Relationship between the number of bacteria added to soil or seeds and their abundance and distribution in the rhizosphere of alfalfa

A study was conducted of the relationship between the density of several bacterial strains introduced into soil or onto seeds and their abundance in the rhizosphere of alfalfa. The abundance of six species in the rhizosphere was directly correlated with the density of bacteria initially added to soil. The density of six species in the rhizosphere of 15-day-old plants also was directly correlated with the density of each strain in nonrhizosphere soil. Tests of seven species added to soil at four inoculum densities showed that bacteria that survived well in the soil attained the highest densities in the rhizosphere and those that survived poorly in the soil were present at the lowest densities in the rhizosphere. Sixteen of 19 bacterial strains added to alfalfa seeds at 10⁷ or 10⁸ cells per g colonized the rhizosphere of 15-day-old plants, but nearly all of the cells were localized in the upper third of the rhizosphere. A study of 12 bacterial strains that failed to colonize the lower part of the rhizosphere if inoculated onto seeds showed that the bacteria colonized the entire rhizosphere of 15-day-old alfalfa plants if initially inoculated throughout the soil. The data suggest that the density of individual bacterial strains in the rhizosphere is dependent on their density in the soil and that seed inoculation only has an effect on the population in the proximal portion of the alfalfa root system.     

Enhancing Transport of Hydrogenophagaflava ENV735 for Bioaugmentation of Aquifers Contaminated with Methyl tert-Butyl Ether

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.     

Potential for cometabolic biodegradation of 1,4-dioxane in aquifers with methane or ethane as primary substrates

The objective of this research was to evaluate the potential for two gases, methane and ethane, to stimulate the biological degradation of 1,4-dioxane (1,4-D) in groundwater aquifers via aerobic cometabolism. Experiments with aquifer microcosms, enrichment cultures from aquifers, mesophilic pure cultures, and purified enzyme (soluble methane monooxygenase; sMMO) were conducted. During an aquifer microcosm study, ethane was observed to stimulate the aerobic biodegradation of 1,4-D. An ethane-oxidizing enrichment culture from these samples, and a pure culture capable of growing on ethane (Mycobacterium sphagni ENV482) that was isolated from a different aquifer also biodegraded 1,4-D. Unlike ethane, methane was not observed to appreciably stimulate the biodegradation of 1,4-D in aquifer microcosms or in methane-oxidizing mixed cultures enriched from two different aquifers. Three different pure cultures of mesophilic methanotrophs also did not degrade 1,4-D, although each rapidly oxidized 1,1,2-trichloroethene (TCE). Subsequent studies showed that 1,4-D is not a substrate for purified sMMO enzyme from Methylosinus trichosporium OB3b, at least not at the concentrations evaluated, which significantly exceeded those typically observed at contaminated sites. Thus, our data indicate that ethane, which is a common daughter product of the biotic or abiotic reductive dechlorination of chlorinated ethanes and ethenes, may serve as a substrate to enhance 1,4-D degradation in aquifers, particularly in zones where these products mix with aerobic groundwater. It may also be possible to stimulate 1,4-D biodegradation in an aerobic aquifer through addition of ethane gas. Conversely, our results suggest that methane may have limited importance in natural attenuation or for enhancing biodegradation of 1,4-D in groundwater environments.     

Aerobic Biodegradation of N-Nitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [¹⁴C]NDMA to ¹⁴CO₂, growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.N-Nitrosodimethylamine (NDMA) is a potent carcinogen that has recently been detected in groundwater, wastewater, and drinking water (1, 2, 17, 18). It forms as a disinfection byproduct in wastewater and drinking water treated with chloramine and other disinfectants (17, 18, 43). NDMA has also been found to be present in aquifers at several military sites that have used 1,1-dimethylhydrazine, a component of liquid rocket propellant that contained NDMA as an impurity (6, 9). Although there is presently no federal maximum contaminant level for NDMA in drinking water, a risk assessment conducted by the U.S. Environmental Protection Agency suggested that concentrations as low as 0.7 ng/liter can increase lifetime cancer risk by 1 × 10⁻⁶ (34). In addition, California currently has a 10 ng/liter notification level for NDMA concentrations in drinking water and has recently recommended an even lower public health goal of 3 ng/liter (3, 20). Thus, the presence of even trace concentrations of this chemical in drinking water represents a potential public health concern.The rates and extents of NDMA biodegradation in natural environments, including surface water, sludges, and soils, are highly variable. In some studies, the compound has been reported to be recalcitrant or only partially biodegraded (16, 30, 31); in others, fairly rapid and extensive biodegradation has been previously observed (2, 13, 22, 40). Few studies have been conducted to examine NDMA biodegradation in groundwater. However, the persistence of NDMA derived originally from 1,1-dimethylhydrazine-based rocket fuel over decades in some groundwater aquifers (e.g., Rocky Mountain Arsenal, CO; former Air Force Plant PJKS, CO; and Aerojet Superfund Site, CA) suggests that this molecule can be very recalcitrant (8, 9, 35). At sites where biodegradation has been observed, the organisms responsible and the microbial degradation pathways are largely unknown.The metabolism of NDMA and other nitrosamines by mammals has received extensive study. NDMA requires metabolic activation to the methyldiazonium ion (a strong alkylating agent) to exert its genotoxic effects (1, 19, 34). This activation reaction is catalyzed by specific isozymes of the cytochrome P-450-dependent mixed-function oxidase system and proceeds through an initial α-hydroxylation reaction. Alternately, NDMA can be oxidized by the P-450 system via a denitrosation route, which does not result in the formation of a highly carcinogenic intermediate (11, 28, 37).The bacterial transformation of NDMA has not been studied in significant detail. Several bacteria expressing broad-specificity monooxygenase enzymes have been reported to degrade NDMA via cometabolism. These include the propanotrophs Rhodococcus sp. strain RHA1 (25, 26) and Rhodococcus ruber ENV425 (29) as well as Mycobacterium vaccae JOB5 (25), the methanotroph Methylosinus trichosporium OB3b (42), and the toluene oxidizer Pseudomonas mendocina KR1 (7). We recently characterized the pathway of NDMA transformation used by P. mendocina KR1, a bacterium that utilizes the enzyme toluene-4-monooxygenase (T4MO) to cometabolically degrade NDMA and other anthropogenic pollutants (7, 38). The pathway of NDMA transformation by KR1 differs from the two pathways described for mammals. A majority of the NDMA metabolized by T4MO in this strain is oxidized to N-nitrodimethylamine (NTDMA) and then further to N-nitromethylamine (NTMA), which accumulates as a terminal product (7).In this report, we describe the pathway used by the propanotroph R. ruber ENV425 to catabolize NDMA. This strain was originally isolated from turf soil, where propane was used as the sole carbon source, and was previously reported to oxidize methyl tertiary-butyl ether and other gasoline oxygenates (27). Our data show that the pathway of NDMA degradation mediated by strain ENV425 differs from that mediated by P. mendocina KR1. Rather, the pathway used for transformation of NDMA by ENV425 appears to be similar to the denitrosation pathway catalyzed by various P-450 isozymes in mammals, resulting in the production of nitric oxide (NO), nitrite, nitrate, formaldehyde, formate, and methylamine (MA) (11, 12, 28, 39). A significant fraction of the carbon in the NDMA molecule was released as CO₂ by strain ENV425, although growth on NDMA could not be confirmed. However, the bacterium was observed to utilize NDMA as well as the NDMA-degradation intermediates MA and nitrate as sources of nitrogen during growth on propane as a sole carbon and energy source.     

