Maturation of mast cell progenitors to mucosal mast cells during allergic pulmonary inflammation in mice.

In contrast to resident constitutive mast cells (CMCs), mucosal MCs (MMCs) appear in the lung and trachea of sensitized mice only following inhalation challenge. We monitored the influx and maturation of MCs by their expression of Kit, FcɛRI, ß7-integrin and side scatter (SSC) by flow cytometry. Influx of MC progenitors (MCps) (FcɛRI(lo), Kit(int), ß7(hi), and SSC(lo)) peaks 1 day after challenges and subsides to baseline by day 7 after challenge. The mature MMCs appear as a distinct population on day 7 and peak at day 14 with higher SSC and FcɛRI expression, but lower ß7 and Kit expression. A distinct transitional population is present between 1 and 7 days after challenge. Maturation occurs more rapidly in the trachea. The resident tracheal CMCs had higher SSC, FcɛRI, and Kit and lower ß7-integrin expression than the MMCs. By histology, the MMCs follow similar kinetics to the flow cytometry-identified mature MMCs and are notably persistent for >42 days. Steroid treatment reduced inflammation and MCp influx but had no effect on established MMCs. Thus, changes in SSC, FcɛRI, and Kit together with the expression of αE/α4:ß7-integrins characterizes the development of induced MMCs from MCps and distinguishes them from resident CMCs in the trachea and large airways.

Aerated biofiltration for simultaneous removal of iron and polycyclic aromatic hydrocarbons from groundwater.

Filters incorporating the principles of biological accumulation have been used in Europe to remove iron from drinking water for many years. The authors of this study hypothesized that a modified biological iron-removing filter could simultaneously degrade polycyclic aromatic hydrocarbons (PAHs) in groundwater impacted by former manufactured gas plant (MGP) operations. The MGP-impacted groundwater obtained for this study had an average total iron concentration of 3.8 mg/L and an average total dissolved PAH concentration of 3.0 mg/L. Naphthalene was the primary PAH, with an average concentration of 2.8 mg/L. The groundwater was passed intermittently through duplicate gravel-media columns at 15 to 30 cm/d for 2 months while filtered air was delivered countercurrently at 4 mL/min. The columns remained partially saturated throughout the study. Flooding, which would have indicated plugging of the columns, was not observed, and the pressure needed to aerate the columns remained constant. Total iron in the effluent was below the detection limit of 0.1 mg/L (97% removal) for 40 days of operation. Removal of total PAHs, primarily two- and three-ring compounds, averaged 99%. This single-stage treatment process represents an economical alternative to biological treatment systems currently available for MGP-impacted groundwater that require pretreatment to remove iron.

Transformation of mono- and dichlorinated phenoxybenzoates by phenoxybenzoate-dioxygenase in pseudomonas pseudoalcaligenes POB310 and a modified diarylether-metabolizing bacterium.

Pseudomonas pseudoalcaligenes POB310 contains genes that encode phenoxybenzoate dioxygenase. The enzyme transforms mono- and dichlorinated phenoxybenzoates to yield protocatechuate that is used as a growth substrate and chlorophenols that are nonmetabolizable. Mass spectral analysis of (18)O metabolites obtained from the protocatechuate 3,4-dioxygenase-deficient mutant, POB310-B1, suggested that the reaction mechanism is a regioselective angular dioxygenation. A cloning vector containing reaction relevant genes (pD30.9) was transferred into Pseudomonas sp. strain B13 containing a modified ortho-cleavage pathway for aromatic compounds. The resultant Pseudomonas sp. strain B13-D5 (pD30.9) completely metabolized 3-(4-chlorophenoxy)benzoate. During growth on 3-phenoxybenzoate, strain B13-D5 (pD30.9) (K(s) = 0.70+/-0.04 mM, mu(max) = 0.45+/-0.03 h(-1), t(d) = 1.5 h, Y = 0.45+/-0.03 g bio- mass x g substrate(-1)) was better adapted to low substrate concentrations, had a faster rate of growth, and a greater yield than POB310 (K(s) = 1.13+/-0.06 mM, mu(max) = 0.31+/-0.02 h(-1), t(d) = 2.2 h, Y = 0.39+/-0.02 g biomass. g substrate(-1)).

Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(pPOB) and two modified Pseudomonas strains.

