Discipline of Microbiology School of Biochemistry, Genetics and Microbiology University of KwaZulu-Natal (Pietermaritzburg campus) Private bag X01 Pietermaritzburg 3209 South Africa
Abstract: The present long-term study (about 1,100 days) monitored the diversity of methanogens during the mesophilic, anaerobic digestion of beet silage. Six fermentor samples were analyzed by ribosomal RNA gene restriction analysis, fluorescence in situ hybridization, and fluorescence microscopy. Hydrogenotrophic methanogens dominated within the population in all samples analyzed. Multidimensional scaling revealed that a rapid decrease in hydraulic retention time resulted in increased species richness, which in turn led to slightly higher CH(4) yields.
Abstract: Beet silage and beet juice were digested continuously as representative energy crops in a thermophilic biogas fermentor for more than 7 years. Fluorescence microscopy of 15 samples covering a period of 650 days revealed that a decrease in temperature from 60 degrees C to 55 degrees C converted a morphologically uniform archaeal population (rods) into a population of methanogens exhibiting different cellular morphologies (rods and coccoid cells). A subsequent temperature increase back to 60 degrees C reestablished the uniform morphology of methanogens observed in the previous 60 degrees C period. In order to verify these observations, representative samples were investigated by amplified rRNA gene restriction analysis (ARDRA) and fluorescence in situ hybridization (FISH). Both methods confirmed the temperature-dependent population shift observed by fluorescence microscopy. Moreover, all samples investigated demonstrated that hydrogenotrophic Methanobacteriales dominated in the fermentor, as 29 of 34 identified operational taxonomic units (OTUs) were assigned to this order. This apparent discrimination of acetoclastic methanogens contradicts common models for anaerobic digestion processes, such as anaerobic digestion model 1 (ADM1), which describes the acetotrophic Euryarchaeota as predominant organisms.
Abstract: From water sampled in the River Elbe, we isolated a bacterial strain able to use the biocidal compound benzylbenzoate as its sole source of carbon and energy under aerobic conditions. This isolate was tentatively assigned to the genus Acinetobacter due to its morphological, physiological and partial SSU rRNA gene sequence properties. The productive bacterial degradation of the biocide benzylbenzoate was demonstrated, and the catabolic sequence was elucidated biochemically. Growth experiments, along with enzymatic studies, demonstrated that strain Acinetobacter sp. AG1 hydrolyzed benzylbenzoate enzymatically to yield benzylalcohol and benzoate. Benzylalcohol was further transformed to benzoate via benzaldehyde. Benzoate was subsequently channeled via catechol into the oxoadipate pathway for further degradation.
Abstract: Polybrominated diphenyl ethers (PBDEs) are common flame-retardant chemicals that are used in diverse commercial products such as textiles, circuit boards, and plastics. Because of the widespread production and improper disposal of materials that contain PBDEs, there has been an increasing accumulation of these compounds in the environment. The toxicity and bioavailability of PBDEs are variable for different congeners, with some congeners showing dioxin-like activities and estrogenicity. The diphenyl ether-utilizing bacterium Sphingomonas sp. PH-07 was enriched from activated sludge of a wastewater treatment plant. In liquid cultures, this strain mineralized 1 g of diphenyl ether per liter completely within 6 days. The metabolites detected and identified by gas chromatography/mass spectrometry (MS) and electrospray ionization/MS analysis corresponded with a feasible degradative pathway. However, the strain PH-07 even catabolized several brominated congeners such as mono-, di-, and tribrominated diphenyl ethers thereby producing the corresponding metabolites.
Abstract: The effect of heavy metals on the degradation of dibenzofuran by Sphingomonas wittichii RW1 was determined in liquid cultures. The results showed that 10mg/L cadmium, mercury and copper not only affected the growth of RW1 with dibenzofuran but also the ability of resting cells to degrade this compound. Growth and degradation were strongly inhibited by mercury, even at 1mg/L, while the inhibitory effect of cadmium and copper at the same concentration or at 5mg/L were negligible. In contrast, arsenic and lead did not affect degradation or growth, even at very high concentrations of 100mg/L. Subsequent analyses additionally revealed that concentrations of arsenic and lead remained unchanged following incubation, while those of cadmium, mercury and copper decreased significantly.
Abstract: Sphingomonas wittichii RW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and after n-butylboronate and N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.
