Biodegradation of o-Benzyl-p-Chlorophenol

The extent of biodegradation of o-benzyl-p-chlorophenol, marketed as a germicide under the name Santophen((R)) 1 (Monsanto Co.), in river water, sewage, and activated sludge was determined. Biodegradation was assessed by use of a colorimetric procedure for phenolic materials, carbon analysis, and CO(2) evolution. In unacclimated river water, 0.1 mg of Santophen 1 per liter was degraded within 6 days. In sewage, 0.5 and 1.0 mg/liter levels of Santophen 1 were degraded in 1 day. Acclimated activated sludge achieved 80% biodegradation of 1.0 mg/liter Santophen 1 in 8 h and 100% in 24 h. When effluent from a semicontinuous activated sludge unit, acclimated to 20 mg of Santophen 1 per liter was used as the inoculum for the CO(2) evolution procedure, 60% of the total theoretical CO(2) was evolved from Santophen 1. Based on the results of these studies, indicating Santophen 1 to be readily biodegraded in at least four biological systems, the continued use of present levels of Santophen 1 should present no significant environmental problems.     

Estimation of Biodegradation Potential of Xenobiotic Organic Chemicals

A method is described to estimate the biodegradation potential of soluble, insoluble, and unknown organic chemicals. The method consists of two stages: (i) generation of a microbial inoculum in a bench scale semicontinuous activated sludge system during which microorganisms are acclimated to test material and the removal of dissolved organic carbon is monitored and (ii) biodegradability testing (CO₂ evolution) in a defined minimal medium containing the test material as the sole carbon and energy source and a dilute bacterial inoculum obtained from the supernatant of homogenized activated sludge generated in the semicontinuous activated sludge system. Removal and biodegradation are measured using nonspecific methods, at initial concentrations of 5 to 10 mg of dissolved organic carbon per liter. Biodegradability data are accurately described by a nonlinear computer model which allows the rate and extent of biodegradation for different compounds to be compared and statistically examined. The evaluation of data generated in the combined removability-biodegradability system allows the biodegradation potential of a variety of xenobiotic organic chemicals to be estimated.     

Role of chemical concentration and second carbon sources in acclimation of microbial communities for biodegradation

A study was conducted to determine the role of concentration of the test chemical, of a second organic compound, and of mutation in the acclimation period before the mineralization of organic compounds in sewage. The acclimation period for the mineralization in sewage of 2 micrograms of 4-nitrophenol (PNP) per liter increased from 6 to 12 days in the presence of 10 mg of 2,4-dinitrophenol per liter. The extension of the acclimation period was equivalent to the time required for mineralization of 2,4-dinitrophenol. In contrast, the time for acclimation for the degradation of 2 micrograms of PNP per liter was reduced when 10 or 100 mg of phenol per liter was added. Lower phenol levels increased the acclimation period to 8 days. The length of the acclimation period for PNP mineralization decreased as the initial concentration of PNP increased from 2 micrograms to 100 mg/liter. The acclimation period for phenol mineralization was lengthened as the phenol concentration increased from 100 to 1,400 mg/liter. The length of the acclimation period for PNP and phenol biodegradation was reproducible, but it varied among replicates for the biodegradation of other nitro-substituted compounds added to sewage or lake water, suggesting that a mutation was responsible for acclimation to these other compounds. The acclimation period may thus reflect the time required for the destruction of toxins, and it also may be affected by the concentration of the test compound or the presence of other substrates.     

Role of chemical concentration and second carbon sources in acclimation of microbial communities for biodegradation.

A study was conducted to determine the role of concentration of the test chemical, of a second organic compound, and of mutation in the acclimation period before the mineralization of organic compounds in sewage. The acclimation period for the mineralization in sewage of 2 micrograms of 4-nitrophenol (PNP) per liter increased from 6 to 12 days in the presence of 10 mg of 2,4-dinitrophenol per liter. The extension of the acclimation period was equivalent to the time required for mineralization of 2,4-dinitrophenol. In contrast, the time for acclimation for the degradation of 2 micrograms of PNP per liter was reduced when 10 or 100 mg of phenol per liter was added. Lower phenol levels increased the acclimation period to 8 days. The length of the acclimation period for PNP mineralization decreased as the initial concentration of PNP increased from 2 micrograms to 100 mg/liter. The acclimation period for phenol mineralization was lengthened as the phenol concentration increased from 100 to 1,400 mg/liter. The length of the acclimation period for PNP and phenol biodegradation was reproducible, but it varied among replicates for the biodegradation of other nitro-substituted compounds added to sewage or lake water, suggesting that a mutation was responsible for acclimation to these other compounds. The acclimation period may thus reflect the time required for the destruction of toxins, and it also may be affected by the concentration of the test compound or the presence of other substrates.     

Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp.

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Biodegradation of phthalic acid esters in river water and activated sludge.

The primary and ultimate biodegradability of phthalic acid, monobutyl phthalate, and five structurally diverse phthalic acid ester plasticizers in river water and activated sludge samples were determined via ultraviolet spectrophotometry, gas chromatography, and CO2 evolution. The compounds studied underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge. When activated sludge acclimated to phthalic acid esters was used as the inoculum for the CO2 evolution procedure, greater than 85% of the total theoretical CO2 was evolved. These studies demonstrate that the phthalic acid ester plasticizers and intermediate degradation products readily undergo ultimate degradation in different mixed microbial systems at concentrations ranging from 1 to 83 mg/liter.     

Biodegradation of p-nitrophenol in an aqueous waste stream by immobilized bacteria.

Microbiological analyses of activated sludge reactors after repeated exposure to 100 mg of p-nitrophenol (PNP) per liter resulted in the isolation of three Pseudomonas species able to utilize PNP as a sole source of carbon and energy. Cell suspensions of the three Pseudomonas sp., designated PNP1, PNP2, and PNP3, mineralized 70, 60, and 45% of a 70-mg/liter dose of PNP in 24, 48, and 96 h, respectively. Mass-balance analyses of PNP residues for all three cultures showed that undegraded PNP was less than 1% (less than 50 micrograms); volatile metabolites, less than 1%; cell residues, 8.4 to 14.9%; and water-soluble metabolites, 1.2 to 6.7%. A mixed culture of all three PNP-degrading Pseudomonas sp. was immobilized by adsorption onto diatomaceous earth biocarrier in a 1.75-liter Plexiglas column. The column was aerated and exposed to a synthetic waste stream containing 629 to 2,513 mg of PNP per liter at flow rates of 2 to 15 ml/min. Chemical loading studies showed that the threshold concentration for acute toxicity of PNP to the immobilized bacteria was 2,100 to 2,500 mg/liter. Further studies at PNP concentrations of 1,200 to 1,800 mg/liter showed that greater than 99 and 91 to 99% removal of PNP was achieved by immobilized bacteria at flow rates of 10 and 12 ml/min, respectively. These values represent hydraulic retention times of 48 to 58 min and PNP removal rates of 0.99 to 1.1 mg/h per g of biocarrier at 25 degrees C under optimal conditions. This study shows the successful use of immobilized bacteria technology to remove high concentrations of PNP from aqueous waste streams.     

Biodegradation of p-nitrophenol in an aqueous waste stream by immobilized bacteria

Microbiological analyses of activated sludge reactors after repeated exposure to 100 mg of p-nitrophenol (PNP) per liter resulted in the isolation of three Pseudomonas species able to utilize PNP as a sole source of carbon and energy. Cell suspensions of the three Pseudomonas sp., designated PNP1, PNP2, and PNP3, mineralized 70, 60, and 45% of a 70-mg/liter dose of PNP in 24, 48, and 96 h, respectively. Mass-balance analyses of PNP residues for all three cultures showed that undegraded PNP was less than 1% (less than 50 micrograms); volatile metabolites, less than 1%; cell residues, 8.4 to 14.9%; and water-soluble metabolites, 1.2 to 6.7%. A mixed culture of all three PNP-degrading Pseudomonas sp. was immobilized by adsorption onto diatomaceous earth biocarrier in a 1.75-liter Plexiglas column. The column was aerated and exposed to a synthetic waste stream containing 629 to 2,513 mg of PNP per liter at flow rates of 2 to 15 ml/min. Chemical loading studies showed that the threshold concentration for acute toxicity of PNP to the immobilized bacteria was 2,100 to 2,500 mg/liter. Further studies at PNP concentrations of 1,200 to 1,800 mg/liter showed that greater than 99 and 91 to 99% removal of PNP was achieved by immobilized bacteria at flow rates of 10 and 12 ml/min, respectively. These values represent hydraulic retention times of 48 to 58 min and PNP removal rates of 0.99 to 1.1 mg/h per g of biocarrier at 25 degrees C under optimal conditions. This study shows the successful use of immobilized bacteria technology to remove high concentrations of PNP from aqueous waste streams.     

