Effect of model sorptive phases on phenanthrene biodegradation: different enrichment conditions influence bioavailability and selection of phenanthrene-degrading isolates

The sorption of organic contaminants by natural organic matter (NOM) often limits substrate bioavailability and is an important factor affecting microbial degradation rates in soils and sediments. We hypothesized that reduced substrate bioavailability might influence which microbial assemblages are responsible for contaminant degradation under enrichment culture conditions. Our primary goal was to characterize enrichments in which different model organic solid phases were used to establish a range of phenanthrene bioavailabilities for soil microorganisms. Phenanthrene sorption coefficients (expressed as log K(D) values) ranged from 3.0 liters kg(-1) for Amberlite carboxylic acid cation-exchange resin (AMB) to 3.5 liters kg(-1) for Biobeads polyacrylic resin (SM7) and 4.2 liters kg(-1) for Biobeads divinyl benzene resin (SM2). Enrichment cultures were established for control (no sorptive phase), sand, AMB, SM7, and SM2 treatments by using two contaminated soils (from Dover, Ohio, and Libby, Mont.) as the initial inocula. The effects of sorption by model phases on the degradation of phenanthrene were evaluated for numerous transfers in order to obtain stable microbial assemblages representative of sorptive and nonsorptive enrichment cultures and to eliminate the effects of the NOM present in the initial inoculum. Phenanthrene degradation rates were similar for each soil inoculum and ranged from 4 to 5 micromol day(-1) for control and sand treatments to approximately 0.4 micromol day(-1) in the presence of the SM7 sorptive phase. The rates of phenanthrene degradation in the highly sorptive SM2 enrichment culture were insignificant; consequently, stable microbial populations could not be obtained. Bacterial isolates obtained from serial dilutions of enrichment culture samples exhibited significant differences in rates of phenanthrene degradation performed in the presence of SM7, suggesting that enrichments performed in the presence of a sorptive phase selected for different microbial assemblages than control treatments containing solid phase phenanthrene.     

Effect of Model Sorptive Phases on Phenanthrene Biodegradation: Molecular Analysis of Enrichments and Isolates Suggests Selection Based on Bioavailability

Reduced bioavailability of nonpolar contaminants due to sorption to natural organic matter is an important factor controlling biodegradation of pollutants in the environment. We established enrichment cultures in which solid organic phases were used to reduce phenanthrene bioavailability to different degrees (R. J. Grosser, M. Friedrich, D. M. Ward, and W. P. Inskeep, Appl. Environ. Microbiol. 66:2695–2702, 2000). Bacteria enriched and isolated from contaminated soils under these conditions were analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified 16S ribosomal DNA segments. Compared to DGGE patterns obtained with enrichment cultures containing sand or no sorptive solid phase, different DGGE patterns were obtained with enrichment cultures containing phenanthrene sorbed to beads of Amberlite IRC-50 (AMB), a weak cation-exchange resin, and especially Biobead SM7 (SM7), a polyacrylic resin that sorbed phenanthrene more strongly. SM7 enrichments selected for mycobacterial phenanthrene mineralizers, whereas AMB enrichments selected for a Burkholderia sp. that degrades phenanthrene. Identical mycobacterial and Burkholderia 16S rRNA sequence segments were found in SM7 and AMB enrichment cultures inoculated with contaminated soil from two geographically distant sites. Other closely related Burkholderia sp. populations, some of which utilized phenanthrene, were detected in sand and control enrichment cultures. Our results are consistent with the hypothesis that different phenanthrene-utilizing bacteria inhabiting the same soils may be adapted to different phenanthrene bioavailabilities.     

Effect of model sorptive phases on phenanthrene biodegradation: molecular analysis of enrichments and isolates suggests selection based on bioavailability

Reduced bioavailability of nonpolar contaminants due to sorption to natural organic matter is an important factor controlling biodegradation of pollutants in the environment. We established enrichment cultures in which solid organic phases were used to reduce phenanthrene bioavailability to different degrees (R. J. Grosser, M. Friedrich, D. M. Ward, and W. P. Inskeep, Appl. Environ. Microbiol. 66:2695-2702, 2000). Bacteria enriched and isolated from contaminated soils under these conditions were analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified 16S ribosomal DNA segments. Compared to DGGE patterns obtained with enrichment cultures containing sand or no sorptive solid phase, different DGGE patterns were obtained with enrichment cultures containing phenanthrene sorbed to beads of Amberlite IRC-50 (AMB), a weak cation-exchange resin, and especially Biobead SM7 (SM7), a polyacrylic resin that sorbed phenanthrene more strongly. SM7 enrichments selected for mycobacterial phenanthrene mineralizers, whereas AMB enrichments selected for a Burkholderia sp. that degrades phenanthrene. Identical mycobacterial and Burkholderia 16S rRNA sequence segments were found in SM7 and AMB enrichment cultures inoculated with contaminated soil from two geographically distant sites. Other closely related Burkholderia sp. populations, some of which utilized phenanthrene, were detected in sand and control enrichment cultures. Our results are consistent with the hypothesis that different phenanthrene-utilizing bacteria inhabiting the same soils may be adapted to different phenanthrene bioavailabilities.     

