Bioremediation of Chlorate and Chromium Contamination with Native Microbial Culture in Cold Climate

Chlorate and hexavalent chromium are two chemicals with adverse health effects that may cause groundwater contamination in industrial areas. The objective of this study was to determine if the native microorganisms collected from a site contaminated with chlorate and chromate can lower the concentration of these chemicals in groundwater to acceptable regulatory levels. Several anaerobic microcosm experiments were conducted with synthetic groundwater (media), native microorganisms, acetate as an electron donor, nitrogen, phosphorus, and minerals. The microorganisms utilized 2200 mg/L acetate to remove 1000 mg/L of chlorate and 3 mg/L of hexavalent chromium entirely from the media provided that the groundwater is supplemented with additional nitrogen and phosphorous (with the Carbon:Nitrogen:Phosphorous molar ratio of 100:10:5). The added trace minerals solution prepared based on American Type Culture Collection (ATCC) 1191 medium did not improve the remediation process. Native microbial culture derived from the contaminated site removed the chlorate and chromate from the synthetic groundwater at 20 °C in about 40 days. The same removal was achieved at 10 °C, but in a longer timespan of 80 days. This work confirmed the importance of ensuring the presence of sufficient N and P to stimulate chlorate‐ and chromate‐reducing bacteria in the groundwaters.


Introduction
Groundwater is a vital drinking water resource that can be contaminated by anthropogenic pollutants.With increasing industrial development, the quantity of released chemicals into the environment increases.Oxyanions with the generic formula of A x O y z− (where A represents a chemical element and O represents an oxygen atom) are compounds with various applications in the industry.There are several contaminated sites associated with past/historical operational practices and a lack of controls, which are now mandatory.Oxyanions are very soluble in water and move with the flow of groundwater.Their release in groundwater (e.g., through agricultural herbicides and defoliants, or industrial effluent, waste, and/or discharge) may result in potential risk to human and/or environmental health, requiring effective and efficient in-situ treatments (Adegoke et al. 2013;Sarria et al. 2019;Weidner and Ciesielczyk 2019).Chlorate and chromium are common co-contaminants in groundwater at pulp and paper mills, sodium chlorate facilities, and electroplating industries.Given the scale of some of the con-tamination plumes, these sites often go unremediated (e.g., brownfields), or use costly, ex-situ techniques to control groundwater contamination (e.g., pump and treat).Stimulating in-situ bioremediation in groundwater can provide a cost-effective, sustainable process, with minimal inputs required for optimizing conditions for decontamination.Insitu remediation approaches for chlorate may not be applicable for other co-contaminants (e.g., hexavalent chromium) and may result in the undesirable mobilization of other metals (e.g., arsenic, barium) from native soils and/or associated with historical brine sludge waste.The efficacy of bioremediation of an oxyanion, such as chlorate in the presence of chromium has not been evaluated, especially for cold climate conditions predominant in Canada.
A sodium chlorate production facility located in Canada's Prairie province of Manitoba is one of the world's largest and highest-producing sodium chlorate facilities.Since the 1960s, the facility has manufactured sodium chlorate by processing a brine solution (NaCl) through electrolysis to produce a purified crystalline powder that is readily soluble in water.The end product, sodium chlorate, is transported off-site in rail cars for use as an industrial bleaching agent in the pulp and paper industry.Due to historical waste storage practices and ongoing operations, groundwater at the site has been impacted by both chlorate and hexavalent chromium, used in the electrolysis production process.
Sample collection site.The site location is considered a cold climate site (Stempvoort and Biggar 2008), with an annual average air temperature of 2.2 ± 1.1 °C (Government of Canada 2017) and a yearly average groundwater temperature of 7.2 ± 1.2 °C (Province of Manitoba 2017).
Investigations at the site have determined that surficial sediments consist of till and alluvium, and glaciofluvial deposits (coarse sand and gravel).Distinct soil types have been encountered at the site, including a top layer of sand and gravel (ranging in thickness between 3 and 7 m) followed by a clay till (2 to 4 m in thickness), a 1 to 2 m thick gravel layer, and finally followed by a till layer of undetermined thickness.
Groundwater flow direction has consistently been northeast towards the Nearby Unnamed River with an average horizontal hydraulic gradient of 0.031 m/m and an average hydraulic conductivity of 1.8 × 10 −5 m/s.Depth to groundwater has ranged from 2.2 to 6 m depending on the proximity to the nearby river and the monitoring well aquifer zone.As the Nearby Unnamed River water levels fluctuate with seasonal flooding events and regional precipitation, it is possible that the contaminated groundwater discharge directly into the river.
The current impacts on the groundwater at the site are related to the historical brine sludge disposal and historical site operations and consist of a co-mingled plume with elevated values for electrical conductivity and elevated concentrations of chlorate, and chromium (including hexavalent chromium).Maximum and average chlorate concentrations (4200 and 1400 mg/L, respectively) and chromium concentrations (3.02 and 0.75 mg/L, respectively) have been measured in the downgradient plume.Analytical results have shown that most of the chromium observed is in the hexavalent chromium form.
Although there is no particular regulatory value for chemical concentration in Canada's recreational water criteria due to insufficient data, chlorate is regulated to be less than 30 mg/L for freshwater aquatic life in British Columbia (Health Canada 2012) (British Columbia Ministry of Environment 2002).In the Canadian Drinking Water Guidelines, the maximum allowable concentration for chlorate in drinking water is 1 mg/L and for total chromium is 0.05 mg/L (Health Canada 2022).The regulatory limit on concentration of chromium is due to health effects of hexavalent chromium (Health Canada 2022).

