractions were performed in triplicates). Solid and empty bars represent experiments with pure quarts sand aggregates and ferrihydrite-coated sand aggregates, respectively. The four grey shades represent different concentration combinations of selenate (Lo: 0.25 mm, Hi: 0.5 mm) and acetate (Lo: 0.3 mm, Hi: 1.2 mm) in the input solution. Selenate solid phase concentrations where relatively homogenous throughout individual aggregates with no significant difference observed between aggregate sections (Figure 4-A). The overall solid phase selenium concentrations were proportional to the input selenate concentrations and were higher for aggregates composed of ferrihydrite-coated sand as compared to those composed of pure quartz sand: the average solid phase selenate concentrations were 0.08±0.01 μmol/g, 0.17±0.01 μmol/g, 0.15±0.01 μmol/g, and 0.32±0.02 μmol/g, for quartz aggregate with 0.25 mm, quartz aggregate with 0.5 mm, ferrihydrite aggregates with 0.25 mm and ferrihydrite aggregates with 0.5 mm selenate in the input solution, respectively. 6

7 Solid phase selenate concentration [umol/g] Solid phase selenite concentration [umol/g] Impact of Aggregate-scale Heterogeneity of Transport and Biogeochemical Processes on For solid phase selenite concentrations the large internal variability obscured any differences that may have been caused by investigated experimental factors and also the differences between exterior and mid sections of aggregates were not statistically significant. Core aggregate sections however consistently displayed solid phase selenite concentrations below the detection limit Sand: Lo selenate, Lo acetate Sand: Hi selenate, Lo acetate Sand: Lo selenate, Hi acetate Sand: Hi selenate, Hi acetate 0.6 Ferri: Hi selenate, Lo acetate Ferri: Lo selenate, Hi acetate Ferri: Hi selenate, Hi acetate Exterior E: total ( Se-Se(IV) mm) Mid M: section total Se-Se(IV) (5-9mm) C: Core total (0-5mm) Se-Se(IV) 0.0 Exterior E: ( Se(IV) mm) Mid section M: Se(IV) (5-9mm) Core C: Se(IV) (0-5mm) Figure 4: Solid phase (A) selenate concentrations (computed as difference between total Se and Se(IV) concentrations) and (B) selenite concentrations for the three concentric sections of aggregates from aerobic T. selenatis flow-through experiments. Error bars represent ±1 standard deviation from the mean (extractions were performed in triplicates). Solid and empty bars represent experiments with pure quarts sand aggregates and ferrihydrite-coated sand aggregates, respectively. The four grey shades represent different concentration combinations of selenate (Lo: 0.25 mm, Hi: 0.5 mm) and acetate (Lo: 0.3 mm, Hi: 1.2 mm) in the input solution. Discussion Impact of variable factors on average selenite export rates The decrease in selenite export rates for lower concentrations of selenate or of the carbon source in the input solution, observed for both E. cloacae and T. selenatis under aerobic as well as anaerobic conditions is consistent with Monod kinetics for rates of Se reduction. The preliminary reactive transport model parameterized with Monod constants for E. cloacae obtained from the literature (MA et al., 2007) matches the quasi steady-state in selenite export from the experimental breakthrough curves for the anaerobic quartz sand set-up (Figure 5), indicating that the impact of input concentrations on Se reduction rates in the aggregate-reactor system is adequately explained by Monod kinetics for the reaction in conjunction with transport of reactants as implemented in the model. No model expression for the impact of oxygen on Se reduction in our systems is presently available, however the decrease in reduction rates as evidenced by the lowered selenite export rates is in agreement with expectations, since Se-reduction by E. cloacae is known to decrease in the presence of oxygen (LOSI and FRANKENBERGER, 1998; YEE and KOBAYASHI, 2008). 7