Degradation of Mono-Fluorophenols by an Acclimated Activated Sludge

Acclimated activated sludge was examined for its ability to degrade mono-fluorophenols as the sole carbon source in aerobic batch cultures. The acclimated activated sludge degraded fluorophenol efficiently. It degraded 100 mg/l 3-fluoropheno and 4-fluorophenol in 16 h with, respectively, 99.85% and 99.91% fluoride anion release and it degraded 50 mg/l 2-fluorophenol in 15 h with 99.26% fluoride anion release. The aerobic biodegradability of the mono-fluorophenols decreased in the order: 4-fluorophenol > 3-fluorophenol > 2-fluorophenol, resulting mainly from a different octanol/water partition coefficient and different steric parameter of the fluorophenols. The mechanism study revealed that the initial step in the aerobic biodegradation of mono-fluorophenols by the activated sludge was their transformation to fluorocatechol. Following transformation of the fluorophenol to fluorocatechol, ring cleavage by catechol 1, 2-dioxygenases proceeded via an ortho-cleavage pathway, then defluorination occurred.     

Improving Biodegradation of VOCs in Soil by Controlling Volatilization

The use of microbial inoculum and a hydrocarbon adsorbent as a soil amendment was examined to improve bioremediation efficacy of soil contaminated by volatile hydrocarbons. Biodegradation and volatilization losses of VOCs were assessed under contained composting in the laboratory and technical scales. Rhodococcus opacus GM-14, a degrader of a multitude of different hydrocarbons was used as an inoculum and activated carbon as a VOC adsorbent on a laboratory scale. Inoculating soil with R. opacus (0.02 mg R. opacus biomass per 1 mg of benzene) reduced volatilization of benzene from 80% to 40%. Amending the soil with activated carbon reduced volatilization of benzene to 15% and further to 4% when used together with R. opacus. Both amendments promoted mineralization when used separately but slowed down the mineralization when combined. Activated carbon improved the biodegradation of VOCs also during technical scale compostings (700-1100 kg of soil with 1.6-2.4 kg of VOC) from 30-40% to 86% and reduced volatilization from 40-50% to 2-5%. Reduction of VOC volatilization by use of the activated carbon improved the efficiency of VOC biodegradation on a technical scale. The activated carbon addition improves the occupational safety at the contaminated site and during transport.     

Chemical Disinfection of Holding-Tank Sewage

A number of chemical disinfectants were evaluated for their bactericidal and virucidal effectiveness in holding-tank sewage. It was found that the disinfection efficiencies of formaldehyde, benzalkonium chloride, cetylpyridinium chloride, and methylene blue were markedly improved if the pH of the sewage was raised from 8.0 to 10.5. When formaldehyde, benzalkonium chloride, and methylene blue were tested with either 2-week holding times with no sewage additions or 10-day holding times with daily sewage additions, disinfection effectiveness was maintained as long as the sewage pH was kept at 10.5 and the disinfectant concentration was kept at 100 mg/liter or more. Calcium hypochlorite, zinc sulfate, and phenol were found to be relatively ineffective disinfectants for holding-tank sewage.     

Activated sludge encapsulation in gellan gum microbeads for gasoline biodegradation

A two-phase dispersion technique, termed emulsification–internal gelation, is proposed for encapsulation of activated sludge in gellan gum microbeads. The influence of emulsion parameters on size distribution of microbeads was investigated. Mean diameter of microbeads varied within a range of 34–265 µm as a descending function of emulsion stirring rate (1,000–5,000 rpm), emulsification time (10–40 min), and emulsifier concentration (0–0.1% w/w), and as an ascending function of disperse phase volume fraction (0.08–0.25). Encapsulated sludge expressed a high biodegradation activity compared with non-encapsulated sludge cultures even at 4.4 times lower level of overall biomass loading. Over 90% of gasoline at an initial concentration of 35 and 70 mg l^–1 was removed by both encapsulated and non-encapsulated sludge cultures in sealed serum bottles within 7 days. Encapsulation of activated sludge in gellan gum microbeads enhanced the biological activity of microbial populations in the removal of gasoline hydrocarbons. The results of this study demonstrated the feasibility of the production of size-controlled gellan gum-encapsulated sludge microbeads and their use in the biodegradation of gasoline.     

