Aerobic biodegradation of 1,2-dichloroethane and 1,3-dichloropropene by bacteria isolated from a pulp mill wastewater effluent in South Africa

Large volumes of chlorinated aliphatic hydrocarbons are produced annually for a variety of industrial and commercial uses. They therefore constitute common contaminants of soil and groundwater causing serious environmental and human health problems. In this study, three bacteria were isolated from a pulp mill wastewater effluent in South Africa by culture enrichment technique and characterized for their ability to degrade 1,2-dichloroethane (1,2-DCE) and 1,3-dichloropropene (1,3-DCP). Specific growth rate constants of the organisms ranged between 0.864∼1.094 and 0.530∼0.585 d⁻¹ in 1.2-DCE and 1,3-DCP, respectively, while the degradation rate constant of the compounds ranged variously between 0.33 and 1.006 d⁻¹, with 1,2-DCE generally better utilized than 1,3-DCP. Gas chromatographic analysis revealed up to 75 and 80% removal of 1,2-DCE and 1,3-DCP, respectively, above that observed in the control bottles. These organisms also demonstrated high haloalkane dehalogenase activities with specific dehalogenase activities ranging between 0.25∼0.31 U (mg protein)⁻¹. Analysis of their 16S rRNA gene sequences revealed that they belong to the generaPaenibacillus, Bacillus, andMicrobacterium.     

Enrichment, isolation, and characterization of 4-chlorophenol-degrading bacterium Rhizobium sp. 4-CP-20

The objectives of this research were to monitor the variations of species in mixed cultures during the enrichment period, isolate species and identify and characterize the pure 4-chlorophenol (4-CP) degrading strains from enriched mixed cultures. Strain Rhizobium sp. 4-CP-20 was isolated from the acclimated mixed culture. The DGGE result indicated that strain Rhizobium sp. 4-CP-20 was undetectable at the beginning but detectable after 2 weeks of enrichment. The optimum growth temperatures for Rhizobium sp. 4-CP-20 were both 36°C using 350 mg l⁻¹ glucose or sodium acetate as the substrate. The optimum pH range for degrading 100 mg l⁻¹ 4-CP was between 6.89 and 8.20. Strain Rhizobium sp. 4-CP-20 could degrade 4-CP completely within 3.95 days, as the initial 4-CP concentration was 100 mg l⁻¹. If the initial 4-CP concentration was higher than 240 mg l⁻¹, the growth of bacterial cells and the activity of degrading 4-CP were both inhibited.     

Microbial consortia for the aerobic degradation of aromatic compounds in olive oil mill effluent

Aerobic consortia that grow on olive oil mill effluent (OOME) were obtained by enrichment. Several cultures were capable of metabolizing monoaromatic compounds, supplied as the sole carbon source at 2 g L^–1. Some consortia degraded mixtures of seven aromatics (4 g L^–1) after 1 week of incubation at 32°C. The consortia were also active against monoaromatics of the undiluted OOME. This reduced the inhibitory effect of phenolic compounds prior to the anaerobic digestion of OOME at batch scale. No inhibition of the anaerobic microbial populations was noticed with treated OOME. From the most active consortium, nine different bacterial strains were isolated and shown to grow on simple aromatic compounds. Removal of 50% of the initial chemical oxygen demand and degradation of almost all of the simple aromatics in undiluted OOME was obtained with reconstituted bacterial mixtures. A slight reduction in colouration was due to adsorption of coloured compounds to bacterial cells. Presumably, the consortia could not reduce and degrade the coloured compounds in OOME.     

Enrichment and kinetics of biodegradation of 1-naphthylamine in activated sludge

Sequencing-batch reactors were used to develop an activated sludge enrichment culture capable of degrading 1-naphthylamine (1NA). Approximately 5 months acclimation with salicylic acid (1600 mg l^–1) as the primary source of carbon were required to obtain an enrichment culture able to degrade even small quantities of 1NA. After an additional 4 months acclimation, during which the concentration of salicyclic acid was decreased to 50 mg l^–1, a culture developed that degraded 1NA concentrations as high as 300 mg l^–1. Kinetic determinations showed that 1NA degradation (in the presence of salicylate) followed Michaelis-Menten kinetics with K _m and V _m values of 32.5±2.2 mg l^–1 and 375±18 ng 1NA mg^–1 cells h^–1, respectively. The same enrichement was able to degrade 1NA when present as the sole source of carbon and energy and to convert approximately 87% to CO₂.     

Microbial oxidation of isoprene, a biogenic foliage volatile and of 1,3-butadiene, an anthropogenic gas

Nocardia strains that were able to degrade isoprene were isolated from several locations using enrichment cultures with isoprene or 1,3-butadiene as the sole carbon and energy source. Specific growth rates of representative isolates on isoprene and 1,3-butadiene ranged from 0.05 to 0.2 h⁻¹. The initial oxygenation of both 1,3-butadiene and isoprene was mediated by mono-oxygenases which converted these alkadienes into the respective epoxyalkanes.     

