Degradation of 1,4-dichlorobenzene by a Pseudomonas sp.

A Pseudomonas species able to degrade p-dichlorobenzene as the sole source of carbon and energy was isolated by selective enrichment from activated sludge. The organism also grew well on chlorobenzene and benzene. Washed cells released chloride in stoichiometric amounts from o-, m-, and p-dichlorobenzene, 2,5-dichlorophenol, 4-chlorophenol, 3-chlorocatechol, 4-chlorocatechol, and 3,6-dichlorocatechol. Initial steps in the pathway for p-dichlorobenzene degradation were determined by isolation of metabolites, simultaneous adaptation studies, and assay of enzymes in cell extracts. Results indicate that p-dichlorobenzene was initially converted by a dioxygenase to 3,6-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene, which was converted to 3,6-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,6-dichlorocatechol was by a 1,2-oxygenase to form 2,5-dichloro-cis, cis-muconate. Enzymes for degradation of haloaromatic compounds were induced in cells grown on chlorobenzene or p-dichlorobenzene, but not in cells grown on benzene, succinate, or yeast extract. Enzymes of the ortho pathway induced in cells grown on benzene did not attack chlorobenzenes or chlorocatechols.     

Degradation of 1,4-dichlorobenzene by a Pseudomonas sp

A Pseudomonas species able to degrade p-dichlorobenzene as the sole source of carbon and energy was isolated by selective enrichment from activated sludge. The organism also grew well on chlorobenzene and benzene. Washed cells released chloride in stoichiometric amounts from o-, m-, and p-dichlorobenzene, 2,5-dichlorophenol, 4-chlorophenol, 3-chlorocatechol, 4-chlorocatechol, and 3,6-dichlorocatechol. Initial steps in the pathway for p-dichlorobenzene degradation were determined by isolation of metabolites, simultaneous adaptation studies, and assay of enzymes in cell extracts. Results indicate that p-dichlorobenzene was initially converted by a dioxygenase to 3,6-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene, which was converted to 3,6-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,6-dichlorocatechol was by a 1,2-oxygenase to form 2,5-dichloro-cis, cis-muconate. Enzymes for degradation of haloaromatic compounds were induced in cells grown on chlorobenzene or p-dichlorobenzene, but not in cells grown on benzene, succinate, or yeast extract. Enzymes of the ortho pathway induced in cells grown on benzene did not attack chlorobenzenes or chlorocatechols.     

Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event.

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Aromatic Compounds on Cellular Fatty Acid Composition of Rhodococcus opacus

In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds.     

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1

Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds. 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway. All enzymes necessary to convert 1,4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1,3-dichlorobenzene degradation. Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X. flavus 14p1. Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism. Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities. As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth. Presumably a toxic compound was formed from one of the intermediates. Received: 10 June 1996 / Revision received: 23 December 1996 / Accepted: 18 January 1997     

Heterogeneity of Rhodococcus opacus 1CP as a response to stress induced by chlorophenols

Dissociation of Rhodococcus opacus 1CP during culturing in different media (containing phenol and its monochlorinated derivatives as the sole source of carbon and energy) was studied. Three variants of strain 1CP (S1, S2, and R) differing in the morphology of cells and colonies, lipid composition, and manner of growth on phenol and monochlorophenols were isolated. It was shown that 2- and 4-chlorophenols were most actively degraded by the smooth (S) forms of the culture, and that the rough (R) form predominated when the culture was grown in a rich medium. The S forms differed from the R forms of the strain by an increased content of cardiolipin, fatty acids, and phosphatidylethanolamine.     

Degradation of Pyrene, Benz[a]anthracene, and Benzo[a]pyrene by Mycobacterium sp. Strain RJGII-135, Isolated from a Former Coal Gasification Site

Volume 62, no. 1, p. 14, column 1, line 20: "250 (mu)g each of yeast extract, peptone, and soluble starch ml(sup-1)" should read "250 (mu)g each of yeast extract, peptone, and soluble starch liter(sup-1)." [This corrects the article on p. 13 in vol. 62.].     

