Environmental distribution of the trichloroethene reductive dehalogenase gene (tceA) suggests lateral gene transfer among Dehalococcoides

The trichloroethene reductive dehalogenase gene (tceA) of Dehalococcoides spp. was detected in 12 of 21 trichloroethene-to-ethene dechlorinating enrichment cultures established from aquifer and sediment samples collected from diverse geographic locations in the USA. Analysis of the tceA chromosomal regions indicated that the tceA genes shared greater than 95% sequence identity, and all shared identical tceAB spacer sequences and tceB genes downstream of tceA. A putative transposable element (PTE) was present 1077 bp downstream of the tceB stop codon in three of eight chromosomal regions analyzed. Sequence identity was interrupted downstream of tceB and upstream or downstream of the PTE, suggesting that intrachromosomal or interchromosomal transfer of tceAB had occurred.     

Molecular characterization of microbial populations at two sites with differing reductive dechlorination abilities

This study compares three molecular techniques, including terminal restriction fragment length polymorphism (T-RFLP), RFLP analysis with clone sequencing, and quantitative PCR (Q-PCR) for surveying differences in microbial communities at two contaminated field sites that exhibit dissimilar chlorinated solvent degradation activities. At the Idaho National Engineering and Environmental Laboratory (INEEL), trichloroethene (TCE) was completely converted to ethene during biostimulation with lactate. At Seal Beach, California, perchloroethene (PCE) was degraded only to cis-dichloroethene (cDCE) during biostimulation but was degraded to ethene after bioaugmentation with a dechlorinating culture containing Dehalococcoides strains. T-RFLP analysis showed that microbial community composition differed significantly between the two sites, but was similar within each site among wells that had low or no electron donor exposure. Analysis of INEEL clone libraries by RFLP with clone sequencing revealed a complex microbial population but did not identify any Dehalococcoides strains. Q-PCR targeting the 16S rRNA gene of Dehalococcoides strains – known for their unique capability to dechlorinate solvents completely to ethene – revealed a significant population at INEEL, but no detectable population at Seal Beach prior to bioaugmentation. Detection of Dehalococcoides by Q-PCR correlated with observed dechlorination activity and ethene production at both sites. Q-PCR showed that Dehalococcoides was present in even the pristine well at INEEL, suggesting that the difference in dechlorination ability at the two sites was due to the initial absence of this genus at Seal Beach. Of the techniques tested, Q-PCR quantification of specific dechlorinating species provided the most effective and direct prediction of community dechlorinating potential.     

Theoretical investigation of the sequential reductive dechlorination pathways of chlorobenzenes and chloroanilines

The distribution of product isomers during the sequential reductive dechlorination of pentachloroaniline (PCA) and pentachlorobenzene (PeCB) was examined based on calculated thermodynamic, chromatographic, and electronic properties and then compared to the product distribution achieved by enrichment cultures. The dechlorination pathway analysis based on free energy considerations matched 78% and 67% of the experimental results for the sequential reductive dechlorination of chlorobenzenes (CBs) and chloroanilines (CAs), respectively. Chromatographic properties of CBs and CAs were able to explain some but not all of the reactions in the observed dechlorination pathways. Correlations between the observed dechlorination pattern and electronic properties of the parent compounds were able to explain most of the formation of the observed products. Experimentally observed sequential reductive dechlorination of CBs and CAs were similar to predicted dechlorination pathways based on the charge differential values calculated for the carbon–chloride bonds. Chlorine atoms were removed from the carbon atom that has the highest charge differential or the second highest charge differential. However, although thermodynamic, electronic as well as chromatographic properties of the CBs and CAs are certainly important factors, they may not be sufficient to completely describe the sequential microbial reductive dechlorination. Enzymatic specificity, as well as other factors (i.e., culture acclimation, environmental factors) should be considered for the interpretation of observed sequential reductive dehalogenation pathways of haloorganic compounds. This work provides the most comprehensive analysis to date of theoretical factors that control the sequential reductive chlorination of two homologous series of single‐ring chloroaromatic species. Biotechnol. Bioeng. 2010; 105: 574–587. © 2009 Wiley Periodicals, Inc.     

Molecular characterization of polychlorinated biphenyl-dechlorinating populations in contaminated sediments

Polychlorinated biphenyl (PCB)-dechlorinating microorganisms were characterized in PCB-contaminated sediments using amplified ribosomal DNA restriction analysis (ARDRA). The sediments were prepared by spiking Aroclor 1248 into PCB-free sediments, and were inoculated with microorganisms eluted from St. Lawrence River sediments. PCB-free sediments inoculated with the same inoculum served as the control. Four restriction fragment length polymorphism (RFLP) groups in the eubacterial and two in the archaeal domain were found exclusively in PCB-spiked sediment clone libraries. Sequence analysis of the four eubacterial clones showed homology to Escherichia coli, Lactosphaera pasteurii, Clostridium thermocellum, and Dehalobacter restrictus. The predominant archaeal sequence in the PCB-spiked sediment clone library was closely related to Methanosarcina barkeri, which appear to support earlier findings that methanogens are involved in PCB dechlorination. When the dot-blot hybridization was performed between the sediment DNA extract and the probes designed with eubacterial RFLP groups, the intensity of two of eubacterial RFLP groups, which showed high sequence homology to C. pascui and D. restrictus, was highly correlated with the number of dechlorinating microorganisms suggesting these two members intend to contribute to PCB dechlorination.     

The Impact of Sediment Characteristics on PCB-dechlorinating Cultures: Implications for Bioaugmentation

Polychlorinated Biphenyl (PCB)-dechlorinating cultures with complimentary activities, previously derived from estuarine Baltimore Harbor (B), marine Palos Verdes (P), and riverine Hudson River (H) sediments, were mixed and then inoculated into sterile sediments from the same sources. In the treatments containing sterile B sediment, the different inocula had limited impact on the bacterial community development and on dechlorination patterns, all of which were similar. In treatments containing sterile P or H sediment, however, different inocula resulted in significantly different PCB dechlorination patterns and bacterial communities. The B sediment appeared to support not only the most extensive and rapid dechlorination of the three sediments, but also supported a more diverse bacterial community. This was thought to be a result of nutritional richness, as it was high in organic carbon and micronutrients such as zinc and cobalt. Although mixing three PCB-dechlorinating cultures was able to produce a culture capable of enhanced PCB-dechlorinating activity as compared to single cultures, some activities were lost upon culture transfer. This indicates that care must be taken to establish robust PCB-dechlorinating cultures capable of extensive dechlorination prior to pursuing bioaugmentation. In addition, our results indicate that the concentration and availability of macro-and micronutrients could have a significant impact on the microbial community structure, and thus a thorough characterization of the sediment at contaminated sites is essential for implementing bioaugmentation for PCB bioremediation.     

Structure analysis and performance of a microbial community from a contaminated aquifer involved in the complete reductive dechlorination of 1,1,2,2-tetrachloroethane to ethene

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.     

Managing methanogens and homoacetogens to promote reductive dechlorination of trichloroethene with direct delivery of H2 in a membrane biofilm reactor

A study with H(2)-based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H(2) delivery in ex-situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH-increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start-up by creating H(2) limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start-up. This was corroborated by the methanogenesis functional gene mcrA, which was 1-2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides-like morphology in the fiber biofilm. These results support that direct addition of H(2) can allow for efficient and complete reductive dechlorination, and they shed light into how H(2)-fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex-situ bioremediation of chlorinated ethenes.     

