Microbial Isotopic Fractionation of Perchlorate Chlorine

Perchlorate contamination can be microbially respired to innocuous chloride and thus can be treated effectively. However, monitoring a bioremediative strategy is often difficult due to the complexities of environmental samples. Here we demonstrate that microbial respiration of perchlorate results in a significant fractionation (~−15‰) of the chlorine stable isotope composition of perchlorate. This can be used to quantify the extent of biotic degradation and to separate biotic from abiotic attenuation of this contaminant.     

Peptide biomarkers as evidence of perchlorate biodegradation

Perchlorate is a known health hazard for humans, fish, and other species. Therefore, it is important to assess the response of an ecosystem exposed to perchlorate contamination. The data reported here show that a liquid chromatography-mass spectrometry-based proteomics approach for the detection of perchlorate-reducing enzymes can be used to measure the ability of microorganisms to degrade perchlorate, including determining the current perchlorate degradation status. Signature peptides derived from chlorite dismutase (CD) and perchlorate reductase can be used as biomarkers of perchlorate presence and biodegradation. Four peptides each derived from CD and perchlorate reductase subunit A (PcrA) and seven peptides derived from perchlorate reductase subunit B (PcrB) were identified as signature biomarkers for perchlorate degradation, as these sequences are conserved in the majority of the pure and mixed perchlorate-degrading microbial cultures examined. However, chlorite dismutase signature biomarker peptides from Dechloromonas agitata CKB were found to be different from those in other cultures used and should also be included with selected CD biomarkers. The combination of these peptides derived from the two enzymes represents a promising perchlorate presence/biodegradation biomarker system. The biomarker peptides were detected at perchlorate concentrations as low as 0.1 mM and at different time points both in pure cultures and within perchlorate-reducing environmental enrichment consortia. The peptide biomarkers were also detected in the simultaneous presence of perchlorate and an alternate electron acceptor, nitrate. We believe that this technique can be useful for monitoring bioremediation processes for other anthropogenic environmental contaminants with known metabolic pathways.     

Environmental factors that control microbial perchlorate reduction

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter(-1) were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.     

A Comparative Toxicological Assessment of Perchlorate and Thiocyanate Based on Competitive Inhibition of Iodide Uptake as the Common Mode of Action

Thiocyanate and perchlorate are known to competitively inhibit thyroidal iodide uptake at the sodium-iodide symporter. Estimates of their relative potencies have recently been refined; thiocyanate is 15 times less potent than perchlorate on a serum concentration basis. Numerous studies have been published relating serum thiocyanate concentrations (or surrogate measures) with thyroid function in various populations including pregnant women and neonates in regions with varying degrees of iodine deficiency. Fifteen published studies were located that relate serum thiocyanate concentrations with thyroid function. In the absence of severe iodine deficiency or iodine excess, adverse thyroidal effects occur with chronic serum thiocyanate concentrations ≥ 200 μ mol/L whereas non-adverse effects are observed with concentrations in the range of 65–85 μ mol/L. No adverse or non-adverse effects are observed at serum concentrations below 50 μ mol/L, even among sensitive subpopulations. Recently, studies relating serum perchlorate concentrations with perchlorate dose have become available, thus making it possible to predict the perchlorate dose associated with a serum perchlorate concentration. Serum thiocyanate concentrations found to induce non-adverse or adverse thyroid effects can thereby be used to predict the perchlorate concentration and thus the perchlorate dose that would be expected to induce similar effects. To place a perspective on environmental perchlorate exposure, a serum thiocyanate concentration of 50 μ mol/L is equivalent to a serum perchlorate concentration of 3.3 μ mol/L in terms of iodine uptake inhibition. This serum perchlorate concentration would require a perchlorate dose of 0.27 mg/kg-day, or a drinking water equivalent level of 9 mg/L using standard default assumptions of a 70 kg adult drinking 2 liters of water daily.     

Environmental Factors That Control Microbial Perchlorate Reduction

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter⁻¹ were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.     

