Degradation of phenol under meso- and thermophilic, anaerobic conditions

Based on the results of preliminary studies on phenol degradation under mesophilic conditions with a mixed methanogenic culture, we proposed a degradation pathway in which phenol is fermented to acetate: Part of the phenol is reductively transformed to benzoate while the rest is oxidised, forming acetate as end product. According to our calculations, this should result in three moles of phenol being converted to two moles of benzoate and three moles of acetate (3 phenol + 2 CO2 + 3 H2O --> 3 acetate + 2 benzoate): To assess the validity of our hypothesis concerning the metabolic pathway, we studied the transformation of phenol under mesophilic and thermophilic conditions in relation to the availability of hydrogen. Hence, methanogenic meso- and thermophilic cultures amended with phenol were run with or without an added over-pressure of hydrogen under methanogenic and non-methanogenic conditions. Bromoethanesulfonic acid (BES) was used to inhibit methanogenic activity. In the mesophilic treatments amended with only BES, about 70% of the carbon in the products found was benzoate. During the course of phenol transformation in these BES-amended cultures, the formation pattern of the degradation products changed: Initially nearly 90% of the carbon from phenol degradation was recovered as benzoate, whereas later in the incubation, in addition to benzoate formation, the aromatic nucleus degraded completely to acetate. Thus, the initial reduction of phenol to benzoate resulted in a lowering of H2 levels, giving rise to conditions allowing the degradation of phenol to acetate as the end product. Product formation in bottles amended with BES and phenol occurred in accordance with the hypothesised pathway; however, the overall results indicate that the degradation of phenol in this system is more complex. During phenol transformation under thermophilic conditions, no benzoate was observed and no phenol was transformed in the BES-amended cultures. This suggests that the sensitivity of phenol transformation to an elevated partial pressure of H2 is higher under thermophilic conditions than under mesophilic ones. The lack of benzoate formation could have been due to a high turnover of benzoate or to a difference in the phenol degradation pathway between the thermophilic and mesophilic cultures.     

Dehalogenation of dichloroethene in a contaminated soil: fatty acids and alcohols as electron donors and an apparent requirement for tetrachloroethene

Environmental soil contamination at an industrial site in Marion, Ohio (USA) with tetrachloroethene (perchloroethene, PCE) resulted in residual cis-1, 2-dichloroethene (DCE) contamination that had not declined after more than 15 years. Microcosm slurries containing 2.6% soil from this site were supplemented with different electron donors, i.e., individual fatty acids or alcohols. None of the microcosms supported complete DCE dechlorination, unless PCE was added to the microcosm at initiation. The addition of fresh PCE resulted in the dehalogenation of PCE to DCE in the microcosms supplemented with fatty acids having an even number of carbon atoms (acetate, butyrate, and caproate), but not in those with an odd number of carbon atoms (formate, propionate, and valerate), where negligible or no activity was detected. No significant further DCE degradation was observed in any of the microcosms supplied with fatty acids as electron donors. Microcosms supplemented with freshly added PCE bioconverted PCE to DCE and completely dehalogenated both the ex-novo and soil-supplied DCE within 60 days, but only if alcohols having an even number of carbon atoms (ethanol or butanol) were also added as electron donors. Odd-numbered alcohols either did not produce dehalogenation (as with methanol) or only dehalogenated PCE to DCE (as with propanol).     

Complete degradation of tetrachloroethene by combining anaerobic dechlorinating and aerobic methanotrophic enrichment cultures

