Isolation and use of perchlorate and nitrate reducing bacteria

Details Isolation and use of perchlorate and nitrate reducing bacteria Isolation and use of perchlorate and nitrate reducing bacteria

2000-11-16

2002-07-23

Beisner, William H. (Department: 1744)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Destruction of hazardous or toxic waste

C435S252100, C210S610000, C210S611000

Reexamination Certificate

active

06423533

ABSTRACT:

FIELD OF INVENTION
This invention relates to perchlorate and nitrate metabolizing bacteria, their isolation and method of use. More particularly, this invention relates to bacteria useful in the remediation of perchlorate and nitrate contaminated materials and means and materials to enhance such remediation.
BACKGROUND
The use of bacteria to treat perchlorate and nitrate contaminated material such as soil and water is described in the “Description of Related Art” of U.S. Pat. No. 6,077,429 of William T. Frankenberger, Jr. and David Herman, issued Jun. 20, 2000, entitled
Bacterial Removal of Perchlorate and Nitrate,
which patent is incorporated herein by reference. As discussed by Coates, Michaelidou, Bruce, O'Connor, Crespi, and Achenbach, in
Ubiquity and Diversity of Dissimilatory
(
Per
)
chlorate-Reducing Bacteria,
in
Applied and Environmental Microbiology,
December, 1999, only six organisms capable of obtaining energy for growth by the metabolism of compounds containing oxyanions of chlorine, such as perchlorates, had previously been identified at the time of Coates et al. writing, even though the use of such microbes to reduce such compounds has been known for more than fifty years. The Coates et al. article is incorporated herein by reference.
Coates et al. point out that the discovery of a phylogenitically diverse group of organisms that had evolved with the ability to couple growth to the reduction of perchlorate was unexpected. Also unexpected was the discovery of perchlorate metabolizing bacteria in environments free of perchlorates. Frankenberger et al. teach that the presence of nitrate inhibits the reduction of perchlorate by a perchlorate metabolizing bacterium.
Perchlorate is a strong oxidizing agent in its associated form and is principally manufactured as the oxidizing component in propellants and explosives. In its aqueous ionic form, the perchlorate oxyanion is extremely stable and mobile, making effective treatment difficult and expensive. It has been estimated that under typical ground and surface water conditions, the perchlorate anion may persist for decades. As the concern of perchlorate in groundwater has taken on new importance nationwide, multiple studies are currently being conducted that focus on improved analytical methods, human health assessments, ecological impact assessments, and improved treatment technologies.
It has long been known that perchlorate has the potential to perturbate the mammalian hypothalamic-pituitary-thyroid axis. Specifically, perchlorate inhibits thyroid iodide anion uptake through the action of competitive binding. This leads to reduced T3 and T4 thyroid hormones, resulting in excess Thyroid Stimulating Hormone (TSH) by the pituitary gland (Anbar et al., 1959; Stanbury and Wyngaarden, 1952; Wolff, J., 1998). Prolonged perturbations may ultimately result in thyroid neoplasia, especially in sensitive rodent species. The California Department of Health Services has adopted an action level of 18 &mgr;g L
−1
perchlorate in drinking water.
A 4.4 acre constructed wetland system, at the Apache Powder Superfund Site (APS) in Cochise County, Arizona, referred to as the Northern Area Remediation System (NARS), consisting of three primary denitrification cells, an aerobic nitrification cell, and a final denitrification cell, is intended to denitrify high levels of nitrate found in the shallow aquifer. Previous engineering design and modeling efforts for NARS did not anticipate the presence of perchlorate. Therefore, after perchlorate discovery at APS in 1998, the inventors established the current study to investigate the possibility that perchlorate would interfere with or diminish the capability of the NARS to treat nitrate containing groundwater.
Data regarding the effects of perchlorate on denitrification is generally limited. Herman and Frankenberger, Jr. (1999), using a bacterial isolate known as perclace, found a decrease in the rate of denitrification when the concentrations of nitrate and perchlorate were equal at 1 mM, requiring 48 h for complete reduction. However, when perchlorate levels were reduced to 0.089 mg L
−1
, complete nitrate reduction required only 24 h. However, Herman and Frankenberger, Jr. (1999) focused on a single bacterial isolate and not on the assemblage of microorganisms known to denitrify. One aspect of the current study focused on determining the potential effects of high levels of perchlorate on a mixed inoculant sampled from an operating wetland wastewater treatment system.
Dendooven and Anderson (1994) reported that in the presence of perchlorate, nitrous oxide production was low during the first 3-4 h, then increased sharply at 4 h and held constant for the next 20 h. After 24 h, all of the nitrate was reduced and very little nitrous oxide was produced. They suggest the lag time was due to two factors, the persistence of oxygen which delayed the de-repression of the reduction enzyme system and the kinetics of the denitrification process.
Strategies for the removal of perchlorate based on adsorption by activated carbon or use of reverse osmosis and ion exchange have not shown remediation solutions as promising as biological processes. Microorganisms utilization to degrade perchlorate in anaerobic or microaerophylic conditions to innocuous end-product, namely chloride are by far the most promising perchlorate remediation technology. Such biological treatment can be further used for the simultaneous treatment of perchlorate and nitrate. Wetlands typically contain extensive anaerobic or microaerophylic environments due to natural decomposition of plants, algae, fungi, bacteria and other organic material. Therefore, the current study also initiated preliminary treatability experiments designed to determine if enriched or non-enriched wetland derived cultures are capable of perchlorate reduction and to determine baseline kinetics.
Identified perchlorate reducers fall into several different categories. Coates et al. (1999) investigated six different environments including pristine soil, paper mill waste sludge, heavy metal contaminated aquatic sediments, hydrocarbon contaminated lake sediments, hydrocarbon contaminated soils, and animal waste treatment sludge. They recovered perchlorate reducers from all six environments. Coates et al. (1999) isolated 13 (per)chlorate reducing bacteria (CIRB), eight of which were characterized. Collectively, they represent broad phylogenetic diversities. All of the isolates were members of Proteobacteria. These bacteria were typified as being motile, gram-negative, non-fermentative, and facultative anaerobes. Their optimum growth occurred at 35° C., pH 7.5 and 1% NaCl. All could utilize acetate, propionate, isobutyrate, butyrate, valerate, malate, fumerate and lactate as electron donors, while none could utilize methanol, catechol, glycerol, citrate, glucose or hydrogen. All of the characterized bacteria could use chlorate and oxygen as electron acceptors, but could not utilize sulfate, selenate, fumerate, malate, Mn(IV), or Fe(III). Coates et al. (1999) suggests these genera may be the dominant perchlorate reducing bacteria in the environment. Coates et al. (1999) identified and named two species of perchlorate reducers in the &bgr; sub-division of Proteobacteria,
Dechlorimonas agitatus
and
Dechlorosoma suilla.
In a related study, Michaelidou et al. (1999) isolated two Proteobacteria strains from a swine waste lagoon. They were both typified by being non-fermentative, mesophilic, motile, gram-negative bacteria. One strain, designated as PS, was rod-shaped and 0.2 &mgr;m by 2 &mgr;m in length and placed within the &bgr; sub-division of Proteobacteria. Nearly complete 16S rRNA sequencing indicated that the closest known relative was
Rhodocyclus tenuis.
The second strain, designated as WD, was placed into the &agr; sub-division and shared 94.6% similarity to
Magnetospirillum gryphiswaldense.
Strain WD grew as a spirillum, but did not produce magnetosomes when grown in iron based media and there was no indication of magnetotaxis. Malmqvist e

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