Empirical model for the autotrophic biodegradation of thiocyanate in an activated sludge reactor

AIMS: The aim of this investigation was to develop an empirical model for the autotrophic biodegradation of thiocyanate using an activated sludge reactor. METHODS AND RESULTS: The methods used for this purpose included the use of a laboratory scale activated sludge reactor unit using thiocyante feed concentrations from 200 to 550 mg x l(-1). Reactor effluent concentrations of <1 mg x l(-1) thiocyanate were consistently achieved for the entire duration of the investigation at a hydraulic retention time of 8 h, solids (biomass) retention of 18 h and biomass (dry weight) concentrations ranging from 2 to 4 g x l(-1). A biomass specific degradation rate factor was used to relate thiocyanate degradation in the reactor to the prevailing biomass and thiocyanate feed concentrations. A maximum biomass specific degradation rate of 16 mg(-1) x g(-1) x h(-1) (mg thiocyanate consumed per gram biomass per hour) was achieved at a thiocyanate feed concentration of 550 mg x l(-1). The overall yield coefficient was found to be 0.086 (biomass dry weight produced per mass of thiocyanate consumed). CONCLUSION: Using the results generated by this investigation, an empirical model was developed, based on thiocyanate feed concentration and reactor biomass concentration, to calculate the required absolute hydraulic retention time at which a single-stage continuously stirred tank activated sludge reactor could be operated in order to achieve an effluent concentration of <1 mg x l(-1). The use of an empirical model rather than a mechanistic-based kinetic model was proposed due to the low prevailing thiocyanate concentrations in the reactor. SIGNIFICANCE AND IMPACT OF THE STUDY: These results represent the first empirical model, based on a comprehensive data set, that could be used for the design of thiocyanate-degrading activated sludge systems.     

Mutation of Candida tropicalis by irradiation with a He-Ne laser to increase its ability to degrade phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 x 10(7) cells ml(-1) were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter(-1) phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter(-1). The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Measurement of the effects of cadmium stress on protozoan grazing of bacteria (bacterivory) in activated sludge by fluorescence microscopy.

The effect of cadmium stress on protozoan bacterivory in sewage sludge was measured by experimentally exposing sludge communities to 0 to 150 mg of Cd per liter for up to 6 h and then determining the rates of protozoan grazing on bacteria, using a double-staining technique and epifluorescence microscopy. Bacterivory was measured by incubating the sludge with fluorescently labeled bacterium-sized latex beads and directly observing ingestion of the beads and bacterial cells in the sludge by epifluorescence microscopy of preserved samples. Staining with 4',6-diamidino-2-phenylindole and acridine orange permitted the simultaneous determination of protozoan numbers and bacterivory activity as estimated by the number of bacterial cells and bacterium-sized latex beads ingested by the representative ciliate Aspidisca costata. Enumeration with latex beads proved to be an effective way of estimating bacterivory in sludges subjected to heavy-metal stress. This technique should prove useful for determining the effects of other chemical stresses on protozoan numbers and bacterivory in organic-rich environments. Although the number of protozoa declined significantly only after exposure to 100 mg of Cd per liter for 4 h, grazing, as indicated by bead ingestion, was significantly inhibited by Cd concentrations of greater than 25 mg/liter in less than 1 h, and exposure to 100 mg of Cd per liter effectively stopped protozoan grazing within 1 h of exposure. Protozoan ingestion of latex beads and bacteria was inversely correlated to Cd concentration and exposure time. The reduction of protozoan bacterivory by Cd provides a possible explanation for the increase in suspended bacteria in the effluents of metal-stressed treatment facilities.     

