Chlorophenol degradation coupled to sulfate reduction.

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Chlorophenol degradation coupled to sulfate reduction

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Biodegradation of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by dichlorophenol-adapted microorganisms from freshwater,anaerobic sediments

Reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated in anaerobic sediments by non-adapted microorganisms and by microorganisms adapted to either 2,4- or 3,4-dichlorophenol (DCP). The rate of dechlorination of 2,4-D was increased by adaptation of sediment microorganisms to 2,4-DCP while dechlorination by sediment microorganisms adapted to 3,4-DCP displayed a lag phase similar to non-adapted sediment slurries. Both 2,4- and 3,4-DCP-adapted microorganisms produced 4-chlorophenoxyacetic acid by ortho-chlorine removal. Lag phases prior to dechlorination of the initial addition of 2,4,5-T by DCP-adapted sediment microorganisms were comparable to those from non-adapted sediment slurries. However, the rates of dechlorination increased upon subsequent additions of 2,4,5-T. Biodegradation of 2,4,5-T by sediment microorganisms adapted to 2,4- and/ or 3,4-DCP produced 2,5-D as the initial intermediate followed by 3-chlorophenol and phenol indicating a para > ortho > meta order of dechlorination. Dechlorination of 2,4,5-T, by either adapted or non-adapted sediment microorganisms, progressed without detection of 2,4,5-trichlorophenol as an intermediate.     

Degradation ofortho-chlorophenol,para-chlorophenol, and 2,4-dichlorophenoxyacetic acid by the bacterial community of anaerobic sludge

The bacterial community of anaerobic sludge could degradeo-chlorophenol,p-chlorophenol, and 2,4-dichlorophenoxyacetic acid at concentrations as high as 100 mg/l. The time needed for the degradation of a given chlorinated phenol derivative increased 1.5- to 2-fold upon a twofold increase in its concentration (from 50 to 100 mg/l). The duration of the adaptation period depended on the compound studied and on its concentration. The degradation of 2,4-dichlorophenoxyacetic acid proceeded via 2,4-dichlorophenol andp-chlorophenol as intermediates; the degradation ofo-chlorophenol occurred with the formation of phenol. The dynamics ofp-chlorophenol degradation and chloride ion accumulation were studied.     

Versatile aromatic compound-degrading capacity and microdiversity of <Emphasis Type="Italic">Thauera</Emphasis> strains isolated from a coking wastewater treatment bioreactor

Bacteria of the Thauera genus have been described as important aromatic compound degraders and have attracted increased attention. In this study, three Thauera strains (Q4, Q20-C, and 3–35) were isolated from a coking wastewater treatment plant (WWTP) with a high abundance of Thauera. The 16S rRNA, nitrite reductase, and phenol hydroxylase (LmPH) genes and pollutant-degrading capacity of these strains were characterized and compared. Their 16S rRNA gene sequences were identical, but the genomic structures differed, as demonstrated by distinct enterobacterial repetitive intergenic consensus sequence PCR profiles with a similarity of less than 0.65. The analysis of degradation of coking wastewater by these strains showed that most of the main organic pollutants—phenol, methylphenol, and indole, but not quinoline—were degraded under aerobic conditions. These strains contained different LmPHs genes and showed different phenol degradation rates (Q4 > 3–35 > Q20-C). The presence of a microdiversity of Thauera spp. implies the existence of various finely differentiated niches in the industrial WWTP. The capacity of the Thauera strains to degrade a wide spectrum of aromatic compounds suggests their potential in bioremediation applications targeting aromatic pollutant-containing wastewater.     

Regiospecific dechlorination of pentachlorophenol by dichlorophenol-adapted microorganisms in freshwater, anaerobic sediment slurries.

The reductive dechlorination of pentachlorophenol (PCP) was investigated in anaerobic sediments that contained nonadapted or 2,4- or 3,4-dichlorophenol (DCP)-adapted microbial communities. Adaptation of sediment communities increased the rate of conversion of 2,4- or 3,4-DCP to monochlorophenols (CPs) and eliminated the lag phase before dechlorination was observed. Both 2,4- and 3,4-DCP-adapted sediment communities dechlorinated the six DCP isomers to CPs. The specificity of chlorine removal from the DCP isomers indicated a preference for ortho-chlorine removal by 2,4-DCP-adapted sediment communities and for para-chlorine removal by 3,4-DCP-adapted sediment communities. Sediment slurries containing nonadapted microbial communities either did not dechlorinate PCP or did so following a lag phase of at least 40 days. Sediment communities adapted to dechlorinate 2,4- or 3,4-DCP dechlorinated PCP without an initial lag phase. The 2,4-DCP-adapted communities initially removed the ortho-chlorine from PCP, whereas the 3,4-DCP-adapted communities initially removed the para-chlorine from PCP. A 1:1 mixture of the adapted sediment communities also dechlorinated PCP without a lag phase. Dechlorination by the mixture was regiospecific, following a para greater than ortho greater than meta order of chlorine removal. Intermediate products of degradation, 2,3,5,6-tetrachlorophenol, 2,3,5-trichlorophenol, 3,5-DCP, 3-CP, and phenol, were identified by a combination of cochromatography (high-pressure liquid chromatography) with standards and gas chromatography-mass spectrometry.     

Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells of Alcaligenes sp. A7-2

Summary The biodegradation of the three isomeric monochlorophenols 2-(2CP), 3- (3CP) and 4-chlorophenol (4CP) and phenol by the constructed strain Alcaligenes sp. A7-2 was investigated. Mineralization took place in the order: phenol >4CP >2CP >3CP, whereas 3CP was mineralized only co-metabolically. In substrate mixtures with phenol, degradation of 4CP was decelerated but degradation of 2CP was accelerated. Free cells in batch culture showed biphasic growth with an equimolar mixture of 2CP and 4CP as substrates, perhaps due to diauxie. Degradation patterns obtained with free cells in batch culture were confirmed with immobilized cells in continuous culture. Immobilized cells of Alcaligenes sp. A7-2 built up a biofilm on the lava that was used as filling material in the packed-bed reactors. The continuous cultures remained stable despite increasing input rates of chlorophenol and phenol mixtures up to 1.16 mMo1.1^–1.h^–1 for several weeks. Correspondence to: H.-J. Rehm     

Inhibition of benzene,toluene, phenol and benzoate in single and combination on Anammox activity: implication to the denitrification–Anammox synergy

A previous study demonstrated that denitrification synergized with Anammox could accelerate the anaerobic degradation of benzene. The inhibitory effects of benzene, toluene, phenol and benzoate in single and combination on Anammox activity were investigated by short-term batch tests. The results indicated that the inhibition of single compounds on Anammox could be well fitted with the extended non-competitive and Luong inhibition kinetic models. The inhibitions of the individual compound were in order as follows: benzene?>?toluene?>?phenol?>?benzoate. The joint inhibitions of bi-component mixtures of benzene with toluene, benzene with phenol and benzene with benzoate on Anammox activity were additive; the joint inhibition of a tri-component mixture (benzene, toluene and phenol) was partly additive; and the joint inhibition of a multicomponent mixture (benzene, toluene, phenol and benzoate) was synergistic. The effect of benzoate on the denitrification–Anammox synergy for benzene degradation was evaluated using a long-term test. Although the average rate of benzene degradation decreased by 13% with the addition of 10 mg L^?1 benzoate, the average rate of NO₃^? and NH₄⁺ increased by approximately 1- and 0.56-fold, respectively, suggesting that benzoate favors the stability of the denitrification–Anammox synergy. The carboxylation of benzene would be a more favorable pathway for the anaerobic degradation of benzene under denitrification synergized with Anammox.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

Anaerobic treatment of 2,4,6-trichlorophenol in an expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactor at 15 degrees C

Expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactors were operated at 15 degrees C for the treatment of 2,4,6-trichlorophenol (TCP)-containing volatile fatty acid (VFA)-based wastewaters. The seed sludge used as inoculum for the control (no TCP) and test reactor was unexposed to chlorophenols (CPs) prior to the 425-day trial. TCP supplementation to the feed at 50 mg TCPl(-1) partially inhibited the anaerobic degradation of the VFA feed measured as COD removal efficiency. However, the withdrawal and subsequent application of stepwise increments to the TCP loading resulted in steady COD removal. Terminal restriction fragment length polymorphism analysis showed Methanosaeta-like Archaea in the control reactor over the experimental period. Different methanogenic populations were detected in the test reactor and responded to the changes in feed composition. Bacterial community analyses indicated changes in the community structure over time and suggested the presence of Campylobacter-like, Acidimicrobium-like and Heliophilum-like organisms in the samples. TCP mineralisation was by a reductive dechlorination pathway through 2,4-dichlorophenol (DCP) and 4-chlorophenol (4-CP) or 2-chlorophenol (2-CP). CP degradation rates in sludge granules from the lower chamber of the hybrid EGSB-AF reactor was in the order TCP > DCP > 4-CP > 2-CP. However, a biodegradability order of lower CPs > TCP was observed in fixed-film biomass taken from the upper reactor chamber, thus reflecting the role of this reactor section in the metabolism of residual lower CPs from the lower sludge-bed stage of operation.     

Reductive dechlorination of chlorophenols in slurries of low-organic-carbon marine sediments and subsurface soils

The reductive dechlorination of 2,4- and 3,4-dichlorophenol (DCP) was studied in slurries of marine sediments and subsurface soils with dissolved organic carbon concentrations less than 1 ppm. Dechlorination was markedly greater in marine sediment slurries than in subsoil slurries, although similar products were observed in each case. From 25% to 98% of the 2,4- and 3,4-DCP (6.5 μm/l) added to most marine slurries was converted to 4- and 3-chlorophenol (CP) respectively, within 30 weeks. In contrast 2,4-DCP was dechlorinated to 4-CP (>90%) in only 1 of 24 replicate subsoil slurries after 32 weeks of incubation. Dechlorination was observed within 2 weeks when yeast extract was added to subsoil slurries; yeast extract additions also stimulated dechlorination in marine sediments but to a lesser extent. The intermediate monochlorophenol products did not persist in marine slurries but did persist in the subsoil slurries. It was concluded that the total organic carbon at a site is not always a good predictor of the site's ability to support dechlorination activity. Received: 3 December 1996 / Received revision: 28 February 1997 / Accepted: 7 March 1997     

Degradation of Aniline by <Emphasis Type="Italic">Delftia tsuruhatensis</Emphasis> 14S in Batch and Continuous Processes

A Delftia tsuruhatensis strain capable of consuming aniline as the sole source of carbon, nitrogen, and energy at concentrations of up to 3200 mg/l was isolated from activated sludge of the sewage disposal plants of OAO Volzhskii Orgsintez. The strain grew on catechol and p-hydroxybenzoic acid but did not consume phenol, 2-aminophenol, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, 2-nitroaniline, 2-chlorophenol, or aminobenzoate. Aniline is degraded by cleavage of the catechol aromatic ring at the ortho position. Cells were immobilized on polycaproamide fiber. It was shown that the strain degraded aniline at 1000 mg/l in a continuous process over a long period of time.     

Production of doubled haploid plants from anther cultures of borage (<Emphasis Type="Italic">Borago officinalis</Emphasis> L.) by the application of chemical and physical stress