Abstract: The bacterial catabolism of a technical formulation of alkylsulfonic acid phenyl esters employed as a general purpose plasticizer, Mesamoll, was elucidated. Two strains of the genus Rhodococcus were found able to utilize this substrate mixture as sole source of carbon and energy. Growth experiments along with enzymatic measurements indicated that both strains utilized the phenol that was released from the corresponding alkylsulfonic acid phenyl ester-probably by enzymatic hydrolysis catalyzed by esterases-via the ortho-pathway. By GC/MS analysis it was demonstrated that those alkyl chain homologues with substituents present at or close to the end of the aliphatic backbone (i.e., 2-tetradecylsulfonic acid phenyl ester) are degraded, while those with substituents close to the center of the alkyl chain (i.e., 7-tetradecylsulfonic acid phenyl ester) are rather persistent.
Abstract: We evaluated the High Production Volume (HPV)-compound 1,2,3-trichloropropane regarding its potential environmental impact by exploiting available data and by using the applicable EU guidelines for the risk assessment of new and existing substances. Consequently, a risk quotient of <1 was established by employing the PEC (predicted environmental concentration) and the PNEC (predicted no effect concentration) estimated from the data at our disposal. We do assume that a severe risk for the aquatic environment is not to be expected although long-term toxic effects cannot be entirely excluded for this compartment. No data were identified concerning the toxic effects exhibited by this compound upon terrestrial invertebrates or higher plants. Unfortunately, available ecotoxicological studies evaluating the inhibition of soil bound microbial activity by 1,2,3-trichloropropane were not sufficient to support a reliable risk characterization for the terrestrial compartment.
Abstract: The high production volume (HPV)-compound, p-chloroaniline, was evaluated with respect to its ecotoxicological impact upon aquatic systems. This was accomplished by employing the applicable EU-guidelines for the risk assessment of new and existing substances and by using available data. Accordingly, a risk quotient was obtained by employing a PEC and a PNEC derived from available data. We do presume that, in general, this compound does not pose a serious risk for the aquatic environment. However, by accidental spill or under conditions unfavourable for biotic or abiotic decomposition, p-chloroaniline might pose a potential risk for sensitive aquatic species.
Abstract: The role of green microalgae in the biotransformation of naphthalene (a polycyclic aromatic hydrocarbon) and diaryl ethers was investigated using axenic cultures of Chlorella vulgaris and two environmental isolates, Scenedesmus SI1 and Ankistrodesmus SI2. Biotransformation experiments with dense cell cultures showed that these three green algae transformed toxic xenobiotics to more polar metabolites. Chlorella vulgaris metabolized naphthalene to 1-naphthol (0.36-0.65%). Ankistrodesmus SI2 biotransformed dibenzofuran to six metabolites (total over 7%), three of which (possibly four) were identified as monohydroxylated dibenzofurans, the remaining two may be dihydroxylated derivatives. Scenedesmus SI1 biotransformed dibenzo-p-dioxin to three metabolites, one of which was tentatively identified as 2-hydroxydibenzo-p-dioxin (approximately 3.8%), the remainder may be dihydroxylated derivatives. This is the first time that the biotransformation of diaryl ethers by green microalgae has been investigated.
Abstract: Aerobic biotransformation of the diaryl ethers 2,7-dichlorodibenzo-p-dioxin and 1,2,3,4-tetrachlorodibenzo-p-dioxin by the dibenzo-p-dioxin-utilizing strain Sphingomonas wittichii RW1, producing corresponding metabolites, was demonstrated for the first time. Our strain transformed 2,7-dichlorodibenzo-p-dioxin, yielding 4-chlorocatechol, and 1,2,3,4-tetrachlorodibenzo-p-dioxin, producing 3,4,5,6-tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol; all of these compounds were unequivocally identified by mass spectrometry both before and after N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization by comparison with authentic standards. Additional experiments showed that strain RW1 formed a second metabolite, 2-methoxy-3,4,5,6-tetrachlorophenol, from the original degradation product, 3,4,5,6-tetrachlorocatechol, by methylation of one of the two hydroxy substituents.
Abstract: The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and (1)H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.