Biodegradation of 2,4-dichlorophenol by a <Emphasis Type="Italic">Bacillus</Emphasis> consortium

As part of our effort at establishing microbial consortia of relevance for the bioremediation of xenobiotics polluted environments in Mexico, we assessed the aerobic biodegradation of 2,4-dichlorophenol (2,4-DCP) by a consortium of four Bacillus species that were isolated from a polluted soil by enrichment using a mixture of chlorophenols. The bacterial consortium effectively biodegraded 2-chlorophenol, 3-chlorophenol and 2,4-dichlorophenol at degradation rates of between 1.7 and 6.7 μmoles l⁻¹ h⁻¹. In the presence of NH₄Cl or KNO₂ as nitrogen sources_, 2,4-DCP was variously degraded. Under both conditions, cell biomass attained highest values of 350 and 450 mg l⁻¹ respectively, while the amounts of 2,4-DCP metabolized in 21 days reached peak values of 2.1 and 2.5 mM representing between 70 and 85% degradation respectively. Chloride releases during the same period were highest at 4.7 mM and 5.3 mM in the presence of the two nitrogen sources. The presence of free-chloride in the culture medium had a significant impact on the catabolism of 2,4-dichlorophenol.     

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Characterization of a strain of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide)

Bacteria capable of degrading the pesticide, cadusafos, were isolated from agricultural soil using an enrichment method. In this way, five distinct cadusafos-degrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis, as well as 16S rRNA sequencing. Strain PC1 exhibited the greatest cadusafos degradation rate and was consequently selected for further investigation. Degradation of cadusafos by strain PC1 was rapid at 20 and 37°C, but was greatly reduced (~1.5-fold) by the presence of carbon sources. Strain PC1 was able to effectively degrade cadusafos in sterilized soil using low inoculum levels. The maximum degradation rate of cadusafos (V _max ) was calculated as 1.1 mg l⁻¹ day⁻¹, and its saturation constant (K _s ) was determined as 2.5 mg l⁻¹. Bacteria such as strain PC1, that use cadusafos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.     

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein⁻¹) and also had the highest affinity for 1,3-DCP (K _m, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k ₁) of 1.13 h⁻¹ . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl⁻¹. Received: 30 April 1996 / Received revision: 30 July 1996 / Accepted: 5 August 1996     

Isolation and characterization of phenol-degrading yeasts from an oil refinery wastewater in Brazil

This study investigated the aerobic degradation of phenol by yeast strains isolated from an oil refinery wastewater from the Northeast of Brazil. The samples displayed low fungal diversity, as only yeast colonies were detected on Sabouraud dextrose agar containing chloramphenicol 0.05% (w/v). Among the isolates, three yeast strains were selected to be evaluated for their potential for degrading high phenol concentrations. These species were identified through morphological and biochemical characteristics as Candida tropicalis, C. rugosa, and Pichia membranaefaciens. Although the strains were able to degrade the phenol concentration present in the wastewater, which was 7 mg l⁻¹, only C. tropicalis was capable of growing at high concentrations of phenol such as 500 mg l⁻¹ and 1,000 mg l⁻¹ in a mineral medium containing this pollutant as the only carbon source. C. rugosa and P. membranaefaciens were inhibited in the presence of 500 mg l⁻¹ of phenol. However, a longer incubation time was needed for C. tropicalis strain to degrade 1,000 mg l⁻¹ of phenol compared to the time required to degrade 500 mg l⁻¹. Moreover, the strain released a significant amount of polysaccharide biosurfactant in the medium probably to minimize the toxic effect of the high phenol concentration. When challenged with 1,500 and 2,000 mg l⁻¹ of phenol, C. tropicalis was unable to grow at the tested conditions. The results indicate that this strain of C. tropicalis can be considered both a good phenol-degrader and biosurfactant-producer. Application of this strain might be useful in bioremediation activities or treatment of phenol-polluted wastewater.     

Evaluation of the biodegradation potential of 1,4-dioxane in river, soil and activated sludge samples

To evaluate the biodegradation potential of 1,4-dioxane in natural environments, a total of 20 environmental samples including river water, activated sludge, soil from the drainage area of a chemical factory and garden soil were subjected to a 1,4-dioxane degradation test. The five soil samples from the drainage area of the chemical factory were capable of reducing 100 mg l^?1 of 1,4-dioxane to below the detection limit (0.8 mg l^?1) within 33 days. In one activated sludge sample, 100 mg l^?1 of 1,4-dioxane decreased by 69% within 14 days via cometabolic degradation in the presence of 100 mg l^?1 of tetrahydrofuran (THF). The ability of all samples to degrade 1,4-dioxane degradation with or without THF increased after repeated enrichment, except for one soil sample from the drainage area of the chemical factory that was no longer able to degrade 1,4-dioxane after the third cycle of enrichment. However, most of the samples (14/20) were not able to degrade 1,4-dioxane degradation. Thus, it can be concluded that the potential for 1,4-dioxane degradation is not ubiquitously distributed in natural environment.     

