Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB-contaminated sediment

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate. Growth on 5 g l⁻¹ glucose alone resulted in the highest stimulation of the growth of bacterial strains, whereas grown on 10 mg l⁻¹, 100 mg l⁻¹, 1 g l⁻¹, or 5 g l⁻¹ biphenyl did not effected the bacterial growth. None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown on glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at concentrations of 1 or 5 g l⁻¹ did not increase total PCB degradation, but stimulated the degradation of highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was significantly lower for cells grown on 5 g l⁻¹ biphenyl independently on glucose addition. The highest degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of bioremediation.     

The adaptation responses of bacterial cytoplasmic membrane fluidity in the presence of environmental stress factors — polychlorinated biphenyls and 3-chlorobenzoic acid

Only bacteria sufficiently resistant to the toxic compounds in their environment can be used for the efficient biodegradation process in order to eliminate a widespread contamination by polychlorinated biphenyls (PCBs). The presence of PCBs results in bacterial controlled rigidification of cytoplasmic membrane. The four bacterial isolates from long-term PCB-contaminated soil (Alcaligenes xylosoxidans, Pseudomonas stutzeri) and sediment (Ochrobactrum anthropi, Pseudomonas veronii) have been used to select the strain most adapted to the PCBs, i.e. with efficient changes in the membrane phospholipid fatty acids. PCBs and their toxic degradation products — the 3-chlorobenzoic acids (3-CBA as the most toxic one) — were added separately to the liquid medium with glucose in two experimental sets: at lag phase and in stationary phase of bacterial growth in order to evaluate the effects of chemicals to cytoplasmic membrane. The main parameter — the changes in fatty acids composition (in the total lipids and the main membrane phospholipid phosphatidyletanolamine) were studied. 3-CBA caused growth inhibition when added at lag phase. However, when added during the stationary growth, inhibition was not observed. Similarly, after addition of PCBs to the stationary growth culture, inhibition of growth was not observed with all tested strains (except for P. stutzeri). This fact indicates the importance of time contact of bacteria during growth phase with xenobiotics. O. anthropi and A. xylosoxidans appeared to be the most adapted to the presence of PCBs (with sufficient membrane adaptation), active under the adverse conditions, and able to survive in the contaminated environment.     

The effect of polychlorinated biphenyls (PCBs) on the membrane lipids of Pseudomonas stutzeri

In this study we examined the effect of polychlorinated biphenyls (PCBs) on biomass production of a PCB-degrading Pseudomonas stutzeri, and on the fatty acid profile of its major membrane lipids. Growth based on biomass weight was stimulated when PCBs were added at the time of inoculation, but PCB addition three days after inoculation led to a significant decrease in biomass. Simultaneous addition of PCBs plus biphenyl or PCBs plus carvone negatively affected P. stutzeri biomass (addition of biphenyl or carvone at the time of inoculation and PCBs to three-day-old culture). In the presence of PCBs alone the amount of the prevalent fatty acids C16:0 and C17-cyclopropyl fatty acid (C17-CP) of P. stutzeri in total and neutral lipids was significantly reduced. When PCBs were added together with carvone (carvone at the time of inoculation and PCBs after three days) a significant reduction of these fatty acids was obtained, but, in addition, oleic, cis-vaccenic, and cyclononadecanic (C19-CP) acids were increased. When PCBs were combined to biphenyl the prevalent fatty acids were reduced and oleic, cis-vaccenic, and cyclononadecanic acids were increased in total and neutral lipids. Addition of 3-chlorobenzoic acid led to a significant growth inhibition and to the production of oleic and cis-vaccenic acids in the membrane fraction phosphatidylcholine.     