Abstract: The microbial degradation of aromatic pollutants has been well characterized over a period of more than 30 years. The microbes of most interest have been bacteria and fungi. Only relatively recently has the question of how algae figure in the catabolism of these compounds attracted a degree of interest. The aim of this review is to highlight the biodegradative capabilities of microalgae on aromatic compounds, ranging from simple monocyclic to more complex polycyclic pollutants. This paper will briefly encompass studies which have investigated the growth on and the oxidation of these compounds by algae, as well as a more derailed characterization of the catabolic sequences involved in the transformation of these compounds. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Abstract: A bacterial isolate, tentatively identified as Pseudomonas sp. strain TR3, was found to utilize the diaryl ester phenylbenzoate as sole source of carbon and energy. This strain has the ability to productively degrade phenylbenzoate and some substituted derivatives by a catabolic sequence which was characterized biochemically. The biodegradation of phenylbenzoate is thus initiated by an inducible esterase, effectively hydrolyzing the diaryl esters to produce stoichiometric amounts of two monoaromatic metabolites, identified as benzoate and phenol in the case of phenylbenzoate. The diaryl ester p-tolylbenzoate was hydrolyzed to yield benzoate and 4-methylphenol while 4-chlorophenylbenzoate gave rise to the production of benzoate and 4-chlorophenol. These monoaromatic catabolites were further degraded via the oxoadipate pathway.
Abstract: Due to their physicochemical and toxicological properties, polychlorinated dibenzofurans are regarded as a class of compounds providing reason for serious environmental concern. While the nonhalogenated basic structure dibenzofuran is effectively mineralized by appropriate bacterial strains, its polychlorinated derivatives are not. To elucidate the ability of the strain Sphingomonas sp RW1 to metabolize some of these chlorinated derivatives, we performed turnover experiments using 2,7-dichloro- and 2,4,8-trichlorodibenzofuran. As indicated by the oxygen-uptake rates determined for these two chlorinated dibenzofurans, Sphingomonassp RW1 can catabolize these chlorinated dibenzofurans yielding small quantities of oxidation products, which we isolated and subsequently characterized employing GC/MS and (1)H- as well as (13)C-NMR spectroscopy. In the case of 2,7-dichlorodibenzofuran, two metabolites accumulated, which we identified as 6-chloro- and 7-chloro-2-methyl-4H-chromen-4-one. The single metabolite isolated from the turnover experiments performed with 2,4,8-trichlorodibenzofuran was unequivocally identified as 6,8-dichloro-2-methyl-4H-chromen-4-one.
Abstract: Scedosporium apiospermum, a recently isolated phenol-degrading hyphomycete, was shown to be able to productively utilise the diaryl ester phenylbenzoate as its sole source of carbon and energy. The characterisation of degradation intermediates together with the detection of the corresponding catabolic enzymes in crude extracts enabled us to propose a pathway for the degradation of this diaryl ester. According to our results, an inducible esterase initiated the biodegradation of phenylbenzoate by hydrolysing the ester bond to yield stoichiometric amounts of phenol and benzoate. While phenol was catabolised via catechol and hydroxyhydroquinone, the benzoate was further degraded via the protocatechuate branch of the ortho-pathway. In addition, the fungus utilised p-tolylbenzoate and 4-chlorophenylbenzoate by employing similar catabolic sequences.
Abstract: A hyphomycete with the ability to utilize phenol and p-cresol as carbon and energy source was isolated from soil and subsequently identified as Scedesporium apiospermum. The identification of degradation metabolites and the detection of the corresponding catabolic enzymes in crude extracts enabled us to propose different pathways for the degradation of both phenol and p-cresol in this organism. Generally, the catabolism proceeded via three different dihydroxylated intermediates (catechol, hydroxyhydroquinone and protocatechuate) which were intradiolically cleaved by the corresponding inducible dioxygenases and further catabolized via the 3-oxoadipate pathway.
Abstract: The diphenyl ether degrading bacterial strain Sphingomonas sp. SS3, although not able to utilize 4-hydroxy- and 4,4'-dihydroxydiphenyl ether as sole sources of carbon and energy, is nevertheless capable of transforming these diaryl ethers into mononuclear hydroxylated aromatic compounds. The cleavage of the ether-bridge is prompted by an angular dioxygenation, yielding an unstable hemiacetal which spontaneously decays into catechol, phenol, 1,4-dihydroxy- and 1,2,4-trihydroxybenzene in the case of 4-hydroxydiphenyl ether, and 1,4-dihydroxy- and 1,2,4-trihydroxybenzene in the case of 4,4'-dihydroxydiphenyl ether, respectively. Although catechol and phenol are utilized as carbon sources, 1,4-dihydroxy- and 1,2,4-trihydroxybenzene are not. The 1,2,4-trihydroxybenzene proved able to inhibit growth of Sphingomonas sp. strain SS3, probably explaining why this organism is not able to grow at the expense of these p-hydroxylated diphenyl ethers.
