Biological Reduction of Perchlorate from Spent Modified Reed Mediated by an Enriched Microbial Culture

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Introduction
Quaternary amine-modifed reed (MR) can be used as an adsorbent to remove perchlorate rapidly from aqueous solutions through electrostatic interactions [1], but then perchlorate detoxifcation is needed to prevent environmental contamination.At high pH values, MR electrostatic interactions weaken, allowing perchlorate to desorb and leach into ground or surface water [2].Because reed is a grazing plant [3], consumption of perchlorate-laden MR by animals low on the food chain can lead to human exposure, possibly causing health efects [1].
Although adsorbed perchlorate can be destroyed by the incineration of spent MR, such an approach is not sustainable because it generates air pollution and has a high capital cost.Alternatively, it is possible to pursue a regeneration approach given that synthetic ion-exchange resins can be regenerated [4,5].However, MR regeneration is not economically feasible because MR is an agricultural byproduct and constitutes a waste stream if not fully utilized [6].Applying regeneration methods similar to those used for synthetic ion-exchange resins would produce brine with high perchlorate and salt concentrations, which would require further treatment before it could be disposed of [7,8].
Perchlorate-laden MR can be treated biologically.Certain species of bacteria can utilize perchlorate for respiration under anaerobic conditions; the end products of such reactions are chloride ions and oxygen [7].Most of these microorganisms are ubiquitous in the environment and have simple nutritional requirements [9].Anaerobic biodegradation of perchlorate is possible owing to its favorable electrochemical properties [10].Te perchlorate anion (ClO) is an ideal electron acceptor for microbial metabolism, in part because it contains chlorine in its highest oxidation state, at which all valence electrons have been lost [11].
Half-reactions that include perchlorate (ClO), chlorate (ClO), chlorite (ClO), and chloride (Cl − ) can be combined to derive the Gibbs free energy associated with the reduction of perchlorate to chloride as follows: Although the above reaction is thermodynamically favorable [12], it is not kinetically spontaneous under ambient conditions.For the forward reaction to proceed, a kinetic barrier has to be overcome with energy or an electron source [13].Te biological reduction of perchlorate is kinetically favorable when a suitable electron donor and carbon source are supplied to sustain bacterial respiration and growth [14].
Brewer's yeast (Saccharomyces cerevisiae, herein referred to as yeast) can be used for perchlorate degradation.Yeast has a lower cost and greater perchlorate reduction activity (mg-perchlorate per mg-electron donor) than acetate, citrate, ethanol, formate, fumarate, glucose, molasses, or succinate [15].In addition, yeast has a stimulatory efect on degradation owing to its abundance of vitamins and trace minerals [16].
Te following respiration pathway (Figure 1) has been proposed for anaerobic perchlorate biodegradation with yeast as the electron donor, wherein perchlorate is converted into chlorate and then chlorite by reductase enzymes.Chlorite is reduced to chloride and oxygen by chlorite dismutase enzymes.Te complete reduction cycle is as follows: ClO 4 [18].Perchlorate recovery with microwave-assisted extraction (MAE) has been employed extensively in sample digestion studies conducted to quantitate targeted elements in the solid and liquid phases.Procedurally, MAE involves the addition of acid at an elevated temperature and pressure to attain complete decomposition of the sample matrix [19].MAE takes advantage of the high microwave energy absorption of water, and acid maximizes the conversion of microwaves to heat.Closed vessels are pressurized to avoid contamination and prevent the escape of volatile elements.High pressure during extraction reduces the digestion time and the amount of reagents required for digestion.Numerous parameters afect extraction efciency, including sample mass, acid type, reagent quantities, temperature, pressure, and extraction time [20].We used nitric acid in MAE owing to its simple handling, ease of adjustment of concentration, facility of purifcation, and oxidative capacity for organic compounds [21].Microwave-assisted plant digestion [22] has been enhanced with the addition of a small volume of hydrogen peroxide as an auxiliary oxidant [23,24].
Te primary aim of this work was to assess the biodegradability of perchlorate from spent MR using enriched microbial culture (EMC) preparations.Te microbial culture was enriched to utilize perchlorate as an electron acceptor and brewer's yeast as an electron donor.Environmental parameters that afect biodegradation, including pH, dissolved oxygen (DO), and oxidationreduction potential (ORP), were controlled and recorded.Kinetics experiments were performed to determine biodegradation rates and the time needed for complete removal, defned as a level below the minimum detection limit of perchlorate (0.1 mg/L).Our secondary aim was to develop an MAE-based method that allows accurate quantifcation of total perchlorate concentrations, including in biomass substances such as EMCs or MR.MAE efciency was optimized by varying HNO 3 and H 2 O 2 concentrations at a constant volume.Together, these aims are intended to demonstrate the achievability of biodegrading MR-adsorbed perchlorate.Furthermore, the ability to measure total perchlorate concentration improves the accuracy of kinetic models predicting biodegradation rates and residence times, an advancement that is essential for improving full-scale treatment system design.
Herein, we propose a new process that combines ionexchange with biological reduction by putting perchlorateladen MR in contact with an EMC tailored to reduce perchlorate.Such biodegradation represents a practical spent-MR treatment option, and adsorption followed by perchlorate biodegradation could be integrated into an inexpensive yet efective perchlorate removal system that generates no hazardous waste.
Te total exchange capacity of reed materials is associated with their nitrogen content, which increases from 0.9% in GR to 7.78% in our MR [27].
Biological treatment allows for low-cost selective perchlorate removal [27].To integrate ion-exchange and biological perchlorate reduction, after perchlorate has been  removed from water via an ion exchanger, the perchlorateladen spent exchanger is exposed to perchlorate-respiring bacteria under anaerobic conditions.Successful biological treatment of perchlorate from spent quaternary-aminefunctionalized ion exchangers has been demonstrated [28].Agricultural processes generate waste materials that can be used as substrates for bacterial growth.Although biodegradation using various electron donors has been investigated as a means of removing perchlorate from synthetic ion exchangers [18], the biodegradation of perchlorate adsorbed on functionalized natural adsorbents synthesized from agricultural waste has not been demonstrated previously.Heterotrophic bacteria can reduce perchlorate to chloride in water treatment applications [14].Because the biodegradation of perchlorate is an economically sound and green process, generating benign endproducts such as chloride ions and oxygen, biodegradative MR processes merit systematic investigation.

