Biostimulation Experiments Research Articles

Selection of reference genes for expression profiling in biostimulation research of soybean

BackgroundPlant biostimulants constitute a promising environmentally friendly alternative for increasing crop yield and tolerance to unfavorable conditions. Among various types of such formulations, botanical extracts are gaining more recognition as products supporting plant performance. Moreover, novel tools such as cold-plasma or low-pressure microwave plasma discharge are being proposed as techniques that might improve their efficacy. Elucidation of the biostimulant’s mode of action requires complex research at a molecular level. Transcriptional changes occurring after biostimulant spraying might be investigated using RT-qPCR. However, this technique requires data normalization against stable endogenous controls.ResultsHere, we tested the expression stability of ten candidate genes in soybean plants exposed to various biostimulants treatment. Selection of the best-performing reference genes was conducted using four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). According to the obtained results, Bic-C2 (RNA-binding protein Bicaudal-C) and CYP (cyclophilin type peptidyl-prolyl cis–trans isomerase) showed highest expression stability, while expression of EF1B (elongation factor 1-beta) fluctuated the most among a tested set of candidate genes.ConclusionsOverall, we recommend using Bic-C2 together with CYP for the RT-qPCR data normalization in soybean biostimulation experiments. To our best knowledge, this is the first comprehensive study of reference genes stability in plants subjected to biostimulant treatment. The results of this study will aid in further biostimulant research in crop plants, facilitating analyses performed on the transcriptional level.Graphical

Response of petroleum-contaminated soil to chemical oxidation combined with biostimulation

In this study, a microcosm experiment was conducted to investigate the effects of Na2S2O8 preoxidation combined with biostimulation on petroleum-contaminated soil remediation. The response of microbial community during this process was explored using BIOLOG ECO microplate carbon utilization method and 16 s rDNA high-throughput sequencing. The results showed that use of 10 mg/g Na2S2O8 removed 19.8 % of the petroleum hydrocarbons, reduced soil biotoxicity and did not affect soil microbial activity compared to other concentrations. Therefore, sodium persulfate of ca. 10 mg/g was used to oxidize petroleum in soil before the biostimulation experiment with organic and inorganic fertilizers. Our finding showed that the content of total petroleum hydrocarbons (TPHs) in soil was reduced by 43.3 % in inorganic fertilizer treatment after 60 days. The results of BIOLOG ECO microplate carbon utilization analysis and 16 S rDNA high-throughput sequencing further confirmed that biostimulation quickly restored the microbial activities in oxidant treated soil. The main marker bacteria in chemical oxidation combined with biostimulation remediation were Arthrobacter and Paenarthrobacter, and their relative abundances were both significantly negatively correlated with the content of petroleum hydrocarbons in soil.

Cerium oxide and neodymium oxide phytoextraction by ryegrass in bioenhanced hydroponic environments

Sustainable technologies for the recovery of rare earth elements (REE) from waste need to be developed to decrease the volume of ore mining extractions and its negative environmental consequences, while simultaneously restoring previously impacted lands. This is critical due to the extensive application of REE in everyday life from electronic devices to energy and medical technologies, and the dispersed distribution of REE resources in the world. REE recovery by plants has been previously studied but the feasibility of REE phytoextraction from a poorly soluble solid phase (i.e., nanoparticles) by different plant species has been rarely investigated. In this study, the effect of biostimulation and bioaugmentation on phytorecovery of REE nanoparticles (REE-NP) was investigated by exposing ryegrass seeds to REE-NP in hydroponic environments. This was studied in two sets of experiments: bioaugmentation (using CeO2 nanoparticles and Methylobacterium extorquens AM1 pure culture), and biostimulation (using CeO2 or Nd2O3 nanoparticles and endogenous microorganisms). Addition of M. extorquens AM1 in bioaugmentation experiment including 500 mg/L CeO2 nanoparticles could not promote the nanoparticles accumulation in both natural and surface-sterilized treatments. However, it enhanced the translocation of Ce from roots to shoots in sterile samples. Moreover, another REE-utilizing bacterium, Bacillus subtilis, was enriched more than M. extorquens in control samples (no M. extorquens AM1), and associated with 52% and 14% higher Ce extraction in both natural (165 μg/gdried-plant) and surface-sterilized samples (136 μg/gdried-plant), respectively; showing the superior effect of endogenous microorganisms’ enrichment over bioaugmentation in this experiment. In the biostimulation experiments, up to 705 μg/gdried-plant Ce and 19,641 μg/gdried-plant Nd could be extracted when 500 mg/L REE-NP were added. Furthermore, SEM-EDS analysis of the surface and longitudinal cross-sections of roots in Nd2O3 treatments confirmed surface and intracellular accumulation of Nd2O3-NP. These results demonstrate stimulation of endogenous microbial community can lead to an enhanced REE phytoaccumulation.