Direct measurement of perchlorate exposure biomarkers in a highly exposed population: a pilot study

Exposure to perchlorate is ubiquitous in the United States and has been found to be widespread in food and drinking water. People living in the lower Colorado River region may have perchlorate exposure because of perchlorate in ground water and locally-grown produce. Relatively high doses of perchlorate can inhibit iodine uptake and impair thyroid function, and thus could impair neurological development in utero. We examined human exposures to perchlorate in the Imperial Valley among individuals consuming locally grown produce and compared perchlorate exposure doses to state and federal reference doses. We collected 24-hour urine specimen from a convenience sample of 31 individuals and measured urinary excretion rates of perchlorate, thiocyanate, nitrate, and iodide. In addition, drinking water and local produce were also sampled for perchlorate. All but two of the water samples tested negative for perchlorate. Perchlorate levels in 79 produce samples ranged from non-detect to 1816 ppb. Estimated perchlorate doses ranged from 0.02 to 0.51 μg/kg of body weight/day. Perchlorate dose increased with the number of servings of dairy products consumed and with estimated perchlorate levels in produce consumed. The geometric mean perchlorate dose was 70% higher than for the NHANES reference population. Our sample of 31 Imperial Valley residents had higher perchlorate dose levels compared with national reference ranges. Although none of our exposure estimates exceeded the U. S. EPA reference dose, three participants exceeded the acceptable daily dose as defined by bench mark dose methods used by the California Office of Environmental Health Hazard Assessment.     

The use of sodium perchlorate in deproteinization during the preparation of nucleic acids (Short Communication)

Sodium perchlorate in high concentrations will remove from solution the detergent sodium dodecyl sulphate and protein complexed with it. This and the failure of proteins to be precipitated by ethanol from solutions containing a high concentration of sodium perchlorate can be utilized as efficient, rapid and simple deproteinization procedures during the preparation of nucleic acids.     

Characterization of a microbial community capable of reducing perchlorate and nitrate in high salinity

Denitrifying up-flow packed-bed bioreactors fed with perchlorate and nitrate allowed for the examination of the impact of a variety of salt conditions (up to 10% w/v NaCl) on the complete perchlorate and nitrate removal capacity of the reactor using activated sludge taken from a municipal wastewater treatment plant. Based on the evaluation of the microbial community in the bioreactor by cloning analysis, Clostridium sp. and a Rhodocyclaceae bacteria were identified as the dominant clones. This suggests that they may be tolerant to high salt and can reduce both nitrate and perchlorate in such conditions.     

Bioremediation of Chlorate or Perchlorate Contaminated Water Using Permeable Barriers Containing Vegetable Oil

A scale model of an in situ permeable barrier, formed by injecting vegetable oil onto laboratory soil columns, was used to remove chlorate and perchlorate from flowing groundwater. The hypothesis that trapped oil would serve as a substrate enabling native microorganisms to reduce chlorate or perchlorate to chloride as water flowed through the oil-rich zone had merit. Approximately 96% of the 0.2 mM chlorate and 99% of the 0.2 mM perchlorate present in the water was removed as water was pumped through columns containing vegetable oil barriers. The product formed was chloride. When nitrate at 1.4 mM was added to the water, both nitrate and chlorate were removed. High concentrations of chlorate or perchlorate can be treated; 24 mM chlorate and 6 mM perchlorate were completely reduced to chloride during microcosm incubations. Microorganisms capable of reducing perchlorate are plentiful in the environment. Received: 19 December 2001 / Accepted: 25 January 2002     

Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars

In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.     