Conversion of glycerol to 1,3-propanediol by a newly isolated thermophilic strain

Of 60 different thermophilic enrichment cultures, 16 converted glycerol anaerobically to 1,3-propanediol. Two PD-forming strains were further enriched, isolated, and characterised. For the most active strain, AT1, the optimal cultivation parameters for pH and temperature were determined as 5.8 to 6.0 and 58°C, respectively. In batch-fermentations with AT1, 6.4 g propanediol per litre was formed with a productivity of 0.17 g l^–1 h^–1.     

Synergistic utilization of dichloroethylene as sole carbon source by bacterial consortia isolated from contaminated sites in Africa

The widespread use and distribution of chloroethylene organic compounds is of serious concern owing to their carcinogenicity and toxicity to humans and wildlife. In an effort to develop active bacterial consortia that could be useful for bioremediation of chloroethylenecontaminated sites in Africa, 16 combinations of 5 dichloroethylene (DCE)-utilizing bacteria, isolated from South Africa and Nigeria, were assessed for their ability to degradecis- andtrans-DCEs as the sole carbon source. Three combinations of these isolates were able to remove up to 72% of the compounds within 7 days. Specific growth rate constants of the bacterial consortia ranged between 0.465 and 0.716 d⁻¹ while the degradation rate constants ranged between 0.184 and 0.205 d⁻¹, with 86.36–93.53 and 87.47–97.12% of the stoichiometric-expected chloride released during growth of the bacterial consortia, incis- andtrans-DCE, respectively. Succession studies of the individual isolates present in the consortium revealed that the biodegradation process was initially dominated byAchromobacter xylosoxidans and subsequently byAcinetobacter sp. andBacillus sp., respectively. The results of this study suggest that consortia of bacteria are more efficient than monocultures in the aerobic biodegradation of DCEs, degrading the compounds to levels that are up to 60% below the maximum allowable limits in drinking water.     

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μ_max of 0.094 h⁻¹ and S _m of 1,435 mg l⁻¹. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.     

Degradation of phenanthrene,fluorene and fluoranthene by pure bacterial cultures

Summary Bacterial mixed cultures able to degrade the polycyclic aromatic hydrocarbons (PAH) phenanthrene, fluorene and fluoranthene, were obtained from soil using conventional enrichment techniques. From these mixed cultures three pure strains were isolated:Pseudomonas paucimobilis degrading phenanthrene;P. vesicularis degrading fluorene andAlcaligenes denitrificans degrading fluoranthene. The maximum rates of PAH degradation ranged from 1.0 mg phenanthrene/ml per day to 0.3 mg fluoranthene/ml per day at doubling times of 12 h to 35 h for growth on PAH as sole carbon source. The protein yield during PAH degradation was about 0.25 mg/mg C for all strains. Maximum PAH oxidation rates and optimum specific bacterial growth were obtained near pH 7.0 and 30°C. After growth entered the stationary phase, no dead end-products of PAH degradation could be detected in the culture fluid.     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Cross-breeding of selected and mutated homokaryotic strains of Phanerochaete chrysosporium K-3: New cellulase deficient strains with increased ability to degrade lignin

Summary A strain of Phanerochaete chrysosporium, designated strain K-3, was isolated from a monosporous conidiospore culture of Sporotrichum pulverulentum. This strain produces fruit bodies with only four sterigmata. From basidiospores of this culture, the homokaryotic strain 31 with high lignin degrading capacity was selected and subjected to ultraviolet irradiation to obtain cellulase deficient (Cel^-) strains. By cross-breeding one of these Cel^- variants with selected Cel⁺ homokaryotic strains from K-3 with high lignin degrading capacity, new Cel^- mutants were isolated which exceeded K-3 in their capacity to degrade lignin.The Cel^- strains were totally incapable of degrading cellulose but were able to degrade xylan. Evolution of ¹⁴CO₂ from ¹⁴C-ring-labelled synthetic lignin a dehydrogenation polymerizate (DHP) was used to screen for strains with high lignin degrading capacity.Studies of weight loss on birch and spruce wood revealed that the weight losses caused by strain K-3 exceeded, in all cases, those caused by the Cel^- strains. However, higher lignin losses in birch wood were obtained with several of the Cel^- strains than with the K-3 strain. After 2 weeks, one strain caused a lignin loss in birch wood of 21% of the initial amount of lignin, while with another strain there was, after 3 weeks incubation, a 28.5% decrease in the lignin content.     

Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

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.     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid