Implementation of in situ aerobic cometabolism for groundwater treatment: State of the knowledge and important factors for field operation.

In situ aerobic cometabolism of groundwater contaminants has been demonstrated to be a valuable bioremediation technology to treat many legacy and emerging contaminants in dilute plumes. Several well-designed and documented field studies have shown that this technology can concurrently treat multiple contaminants and reach very low cleanup goals. Fundamentally different from metabolism-based biodegradation of contaminants, microorganisms that cometabolically degrade contaminants do not obtain sufficient carbon and energy from the degradation process to support their growth and require an exogenous growth supporting primary substrate. Successful applications of aerobic cometabolic treatment therefore require special considerations beyond conventional in situ bioremediation, such as competitive inhibition between growth-supporting primary substrate(s) and contaminant non-growth substrates, toxic effects resulting from contaminant degradation, and differences in microbial population dynamics exhibited by biostimulated indigenous consortia versus bioaugmentation cultures. This article first provides a general review of microbiological factors that are likely to affect the rate of aerobic cometabolic biodegradation. We subsequently review fourteen well documented field-scale aerobic cometabolic bioremediation studies and summarize the underlying microbiological factors that may affect the performance observed in these field studies. The combination of microbiological and engineering principles gained from field testing leads to insights and recommendations on planning, design, and operation of an in situ aerobic cometabolic treatment system. With a vision of more aerobic cometabolic treatments being considered to tackle large, dilute plumes, we present several novel topics and future research directions that can potentially enhance technology development and foster success in implementing this technology for environmental restoration.

Enhancing predictive power by unamalgamating multi-item scales.

The generally small but touted as "statistically significant" correlation coefficients in the social sciences jeopardize theory testing and prediction. To investigate these small coefficients' underlying causes, traditional equations such as Spearman's (1904) classic attenuation formula, Cronbach's (1951) alpha, and Guilford and Fruchter's (1973) equation for the effect of additional items on a scale's predictive power are considered. These equations' implications differ regarding large interitem correlations enhancing or diminishing predictive power. Contrary to conventional practice, such correlations decrease predictive power when treating items as multi-item scale components but can increase predictive power when treating items separately. The implications are wide-ranging. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Diyne inactivators and activity-based fluorescent labeling of phenol hydroxylase in Pseudomonas sp. CF600.

An activity-based labeling (ABL) approach was investigated for the phenol-oxidizing bacterium, Pseudomonas sp. CF600. Phenol-grown cells were exposed to several different terminal diynes, and following cell breakage, extracts of these cells were added to copper-catalyzed alkyne/azide cycloaddition reactions containing Alexa Fluor 647 azide. Analysis of total cell proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and near-infrared scanning demonstrated covalent fluorescent labeling of a 58- and a 34-kDa polypeptide in all diyne-treated cell types. Further studies using 1,4-diethynylbenzene (DEB) demonstrated that these labeled polypeptides were consistently detected in cells grown on substrates that exhibited phenol-dependent O2 uptake activity but not observed when cells were grown on substrates such as dextrose or catechol that did not support this activity. Fluorescent labeling of the two polypeptides in DEB-treated, phenol-grown cells was time dependent and was inhibited by several known substrates for phenol hydroxylase. These results suggest that diverse diynes act as mechanism-based inactivators of phenol hydroxylase in Pseudomonas sp. CF600 and that this effect can be exploited by ABL approaches to selectively label the major 58- and 34-kDa subunits of the hydroxylase component of this complex enzyme.

Pilot-scale biofiltration of 1,4-dioxane at drinking water-relevant concentrations.