Perchlorate Remediation by Electrokinetic Extraction and Electrokinetic Injection of Substrates

Perchlorate (ClO₄^-) contamination of groundwater has recently become a major concern across the nation. Electrokinetic (EK) extraction with the simultaneous EK injection of organic material to promote degradation could allow for the efficient removal of perchlorate while simultaneously promoting degradation of perchlorate. Column experiments were conducted to evaluate the technology. Lactate and glycine served as organic substrates to promote degradation after injection into the columns as well as maintaining the pH near neutral. Removal of perchlorate from contaminated materials kaolin, sand, and a natural soil historically contaminated by perchlorate was controlled by the ionic flux of perchlorate and not by transport from the osmotic flux which was only significant for kaolin experiments. Perchlorate was removed from contaminated sand and clay below our detection limits (5 ppb). Both lactic acid and glycine were successfully injected into clay and a sand matrix. Results from a contaminated site soil indicate that the Chemical Oxygen Demand was increased after electrokinetic injection of glycine and lactate. Experiments using soil from a contaminated site confirmed that EK can be used to both remove perchlorate and stimulate bioremediation by the injection of lactate or glycine. The use of EK technology to both remove and provide for continued source removal by bioremediation offers a potential new tool to treat low permeability systems.     

Perchlorate Remediation by Electrokinetic Extraction and Electrokinetic Injection of Substrates

Perchlorate (ClO4?) contamination of groundwater has recently become a major concern across the nation. Electrokinetic (EK) extraction with the simultaneous EK injection of organic material to promote degradation could allow for the efficient removal of perchlorate while simultaneously promoting degradation of perchlorate. Column experiments were conducted to evaluate the technology. Lactate and glycine served as organic substrates to promote degradation after injection into the columns as well as maintaining the pH near neutral. Removal of perchlorate from contaminated materials kaolin, sand, and a natural soil historically contaminated by perchlorate was controlled by the ionic flux of perchlorate and not by transport from the osmotic flux which was only significant for kaolin experiments. Perchlorate was removed from contaminated sand and clay below our detection limits (5 ppb). Both lactic acid and glycine were successfully injected into clay and a sand matrix. Results from a contaminated site soil indicate that the Chemical Oxygen Demand was increased after electrokinetic injection of glycine and lactate. Experiments using soil from a contaminated site confirmed that EK can be used to both remove perchlorate and stimulate bioremediation by the injection of lactate or glycine. The use of EK technology to both remove and provide for continued source removal by bioremediation offers a potential new tool to treat low permeability systems.     

Investigation of Factors Affecting the Bioregeneration Process for Perchlorate-Laden Gel-Type Anion-Exchange Resin

Ion exchange is the most common process for perchlorate removal from waters. Selective ion-exchange resins are widely used for perchlorate removal from waters, but are incinerated after one-time use, making the ion-exchange process incomplete for perchlorate removal. As perchlorate ions are readily biodegradable, direct contact of spent ion-exchange resins with perchlorate-reducing bacteria for its regeneration has been studied recently. In this research, some factors affecting the bioregeneration of perchlorate-laden gel-type anion-exchange resin were investigated. Bioregeneration is a sustainable process when compared to one-time use of resin and disposal by incineration. Batch bioregeneration experiments were performed to determine (a) the effect of initial perchlorate load in the resin, (b) the effect of microbial concentration, and (c) the effect of nitrate load on the degradation of perchlorate in the resin bead. The results of the bioregeneration tests suggested that the bioregeneration process may be controlled by both kinetics and diffusion. Higher perchlorate load in the resin had a positive effect on perchlorate degradation rates, whereas varying microbial concentration did not have a significant effect on perchlorate degradation in gel-type resin. The presence of nitrate suppressed perchlorate degradation initially, but once all nitrate was utilized, perchlorate degradation took place.     

Biotic resistance to exotic invasions: its role in forest ecosystems,confounding artifacts,and future directions

Biotic resistance, the ability of communities to resist exotic invasions, has long attracted interest in the research and management communities. However, inconsistencies exist in various biotic resistance studies and less is known about the current status and knowledge gaps of biotic resistance in forest ecosystems. In this paper, we provide a brief review of the history and mechanisms of the biotic resistance hypothesis, and summarize the central topics and knowledge gaps related to biotic resistance with a special emphasis on forest ecosystems. Overall, although the amount of research efforts on biotic resistance in forest ecosystems has increased since the mid-2000s, aspects such as resistance to exotic pests and pathogens remain understudied. In addition, we synthesize ecological and statistical explanations of observed inconsistencies and provide suggestions for future research directions. Some of the observed inconsistencies on biotic resistance can be attributed to (1) the interactive or additive effects of other ecological processes and (2) the statistical artifacts of modifiable areal unit problem. With the advancement of new statistical knowledge and tools, along with availability of big data, biotic resistance research can be greatly improved with the simultaneous consideration of key ecological processes, the attention to various scales involved, and the addition of understudied systems.     

Perchlorate-reducing microorganisms isolated from contaminated sites

An extensive microcosm survey of perchlorate-contaminated sites was undertaken to assess the ability of indigenous microorganisms to degrade perchlorate. Samples from 12 contaminated sites and from one pristine location were analysed. Perchlorate was degraded to below detection limit in all electron donor-amended microcosms. Perchlorate-reducing microorganisms (PRMs) were numerous at most of these sites. Sixteen distinct PRMs were isolated that were phylogenetically related to either Dechloromonas in the Beta Proteobacteria (9/16 isolates) or to Azospirillum in the Alpha Proteobacteria (7/16 isolates). The majority of previously isolated PRMs are in the Beta Proteobacteria related to Dechloromonas or Dechlorosoma. This study indicates that PRMs of the genus Azospirillum may be more prevalent at contaminated sites than the current record of isolates suggests. Cell yields, electron donor to perchlorate ratios and maximum specific growth rates were similar among the isolates and similar to the few previously published values. However, the Monod half-saturation constants for perchlorate for the two Azospirillum isolates characterized were lower than those measured for other genera, suggesting that they may be more effective at low concentrations of perchlorate. These results extend the current understanding of PRMs from diverse environments and provide added confidence that microbial perchlorate reduction is ubiquitous, even at highly contaminated sites, and can be harnessed effectively for bioremediation.     

Modelling salinity inhibition effects during biodegradation of perchlorate

AIMS: To determine the mathematical kinetic rates and mechanisms of acclimated perchlorate (ClO)-reducing microbial cultures by incorporating a term to relate the inhibitory effect of high salinity during biological reduction of concentrated perchlorate solutions. METHODS AND RESULTS: Salt toxicity associated with the biodegradation of concentrated perchlorate (200, 500, 1100, 1700 and 2400 mg l(-1) as ClO) was investigated using two microbial cultures isolated from a domestic wastewater treatment plant [return activated sludge (RAS) and anaerobic digester sludge (ADS)]. Experiments were performed in wastewaters containing various sodium chloride concentrations, ranging from 0% to 4.0% (w/v) NaCl (ionic strength: 0.14-0.82 mol l(-1), total dissolved solids: 5.3-42.6 g l(-1)) at near-neutral values of pH (6.7-7.8). Perchlorate biodegradation was stimulated through stepwise acclimation to high salinity. The ADS culture was capable of reducing perchlorate at salinities up to 4% NaCl, while the RAS culture exhibited complete inhibition of perchlorate degradation at 4% NaCl, probably resulting from either a toxic effect or enzyme inactivation of the perchlorate-reducing microbes. Further, a kinetic growth model was developed based on experimental data in order to express an inhibition function to relate specific growth rate and salinity. CONCLUSIONS: Biological reduction of concentrated perchlorate wastewaters using either acclimated RAS or ADS cultures is feasible up to 3% or 4% NaCl, respectively. In addition, the kinetic model including a salinity inhibition term should be effective in many practical applications such as improving reactor design and management, furthering the understanding of high salinity inhibition, and enhancing bioremediation under high salinity loading conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Applications of these findings in water treatment practice where ion exchange or membrane technologies are used to remove perchlorate from water can have the potential to increase the overall attractiveness of these processes by eliminating the need to dispose of a concentrated perchlorate solution.     

