Biodegradation and photolysis of pentachlorophenol in artificial freshwater streams.

The biodegradation, photolysis, and adsorption of pentachlorophenol (PCP) in outdoor, aquatic environments were examined with man-made channels built by the U.S. Environmental Protection Agency at a field station on the Mississippi River near Monticello, Minn. Four channels were used, each channel being approximately 520 m long and receiving river water that flowed through the channels for about 10 h before reentering the river. The channels were dosed continuously during the summer of 1982 with various concentrations of PCP (approximately 0, 48, 144, and 432 micrograms/liter). We monitored the biotic and abiotic degradation of PCP in these channels for approximately 16 weeks. Photolysis of PCP was rapid at the water surface, but greatly attenuated with depth. Depending on sunlight conditions, photolysis accounted for a 5 to 28% decline in initial PCP concentration. Adsorption of PCP by sediment and uptake by biota accounted for less than 15% and probably less than 5% in unacclimated water. Microbial degradation of PCP became significant about 3 weeks after the initiation of dosing and eventually became the primary mechanism of PCP removal, accounting for a 26 to 46% (dose-dependent) decline in initial PCP. Most of the PCP-mineralizing microorganisms that developed in the channels were either attached to surfaces (e.g., rocks and macrophytes) or associated with surface sediments. Total bacterial numbers (direct microscopic counts) in the various channels were not affected significantly by PCP concentrations of micrograms per liter. Numerous strains of bacteria able to grow at the expense of PCP were isolated from the adapted channels. The experiments reported here will help predict the responses of flowing aquatic ecosystems to contamination by biocides such as pentachlorophenol.     

Response of the microflora in outdoor experimental streams to pentachlorophenol: compartmental contributions

Outdoor artificial streams were treated continuously with pentachlorophenol (PCP) for 88 days during the summer of 1983. The contributions of different stream compartments (microbial habitats) to microbial degradation of PCP were determined in a stream treated with 144 micrograms of PCP per liter. The 488-m long stream was composed of mud-bottomed pools alternating with gravel riffles. PCP loss in the stream attributable to microbial degradation after an adaptation period was in the range of 55 to 74%. Contributions to PCP loss were determined for rock surface (epilithic), macrophyte surface (epiphytic), sedimentary, and water column communities by measuring rates of PCP disappearance in stream water, containing ambient concentrations of PCP, in contact with representative compartmental samples. The specific capability, in units of micrograms of PCP per hour per square meter of stream cross-sectional area (macrophytes at maximum plant density, water column at mean depth, upper 10-cm layer of gravel), followed the order rock surface much greater than macrophytes greater than sediment approximately equal to water column. The compartmental contribution to total stream losses in units of grams per hour followed the same order, although the differences were smaller. The rate of PCP disappearance in the water column above sediment cores followed the order oxygen-rich greater than oxygen-poor approximately equal to anaerobic greater than sorption-only conditions. The large difference in specific capability between the rock surface and sediment compartments could be attributed to oxygen deficiency (because of chemical and biological oxygen demand) in the sediments. Free-floating and particle-attached organisms in the water column were important to PCP biodegradation.     

Response of the microflora in outdoor experimental streams to pentachlorophenol: compartmental contributions.

Outdoor artificial streams were treated continuously with pentachlorophenol (PCP) for 88 days during the summer of 1983. The contributions of different stream compartments (microbial habitats) to microbial degradation of PCP were determined in a stream treated with 144 micrograms of PCP per liter. The 488-m long stream was composed of mud-bottomed pools alternating with gravel riffles. PCP loss in the stream attributable to microbial degradation after an adaptation period was in the range of 55 to 74%. Contributions to PCP loss were determined for rock surface (epilithic), macrophyte surface (epiphytic), sedimentary, and water column communities by measuring rates of PCP disappearance in stream water, containing ambient concentrations of PCP, in contact with representative compartmental samples. The specific capability, in units of micrograms of PCP per hour per square meter of stream cross-sectional area (macrophytes at maximum plant density, water column at mean depth, upper 10-cm layer of gravel), followed the order rock surface much greater than macrophytes greater than sediment approximately equal to water column. The compartmental contribution to total stream losses in units of grams per hour followed the same order, although the differences were smaller. The rate of PCP disappearance in the water column above sediment cores followed the order oxygen-rich greater than oxygen-poor approximately equal to anaerobic greater than sorption-only conditions. The large difference in specific capability between the rock surface and sediment compartments could be attributed to oxygen deficiency (because of chemical and biological oxygen demand) in the sediments. Free-floating and particle-attached organisms in the water column were important to PCP biodegradation.     

Potential for thermophilic (50 degrees C) anaerobic dechlorination of pentachlorophenol in different ecosystems

Thermophilic (50 degrees C) anaerobic biodegradation of pentachlorophenol (PCP) was investigated by using different inocula from natural ecosystems and anaerobic digesters. The inocula tested were three freshwater sediments, four anaerobic sewage sludge samples from digesters treating sludge from wastewater plants with various industrial inputs, and digested manure from an anaerobic reactor. Only one digested-sludge sample and the manure sample were from thermophilic environments. The initial PCP concentration was 7.5 or 37.5 microM. After 8 months, PCP had disappeared from the sediment samples and various, less chlorinated intermediates were present. Additions of extra PCP were degraded within 4 weeks, and a maximal observed dechlorination rate of 1.61 mumol/liter/day in the vials with addition of 7.5 microM PCP and 7.50 mumol/liter/day in the vials with addition of 37.5 microM PCP were measured for a freshwater sediment. In contrast, only 2.8 to 17.5% of the initial PCP added had disappeared from the sludge samples after 8 months of incubation. The complex pattern of intermediates formed indicated that the dechlorination of PCP proceeded via different pathways, involving at least two different populations in the dechlorination processes.     

Potential for thermophilic (50 degrees C) anaerobic dechlorination of pentachlorophenol in different ecosystems.

Thermophilic (50 degrees C) anaerobic biodegradation of pentachlorophenol (PCP) was investigated by using different inocula from natural ecosystems and anaerobic digesters. The inocula tested were three freshwater sediments, four anaerobic sewage sludge samples from digesters treating sludge from wastewater plants with various industrial inputs, and digested manure from an anaerobic reactor. Only one digested-sludge sample and the manure sample were from thermophilic environments. The initial PCP concentration was 7.5 or 37.5 microM. After 8 months, PCP had disappeared from the sediment samples and various, less chlorinated intermediates were present. Additions of extra PCP were degraded within 4 weeks, and a maximal observed dechlorination rate of 1.61 mumol/liter/day in the vials with addition of 7.5 microM PCP and 7.50 mumol/liter/day in the vials with addition of 37.5 microM PCP were measured for a freshwater sediment. In contrast, only 2.8 to 17.5% of the initial PCP added had disappeared from the sludge samples after 8 months of incubation. The complex pattern of intermediates formed indicated that the dechlorination of PCP proceeded via different pathways, involving at least two different populations in the dechlorination processes.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp.

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium.

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Performance of anaerobic granules for degradation of pentachlorophenol.

Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28 degrees C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99% was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14C]PCP to 14CH4 and 14CO2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP.     

Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Biodegradation of pentachlorophenol in a continuous anaerobic reactor augmented with Desulfitobacterium frappieri PCP-1.

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader, Desulfitobacterium frappieri PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10(6) to 10(10) cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Kinetics of microbial growth on pentachlorophenol

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Kinetics of microbial growth on pentachlorophenol.

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.