1,4-Dioxane is a drinking water contaminant of emerging concern. Because conventional and many advanced drinking water treatment technologies are ineffective for 1,4-dioxane removal, cost-effective technologies for the removal of 1,4-dioxane at drinking water-relevant concentrations are needed. In this research, a gravity-fed, cometabolic biofiltration system was developed to degrade 1,4-dioxane that was spiked into coagulated, settled surface water at a concentration of ∼10 µg/L. Objectives were to determine whether cometabolic degradation of trace levels of 1,4-dioxane can be sustained using n-butane as primary substrate and whether filter media properties and empty bed contact time (EBCT) affect biofiltration efficiency. A mixed culture of bacteria derived from the Cape Fear River basin and previously enriched using isobutane served as inoculum for biologically active filters. Two granular activated carbons (GACs) with different grain sizes and one carbonaceous resin were used as attachment media, and n-butane served as the primary substrate for biologically active filters. Non-inoculated controls with the same media were evaluated in parallel to distinguish between biological and adsorptive removals of 1,4-dioxane. For the duration of the pilot study (>3 months), 1,4-dioxane was degraded in inoculated biofilters receiving n-butane. In control filters containing larger and smaller grain GAC, 1,4-dioxane broke through completely within 750 and 1250 bed volumes, respectively, corresponding to 15 to 30 days of operation at an EBCT of 30 min. 1,4-Dioxane removal increased with increasing EBCT in all biologically active filters. At an EBCT of 30 min, the biologically active GAC filter containing the larger-grain GAC removed on average 87% of 1,4-dioxane at pseudo steady-state. When the hydraulic loading rate was decreased to achieve an overall EBCT of 60 min, 1,4-dioxane was removed to <1 µg/L in the biologically active GAC filter containing the larger-grain GAC. Activity-based labeling showed the presence of catalytically active monooxygenases in backwash water from biologically active filters that degraded 1,4-dioxane. Amplicon sequencing results showed that while taxa shifted after the initial inoculation of biologically active filters, taxa in biologically active filters remained more similar to the inoculum than those in the non-inoculated control filters. Overall, results of this research demonstrate that cometabolic degradation of 1,4-dioxane at trace levels is possible for extended periods of time in inoculated biofilters that receive n-butane as primary substrate.

The Ethics and Politics of Academic Knowledge Production: Thoughts on the Future of Business Ethics.

To commemorate 40 years since the founding of the Journal of Business Ethics, the editors in chief of the journal have invited the editors to provide commentaries on the future of business ethics. This essay comprises a selection of commentaries aimed at creating dialogue around the theme The Ethics and Politics of Academic Knowledge Production. Questions of who produces knowledge about what, and how that knowledge is produced, are inherent to editing and publishing academic journals. At the Journal of Business Ethics, we understand the ethical responsibility of academic knowledge production as going far beyond conventions around the integrity of the research content and research processes. We are deeply aware that access to resources, knowledge of the rules of the game, and being able to set those rules, are systematically and unequally distributed. One could ask the question "for whom is knowledge now ethical'"? (See the Burrell commentary.) We have a responsibility to address these inequalities and open up our journal to lesser heard voices, ideas, and ways of being. Our six commentators pursue this through various aspects of the ethics and politics of academic knowledge production. Working with MacIntyre's scheme of practices and institutions, Andrew West provides commentary on the internal good of business ethics learning and education. Inviting us to step out of the cave, Christopher Michaelson urges a clear-eyed, unblinking focus on the purposes and audiences of business ethics scholarship. As developmental editor, Scott Taylor uncovers some of the politics of peer review with the aim of nurturing of unconventional research. Mike Hyman presents his idiosyncratic view of marketing ethics. In the penultimate commentary, Julie Nelson attributes difficulties in the academic positioning of the Business Ethics field to the hegemony of a masculine-centric model of the firm. And finally, Gibson Burrell provides a powerful provocation to go undercover as researcher-investigators in a parallel ethics of the research process.

Single-well push-pull tests evaluating isobutane as a primary substrate for promoting in situ cometabolic biotransformation reactions.

