D. L. Sorensen

Ultraviolet inactivation of selected bacteria and viruses with photoreactivation of the bacteria

Abstract u.v. dose/survival response curves were developed for E. coli, S. faecalis , poliovirus and reovirus. The influence of photoreactivation on the bacterial curves was examined. A maximum photoreactivation of 3.4 and 2.4 log of u.v. inactivated E. coli and S. faecalis populations, respectively, was observed. When allowing for photoreactivation, the dose required for 99.9% inactivation of the bacteria was approximately twice that required when photoreactivation was not considered. Reovirus, a double-standard RNA virus, was found to be significantly more resistant to u.v. radiation than poliovirus, a single-stranded RNA virus. The viruses in general, were found to be more resistant to u.v. radiation than the bacteria.

Isolation and Characterization of Polycyclic Aromatic Hydrocarbon–Degrading Mycobacterium Isolates from Soil

Bioremediation of soils contaminated with wood preservatives containing polycyclic aromatic hydrocarbons (PAHs) is desired because of their toxic, mutagenic, and carcinogenic properties. Creosote wood preservative–contaminated soils at the Champion International Superfund Site in Libby, Montana currently undergo bioremediation in a prepared-bed land treatment unit (LTU) process. Microbes isolated from these LTU soils rapidly mineralized the 14C-labeled PAH pyrene in the LTU soil. Gram staining, electron microscopy, and 16S rDNA-sequencing revealed that three of these bacteria, JLS, KMS, and MCS, were Mycobacterium strains. The phylogeny of the 16S rDNA showed that they were distinct from other Mycobacterium isolates with PAH-degrading activities. Catalase and superoxide dismutase (SOD) isozyme profiles confirmed that each isolate was distinct from each other and from the PAH-degrading mycobacterium, Mycobacterium vanbaalenii sp. nov, isolated from a petroleum-contaminated soil. We find that dioxygenase genes nidA and nidB are present in each of the Libby Mycobacterium isolates and are adjacent to each other in the sequence nidB-nidA, an order that is unique to the PAH-degrading mycobacteria.

Clostridium perfringens as a point source indicator in non-point polluted streams

Abstract Clostridium perfringens spores were evaluated as an indicator of point source pollution in small streams whose bacteriological water quality was influenced by both point and non-point sources. Samples were collected from small streams (the Rio San Jose, New Mexico and Worm Creek, Idaho/Utah); the Grants, New Mexico; Preston, Idaho; Hyrum, Utah; and San Jose/Santa Clara, California; sewage treatment plant effluents; and the Franklin, Idaho; Logan, Utah; and Wellsville, Utah: facultative sewage lagoons. In both of the small streams, C. perfringens spores could be detected, in decreasing concentrations, for more than 10 km downstream from municipal wastewater treatment plant effluents. Coliform and fecal streptococci concentrations varied widely along the streams in apparent response to non-point sources. Samples of cow, horse and sheep feces; farmlot runoff; oxbow lakes that receive fecal material from animal feeding facilities; and from animal grazing area soil were low in C. perfringens spores, but contained high concentrations of other fecal indictor bacteria. C. perfringens spores appear to be a sensitive indicator for microorganisms entering streams with municipal wastewater, even when agricultural non-point sources of fecal indicator bacteria are important in the receiving stream. Samples of effluents taken from sewage treatment plants with sand filtration processes, and from facultative sewage lagoons, indicate that C. perfringens spores concentrations may be low in these effluents.

Polycyclic Aromatic Hydrocarbon Biodegradation as a Function of Oxygen Tension in Contaminated Soil

Abstract Laboratory tests were conducted to determine the effect of soil gas oxygen concentration on the degradation and mineralization of spiked 14C-pyrene and nonspiked 16 priority pollutant polycyclic aromatic hydrocarbons (PAH) present in the soil. The soil used for the evaluation was taken from a prepared-bed land treatment unit at the Champion International Superfund Site in Libby, Montana. This soil was contaminated with wood preserving wastes including creosote (composed primarily of polycyclic aromatic hydrocarbons and pentachlorophenol). Degradation rates of 14C-pyrene and PAH compounds were found to be enhanced under soil gas oxygen concentrations between 2% and 21% in the contaminated soil. Between 45% and 55% of 14C-pyrene spiked onto the soil was mineralized after 70 days at soil gas oxygen levels between 2% and 21%. No statistically significant mineralization was found to occur at 0% oxygen concentrations. Mineralization of 14C-pyrene in contaminated soil poisoned with mercuric chloride was determined to be less than 0.5%. Degradation of indigenous nonradiolabeled PAH in non-poisoned soil was statistically significantly greater than in poisoned soil. These results indicated that the degradation of 14C-pyrene and PAH compounds was biological and would occur under low oxygen concentrations. For example, the use of soil aeration technology in order to achieve continued treatment for buried lifts of soil while new lifts are added will decrease the total time for soil remediation of the prepared-bed.