Potential for polychlorinated biphenyl biodegradation in sediments from Indiana Harbor and Ship Canal

Polychlorinated biphenyls (PCBs) are carcinogenic, persistent, and bioaccumulative contaminants that pose risks to human and environmental health. In this study, we evaluated the PCB biodegradation of sediments from Indiana Harbor and Ship Canal (IHSC), a PCB-contaminated site (average PCB concentration = 12,570 ng/g dw). PCB congener profiles and bacterial community structure in a core sediment sample (4.57 m long) were characterized. Analysis of vertical PCB congener profile patterns in sediment and pore water strongly suggests that in situ dechlorination occurred in sediments. However, 16S rRNA genes from putative PCB-dechlorinating Chloroflexi were relatively more abundant in upper 2 m sediments, as were genes indicative of aerobic biodegradation potential (i.e. biphenyl dioxygenase (bphA)). Characterization of the bacterial community by terminal restriction fragment length polymorphism and comparison of these with sediment and pore water PCB congener profiles with the Mantel test revealed a statistical correlation (p < 0.001). Sequences classified as Acinetobacter and Acidovorax were highly abundant in deep sediments. Overall, our results suggest that PCB dechlorination has already occurred, and that IHSC sediments have the potential for further aerobic and anaerobic PCB biodegradation.     

<Emphasis Type="Italic">Carex vaginata</Emphasis> — new species in the Western Carpathians

The first locality of Carex vaginata Tausch from the Western Carpathians is reported. This glacial relic was found at a refugial site in the Mt. Kubínska hoľa (the Oravská Magura Mts, Slovakia), in the saddle peat bog at 1,312 m a.s.l.     

DNA-Stable Isotope Probing Integrated with Metagenomics for Retrieval of Biphenyl Dioxygenase Genes from Polychlorinated Biphenyl-Contaminated River Sediment

Stable isotope probing with [¹³C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from [¹³C]DNA after a 14-day incubation with [¹³C]biphenyl revealed the dominant organisms to be members of the genera Achromobacter and Pseudomonas. A library built from PCR amplification of genes for aromatic-ring-hydroxylating dioxygenases from the [¹³C]DNA fraction revealed two sequence groups similar to bphA (encoding biphenyl dioxygenase) of Comamonas testosteroni strain B-356 and of Rhodococcus sp. RHA1. A library of 1,568 cosmid clones was produced from the [¹³C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic dioxygenase primers, contained genes of biphenyl dioxygenase subunits bphAE, while the rest of the clone''s sequence was similar to that of an unknown member of the Gammaproteobacteria. A discrepancy in G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and para-only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed dioxygenase gene organization in nature.Commercially used polychlorinated biphenyls (PCBs), which are mixtures of more than 60 individual chlorinated biphenyl congeners, are among the most persistent anthropogenic chemical pollutants that threaten natural ecosystems and human health (1). Numerous biphenyl-degrading microorganisms have been isolated and studied, especially for the range of PCB congeners that they degrade. Research has been primarily focused on the biodegradative pathways and the biphenyl dioxygenases responsible for initial PCB oxidation by isolated bacteria (14, 27). Knowledge, however, is limited concerning the indigenous microbial populations that metabolize PCBs in the environment. Stable isotope probing (SIP) coupled with metagenomics is one approach to more directly explore which organisms and genetic information may be involved in PCB degradation in PCB-contaminated sites.SIP was developed to separate and concentrate the nucleic acids or fatty acids of microbial populations that metabolize and, hence, assimilate the isotopically labeled substrates into new cell material (4, 5, 28). Recently, the active PCB degraders in a biofilm community on PCB droplets were revealed as Burkholderia species by using DNA-SIP (32). In another DNA-SIP study, 75 different genera that acquired carbon from [¹³C]biphenyl were found in the PCB-contaminated root zone of a pine tree (22). In addition, that heavy [¹³C]DNA fraction revealed new dioxygenase sequences and possible PCB degradation pathways from GeoChip (16) results and from PCR-amplified sequences obtained by using primers targeting aromatic-ring-hydroxylating dioxygenase (ARHD) genes (22).A major hurdle in using DNA-SIP for metagenomic analyses (9) is the very small amount of heavy DNA that is produced and, hence, recovered, making library construction difficult. Two studies have shown the feasibility of DNA-SIP for metagenomic analyses for C-1 compound-utilizing communities, but they first increased the amount of the heavy DNA fraction by multiple-displacement amplification (6, 10) or enriched the community by growth in sediment slurries. (18).In this study, we used [¹³C]biphenyl to probe for potential PCB-degrading populations in a PCB-contaminated river sediment and to recover genes potentially involved in the critical first step of PCB degradation, the dioxygenase attack. We found a 31.8-kb cosmid clone that contained a biphenyl dioxygenase sequence (bphAE) and demonstrated its activity on PCBs.     