PCB decomposition promoted by sugarcane bagasse organic waste

This work aimed to explore the changes in the bacterial community composition of soil contaminated with high concentration of polychlorinated biphenyls (PCBs) treated by biostimulation with sugarcane bagasse. Non-sterilized soil samples were spiked with transformer oil contaminated with PCB 18, 52, 66, 87,101, 110, 153, 180, and 187. The biostimulation experiment was performed by mixing non-sterilized PCB-contaminated soil with sterilized sugarcane bagasse and incubating the mixture 90 d in darkness at 25 ± 1 °C. The results showed that percentage removal of 88 % was similar for all PCB congeners in biostimulated soil, independently of their degree of chlorination, and it was accompanied by a significant change in bacterial community composition. Relative abundance of the bacterial communities at the phylum level in biostimulation treatment showed they were conquered by Proteobacteria and Firmicutes, followed by Acidobacteria and Actinobacteria.Bacteriall diversity increased in the biostimulation system due to the enrichment in genera possessing PCB remediation capacity which demonstrated the boosting potential of sugarcane bagasse in soil with high concentration of PCBs.

Spectral Induced Polarization (SIP) of Denitrification‐Driven Microbial Activity in Column Experiments Packed With Calcareous Aquifer Sediments

AbstractSpectral Induced Polarization (SIP) has been suggested as a non‐invasive monitoring proxy for microbial processes. Under natural conditions, however, multiple and often coupled polarization processes co‐occur, impeding the interpretation of SIP signals. In this study, we analyze the sensitivity of SIP to microbially‐driven reactions under quasi‐natural conditions. We conducted flow‐through experiments in columns equipped with SIP electrodes and filled with natural calcareous, organic‐carbon‐rich aquifer sediment, in which heterotrophic denitrification was bio‐stimulated. Our results show that, even in the presence of parallel polarization processes in a natural sediment under field‐relevant geochemical conditions, SIP is sufficiently sensitive to microbially‐driven changes in electrical charge storage. Denitrification yielded an increase in imaginary conductivity of up to 3.1 (+140%) and the formation of a distinct peak between 1 and 10 Hz, that matched the timing of expected microbial activity predicted by a reactive transport model fitted to solute concentrations. A Cole‐Cole decomposition allowed separating the polarization contribution of microbial activity from that of cation exchange, thereby helping to locate microbial hotspots without the need for (bio)geochemical data to constrain the Cole‐Cole parameters. Our approach opens new avenues for the application of SIP as a rapid method to monitor a system's reactivity in situ. While in preceding studies the SIP signals of microbial activity in natural sediments were influenced by mineral precipitation/dissolution reactions, the imaginary conductivity changes measured in the biostimulation experiments presented here were dominated by changes in the polarization of the bacterial cells rather than a reaction‐induced alteration of the abiotic matrix.