Abstract: Here we report that the bacterial catabolism of 4-hydroxy-3,5-dimethylbenzoic acid 1 takes a different course in Rhodococcus rhodochrous N75 and Pseudomonas sp. strain HH35. The former organism accumulates a degradation metabolite of the acid which we isolated and identified as 2,6-dimethylhydroquinone 2. The latter bacterial strain converts the acid and the hydroquinone into a dead-end metabolite. This novel compound was characterised unequivocally by mass spectrometry and 1H and 18C NMR and UV spectroscopy as 4-acetonyl-4-hydroxy-2-methylbut-2-en-1,4-olide 4, a cyclic tautomer of (3-methylmaleyl)acetone, which exists as the enol carboxylate form 8 in aqueous solution.
Abstract: The structurally related polyhalogenated diaryl ethers such as diphenyl ethers (DEs), dibenzofurans (DFs), and dibenzo-p-dioxins (DDs) are regarded, due to their physicochemical and toxicological properties, as a class of compounds giving reason for serious environmental concern. While the nonhalogenated basic structures are biodegradable under aerobic conditions, there is the need for rather specialized strains to mineralize the halogenated derivatives. Certain halogenated metabolites might cause serious problems such as having inhibitory effects upon the degradation. Anaerobic methanogenic consortia do have the ability to almost completely dehalogenate even polyhalogenated congeners. It has been shown that certain fungi are capable of transforming chlorinated DFs and DDs by the activity of nonspecific enzymes such as lignin-peroxidases. (C) 1997 Elsevier Science Inc.
Abstract: The CD and UV spectra of a homochiral set of nine microbial muconolactones (5-oxo-2,5-dihydrofuran-2-acetic acids) 6 are recorded and the observed Cotton effects correlated with the absolute configurations.
Abstract: 3,4-Dimethylbenzoic acid and 3,5-dimethylbenzoic acid were both oxidised by 4-methylbenzoate (p-toluate)-grown cells of Rhodococcus rhodochrous N75 via the ortho-pathway through the intermediates 3,4- and 3,5-dimethylcatechol, respectively. Owing to the formation of the two novel dead-end metabolites, 4-carboxymethyl-2,3-dimethylbut-2-en-1,4-olide and 4-carboxymethyl-2,4-dimethylbut-2-en-1,4-olide from these substrates, 3,4- and 3,5-dimethylbenzoate did not serve as growth substrates for the strain.
Abstract: The bacterium Sphingomonas sp. strain SS33, obtained from parent diphenyl ether-mineralizing strain SS3 (S. Schmidt, R.-M. Wittich, D. Erdmann, H. Wilkes, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:2744-2750, 1992) after several weeks of adaptation on 4,4'-difluorodiphenyl ether as the new target compound, also utilized 4,4'-dichlorodiphenyl ether for growth. Intermediary halocatechols were also mineralized via the ortho pathway by type I enzymes. 4,4'-Dibromodiphenyl ether was not used as a carbon source although transformation by resting cells yielded mononuclear haloaromatic compounds, such as 4-bromophenol and 4-bromocatechol. The same was true for the conversion of 2,4-dichlorodiphenyl ether, which yielded the respective (halo-) phenols and (halo-) catechols.
Abstract: The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.
Abstract: The bacterium Sphingomonas sp. SS31, which was obtained from the diphenyl ether-degrading strain Sphingomonas sp. SS3 by an adaptation process, utilized 3-methyldiphenyl ether for growth in addition to diphenyl ether. The initial enzymatic attack onto this compound proceeded by a regioselective, but non-specific dioxygenation at the carbon carrying the ether bridge and the adjacent carbon of the unsubstituted as well as the methyl-substituted aromatic nucleus. Upon spontaneous decomposition, the resulting unstable hemiacetal structure yielded 3-methylphenol and catechol, or phenol, 3-methylcatechol, and 4-methylcatechol, respectively. Phenol and 3-methylphenol were oxidized to the corresponding catechols which, after subsequent ortho-cleavage, were channeled into the oxoadipate pathway.