−
) as an oxyanion is known for its strong ability to oxidize materials.This ion exists in various compounds like sodium chlorate, calcium chlorate, potassium chlorate, and magnesium chlorate, produced for different purposes in chemical manufacturing.Sodium chlorate is one of the most common salts used in agriculture as a herbicide and in industry for bleaching papers or generating antiseptic water products (US EPA 2016).
Chloride and its inorganic oxidation states form five species ClO 4 − (perchlorate)/ClO 3 − (chlorate)/ClO 2 − (chlorite)/ClO − (hypochlorite)/Cl − (chloride).Chloride and perchlorate are the most stable forms, while hypochlorite and chlorite are unstable.Chlorate is also considered a chemically stable specie in the environment, and it can form when a perchlorate molecule loses an oxygen atom in the degrada-tion process (Coates and Achenbach 2004;EnviroGulf Consulting 2007).The natural occurrence of chloride oxyanions is not common, and they are mostly the result of human activities on the earth's crust (Bruce et al. 1999).
Several methods with different approaches have been used to remove perchlorate from contaminated sites and water.These techniques are categorized into three major groups: abiotic remediation technologies like ion exchange or activated carbon systems, abiotic reduction technologies such as chemical or electrochemical reduction, and biological remediation methods (ITRC 2007;Taraszki 2009).
Researchers primarily focus on perchlorate reduction, a more common challenge globally.Perchlorate research is applicable for chlorate remediation, since chlorate forms in perchlorate reduction steps and is reduced under similar conditions (Bruce et al. 1999;Taraszki 2009;Carlström et al. 2015).
Perchlorate and chlorate act as electron acceptors in a series of biological reactions in the environment to reduce to other oxyanions, and, finally, to chloride as the most stable species (Malmqvist et al. 1991;Kotlarz et al. 2016).Several studies have shown that the microorganisms which can reduce chlorine oxyanions in the presence of suitable electron donors naturally exist in different anoxic microbial ecosystems such as soil, groundwater aquifers, sediment, sludge, animal waste, and hypersaline soils (Achenbach et al. 2001) (Hatzinger et al. 2002) (Wu et al. 2001) (Bardiya andBae 2005) (Acevedo-Barrios et al. 2019).
Many electron donors have been tested for biodegradation of (per)chlorate.It is verified that microorganisms utilize organic carbon and start the reduction route in the presence of organic carbon.Perchlorate and chlorate are used as the terminal electron acceptors for the oxidation of a wide range of defined organic carbon compounds, including methane, acetate, methanol, glucose, saccharose, and succinate, as well as vegetable oil, starch, yeast extract, and even chicken litter (Hunter 2002;Luo et al. 2015;Kannepalli and Farrish 2016;Lian et al. 2017).The reduction process of perchlorate starts with transferring the electron and converting the ion to chlorate, then chlorite and hypochlorite and finally ends with chloride ion (Rikken et al. 1996).
"Perchlorate-reducing bacteria" (PRB) are a phylogenetically diverse group of microorganisms capable of growth by respiring perchlorate as the sole electron acceptor (Kotlarz et al. 2016).