Highly Active Microbial Communities in the Ice and Snow Cover of High Mountain Lakes

An exploratory study carried out in Pyrenean and Alpine lakes shows that a rich, active microbial community lives in the slush layers of the winter cover of such lakes in spite of the low temperature and the seasonal occurrence of the habitat. Bacteria were very diverse in morphology, with filaments reaching up to 100 (mu)m long; flagellates, both autotrophic (chrysophytes, cryptophytes, dinoflagellates, and volvocales) and heterotrophic, and ciliates were abundant, reaching biovolume values up to 2.7 x 10(sup6) (mu)m(sup3) ml(sup-1). Species composition was very variable, with dominance depending on date and depth. Although many species were typical of lake plankton communities, some were restricted to the slush, for instance the predatory ciliates Dileptus sp. and Lacrymaria sp., and others were restricted to the surface pools, such as the snow algae Chlamydomonas nivalis. Microbial biomasses and usually bacterial and algal activities were greater in the slush layers than in the lake water. Photosynthesis rate in the upper cover layers reached values up to 0.5 (mu)g of C liter(sup-1) h(sup-1), and high bacterial activities up to 226 pmol of leucine incorporated liter(sup-1) h(sup-1) and 25 pmol of thymidine incorporated liter(sup-1) h(sup-1) were measured. For most species, lake water flooding the ice and snow cover could provide an inoculum. Differential growth depending on the environmental conditions (nutrients, organic matter, light) of a particular slush layer could provide dominance of different groups or species. However, there was no obvious colonizing mechanism for those species not appearing either in plankton or in communities on top of the snowpack.     

Growth of Rhodococcus opacus on mixtures of monohalogenated benzenes and phenols

The growth of Rhodococcus opacus GM-14 on mixtures of 2-chloro- and 2-bromophenol, of 4-chloro, 4-bromo-, and 4-iodophenol, and of chloro-, bromo-, and iodobenzenes was accompanied by consumption of the substrates and excretion of halogen ions to the medium. During the growth on monochlorophenols, the substrates were consumed sequentially in the following order: 4-chloro-, 3-chloro-, and then 2-chlorophenol. Chlorine ions were excreted in a two-phase manner in amounts comprising 79% of the theoretical yield. The diauxic growth of R. opacus GM-14 can be explained by the existence in this bacterium of two modified metabolic pathways for the ortho-cleavage of halogenated pyrocatechols. The first pathway included 4-halogeno- or dihalogenopyrocatechols as intermediates, whereas the second pathway included 3-halogenopyrocatechols.     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations.

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

A New In Vivo Fluorimetric Technique To Measure Growth of Adhering Phototrophic Microorganisms

We developed a noninvasive rapid fluorimetric method for the investigation of growth of adhering (benthic) phototrophic microorganisms. The technique is based on the sensitive detection of the in vivo fluorescence of chlorophylls chlorophyll a and bacteriochlorophyll a and monitors increases in signal over time as an indicator for growth. The growth fluorimeter uses modulated excitation light of blue-light-emitting diodes and a photodiode as the detector. The light-emitting diodes are mounted geometrically in an aluminum housing for efficient and uniform illumination of the bottoms of the growth containers. The fluorimeter was characterized with respect to detection limit and dynamic range. This system is capable of resolving in vivo chlorophyll a concentrations of 0.5 (mu)g liter(sup-1) in cyanobacteria and 0.03 (mu)g liter(sup-1) in diatoms as well as in vivo bacteriochlorophyll a concentrations in phototrophic bacteria of 0.3 (mu)g liter(sup-1), which points to an extremely high sensitivity compared with that of similar available techniques. Thus, the new fluorimeter allows the determination of growth at extremely low cell densities. The instrument was used successfully to measure the growth of several adhering isolates of the filamentous cyanobacterium Microcoleus chthonoplastes from benthic microbial mats in seawater of different salinities. The data obtained demonstrate broad growth responses for all strains, which thus can be characterized as euryhaline organisms.     

