Maximizing microbial degradation of perchlorate using a genetic algorithm: Media optimization.

Microbial communities are under constant influence of physical and chemical components in ecosystems. Shifts in conditions such as pH, temperature or carbon source concentration can translate into shifts in overall ecosystem functioning. These conditions can be manipulated in a laboratory setup using evolutionary computation methods such as genetic algorithms (GAs). In work described here, a GA methodology was successfully applied to define sets of environmental conditions for microbial enrichments and pure cultures to achieve maximum rates of perchlorate degradation. Over the course of 11 generations of optimization using a GA, we saw a statistically significant 16.45 and 16.76-fold increases in average perchlorate degradation rates by Dechlorosoma sp. strain KJ and Dechloromonas sp. strain Miss R, respectively. For two bacterial consortia, Pl6 and Cw3, 5.79 and 5.75-fold increases in average perchlorate degradation were noted. Comparison of zero-order kinetic rate constants for environmental conditions in GA-determined first and last generations of all bacterial cultures additionally showed marked increases.

Sterilization of biological pathogens using supercritical fluid carbon dioxide containing water and hydrogen peroxide.

Novel noninvasive techniques for the removal of biological contaminants to generate clean or sterile materials are in demand by the medical, pharmaceutical and food industries. The sterilization method described here uses supercritical fluid carbon dioxide (SF-CO(2)) containing 3.3% water and 0.1% hydrogen peroxide (v/v/v) to achieve from four to eight log viability reduction of all tested microbial species, including vegetative cells, spores and biofilms. The sterilization method employs moderate pressure and temperature (80 atm, 50°C) and a short (30-minute) treatment time. The procedure kills various opportunistic pathogens that often persist in biofilm structures, fungal spores commonly associated with nosocomial infections, and Bacillus pumilus SAFR-032 endospores that are notoriously hard to eradicate by conventional sterilization techniques.

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase.

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

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.

Effect of organic loading and retention time on dairy manure fermentation.

The investigations presented and discussed herein establish an enhanced understanding on volatile fatty acid (VFA) production as a function of dairy manure fermenter organic loading (OL) and retention time (RT), first through a factorial of 64 fermentation potential (FP) batch tests, followed by analysis of a continuously operated pilot-scale fermenter. The maximum observed net FP - 0.103 mg VFA produced (as COD) (mg VS applied)(-1) - occurred at an OL of 40.7 g VS L(-1) and at a RT of 6 days. The pilot-scale fermenter exhibited an average yield of 0.09 mg VFA (as COD) synthesized (mg VS applied)(-1), with average effluent total VFA concentrations of 6398 mg VFA (as COD) L(-1). The research demonstrates that FP tests are an effective method to optimize continuously operated dairy manure fermenters, and that dairy manure fermentation can yield large quantities of organic acids at short RTs and high OL rates.

Simultaneous abiotic reduction-biotic oxidation in a microbial-MnO2-catalyzed Fenton-like system.

The possibility of simultaneous activity of superoxide-mediated transformations and heterotrophic aerobic bacterial metabolism was investigated in catalyzed H(2)O(2) propagations (CHP; i.e., modified Fenton's reagent) systems containing Escherichia coli. Two probe compounds were used: glucose for the detection of heterotrophic metabolism of E. coli, and tetrachloromethane (CCl(4)) for the detection of superoxide generated in a MnO(2)-catalyzed CHP system. In the MnO(2)-catalyzed CHP system without bacteria, only CCl(4) loss was observed; in contrast, only glucose degradation occurred E. coli systems without CHP reagents. In combined microbial-MnO(2) CHP reactions, loss of both probes was observed. Glucose assimilation decreased and CCl(4) transformation increased as a function of H(2)O(2) concentration. Central composite rotatable experimental designs were used to determine that the conditions providing maximum simultaneous abiotic-biotic reactions were a biomass level of 10(9)CFU/mL, 0.5mM H(2)O(2), and 0.5 g MnO(2). These results demonstrate that bacterial metabolism can occur in the presence of superoxide-mediated transformations. Such coexisting reactions may occur when H(2)O(2) is injected into MnO(2)-rich regions of the subsurface as a microbial oxygen source or for in situ oxidation; however, process control of such coexisting transformations may be difficult to achieve in the subsurface due to heterogeneity. Alternatively, hybrid abiotic reduction-biotic oxidation systems could be used for the treatment of industrial effluents or dilute solvent wastes that contain traces of highly halogenated compounds.

A method for assaying perchlorate concentration in microbial cultures using the fluorescent dye resazurin.