Reagents.
Tap water left for 24 h at room temperature (RT; 25 ± 2 °C) was used for all experiments.Deionized (DI) water (18.2MΩcm, Millipore, Cambridge, MA) was used for stock preparation, sample dilution, and apparatus cleaning.All chemicals were of the American Chemical Society grade.A perchlorate spiking solution was prepared with sodium perchlorate monohydrate (NaClO 4 •H 2 O) from Fisher Scientifc (Pittsburgh, PA).Nitric acid (70% HNO 3 ) and hydrogen peroxide (30% H 2 O 2 ) were purchased from Fisher Scientifc.Working standards were prepared almost daily from a stock solution.Hydrochloric acid (HCl) and sodium hydroxide (NaOH) solutions were purchased from Sigma-Aldrich (St. Louis, MO).All glassware and plasticware were acid-washed with 50% HNO 3 , rinsed with DI water, and airdried before use.
Chromeleon ® version 6.4 software was used for instrument control and data collection.All instrument components and software were produced by Dionex Corporation (Sunnyvale, CA).Te eluent, potassium hydroxide, was set to 50 mM at a fow rate of 1.0 mL/min.Te current suppressor was set to 100 mA.Te sample injection volume was 250 µL, and the running time was 18 min.Perchlorate calibration standards (1-100 mg/L) were prepared by diluting stock solutions with DI water.IC analyses were performed immediately after sample collection to prevent any biodegradation caused by microorganisms not removed by centrifugation.