Hexavalent Chromium
As a transition metal with six valence electrons, chromium can occupy different oxidation states from −4 to +6.Still, there are only three stable forms in nature: Cr 0 (or metallic chromium), Cr(III), and Cr (VI) (Guertin et al. 2005).Chromium deposits in the earth's crust are in the form of trivalent chromium, and high concentration of hexavalent chromium is typically a result of human activity.Because of the specific properties of chromium ions, they are used extensively in different industries for pigments and dyes, catalysts, tanning, and wood preservation (Lunk 2015).
Chromium (III) is considered a micronutrient necessary to metabolize sugar and fat with a safe daily intake of 50 to 200 μg (Anderson 1997).Even though a regular diet may not contain the minimum level of that range, it is not recommended to supplement the diet with Chromium (III) (Panchal et al. 2017).Trivalent chromium does not show any toxic effect, skin allergic reaction or asthma if the dose is less than 5 g/kg body weight (WHO 2009).Reducing chromium to a lower oxidation state decreases its toxicity in contaminated environments (Guertin et al. 2005).
Chromium (VI) can participate in several chemical and biological reactions in nature.Studies showed that chromium-reducing bacteria are ubiquitous in soil and groundwater samples, facilitating the reduction process under aerobic and anaerobic conditions (Kamaludeen et al. 2003).These microorganisms can utilize organic carbon from the environment as electron donors and transform hexavalent chromium to the innocuous trivalent form.Molasses, lactate, glycerol, acetate, yeast extract, gluconate, or fructose are some commercially available electron donors that could be added to the environment if the organic matter concentration is not sufficient (Rahman and Thomas 2021).
Acetate is widely used by organisms capable of anaerobic respiration.It can be measured directly, which makes it easy to monitor its consumption to keep track of the carbon ratio.
Besides the electron donor materials, adding other nutrients microorganisms need may increase the rate of biodegradation.Trace minerals are essential for the growth of all life forms, and supplementation is necessary for the growth of organisms in the laboratory in defined media (Froese and Sparling 2021).Nitrogen and phosphorous are two essential elements for making the building blocks of living organisms, and they have been shown to be biomass-limiting elements at some perchlorate remediation sites (Wang et al. 2013).Temperature shown to limit the development of biodegradation activity of some processes (Bardiya and Bae 2011).
While PRBs (perchlorate-reducing bacteria) are specific organisms that can reduce chlorate with their specific enzymes, hexavalent chromium reduction is unspecific and could be done by variety of microorganisms.Chromium biological reduction is facilitated by non-specific enzymes (mostly called oxidoreductases) that can transfer electrons to the outer valence layer of metallic ions (Rahman and Thomas 2021).

Objectives
The main hypothesis is that presence of electron donors and available nutrients in in-situ bioremediation are limiting factors to reduce chlorate and hexavalent chromium simultaneously.The following objectives were determined to examine this hypothesis: • Determining the ability of native microorganisms to degrade chlorate and hexavalent chromium to identify the necessity of bioaugmentation or ex-situ bioremediation as an alternative option for decontaminating the site.• Analyzing the effects of electron donor (acetate) and nutrients as the limiting factors of biological stabilization of chlorate and chromate.
• Investigating the impact of low temperate on the in-situ bioremediation.
The experiments were designed to: firstly, show the effect of adding electron donors and other nutrients, including micronutrients (mineral's solution) and macronutrients (nitrogen and phosphorus sources) to the environment containing native microorganisms from the site; and secondly, investigate whether the reduction is possible at lower temperature.