Measurement of the effects of cadmium stress on protozoan grazing of bacteria (bacterivory) in activated sludge by fluorescence microscopy

The effect of cadmium stress on protozoan bacterivory in sewage sludge was measured by experimentally exposing sludge communities to 0 to 150 mg of Cd per liter for up to 6 h and then determining the rates of protozoan grazing on bacteria, using a double-staining technique and epifluorescence microscopy. Bacterivory was measured by incubating the sludge with fluorescently labeled bacterium-sized latex beads and directly observing ingestion of the beads and bacterial cells in the sludge by epifluorescence microscopy of preserved samples. Staining with 4',6-diamidino-2-phenylindole and acridine orange permitted the simultaneous determination of protozoan numbers and bacterivory activity as estimated by the number of bacterial cells and bacterium-sized latex beads ingested by the representative ciliate Aspidisca costata. Enumeration with latex beads proved to be an effective way of estimating bacterivory in sludges subjected to heavy-metal stress. This technique should prove useful for determining the effects of other chemical stresses on protozoan numbers and bacterivory in organic-rich environments. Although the number of protozoa declined significantly only after exposure to 100 mg of Cd per liter for 4 h, grazing, as indicated by bead ingestion, was significantly inhibited by Cd concentrations of greater than 25 mg/liter in less than 1 h, and exposure to 100 mg of Cd per liter effectively stopped protozoan grazing within 1 h of exposure. Protozoan ingestion of latex beads and bacteria was inversely correlated to Cd concentration and exposure time. The reduction of protozoan bacterivory by Cd provides a possible explanation for the increase in suspended bacteria in the effluents of metal-stressed treatment facilities.     

Biodegradation, decolourisation and detoxification of textile wastewater enhanced by advanced oxidation processes

Recently, an increasing application of so called advanced oxidation processes (AOPs) to industrial wastewater has been observed. In particular, an integrated approach of biological and chemical treatment of wastewater is advantageous conceptually. The subject of our study was synthetic wastewater, simulating effluents from knitting industry. The wastewater contained components that are very often used in Polish textile industry: an anionic detergent Awiwaz KG conc., a softening agent Tetrapol CLB and an anthraquinone dyestuff-Acid Blue 40, CI 2125. The toxicity of the detergents and the dye was determined in terms of effective concentration EC50 using mixed cultures of activated sludge as well as pure culture of luminescent bacteria Vibrio fischerii NRRLB-11177. The dye did not undergo biodegradation without AOPs pretreatment, therefore a degree of its removal (decolourisation) by the AOPs has been determined and its bio-sorption properties on the flocks of activated sludge have been studied. The dye adsorption onto flocks of activated sludge was described by Henry's isotherm. Our investigations focussed on the influence of various oxidants like O3, H2O2 and UV light on biodegradation of single components aqueous solution as well as of the whole textile wastewater. The results of kinetic measurements of the biodegradation (by means of acclimated activated sludge) was described by Monod type of kinetic equation. The experimental evidence of the positive effect of chemical oxidation pretreatment on the biodegradation of recalcitrant compounds was quantified by estimation of the kinetic parameters of the Monod equation. Due to the AOPs pretreatment a decrease of the Monod constant and an increase of maximal specific growth rate was observed. The activity of degradative enzymes of activated sludge was assayed by the methods of 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride test.     

Activated sludge degradation of adipic acid esters.

The biodegradability of three aliphatic adipic acid diesters and a 1,3-butylene glycol adipic acid polyester was determined in acclimated, activated sludge systems. Rapid primary biodegradation from 67 to 99+% was observed at 3- and 13-mg/liter feed levels for di-n-hexyl adipate, di(2-ethylhexyl) adipate, and di(heptyl, nonyl) adipate in 24 h. When acclimated, activated sludge microorganisms were employed as the seed for two carbon dioxide evolution procedures, greater than 75% of the theoretical carbon dioxide was evolved for the three diesters and the polyester in a 35-day test period. The essentially complete biodegradation observed in these studies suggests that these esters would not persist when exposed to similar mixed microbial populations in the environment.     

Activated sludge degradation of adipic acid esters.

The biodegradability of three aliphatic adipic acid diesters and a 1,3-butylene glycol adipic acid polyester was determined in acclimated, activated sludge systems. Rapid primary biodegradation from 67 to 99+% was observed at 3- and 13-mg/liter feed levels for di-n-hexyl adipate, di(2-ethylhexyl) adipate, and di(heptyl, nonyl) adipate in 24 h. When acclimated, activated sludge microorganisms were employed as the seed for two carbon dioxide evolution procedures, greater than 75% of the theoretical carbon dioxide was evolved for the three diesters and the polyester in a 35-day test period. The essentially complete biodegradation observed in these studies suggests that these esters would not persist when exposed to similar mixed microbial populations in the environment.