 Degradation of tetrachloroethene (perchloroethylene, PCE) was investigated by combining the metabolic abilities of anaerobic bacteria, capable of reductive dechlorination of PCE, with those of aerobic methanotrophic bacteria, capable of co-metabolic degradation of the less-chlorinated ethenes formed by reductive dechlorination of PCE. Anaerobic communities reductively dechlorinating PCE, trichloroethene (TCE) and dichloroethenes were enriched from various sources. The maximum rates of dechlorination observed for various chloroethenes in these batch enrichments were: PCE to TCE (341 μmol l^-1 day^-1), TCE to cis-dichloroethene (159 μmol l^-1 day^-1), cis-dichloroethene to chloroethene (99 μmol l^-1 day^-1) and trans-dichloroethene to chloroethene (22 μmol l^-1 day^-1). A mixture of these enrichments was inoculated into an anoxic fixed-bed upflow column. In this column PCE was converted mainly into cis-1, 2-dichloroethene, small amounts of TCE and chloroethene, and chloride. Enrichments of aerobic methanotrophic bacteria were grown in an oxic fixed-bed downflow column. Less-chlorinated ethenes, formed in the anoxic column, were further metabolized in this oxic methanotrophic column. On the basis of analysis of chloride production and the disappearance of chlorinated ethenes it was demonstrated that complete degradation of PCE was possible by combining these two columns. Operation of the two-column system under various process conditions indicated that the sensitivity of the methanotrophic bacteria to chlorinated intermediates represented the bottle-neck in the sequential anoxic/oxic degradation process of PCE. Received: 24 October 1994 / Received revision: 20 January 1995 / Accepted: 23 January 1995     

Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket Reactors by Incorporation of Dehalospirillum multivorans into Granular Sludge

Dechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium Dehalospirillum multivorans into granular sludge. This reactor was compared to the reference 1 (R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2 (R2) reactor, containing only living granular sludge. All three reactors were fed mineral medium containing 3 to 57 μM PCE, 2 mM formate, and 0.5 mM acetate and were operated under sterile conditions. In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor. The R2 reactor, with no inoculation, produced only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes. No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum. During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of D. multivorans, indicating that this bacterium was immobilized in the living and autoclaved granular sludge. In contrast, the R2 reactor, with no inoculation of D. multivorans, only converted PCE to TCE under the same conditions. Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against D. multivorans. In granules obtained from the R1 reactor, D. multivorans grew mainly in microcolonies located in the centers of the granules, while in the test reactor, the bacterium mainly covered the surfaces of granules.     

Production of aromatic compounds during methanogenic degradation of straw in rice field soil

Production of CH(4) in anoxic rice field soil is stimulated by the addition of rice straw. Previous experiments showed that acetate and propionate are the most important intermediates of the carbon flow to CH(4), and accumulate if CH(4) production is inhibited by 2-bromoethanesulfonate (BES). However, some unidentified compounds were found to accumulate in addition. We now identified them as benzoate, phenylpropionate, and phenylacetate by comparison of the retention times in HPLC chromatograms with authentic standards and by mass spectrometry. These aromatic compounds accumulated only to concentrations <100 microM, especially in soil amended with rice straw (stem, sheath or blade straw). Phenylpropionate and benzoate were the most abundant aromatic intermediates contributing up to 4% to total CH(4) production. Phenylacetate, on the other hand, contributed very little (<0.3%). Gibbs free energies (DeltaG) were calculated for different anaerobic degradation pathways of the aromatic compounds at the actual incubation conditions. Conversion of benzoate to acetate, CO(2) and H(2) was strongly exergonic (DeltaG = -86 kJ mol(-1)) under methanogenic conditions, but became less exergonic (DeltaG = -30 kJ mol(-1)) when CH(4) production was inhibited. The primary oxidation of phenylpropionate was only exergonic for alpha-oxidation (i.e. phenylacetate as product) but not for beta-oxidation (i.e. benzoate as product). However, the DeltaG values for the complete degradation of phenylpropionate to acetate, CO(2) and H(2) were similar for both pathways and were also similar to those of benzoate degradation. Collectively, the results suggest that aromatic compounds are minor intermediates of anaerobic degradation of organic matter in rice field soil, and are syntrophically degraded by coupling to methanogenesis.     

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing,strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H₂ as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H₂, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H₂. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160M), a dechlorination rate of about 50 nmol min^-1 (mg cell protein)^-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.Abbreviations PCE Perchloroethylene, tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon     

Growth and final product formation by <Emphasis Type="Italic">Bifidobacterium infantis</Emphasis> in aerated fermentations