Arsenic(V) Reduction in Relation to Iron(III) Transformation and Molecular Characterization of the Structural and Functional Microbial Community in Sediments of a Basin-Fill Aquifer in Northern Utah

ABSTRACT Basin-fill aquifers of the Southwestern United States are associated with elevated concentrations of arsenic (As) in groundwater. Many private domestic wells in the Cache Valley Basin, UT, have As concentrations in excess of the U.S. EPA drinking water limit. Thirteen sediment cores were collected from the center of the valley at the depth of the shallow groundwater and were sectioned into layers based on redoxmorphic features. Three of the layers, two from redox transition zones and one from a depletion zone, were used to establish microcosms. Microcosms were treated with groundwater (GW) or groundwater plus glucose (GW+G) to investigate the extent of As reduction in relation to iron (Fe) transformation and characterize the microbial community structure and function by sequencing 16S rRNA and arsenate dissimilatory reductase (arrA) genes. Under the carbon-limited conditions of the GW treatment, As reduction was independent of Fe reduction, despite the abundance of sequences related to Geobacter and Shewanella, genera that include a variety of dissimilatory iron-reducing bacteria. The addition of glucose, an electron donor and carbon source, caused substantial shifts toward domination of the bacterial community by Clostridium-related organisms, and As reduction was correlated with Fe reduction for the sediments from the redox transition zone. The arrA gene sequencing from microcosms at day 54 of incubation showed the presence of 14 unique phylotypes, none of which were related to any previously described arrA gene sequence, suggesting a unique community of dissimilatory arsenate-respiring bacteria in the Cache Valley Basin.

Arsenic(V) Reduction in Relation to Iron(III) Transformation and Molecular Characterization of the Structural and Functional Microbial Community in Sediments of a Northern Utah, Basin-Fill Aquifer

ABSTRACT Basin-fill aquifers of the Southwestern United States are associated with elevated concentrations of arsenic (As) in groundwater. Many private domestic wells in the Cache Valley Basin, UT, have As concentrations in excess of the U.S. EPA drinking water limit. Thirteen sediment cores were collected from the center of the valley at the depth of the shallow groundwater and were sectioned into layers based on redoxmorphic features. Three of the layers, two from redox transition zones and one from a depletion zone, were used to establish microcosms. Microcosms were treated with groundwater (GW) or groundwater plus glucose (GW+G) to investigate the extent of As reduction in relation to iron (Fe) transformation and characterize the microbial community structure and function by sequencing 16S rRNA and arsenate dissimilatory reductase (arrA) genes. Under the carbon-limited conditions of the GW treatment, As reduction was independent of Fe reduction, despite the abundance of sequences related to Geobacter and Shewanella, genera that include a variety of dissimilatory iron-reducing bacteria. The addition of glucose, an electron donor and carbon source, caused substantial shifts toward domination of the bacterial community by Clostridium-related organisms, and As reduction was correlated with Fe reduction for the sediments from the redox transition zone. The arrA gene sequencing from microcosms at day 54 of incubation showed the presence of 14 unique phylotypes, none of which were related to any previously described arrA gene sequence, suggesting a unique community of dissimilatory arsenate-respiring bacteria in the Cache Valley Basin.

Development of a catabolically significant genetic probe for polycyclic aromatic hydrocarbon-degrading mycobacteria in soil.

A gene probe for the detection of polycyclic aromatic hydrocarbon (PAH) induced nidB and nidA dioxygenase genes has been designed from Mycobacteria JLS, KMS, and MCS. The probe detects a catabolic gene involved in the initial steps of PAH biodegradation in mycobacteria. The gene probe is comprised of three PCR primer sets designed to detect the genes that code for two subunits of the PAH induced dioxygenase enzyme within PAH-degrading mycobacteria. The probe was built by combining three primer sets with a DNA extraction procedure that was designed to lyse the gram-positive mycobacteria cells while in the soil matrix and remove PCR inhibitors. The probe was tested on PAH contaminated soils undergoing bioremediation through landfarming and uncontaminated soils from the same site. The PAH gene probe results demonstrate that the dioxygenase genes can be detected in soils. Sequencing the nidA and nidBPCR products verified that the genes were detected in soil. Comparisons of the sequences obtained from the soil probe to seven known nid gene sequences from various PAH-degrading mycobacteria showed between 97 and 99% nucleotide matches with the nidB gene and 95 and 99% matches with the nidA gene.