Pseudomonas pseudoalcaligenes POB310(pPOB) and Pseudomonas sp. strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium. Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB. Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB. Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%). Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10(6) to 10(8) CFU/g (dry weight) of soil. Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant. Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil. This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria. In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to Pseudomonas sp. strain B13 occurred at frequencies of 5 x 10(-7) and 10(-1) transconjugant per donor, respectively. Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil. Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.

Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin from soils inoculated with Sphingomonas sp. strain RW1.

Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin (2-CDD) (10 ppm each) from soil microcosms to final concentrations in the parts-per-billion range was affected by the addition of Sphingomonas sp. strain RW1. Rates and extents of removal were influenced by the density of RW1 organisms. For 2-CDD, the rate of removal was dependent on the content of soil organic matter (SOM), with half-life values ranging from 5.8 h (0% SOM) to 26.3 h (5.5% SOM).

Cloning, nucleotide sequence, and expression of the gene encoding a novel dioxygenase involved in metabolism of carboxydiphenyl ethers in Pseudomonas pseudoalcaligenes POB310.

Pseudomonas pseudoalcaligenes strain POB310 degrades 3- and 4-carboxydiphenyl ether. The initial reaction involves an angular dioxygenation yielding an unstable hemiacetal that spontaneously decays to phenol and protocatechuate. We cloned a DNA fragment containing the gene encoding the initial dioxygenase from an unstable, self-transmissible plasmid. Sequence analysis revealed two open reading frames encoding proteins with putative molecular masses of 46.3 and 33.6 kDa. The deduced amino acid sequences showed homologies to oxygenase and reductase subunits of aromatic ring-activating dioxygenases, and contained regions identical to consensus sequences that bind chloroplast-like and Rieske-type [2Fe2S] clusters, suggesting that the initial dioxygenase is a class IA aromatic ring-activating dioxygenase system. Initial dioxygenase activity was induced in bacteria grown in M9 minimal medium containing 3- or 4-carboxydiphenyl ether or phenol as carbon source, indicating that the regulation is dependent on the phenol pathway. The maximal specific activity was measured at the beginning of the exponential growth phase.

Tetrameric structure and cellular location of catechol 2,3-dioxygenase.

Catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the TOL plasmid of Pseudomonas putida (pWWO) was investigated by electron microscopy. Negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. Monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in Escherichia coli (pAW31) and P. putida (pWWO), using the protein A-gold technique carried out as a post-embedding immunoelectron microscopy procedure. Our in situ labeling studies revealed a cytoplasmic location of the catechol 2,3-dioxygenase in both cell types.

Efficacy in aquatic microcosms of a genetically engineered pseudomonad applicable for bioremediation.

A genetically engineered microorganism (GEM), Pseudomonas sp. B13 FRI (pFRC20P) (abbreviated FR120), has previously been engineered to simultaneously mineralize mixtures of methylated and chlorinated benzoic acids and phenols through a modified ortho cleavage pathway. In this study, its performance was investigated both in different types of aquatic microcosms and in pure culture to determine (1) if under simulated in situ conditions the genetically engineered pathway effectively removes mixtures of model pollutants simultaneously, quickly, and completely; (2) where the optimum pollutant concentration range for this activity lies; and (3) how physical, chemical, and biological factors in the microcosms influence degradation rates. Growth and degradation parameters of FR 120 in pure culture were determined with 3-chlorobenzoate (3CB), 4-methylbenzoate (4MB), and equimolar mixtures of both as carbon sources. These substrates were degraded simultaneously, albeit with different degradation velocities, by FR120. The optimum growth concentrations for 3CB and 4MB were 3.0 mm and 2.1 mM, respectively, and the inhibition constants (Ki) were 11 mm (3CB) and 6 mm (4MB). The pathway was induced at low concentrations of substrate (> 1 [µM). The first order degradation constants (kl) were determined with respect to substrate concentration, cell density, and temperature. In aquatic microcosms inoculated with FR120, first order degradation constants and half lives of target chemicals were calculated based on the total amount of aromatics recovered. Half lives ranged from 1.3 days to 3.0 days, depending on the target chemical and the type of microcosm. Degradation constants determined in pure culture were extrapolated to the densities of FR120, substrate concentrations, and temperature occurring in the microcosm experiments, and used to calculate theoretical half lives. In water microcosms, theoretical and observed half lives corresponded well, indicating that FR120 functioned optimally in this environment. In whole core sediment microcosms, and especially at low cell densities, the observed degradation activity was in some cases considerably higher than expected from pure culture degradation rates. This suggests that environmental conditions in the sediment were more favorable to the degradation of substituted aromatics than those in pure culture. The physiological characteristics of FR120 and its performance in aquatic microcosms make it a good candidate for bioremediation at sites contamninated with mixtures of chlorinated and methylated aromatics.

A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs).

In this report we describe the development and construction of a DNA module which encodes bph genes for the metabolism of PCBs and which is capable of stable integration into the chromosome of Gram negative bacteria. Introduction of the bph-module into Pseudomonas putida KT2442, Pseudomonas sp. strain B13 and its genetically engineered derivative B13FR1 expanded the biodegradative ability of these strains to include biphenyl and 4-chlorobiphenyl. The bph operon was stably inherited under laboratory conditions. Behavior of the genetically engineered strains was evaluated under simulated natural habitat conditions in lake sediment microcosms with respect to survival and removal of 4-chlorobiphenyl. The genetically engineered strains persisted under these conditions and were effective in degrading 4-chlorobiphenyl over a five day incubation period.

Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms.

Pseudomonas cepacia G4 degrades trichloroethylene (TCE) via a degradation pathway for aromatic compounds which is induced by substrates such as phenol and tryptophan. P. cepacia G4 5223 PR1 (PR1) is a Tn5 insertion mutant which constitutively expresses the toluene ortho-monooxygenase responsible for TCE degradation. In groundwater microcosms, phenol-induced strain G4 and noninduced strain PR1 degraded TCE (20 and 50 microM) to nondetectable levels (< 0.1 microM) within 24 h at densities of 10(8) cells per ml; at lower densities, degradation of TCE was not observed after 48 h. In aquifer sediment microcosms, TCE was reduced from 60 to < 0.1 microM within 24 h at 5 x 10(8) PR1 organisms per g (wet weight) of sediment and from 60 to 26 microM over a period of 10 weeks at 5 x 10(7) PR1 organisms per g. Viable G4 and PR1 cells decreased from approximately 10(7) to 10(4) per g over the 10-week period.

Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate.

A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G + C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450RR1 was induced by 2-ethoxyphenol, and cytochrome P-450RR2 was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (KS = 4.2 +/- 0.3 microM) and 2-methoxyphenol (KS = 2.0 +/- 0.1 microM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450RR2 by both 4-methoxybenzoate (KS = 2.1 +/- 0.1 microM) and 4-ethoxybenzoate (KS = 1.6 +/- 0.1 microM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-450RR2, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of aquatic sediment microcosms and their use in assessing possible effects of introduced microorganisms on ecosystem parameters.

In this paper we describe a sediment microcosm system consisting of 20 undisturbed, layered sediment cores with overlying site water which are incubated under identical conditions of temperature, light, stirring rate of overlying water, and water exchange rate. Ecosystem parameters (nutrient level, photosynthetic potential, community structure of heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients) of the laboratory microcosms and the source ecosystem were compared and shown to be indistinguishable for the first 2 weeks. In weeks 3 and 4, small differences were detectable in the nutrient level, community structure of heterotrophic bacteria, and thymidine incorporation rate. However, the photosynthetic potential, depth profiles of heterotrophic bacterial community structure, and oxygen microgradients were maintained throughout the incubation period and did not differ between laboratory microcosms and the source ecosystem. The microcosm system described here would thus appear to be a valid model of aquatic sediments for up to 4 weeks; the actual period would depend on the sediment source and incubation temperature. The validated systems were used with Rhine river sediment to assess possible effects on ecosystem parameters of Pseudomonas sp. strain B13 FR1(pFRC20P), a genetically engineered microorganism (GEM) that had been constructed to degrade mixtures of halo- and alkylbenzoates and -phenols. The GEM survived in the surface sediment at densities of 5 x 10(4) to 5 x 10(5)/g (dry weight) for 4 weeks and degraded added chloro- and methylaromatics. The GEM did not measurably influence ecosystem parameters such as photosynthesis, densities of selected heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients. Thus, the microcosm system described here would seem to be useful for the study of the ecology of biodegradation and the fate and effect of microorganisms introduced into the environment.

Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms.

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-Döbler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.

Bioenergetic conditions of butyrate metabolism by a syntrophic, anaerobic bacterium in coculture with hydrogen-oxidizing methanogenic and sulfidogenic bacteria.