Cibotium barometz is an endangered tree fern, used both as ornamental plant and traditional Chinese medicinal plant. In this study, an effective in vitro propagation protocol was obtained through formation of green globular bodies (GGBs) from in vitro juvenile sporophytes. The effect of plant growth regulators (PGRs) on GGB induction and multiplication, as well as mineral salt concentration and active charcoal (AC) on plantlet regeneration from GGBs was evaluated. Thidiazuron (TDZ; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea) had a significant effect on GGB induction and multiplication (P?<?0.001), while a-naphthaleneacetic acid (NAA) did not (P?>?0.05). GGB induction rate was above 80?% on 1/2 Murashige and Skoog (MS) media supplemented with TDZ (1.0 mg L^??1) and NAA (0.1, 0.3 or 0.5 mg L^??1). The same media were also optimal for GGB multiplication. GGBs cultured on 1/4 MS media supplemented with 0.1 or 0.2?% (w/v) AC showed a high rate of GGB development into plantlets above 90?%. 1/2 MS media supplemented with 0.1 or 0.2?% AC were the most effective for plantlet growth. Regenerated plantlets were successfully acclimatized (80?%) in greenhouse conditions. Morphological and histological analysis revealed that C. barometz GGBs was a yellow-green globular structure composed of the single GGB with meristems and hair-like structures, and new single GGBs were initiated from the epidermal cells of meristem zone.     

Widespread Distribution of <Emphasis Type="Italic">Dehalococcoides mccartyi</Emphasis> in the Houston Ship Channel and Galveston Bay,Texas, Sediments and the Potential for Reductive Dechlorination of PCDD/F in an Estuarine Environment

Sediments in the Houston Ship Channel and upper Galveston Bay, Texas, USA, are polluted with polychlorinated dibenzo-p-dioxins/furans (PCDD/F; ≤46,000 ng/kg dry weight (wt.)) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener, contributing >50 % of the total toxic equivalents (TEQ) at most locations. We measured PCDD/F concentrations in sediments and evaluated the potential for enhanced in situ biodegradation by surveying for Dehalococcoides mccartyi, an obligate organohalide respiring bacterium. Dehalococcoides spp. (98 % similar to D. mccartyi) and 22 other members of the class Dehalococcoidia were predominant 16S ribosomal RNA (rRNA) phylotypes. Dehalococcoides spp. were also present in the active fraction of the bacterial community. Presence/absence PCR screening detected D. mccartyi in sediment cores and sediment grab samples having at least 1 ng/kg dry wt. TEQ at salinities ranging from 0.6 to 19.5 PSU, indicating that they are widespread in the estuarine environment. Organic carbon-only and organic carbon + sulfate-amended sediment microcosm experiments resulted in ～60 % reduction of ambient 2,3,7,8-TCDD in just 24 months leading to reductions in total TEQs by 38.4 and 45.0 %, respectively, indicating that 2,3,7,8-TCDD degradation is occurring at appreciable rates.     

The anaerobic degradation of 3-chloro-4-hydroxybenzoate in freshwater sediment proceeds via either chlorophenol or hydroxybenzoate to phenol and subsequently to benzoate.

To study the anaerobic degradation of the chimera 3-chloro-4-hydroxybenzoate (3-Cl,4-OHB), anaerobic freshwater sediment samples from the vicinity of Athens, Ga., were adapted for the transformation of 4-hydroxybenzoate (4-OHB), 3-chlorobenzoate (3-CB), 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). In nonadapted samples, both 4-OHB (product of aryl dechlorination) and 2-CP (product of aryl decarboxylation) were observed as intermediates in the transformation of 3-Cl,4-OHB to phenol. The accumulated phenol was subsequently transformed to benzoate, an intermediate in the conversion to methane and CO2. In 4-OHB-adapted samples (i.e., samples adapted for aryl decarboxylation), 2-CP was the first intermediate which was subsequently dechlorinated to phenol. In 3-CB-adapted samples (i.e., samples adapted for meta-chlorobenzoate dehalogenation), 3-Cl,4-OHB was stoichiometrically dechlorinated to 4-OHB. In 2-CP-adapted samples (i.e., samples adapted for ortho-chlorophenol dehalogenation), 4-OHB was the first major intermediate. Furthermore, 3-CB was not dechlorinated in 2-CP-adapted sediment samples, suggesting the possibility that different 3-Cl,4-OHB dechlorinating systems were induced in the 2-CP- and 3-CB-adapted sediments. Adaptation of sediment samples for dechlorination of 2,4-DCP did not lead to adaptation for dechlorination of 3-Cl,4-OHB. However, 3-Cl,4-OHB was dechlorinated to 4-OHB in our stable, sediment-free 2,4-DCP-dechlorinating enrichment, isolated previously from the same environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The anaerobic degradation of 3-chloro-4-hydroxybenzoate in freshwater sediment proceeds via either chlorophenol or hydroxybenzoate to phenol and subsequently to benzoate.

To study the anaerobic degradation of the chimera 3-chloro-4-hydroxybenzoate (3-Cl,4-OHB), anaerobic freshwater sediment samples from the vicinity of Athens, Ga., were adapted for the transformation of 4-hydroxybenzoate (4-OHB), 3-chlorobenzoate (3-CB), 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). In nonadapted samples, both 4-OHB (product of aryl dechlorination) and 2-CP (product of aryl decarboxylation) were observed as intermediates in the transformation of 3-Cl,4-OHB to phenol. The accumulated phenol was subsequently transformed to benzoate, an intermediate in the conversion to methane and CO2. In 4-OHB-adapted samples (i.e., samples adapted for aryl decarboxylation), 2-CP was the first intermediate which was subsequently dechlorinated to phenol. In 3-CB-adapted samples (i.e., samples adapted for meta-chlorobenzoate dehalogenation), 3-Cl,4-OHB was stoichiometrically dechlorinated to 4-OHB. In 2-CP-adapted samples (i.e., samples adapted for ortho-chlorophenol dehalogenation), 4-OHB was the first major intermediate. Furthermore, 3-CB was not dechlorinated in 2-CP-adapted sediment samples, suggesting the possibility that different 3-Cl,4-OHB dechlorinating systems were induced in the 2-CP- and 3-CB-adapted sediments. Adaptation of sediment samples for dechlorination of 2,4-DCP did not lead to adaptation for dechlorination of 3-Cl,4-OHB. However, 3-Cl,4-OHB was dechlorinated to 4-OHB in our stable, sediment-free 2,4-DCP-dechlorinating enrichment, isolated previously from the same environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower <Emphasis Type="Italic">Griffinia liboniana</Emphasis>

Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0?±?1.9%) and numbers of differentiated buds (3.7?±?0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L^?1 2,4-D and 1.0 mg L^?1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9?±?2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L^?1 6-BA and 1.5 mg L^?1 2,4-D, with an average of 28.0?±?2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments.

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Trends in accumulation of ephedrine in callus cultures of <Emphasis Type="Italic">Ephedra major</Emphasis> Host

Ephedra major Host, a medicinal plant, belongs to the family of Ephedraceae. Ephedrine is the main alkaloid in Ephedra, which has different medicinal properties. However, the amount of ephedrine in plant material is low and callus culture can be a way to increase the alkaloid content. The aim of this research was to compare Murashige and Skoog (MS) and Gamborg’s B5 culture media for callus induction and ephedrine production. For this purpose, stem explants were cultured on MS or B5 media containing 0.0, 0.5, 1.0, 2.0, or 3.0 mg L^?1 of kinetin (Kin) either alone or in combination with 0.0, 0.5, 1.0, or 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and/or naphthalenacetic acid (NAA), in five replicates. MS medium containing 1.0 or 2.0 NAA and 0.5 mg L^?1 Kin were the most effective for callus induction. The highest percentage of callus induction (100%) on B5 culture medium was obtained with 2.0 2,4-D and 0.5 mg L^?1 Kin treatments. The results showed that there was no significant difference between MS and B5 media for callus induction, and fresh and dry weight production. High-performance liquid chromatography was conducted for the identification and quantification of ephedrine in the obtained callus. The highest level of ephedrine (7.38 mg g^?1 DW) was found in callus grown on MS medium containing 0.5 mg L^?1 of 2,4-D. The results revealed that ephedrine can accumulate in callus cultures to levels much higher than in E. major wild plants.