Activity of Toluene-degrading Pseudomonas putida in the early growth phase of a biofilm for waste gas treatment

A biological trickling filter for treatment of toluene-containing waste gas was studied. The overall kinetics of the biofilm growth was followed in the early growth phase. A rapid initial colonization took place during the first three days. The biofilm thickness increased exponentially, whereas the incease of active biomass and polymers was linear. In order to investigate the toluene degradation, various toluene degraders from the multispecies biofilm were isolated, and a Pseudomonas putida was chosen as a representative of the toluene-degrading population. A specific rRNA oligonucleotide probe was used to follow the toluene-degrading P. putida in the multispecies biofilm in the filter by means of number and cellular rRNA content. P. putida appeared to detach from the biofilm during the first three days of growth, after which P. putida was found at a constant level of 10% of the active biomass in the biofilm. Based on the rRNA content, the in situ activity was estimated to be reduced to 20% of cells grown at maximum conditions in batch culture. The toluene degraded by P. putida was estimated to be a minor part (11%) of the overall toluene degradation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 131-141, 1997.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150

The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled. Both of these compounds can serve as the sole source of carbon and energy for this microorganism. The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol. Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate. Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene. Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150. Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions.     

Design of a large-scale surface-aerated bioreactor for biomass production using a VOC substrate

The design of a large-scale bioreactor for the production of bacterial biomass adapted to the biodegradation of volatile organic compounds was carried out. The bioreactor model used integrated the microbial kinetics and fluid dynamics described by the compartment model approach. The process conditions and kinetic parameters were adopted from the laboratory experimental study of (León, E., Seignez, C., Adler, N., Péringer, P., 1999. Growth inhibition of biomass adapted to the degradation of toluene and xylenes in mixture in a batch reactor with substrates supplied by pulses. Biodegradation 10, 245-250). The performance of the pulsed-batch stirred bioreactor under surface aeration conditions was simulated for different mixing configurations and conditions such as the impeller diameter, number of impellers, stirring speed, and oxygen pressure. The simulations were used for the cost analysis which resulted in the optimal design of the bioreactor.     

Biodegradation of paint stripper solvents in a modified gas lift loop bioreactor

Paint stripping wastes generated during the decontamination and decommissioning of former nuclear facilities contain paint stripping organics (dichloromethane, 2-propanol, and methanol) and bulk materials containing paint pigments. It is desirable to degrade the organic residues as part of an integrated chemical-biological treatment system. We have developed a modified gas lift loop bioreactor employing a defined consortium of Rhodococcus rhodochrous strain OFS and Hyphomicrobium sp. DM-2 that degrades paint stripper organics. Mass transfer coefficients and kinetic constants for biodegradation in the system were determined. It was found that transfer of organic substrates from surrogate waste into the air and further into the liquid medium in the bioreactor were rapid processes, occurring within minutes. Monod kinetics was employed to model the biodegradation of paint stripping organics. Analysis of the bioreactor process was accomplished with BIOLAB, a mathematical code that simulates coupled mass transfer and biodegradation processes. This code was used to fit experimental data to Monod kinetics and to determine kinetic parameters. The BIOLAB code was also employed to compare activities in the bioreactor of individual microbial cultures to the activities of combined cultures in the bioreactor. This code is of benefit for further optimization and scale-up of the bioreactor for treatment of paint stripping and other volatile organic wastes in bulk materials. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 163-169, 1997.     

Biodegradation of chlorferon and diethylthiophosphate by consortia enriched from waste cattle dip solution

Chlorferon and diethylthiophosphate (DETP) are the hydrolysis products of coumaphos, an organophosphate pesticide. In this research, two consortia of bacterial cultures, one responsible for degrading chlorferon and the other for degrading DETP, were selectively enriched from waste cattle dip solution. The enriched cultures were used as inocula to grow biomass for biodegradation studies. For chlorferon degradation, the optimum biomass concentration was found to be 80g/L, and pH 7.5 was selected as the optimal operating pH. Chlorferon degradation was characterized by substrate inhibition kinetics with parameter values estimated to be V(m)=0.062+/-0.011mg/(g-biomass)h, K(m)=21+/-7mg/L, and K(Si)=118+/-45mg/L. For DETP degradation, the optimum biomass concentration was found to be 60g/L, and the optimum pH was in the range of 7.5-8. DETP degradation was characterized by Michaelis-Menten kinetics with parameter values estimated to be V(m)=1.52+/-0.10mg/(g-biomass)h and K(m)=610+/-106mg/L.     

A possible mechanism of metabolic regulation in Gibberella fujikuroi using a mixed carbon source of glucose and corn oil inferred from analysis of the kinetics data obtained in a stirrer tank bioreactor

A nonstructured model was used to study the dynamics of gibberellic acid production in a stirred tank bioreactor. Experimental data were obtained from submerged batch cultures of Gibberella fujikuroi (CDBB H‐984) grown in varying ratios of glucose‐corn oil as the carbon source. The nitrogen depletion effect was included in mathematical model by considering the specific kinetic constants as a linear function of the normalized nitrogen consumption rate. The kinetics of biomass growth and consumption of phosphate and nitrogen were based on the logistic model. The traditional first‐order kinetic model was used to describe the specific consumption of glucose and corn oil. The nitrogen effect was solely included in the phosphate and corn oil consumption and biomass growth. The model fit was satisfactory, revealing the dependence of the kinetics with respect to the nitrogen assimilation rate. Through simulations, it was possible to make diagrams of specific growth rate and specific rate of substrate consumptions, which was a powerful tool for understanding the metabolic interactions that occurred during the various stages of fermentation process. This kinetic analysis provided the proposal of a possible mechanism of regulation on growth, substrate consumptions, and production of gibberellic acid (GA₃) in G. fujikuroi. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1169–1180, 2013     

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.     

Controlled biomass formation and kinetics of toluene degradation in a bioscrubber and in a reactor with a periodically moved trickle-bed

The kinetics of degradation of toluene from a model waste gas and of biomass formation were examined in a bioscrubber operated under different nutrient limitations with a mixed culture. The applicability of the kinetics of continuous cultivation of the mixed culture was examined for a special trickle-bed reactor with a periodically moved filter bed. The efficiency of toluene elimination of the bioscrubber was 50 to 57% and depended on the toluene mass transfer as evident from a constant productivity of 0.026 g dry cell weight/L . h over the dilution rate. Under potassium limitation the biomass productivity was reduced by 60% to 0.011 g dry cell weight/L . h at a dilution rate of 0.013/h. Conversely, at low dilution rates the specific toluene degradation rates increased. Excess biomass in a trickle-bed reactor causes reduction of interfacial area and mass transfer, and increase in pressure drop. To avoid these disadvantages, the trickle-bed was moved periodically and biomass was removed with outflowing medium. The concentration of steady state biomass fixed on polyamide beads decreased hyperbolically with the dilution rate. Also, the efficiency of toluene degradation decreased from 72 to 56% with increasing dilution rate while the productivity increased. Potassium limitation generally caused a reduction in biomass, productivity, and yield while the specific degradation increased with dilution rate. This allowed the application of the principles of the chemostat to the trickle-bed reactor described here, for toluene degradation from waste gases. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 686-692, 1997.     