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium Methanospirillum hungatei PM-1 or Desulfovibrio sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H(2) uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (DeltaG') and the butyrate degradation rate (v) were greater for NSF-2-Desulfovibrio sp. strain PS-1 (DeltaG' = -17.0 kJ/mol; v = 0.20 mM/h) than for NSF-2-M. hungatei PM-1 (DeltaG' = -3.8 kJ/mol, v = 0.12 mM/h). The DeltaG' value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable DeltaG' resulted from the close coupling of the rates of butyrate and H(2) oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when Desulfovibrio sp. strain PS-1 was added as a new H(2) sink. When the specific activity of H(2) consumption was increased by adding higher densities of the Desulfovibrio sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H(2) pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H(2) consumption, not the type of H(2) consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical V(max).

Fate of genetically-engineered bacteria in activated sludge microcosms.

The conclusions that can be derived from this study regarding the fate of the GEMs and their ability to degrade added pollutants are as follows: 1. Both GEMs were able to survive in the microcoms. Because Pseudomonas sp. B13 has been cultured for a long time in the laboratory, it was not expected to survive well in the microcosm. Surprisingly, it and the derivative GEMs persisted at a high population level of approximately 10(5) bacteria/ml. Pure culture studies had demonstrated an ability of FR1(pFRC20P) to readily degrade simultaneous mixtures of 3CB and 4MB. In the microcosms, however, the GEM did not perform as well as expected, particularly when confronted with a shock load of a 3CB and 4MB mixture. Thus, the microcosm studies may be of potential help for making predictions concerning environmental applications of GEMs. 2. Pseudomonas sp. B13 derivative strains FR1 and FR1(pFRC20P) were able to degrade low concentrations of substituted benzoates within the complex ecosystem of the activated sludge microcosm. A good deal of information concerning the degradation pathway for aromatics by Pseudomonas sp. B13 was already known. This allowed for the construction of the stable, regulated pathways for the degradation of substituted aromatic compounds in both GEMs and indicates that the construction of similar GEMs for the degradation of environmental pollutants is a promising experimental strategy. 3. There was not any demonstrable, adverse effect of GEM addition to the microbial population level in the microcosm. The GEMs were even able to function in a protective manner for the indigenous populations by buffering them against the adverse effects of addition of substituted benzoates. In contrast, for microcosms lacking GEM addition, a wash-out of the bacterial population in the microcosm occurred (data not shown). 4. Lateral transfer of new genetic information (xylXYZLS) from the GEM chromosome to indigenous microorganisms was not detected, whereas transfer of the hybrid, mobilizable pFRC20P carrying the gene for lactone isomerase did apparently occur. In this particular case, transfer may have been beneficial for the community as a whole if it increased the ecosystem's ability to cope with the presence of toxic pollutants. As more GEMs are constructed for specific biotechnological applications the diversity and complexity of microcosms used to study their fate and function will increase. The ability of such studies to predict a priori the fate of these microorganisms will help to develop strategies both to decrease the risks associated with introducing GEMs into the environment and to increase and regulate the capacity of GEMs to degrade toxic pollutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp.

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.

Kinetics of phenol biodegradation by an immobilized methanogenic consortium.

A phenol-degrading methanogenic enrichment was successfully immobilized in agar as shown by the stoichiometric conversion of phenol to CH(4) and CO(2). The enrichment contained members of three physiological groups necessary for the syntrophic mineralization of phenol: a phenol-oxidizing bacterium, a Methanothrix-like bacterium, and an H(2)-utilizing methanogen. The immobilization technique resulted in the cells being embedded in a long, thin agar strand (1 mm in diameter by 2 to 50 cm in length) that resembled spaghetti. Immobilization had three effects as shown by a comparative kinetic analysis of phenol degradation by free versus immobilized cells. (i) The maximum rate of degradation was reduced from 14.8 to 10.0 mug of phenol per h; (ii) the apparent K(m) for the overall reaction was reduced from 90 to 46 mug of phenol per ml, probably because of the retention of acetate, H(2) and CO(2) in the proximity of immobilized methanogens; and (iii) the cells were protected from substrate inhibition caused by high concentrations of phenol, which increased the apparent K(i) value from 900 to 1,725 mug of phenol per ml. Estimates for the kinetic parameters K(m), K(i), and V(max) were used in a modified substrate inhibition model that simulated rates of phenol degradation for given phenol concentrations. The simulated rates were in close agreement with experimentally derived rates for both stimulatory and inhibitory concentrations of phenol.