Microbial Culture
Enrichment.An enriched microbial culture (EMC) was prepared from activated sludge/mixed liquor samples sourced from the Little Ferry wastewater treatment plant (Bergen County, NJ).To remove residual chemicals and coarse particles, the sludge was rinsed with oxygen-free tap water, which had been purged for 20 min with nitrogen gas (AGL Welding Co., Clifton, NJ).Te original culture contained heterotrophic bacteria known to degrade perchlorate.
Enrichment was performed in a sealed 4-L Erlenmeyer fask bioreactor (Pyrex ® , Corning Inc., Corning, NY).Te culture was grown gradually with regular feedings of perchlorate (electron acceptor) and yeast (electron donor and carbon source) until the total culture suspension reached a fnal volume of 4.0 L. Yeast was added at a 3 : 2 yeast-toperchlorate ratio (mass/mass), more than the stoichiometric equivalent, to ensure that low levels of electrons or carbon would not limit perchlorate degradation.Among the electron donors commonly used for anaerobic perchlorate degradation, yeast (0.1 mg/L) has the lowest cost and the highest perchlorate reduction potential [15].To promote microbial growth, aqueous perchlorate was added from the stock solution to attain a fnal ClO concentration of 800 mg/ L. No additional nutrients were added.
Continuous mixing was performed with a magnetic agitator (Isotemp, Fisher Scientifc, USA) at 50 rpm at RT. pH, oxidation-reduction potential, dissolved oxygen (mg/L), conductivity (µS/cm), and temperature ( °C) were monitored regularly to ensure favorable perchlorate reduction conditions.Parameters were measured directly from the reactor with a multiparameter meter (Termo Orion 920+, Termo Fisher Scientifc, Waltham, MA).
A neutral pH (7.0 ± 0.5) was maintained with 0.1 M HCl and 0.1 M•NaOH because maximum perchlorate reduction occurs at a pH near neutral (6.8-7.2) and moderate temperature (20-40 °C) [29].Anaerobic conditions were maintained by purging the reactor with nitrogen gas (AGL Welding Co., Clifton, NJ), and monitored by measuring DO (mg/L).Conductivity measurements were taken as an indirect measure of biomass concentration.Sludge was washed with DI water when conductivity exceeded 100 µS/cm, a level above which we obtain low levels of perchlorate removal, possibly due to the accumulation of chloride ions.In general, perchloratereducing microorganisms have low halotolerance.
ORP is a measure of perchlorate reduction performance.ORP measurements were taken with an epoxy electrode flled with silver-silver chloride (Ag-AgCl) is an internal reference solution (Termo Fisher Scientifc, Waltham, MA).Readings were reported as E h values (mV) relative to a standard hydrogen electrode.Te measuring electrode was calibrated to read E h � 420 mV at 25 °C for an ORP calibration standard solution (Cat.No. 967901, Termo Fisher Scientifc, USA).Values were recorded after 15 min of electrode immersion, during which the electrode readings stabilized (≤2.0 mV fuctuation per 5-min period).
Triplicate samples were withdrawn periodically from the bioreactor for perchlorate degradation measurement.Bacteria were recovered by centrifugation; homogeneous sludge samples were collected in 1.5 mL microcentrifuge tubes (Fisher Scientifc, USA), then placed in a Micromax ® cen- trifuge (Int'l Equipment Co., Needham, MA) at 10,000 rpm for 10 min.After centrifuging, 1.0 mL was withdrawn from the supernatant and placed in a 2-mL clear glass vial (Varian, Walnut Creek, CA) for IC.

