Degradation of polychlorinated biphenyls by mixed microbial cultures.

Three different enriched mixed cultures capable of degrading polychlorinated biphenylas were isolated from two soil samples and a river sediment, respectively. The predominant organisms found in all three mixed cultures were Alcaligenes odorans, Alcaligenes dentrificans, and an unidentified bacterium. The polychlorinated biphenyl isomers that were more water soluble and had lower chlorination were not only degraded at a faster rate than those that were less water soluble and had higher chlorination, but were also more completely utilized by these mixed cultures. This resulted in the presence in the environment of polychlorinated biphenyl residues consisting mainly of higher-chlorinated isomers. A form of cometabolism of polychlorinated biphenyls was also found with these cultures in the presence of acetate as the cosubstrate.     

Degradation of polychlorinated biphenyls by mixed microbial cultures.

Three different enriched mixed cultures capable of degrading polychlorinated biphenylas were isolated from two soil samples and a river sediment, respectively. The predominant organisms found in all three mixed cultures were Alcaligenes odorans, Alcaligenes dentrificans, and an unidentified bacterium. The polychlorinated biphenyl isomers that were more water soluble and had lower chlorination were not only degraded at a faster rate than those that were less water soluble and had higher chlorination, but were also more completely utilized by these mixed cultures. This resulted in the presence in the environment of polychlorinated biphenyl residues consisting mainly of higher-chlorinated isomers. A form of cometabolism of polychlorinated biphenyls was also found with these cultures in the presence of acetate as the cosubstrate.     

Aerobic biodegradation of dinitrotoluenes in batch systems by pure and mixed cultures

Biodegradation characteristics of 2,4- and 2,6-dinitrotoluenes (DNTs) individually by pure strains and defined mixed cultures obtained from a mixed culture isolated from a slate packed bed bioreactor is described. Batch degradation experiments were carried out with free cells in submerged cultivations. The degradation rate and efficiency of five best individual bacterial strains, bacterial consortia comprising three and five of these strains, and the complete mixed culture were evaluated and compared. All the strains showed ability to degrade both the DNTs. All but one strain degraded both DNTs at the same rate. The degradation rate as well as the degradation efficiency by the mixed cultures was higher than that by the individual strains. The complete mixed culture showed 15-20x higher degradation rate than the individual bacterial strains.     

Anaerobic digestion of cellulose by pure and mixed bacterial cultures

Summary By enrichment technique, nine anaerobic mixed bacterial cultures were isolated, five of which showed stable cellulolysis. All cultures fermented cellulose and produced different fermentative products. Mixed culture BOC 25 yielded major levels of acetate and ethanol (39.6 and 12.0 mmol/l, respectively) and minor levels of propionate (2.5 mmol/l) and digested filter paper cellulose to the extent of 32.5% w/v. BOC 25 digested cellulosic and lignocellulosic substrates and produced filter paper cellulase, carboxymethyl cellulase, Avicelase and -glucosidase. Strain DC 25, a cellulolyticClostridium was purified from one of the mixed cultures. The fermentation products of DC 25 from cellulose, cellobiose or glucose were ethanol, acetate, formate, H₂ and CO₂.     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures.

The microbial degradation of phenol by pure and mixed cultures of Pseudomonas putida was studied in batch, phenol-stat, and continuous culture systems. In the continuous culture runs, both steady state and transient experiments were performed. From these experiments, a model for the kinetic behavior of the organisms was evolved and an analysis performed on the stability and dynamic behavior of pure and mixed cultures. The results indicate that it should be possible to achieve phenol removal from wastewaters down to levels of 1-2 ppm in a single state system. However, because of the effect of substrate inhibition on kinetic behavior of the microorganisms, long lasting transients can occur. The transient behavior of such systems cannot be solely determined from mumax or Ks parameters, but must include a consideration of the transient size and response characteristic of the organism.     

Effects of media composition on substrate removal by pure and mixed bacterial cultures

Continuous culture experiments with identical experimental designs were run with a mixed microbial community of activated sludge origin and an axenic bacterial culture derived from it. Each culture received 2-chlorophenol (2-CP) at a concentration of 160 mg/L as COD and L-lysine at a concentration of 65 mg/L as COD. A factorial experimental design was employed with dilution rate and media composition as the two controlled variables. Three dilution rates were studied: 0.015, 0.0325, and 0.05 h^–1. Media composition was changed by adding four biogenic compounds (butyric acid, thymine, glutamic acid and lactose) in equal COD proportions at total concentrations of 0, 34, 225, and 1462 mg/L as COD. The measured variables were the effluent concentrations of 2-CP as measured by the 4-aminoantipyrene test and lysine as measured by the o-diacetylbenzene procedure. The results suggest that community structure and substrate composition play important roles in the response of a microbial community to mixed substrates. The addition of more biogenic substrates to the axenic culture had a deleterious effect on the removal of both lysine and 2-CP, although the effect was much larger on lysine removal. In contrast, additional substrates had a positive effect on the removal of 2-CP by the mixed community and much less of a negative effect on the removal of lysine. The dilution rate at which the cultures were growing had relatively little impact on the responses to the additional substrates.Abbreviations COD chemical oxygen demand - 2-CP 2-chlorophenol - DOC dissolved organic carbon - MDL method detection limit - SS suspended solids     

Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.

Of 36 pure isomers (chlorine numbers 1 to 5) of polychlorinated biphenyls examined, 23 compounds were metabolized by Alcaligenes sp. strain Y42, and 33 compounds were metabolized by Acinetobacter sp. strain P6. The major pathway of many polychlorinated biphenyl isomers examined was considered to proceed through 2',3'-dihydro-2',3'-diol compounds, concomitant dehydrogenated 2',3'-dihydroxy compounds, subsequently the 1',2'-meta-cleavage compounds (chlorinated derivatives of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids), and then chlorobenzoic acids. The meta-cleavage products were usually converted to chlorobenzoic acids upon further incubation in many polychlorinated biphenyls, but they accumulated specifically in the metabolism of 2,4'-, 2,4,4'-, and 2,5,4'-chlorobiphenyls, which are all chlorinated at the 2,4'-position in the molecules in common. Dihydroxy compounds accumulated mainly in the metabolism of 2,6-, 2,3,6-, 2,4,2',5'-, 2,5,2',5'-, and 2,4,5,2',5'-chlorobiphenyls by Acinetobacter sp. P6. The 2,3,2',3'-, 2,3,2',5'-, and 2,4,5,2',3'-chlorobiphenyls, which are chlorinated at the 2,3-position of one of the rings, were metabolized in a different fashion. Two major metabolites of a chlorobenzoic acid and an unknown compound accumulated always in the metabolism of this group of polychlorinated biphenyls. 2,4,6-Trichlorobiphenyl was metabolized quite differently between the two organisms. Alcaligenes sp. Y42 metabolized this compound very slowly to trichlorobenzoic acid by the major oxidative route. In contrast, Acinetobacter sp. P6 metabolized it to a trihydroxy compound via a dihydroxy compound.     

Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.

Of 36 pure isomers (chlorine numbers 1 to 5) of polychlorinated biphenyls examined, 23 compounds were metabolized by Alcaligenes sp. strain Y42, and 33 compounds were metabolized by Acinetobacter sp. strain P6. The major pathway of many polychlorinated biphenyl isomers examined was considered to proceed through 2',3'-dihydro-2',3'-diol compounds, concomitant dehydrogenated 2',3'-dihydroxy compounds, subsequently the 1',2'-meta-cleavage compounds (chlorinated derivatives of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids), and then chlorobenzoic acids. The meta-cleavage products were usually converted to chlorobenzoic acids upon further incubation in many polychlorinated biphenyls, but they accumulated specifically in the metabolism of 2,4'-, 2,4,4'-, and 2,5,4'-chlorobiphenyls, which are all chlorinated at the 2,4'-position in the molecules in common. Dihydroxy compounds accumulated mainly in the metabolism of 2,6-, 2,3,6-, 2,4,2',5'-, 2,5,2',5'-, and 2,4,5,2',5'-chlorobiphenyls by Acinetobacter sp. P6. The 2,3,2',3'-, 2,3,2',5'-, and 2,4,5,2',3'-chlorobiphenyls, which are chlorinated at the 2,3-position of one of the rings, were metabolized in a different fashion. Two major metabolites of a chlorobenzoic acid and an unknown compound accumulated always in the metabolism of this group of polychlorinated biphenyls. 2,4,6-Trichlorobiphenyl was metabolized quite differently between the two organisms. Alcaligenes sp. Y42 metabolized this compound very slowly to trichlorobenzoic acid by the major oxidative route. In contrast, Acinetobacter sp. P6 metabolized it to a trihydroxy compound via a dihydroxy compound.     

Dihexyl sulfosuccinate biodegradation by mixed cultures

The primary biodegradation of dihexyl sulfosuccinate has been demonstrated using two single cultures and three mixed bacterial cultures ofComamonas terrigena. Primary biodegradation was found in all variants. The highest biotransformation rate and efficiency was observed in one of the single strains. No synergistic effect on degradation was found in mixed cultures.     

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

Biotransformation of trichloroethene by pure bacterial cultures

From natural samples 11 isolates able to remove trichloroethene (CCl₂CHl) from an aqueousenvironment were obtained which were capable of cometabolic degradation of CCl₂CHCl by an enzyme system for phenol degradation. At an initial CCl₂CHCl concentration of 1 mg/L, the resting cells of particular cultures degraded 33–94% CCl₂CHCl during 1 d and their transformation capacity ranged from 0.3 to 3.1 mg CCl₂CHCl per g organic fraction. An analysis of a mixed phenol-fed culture with an excellent trichloroethene-degrading ability found a markedly minority isolate represented in the consortium to be responsible for this property. This culture degraded CCl₂CHCl even at a low inoculum concentration and attained a transformation capacity of 14.7 mg CCl₂CHCl per g. The increase in chloride concentration after degradation was quantitative when compared with the decrease in organically bound chlorine. The degree of CCl₂CHCl degradation was affected by Me₂S₂; this substance can significantly reduce the degrading ability of some tested cultures (>60%); however, it does not cause this inhibition with others.     

Efficient biodegradation of high-molecular-weight polyethylene glycols by pure cultures of Pseudomonas stutzeri.

Biodegradation of polyethylene glycols (PEGs) of up to 13,000 to 14,000 molecular weight has been shown to be performed by a river water bacterial isolate (strain JA1001) identified as Pseudomonas stutzeri. A pure culture of strain JA1001 grew on PEG 1000 or PEG 10000 at 0.2% (wt/vol) as a sole source of carbon and energy with a doubling time of 135 or 150 min, respectively. Cultures metabolized 2 g of polymer per liter in less than 24 h and 10 g/liter in less than 72 h. The limit of 13,500 molecular weight in the size of the PEG sustaining growth and the presence of a PEG-oxidative activity in the periplasmic space indicated that PEGs cross the outer membrane and are subsequently metabolized in the periplasm. PEG oxidation was found to be catalyzed by PEG dehydrogenase, an enzyme that has been shown to be a single polypeptide. Characterization of PEG dehydrogenase revealed glyoxylic acid as the product of the PEG-oxidative cleavage. Glyoxylate supported growth by entering the cell and introducing its carbons in the general metabolism via the dicarboxylic acid cycle, as indicated by the ability of strain JA1001 to grow on this compound and the presence of malate synthase, the first enzyme in the pathway, in extracts of PEG-grown cells.     

Aerobic biodegradation of methyl tert-butyl ether (MTBE) by pure bacterial cultures isolated from contaminated soil

Summary Methyl tert-Butyl Ether (MTBE) has been used in gasoline as a substitute for lead-based additives, which have been demonstrated to be toxic. MTBE however, is persistent in soil and water, showing high affinity for water and low affinity for soil, and has become an important contaminant. Therefore, the aim of this work was to isolate and identify soil microorganisms capable of degrading MTBE. Two samples were taken from a gasoline-contaminated soil at a service station and 59 different bacterial strains were isolated by enrichment culture with three consecutive selective transfers. Biochemical and morphological characterization of the bacterial isolates classified them into the following groups: Bacillus, Rhodococcus, Micrococcus, Aureobacterium and Proteus. Twelve strains were selected for evaluation of MTBE biodegradation depending on visual growth and biomass production of the isolates in minimal salt broth. Six strains significantly reduced MTBE concentration (22–37%) compared to an abiotic control after 5 days of incubation. Although it has been considered that MTBE is degraded mainly by cometabolism, our results demonstrate that these microorganisms are able to reduce MTBE concentration when MTBE is the sole source of carbon.     

Efficient biodegradation of high-molecular-weight polyethylene glycols by pure cultures of Pseudomonas stutzeri.