Biostimulation is a valuable tool to assess pesticide biodegradation capacity of groundwater microorganisms

Groundwater is the main source for drinking water production globally. Groundwater unfortunately can contain micropollutants (MPs) such as pesticides and/or pesticide metabolites. Biological remediation of MPs in groundwater requires an understanding of natural biodegradation capacity and the conditions required to stimulate biodegradation activity. Thus, biostimulation experiments are a valuable tool to assess pesticide biodegradation capacity of field microorganisms. To this end, groundwater samples were collected at a drinking water abstraction aquifer at two locations, five different depths. Biodegradation of the MPs BAM, MCPP and 2,4-D was assessed in microcosms with groundwater samples, either without amendment, or amended with electron acceptor (nitrate or oxygen) and/or carbon substrate (dissolved organic carbon (DOC)). Oxygen + DOC was the most successful amendment resulting in complete biodegradation of 2,4-D in all microcosms after 42 days. DOC was most likely used as a growth substrate that enhanced co-metabolic 2,4-D degradation with oxygen as electron acceptor. Different biodegradation rates were observed per groundwater sample. Overall, microorganisms from the shallow aquifer had faster biodegradation rates than those from the deep aquifer. Higher microbial activity was also observed in terms of CO2 production in the microcosms with shallow groundwater. Our results seem to indicate that shallow groundwater contains more active microorganisms, possibly due to their exposure to higher concentrations of both DOC and MPs. Understanding field biodegradation capacity is a key step towards developing further bioremediation-based technologies. Our results show that biostimulation has real potential as a technology for remediating MPs in aquifers in order to ensure safe drinking production.

Enhancing biogenic methane generation in coalbed methane reservoirs – Core flooding experiments on coals at in-situ conditions

Stimulation of microbial methanogenesis on intact coal under in-situ conditions is demonstrated in a series of 18 anaerobic core flooding experiments. The experiments used core samples from producing coalbed methane fields in the Surat Basin and Bowen Basin, with formation water containing methanogenic microbial consortia sourced from within the matching basin. The formation water was amended with nutrients containing phosphorus and nitrogen before being used in core flooding experiments of between 6 and 20 weeks duration. Since the core floods used degassed coal core at reservoir pressure, gas generated during the core flood could be adsorbed and retained by the coal. Therefore the quantity of methane generated was determined at the end of the experiment by decreasing the pore pressure and measurement of the volume of gas desorbed from the core sample. The amount of methane generated, averaged on a per week basis, varied considerably across the different core floods but was up to 7.75 μmol methane per gram of coal per week, comparable to previous studies where biostimulation experiments were conducted on crushed coal particles. During the core floods, the nutrient concentration was measured periodically in inflow and outflow water samples and the rates of nutrient consumption determined. The concentration of methane dissolved in the water samples was also determined and used as an indicator during a core flood of the relative rate of methanogenesis. Plateaus in gas generation were observed in the majority of core floods despite constant nutrient supply, indicating nutrient availability is not the single limiting factor to sustained methanogenesis. Higher gas generating core floods displayed higher rates of utilisation of phosphorus, underlining its importance in stimulation of microbial activity, while nutrient utilisation and gas generation measured in the absence of continuous nutrient delivery suggests the possibility of injection and production from a single CBM well. Results demonstrate the ability to stimulate gas generation on intact coal under reservoir conditions, an important step in development of technology for increasing gas content in depleted or undersaturated CBM fields.

D-optimal Design Application to Study Enhanced Biostimulation of Used Motor Oil Contaminated Soil

The indiscriminate disposal of fresh and used motor oil into the environment (land and water) is a common practice in Nigeria and this practice has contributed to its soil contamination and makes up a greater percentage of polluted ground in the world. Over the years, the contamination of auto-mechanic workshops by used oil and fresh oil has been left uncared for, Soil contaminated with fresh and used motor oil creates a serious effect on plant tissues, soil components, and its microorganisms, human and other animal health. Optimization of a biostimulation process used in the treatment of used motor oil contaminated soil has been carried out. D-optimal design of response surface methodology with one numerical factor and a categorical factor at two levels was employed to generate 10 experimental runs for the biostimulation system. The input variables of the system were palm bunch ash (a stimulant) and pollution level. The two levels of the latter factor considered were 5% and 10 % (w/w) pollution level, while the response of the system was oil and grease (O&G) removal. The biostimulation experiments were carried out according to the RSM design, and the experimental data obtained were analyzed using the quadratic model. The results of the analyses showed that the developed quadratic model was able to represent the system with both input factors affecting the system significantly since p-values for the model was found to be less than 0.05. The results also showed that O&G removal was affected by both the PBA and the pollution level. It was also observed from the numerical optimisation carried out that 100 % and 83.36 % O&G removal was attained at 5 % and 10 % pollution level respectively after ten weeks of the study. The findings of this work revealed that palm bunch ash (PBA) can be used as a stimulant for the bioremediation of used motor oil contaminated soil. Thus, its application in the treatment of oil-contaminated soil is highly recommended.