Phosphorus Accumulation and Sorption in Calcareous Soil under Long-Term Fertilization

Application of phosphorus (P) fertilizers to P-deficient soils can also result in P accumulation. In this study, soil P status and P uptake by apple trees were investigated in 5-, 10-, and 15-year-old orchards in the semi-arid Loess Plateau, China, and subset soils with different soil P statuses (14–90 Olsen-P mg kg⁻¹) were selected to evaluate the characteristic P adsorption. Due to the low P-use efficiency (4–6%), total soil P increased from 540 mg kg⁻¹ to 904 mg kg⁻¹, Olsen-P ranged from 3.4 mg kg⁻¹ to 30.7 mg kg⁻¹, and CaCl₂-P increased from less than 0.1 mg kg⁻¹ to 0.66 mg kg⁻¹ under continuous P fertilization. The P sorption isotherms for each apple orchard were found to fit the Langmuir isotherm model (R ² = 0.91–0.98). K (binding energy) and Q _m (P sorption maximum) decreased, whereas DPS (degree of phosphorus sorption) increased with increasing P concentration. CaCl₂-P increased significantly with the increase of Olsen-P, especially above the change point of 46.1 mg kg⁻¹. Application of surplus P could result in P enrichment in P-deficient soil which has high P fixation capacity, thus posing a significant environmental risk.     

Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities (H. Futamata, S. Harayama, and K. Watanabe, Appl. Environ. Microbiol. 67:4671-4677, 2001). To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter⁻¹ day⁻¹) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter⁻¹ day⁻¹ (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.     

Two-Stage Mineralization of Phenanthrene by Estuarine Enrichment Cultures

The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring ¹⁴CO₂ production from low concentrations (0.5 mg liter⁻¹) of radiolabeled phenanthrene. Here, mineralization of ¹⁴C-labeled 1H2NA could explain the incremental ¹⁴CO₂ produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications.     

Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil

We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g⁻¹). Addition of the surfactant at a concentration below the CMC′ (2 mg g⁻¹) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g⁻¹), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g⁻¹) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.     

Impact of Different In Vitro Electron Donor/Acceptor Conditions on Potential Chemolithoautotrophic Communities from Marine Pelagic Redoxclines

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO₂ dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO₂ dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO₂ dark fixation rates, ranging from 0.7 to 1.4 μmol liter⁻¹ day⁻¹, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO₂ dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter⁻¹ CO₂ day⁻¹. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter⁻¹ NO₃⁻ day⁻¹. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.     

PAH Degradation Capacity of Soil Microbial Communities—Does It Depend on PAH Exposure?

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs ≈ 0.1 mg kg^?1) to heavily polluted industrial sites (total PAHs ≈ 400 mg kg^?1). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of ¹⁴C-labeled spikes. The time to 10% mineralization of added ¹⁴C phenanthrene and ¹⁴C pyrene was inversely correlated with the PAH content of the soils. Substantial ¹⁴C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. ¹⁴C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little ¹⁴C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.     

Tin and Tin-Resistant Microorganisms in Chesapeake Bay

Sediment and water samples from nine stations in Chesapeake Bay were examined for tin content and for microbial populations resistant to inorganic tin (75 mg of Sn liter⁻¹ as SnCl₄·5H₂O) or to the organotin compound dimethyltin chloride [15 mg of Sn liter⁻¹ as (CH₃)₂SnCl₂]. Tin concentrations in sediments were higher (3.0 to 7.9 mg kg⁻¹) at sites impacted by human activity than at open water sites (0.8 to 0.9 mg kg⁻¹), and they were very high (239.6 mg kg⁻¹) in Baltimore Harbor, which is impacted by both shipping and heavy industry. Inorganic tin (75 mg Sn liter⁻¹) in agar medium significantly decreased viable counts, but its toxicity was markedly reduced in liquid medium; it was not toxic in medium solidified with silica gel. Addition of SnCl₄·5H₂O to these media produced a tin precipitate which was not involved in the metal's toxicity. The data suggest that a soluble tin-agar complex which is toxic to cells is formed in agar medium. Thus, the toxicity of tin depends more on the chemical species than on the metal concentration in the medium. All sites in Chesapeake Bay contained organisms resistant to tin. The microbial flora was more sensitive to (CH₃)₂SnCl₂ than to SnCl₄·5H₂O. The elevated level of tin-resistant microorganisms in some aeas not containing unusually high tin concentrations suggests that factors other than tin may participate in the selection for a tin-tolerant microbial flora.     