Fermentation conditions were developed to allow Bifidobacterium infantis to grow in the presence of air. Batch fermentations in TPYG medium, starting from anoxic conditions followed by the application of low airflow rates [0.02–0.1 air volume, per liquid media volume, per minute (vvm)], were analyzed for growth, oxygen uptake, and product formation by the bacterium. Under all aerated fermentations, B. infantis showed high aerotolerance, with a maximum oxygen-specific consumption rate of 0.34 mmol oxygen per gram dry cell weight per hour in the presence of 0.06 vvm. Similar growth yields were obtained under oxic and anoxic conditions (0.11–0.13 and 0.11 g dry cell weight per mmol glucose, respectively). Oxygen also influenced metabolite formation since lactate production and its molar relation to acetate increased and formate decreased with aeration rate. Under anoxic conditions, a maximum concentration of 8.1 mM lactate and an acetate/lactate ratio of 3.5:1 were obtained, while under oxic conditions the lactate concentration increased more than two-fold and the acetate/lactate molar ratio decreased to 1.5:1. The possibility of balancing acetate/lactate molar ratios for organoleptic purposes as well as for obtaining good growth under microaerated conditions was demonstrated.     

Scale-up issues for in situ anaerobic tetrachloroethene bioremediation

For the full scale implementation of in situ anaerobic bioremediation of tetrachloroethene (PCE) in groundwater, the following issues must be addressed: which organic substrates at which concentration would be most effective in promoting dechlorination and are economical; how far the substrate, electron acceptor, and nutrients can be transported in the aquifer; and the placement of delivery and recovery wells for distributing these amendments. In a microcosm study, almost all of the tested inexpensive substrates supported reductive dechlorination of PCE through vinyl chloride (VC) under methanogenic conditions. A minimum of about 60 mg L⁻¹ of organic carbon was needed to dechlorinate 23 μM PCE with a single feeding. In a second microcosm study dechlorination stopped at 1,2-dichloroethene (DCE) in microcosms fed higher concentrations of several substrates. At the highest concentrations the substrates inhibited DCE production. Three field tracer tests were conducted to evaluate methods to distribute the amendments across the aquifer. The natural groundwater gradient is not sufficient to distribute substrate evenly. Groundwater injection at 60 times the natural flux rate increased the distribution of substrate. A mixing strategy of cross-gradient injection further increased the distribution of the substrate. Ammonia-nitrogen, sulfate, and phosphate were retarded relative to the substrate and inorganic tracer. Received 30 October 1995/ Accepted in revised form 07 June 1996     

Properties of tetrachloroethene and trichloroethene dehalogenase of Dehalospirillum multivorans

Some properties of tetrachloroethene and trichloroethene dehalogenase of the recently isolated, tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, were studied with extracts of cells grown on pyruvate plus fumarate. The dehalogenase catalyzed the oxidation of reduced methyl viologen with tetrachloroethene (PCE) or trichloroethene (TCE) as electron acceptor. All other artificial or physiological electron donors tested were ineffective. The PCE and TCE dehalogenase activity was insensitive towards oxygen in crude extracts. When extracts were incubated under anoxic conditions in the presence of titanium citrate as reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (50 M). Inactivation did not occur in the absence of titanium citrate. The activity of propyl-iodide-treated extracts was restored almost immediately by illumination. The dehalogenase was inhibited by cyanide. The inhibition profile was almost the same under oxic and anoxic conditions independent of the presence or absence of titanium citrate. In addition, N₂O, nitrite, and ethylene diamine tetra-acetate (EDTA) were inhibitors of PCE and TCE dehalogenase. Carbon monoxide and azide had no influence on the dehalogenase activity. Trans-1,2-dichloroethene or 1,1-dichloroethene, both of which are isomers of the dechlorination product cis-1,2-dichloroethene, neither inhibited nor inactivated the dehalogenase. PCE and TCE dechlorination appeared to be mediated by the same enzyme since the inhibitors tested had nearly the same effects on the PCE and TCE dehalogenating activity. The data indicated the involvement of a corrinoid and possibly of an additional transition metal in reductive PCE and TCE dechlorination.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE Dichloroethene - EDTA Ethylene diamine tetra-acetate - MV Methyl viologen - BV Benzyl viologen - PI Propyl iodide, 1-iodopropane - TC Titanium(III) citrate     

A Comparison of Complex Electron Donors for Anaerobic Dechlorination of PCE

The potential of sugar, flour, corn steep liquor, molasses, non-fat milk, and whey to serve as electron donors for anaerobic dechlorination of tetrachloroethene (PCE) was examined. The electron donors were compared based on acclimation time, the extent of PCE dechlorination achieved, the minimum electron donor dose necessary to achieve PCE removal, and unit cost. The time required to achieve routine dechlorination of PCE (to any daughter product) for each donor was (in days): corn steep liquor (10), milk (10), whey (10), methanol (12), molasses (14), sugar (26), flour (30). Ethene production was achieved by milk-, whey-, and methanol-fed cultures, whereas the other donors did not facilitate ethene production over a 135-day period. Corn steep liquor-, whey-, molasses-, and sugar-fed cultures needed five times the stoichiometric amount (e.g., donor per eq PCE to ethene) to facilitate PCE conversion to dichloroethene (DCE). Cultures fed milk and flour needed 20 times the stoichiometric amount, and methanol-fed cultures required 50 times the stoichiometric amount, perhaps due to competition from methanogenic organisms. Minimum laboratory-scale electron donor costs to achieve stoichiometric conversion of PCE to DCE are ($ per pound [lb] PCE) whey (0.04), molasses (0.07), milk (0.14), corn steep liquor (0.19), sugar (0.38), methanol (0.58), and flour (1.30).     

The impact of feed composition on biodegradation of benzoate under cyclic (aerobic/anoxic) conditions

The response of a mixed microbial culture to different feed compositions, that is, containing benzoate and pyruvate as sole carbon sources at different levels, was studied in a chemostat with a 48-h hydraulic residence time under cyclic aerobic and anoxic (denitrifying) conditions. The cyclic bacterial culture was well adapted to different feed compositions as evidenced by the lack of accumulation of benzoate or pyruvate in the chemostat. Both the benzoate-degrading capabilities and the in vitro catechol 2,3-dioxygenase (C23DO) activities of the cyclic bacterial cultures were in direct proportion to the flux through the chemostat of the substrate degraded by the pathway containing C23DO, with some exceptions. The quantity of C23DO showed a transient decrease during the initial portion of the aerobic period before returning to the level present during the anoxic period. That decrease was most likely caused by the production of H(2)O(2) by the cells upon being returned to aerobic conditions.     

Influence of Hydraulic Retention Time on Extent of PCE Dechlorination and Preliminary Characterization of the Enrichment Culture

The extent of tetrachloroethene (PCE) dechlorination in two chemostats was evaluated as a function of hydraulic retention time (HRT). The inoculum of these chemostats was from an upflow anaerobic sludge blanket (UASB) reactor that rapidly converts PCE to vinyl chloride (VC) and ethene. When the HRT was 2.9 days, PCE was converted only to cis-dichloroethene (cDCE). When the HRT was 11 days, the end products were VC and ethene. Further studies showed that the dechlorinating microbial community in the UASB reactor contained two distinct populations, one of which converted PCE to cDCE and the other cDCE to VC and ethene. Methanogenic activity was very low in these cultures. The cDCE dechlorinating culture apparently has a lower growth rate than the PCE dechlorinating culture, and as a result, at a shorter HRT, the cDCE dechlorinating culture was washed out from the system leading to incomplete dechlorination of PCE. Both enrichment cultures used pyruvate or hydrogen as electron donors for dechlorination. Acetate was the carbon source (but not energy source) when hydrogen was used. Both cultures had undefined nutrient requirements and needed supplements of cell extract obtained from the mixed culture in the UASB reactor. However, the two cultures were different in their response to the addition of an inhibitor of methanogenesis (2-bromoethanesulfonate [BES]). BES inhibited the dechlorinating activity of the enriched cDCE dechlorinating culture, but had no influence on the PCE dechlorinating culture. Preliminary studies on BES inhibition are presented.     

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real‐time RT‐PCR analysis revealed the intrinsic self‐regulation of GCD and GAD. Utilizing this self‐regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2‐ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm², 0.27 ± 0.02 mmol/g_CDW/hr and 0.25 ± 0.02 mmol/g_CDW/hr, which were 327%, 477%, and 644% of the values of wild‐type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.     

Novel uncultured Chloroflexi dechlorinate perchloroethene to trans-dichloroethene in tidal flat sediments

The marine environment represents a rich source of bio- and geogenically produced organohalogens, including the common pollutant perchloroethene (PCE). However, diversity and function of marine chloroethene-dechlorinating microorganisms are largely unknown. Here, we have studied the activity and composition of a tidal flat sediment bacterial and archaeal community from the North Sea exposed to low concentrations of PCE. After 2 weeks of incubation, PCE was rapidly dechlorinated via trichloroethene to dichloroethene (DCE). Unexpectedly, these microcosms produced 3.5-fold more trans-DCE than cis-DCE. The actively dechlorinating microbial populations were traced by stable isotope probing of rRNA with (13)C-labelled acetate for 4 days. Terminal restriction fragment length polymorphism fingerprinting and clone libraries of isotopically enriched, 'heavy'(13)C-labelled bacterial 16S rRNA revealed the populations potentially involved in reductive dechlorination. Major clone groups belonged to the Proteobacteria (50.0%; 22.4% delta-, 12.1% gamma-, 6.9% alpha-, 6.9% beta- and 1.7% epsilon-subgroup) and Chloroflexi (29.3%). Populations represented by the two dominant terminal restriction fragments were affiliated with the Dehalococcoidetes (subphylum II of the Chloroflexi), and were exclusively detected in the heavy fraction of the PCE-dechlorinating incubation. The phylogenetically novel, larger population, designated Tidal Flat Chloroflexi Cluster, was closely related to the recently discovered PCE-dechlorinating Lahn Cluster bacteria from anoxic river sediment but more distantly related to canonical Dehalococcoides spp. (92-94% sequence identity). The second population was closely related to 'Dehalobium chlorocoercia DF-1'. Both populations appear to be responsible for reductive dechlorination of highly chlorinated ethenes to predominantly trans-DCE in tidal flat sediment incubations.     

Amino acids in the rat liver and plasma and some metabolites in the liver after sodium benzoate treatment

The effect of sodium benzoate administration on amino acids in the liver and plasma and various metabolites in the liver was studied. Changes in glutamine and ornithine were noted only at a higher dose (10 mmol/kg body wt) of benzoate, whereas even a lower dose caused a significant decrease in glycine, serine, and alanine levels of plasma and liver. A dose- and time-dependent decrease in glycine levels was studied. A decrease of up to 50% in the glycine concentration may limit its own transport into mitochondria and availability for the formation of hippurate. A decrease in alanine may have resulted from stimulation of gluconeogenesis from alanine, by increased ammonia. Among the metabolites studied, ATP and acetyl-CoA decreased and ammonia increased significantly even at a lower dose (5 mmol/kg body wt) of benzoate. The compounds that require ATP for their synthesis such as N-acetylglutamate and glutamine decreased significantly only at the higher dose of benzoate, whereas urea and glutathione levels were unaffected under our experimental conditions.     

Reductive dechlorination of Tri- and tetrachloroethylenes depends on transition from aerobic to anaerobic conditions.

Aerobic enrichment cultures from contaminated groundwaters dechlorinated trichloroethylene (TCE) (14.6 mg/liter; 111 mumol/liter) and tetrachloroethylene (PCE) (16.2 mg/liter; 98 mumol/liter) reductively within 4 days after the transition from aerobic to anaerobic conditions. The transformation products were equimolar amounts of cis-1,2-dichloroethylene and traces of 1,1-dichloroethylene. No other chlorinated product and no methane were detected. The change was accompanied by the release of sulfide, which caused a decrease in the redox potential from 0 to -150 mV. In sterile control experiments, sulfide led to the abiotic formation of traces of 1,1-dichloroethylene without cis-1,2-dichloroethylene production. The reductive dechlorination of PCE via TCE depended on these specific transition conditions after consumption of the electron acceptor oxygen or nitrate. Repeated feeding of TCE or PCE to cultures after the change to anaerobic conditions yielded no further dechlorination. Only aerobic subcultures with an air/liquid ratio of 1:4 maintained dechlorination activities; anaerobic subcultures showed no transformation. Bacteria from noncontaminated sites showed no reduction under the same conditions.     