Specific removal of chlorine from the ortho-position of halogenated benzoic acids by reductive dechlorination in anaerobic enrichment cultures

Anaerobic enrichment cultures catalysing the reductive dechlorination of chlorinated benzoic acids were obtained from three fresh-water sediments collected from seven different locations. Sub-cultures from these enrichments specifically removed ortho-substituted chlorine from 2,3,6-, 2,3,5- and 2,4,6-trichlorobenzoic acid, yielding chloride and 2,5-, 3,5-, and 2,4-dichlorobenzoic acids, respectively. These reductive dehalogenations were stimulated by the addition of benzoate and/or volatile organic acids. In one of these enrichments dehalogenation of ortho- and/or para-chlorine substituents was also observed from 2,3-, 2,4-, 2,5-, and 3,4-dichlorobenzoic acid, yielding 3- and 4-chlorobenzoate. Removal of meta-chlorines was not observed in any of the enrichments.     

Comparative study of methanol, butyrate, and hydrogen as electron donors for long-term dechlorination of tetrachloroethene in mixed anerobic cultures

This study examined the ability of different electron donors (i.e., hydrogen, methanol, butyrate, and yeast extract) to sustain long-term (500 days) reductive dechlorination of tetrachloroethene (PCE) in anerobic fill-and-draw bioreactors operated at 3:1 donor:PCE ratio (defined on a total-oxidation basis for the donor). Initially (i.e., until approximately day 80), the H(2)-fed bioreactor showed the best ability to completely dechlorinate the dosed PCE (0.5 mmol/L) to ethene whereas, in the presence of methanol, butyric acid or no electron donor added (but low-level yeast extract), dechlorination was limited by the fermentation of the organic substrates and in turn by H(2) availability. As the study progressed, the H(2)-fed reactor experienced a diminishing ability to dechlorinate, while more stable dechlorinating activity was maintained in the reactors that were fed organic donors. The initial diminished ability of the H(2)-fed reactor to dechlorinate (after about 100 days), could be partially explained in terms of increased competition for H(2) between dechlorinators and methanogens, whereas other factors such as growth-factor limitation and/or accumulation of toxic and/or inhibitory metabolites were shown to play a role for longer incubation periods (over 500 days). In spite of decreasing activity with time, the H(2)-fed reactor proved to be the most effective in PCE dechlorination: after about 500 days, more than 65% of the added PCE was dechlorinated to ethene in the H(2)-fed reactor, versus 36%, 22%, and <1% in the methanol-fed, butyrate-fed, and control reactors, respectively.     

Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri.

Cobalamin and the native and diepimeric forms of factor F430 catalyzed the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene or chloroethane (CA) in a buffer with Ti(III) citrate as the electron donor. Ethylene was the major product in the cobalamin-catalyzed transformation, and the ratio of ethylene to CA formed was 25:1. Native F430 and 12,13-di-epi-F430 produced ethylene and CA in ratios of about 2:1 and 1:1, respectively. Cobalamin dechlorinated 1,2-DCA much faster than did factor F430. Dechlorination rates by all three catalysts showed a distinct pH dependence, correlated in a linear manner with the catalyst concentration and doubled with a temperature increase of 10 degrees C. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethylene and CA with Ti(III) citrate as the reductant. The catalytic components in boiled extracts were heat and oxygen stable and had low molecular masses. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic and part had a hydrophobic character. These chemical properties of the dechlorinating components and spectral analysis of boiled extracts indicated that corrinoids or factor F430 was responsible for the dechlorinations. The ratios of 3:1 to 7:1 of ethylene and CA formed by cell extracts suggested that both cofactors were concomitantly active.     

Further evidence for the regulation of bacterial populations in soil by protozoa

After the addition to soil of large numbers of a cowpea Rhizobium strain, the population declined steadily until the numbers reached about 10⁷/g, and the protozoa rose to about 10⁴/g. When indigenous protozoa were suppressed by the addition of actidione to the soil, the density of the test rhizobium did not fall initially, but its abundance declined to about 10⁷/g when actidione-resistant protozoa arose in significant numbers. The addition to actidione-treated soil of an antibiotic-resistant strain of Paramecium led to a rapid decrease in the population of the rhizobium, the density reaching essentially the same value as in soil receiving neither the drug nor the paramecia. The same changes occurred with Xanthomonas campestris as test prey except that its numbers fell to about 10⁵/g of soil. These data provide further evidence for the key role of protozoa in controlling the abundance of populations of certain bacteria introduced into soil.     