Biodegradation and evaporation of polychlorinated biphenyls (PCBs) in liquid media

During microbial degradation of PCBs in a liquid medium, two processes influence the PCB concentration in the medium simultaneously: biodegradation and evaporation. The physical loss of PCB due to evaporation frequently causes false positive results in biodegradation experiments. Therefore, if only PCBs are monitored, the determination of the PCB concentration in both liquid and gaseous phases is necessary for a correct appraisal of biodegradation. The kinetics of PCB evaporation and biodegradation were monitored and described by a simple mathematical model. The evaporation and biodegradation rate constants for individual PCB congeners were determined for PCB degradation in liquid medium byPseudomonas stutzeri andAlcaligenes xylosoxidans, both isolated from a longterm PCB-contaminated soil.Symbols a ₁,b ₁,a ₂,b ₂ fitting parameters - c ₀ initial concentration of PCB congener in liquid medium - c _l concentration of PCB congener in liquid medium - c _ev concentration of PCB congener in sorbent - k _ev rate constant of PCB congener evaporation - k _met rate constant of PCB congener metabolization - n _s amount of PCB congener in sorbent - t _1/2 half-time of evaporation - V _t volume of liquid medium     

Effects of co-inoculation of two different plant growth-promoting bacteria on duckweed

Aseptic Lemna minor was soaked for 4 h in pond water where wild L. minor was naturally flourishing. Seven of the eight surface-colonizing bacterial strains were found capable of promoting the growth of L. minor. This high appearance of plant growth-promoting bacteria (PGPB) suggests that association of environmental bacteria is generally beneficial rather than harmful for host plants. One of the PGPB, Pseudomonas sp. Ps6, enhanced the growth of L. minor by 2–2.5-fold in 10 days. This activity was higher than that previously reported for Acinetobacter calcoaceticus P23, which enhanced growth of L. minor by 1.5–2-fold. Ps6 mostly adhered to and colonized the root rather than the frond, a leaf-like structure of duckweed where P23 preferentially adheres. It was expected that these two strains can share niches, coexist, and enhance the growth of duckweed additively upon co-inoculation. However, contrary to expectation, the growth of L. minor was enhanced by only 2.3-fold by co-inoculation of these two bacteria. P23 lowered the initial adhesion of Ps6 cells by 98.2% on the fronds and by 79.5% on the roots. However, initial adhesion of P23 cells to the roots increased dramatically, by 47.2-fold, following co-inoculation with Ps6. However, the number of P23 cells decreased dramatically to 0.7% on the root and to 3.6% on the frond after 10 days, whereas Ps6 cells increased by 12.5-fold on the frond and kept 69% on the root, thereby eventually restoring the population on the plant surfaces. Because duckweed is the fastest growing vascular plant and it is easy to grow an aseptic and axenic plant, the duckweed/bacteria co-culture system will be a model platform for studying multiple interactions among host plants and the associated bacteria.     

Additive and Synergistic Growth-inhibiting Properties of the Canaline-Urea Cycle Amino Acids

Growth studies with Lemna minor revealed the additive and synergistic growth-inhibiting properties of the canaline-urea cycle amino acids. Simultaneous canavanine and canaline treatment caused an additive reduction in frond production. Ureidohomoserine interacted with canaline or canavanine to affect synergistically L. minor growth by enhancing individual canavanine or canaline toxicity and increasing the additive growth reduction caused by canavanine plus canaline. The ornithineurea cycle amino acids effectively counteracted both the additive and synergistic growth-inhibiting properties of the canaline-urea cycle compounds.     