Characterization of the Rhodococcus sp. MK1 strain and its pilot application for bioremediation of diesel oil-contaminated soil.

Petroleum hydrocarbons and derivatives are widespread contaminants in both aquifers and soil, their elimination is in the primary focus of environmental studies. Microorganisms are key components in biological removal of pollutants. Strains capable to utilize hydrocarbons usually appear at the contaminated sites, but their metabolic activities are often restricted by the lack of nutrients and/or they can only utilize one or two components of a mixture. We isolated a novel Rhodococcus sp. MK1 strain capable to degrade the components of diesel oil simultaneously. The draft genome of the strain was determined and besides the chromosome, the presence of one plasmid could be revealed. Numerous routes for oxidation of aliphatic and aromatic compounds were identified. The strain was tested in ex situ applications aiming to compare alternative solutions for microbial degradation of hydrocarbons. The results of bioaugmentation and biostimulation experiments clearly demonstrated that - in certain cases - the indigenous microbial community could be exploited for bioremediation of oil-contaminated soils. Biostimulation seems to be efficient for removal of aged contaminations at lower concentration range, whereas bioaugmentation is necessary for the treatment of freshly and highly polluted sites.

Bioremediation of contaminated marine sediments can enhance metal mobility due to changes of bacterial diversity

Bioremediation strategies applied to contaminated marine sediments can induce important changes in the mobility and bioavailability of metals with potential detrimental consequences on ecosystem health. In this study we investigated changes of bacterial abundance and diversity (by a combination of molecular fingerprinting and next generation sequencing analyses) during biostimulation experiments carried out on anoxic marine sediments characterized by high metal content. We provide evidence that the addition of organic (lactose and/or acetate) and/or inorganic compounds to contaminated sediments determines a significant increase of bacterial growth coupled with changes in bacterial diversity and assemblage composition. Experimental systems supplied only with organic substrates were characterized by an increase of the relative importance of sulfate reducing bacteria belonging to the families Desulfobacteraceae and Desulfobulbaceae with a concomitant decrease of taxa affiliated with Flavobacteriaceae. An opposite effect was observed in the experimental treatments supplied also with inorganic nutrients. The increase of bacterial metabolism coupled with the increase of bacterial taxa affiliated with Flavobacteriaceae were reflected in a significant decrease of Cd and Zn associated with sedimentary organic matter and Pb and As associated with the residual fraction of the sediment. However, independently from the experimental conditions investigated no dissolution of metals occurred, suggesting a role of bacterial assemblages in controlling metal solubilization processes. Overall results of this study have allowed to identify key biogeochemical interactions influencing the metal behavior and provide new insights for a better understanding of the potential consequences of bio-treatments on the metal fate in contaminated marine sediments.

Uranium bioreduction rates across scales: biogeochemical hot moments and hot spots during a biostimulation experiment at Rifle, Colorado.

We aim to understand the scale-dependent evolution of uranium bioreduction during a field experiment at a former uranium mill site near Rifle, Colorado. Acetate was injected to stimulate Fe-reducing bacteria (FeRB) and to immobilize aqueous U(VI) to insoluble U(IV). Bicarbonate was coinjected in half of the domain to mobilize sorbed U(VI). We used reactive transport modeling to integrate hydraulic and geochemical data and to quantify rates at the grid block (0.25 m) and experimental field scale (tens of meters). Although local rates varied by orders of magnitude in conjunction with biostimulation fronts propagating downstream, field-scale rates were dominated by those orders of magnitude higher rates at a few selected hot spots where Fe(III), U(VI), and FeRB were at their maxima in the vicinity of the injection wells. At particular locations, the hot moments with maximum rates negatively corresponded to their distance from the injection wells. Although bicarbonate injection enhanced local rates near the injection wells by a maximum of 39.4%, its effect at the field scale was limited to a maximum of 10.0%. We propose a rate-versus-measurement-length relationship (log R' = -0.63 log L - 2.20, with R' in μmol/mg cell protein/day and L in meters) for orders-of-magnitude estimation of uranium bioreduction rates across scales.