Biodegradation of methyl tert-butyl ether by a pure bacterial culture.

Biodegradation of methyl tert-butyl ether (MTBE) by the hydrogen-oxidizing bacterium Hydrogenophaga flava ENV735 was evaluated. ENV735 grew slowly on MTBE or tert-butyl alcohol (TBA) as sole sources of carbon and energy, but growth on these substrates was greatly enhanced by the addition of a small amount of yeast extract. The addition of H(2) did not enhance or diminish MTBE degradation by the strain, and MTBE was only poorly degraded or not degraded by type strains of Hydrogenophaga or hydrogen-oxidizing enrichment cultures, respectively. MTBE degradation activity was constitutively expressed in ENV735 and was not greatly affected by formaldehyde, carbon monoxide, allyl thiourea, or acetylene. MTBE degradation was inhibited by 1-amino benzotriazole and butadiene monoepoxide. TBA degradation was inducible by TBA and was inhibited by formaldehyde at concentrations of >0.24 mM and by acetylene but not by the other inhibitors tested. These results demonstrate that separate, independently regulated genes encode MTBE and TBA metabolism in ENV735.     

Human plasma DSIP decreases at the initiation of sleep at different circadian times

Nocturnal plasma delta sleep-inducing peptide-like immunoreactivity (DSIP-LI) was determined serially in seven healthy male subjects. Time courses during nocturnal sleep (2300-0800 h), nocturnal sleep deprivation (2300-0500 h), and morning recovery sleep (0500–0800 h) after sleep deprivation were compared. A significant decrease in plasma DSIP-LI was found at the transition from wakefulness to sleep in both evening sleep (2300 h) and morning recovery sleep (0500 h). Time courses were accompanied by physiological changes in sleep electroencephalographic slow-wave activity, and in plasma concentrations of cortisol and human growth hormone. No sleep stage specificity was found. It is concluded that DSIP is influenced by the initiation of sleep.     

Rat kidney proximal tubule cells in defined medium: The roles of cholera toxin,extracellular calcium and serum in cell growth and expression of γ-glutamyltransferase

Summary A culture system is described in which rat kidney proximal tubule epithelial cells (RPTE) can be prepared with good yield and high viability and grown in culture under serum-free conditions. The cells require EGF, insulin, cholera toxin and either 1% dialyzed serum or a complex of bovine serum albumin with oleic acid (BSA/OA). The cells can be maintained for long periods of time and express several markers for RPTE. The cells have both alkaline phosphatase and γ-glutamyltransferase activity and respond to parathyroid hormone but not vasopressin. The specific activity of γ-glutamyltransferase decreases when the cells begin to grow, but increases when they reach confluence. Extracellular calcium plays a role in the induction of γ-glutamyltransferase in confluent cells. Cells grown in media containing low calcium, i.e. less than 0.4 mM, have reduced specific activity of γ-glutamyltransferase. Extracellular calcium also alters the morphology of the cells in that cells grown in low calcium are single cells or loose clusters suggesting poor cell-cell contact. When the calcium is raised to 1.0 mM, the cells change their shape and organization to adopt the morphology of cells maintained continuously in 1.0 mM calcium. The cells can be passaged onto plastic surfaces which have been coated with collagen but cannot be subcultured on uncoated or serum coated plastic. This culture system will be a useful model for the investigation of renal carcinogenesis and the role of cell proliferation in that process. This work was supported by grants CA48197 and DK38925 from the National Institutes of Health Editor's Statement This paper reports a method for isolation and, uniquely, multipassage culture of rat proximal tubule epithelial cells. The authors use this culture system to explore some aspects of the physiology of these cells, demonstrating the value of the model.