Sphingobium fuliginis HC3: A Novel and Robust Isolated Biphenyl- and Polychlorinated Biphenyls-Degrading Bacterium without Dead-End Intermediates Accumulation

Biphenyl and polychlorinated biphenyls (PCBs) are typical environmental pollutants. However, these pollutants are hard to be totally mineralized by environmental microorganisms. One reason for this is the accumulation of dead-end intermediates during biphenyl and PCBs biodegradation, especially benzoate and chlorobenzoates (CBAs). Until now, only a few microorganisms have been reported to have the ability to completely mineralize biphenyl and PCBs. In this research, a novel bacterium HC3, which could degrade biphenyl and PCBs without dead-end intermediates accumulation, was isolated from PCBs-contaminated soil and identified as Sphingobium fuliginis. Benzoate and 3-chlorobenzoate (3-CBA) transformed from biphenyl and 3-chlorobiphenyl (3-CB) could be rapidly degraded by HC3. This strain has strong degradation ability of biphenyl, lower chlorinated (mono-, di- and tri-) PCBs as well as mono-CBAs, and the biphenyl/PCBs catabolic genes of HC3 are cloned on its plasmid. It could degrade 80.7% of 100 mg L ⁻¹ biphenyl within 24 h and its biphenyl degradation ability could be enhanced by adding readily available carbon sources such as tryptone and yeast extract. As far as we know, HC3 is the first reported that can degrade biphenyl and 3-CB without accumulation of benzoate and 3-CBA in the genus Sphingobium, which indicates the bacterium has the potential to totally mineralize biphenyl/PCBs and might be a good candidate for restoring biphenyl/PCBs-polluted environments.     

Notes on Oenocarpus (Palmae) in the Colombian Amazon

Five species ofOenocarpus from the Río Caquetá, in the Colombian Amazon, are discussed. Two of themOenocarpus simplex andOenocarpus makeru are described as new.Oenocarpus bacaba var.parvus is shown to be a synonym ofO. balickii; this species andO. minor are recorded for the first time in Colombia. Some comments on the poorly knownO. circumtextus are also given. The new findings support the inclusion ofJessenia inOenocarpus.     

Allocaeculus sandbergensis sp. n. (Acari: Caeculidae), a new prostigmatid mite from Slovakia

In January and in May 2012 three individuals (two adults and one nymph) from the genus Allocaeculus (family Caeculidae) were found in the area of the National nature reserve Devínska Kobyla near Sandberg (SW Slovakia). This mite family has not been recorded in Slovakia yet. Data about its nearest occurrence come from the Czech Republic and Austria. Based on the specimens, Allocaeculus sandbergensis sp. n. is described.     

Polychlorinated biphenyls (PCBs) in fish-eating sea bird—I. Molecular features of PCB isomers and congeners in adipose tissue of male and female razorbills (Alca tarda) of British and Irish coastal waters

1. The concentration of individual PCBs was measured in adipose tissue of male and female razorbills obtained from the Isle of May and the Saltees islands.2. No significant differences were found in the concentration of total PCBs, which showed positive correlations with the concentration of p,p'-DDE in the tissues.3. Enrichment factors were calculated by comparing the concentration of an individual PCB in the tissue with its abundance in commercial mixtures of PCBs.4. Eight PCBs, together accounting for 20–65% of the concentration of total PCBs present, had enrichment factors of > 10. They had the common molecular feature of chlorine atoms at adjacent meta-para positions in at least one of the biphenyl rings.5. Many PCBs had enrichment factors of < 1, suggesting that they had been subjected to metabolism and presumably excretion. They had, in common, the absence of chlorine atoms at the meta-para positions of the biphenyl rings.     

Der Einfluss von Phenäthylpyridinen und Pyridylphenylazetylenen auf die Wurzelatmung vonSinapis alba L.

Aktivita sukcinát-dehydrogenázy SDH a sukcinát-oxydázového systému SO byla mě?ena sestý den po vyklí?ení v homogenátu z ko?en? klí?ních rostlin ho??ice, které byly pěstovány v ?ivném roztoku, obsahujícím některý ze t?í polohových isomer? fenethylpyridinu (IIIA, B, C) nebo pyridylfenylacetylenu (IVA, B, C) v koncentraci 5×10^?4 m. Slou?eniny fenethylpyridinového typu prakticky neovlivňují aktivitu dehydrogenázy kyseliny jantarové SDH aniin vivo, aniin vitro, av?ak pyridylfenylacetyleny aktivitu tohoto enzymuin vivo výrazně stimulují. Naproti tomu oba typy slou?enin v pokusném uspo?ádání jakin vivo, takin vitro pr?kazně inhibují aktivitu sukcinát-oxydázového systému SO. Ú?inek fenethylpyridin? i pyridylfenylacetylen? je tedy pravým opakem ú?innosti trans-styrylpyridin? (typ I).     