Comparing Metabolic Functionalities,Community Structures,and Dynamics of Herbicide-Degrading Communities Cultivated with Different Substrate Concentrations

Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter⁻¹) or high (25 mg liter⁻¹) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.     

Effect of the polycyclic aromatic hydrocarbon phenanthrene on root exudation of Sorghum bicolor (L.) Moench

The objective of this work was to study the effect of two concentrations (10 and 100 mg kg⁻¹) of phenanthrene, a ubiquitous polycyclic aromatic hydrocarbon (PAH), on root exudation of the remediating plant Sorghum bicolor (L.) Moench under controlled conditions in a pot experiment. It was found that the phenanthrene concentration of 10 mg kg⁻¹ did not cause significant effects on plant survival and growth but had little stimulating effect on carbohydrate exudation. The contamination with phenanthrene at 100 mg kg⁻¹ inhibited accumulation of plant shoot and root biomass, decreasing the carboxylic acid, carbohydrate, and amino acid amounts released by sorghum root into the rhizosphere. However, root exudation per unit of root surface was not changed significantly with increasing phenanthrene concentration. There were no differences in qualitative composition of root exudates under the influence of PAH were found. The observed alterations in the ratio between the main root-exuded components are assumed to manifest adaptive alterations occurring in the plant as a response to pollutant stress. The activity of three oxidoreductases (oxidase, peroxidase, and tyrosinase) released by sorghum roots was clearly progressive to the increasing phenanthrene concentration in the substrate. Under the influence of phenanthrene, the population of phenanthrene-degrading microorganisms in sorghum root zone increased, and their share in the total number of culturable heterotrophs increased as well. The main promotional factor was the pollutant; however, the stimulating effect of the plant root exudates was also involved. The increased pollutant-degrading microbial population and activity of the extracellular root enzymes are presumed to be important for the rhizodegradation of PAH.     

Low-Molecular-Weight Sulfonates, a Major Substrate for Sulfate Reducers in Marine Microbial Mats

Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 μM HS⁻ h⁻¹ in a stromatolite mat, 38 and 36 μM HS⁻ h⁻¹ in a salt pond mat, and 27 and 18 μM HS⁻ h⁻¹ in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell⁻¹ h⁻¹. Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.     

Isolation of Soil Bacteria Adapted To Degrade Humic Acid-Sorbed Phenanthrene

The goal of these studies was to determine how sorption by humic acids affected the bioavailability of polynuclear aromatic hydrocarbons (PAHs) to PAH-degrading microbes. Micellar solutions of humic acid were used as sorbents, and phenanthrene was used as a model PAH. Enrichments from PAH-contaminated soils established with nonsorbed phenanthrene yielded a total of 25 different isolates representing a diversity of bacterial phylotypes. In contrast, only three strains of Burkholderia spp. and one strain each of Delftia sp. and Sphingomonas sp. were isolated from enrichments with humic acid-sorbed phenanthrene (HASP). Using [¹⁴C]phenanthrene as a radiotracer, we verified that only HASP isolates were capable of mineralizing HASP, a phenotype hence termed “competence.” Competence was an all-or-nothing phenotype: noncompetent strains showed no detectable phenanthrene mineralization in HASP cultures, but levels of phenanthrene mineralization effected by competent strains in HASP and NSP cultures were not significantly different. Levels and rates of phenanthrene mineralization exceeded those predicted to be supported solely by the metabolism of phenanthrene in the aqueous phase of HASP cultures. Thus, competent strains were able to directly access phenanthrene sorbed by the humic acids and did not rely on desorption for substrate uptake. To the best of our knowledge, this is the first report of (i) a selective interaction between aerobic bacteria and humic acid molecules and (ii) differential bioavailability to bacteria of PAHs sorbed to a natural biogeopolymer.     