高氯酸盐污染及修复的研究进展

戏真做高氯酸盐作为一种甲状腺干扰物质,因其高溶解度、低吸附性及其稳定性,决定了其是一种可快速扩散的持久型污染物,其环境污染问题已引起人们的高度关注。从高氯酸盐污染的危害性出发,阐述了我国可能存在的高氯酸盐污染状况,并对近年来不同介质中高氯酸盐的分析检测技术及前处理方法进行了介绍,同时对已开展的高氯酸盐动物毒理研究、在不同环境介质中的迁移转化和植物积累、及其按生物修复和非生物修复进行分类的修复方法进行了综述。最后,对进一步研究的意义和方向提出建议。     

Mechanistic changes during phytoremediation of perchlorate under different root-zone conditions

Two types of hydroponic bioreactors were used to investigate the mechanisnistic changes during phytoremediation of perchlorate under different root-zone conditions. The bioreactors included: (1) an aerobic ebb-and-flow system planted with six willow trees, and (2) individual willow trees grown in sealed root-zone bioreactors. Rhizosphere probes were used to monitor for the first time during phytoremediation of perchlorate, diurnal swings in oxidation-reduction potential (E(H)), dissolved oxygen (DO), and pH. Radiolabeled (36Cl-labeled) perchlorate was used as a tracer in a subset of the sealed bioreactor experiments to quantify the contribution of phytodegradation and rhizodegradation mechanisms. Rhizodegradation accounted for the removal of 96.1 +/- 4.5% (+/-95% CI) of the initial perchlorate dose in experiments conducted in sealed hydroponic bioreactors with low DO and little or no nitrate N. Meanwhile, the contribution of rhizodegradation decreased to 76 +/- 14% (+/-95% CI) when nitrate (a competing terminal electron acceptor) was provided as the nitrogen source. Slower rates of phytoremediation by uptake and phytodegradation were observed under high nitrate concentrations and aerobic conditions, which allowed perchlorate to persist in solution and resulted in a higher fraction uptake by the plant. Specifically, the rate of removal of perchlorate from bulk solution ranged from 5.4 +/- 0.54 to 37.1 +/- 2.25 mg/L/d (+/-SE) in the absence of nitrate to 1.78 +/- 0.27 to 0.46 +/- 0.02 mg/L/d (+/-SE) at high nitrate concentration. The results of this study indicate that the root-zone environment of plants can be manipulated to optimize rhizodegradation and to minimize undesirable processes such as uptake, temporal phytoaccumulation, and slow phytodegradation during phytoremediation of perchlorate. Rhizodegradation is desired because contaminants resident in plant tissue may remain an ecological risk until completely phytodegraded.     

高氯酸盐胁迫对水稻生长发育和养分吸收的影响

通过土培盆栽试验,研究了0.2、2.0和4.0mmol/kg 3种浓度下高氯酸盐胁迫对两个水稻品种的生长发育和吸收主要养分元素氮、磷、钾的影响。研究发现:(1)高氯酸盐对水稻生长的抑制程度随高氯酸盐处理浓度的增高而加重,且随着处理时间的延长,水稻的受害症状也越来越明显,到分蘖期时,3个浓度组对赣糯香生长的抑制率分别为3.08%、33.39%和39.03%,对IR65598-112-2生长的抑制率分别为9.18%、21.07%和34.97%。(2)各浓度处理组(赣糯香0.2mmol/kg组除外)都显著抑制了两品种水稻的分蘖。(3)各浓度处理组水稻始穗时间都比对照组晚,其中高浓度处理组晚了1个月。(4)IR65598-112-2对高氯酸盐的胁迫表现出更早的反应和对低浓度污染物的敏感性,而赣糯香表现出相对较强的抗性。(5)高氯酸盐对水稻根部的伤害比对地上部的伤害更严重。(6)高氯酸盐处理减少了水稻各器官的生物量,降低了水稻各器官中养分元素总氮和总磷的总积累量。研究表明,高氯酸盐污染可抑制两品种水稻的生长和分蘖,延迟其发育,降低水稻对养分元素的积累,其植物毒性效应与污染物的浓度、处理的延续时间、水稻品种及不同植物器官都有关系。