A Chromosomally Based tod-luxCDABE Whole-Cell Reporter for Benzene, Toluene, Ethybenzene, and Xylene (BTEX) Sensing

A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8. This strain was used to examine the induction of the tod operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents. Since this system contained the complete lux cassette (luxCDABE), bacterial bioluminescence in response to putative chemical inducers of the tod operon was measured on-line in whole cells without added aldehyde substrate. There was an increasing response to toluene concentrations from 30 μg/liter to 50 mg/liter, which began to saturate at higher concentrations. The detection limit was 30 μg/liter. There was a significant light response to benzene, m- and p-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to o-xylene. The transposon insertion was stable and had no negative effect on cell growth.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

Production of Pseudomonas aeruginosa Rhamnolipid Biosurfactants in Heterologous Hosts

The high-level production of rhamnolipid biosurfactants is a unique feature of Pseudomonas aeruginosa and is strictly regulated in response to environmental conditions. The final step in rhamnolipid biosynthesis is catalyzed by the rhlAB genes encoding a rhamnosyltransferase. The expression of the cloned rhlAB genes was studied in heterologous hosts, either under the control of the rhlR and rhlI rhamnolipid regulatory elements or under the control of the tac promoter. A recombinant P. fluorescens strain harboring multiple plasmid-encoded copies of the rhamnolipid gene cluster produced rhamnolipids (0.25 g liter(sup-1)) when grown under nitrogen-limiting conditions. The highest yields (0.6 g liter(sup-1)) and productivities (24 mg liter(sup-1) h(sup-1)) were obtained in a recombinant Pseudomonas putida strain, KT2442, harboring promoterless rhlAB genes fused to the tac promoter on a plasmid. Active rhamnosyltransferase was synthesized, but no rhamnolipids were produced, by recombinant Escherichia coli upon induction of rhlAB gene expression.     

Growth of moderately halophilic bacteria isolated from sea water using phenol as the sole carbon source

Moderately halophilic bacteria utilizing phenol as the sole carbon source were isolated by selective enrichment from sea water. The isolate (Gram-negative motile rods) was identified asDeleya venusta. It grew well in the presence of up to 1600 mg/L of phenol and 8% NaCl under aerobic conditions. When the cells were treated with chloramphenicol prior to the addition of phenol they did not utilize added phenol, even after prolonged incubation. Thus, the enzymes necessary for phenol metabolism appeared to be inducible.     

Dissolution of Barium from Barite in Sewage Sludges and Cultures of Desulfovibrio desulfuricans

High concentrations of total barium, ranging from 0.42 to 1.58 mg(middot)g(sup-1) (dry weight) were found in sludges of two sewage treatment plants near Florence, Italy. Barium concentrations in the suspended matter decreased as redox potential values changed from negative to positive. An anoxic sewage sludge sample was aerated, and 30% of the total barium was removed in 24 h. To demonstrate that barium was solubilized from barite by sulfate-reducing bacteria, a strain of Desulfovibrio desulfuricans was used to study the solubilization of barium from barite under laboratory conditions. During cell growth with different concentrations of barite from 0.01 to 0.3 g(middot)liter(sup-1) (the latter is the MIC) as the only source of sulfates in the cultures, the D. desulfuricans strain accumulated barium up to 0.58 (mu)g(middot)mg(sup-1) (dry weight). Three times the quantity of barium was dissolved by bacteria than in the uninoculated medium (control). The unexpectedly low concentration of soluble barium (1.2 mg of Ba(middot)liter(sup-1)) with respect to the quantity expected (109 mg of Ba(middot)liter(sup-1)), calculated on the basis of the free H(inf2)S evolved from the dissimilatory reduction of sulfate from barite, was probably due to the formation of other barium compounds, such as witherite (BaCO(inf3)) and the transient species barium sulfide (BaS). The D. desulfuricans strain, growing on barite, formed visible aggregates. Confocal microscopy analysis showed that aggregates consisted of bacteria and barite. After 3 days of incubation, several autofluorescent crystals surrounded by a dissolution halo were observed. The crystals were identified as BaS by comparison with the commercial compound.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume.

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.