Mechanistic models of biofilm growth in porous media

AbstractNondestructive acoustics methods can be used to monitor in situ biofilm growth in porous media. In practice, however, acoustic methods remain underutilized due to the lack of models that can translate acoustic data into rock properties in the context of biofilm. In this paper we present mechanistic models of biofilm growth in porous media. The models are used to quantitatively interpret arrival times and amplitudes recorded in the 29 day long Davis et al. (2010) physical scale biostimulation experiment in terms of biofilm morphologies and saturation. The model pivots on addressing the sediment elastic behavior using the lower Hashin‐Shtrikman bounds for grain mixing and Gassmann substitution for fluid saturation. The time‐lapse P wave velocity (VP; a function of arrival times) is explained by a combination of two rock models (morphologies); “load bearing” which assumes the biofilm as an additional mineral in the rock matrix and “pore filling” which assumes the biofilm as an additional fluid phase in the pores. The time‐lapse attenuation (QP−1; a function of amplitudes), on the other hand, can be explained adequately in two ways; first, through squirt flow where energy is lost from relative motion between rock matrix and pore fluid, and second, through an empirical function of porosity (φ), permeability (κ), and grain size. The squirt flow model‐fitting results in higher internal φ (7% versus 5%) and more oblate pores (0.33 versus 0.67 aspect ratio) for the load‐bearing morphology versus the pore‐filling morphology. The empirical model‐fitting results in up to 10% increase in κ at the initial stages of the load‐bearing morphology. The two morphologies which exhibit distinct mechanical and hydraulic behavior could be a function of pore throat size. The biofilm mechanistic models developed in this study can be used for the interpretation of seismic data critical for the evaluation of biobarriers in bioremediation, microbial enhanced oil recovery, and CO2 sequestration.

Degradation kinetics of butyltin compounds during the bioremediation of contaminated harbour sediments

The present study was aimed at the reclamation of harbour sediments contaminated with butyltin compounds (BTCs) by means of biostimulation and/or bioaugmentation experiments carried out in a 5-L slurry bioreactor. In the biostimulation experiment with inorganic nutrients, almost 50% of TBT was degraded in 20 weeks. In the bioaugmentation experiment with a microbial consortium, no significant degradation of TBT was observed. Conversely, bioaugmentation combined with biostimulation led to a decrease of ∼50% in TBT after four weeks. A simple kinetic model fitted to the experimental data of BTC concentration allowed us to estimate that 10 weeks are needed to decrease BTC contamination by 90% using a strategy based on both biostimulation and bioaugmentation, and 29 weeks for a strategy using biostimulation only. Overall, our study indicates that strategies based on biostimulation coupled to bioaugmentation can be effective in significantly reducing the concentration of BTCs over relatively short timescales.

A large column analog experiment of stable isotope variations during reactive transport: I. A comprehensive model of sulfur cycling and δ34S fractionation