Reductive dechlorination of Tri- and tetrachloroethylenes depends on transition from aerobic to anaerobic conditions

Aerobic enrichment cultures from contaminated groundwaters dechlorinated trichloroethylene (TCE) (14.6 mg/liter; 111 mumol/liter) and tetrachloroethylene (PCE) (16.2 mg/liter; 98 mumol/liter) reductively within 4 days after the transition from aerobic to anaerobic conditions. The transformation products were equimolar amounts of cis-1,2-dichloroethylene and traces of 1,1-dichloroethylene. No other chlorinated product and no methane were detected. The change was accompanied by the release of sulfide, which caused a decrease in the redox potential from 0 to -150 mV. In sterile control experiments, sulfide led to the abiotic formation of traces of 1,1-dichloroethylene without cis-1,2-dichloroethylene production. The reductive dechlorination of PCE via TCE depended on these specific transition conditions after consumption of the electron acceptor oxygen or nitrate. Repeated feeding of TCE or PCE to cultures after the change to anaerobic conditions yielded no further dechlorination. Only aerobic subcultures with an air/liquid ratio of 1:4 maintained dechlorination activities; anaerobic subcultures showed no transformation. Bacteria from noncontaminated sites showed no reduction under the same conditions.     

Reductive dechlorination of chlorinated ethenes and 1, 2-dichloroethane by "Dehalococcoides ethenogenes" 195.

"Dehalococcoides ethenogenes" 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Anthocyanin-Rich Purple Corn Extract Inhibit Diabetes-Associated Glomerular Angiogenesis

Diabetic nephropathy (DN) is one of the major diabetic complications and the leading cause of end-stage renal disease. Abnormal angiogenesis results in new vessels that are often immature and play a pathological role in DN, contributing to renal fibrosis and disrupting glomerular failure. Purple corn has been utilized as a daily food and exerts disease-preventive activities. This study was designed to investigate whether anthocyanin-rich purple corn extract (PCE) prevented glomerular angiogenesis under hyperglycemic conditions. Human endothelial cells were cultured in conditioned media of mesangial cells exposed to 33 mM high glucose (HG-HRMC-CM). PCE decreased endothelial expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF)-1α induced by HG-HRMC-CM. Additionally, PCE attenuated the induction of the endothelial marker of platelet endothelial cell adhesion molecule (PECAM)-1 and integrin β3 enhanced in HG-HRMC-CM. Endothelial tube formation promoted by HG-HRMC-CM was disrupted in the presence of PCE. In the in vivo study employing db/db mice treated with 10 mg/kg PCE for 8 weeks, PCE alleviated glomerular angiogenesis of diabetic kidneys by attenuating the induction of VEGF and HIF-1α. Oral administration of PCE retarded the endothelial proliferation in db/db mouse kidneys, evidenced by its inhibition of the induction of vascular endothelium-cadherin, PECAM-1 and Ki-67. PCE diminished the mesangial and endothelial induction of angiopoietin (Angpt) proteins under hypeglycemic conditions. The induction and activation of VEGF receptor 2 (VEGFR2) were dampened by treating PCE to db/db mice. These results demonstrate that PCE antagonized glomerular angiogenesis due to chronic hyperglycemia and diabetes through disturbing the Angpt-Tie-2 ligand-receptor system linked to renal VEGFR2 signaling pathway. Therefore, PCE may be a potent therapeutic agent targeting abnormal angiogenesis in DN leading to kidney failure.     

Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments

BTEX compounds such as benzene are frequent soil and groundwater contaminants that are easily biodegraded under oxic conditions by bacteria. In contrast, benzene is rather recalcitrant under anaerobic conditions. The analysis of anoxic degradation is often hampered by difficult sampling conditions, limited amounts of biomass and interference of matrix compounds with proteomic approaches. In order to improve the procedure for protein extraction we established a scheme consisting of the following steps: dissociation of cells from lava granules, cell lysis by ultrasonication and purification of proteins by phenol extraction. The 2D-gels revealed a resolution of about 240 proteins spots and the spot patterns showed strong matrix dependence, but still differences were detectable between the metaproteomes obtained after growth on benzene and benzoate. Using direct data base search as well as de novo sequencing approaches we were able to identify several proteins. An enoyl-CoA hydratase with cross species homology to Azoarcus evansii, is known to be involved in the anoxic degradation of xenobiotics. Thereby the identification confirmed that this procedure has the capacity to analyse the metaproteome of an anoxic living microbial community.