8 Se reduction by T. selenatis is known to be inhibited in the presence of oxygen (LOSI and FRANKENBERGER, 1997; MACY et al., 1993). Thus, no detectable selenite export would have been expected in aerobic experiments if aggregates were oxygenated throughout. The observed selenite export is an indication for the presence of anaerobic zones within the aggregate in which Se reduction by T. selenatis can proceed despite the oxygenated solution surrounding the aggregate. This result highlights the importance of aggregate scale reactive transport dynamics on bulk reduction rates, since it exemplifies how a system in which no Se reduction would be expected based on bulk conditions can exhibit reduction due to aggregate scale transport limitations. Figure 5: Experimental breakthrough curves based on solution samples collected at the outflow of anaerobic reactors containing pure quartz sand aggregates inoculated with E. cloacae in comparison with modeled breakthrough curves. Reactive transport model and experimental data are shown for all three concentration combinations of selenate and pyruvate in the input solution (empty diamonds/dashed line: 0.25 mm selenate and 0.3 mm pyruvate, grey squares/grey line: 0.5 mm selenate and 0.3 mm pyruvate, black triangles/black line: 0.25 mm selenate and 1.2 mm pyruvate). Model parameterization between scenarios is consistent with cell density as only adjustable parameter. Intra-aggregate variability in solid phase Se concentrations Selenate solid phase concentrations for pure sand aggregates compare well to the amount expected from estimating the dissolved selenate content in the aggregates porosity, assuming that 8

9 the porosity was fully saturated with input solution by the conclusion of the experiment and that no selenate was lost during drying (0.2 μmol/g and 0.1 μmol/g, for aggregate saturated with 0.5 mm and 0.25 mm selenate, respectively). The elevated selenate concentrations in ferrihydritecoated sand aggregates (Figure 4-A) are an indication of surface excess in the presence of a ferrihydrite solid phase, which is in line with expectations since selenate is known to sorb to ferrihydrite, even forming inner sphere complexes (MANCEAU and CHARLET, 1994; SU and SUAREZ, 2000). The proportionality to the input selenate concentration indicates that the aggregates were fully saturated by experiment conclusion. This is not the case for solid phase selenite concentrations (Figure 4-B). There was no detectable selenite in the core sections of analyzed aggregates (T. selenatis, aerobic conditions), which might be an indication that there is no Se reduction taking place at the core of aggregates surrounded by aerobic solution. A possible explanation for this can be that acetate diffusing into the aggregate is consumed in the outer and mid sections of the aggregates. This is more likely in the presence of oxygen, since aerobic respiration (T. selenatis is capable of aerobic respiration (MACY and LAWSON, 1993)) would represent a large additional acetate sink. Aerobic respiration can also explain the formation of anaerobic zones within aggregates surrounded by oxygenated solution, evidenced by detectable selenite export in aerobic T. selenatis experiments (Figure 2). In conjunction with the large intra-section variability observed in selenite solid phase concentrations it can be conjectured that reduction is localized in microzones within the exterior and midsection of aggregates. Since aggregate section material was thoroughly mixed prior to taking the subsamples for extraction these microzones are expected to be significantly smaller than the grain size of sand used in aggregate construction (i.e. smaller than 100 μm). Future work To investigate the spatial variation in selenium reduction within our artificial aggregate systems under anaerobic experiments and in experiments involving E. cloacae we will continue performing solid phase extractions. Additionally, we will attempt to obtain direct evidence for the here postulated microzones of Se reduction in aggregates surrounded by oxygenated solution by making use of methods such as μ-xrf, as well as μ-xas and fluorescence mapping (Advanced Light Source beam time proposal has been resubmitted) that have the potential of resolving them directly. We will also continue work on the reactive transport model to include transport and reactions of oxygen as well as a mechanism of Se reduction inhibition for aerobic set-ups. Model parameterization will be improved and an alternative parameterization for T. selenatis developed. A series of batch experiments will be performed to this end. Sorption of selenite to the ferrihydrite phase will also be incorporated in future model versions. References Balistrieri, L. S. and T. T. Chao Adsorption of selenium by amorphous iron oxyhydroxide and manganese-dioxide. Geochimica et Cosmochimica. Acta. 54:

10 Bosnak, C. P. and L. Davidowski Continuous flow hydride generation using the Optima ICP Field Application Reports. Perkin Elmer Life and Analytical Sciences, Shelton, CT USA. Brooks, P. D Simplified, inexpensive, and easily constructed hydride generator for ICP analysis. Atomic Spectroscopy. 12: 1-3. Duc, M., G. Lefevre, F. Fedoroff, J. Jeanjean, J. C. Rouchaud, F. Monteil-Riviera, J. Dumonceau, and S. Milonjic Sorption of selenium anionic species on apatites and iron oxides from aqueous solutions. Journal of Environmental Radioactivity. 70: Losi, M. E. and W. T. Frankenberger Reduction of selenium oxyanions by Enterobacter cloacae SLD1a-1: Isolation and growth of the bacterium and its expulsion of selenium particles. Applied and Environmental Microbiology. 63: Losi, M. E. and W. T. Frankenberger Reduction of selenium oxyanions by Enterobacter cloacae strain SLD1a-1. In: Environmental Chemistry of Selenium (ed. W. T. Frankenberger, and R. A. Engberg), pp: Marcel Dekker. Ma, J., D. Y. Kobayashi, and N. Yee Chemical kinetic and molecular genetic study of selenium oxyanion reduction by Enterobacter cloacae SLD1a-1. Environmental Science & Technology. 41: Macy, J. M. and S. Lawson Cell Yield (YM) of Thauera selenatis grown anaerobically with acetate plus selenate or nitrate. Archives of Microbiology. 160: Macy, J. M., S. Rech, G. Auling, M. Dorsch, E. Stackebrandt, and L. I. Sly Thaurea selenatis gen. nov. sp. nov., a member of the beta sub-class of Proteobacteria with a novel type of respiration. International Journal of Systematic Bacteriology. 43: Manceau, A. and L. Charlet The mechanism of selenate adsorption on goethite and hydrous ferric-oxide. Journal of Colloid and Interface Science. 168: Pallud, C., M. Kausch, S. Fendorf, and C. Meile. 2010a. Spatial patterns and modeling of reductive ferrihydrite transformation observed in artificial soil aggregates. Environmental Science & Technology. 44: Pallud, C., Y. Masue-Slowey, and S. Fendrof. 2010b. Aggregate-scale spatial heterogeneity in reductive transformation of ferrihydrite resulting from coupled biogeochemical and physical processes. Geochimica et Cosmochimica. Acta. 74: Ridley, H., C. A. Watts, D. J. Richardson, and C. S. Butler Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions. Applied and Environmental Microbiology. 72: Schröder, I., S. Rech, T. Krafft, and J. M. Macy Purification and characterization of the selenate reductase from Thauera selenatis. Journal of Biological Chemistry. 272: Su, C. M. and D. L. Suarez Selenate and selenite sorption on iron oxides: An infrared and electrophoretic study. Soil Science Society of America Journal. 64: Tokunaga, T. K., D. S. Lipton, S. M. Benson, A. W. Yee, J. M. Oldfather, E. C. Duckart, P. W. Johannis, and K. E. Halvorsen Soil selenium fractionation, depth profiles and time trends in a vegetated site at Kesterson Reservoir. Water Air and Soil Pollution. 57-8: Yee, N. and D. Y. Kobayashi Molecular genetics of selenate reduction by Enterobacter cloacae SLD1a-1. Advances in Applied Microbiology. 64:

11 Zhang, L. H. and W. T. Frankenberger Determination of selenium fractionation and speciation in wetland sediments by parallel extraction. International Journal of Environmental Analytical Chemistry. 83: This research was funded by the Kearney Foundation of Soil Science: Understanding and Managing Soil-Ecosystem Functions Across Spatial and Temporal Scales, Mission ( The Kearney Foundation is an endowed research program created to encourage and support research in the fields of soil, plant nutrition, and water science within the Division of Agriculture and Natural Resources of the University of California. 11