Biodegradation of Aqueous Perchlorate.
Te EMC described in Section 2.3 was used in experiments to determine the biodegradation rate of perchlorate dissolved in water.Te biomedium consisted of aged tap water supplemented with perchlorate and yeast.Te experiments were performed in 50 mL polypropylene bottles (BD Falcon, Franklin Lakes, NJ) that had been acid-washed with 50% HNO 3 , triplerinsed with DI water, and then air-dried.
Each bottle was flled with 40 mL of oxygen-free water and 10 mL of EMC (total volume, 50 mL).Aqueous perchlorate was added from the stock solution to attain a fnal ClO 4 -concentration of 30 mg/L; yeast was added at a 3 : 2 yeast to perchlorate ratio.EMC-depleted and no-perchlorate control samples were used.Te former was identical to the experimental sample except that the EMC was sterilized with 0.1% HNO 3 to detect any alternate processes that might be removing perchlorate (i.e., physical adsorption).Te latter control was used to account for any residual perchlorate that might be present in the EMC before perchlorate feeding.
pH was maintained at 7.0 ± 0.5 with 0.1 M HCl and 0.1 M NaOH.Anaerobic conditions were achieved by purging with oxygen-free nitrogen gas for 5 min (GAL Welding, Clifton, NJ).Te experiment bottles were sealed with Tefon ® -lined airtight caps and then placed in a rotary mixer at 50 rpm at RT to initiate treatment.
Triplicate 2 mL samples were collected from each bottle at regular intervals and analyzed for residual and total perchlorate concentrations.Following sample centrifugation, the residual perchlorate concentration in the solution was determined by IC.Deionized water was added to the sample bottles to replace withdrawn volumes and thus maintain the concentrations.Te total perchlorate concentration was determined for the entire contents of each sample bottle using the MAE procedure and then analyzed by IC.

Biodegradation of Perchlorate in Spent MR.
Perchlorate-laden MR (properties listed in Table 1) was placed in contact with EMC and yeast under anaerobic conditions.To prepare perchlorate-laden MR, MR (0.1 g) was added to aged tap water (50 mL) containing perchlorate (30 mg/L).Te pH of each sample was adjusted to 7.0 ± 0.5.Mixing was performed in 50 mL polypropylene bottles (BD Falcon, Franklin Lakes, NJ) on a rotary mixer at 150 rpm (Glas-Col, 2128, Terre Haute, IN) for 24 h to allow equilibrium to be reached at RT.After mixing, the bottles were placed upright on a lab bench for 3 h to allow the MR to settle by gravity; 1.0 mL aliquots of settled biomass were transferred from the bioreactor to seed fresh enrichment cultures.Supernatant samples were placed in 2.0 mL clear glass vials for IC perchlorate analysis.Te remaining supernatant was discarded.
To allow biological degradation, settled spent MR was resuspended immediately in 40 mL of oxygen-free aged water previously purged with nitrogen for 20 min and 10 mL of EMC to obtain a total volume of 50 mL.Yeast (2 g/L) was added as the external carbon source and electron donor.Te aforementioned sterilized and perchlorate-free control samples were prepared.
Neutral pH and anerobic conditions were achieved as described in Section 2.5.Te bottles were sealed with Tefon ® -lined airtight caps and placed on a rotary mixer at 50 rpm at RT. Triplicate 2 mL samples were collected from each bottle at regular intervals, centrifuged, and the supernatant solutions obtained were analyzed for residual and total perchlorate concentrations by IC.Bottle concentrations were maintained by replacing sample volumes with distilled water.Te total perchlorate concentration was determined for entire sample bottles by MAE and analyzed by IC.