Synthetic Groundwater
A synthetic groundwater, as shown in Table 1 was prepared based on site-specific groundwater analytical data.The components of the synthetic groundwater were purchased from Sigma-Aldrich chemicals, and they were dissolved in pure deionized water produced by the Millipore Elix-10 system with Total Organic Carbon (TOC) <10 ppb and resistivity of 10-15 MΩ•cm.Using synthetic groundwater makes it easier to control the concentration of materials since groundwater from the site contains various trace materials with unknown effect on the process (Table S1).
A lower chlorate concentration (1000 mg/L rather than 3900 mg/L) was used in the experiments to observe results in a shorter timeframe while still demonstrating the indigenous microorganisms' capability to reduce the ions.Nitrate and nitrite were not added, and only an anabolic amount of sulphate was added to prevent competition from denitrifying and sulphate-reducing bacteria.Since potassium chromate is a colorful compound, it gives a pale-yellow color to the synthetic groundwater, color change could be a sign of chromate reduction in the media.

Synthetic Media
Synthetic media, the solution used in the microcosms, was prepared by adding the electron donor and nutrients to the synthetic groundwater, as shown in Table 2. Based on the stoichiometry of the chlorate reduction reaction, every 4 mol of chlorate need 3 mol of acetate to complete the catabolic and biosynthetic reactions and produce 1 mole of chloride ion (Rikken et al. 1996).Ammonium phosphate was used as nitrogen and phosphorus sources, and American Type Culture Collection (ATCC) 1191 minerals solution was added to the medium to ensure sufficient trace elements that the bacteria may need.The mineral solution is based on ATCC 1191 medium and is described in Islam et al. (2006).Acetate concentration was selected four times greater than the minimum required, and the C:N:P ratio was 100:10:5 to avoid any limitation of the nutrients.
All the glassware and solutions were sterilized before starting the experiments, and the final synthetic media was filtered through 0.2 μm filters.

Inoculum
The ability of the native microorganisms is essential to any in situ bioremediation process.In this experiment, three possible sources of native microorganisms were available, which were collected on the contaminated site from the depth of four meters: dry soil, wet soil (19% moist), and groundwater.When native microorganisms are transferred to the samples, attention has to be paid to transfer minimum material to microcosms to avoid changing the overall concentration of other constituents.Finally, 4 g of the dry soil, 4 g of the wet soil, and 2 mL of groundwater provided the inoculum for each microcosm which together contribute to 2.5% of each bottle weight.
Since the inoculum used was the microbial community primarily from the soil from the contaminated site, Adam and Duncan's method (Adam and Duncan 2001) for the fluorescein diacetate assay (FDA) was used to verify the microbial activity in the soil.This method has been reported suitable to measure microbial activity in the range for soils, but is unable to detect any microbial activity in the aqueous samples directly.The groundwater's microbial activity was tested using ATP.

Microcosm Design
Three 500 mL bottles were filled with 400 mL of media for each microcosm experiment.
The materials added to synthetic groundwater to prepare the synthetic microcosm media for each experiment are shown in Table 3. "C" stands for carbon source and electron donor, which is acetate."C + M" represents carbon source and minerals solution added to the synthetic groundwater, and "C + NP" shows the treatment containing acetate, nitrogen, and phosphorus source."C + NP + M" bottles contained initial solution, acetate, nitrogen and phosphorous, and micronutrients in their media.There were six bottles with the last composition, three of which remained at room temperature (20°C) with the other samples and three stored in a cold chamber (10°C) to see the effect of a temperature close to the in situ groundwater temperature.
All bottles were capped with rubber disc lids after adding inoculum.They became anaerobic by conducting four cycles of degassing with a vacuum device for 5 min and then purged with nitrogen gas for 2 min.
Bottles were shaken well every day for 30 s to prevent the development of any concentration profile in the media and increase the bioavailability of substrate for microorganisms.
Twelve bottles, including "C," "C + M," "C + NP," and one series of "C + NP + M" media, were stored in a dark place at 20 °C.Three bottles containing the "C + NP + M" media were placed in a cold chamber at 10 °C for 80 days.