Biodegradation of polyethylene glycols (PEGs) of up to 13,000 to 14,000 molecular weight has been shown to be performed by a river water bacterial isolate (strain JA1001) identified as Pseudomonas stutzeri. A pure culture of strain JA1001 grew on PEG 1000 or PEG 10000 at 0.2% (wt/vol) as a sole source of carbon and energy with a doubling time of 135 or 150 min, respectively. Cultures metabolized 2 g of polymer per liter in less than 24 h and 10 g/liter in less than 72 h. The limit of 13,500 molecular weight in the size of the PEG sustaining growth and the presence of a PEG-oxidative activity in the periplasmic space indicated that PEGs cross the outer membrane and are subsequently metabolized in the periplasm. PEG oxidation was found to be catalyzed by PEG dehydrogenase, an enzyme that has been shown to be a single polypeptide. Characterization of PEG dehydrogenase revealed glyoxylic acid as the product of the PEG-oxidative cleavage. Glyoxylate supported growth by entering the cell and introducing its carbons in the general metabolism via the dicarboxylic acid cycle, as indicated by the ability of strain JA1001 to grow on this compound and the presence of malate synthase, the first enzyme in the pathway, in extracts of PEG-grown cells.     

Methyl-beta-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils

The bioremediation of aged polychlorinated biphenyl (PCB)-contaminated soils is adversely affected by the low bioavailability of the pollutants. Randomly methylated-beta-cyclodextrins (RAMEB) were tested as a potential PCB-bioavailability-enhancing agent in the aerobic treatment of two aged-contaminated soils. The soils, contaminated by about 890 and 8500 mg/kg of Aroclor 1260 PCBs, were amended with biphenyl (4 g/kg), inorganic nutrients (to adjust their C:N ratio to 20:1), and variable amounts of RAMEB (0%, 0.5%, or 1.0% [w/w]) and treated in both aerobic 3-L solid-phase reactors and 1.5-L packed-bed loop reactors for 6 months. Notably, significant enhancement of the PCB biodegradation and dechlorination, along with a detectable depletion of the initial soil ecotoxicity, were generally observed in the RAMEB-treated reactors of both soils. RAMEB effects were different in the two soils, depending upon the treatment conditions employed, and generally increased proportionally with the concentration at which RAMEB was applied. RAMEB, which was slowly metabolized by the soil's aerobic microorganisms, was found to markedly enhance the occurrence of the indigenous aerobic, cultivable biphenyl-growing bacteria harboring genes homologous to those of two highly specialized PCB degraders (i.e., bphABC genes of Pseudomonas pseudoalcaligenes KF707 and bphA1A2A3A4BC1 genes of Rhodococcus globerulus P6) and chlorobenzoic acid-degrading bacteria as well as the occurrence of PCBs in the water phase of the soil reactors. These findings indicate that RAMEB enhanced the aerobic bioremediation of the two soils by increasing the bioavailability of PCBs and the occurrence of specialized bacteria in the soil reactors.     

Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene.

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.     

Anaerobic degradation of o-phenylphenol by mixed and pure cultures

Summary An anaerobic enrichment culture that degraded 0.4 mmol/l per day of o-phenylphenol was selected from sediment of a waste water pond of a sugar factory. From the consortium an o-phenylphenol-degrading bacterium, strain B10, was isolated. Strain B10 could not degrade other aromatic substances, including phenylacetic acid, benzoate, o-hydroxybenzoate, p-hydroxybenzoate and phenol. Best growth was observed with glucose, pyruvate, lactate, methanol and H₂/CO₂ as substrates. o-Phenylphenol was slowly degraded if supplied as the only carbon source and was cometabolized in the presence of >5 mmol/l glucose. Strain B10 has not yet been assigned to a known species or family.     

Phosphorus removal by pure and mixed cultures of microorganisms

The ability to remove inorganic phosphate from synthetic wastewater was tested with about 40 microbial strains, and Pseudomonas aeruginosa IAM 1007 was found to give good performance under aerobic conditions. However, the phosphate removal under batch anaerobic/aerobic (A/O) treatment was not satisfactory in pure cultures of several strains including P. aerginosa, and Aceinetobacter calcoaceticus, but the activated sludge from a plant with an A/O process almost depleted the phosphate. Mixed cultures of P. aeruginosa in the presence of the facultativelu anaerobic strains of A-1 or A-8 isolated from the activated sludge showed enhanced phosphate removal. This suggests a symbiotic effect among microbial species on biological removal of inorganic phosphate in the A/O process.