Low concentrations (microg/L) of the perchlorate anion, ClO(4)(-), have been measured in surface and ground water supplies in many locations throughout the United States. Perchlorate is known to affect the function of the thyroid gland in mammals and its toxicity primarily results from its inhibition of thyroid hormone output. The major sources of perchlorate contamination in surface and ground waters are defense contractors, military installations, propellant manufacturers and agriculture. The currently accepted method of perchlorate analysis, recommended by the US EPA, is neither fast nor easy to use and requires purchase of an expensive high performance ion chromatograph (IC). The novel method described here uses dye resazurin to measure perchlorate reduction by bacterial cultures and bacterial consortia in a high-throughput, multi-well, culture plate format. The method is based on the observation that perchlorate reduction and the decrease of resazurin fluorescence occur simultaneously in perchlorate degrading cultures. The bioassays were performed in anaerobic serum bottles or 96-well plates with constant shaking, using a minimal ATCC medium with 10 mM acetate as electron donor/carbon source and 200 ppm perchlorate as an electron acceptor. Fluorescence measurements with excitation at 570 nm and emission at 590 nm were taken in 20 min intervals. Changes in perchlorate concentration were confirmed using IC. Based on the experimental data, a simple model showing the correlation between perchlorate concentration in microbial culture and resazurin fluorescence level was proposed. Other dyes including redox indicators, reactive azo dyes and electron shuttle chemicals were also tested for comparison and were found less useful.

Heavy metals removal from mine runoff using compost bioreactors.

Permeable bioreactors have gained both research and management attention as viable methods for treating mine runoff waters. We examined the operation of a field-scale bioreactor (containing mixed compost, straw and gravel) for treatment of runoff from the Mother Load (ML) mine in northern Idaho, U.S. and compared it to an experimental laboratory-scale reactor, containing a similar matrix and treating similar mine runoff water. In general both reactors were efficient in removing most of the metals assayed, Al, As, Cd, Fe, Ni, Pb and Zn, with the exception of Mn. Both systems showed evidence of bacterial-mediated sulphate reduction and concomitant metal sulphide complexes. However, the experimental laboratory bioreactor showed greater proportions of immobile metals reductions than did the ML bioreactor, presumably due to the greater action of sulphate-reducing bacteria. The major metal removal mechanism in the ML bioreactor was surmised to be adsorption. Differences in metal removal mechanisms between the reactors were hypothesized to be due to fluctuating hydraulic residence times at the ML site, in turn, due to unregulated runoff flow.

Development of drinking water standards for perchlorate in the United States.

Perchlorate, an anion that originates as a contaminant in ground and surface waters, is both naturally occurring and manmade. Because of its toxicity, there has been increased interest in setting drinking water safety standards and in health effects when perchlorate is present at low (parts per billion (ppb)) levels. In January 2009, the EPA issued a heath advisory to assist state and local officials in addressing local contamination of perchlorate in drinking water. The interim health advisory level of 15 micrograms per liter (microg/L), or ppb, is based on the reference dose recommended by the National Research Council (NRC) of the National Academy of Sciences (NAS). This paper describes scope and extent of contaminant issues and a legal process of setting standards for perchlorate concentration in drinking water in the United States of America.

Diversity of aerobic and facultative alkalitolerant and halotolerant endospore formers in soil from the Alvord Basin, Oregon.

Many proteins produced by Bacillus species isolated from extreme environments have been utilized for industrial purposes, as these extreme environments often promote evolution of unique protein properties. The Borax Lake area is unusual due to its geothermal activity, elevated pH, and high arsenic and salt concentrations in its soils. Soils from this region are likely to harbor alkalitolerant, halotolerant, endospore-forming strains that may be of potential ecological and/or commercial interest. The objectives of this study were to develop new PCR primers that could target Bacillus or closely related 16S rRNA genes, to characterize the diversity of alkalitolerant, halotolerant, endospore-forming organisms in the soils surrounding Borax Lake, and to identify novel organisms that may ultimately provide new enzymes for applied use. A three-pronged approach was used to identify such bacteria in soil samples. Organisms were isolated using two different techniques. Finally, metagenomic DNA from soil samples was subjected to 16S rRNA gene amplification using the newly designed primers. Assays were performed to characterize the halotolerance and alkalitolerance of isolates. Four different endospore-forming genera and 22 different species were identified by sequencing their 16S rRNA genes. Twenty-five organisms had 96% or less identity to known organisms. Thus, the newly designed Bacillus-related PCR primer sets proved useful for the detection of new species of endospore-forming bacteria in these unique soils. Results indicate that the collection of strains obtained from the Borax Lake region represents a rich source of alkalitolerant, halotolerant, endospore formers.

Proteomic detection of proteins involved in perchlorate and chlorate metabolism.

Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.