MAE for Perchlorate
Recovery.Samples were digested in a closed-vessel microwave digestion system (Ethos-E, Milestone, Monroe, CT; maximum power, 950 W; frequency, 2.45 GHz).Samples consisted of 0.2 g•MR in 10 mL DI water or biomass suspension, each containing 30 mg/L ClO 4 -from a stock solution.Te samples were placed into 100 mL Teflon ® perfuoroalkoxy-coated vessels and digested with 0.1-2.0mL HNO 3 [30].Te vessels were sealed with a calibrated torque wrench and placed in the microwave to initiate digestion.Te microwave was equipped with a shielded thermocouple temperature probe containing an optical fber with a phosphorus sensor (ATC−400) that was inserted directly into the vessels.Te extraction conditions (percentage power, temperature, pressure, and irradiation time) were controlled with MLS GmbH ™ software (version 05h, HLeutkirch, Germany).
Te microwave instrument's default program for plant digestion was used.Tis program included three steps: (i) preextraction (0 min < t < 5 min), during which the temperature increased gradually to the desired extraction temperature with an 800 W power input; (ii) static extraction (5 min < t < 20 min), during which the temperature was held constant at 120 °C with a 500 W power input; and (iii) ventilation (20 min < t < 35 min), during which the power was of and the vessels were allowed to cool for 15 min.
After cooling, digested samples were collected in 50 mL polypropylene bottles (BD Falcon, Franklin Lakes, NJ) and placed in an Eppendorf model 5810 centrifuge (Eppendorf AG, Hamburg, Germany) at a mixing speed of 10,000 rpm for 10 min to clear the supernatant of minute particles that could clog ion chromatograph tubing.Supernatants were decanted into clean 50 mL polypropylene tubes, and the volumes were recorded.All samples were diluted back to 10 mL, and the pH was measured to ensure near neutrality and thereby prevent chemical damage to instrument components.Finally, 1.0 mL of supernatant was transferred into a 2.0-mL clear glass vial (Varian, Walnut Creek, CA) for IC.
All digestion vessels were cleaned by adding 20 mL of 50% HNO 3 , resealed with a torque wrench, and placed into the microwave, which was run on the same program and steps described earlier for sample digestion.After cooling, the vessels were rinsed with DI water and air-dried on a clean bench for ≥2 h before the next digestion run was performed.

Results and Discussion
After enriching EMCs in batch reactors, we showed that EMCs can degrade in-solution perchlorate efectively and consistently and then proceeded to show that EMC degraded adsorbed perchlorate from spent MR.Tis approach ofers a benign, environmentally friendly, and cost-efective solution to the waste generated by using MR to remove perchlorate from water.

Biodegradation of Perchlorate during Enrichment.
Enrichment was accomplished by feeding EMCs with perchlorate and yeast repeatedly under anaerobic conditions.Te sludge was washed with DI water before each feeding to remove waste (mostly chloride ions) that could hinder perchlorate degradation.Eleven cycles of feeding, reducing the perchlorate to levels below IC detectability, and sludge washing were required to reach stable perchlorate reduction.Environmental conditions with the potential to afect bioreactor performance, including pH, dissolved oxygen (DO), and oxidation-reduction potential (ORP), were controlled to achieve maximum removal.
Enrichment results are shown in Figure 2 and Table 2. Reduction activity could not be detected in the frst 24 h.EMC-mediated reduction of perchlorate from its initial concentration (∼800 mg/L) to undetectable levels was achieved after 5 d ( Table 2, cycle #1), corresponding to a mean removal rate of 0.007 g/L•h.Culture enrichment to restore the original perchlorate and yeast concentrations and nitrogen gas purging of the bioreactor to maintain anaerobic conditions were repeated each time the perchlorate concentration decreased below a detectable level.Te second cycle took only 3 d for perchlorate to fall below a detectable level (Table 2, cycle #2), corresponding to a mean removal rate of 0.01 g/L•h.After 32 d of enrichment, the last cycle achieved complete removal in 24 h (Table 2, cycle #11), corresponding to a mean removal rate of 0.032 g/L•h, a rate more than four times that seen in the frst cycle.By the last cycle, perchlorate biodegradation had reached a steady state.