Trichloroethylene mineralization in a fixed-film bioreactor using a pure culture expressing constitutively toluene ortho -monooxygenase

An aerobic, single-pass, fixed-film bioreactor was designed for the continuous degradation and mineralization of gas-phase trichloroethylene (TCE). A pure culture of Burkholderia cepacia PR1(23)(TOM(23C)), a Tn5transposon mutant of B. cepacia G4 that constitutively expresses the TCE-degrading enzyme, toluene ortho-monooxygenase (TOM), was immobilized on sintered glass (SIRANtrade mark carriers) and activated carbon. The inert open-pore structures of the sintered glass and the strongly, TCE-absorbing activated carbon provide a large surface area for biofilm development (2-8 mg total cellular protein/mL carrier with glucose minimal medium that lacks chloride ions). At gas-phase TCE concentrations ranging from 0.04 to 2.42 mg/L of air and 0.1 L/min of air flow, initial maximum TCE degradation rates of 0.007-0.715 nmol/(min mg protein) (equivalent to 8.6-392.3 mg TCE/L of reactor/day) were obtained. Using chloride ion generation as the indicator of TCE mineralization, the bioreactor with activated carbon mineralized an average of 6.9-10.3 mg TCE/L of reactor/day at 0.242 mg/L TCE concentration with 0.1 L/min of air flow for 38-40 days. Although these rates of TCE degradation and mineralization are two- to 200-fold higher than reported values, TOM was inactivated in the sintered-glass bioreactor at a rate that increased with increasing TCE concentration (e.g., in approximately 2 days at 0.242 mg/L and <1 day at 2.42 mg/L), although the biofilter could be operated for longer periods at lower TCE concentrations. Using an oxygen probe and phenol as the substrate, the activity of TOM in the effluent cells of the bioreactor was monitored; the loss of TOM activity of the effluent cells corroborated the decrease in the TCE degradation and mineralization rates in the bioreactor. Repeated starving of the cells was found to restore TOM activity in the bioreactor with activated carbon and extended TCE mineralization by approximately 34%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 674-685, 1997.     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

Biodegradation of ortho-cresol by a mixed culture of nitrate-reducing bacteria growing on toluene.

A mixed culture of nitrate-reducing bacteria degraded o-cresol in the presence of toulene as a primary growth substrate. No degradation of o-cresol was observed in the absence of toluene or when the culture grew on p-cresol and 2,4-dimethylphenol. In batch cultures, the degradation of o-cresol started after toluene was degraded to below 0.5 to 1.0 mg/liter but continued only for about 3 to 5 days after the depletion of toluene since the culture had a limited capacity for o-cresol degradation once toluene was depleted. The total amount of o-cresol degraded was proportional to the amount of toluene metabolized, with an average yield of 0.47 mg of o-cresol degraded per mg of toluene metabolized. Experiments with [ring-U-14C]o-cresol indicated that about 73% of the carbon from degraded o-cresol was mineralized to CO2 and about 23% was assimilated into biomass after the transient accumulation of unidentified water-soluble intermediates. A mathematical model based on a simplified Monod equation is used to describe the kinetics of o-cresol degradation. In this model, the biomass activity toward o-cresol is assumed to decay according to first-order kinetics once toluene is depleted. On the basis of nonlinear regression of the data, the maximum specific rate of o-cresol degradation was estimated to be 0.4 mg of o-cresol per mg of biomass protein per h, and the first-order decay constant for o-cresol-degrading biomass activity was estimated to be 0.15 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sand administration as an instrument for biofilm control of Pseudomonas putida ATCC 11172 in chemostat cultures

Pseudomonas putida ATCC 11172 was grown in chemostat on L-asparagine or phenol as the sole, limiting carbon and energy source. The growth characteristics of a culture where a biofilm was present, were compared with one where the biofilm was strongly reduced by the grinding and shearing effect of sand suspended in the culture. In the presence of the intact biofilm, the curve of steady-state biomass versus dilution rate diverged greatly from the theoretical pattern predicted by conventional chemostat models. The sand strongly retarded the biofilm formation and to a high degree restored the shape of the biomass versus dilution rate curve to a more conventional pattern. The maximum specific growth rate (mu(max)) could not be calculated from the biofilm cultures. However using the sand cultures, mu(max) was determined to 0.64 h(-1) with L-asparagine as the carbon source and 0.49 h(-1) with phenol which compare favorably with the respective mu(max) values calculated from batch cultures.Incorporation of sand into strongly agitated cultures is recommended as an efficient and simple means of controlling biofilm formation in continuous cultures. The method may enable the gathering of basic kinetic data difficult to obtain in the presence of biofilm.     