Sampling
Samples were taken out from the liquid phase by inserting a clean needle through the rubber cap after 3, 7, 14, 21, 35, 50, 58, and 80 days.Tem milliliter of liquid each time  was withdrawn, filtered (0.45 μm), and tested for hexavalent chromium right after sampling.The rest of the samples were stored in a freezer to analyze the concentration of the other ions.After each sampling process, bottles were purged with nitrogen to ensure no oxygen content remained in the headspace.

Analytical Analysis
Hexavalent chromium analysis was done based on the method 8023 using HACH DR-3900 spectrophotometer (HACH 2023); the detection limit for this method is 0.010 mg/L.Total chromium was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) on a Varian model 725-ES made by Agilent.
Chlorate, chloride, and acetate concentration in the samples were measured using Metrohm 930 Ion Chromatograph (IC) using Standard Methods (APHA 2012).Microbial activity was measured using Fluorescein Diacetate Assay (FDA) method and ATP.The solutions for the FDA were prepared based on Adam and Duncan's procedure (Adam and Duncan 2001), and the final samples were placed in Biochorm Ultraspec 2100 spectrophotometer to measure the fluorescein absorbance.

Results and Discussion
The FDA analysis of soil samples showed that dry soil, wet soil have 0.015, 0.016 mg/L and below detection limit for the groundwater (as an average of triplicate with the standard deviation less than 7%) fluorescein concentrations, respectively.This means microorganisms were present in the soil samples, but this method is unable to detect any microbial activity in the aqueous samples directly.ATP analysis was conducted to test the groundwater sample and it showed the concentration of ATP is 223 pg./L (for a triplicate measurement with a deviation less than 8%), indicating the presence of active microorganisms within the groundwater.

Microcosm Results
After 28 days, the appearance of some microcosms solutions started to change, and the bottles' solutions containing nitrogen and phosphorus source in warm conditions turned cloudy; the rest of the bottles remained clear with the original color, as depicted in Figure 1.For the samples stored at 10 °C this visual change happened after 60 days.
The analyses of the ions in the samples showed that the reduction process of chlorate and chromate occurred simultaneously.Reductions in concentrations of chlorate and hexavalent chromate are depicted in Figure 2a through 2d for all five series in the experiment period and discussed in the subsequent sections.

Chlorate
The initial chlorate concentration of 1000 mg/L was constant in the first 14 days of study for all samples.On day 21, the chlorate content of the samples slightly decreased to 88 and 84% of the initial concentration in C + NP + M and C + NP bottles, respectively, at room temperature.After day 35, the concentration of chlorate was below the detection limit of 1 mg/L.In the C + NP + M microcosms at a lower temperature, the concentration change started after 50 days, and on day 58, it dropped to 770 mg/L.At 80 days, the chlorate concentration of all samples with added nitrogen and phosphorous were below the detection limits of 1 mg/L, while the chlorate content of the solutions that did not receive any nitrogen and phosphorous additives continued unaffected as depicted in Figure 2b.
The results indicate that adding only acetate as an electron donor is insufficient to reduce chlorate and chromate in the microcosms, as their concentration in the "C" series is stable throughout the experiment period.The deficiency of macronutrients in groundwater must be compensated by appropriate bioavailable forms of nitrogen and phosphorous sources.Comparing the "C + M" and "C + NP + M" graphs, Figure 2a and 2b with the "C" and "C + NP" series, reveal that the removal rates and yields are not affected by the minerals solutions indicating that there were sufficient trace minerals included with the inoculum.