Antidepressant-like deliverables from the sea: evidence on the efficacy of three different brown seaweeds via involvement of monoaminergic system

Brown seaweeds exhibit several health benefits in treating and managing wide array of ailments. In this study, the antidepressant-like effect of methaolic extracts from Sargassum swartzii (SS), Stoechospermum marginatum (SM), and Nizamuddinia zanardinii (NZ) was examined in forced swimming test (FST), in rats. Oral administration of SS, SM, and NZ extract (30–60 mg/kg) exhibited antidepressant-like activity in FST by reducing immobility time as compared to control group, without inducing significant change in ambulatory behavior in open field test. In order to evaluate the involvement of monoaminergic system, rats were pretreated with the inhibitor of brain serotonin stores p-chlorophenylalanin (PCPA), dopamine (SCH23390 and sulpiride), and adrenoceptor (prazosin and propranolol) antagonists. Rats receiving treatment for 28 days were decapitated and brains were analyzed for monoamine levels. It may be concluded that the extracts of SS, SM, and NZ produces antidepressant-like activity via modulation of brain monoaminergic system in a rat model.     

Comparison of the diversity of the bacterial communities in the intestinal tract of adult Bactrocera dorsalis from three different populations

Aims: To (i) identify the bacterial communities in the gut of oriental fruit fly (Bactrocera dorsalis) adult and (ii) determine whether the different surroundings and diets influence the bacteria composition. Methods and Results: Polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the oriental fruit fly adult gut. The 16S rDNA cloned libraries from the intestinal tract of laboratory‐reared (LR), laboratory sterile sugar‐reared (LSSR) and field‐collected (FC) populations of oriental fruit fly were compared. Phylogenetic analysis of 16S rDNA revealed that Gammaproteobacteria were dominant in the all samples (73·0–98·3%). Actinobacteria and Firmicutes were judged to be major components of a given library as they constituted 10% or more of the total clones of such library. The Flavobacteria, Deltaproteobacteria, Bacteroidetes and Alphaproteobacteria were observed in small proportions in various libraries. Further phylogenetic analyses indicated common bacterial phylotypes for all three libraries, e.g. those related to Klebsiella, Citrobacter, Enterobacter, Pectobacterium and Serratia. libshuff analysis showed that the bacterial communities of B. dorsalis from the three populations were significantly different from each other (P < 0·0085). Conclusions: (i) The intestinal tract of B. dorsalis adult contains a diverse bacterial community, some of which are stable. (ii) Different environmental conditions and food supply could influence the diversity of the harboured bacterial communities and increase community variations. Significance and Impact of the Study: Comparison of the microbial compositions and common bacterial species found in this paper may be very important for the biocontrol of B. dorsalis.     

A novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers for the enrichment and determination of polychlorinated biphenyls in fish samples

The novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers (rGO@m‐MIPs) were successfully synthesized as adsorbents for six kinds of polychlorinated biphenyls (PCBs) in fish samples. rGO@m‐MIPs was prepared by surface molecular imprinting technique. Besides, Fe₃O₄ nanoparticles (NPs) were employed as magnetic supporters, and rGO@Fe₃O₄ was in situ synthesis. Different from functional monomer and cross‐linker in traditional molecularly imprinted polymer, here, 3,4‐dichlorobenzidine was employed as dummy molecular and poly(ethylene‐co‐vinyl alcohol) was adopted as the imprinted polymers. After morphology and inner structure of the magnetic adsorbent were characterized, the adsorbent was employed for disperse solid phase extraction toward PCBs and exhibited great selectivity and high adsorption efficiency. This material was verified by determination of PCBs in fish samples combined with gas chromatography‐mass spectrometry (GC‐MS) method. According to the detection, the low detection limits (LODs) of PCBs were 0.0035–0.0070 µg l⁻¹ and spiked recoveries ranged between 79.90 and 94.23%. The prepared adsorbent can be renewable for at least 16 times and expected to be a new material for the enrichment and determination of PCBs from contaminated fish samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Marfan syndrome: No evidence for heterogeneity in different populations, and more precise mapping of the gene