Biodegradation of Perchlorate in Spent MR.
After reaching steady-state enrichment conditions, aliquots (10 mL each) of EMC were transferred from the enrichment bioreactor into 50 mL bottles and monitored for total perchlorate concentration with MAE.Four systems were analyzed (Table 3); system 1 was an EMC-sterilized control for system 2, while system 4 was a negative control (no perchlorate added) for system 3. Systems 1-3 contained yeast in a 3 : 2 ratio of yeast to perchlorate.Te overall EMC perchlorate removal percentages from spent MR that were attained are shown for each system in Figure 3. Initial and fnal pH, ORP, and DO measurements for each system are summarized in Table 3.
Negligible perchlorate removal (<5%) was observed in system 1 control samples containing spent MR and 0.1%-HNO 3 sterilized EMC, suggesting that the physical adsorption of perchlorate in our experimental paradigm is insignifcant.In system 2, which contained active (nonsterilized) EMC and spent MR, less than 4% perchlorate removal was observed on day 1 (mean 27.70 ± 0.26 mg/L to 26.55 ± 0.12 mg/L).Te removal rate then increased rapidly, reaching 32.3% on day 2 and 63.90% on day 3.By day 7, the removal rate had reached 98.2%, with a perchlorate residual of only 0.51 ± 0.09 mg/L and an ongoing mean reduction rate of 0.004 g/L•h.
When aqueous perchlorate was exposed to EMC in the absence of MR in System 3, more than half of the perchlorate (54.50%; mean 28.80 ± 0.12 mg/L to 13.10 ± 0.12 mg/L) was removed on day 1.On days 2 and 3, the removal rates were 69.1% and 93.3%, respectively.No perchlorate was detected after 5 d of incubation.In the control system containing EMC only (system 4), no perchlorate was detected, indicating that perchlorate was not present prior to incubation.
Notably, it took only 1 d for EMC to eliminate perchlorate (800 mg/L) from the enrichment bioreactor, but the same culture needed >5 d to degrade 30 mg/L of perchlorate after being transferred to 50 mL bottles (system 3).Tis drop in performance may refect a need for the microorganisms to acclimatize to a sudden environmental change.It is also possible that perchlorate removal was reduced in response to there being greater light exposure in the bottles than in the much larger bioreactor.Exposure to light enables photosynthesis, which generates reduction-dampening oxygen.Upon observing a similar perchlorate removal reduction rate phenomenon, Bardiya and Bae [16] attributed the efect to the presence of residual oxygen after purging with nitrogen.
EMC-mediated perchlorate removal occurred more rapidly when the perchlorate was in an aqueous form (system 3) than when it was adsorbed on MR (system 2).Aqueous perchlorate is free in solution, uniformly distributed, and readily available for microorganisms to consume.Conversely, MR-adsorbed perchlorate is bonded by electrostatic forces, creating two barriers to biodegradation.Firstly, microorganisms have to be transported from the bulk fuid to adsorption sites by mixing, difusion, and/ or microbial movements.Most perchlorate-reducing microorganisms are motile [8].Secondly, there are strong electrostatic forces between positively-charged functional groups on MR and negatively-charged perchlorate ions.Te energy required to overcome these forces could have been supplied by electrons from yeast.Perchlorate reductase and chlorite dismutase act as catalysts, lowering the activation energy needed for bioreduction (respiration) to occur.Te perchlorate removal patterns observed in our experiments are consistent with the possibility that these barriers may have delayed the onset of removal (as suggested by the observed reduction lag) and lowered the magnitude of perchlorate removal.Note.Nitrogen content is 9-fold that of an unmodifed giant reed.