Design, synthesis and biological evaluation of a novel class of anticancer agents: anthracenylisoxazole lexitropsin conjugates.

The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides (AIMs) (While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I. Meyers.) (22-33) are described. The molecules consist of an isoxazole that pre-organizes a planar aromatic moiety and a simple amide and/or lexitropsin-oligopeptide. The new conjugate molecules were prepared via doubly activated amidation modification of Weinreb's amide formation technique, using SmCl(3) as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazolecarboxylic esters. The results of the National Cancer Institute's (NCI) 60 cell line screening assay show a distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these compounds with G-quadruplex (G4) DNA and a structural based rational for the observed selectivity of the AIMs for G4 over B-DNA is presented.

Sterilization of Bacillus pumilus spores using supercritical fluid carbon dioxide containing various modifier solutions.

Supercritical fluid carbon dioxide (SF-CO(2)) with small amounts of chemical modifier(s) provides a very effective sterilization technique that should be useful for destroying microorganism on heat-sensitive devices such as instruments flown on planetary-bound spacecraft. Under a moderate temperature (50 degrees C) and pressure (100 atm), spores of Bacillus pumilus strains ATCC 7061 and SAFR 032 can be effectively inactivated/eliminated from metal surfaces and small electronic devices in only 45 min using optimized modifier concentrations. Modifiers explored in this study included hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide, formic acid, and Triton X-100. During sterilization procedure the modifiers were continuously added to SF-CO(2) in either methanol or water at controlled concentrations. The lowest effective concentrations were established for each modifier. Complete elimination of both types of B. pumilus endospores occurred with an optimal modifier addition of either or 10% methanol containing 12% H(2)O(2) or 12% tert-butyl hydroperoxide in SF-CO(2), or a mixture of 6% H(2)O(2) and 6% tert-butyl hydroperoxide. Using water as the carrier of SF-CO(2) modifier, the complete elimination of spores viability of both B. pumilus strains occurred with an addition of either 3.3% water containing 3% H(2)O(2), or 3.3% water containing 10% methanol and 0.5% formic acid, or 3.3% water containing 10% methanol, 1% formic acid and 2% H(2)O(2).

Using a genetic algorithm to drive a microbial ecosystem in a desirable direction.

The functioning of natural microbial ecosystems is influenced by various biotic and abiotic conditions. The careful experimental manipulation of environmental conditions can drive microbial ecosystems toward exhibiting desirable types of functionality. Such manipulations can be systematically approached by viewing them as a combinatorial optimization problem, in which the optimal configuration of environmental conditions is sought. Such an effort requires a sound optimization technique. Genetic algorithms are a class of optimization methods that should be suitable for such a task because they can deal with multiple interacting variables and with experimental noise and because they do not require an intricate understanding or modelling of the ecosystem of interest. We propose the use of genetic algorithms to drive undefined microbial ecosystems in desirable directions by combinatorially optimizing sets of environmental conditions. We tested this approach in a model system where the microbial ecosystem of a human saliva sample was manipulated in successive steps to display increasing amounts of azo dye decoloration. The results of our experiments indicated that a genetic algorithm was capable of optimizing ecosystem function by manipulating the presence or absence of a set of 10 chemical supplements. Genetic algorithms hold promise for use as a tool in environmental microbiology for the efficient control of the functioning of natural and undefined microbial ecosystems.

Demonstrating the suitability of genetic algorithms for driving microbial ecosystems in desirable directions.

The functioning of natural microbial ecosystems is determined by biotic interactions, which are in turn influenced by abiotic environmental conditions. Direct experimental manipulation of such conditions can be used to purposefully drive ecosystems toward exhibiting desirable functions. When a set of environmental conditions can be manipulated to be present at a discrete number of levels, finding the right combination of conditions to obtain the optimal desired effect becomes a typical combinatorial optimisation problem. Genetic algorithms are a class of robust and flexible search and optimisation techniques from the field of computer science that may be very suitable for such a task. To verify this idea, datasets containing growth levels of the total microbial community of four different natural microbial ecosystems in response to all possible combinations of a set of five chemical supplements were obtained. Subsequently, the ability of a genetic algorithm to search this parameter space for combinations of supplements driving the microbial communities to high levels of growth was compared to that of a random search, a local search, and a hill-climbing algorithm, three intuitive alternative optimisation approaches. The results indicate that a genetic algorithm is very suitable for driving microbial ecosystems in desirable directions, which opens opportunities for both fundamental ecological research and industrial applications.

Transformation and fate of 2,4,6-trinitrotoluene (TNT) in anaerobic bioslurry reactors under various aeration schemes: implications for the decontamination of soils.