The protein euphaseolin inPhaseolinae — A chemotaxonomical study

On the basis of immunochemical analyses of the main reserve protein ofPh. vulgaris, euphaseolin, in numerous cultivars ofPh. vulgaris, in additional 23Phaseolus species, and several representatives of further genera of Viciaceae and on the basis of the comparison of these data with morphological and genetical data the authors propose to separate the sectionEuphaseolus characterized by the presence of the protein euphaseolin. The species characterised by euphaseolin are closely related and capable of being crossed. The proposal requires an additional formal completion from the point of view of the conventions of classical systematies. Further the questions of the taxonomical extent of various protein characters and the problematies of the so-called large and small protein characters are discussed. Na základě imunochemických analys hlavní zásobní bílkovinyPh. vulgaris euphaseolinu u velkého po?tu kultivar?Phaseolus vulgaris, dal?ích 23 druh?Phaseolus a několika zástupc? dal?ích rod?Viciaceae a srovnáváním těchto údaj? s údajl morfologickými a genetickými auto?i navrhují vydělení sekceEuphaseolus, charakterisované p?ítomností bílkoviny euphaseolinu. Druhy charakterisované euphaseolinem jsou si blízce p?íbuzné, jsou k?i?itelné. Návrh vy?aduje je?tě formální doplnění z hlediska zvyklostí klasické systematiky. V ?lánku jsou dále diskutovány otázky taxonomické ?í?e r?zných bílkovinných znak?.     

Differenzialdiagnostische Wirtspflanzen fürAgrobacterium tumefaciens undAgrobacterium rhizogenes

P?i na?í práci s nádorotvornými bakteriemi se ukázalo, ?eBryophyllum diagremontiana L.,Bryophyllum tubiflorum L. aSolanum laciniatum L. jsou vhodné diferen?ní rostliny proAgrobacterium tumefaciens, Agrobacterium rhizogenes a směs obou těchto bakterií.

1. 1.
   NaAgrobacterium tumefaciens reagujeBryophyllum diagremontiana tvorbou nádor? za 32 dní po o?kování a stimulaci r?stu ko?ínk? z okraj? ná dor? (za 52 dn?). UBryophyllum tubiflorum je inkubacni doba nádor? i ko?ínk? 30 dní.Solanum laciniatum má inkuba?ní dobu nádor? 15 dní, ale nedochází stimulaci r?stu ko?ínk?.     
   
   

Frond transformation system mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> for <Emphasis Type="Italic">Lemna minor</Emphasis>

The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8–9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.     

Flow Cytometry Analysis of Changes in the DNA Content of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102: Effect of Metabolites on Cell-Cell Separation

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.     

Polychlorinated biphenyls (PCBs) in fish-eating sea birds—II. Molecular features of PCB isomers and congeners in adipose tissue of male and female puffins (Fratercula arctica), guillemots (Uria aalga), shags (Phalacrocorax aristotelis) and cormorants (Phalacrocorax carbo) of British and Irish coastal waters

1. The concentration of individual PCBs was measured in adipose tissue of male and female puffins, shags, guillemots and cormorants obtained from the Isle of May and the Saltees islands.2. The concentrations of total PCBs showed positive correlations with that ofp,p'-DDE in the tissues.3. Enrichment factors were calculated by comparing the concentration of an individual PCB in the tissue with its abundance in commercial mixtures of PCBs.4. Of the 47 individual PCBs identified five prominent PCBs had enrichment factors considerably > 1 and accounted for approximately 35% of the total concentration of PCBs present. They shared the common molecular feature of chlorine atoms at adjacent meta-para positions in at least one of the biphenyl rings.5. Many PCBs had enrichment factors of <1, which suggested that they had been subjected to metabolism and presumably excretion. They shared the common feature of the absence of chlorine atoms at the meta-para positions of the biphenyl rings.6. These results support strongly the structural “rules” suggested in the preceding paper (Borlakoglu et al., 1990a) for the tendency of individual PCBs to accumulate or to be subjected to metabolism.