Comparison of Planktonic and Biofilm Cultures of Pseudomonas fluorescens DSM 8341 Cells Grown on Fluoroacetate

Comparisons between the physiological properties of Pseudomonas fluorescens biofilm cells grown in a tubular biofilm reactor and planktonic cells grown in a chemostat were performed. Fluoroacetate was the sole carbon source for all experiments. The performance of cells was assessed using cell cycle kinetics and by determining specific fluoroacetate utilization rates. Cell cycle kinetics were studied by flow cytometry in conjunction with the fluorescent stain propidium iodide. Determination of the DNA content of planktonic and biofilm cultures showed little difference between the two modes of growth. Cultures with comparable specific glycolate utilization rates had similar percentages of cells in the B phase of the cell cycle, indicating similar growth rates. Specific fluoroacetate utilization rates showed the performance of planktonic cells to be superior to that of biofilm cells, with more fluoroacetate utilized per cell at similar specific fluoroacetate loading rates. A consequence of this decreased biofilm performance was the accumulation of glycolate in the effluent of biofilm cultures. This accumulation of glycolate was not observed in the effluent of planktonic cultures. Spatial stratification of oxygen within the biofilm was identified as a possible explanation for the overflow metabolism of glycolate and the decreased performance of the biofilm cells.There is a general consensus that the adhesion of microbes to a surface influences bacterial metabolism; however, the experimental results are often contradictory (34). Some studies have compared the physiological status of biofilm and planktonic cells by determining their growth rates, with some investigators (2, 9) reporting increased biofilm growth rates in comparison to planktonic growth rates while others (1) have reported the opposite. Other researchers have compared the influence of adhesion on biofilm metabolic activity to planktonic activity, and as with growth rate, conflicting observations have been reported (16, 31, 11). The objective of the present study was to characterize planktonic cells grown in a chemostat and biofilm cells grown in a tubular biofilm reactor (TBR) and compare their performance with respect to the degradation of the model xenobiotic compound fluoroacetate.Halogenated compounds are extensively used in many applications (refrigeration, lubricants, pharmaceuticals, insecticides, and herbicides) and can be considered significant environmental contaminants. The biodegradation of many chlorinated compounds has been widely reported (5, 26, 28). However, in spite of the increased use of organofluorine compounds in the past 60 years, there is limited information on their degradation (17). Currently, a large fraction of wastewater streams containing fluorinated compounds are incinerated (12). Improved biological waste treatment processes require a deeper understanding of microbial degradation of fluorinated compounds.Some previous studies have focused on the biodegradation of fluorinated aromatic compounds using biofilm reactors (3, 10); however, there have been no studies on the degradation of fluorinated aliphatic compounds in biofilm reactors. Thus, sodium fluoroacetate was chosen as the model xenobiotic to study the efficiency of aliphatic organofluorine degradation in biofilms. It was the first naturally occurring fluorinated compound to be isolated, obtained from the South African shrub Dichapetalum cymosum (23). Fluoroacetate is highly toxic to mammals and has found extensive use as a vertebrate pesticide, particularly in Australia and New Zealand. A number of studies have focused on the isolation and identification of microbial soil isolates with the ability to degrade fluoroacetate (14, 33, 35), and other studies have focused on the mechanism of defluorination (13, 15, 21). However, there has been no research on the degradation of fluoroacetate by biofilm cultures. Biofilm systems appear ideal for the degradation of xenobiotics because of the many reported advantages they have over planktonic cultures. Most microorganisms that have the ability to degrade xenobiotic compounds have comparatively slow growth rates, and biofilm reactors allow the enrichment of these microorganisms independent of hydraulic retention times (36). It has been shown in numerous studies that biofilms are less susceptible than suspended bacteria to changes in environmental conditions such as temperature and pH and the presence of metabolic products and toxic substances (8, 25, 27, 36). The high cell concentrations that can be achieved in biofilm systems in combination with high volumetric flow rates could potentially result in high volumetric productivities without the risk of cell washout.The species Pseudomonas fluorescens has been extensively studied; it commonly exists as a biofilm in natural environments and is ubiquitous in industrial environments (6, 29, 30). The specific strain used here, P. fluorescens DSM 8341, was previously isolated from a soil sample in Western Australia, and in a study with 23 other microbial soil isolates, it was shown to be the most efficient degrader of fluoroacetate when fluoroacetate was the sole carbon source (37). The effect of the environmental factors, pH and temperature, on the biodefluorination of fluoroacetate by P. fluorescens was also determined (38); however, at present there are no reported planktonic growth kinetics established for this strain, nor has it previously been grown as a biofilm. In this context a TBR was employed to investigate the degradation of fluoroacetate by a P. fluorescens biofilm, in conjunction with chemostat studies that were conducted to determine the efficiency of planktonic degradation of the substrate. Specific utilization rates, flow cytometry, and fluorescent microscopy were employed to compare the performance and physiological status of biofilm and planktonic cells grown with fluoroacetate as the sole organic substrate.     