Energetic compounds have been used in a variety of industrial and military applications worldwide leading to widespread environmental contamination. Many of these compounds are toxic and resist degradation by oxidative enzymes resulting in a need for alternative remediation methods. It has been shown that trinitrotoluene (TNT)-contaminated soil subjected to treatment in strictly anaerobic bioreactors results in tight binding of TNT transformation products to soil organic matter. The research presented here examined the fate of TNT and its metabolites in bioreactors under three different aeration regimes. In all treatment regimes, the typical metabolites of aminodinitrotoluenes and diaminonitrotoluenes were observed prior to irreversible binding into the soil fraction of the slurry. Significant transformation of TNT into organic acids or simple diols, as others report in prior work, was not observed in any of the treatments and is an unlikely fate of TNT in anaerobic soil slurries. These results indicate that aeration does not dramatically affect transformation or fate of TNT in reactor systems that receive a rich carbon source but does affect the rate at which metabolites become tightly bound to the soil. The most rapid transformations and lowest redox potentials were observed in reactors in which an aerobic headspace was maintained suggesting that aerobes play a role in establishing conditions that are most conducive to TNT reduction.

Pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas stutzeri KC reduces chromium(VI) and precipitates mercury, cadmium, lead and arsenic.

Interactions of the Pseudomonas stutzeri KC siderophore pyridine-2,6-bis(thiocarboxylic acid) (pdtc) with chromium(VI), mercury(II), cadmium(II), lead(II), and arsenic(III) are described. Pdtc was found to reduce Cr(VI) to Cr(III) in both bacterial cultures and in abiotic reactions with chemically synthesized pdtc. Cr(III) subsequently formed complexes with pdtc and pdtc hydrolysis products, and their presence was confirmed using electrospray ionization-mass spectrometry (ESI-MS). Cr(III):pdtc complexes were found to slowly release Cr(III) as chromium sulfide and possibly Cr(III) oxides. Pdtc also formed poorly soluble complexes with Hg, Cd, Pb, and As(III). Hydrolysis of those complexes led to the formation of their respective metal sulfides as confirmed by energy dispersive X-ray spectroscopy (EDS) elemental analysis. The pdtc-producing strain P. stutzeri KC showed higher tolerance to most of these metals as compared to a pdtc-negative mutant. A novel role of pdtc is postulated as its involvement in providing an extracellular pool of thiols that are used for redox processes in detoxification of the bacterial extracellular environment. These redox processes can be mediated by transition metal:pdtc complexes.

The recent evolution of pentachlorophenol (PCP)-4-monooxygenase (PcpB) and associated pathways for bacterial degradation of PCP.

Man-made polychlorinated phenols such as pentachlorophenol (PCP) have been used extensively since the 1920s as preservatives to prevent fungal attack on wood. During this time, they have become serious environmental contaminants. Despite the recent introduction of PCP in the environment on an evolutionary time scale, PCP-degrading bacteria are present in soils worldwide. The initial enzyme in the PCP catabolic pathway of numerous sphingomonads, PCP-4-monooxygenase (PcpB), catalyzes the para-hydroxylation of PCP to tetrachlorohydroquinone and is encoded by the pcpB gene. This review examines the literature concerning pcpB and supports the suggestion that pcpB/PcpB should be considered a model system for the study of recent evolution of catabolic pathways among bacteria that degrade xenobiotic molecules introduced into the environment during the recent past.

Pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas stutzeri KC reduces and precipitates selenium and tellurium oxyanions.

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H(2)S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.

Bacterial diversity within the planktonic community of an artesian water supply.

Culture and molecular methods were used to describe the planktonic bacterial diversity of an artesian water supply in rural Latah County, Idaho, within the drainage of a small perennial stream, Thorn Creek. The surrounding depth to groundwater at this location is thought to be significant (>100 m), and this transitional zone (basalt-granite) of the Palouse aquifer system is little studied. The water produced by this artesian source is consistent even in years of drought and is of high quality, both mineralogically and microbiologically. A culture-based analysis using 30 media types and four incubation temperatures demonstrated that several metabolic types were present in the water. 16S rRNA gene fragments amplified from the DNA of pooled cultured cells and from the DNA extracted from 1 L of the source water were compared using denaturing gradient gel electrophoresis. The results indicated that the two DNA samples did not have similar 16S rRNA gene compositions and that several uncultured phyla were present in the community DNA sample. These results indicated that large-scale culturing did not accurately represent the structure planktonic community. 16S rRNA gene sequences from 17 different genera were obtained from the community DNA sample; the most abundant were similar to Rhodoferax, Rhodobacter, and Polaromonas species. Sequences related to the Proteo bacteria, Bacteroidetes/Chlorobi, Firmicutes, and Acidobacterium/Fibrobacter divisions were also detected.