This study demonstrates a mechanistic incorporation of the stable isotopes of sulfur within the CrunchFlow reactive transport code to model the range of microbially-mediated redox processes affecting kinetic isotope fractionation. Previous numerical models of microbially mediated sulfate reduction using Monod-type rate expressions have lacked rigorous coupling of individual sulfur isotopologue rates, with the result that they cannot accurately simulate sulfur isotope fractionation over a wide range of substrate concentrations using a constant fractionation factor. Here, we derive a modified version of the dual-Monod or Michaelis–Menten formulation (Maggi and Riley, 2009, 2010) that successfully captures the behavior of the 32S and 34S isotopes over a broad range from high sulfate and organic carbon availability to substrate limitation using a constant fractionation factor. The new model developments are used to simulate a large-scale column study designed to replicate field scale conditions of an organic carbon (acetate) amended biostimulation experiment at the Old Rifle site in western Colorado. Results demonstrate an initial period of iron reduction that transitions to sulfate reduction, in agreement with field-scale behavior observed at the Old Rifle site. At the height of sulfate reduction, effluent sulfate concentrations decreased to 0.5mM from an influent value of 8.8mM over the 100cm flow path, and thus were enriched in sulfate δ34S from 6.3‰ to 39.5‰. The reactive transport model accurately reproduced the measured enrichment in δ34S of both the reactant (sulfate) and product (sulfide) species of the reduction reaction using a single fractionation factor of 0.987 obtained independently from field-scale measurements. The model also accurately simulated the accumulation and δ34S signature of solid phase elemental sulfur over the duration of the experiment, providing a new tool to predict the isotopic signatures associated with reduced mineral pools. To our knowledge, this is the first rigorous treatment of sulfur isotope fractionation subject to Monod kinetics in a mechanistic reactive transport model that considers the isotopic spatial distribution of both dissolved and solid phase sulfur species during microbially-mediated sulfate reduction.

Abiotic U(VI) reduction by sorbed Fe(II) on natural sediments

Laboratory experiments were performed as a function of aqueous Fe(II) concentration to determine the uptake and oxidation of Fe(II), and Fe(II)-mediated abiotic reduction of U(VI) by aquifer sediments from the DOE Rifle field research site in Colorado, USA. Mössbauer analysis of the sediments spiked with aqueous 57Fe(II) showed that 57Fe(II) was oxidized on the mineral surfaces to 57Fe(III) and most likely formed a nano-particulate Fe(III)-oxide or ferrihydrite-like phase. The extent of 57Fe oxidation decreased with increasing 57Fe(II) uptake, such that 98% was oxidized at 7.3 μmol/g Fe and 41% at 39.6 μmol/g Fe, indicating that the sediments had a limited capacity for oxidation of Fe(II). Abiotic U(VI) reduction was observed by XANES spectroscopy only when the Fe(II) uptake was greater than approximately 20 μmol/g and surface-bound Fe(II) was present, possibly as oligomeric Fe(II) surface species. The degree of U(VI) reduction increased with increasing Fe(II)-loading above this level to a maximum of 18% and 36% U(IV) at pH 7.2 (40.7 μmol/g Fe) and 8.3 (56.1 μmol/g Fe), respectively in the presence of 400 ppm CO2. Greater U(VI) reduction was observed in CO2-free systems [up to 44% and 54% at pH 7.2 (17.3 μmol/g Fe) and 8.3 (54.8 μmol/g Fe), respectively] compared to 400 ppm CO2 systems, presumably due to differences in aqueous U(VI) speciation. While pH affects the amount of Fe(II) uptake onto the solid phase, with greater Fe(II) uptake at higher pH, similar amounts of U(VI) reduction were observed at pH 7.2 and 8.3 for a similar Fe(II) uptake. Thus, it appears that abiotic U(VI) reduction is controlled primarily by sorbed Fe(II) concentration and aqueous U(VI) speciation. The range of Fe(II) loadings tested in this study are within the range observed in biostimulation experiments at the Rifle site, suggesting that Fe(II)-mediated abiotic U(VI) reduction could play a significant role in field settings.

Evaluation of a Genome-Scale In Silico Metabolic Model for Geobacter metallireducens by Using Proteomic Data from a Field Biostimulation Experiment

Accurately predicting the interactions between microbial metabolism and the physical subsurface environment is necessary to enhance subsurface energy development, soil and groundwater cleanup, and carbon management. This study was an initial attempt to confirm the metabolic functional roles within an in silico model using environmental proteomic data collected during field experiments. Shotgun global proteomics data collected during a subsurface biostimulation experiment were used to validate a genome-scale metabolic model of Geobacter metallireducens-specifically, the ability of the metabolic model to predict metal reduction, biomass yield, and growth rate under dynamic field conditions. The constraint-based in silico model of G. metallireducens relates an annotated genome sequence to the physiological functions with 697 reactions controlled by 747 enzyme-coding genes. Proteomic analysis showed that 180 of the 637 G. metallireducens proteins detected during the 2008 experiment were associated with specific metabolic reactions in the in silico model. When the field-calibrated Fe(III) terminal electron acceptor process reaction in a reactive transport model for the field experiments was replaced with the genome-scale model, the model predicted that the largest metabolic fluxes through the in silico model reactions generally correspond to the highest abundances of proteins that catalyze those reactions. Central metabolism predicted by the model agrees well with protein abundance profiles inferred from proteomic analysis. Model discrepancies with the proteomic data, such as the relatively low abundances of proteins associated with amino acid transport and metabolism, revealed pathways or flux constraints in the in silico model that could be updated to more accurately predict metabolic processes that occur in the subsurface environment.

Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth

Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L(2,3) absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)-Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.

Reductive Dechlorination of 2,3,4-Chlorobiphenyl by Biostimulation and Bioaugmentation

Abstract Dechlorination ability of indigenous microorganisms from seven stream sediments collected around Bangkok and its vicinity in Thailand was tested by amended with seven polychlorinated biphenyl congeners (PCBs) including 2,3,4-chlorobiphenyl (234-CBp), 22′5-CBp, 24′5-CBp, 22′35′-CBp, 22′45-CBp, 23′44′5-CBp, and 22′34′5′6-CBp. After 2–22 weeks of incubation, 234-CBp dechlorination could be initiated and dechlorinated to 24-CBp in all seven sediment slurry without any further enrichment, with dechlorination completed within 28 weeks. This is the first evidence indicating that 234-CBp dechlorination consortium actually existed in the natural streams of Thailand and could perform the 234-CBp dechlorination in the fresh sediment slurry without any nutrient amendment. The lag phase of 234-CBp was shortest in the sediment slurry collected and prepared from the Hua Lum Poo Canal (HLP), which was sporadically contaminated with hexachlorobenzene. In biostimulation experiments, HLP nutrient-strengthened sedim...

Physicochemical Heterogeneity Controls on Uranium Bioreduction Rates at the Field Scale

It has been demonstrated in laboratory systems that U(VI) can be reduced to immobile U(IV) by bacteria in natural environments. The ultimate efficacy of bioreduction at the field scale, however, is often challenging to quantify and depends on site characteristics. In this work, uranium bioreduction rates at the field scale are quantified, for the first time, using an integrated approach. The approach combines field data, inverse and forward hydrological and reactive transport modeling, and quantification of reduction rates at different spatial scales. The approach is used to explore the impact of local scale (tens of centimeters) parameters and processes on field scale (tens of meters) system responses to biostimulation treatments and the controls of physicochemical heterogeneity on bioreduction rates. Using the biostimulation experiments at the Department of Energy Old Rifle site, our results show that the spatial distribution of hydraulic conductivity and solid phase mineral (Fe(III)) play a critical role in determining the field-scale bioreduction rates. Due to the dependence on Fe-reducing bacteria, field-scale U(VI) bioreduction rates were found to be largely controlled by the abundance of Fe(III) minerals at the vicinity of the injection wells and by the presence of preferential flow paths connecting injection wells to down gradient Fe(III) abundant areas.

Applications and implications of direct groundwater velocity measurement at the centimetre scale

Three projects involving point velocity probes (PVPs) illustrate the advantages of direct groundwater velocity measurements. In the first, a glacial till and outwash aquifer was characterized using conventional methods and multilevel PVPs for designing a bioremediation program. The PVPs revealed a highly conductive zone that dominated the transport of injected substances. These findings were later confirmed with a natural gradient tracer test. In the second, PVPs were used to map a groundwater velocity field around a dipole recirculation well. The PVPs showed higher than expected velocities near the well, assuming homogeneity in the aquifer, leading to improved representations of the aquifer heterogeneity in a 3D flow model, and an improved match between the modelled and experimental tracer breakthrough curves. In the third study, PVPs detected subtle changes in aquifer permeability downgradient of a biostimulation experiment. The changes were apparently reversible once the oxygen source was depleted, but in locations where the oxygen source lingered, velocities remained low. PVPs can be a useful addition to the hydrogeologist's toolbox, because they can be constructed inexpensively, they provide data in support of models, and they can provide information on flow in unprecedented detail.