Simultaneous Growth on Citrate Reduces the Effects of Iron Limitation during Toluene Degradation in Pseudomonas

Rhizoremediation has been suggested as an attractive bioremediation strategy for the effective breakdown of pollutants in soil. The presence of plant root exudates such as organic acids, sugars, and amino acids that may serve as carbon sources or biosynthetic building blocks and the limited bioavailability of iron may influence the degradation of pollutants in the rhizosphere. To test the effect of such compounds on hydrocarbon degradation, trace concentrations of yeast extract or mixtures of organic acids and amino acids were added to continuous cultures of Pseudomonas putida mt2 and P. putida WCS358 (TOL) growing on toluene. By addition of these compounds increased growth yields and higher specific growth rates on toluene were obtained. The effects of iron limitation on the substrate utilization pattern of both strains were tested by growing the strains on a mixture of toluene and the readily degradable carbon source citrate while the iron concentration was varied. Simultaneous use of both substrates under carbon-limited as well as iron-limited conditions was observed. Growth yields were less reduced and iron requirement was lower during iron-limited growth in the toluene + citrate grown cultures compared to cultures in which toluene was used as the sole carbon source. The kinetic properties of the cells for toluene degradation were less hampered by the lack of iron when citrate was used as an additional carbon source. The results indicate that the availability of low concentrations of natural organic compounds, such as produced in the rhizosphere, may positively influence the degradative performance of hydrocarbon-degrading bacteria.     

Kinetics of toluene degradation by a nitrate-reducing bacterium isolated from a groundwater aquifer

Groundwater from a xylene-contaminated acquifer was enriched in the laboratory in the presence of toluene, xylenes, ethylbenzene, and benzene. A pure culture that degrades toluene and m-xylene under nitrate-reducing conditions was isolated. Fatty acid analysis, 16S rRNA sequencing, and morphological traits indicate that the isolate was a strain of Azoarcus tolulyticus. The kinetics of toluene degradation under nitrate-reducing conditions by this isolate was determined. Nitrate reduction does not proceed beyond nitrite. Nitrate and toluene are substrate limiting at low concentrations, whereas toluene, nitrate, and nitrite are inhibitory at high concentrations. Several inhibition models were compared to experimental data to represent inhibition by these substrates. A kinetic model for toluene and nitrate degradation as well as for cell growth and nitrite production was developed and compared to experimental data. The results of this work may find important application in the remediation of groundwater aquifers contaminated with aromatic hydrocarbons. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 82-90, 1997.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.