Marfan syndrome is a dominantly inherited connective tissue disorder with manifestations in the cardiovascular, ocular, and skeletal systems. The diagnosis is hampered by both high variability in the phenotypic expression and late manifestation of symptoms. The cause of Marfan syndrome remains unknown, but our group has recently reported the genetic linkage of Marfan syndrome to a polymorphic marker on chromosome 15. To analyze the possible heterogeneity behind Marfan syndrome, we have performed linkage analyses for four chromosome 15 markers in 17 families from five different populations: Scottish, English, Swiss, American, and Finnish. By combining the linkage data of all the studied families into a LINKMAP analysis we obtained a maximal LOD score of 11.2, which maps the Marfan syndrome locus between D15S25 and D15S45 on the long arm of chromosome 15. The data reveal no evidence for genetic heterogeneity behind Marfan syndrome and provide us with a more precise location of both the Marfan syndrome locus and flanking markers. This information will provide the basis for the DNA diagnostics of Marfan syndrome in the future.     

Regulation of myofibrillar accumulation in chick muscle cultures: evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

The effect of calcium on myofibrillar turnover in primary chick leg skeletal muscle cultures was examined. Addition of the calcium ionophore A23187 at subcontraction threshold levels (0.38 microM) increased significantly rates of efflux of preloaded 45Ca+2 but had no effect on total protein accumulation. However, A23187 as well as ionomycin caused decreased accumulation of the myofibrillar proteins, myosin heavy chain (MHC), myosin light chain 1f (LC1f), 2f (LC2f), alpha-actin (Ac), and tropomyosin (TM). A23187 increased the degradation rate of LC1f, LC2f, and TM after 24 h. In contrast, the calcium ionophore caused decreased degradation of Ac and troponin-C and had no effect on the degradation of MHC, troponin-T, troponin-I, or alpha, beta-desmin (Dm). In addition, A23187 did not alter degradation of total myotube protein. The ionophore had little or no effect on the synthesis of total myotube proteins, but caused a marked decrease in the synthesis of MHC, LC1f, LC2f, Ac, TM, and Dm after 48 h. The mechanisms involved in calcium-stimulated degradation of the myofibrillar proteins were also investigated. Increased proteolysis appeared to involve a lysosomal pathway, since the effect of the Ca++ ionophore could be blocked by the protease inhibitor leupeptin and the lysosomotropic agents methylamine and chloroquine. The effects of A23187 occur in the presence of serum, a condition in which no lysosomal component of overall protein degradation is detected. The differential effect of A23187 on the degradative rates of the myofibrillar proteins suggests a dynamic structure for the contractile apparatus.     

Cytochemical evidence of an organized microtubular cytoskeleton in Xenopus laevis oocytes: involvement in the segregation of mitochondrial populations.

An organized microtubular cytoskeleton was discovered in the cytoplasm of Xenopus laevis oocytes. The microtubules were observed in 10- to 30-micron cryostat sections by indirect immunoperoxidase labeling using an antibody to tubulin. A gradual extraction of cells with a nonionic detergent was essential for good penetration of the antibody into the cells. In the cytoplasm of all previtellogenic oocytes, a dense network of criss-crossed long microtubules was associated in a basket-like structure surrounding the mitochondrial mass. At the beginning of vitellogenesis, the network meshes enlarged, while clusters of mitochondria migrated, in close association with microtubule bundles. At the beginning of vitellogenesis, the reorganization of the microtubular network, mostly in the vegetal hemisphere, occurred during the segregation of the mitochondrial populations. Reorganization is characterized by (1) a temporary enlargement of the network and close association of mitochondrial clusters with microtubular bundles, and (2) a progressive organization of a ring-shaped microtubular structure in the crown elaboration area. It is hypothesized that these modifications of the microtubular cytoskeleton contribute to the maintenance of cell shape and the polarized organization of the cell.