A series of single-well push-pull tests (SWPPTs) were performed to investigate the efficacy of isobutane (2-methylpropane) as a primary substrate for in situ stimulation of microorganisms able to cometabolically transform common groundwater contaminants, such as chlorinated aliphatic hydrocarbons and 1,4-dioxane (1,4-D). In biostimulation tests, the disappearance of isobutane relative to a nonreactive bromide tracer indicated an isobutane-utilizing microbial community rapidly developed in the aquifer around the test well. SWPPTs were performed as natural drift tests with first-order rates of isobutane consumption ranging from 0.4 to 1.4 day-1. Because groundwater contaminants were not present at the demonstration site, isobutene (2-methylpropene) was used as a nontoxic surrogate to demonstrate cometabolic activity in the subsurface after biostimulation. The transformation of isobutene to isobutene epoxide (2-methyl-1,2-epoxypropane) illustrates the epoxidation process previously shown for common groundwater contaminants after cometabolic transformation by alkane-utilizing bacteria. The rate and extent of isobutene consumption and the formation and transformation of isobutene epoxide were greater in the presence of isobutane, with no evidence of primary substrate inhibition. Modeled concentrations of isobutane-utilizing biomass in microcosms constructed with groundwater collected before and after each SWPPT offered additional evidence that the isobutane-utilizing microbial community was stimulated in the aquifer. Experiments in groundwater microcosms also demonstrated that the isobutane-utilizing bacteria stimulated in the subsurface could cometabolically transform a mixture of co-substrates including isobutene, 1,1-dichloroethene, cis-1,2-dichloroethene, and 1,4-D with the same co-substrate preferences as the bacterium Rhodococcus rhodochrous ATCC strain 21198 after growth on isobutane. This study demonstrated the effectiveness of isobutane as primary substrate for stimulating in situ cometabolic activity and the use of isobutene as surrogate to investigate in situ cometabolic reactions catalyzed by isobutane-stimulated bacteria.

Draft Genome Sequences of Four Aerobic Isobutane-Metabolizing Bacteria.

Here, we report the draft genome sequences of four aerobic gaseous alkane-oxidizing bacteria isolated from soil by enrichment culture using isobutane (2-methylpropane) as the sole carbon and energy source. The sequences all reveal microorganisms with multiple alkane-oxidizing monooxygenases, including soluble di-iron monooxygenases (SDIMOs), copper-containing monooxygenases (CuMMOs), and alkane hydroxylases (AHs).

Long-term cometabolic transformation of 1,1,1-trichloroethane and 1,4-dioxane by Rhodococcus rhodochrous ATCC 21198 grown on alcohols slowly produced by orthosilicates.

Long-term cometabolic transformation of 1,1,1-trichlorethane (1,1,1-TCA) and 1,4-dioxane (1,4-D) was achieved using slow release compounds (SRCs) as growth substrates for pure cultures of Rhodococcus rhodochrous ATCC 21198 (ATCC strain 21198). Resting cell transformation tests showed 1,4-D transformation occurred without a lag phase for cells grown on 2-butanol, while an induction period of several hours was required for 1-butanol grown cells. These observations were consistent with activity-based labeling patterns for monooxygenase hydroxylase components and specific rates of tetrahydrofuran (THF) degradation. 1,1,1-TCA and 1,4-D degradation rates for alcohol-grown cells were slower than those for cells grown on gaseous alkanes such as isobutane. Batch metabolism and degradation tests were performed, in the presence of 1,1,1-TCA and 1,4-D, with the growth of ATCC strain 21198 on alcohols produced by the hydrolysis of orthosilicates. Three orthosilicates were tested: tetrabutylorthosilicate (TBOS), tetra-s-butylorthosilicate (T2BOS), and tetraisopropoxysilane (T2POS). The measured rates of alcohol release in poisoned controls depended on the orthosilicate structure with TBOS, which produced a 1° alcohol (1-butanol), hydrolyzing more rapidly than T2POS and T2BOS, that produced the 2° alcohols 2-butanol and 2-propanol, respectively. The orthosilicates were added as light non-aqueous phase liquids (LNAPLs) with ATCC strain 21198 and formed dispersed droplets when continuously mixed. Continuous rates of oxygen (O2) consumption and carbon dioxide (CO2) production confirmed alcohol metabolism by ATCC strain 21198 was occurring. The rates of metabolism (TBOS > T2POS > T2BOS) were consistent with the rates of alcohol release via abiotic hydrolysis. 1,4-D and 1,1,1-TCA were continuously transformed in successive additions by ATCC strain 21198 over 125 days, with the rates highly correlated with the rates of metabolism. The metabolism of the alcohols was not inhibited by acetylene, while transformation of 1,4-D and 1,1,1-TCA was inhibited by this gas. As acetylene is a potent inactivator of diverse bacterial monooxygenases, these results suggest that monooxygenase activity was required for the observed cometabolic transformations but not for alcohol utilization. Alcohol concentrations in the biologically active reactors were maintained below the levels of detection, indicating they were metabolized rapidly after being produced. Much lower rates of O2 consumption were observed in the reactors containing T2BOS, which has benefits for in-situ bioremediation. The results illustrate the importance of the structure of the SRC when developing passive aerobic cometabolic treatment systems.

Co-encapsulation of slow release compounds and Rhodococcus rhodochrous ATCC 21198 in gellan gum beads to promote the long-term aerobic cometabolic transformation of 1,1,1-trichloroethane, cis-1,2-dichloroethene and 1,4-dioxane.

Rhodococcus rhodochrous ATCC 21198 (strain ATCC 21198) was successfully co-encapsulated in gellan gum beads with orthosilicates as slow release compounds (SRCs) to support aerobic cometabolism of a mixture of 1,1,1-trichloroethane (1,1,1-TCA), cis-1,2-dichloroethene (cis-DCE), and 1,4-dioxane (1,4-D) at aqueous concentrations ranging from 250 to 1000 µg L-1. Oxygen (O2) consumption and carbon dioxide (CO2) production showed the co-encapsulated cells utilized the alcohols that were released from the co-encapsulated SRCs. Two model SRCs, tetrabutylorthosilicate (TBOS) and tetra-s-butylorthosilicate (T2BOS), which hydrolyze to produce 1- and 2-butanol, respectively, were encapsulated in gellan gum (GG) at mass loadings as high as 10% (w/w), along with strain ATCC 21198. In the GG encapsulated beads, TBOS hydrolyzed 26 times faster than T2BOS and rates were ∼4 times higher in suspension than when encapsulated. In biologically active reactors, the co-encapsulated strain ATCC 21198 effectively utilized the SRC hydrolysis products (1- and 2-butanol) and cometabolized repeated additions of a mixture of 1,1,1-TCA, cis-DCE, and 1,4-D for over 300 days. The transformation followed pseudo-first-order kinetics. Vinyl chloride (VC) and 1,1-dichloroethene (1,1-DCE) were also transformed in the reactors after 250 days. In the long-term treatment, the batch reactors with co-encapsulated T2BOS GG beads achieved similar transformation rates, but at much lower O2 consumption rates than those with TBOS. The results demonstrate that the co-encapsulation technology can be a passive method for the cometabolic treatment of dilute groundwater plumes.

Aerobic cometabolism of 1,4-dioxane by isobutane-utilizing microorganisms including Rhodococcus rhodochrous strain 21198 in aquifer microcosms: Experimental and modeling study.

Aerobic cometabolism of the emerging contaminant 1,4-dioxane (1,4-D) by isobutane-utilizing microorganisms was assessed in pure culture and aquifer microcosm studies. The bacterium Rhodococcus rhodochrous strain ATCC 21198 transformed low, environmentally-relevant concentrations of 1,4-D when grown on isobutane. Microcosms were constructed with aquifer solids from Fort Carson, Colorado, a site contaminated with 1,4-D and trichloroethene (TCE). Multiple additions of isobutane and 1,4-D over 300 days were transformed in microcosms biostimulated with isobutane and microcosms bioaugmented with strain 21198. Results showed that, over time and with sufficient inorganic nutrients, biostimulation of native microorganisms with isobutane was just as effective as bioaugmentation with strain 21198 to achieve 1,4-D transformation in the microcosms. The presence of TCE at 0.2 mg/L did not inhibit 1,4-D transformation, though TCE itself was not readily transformed. An iterative process was used to determine kinetic parameter values to fit Michaelis-Menten/Monod models to experimental data for simultaneous isobutane utilization, biomass growth, and cometabolic transformation of 1,4-D. Parameter optimization resulted in good model fit to the data over multiple transformations of isobutane and 1,4-D in both short- and long-term experiments. Results suggest low concentrations of 1,4-D studied in the microcosms were cometabolically transformed according to a pseudo first-order rate of 0.37 L/mg TSS/day of 21198. Isobutane consumption was modeled with a maximum rate of 2.58 mg/mg TSS/day and a half saturation constant of 0.09 mg/L. 1,4-D transformation was competitively inhibited by the presence of isobutane and transformation rates were significantly reduced when inorganic nutrients were limiting. Simulations of the repeated additions found a first-order microbial endogenous decay coefficient of 0.03 day-1 fit the alternating periods of active transformation and stagnation between isobutane and 1,4-D additions over approximately one year. The model fitting process highlighted the importance of determining kinetic parameters from data representing low concentrations typically found in the environment.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with ß-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.

Metabolism of 2-methylpropene (isobutylene) by the aerobic bacterium Mycobacterium sp. strain ELW1.

An aerobic bacterium (Mycobacterium sp. strain ELW1) that utilizes 2-methylpropene (isobutylene) as a sole source of carbon and energy was isolated and characterized. Strain ELW1 grew on 2-methylpropene (growth rate = 0.05 h(-1)) with a yield of 0.38 mg (dry weight) mg 2-methylpropene(-1). Strain ELW1 also grew more slowly on both cis- and trans-2-butene but did not grow on any other C2 to C5 straight-chain, branched, or chlorinated alkenes tested. Resting 2-methylpropene-grown cells consumed ethene, propene, and 1-butene without a lag phase. Epoxyethane accumulated as the only detected product of ethene oxidation. Both alkene consumption and epoxyethane production were fully inhibited in cells exposed to 1-octyne, suggesting that alkene oxidation is initiated by an alkyne-sensitive, epoxide-generating monooxygenase. Kinetic analyses indicated that 1,2-epoxy-2-methylpropane is rapidly consumed during 2-methylpropene degradation, while 2-methyl-2-propen-1-ol is not a significant metabolite of 2-methylpropene catabolism. Degradation of 1,2-epoxy-2-methylpropane by 2-methylpropene-grown cells led to the accumulation and further degradation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate, two sequential metabolites previously identified in the aerobic microbial metabolism of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Growth of strain ELW1 on 2-methylpropene, 1,2-epoxy-2-methylpropane, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate was fully inhibited when cobalt ions were omitted from the growth medium, while growth on 3-hydroxybutyrate and other substrates was unaffected by the absence of added cobalt ions. Our results suggest that, like aerobic MTBE- and TBA-metabolizing bacteria, strain ELW1 utilizes a cobalt/cobalamin-dependent mutase to transform 2-hydroxyisobutyrate. Our results have been interpreted in terms of their impact on our understanding of the microbial metabolism of alkenes and ether oxygenates.

Children with special health care needs: exploring the relationships between patients' level of functioning, their oral health, and caregivers' oral health-related responses.

PURPOSE: The purposes of this study were to increase dentists' understanding of how to best engage parents and their children with special health care needs (SHCN) in oral health promotion efforts and explore the relationships between these patients' level of functioning and oral health and their parents' comfort concerning oral health promotion. METHODS: Survey data were collected from 154 caregivers of SHCN children. Children's oral health data were obtained from their clinical charts. RESULTS: The patients' level of functioning ranged from the lowest to the highest regarding their ability to listen/understand, talk, relate to others, care for themselves, play with others, and participate in physical activities. Children's gingival health was correlated with their ability to talk (r=-.12; P<.05). Their oral hygiene score correlated with their ability to talk (r=.18; P<.05) and their skills in social play interactions (r=.21; P<.05). The parents' comfort level concerning oral health promotion correlated positively with their child's level of functioning. Parents' interest in receiving oral health instruction correlated positively with their child's level of functioning. CONCLUSIONS: Understanding patient's level of functioning might predict the degree to which parents actually engage in oral health promotion efforts and are interested in oral health-related education.

RNAi silencing of a cytochrome P450 monoxygenase disrupts the ability of a filamentous fungus, Graphium sp., to grow on short-chain gaseous alkanes and ethers.

Graphium sp. (ATCC 58400), a filamentous fungus, is one of the few eukaryotes that grows on short-chain alkanes and ethers. In this study, we investigated the genetic underpinnings that enable this fungus to catalyze the first step in the alkane and ether oxidation pathway. A gene, CYP52L1, was identified, cloned and functionally characterized as an alkane-oxidizing cytochrome P450 (GSPALK1). Analysis of CYP52L1 suggests that it is a member of the CYP52 cytochrome P450 family, which is comprised of medium- and long-chain alkane-oxidizing enzymes found in yeasts. However, phylogenetic analysis of GSPALK1 with other CYP52 members suggests they are not closely related. Post-transcriptional ds-RNA-mediated gene silencing of CYP52L1 severely reduced the ability of this fungus to oxidize alkanes and ethers, however, downstream metabolic steps in these pathways were unaffected. Collectively, the results of this study suggest that GSPALK1 is the enzyme that catalyzes the initial oxidation of alkanes and ethers but is not involved in the later steps of alkane or ether metabolism.

Children with Special Health Care Needs: Exploring the Relationships between Patients' Level of Functioning, Their Oral Health, and Caregivers' Oral Health-related Responses.

PURPOSE: The purposes of this study were to increase dentists' understanding of how to best engage parents and their children with special health care needs (SHCN) in oral health promotion efforts and explore the relationships between these patients' level of functioning and oral health and their parents' comfort concerning oral health promotion. METHODS: Survey data were collected from 154 caregivers of SHCN children. Children's oral health data were obtained from their clinical charts. RESULTS: The patients' level of functioning ranged from the lowest to the highest regarding their ability to listen/understand, talk, relate to others, care for themselves, play with others, and participate in physical activities. Children's gingival health was correlated with their ability to talk (r=-.12; P<.05). Their oral hygiene score correlated with their ability to talk (r=.18; P<.05) and their skills in social play interactions (r=.21; P<.05). The parents' comfort level concerning oral health promotion correlated positively with their child's level of functioning. Parents' interest in receiving oral health instruction correlated positively with their child's level of functioning. CONCLUSIONS: Understanding patient's level of functioning might predict the degree to which parents actually engage in oral health promotion efforts and are interested in oral health-related education.

Biodegradation of gasoline ether oxygenates.

Ether oxygenates such as methyl tertiary butyl ether (MTBE) are added to gasoline to improve fuel combustion and decrease exhaust emissions. Ether oxygenates and their tertiary alcohol metabolites are now an important group of groundwater pollutants. This review highlights recent advances in our understanding of the microorganisms, enzymes and pathways involved in both the aerobic and anaerobic biodegradation of these compounds. This review also aims to illustrate how these microbiological and biochemical studies have guided, and have helped refine, molecular and stable isotope-based analytical approaches that are increasingly being used to detect and quantify biodegradation of these compounds in contaminated environments.

Preparing Science-Trained Professionals for the Biotechnology Industry: A Ten-Year Perspective on a Professional Science Master's Program.

The biotechnology industry has a need for business-savvy scientists; however, this is not the way scientists are traditionally trained at universities and colleges. To address this need, universities have developed Professional Science Master's (PSM) degree programs that offer advanced training in a technical field along with professional skills development through team-based projects and internships. Nearly ten years ago, the Department of Microbiology at NCSU started a PSM program in Microbial Biotechnology (MMB). This article provides an overview of the MMB program, and shares some of the lessons that we have learned.

Identification of tertiary butyl alcohol (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable isotope probing.

Biodegradation of the gasoline oxygenates methyl tertiary-butyl ether (MTBE) and ethyl tertiary-butyl ether (ETBE) can cause tertiary butyl alcohol (TBA) to accumulate in gasoline-impacted environments. One remediation option for TBA-contaminated groundwater involves oxygenated granulated activated carbon (GAC) reactors that have been self-inoculated by indigenous TBA-degrading microorganisms in ground water extracted from contaminated aquifers. Identification of these organisms is important for understanding the range of TBA-metabolizing organisms in nature and for determining whether self-inoculation of similar reactors is likely to occur at other sites. In this study (13)C-DNA-stable isotope probing (SIP) was used to identify TBA-utilizing organisms in samples of self-inoculated BioGAC reactors operated at sites in New York and California. Based on 16S rRNA nucleotide sequences, all TBA-utilizing organisms identified were members of the Burkholderiales order of the ß-proteobacteria. Organisms similar to Cupriavidus and Methylibium were observed in both reactor samples while organisms similar to Polaromonas and Rhodoferax were unique to the reactor sample from New York. Organisms similar to Hydrogenophaga and Paucibacter strains were only detected in the reactor sample from California. We also analyzed our samples for the presence of several genes previously implicated in TBA oxidation by pure cultures of bacteria. Genes Mpe_B0532, B0541, B0555, and B0561 were all detected in (13)C-metagenomic DNA from both reactors and deduced amino acid sequences suggested these genes all encode highly conserved enzymes. One gene (Mpe_B0555) encodes a putative phthalate dioxygenase-like enzyme that may be particularly appropriate for determining the potential for TBA oxidation in contaminated environmental samples.

Oxidation of gasoline oxygenates by closely related non-haem-iron alkane hydroxylases in Pseudomonas mendocina KR1 and other n-octane-utilizing Pseudomonas strains.

Pseudomonas mendocina KR1 oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) to tertiary butyl alcohol (TBA) during growth on C5 -C8 n-alkanes. We have further explored oxidation of ether oxygenates by this strain to help identify the enzyme that catalyses these reactions. High levels of MTBE-oxidizing activity occurred in resting cells grown on C5 -C8 n-alkanes. Lower activities occurred in cells grown on longer-chain n-alkanes (C9 -C11 ) and 1°-alcohols (C5 -C10 ). N-octane-grown cells also oxidized tertiary amyl methyl ether (TAME) to tertiary amyl alcohol (TAA), but did not oxidize ethyl tertiary butyl ether (ETBE), TBA or TAA. A 39 kDa polypeptide in whole cell extracts of n-octane-grown cells strongly cross-reacted with an anti-AlkB polyclonal antiserum in an SDS-PAGE/immunoblot. This polypeptide was absent or less abundant in cells grown on dextrose, dextrose plus dicyclopropylketone or 1-octanol. N-octane-grown cells of Pseudomonas aeruginosa strains KSLA-473 and ATCC 17423 oxidized MTBE and TAME but not ETBE. N-hexadecane-grown cells of these strains and strain PAO1 did not oxidize any of the oxygenates tested. Our results indicate ether oxygenate-degrading activity in alkane-utilizing pseudomonads is consistently observed with close homologues of the GPo1 non-haem-iron alkane hydroxylases but is otherwise not a consistent catalytic feature of these diverse enzymes.

Effects of gasoline components on MTBE and TBA cometabolism by Mycobacterium austroafricanum JOB5.

In this study we have examined the effects of individual gasoline hydrocarbons (C(5-10,12,14) n-alkanes, C(5-8) isoalkanes, alicyclics [cyclopentane and methylcyclopentane] and BTEX compounds [benzene, toluene, ethylbenzene, m-, o-, and p-xylene]) on cometabolism of methyl tertiary butyl ether (MTBE) and tertiary butyl alcohol (TBA) by Mycobacterium austroafricanum JOB5. All of the alkanes tested supported growth and both MTBE and TBA oxidation. Growth on C(5-8) n-alkanes and isoalkanes was inhibited by acetylene whereas growth on longer chain n-alkanes was largely unaffected by this gas. However, oxidation of both MTBE and TBA by resting cells was consistently inhibited by acetylene, irrespective of the alkane used as growth-supporting substrate. A model involving two separate but co-expressed alkane-oxidizing enzyme systems is proposed to account for these observations. Cyclopentane, methylcyclopentane, benzene and ethylbenzene did not support growth but these compounds all inhibited MTBE and TBA oxidation by alkane-grown cells. In the case of benzene, the inhibition was shown to be due to competitive interactions with both MTBE and TBA. Several aromatic compounds (p-xylene > toluene > m-xylene) did support growth and cells previously grown on these substrates also oxidized MTBE and TBA. Low concentrations of toluene (<10 microM) stimulated MTBE and TBA oxidation by alkane-grown cells whereas higher concentrations were inhibitory. The effects of acetylene suggest strain JOB5 also has two distinct toluene-oxidizing activities. These results have been discussed in terms of their impact on our understanding of MTBE and TBA cometabolism and the enzymes involved in these processes in mycobacteria and other bacteria.