Dynamics of microbial community during bioremediation of phenanthrene and chromium(VI)-contaminated soil microcosms

The combined effect of phenanthrene and Cr(VI) on soil microbial activity, community composition and on the efficiency of bioremediation processes has been studied. Biometer flask systems and soil microcosm systems contaminated with 2,000 mg of phenanthrene per kg of dry soil and different Cr(VI) concentrations were investigated. Temperature, soil moisture and oxygen availability were controlled to support bioremediation. Cr(VI) inhibited the phenanthrene mineralization (CO₂ production) and cultivable PAH degrading bacteria at levels of 500–2,600 mg kg⁻¹. In the bioremediation experiments in soil microcosms the degradation of phenanthrene, the dehydrogenase activity and the increase in PAH degrading bacteria counts were retarded by the presence of Cr(VI) at all studied concentrations (25, 50 and 100 mg kg⁻¹). These negative effects did not show a correlation with Cr(VI) concentration. Whereas the presence of Cr(VI) had a negative effect on the phenanthrene elimination rate, co-contamination with phenanthrene reduced the residual Cr(VI) concentration in the water exchangeable Cr(VI) fraction (WEF) in comparison with the soil microcosm contaminated only with Cr(VI). Clear differences were found between the denaturing gradient gel electrophoresis (DGGE) patterns of each soil microcosm, showing that the presence of different Cr(VI) concentrations did modulate the community response to phenanthrene and caused perdurable changes in the structure of the microbial soil community.     

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO₄²⁻ reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day⁻¹ for sulfate reduction and from 0.13 to 0.60 day⁻¹ for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Evaluation of the mutagenicity and antimutagenicity of Ziziphus joazeiro Mart. bark in the micronucleus assay

The aim of this study was to evaluate the mutagenicity (clastogenicity/aneugenicity) of a glycolic extract of Ziziphus joazeiro bark (GEZJ) by the micronucleus assay in mice bone marrow. Antimutagenic activity was also assessed using treatments associated with GEZJ and doxorubicin (DXR). Mice were evaluated 24–48 h after exposure to positive (N-nitroso-N-ethylurea, NEU - 50 mg.kg⁻¹ and DXR - 5 mg.kg⁻¹) and negative (150 mM NaCl) controls, as well as treatment with GEZJ (0.5–2 g.kg⁻¹), GEZJ (2 g.kg⁻¹) + NEU and GEZJ (2 g.kg⁻¹) + DXR. There were no significant differences in the frequencies of micronucleated polychromatic erythrocytes in mice treated with GEJZ and GEJZ + DXR compared to the negative controls, indicating that GEZJ was not mutagenic. Analysis of the polychromatic:normochromatic erythrocyte ratio revealed significant differences in the responses to doses of 0.5 g.kg⁻¹ and 1–2 g.kg⁻¹ and the positive control (NEU). These results indicated no systemic toxicity and moderate toxicity at lower and higher doses of GEZJ. The lack of mutagenicity and systemic toxicity in the antimutagenic assays, especially for treatment with GEZJ + DXR, suggested that phytochemical compounds in Z. joazeiro bark attenuated DXR-induced mutagenicity and the moderate systemic toxicity of a high dose of Z. joazeiro bark (2 g.kg⁻¹). Further studies on the genotoxicity of Z. joazeiro extracts are necessary to establish the possible health risk in humans and to determine the potential as a chemopreventive agent for therapeutic use.     

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1–18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.     

Modulation of Affinity of a Marine Pseudomonad for Toluene and Benzene by Hydrocarbon Exposure

Trace (microgram liter⁻¹) quantities of either toluene or benzene injected into an amino-acid-limited continuous culture of Pseudomonas sp. strain T2 were utilized immediately with affinities of 2.6 and 6.8 liters g of cells⁻¹ h⁻¹, respectively, and yielded large amounts of organic products, carbon dioxide, and cells. The immediate utilization of hydrocarbons by hydrocarbon-deprived organisms helps to establish the nutritional value of nonpolar substrates in the environment. The observation of small Michaelis constants for toluene transport led to tests of metabolic competition between hydrocarbons; however, competitive inhibition of toluene metabolism was not found for benzene, naphthalene, xylene, dodecane, or amino acids. Benzene and terpenes were inhibitory at milligram liter⁻¹ concentrations. Toluene was metabolized by a strongly inducible system when compared with benzene. The capacity of toluene to effect larger affinity values increased with exposure time and concentration. The kinetics of induction suggested saturation phenomena, resulting in an induction constant, K_ind, of 96 μg of toluene liter⁻¹. Maximal induction of amino-acid-grown cells required about 80 h, with the affinity reaching 317 liters g of cells⁻¹ h⁻¹.