Chloride
Chloride is the final product of the chlorate reduction process; therefore, it is an appropriate indicator to track the reaction chain completion.This ion's concentrations showed an insignificant change in the first 21 days and then soared to 320 mg/L in the "C + NP" group and 355 mg/L in the "C + NP + M" series when no detectible chlorate was left in warm conditions.Bottles in the cold chamber at 10 °C, had not quite reached completion by the last sampling time (80 days).The other two groups, "C" and "C + M," steadily followed a flat line for chlorate and chloride ion since microorganisms did not transform the ions, as shown in Figure 2c.

Hexavalent Chromium
The initial concentration of hexavalent chromium of 3 mg/L remained unchanged in the first 3 weeks of the experiments.This trend continued similarly for the series without nitrogen and phosphorus containing only acetate or acetate plus minerals solution after 80 days.Chromate concentrations dropped after 35 days to 0.65 and 0.83 (as an average of the triplicates) for C + NP and C + NP + M solutions at 20 °C, and it was completely reduced on day 50.The concentration of hexavalent chromium in the samples at 10 °C slightly changed after 58 days and then decreased rapidly at day 80, as is shown in Figure 2a.
After observing Cr (VI) ion reduction in some bottles, samples were taken from all the series, filtered through 0.45-μm filters to measure the total chromium content with an ICP machine suitable for measuring metal content in aqueous phase.Figure 3 shows total chromium concentration using ICP.The graph compares the microcosm groups on days 35, 50, and 80 that indicate that chromium was transformed from soluble Cr (VI) that passed the filter to an insoluble jelly-like precipitate of Cr (III) that was retained by the 0.45 μm filter.

Substrate Consumption and Molar Ratios
Comparing the molar trends for solutions where bioremediation occurred reveals substrate consumption and process completion information.These graphs are shown in Figure 4 a-c.The chlorate concentration decreases in all three microcosms series, while chloride concentration increases.The trends for these two ions prove that the microorganisms utilized acetate, added as the electron donor and a carbon source to the microcosm's media.The chlorate reduced to chloride rapidly once sufficient active biomass was available.In the microcosms without added N and P, the acetate consumption after 80 days of incubation was about 7.26 mM.In Figure 2, the concentration of acetate dropped from an initial concentration of 2200 mg/L to 1719 mg/L in the "C" microcosms and 1662 mg/L in the "C + M" microcosms.However, in the microcosms containing the acetate with nitrogen and phosphorous source, the acetate consumption was higher, especially after day 21, when the chlorate concentration started decreasing significantly (Figure 4a and 4b).
The reduction in concentrations of acetate was much greater than the stoichiometric consumption of acetate predicted from the reduction of chlorate.Each mole of chlorate can oxidize ¾ of a mole of acetate to bicarbonate.With the addition of 12 mM of chlorate, we would have expected to consume 9 mM of acetate when all of the chlorate was consumed.On day 35, when chlorate was not detected in the microcosms amended with N and P, 27.26 mM acetate was consumed for the "C + NP" samples at room temperature, 25.06 mM acetate was consumed in "C + NP + M" samples, and 25.51 mM for the samples at 10°C after 80 days.Considering these values, the minimum amount of the electron donor that must be added to complete chlorate removal is about three times higher than the stoichiometric ratios.While ¾ of the mole covers the electron demand of the remediation process, the rest is most likely utilized as a carbon source to create biomass of the microorganisms that reduced the chlorate and other microorganisms.

Mass Balances Chlorate/Chloride Mass Balance
The initial concentration of chlorine atoms in the solution was 11.97 mM, the same as the chlorate concentration.On a molar basis, each mole of chlorate should result in a mole of chloride ion when reduction of chlorate to chloride is complete.In the treatments without added N and P, more than 90% of the initial concentration of chlorate was present as chlorate or chloride throughout the experiment.For the bottles that did not have enough time to reach the final stage of chlorate transformation, this content comes down to around 80% for microcosms with C + N + P on day 50 and for microcosms with C + N + P + M on days 35 and 50 and for microcosms with C + N + P + M, Cold on the day 80.This value for C + N + P samples at day 35 is even lower and stands at 75% as an average for three bottles.The formation of chlorite and hypochlorite as intermediates before chloride is a possible reason for this deviation (chlorite and hypochlorite were not measured).Figure 5 illustrates the ratio of measured chlorate and chloride to total chlorine in all microcosms.