Journal of Chemistry
A decrease in ORP indicates a more reduced state.ORP values decreased for all four systems over time (Table 3), with the greatest decrease being observed for EMC with spent MR (system 2; initial mean ORP-day 7 mean ORP � 57.21 ± 0.60 mV).Much more modest magnitudes of ORP decrease were observed for the remaining systems (1, 3, and 4; see Table 3).In all four systems, the ORP was maintained far below the −110 mV threshold for perchlorate reduction [31,32].
A decrease in ORP also indicates more anaerobic conditions, a relationship that was confrmed by the decrease in DO measured at the end of the incubation period compared to the initial DO measurement.Similarly, Rikken et al. [33] observed oxygen depletion concomitant with perchlorate degradation in a system that used acetate as an electron donor and bacteria from activated sludge as perchloratereducing microorganisms.A few data points were omitted when sludge conditions were not stable. 6 Journal of Chemistry Similar to ORP, pH decreased over time in all four systems, with the greatest decrease occurring in system 2. Okeke et al. [15] observed a decrease in pH to more acidic levels during the biogeneration of spent ion-exchange resin saturated with perchlorate, and they attributed it to fermentation yielding organic acids.Anaerobic respiration and fermentation are distinct forms of energy metabolism.In anaerobic respiration, organisms transport electrons from an electron donor to an external (exogenous) electron acceptor in an electron transport chain that functions to convert chemical energy into an electrochemical potential.In fermentation, there is no external electron acceptor; microorganisms produce their own electron acceptor to maintain a reduction-oxidation balance.
None of our experimental parameters identify specifcally the type of energy metabolism that took place.Notwithstanding, because perchlorate is nonorganic and generally exogenous, its presence points to the occurrence of anaerobic respiration rather than fermentation.Although it is possible, at least in theory, that fermentation occurred after the microorganisms had exhausted the available perchlorate, the existence of microorganisms that switch from anaerobic respiration to fermentation depending on the availability of electron acceptors has not been described.Although fermentation cannot be excluded as a possible contributing factor completely, anaerobic respiration provides a parsimonious explanation for the decreases in pH observed in our experiments.Journal of Chemistry

Stages of Perchlorate Removal from Spent MR by EMC.
Tree stages of perchlorate removal of varying slopes were observed in system 2, which contained nonsterilized EMC and spent MR (Figure 3).Stage 1 (t ≤ 1 d), characterized by a gentle slope, represents a lag or a stationary phase where no signifcant removal occurred, which we have posited to be a period of EMC acclimatization.Stage 2 (1 d < t ≤ 4 d) has a steep slope, refecting a rapid biodegradation process that removes most of the perchlorate (85-90%).Finally, stage 3 (4 d < t ≤ 7 d) has a decreasing slope, refecting a diminishing biodegradation rate.We propose two reasonable postulates to explain the shape of the last phase.
In the frst postulate, stage 3 refects perchlorate rearrangement throughout MR adsorption sites.It can be assumed that perchlorate ions located at the MR monolayer (outermost layer of the MR sphere) tend to be degraded frst because they are the most exposed.Te degraded perchlorate ions may be efciently replaced by perchlorate ions from an inner layer of the MR sphere until most of the adsorbed perchlorate is degraded (∼98%).In this context, it is also assumed that the removal rate is positively dependent on concentration, which has reached quite low levels at the transition from stage 2 to stage 3. Despite there being a high perchlorate concentration in stage 1, the activity of the recently physically transferred microorganisms was neither stable nor optimized.In stage 3, when the microorganisms had acclimatized, the degradation rate of adsorbed perchlorate may have become slow due to becoming dependent on difusion (Figure 3).
A second postulate that may explain the diminishing biodegradation rate in stage 3 is the possibility that perchlorate desorption from MR may follow a core shrinking model wherein the outermost layer of MR-bound perchlorate is frst degraded, leaving the next closest layer relatively more exposed for degradation.Tis process may continue through a series of shrinking layers, as depicted in the shrinking core model shown in Figure 4(a)-4(f).Briefy, the frst stage is a stationary phase in which little perchlorate degradation occurs, possibly due to reacclimatization of the microorganisms and transport through the bulk layer to the monolayer of the MR.In the second stage, microorganisms can access readily available perchlorate at the monolayer, and the biodegradation rate is maximal as long as perchlorate availability is not limiting.As the perchlorate in the monolayer is consumed, the reaction moves to an inner layer beneath the monolayer, which has a smaller radius than the outer radius and therefore holds fewer perchlorate molecules.As the reaction moves progressively inward towards the core of the MR particle, desorption of perchlorate slows due to decreasing perchlorate availability, constituting the transition to stage 3.At this stage, the biodegradation rate is controlled by the rate at which perchlorate is desorbed from MR.At the end of the incubation period, ∼2% of the original perchlorate concentration (0.51 mg/L) remains adsorbed on MR and has not been degraded.No explanation was found in the literature for retained perchlorate.It could be that the remaining perchlorate is too tightly bound to the innermost layer of MR to escape through small pores.Alternatively, or in addition, perchlorate-consuming microorganisms may have died due to chloride accumulation from the reaction.Ideally, digestion should result in the complete decomposition of organic material using the minimum amounts of reagents, which should be as dilute as possible to minimize acidity and thus prevent deterioration of analytical instruments.To achieve this goal, one can either add small volumes of concentrated nitric acid or, more commonly for biological sample digestion applications, add a diluted nitric acid solution.