Fate of Pyrene in Contaminated Soil Amended with Alternate Electron Acceptors

Creosote-contaminated soil samples from the Libby Ground Water Contamination Superfund Site in Libby, MT, were amended with the potential alternate electron acceptors (AEA) nitrate (KNO3), manganese oxide (MnO2), and amorphous iron oxyhydroxide (FeOOH) and incubated at low oxygen tensions (0-6% O2). The fate of 14C-pyrene was evaluated with respect to the different soil amendments. The fate of 14C from the radiolabeled pyrene with regard to mineralization and bound residue formation within soil humic fractions was not significantly different from controls for the iron and manganese amended soils. Nitrate amendments appeared to stimulate 14C-pyrene mineralization at a level of 170 mg NO3-N kg(-1), and inhibit mineralization at 340 mg NO3-N kg(-1). The stimulatory effect did not appear to be the result of nitrate serving as an electron acceptor. Although AEA amendments did not significantly affect the rate or extent of 14C-pyrene mineralization, results of oxygen-deprived incubations (purged with N2) indicate that AEA may be utilized by the microbial community in the unsaturated contaminated soil system.

Pentachlorophenol and phenanthrene biodegradation in creosote contaminated aquifer material

Contamination of the subsurface environment at the Libby Superfund Site, Montana, includes polycyclic aromatic hydrocarbons and f1p4achlorophenol due to accidental spills and improper disposal of wood preserving wastes. Biodegradation is a treatment technology gaining wide application in the treatment of hazardous waste sites. A microcosm study was conducted to evaluate the effect of temperature, sampling depth, nutrient addition, and oxygen on the biodegradation potential of phenanthrene and pentachlorophenol in aquifer samples using radiolabeled chemicals. Mineralization of phenanthrene reached 14% but was less than 1% for pentachlorophenol over the 56 day incubation period. Phenanthrene mineralization in microcosms at 10 degrees C was not significantly different from those at 20 degrees C. This may have been due to microbial community acclimation to lower temperatures at the site. Average volatilization was less than 2% for both phenanthrene and pentachlorophenol. After 56 days, most of the radiolabeled chemical was either solvent extractable or soil bound.

New Arsenate Reductase Gene (arrA) PCR Primers for Diversity Assessment and Quantification in Environmental Samples

ABSTRACT The extent of arsenic contamination in drinking water and its potential threat to human health have resulted in considerable research interest in the microbial species responsible for arsenic reduction. The arsenate reductase gene (arrA), an important component of the microbial arsenate reduction system, has been widely used as a biomarker to study arsenate-reducing microorganisms. A new primer pair was designed and evaluated for quantitative PCR (qPCR) and high-throughput sequencing of the arrA gene, because currently available PCR primers are not suitable for these applications. The primers were evaluated in silico and empirically tested for amplification of arrA genes in clones and for amplification and high-throughput sequencing of arrA genes from soil and groundwater samples. In silico, this primer pair matched (≥90% DNA identity) 86% of arrA gene sequences from GenBank. Empirical evaluation showed successful amplification of arrA gene clones of diverse phylogenetic groups, as well as amplification and high-throughput sequencing of independent soil and groundwater samples without preenrichment, suggesting that these primers are highly specific and can amplify a broad diversity of arrA genes. The arrA gene diversity from soil and groundwater samples from the Cache Valley Basin (CVB) in Utah was greater than anticipated. We observed a significant correlation between arrA gene abundance, quantified through qPCR, and reduced arsenic (AsIII) concentrations in the groundwater samples. Furthermore, we demonstrated that these primers can be useful for studying the diversity of arsenate-reducing microbial communities and the ways in which their relative abundance in groundwater may be associated with different groundwater quality parameters. IMPORTANCE Arsenic is a major drinking water contaminant that threatens the health of millions of people worldwide. The extent of arsenic contamination and its potential threat to human health have resulted in considerable interest in the study of microbial species responsible for the reduction of arsenic, i.e., the conversion of AsV to AsIII. In this study, we developed a new primer pair to evaluate the diversity and abundance of arsenate-reducing microorganisms in soil and groundwater samples from the CVB in Utah. We observed significant arrA gene diversity in the CVB soil and groundwater samples, and arrA gene abundance was significantly correlated with the reduced arsenic (AsIII) concentrations in the groundwater samples. We think that these primers are useful for studying the ecology of arsenate-reducing microorganisms in different environments.