Chromium Mass Balance
The chromium initial concentration in the synthetic groundwater in the microcosms media was 3 mg/L.Figure 6 compares chromium concentrations in the filtrate of samples from the microcosms (after 50 days incubation) and recovered chromium from the solid phase retained in the filter paper.The digested filter papers used to filter water samples from the microcosms showed each filter could affect the chromium result as much as 0.022 mg/L on each sample; this is lower than the ICP machine's detection limit and negligible.C + N + P + M sample at room temperature has the highest recovery rate, 2.83 mg/L, and C + N + P + M, cold, and C + N + P follow that by 2.74 and 2.69 mg/L, respectively.The result provides an acceptable range of recovery rate of 92% as an average for the method and proves that the reduced Cr(VI) is not in soluble form and remains in the filter with the biomass and soil particles.

ATP Analysis
The concentration of ATP (adenosine triphosphate) in the microcosm water samples was measured after 50 and 80 days, and the results are depicted in Figure 7.The concentration of ATP in groundwater from the site that was used as an inoculum was 223 pg./L.In the "C" and "C + M" treatments, the concentration after 50 and 80 days varied between 885 and 2104 pg./L, roughly 10-fold higher.In the "C + NP", the "C + NP + M" and "C+NP+M (Cold) treatments, the concentration of ATP was more than 100fold higher.Although "C" and "C + M" mediums show a minor increase of ATP over time, the difference between them and samples containing nitrogen and phosphorous sources is meaningful.After 50 and 80 days of incubation, there is higher metabolic activity, as reflected by ATP content, in the biomass of the microcosms containing nitrogen and phosphorous relative to those not containing these nutrients.The elevated concentrations of the ATP analysis result for the microcosms at 10 °C compared to their initial values, confirm that lowering the temperature only slows down the growth rate of the biomass without decreasing the ultimate microbial activity potential.

Conclusion
Bioremediation of two ions, hexavalent chromium and chlorate was investigated in laboratory microcosms.Both ions were removed from the samples by native microorganisms collected from the contaminated site's soil and groundwater.These microorganisms utilized acetate as an electron donor and carbon source to reduce the ions below the acceptable limits under the anaerobic condition at 10 °C and 20 °C.The reduction process needed nitrogen and phosphorous addition to complete, and the samples that contained the electron donor without these macronutrients did not show any significant reduction.Micronutrient solutions prepared based on ATCC 1191 minerals recipe did not affect the remediation rate in any samples, indicating that the soil added with the inoculum provided sufficient micronutrients.
The final product of chlorate reduction, chloride, was measured and produced proportionally in the experiment, indicating reaction completion.Acetate was consumed in excess of its theoretical stoichiometric demand for chlorate reduction in the microcosms since it since it could be also used as a carbon source to produce biomass.
Hexavalent chromium had the same fate in these experiments and was reduced to chromium (III) by the microorganisms in the presence of the electron donor and macronutrients.The reaction rate for chromate is substantially slower than chlorate.After 35 days of incubation at 20 °C, 1000 mg/L of chlorate was entirely removed, but only 75% of 3 mg/L of hexavalent chromium was removed.At lower temperature (10 °C) the removal occurred at a slower pace.Final concentration of chlorate and chromate in cold conditions reach regulatory levels in a longer time (80 days) with the same nutrients' addition.
Overall bioremediation of groundwater contaminated with chlorate and chromate is possible with native microorganisms provided suitable electron donor, nitrogen, and

Figure 5 .
Figure 5. Ratio of measured chlorate and chloride to total chlorine.
. This bioremediation is possible in cold climate conditions.