Perchlorate
All samples to which H 2 O 2 was added produced a popping sound during digestion, possibly due to pressure build-up [30].Tese samples were found to be burned, and they were discarded without analysis to avoid damaging the equipment.Having performed successful digestions without exogenous H 2 O 2 , Veschetti et al. [24] concluded that efcient sample digestion could be performed with nitric acid.Te digestate produced with samples digested with water in place of H 2 O 2 was clear with no solid residue.Perchlorate recovery values are shown in Figure 5 and Table 4.

Conclusions
Tis study demonstrated that perchlorate can be efectively removed from spent MR by EMC-mediated anaerobic biodegradation, an efcient, cost-efective, and environmentally friendly method that complements ion exchange and incineration treatment methods.With optimization, perchlorate removal can proceed rapidly.Tis study describes a newly developed MAE-facilitated method for quantifying perchlorate in biomass, including MR. Extraction efciency was optimized and reached 90% recovery (±5%) by varying the concentration of nitric acid added to each sample type.Figure 5: Perchlorate recovery values obtained showing dependence on the volume of nitric acid added.Te total volume added was constant at 3.0 mL.For example, if 0.2 mL of concentrated nitric acid (∼15.9)M was added, 2.8 mL of water was also added.

Figure 1 :
Figure 1: Pathway of perchlorate reduction by perchloratereducing bacteria as discussed by Trash et al. [17].

Figure 4 :
Figure 4: Shrinking core model of perchlorate biodegradation from spent MR (drawn in SmartDraw).(a) Stationary phase (0-24 h): negligible removal, putatively due to EMC reacclimatization.(b) Transport of EMC from the bulk fuid to the MR surface.(c) EMC utilization of perchlorate at readily available monolayers (limited by reduction reaction rate).(d) Subsequent EMC utilization of perchlorate beneath the monolayer, which has to difuse through pores and has reduced availability (smaller radius; trapped in pores).Removal slows, becoming dependent on the perchlorate difusion rate.(e) Continued decline in the removal rate until all available perchlorate has been removed.(f ) Te removal rate is reduced considerably until it reaches a minimum.Trapped perchlorate is not recovered.

Table 2 :
Perchlorate removal during EMC enrichment and during the establishment of steady-state removal.

Table 3 :
Environmental parameters for the biological removal of perchlorate, ClO 4 − , from spent MR with EMC over 7 d at 25 °C.
Recovery with MAE.Studies were conducted to determine the efciency (recovery) of perchlorate extraction from MR by EMCs following MAE.Efciency was optimized by varying concentrations of HNO 3 and H 2 O 2 .