Influence of easily degradable naturally occurring carbon substrates on biodegradation of monosubstituted phenols by aquatic bacteria

The influence of readily degradable, naturally occurring carbon substrates on the biodegradation of several monosubstitued phenols (m-cresol, m-aminophenol, p-chlorophenol) was examined. The natural substrate classes used were amino acids, carbohydrates, and fatty acids. Samples of the microbial community from Lake Michie, a mesotrophic reservoir, were adapted to different levels of representatives from each natural substrate class in chemostats. After an extended adaptation period, the ability of the microbial community to degrade the monosubstituted phenols was determined by using a radiolabeled substrate uptake and mineralization method. Several microbiological characteristics of the communities were also measured. Adaptation to increasing concentrations of amino acids, carbohydrates, or fatty acids enhanced the ability of the microbial community to degrade all three phenols. The stimulation was largest for m-cresol and m-aminophenol. The mechanism responsible for the enhancement of monosubstituted phenol metabolism was not clearly identified, but the observation that adaptation to amino acids also increased the biodegradation of glucose and, to a lesser extent, naphthalene suggests a general stimulation of microbial metabolism. This study demonstrates that prior exposure to labile, natural substrates can significantly enhance the ability of aquatic microbial communities to respond to xenobiotics.     

Effect of microbial concentration on biodegradation rates of phenols

Summary Biodegradation rates of 12 phenols were measured with respect to acclimated microbial biomass ranging from 2.3×10⁴ to 2.3×10⁸ cells/l. Rates ranged between 0.02 mg l^–1 day^–1 for 1.6 mg/lp-bromophenol exposed to 2.3×10⁴ cells/l and 1.41 mg l^–1 day^–1 for 3.2 mg/lp-methylphenol exposed to 2.3×10⁸ cells/l. Generally, rates for all phenols were first-order in substrate concentration and zero-order in biomass concentration. Bromophenol biodegradation was preceded by lag periods of varying lengths and to a small extent the rate was dependent on microbial biomass. Results from this study suggest chemical biodegradation generally exhibits pseudo-first-and occasionally, second-order kinetics.     

Influence of sludge adaptation on biodegradation of phenol

Summary The effect of sludge adaptation on biodegradation of phenol was studied. The adapted sludge was obtained in a fermenter by stepwise increase in the concentration of phenol. The microorganisms within sludge, adapted to the utilization of phenol, subsequently degraded phenol more rapidly (14 mg/l/day) than organisms within unadapted sludge (8.8 mg/l/day). When the phenol concentration was increased, the organisms within adapted sludge grew almost without change up to 500 mg/phenol/l while a sharp decline in growth was observed for organisms within the unadapted sludge. Microbial isolates in the adapted sludge differed in their phenol-degrading capacity showing that the adaptation ability to degrade phenol was dependent on the presence of specific microorganisms. Among the isolates,Pseudomonas sp. andArthrobacter sp. showed the higher degradation rates. However, they were inferior to that of the adapted sludge, suggesting the existence of a synergistic effect between the organisms present.

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Influence de l'adaptation de la boue sur la biodégradation du phénol

Résumé On étudie l'effet de l'adaptation de la boue sur la biodégradation du phénol. La boue adaptée est obtenue en fermenteur par l'augmentation étagée de la concentration en phénol. Les microorganismes dans la boue, adaptés à l'utilisation du phénol, dégradent par la suite plus rapidement le phénol (14 mg par litre par jour) que les microorganismes dans la boue non adaptée (8.8 mg par litre par jour). Quand on augmente la concentration en phénol, les microorganismes dans la boue adaptée croissent quasi sans modification jusqu'à 500 mg de phénol par litre, tandis qu'on observe une diminution remarquable de la croissance chez les microorganismes dans la boue non adaptée. Les souches microbiennes isolées à partir de la boue adaptée démontrent des capacités différentes de dégradation du phénol, ce qui montre que le capacité adaptée de dégrader le phénol dépend de la présence de microorganismes spécifiques. Parmi les isolats,Pseudomonas sp. etArthrobacter sp. démontrent les vitesses de dégradation les plus élevées. Toutefois, celles-ci sont inférieures à celles de la boue adaptée, ce qui suggère l'existence d'un effet synergique entre les microorganismes présents.

     

Influence of inorganic and organic nutrients on aerobic biodegradation and on the adaptation response of subsurface microbial communities.

The influence of inorganic and organic amendments on the mineralization of ethylene dibromide, p-nitrophenol, phenol, and toluene was examined in subsurface soil samples from a pristine aquifer near Lula, Okla. The responses indicate that the metabolic abilities and nutrient requirements of groundwater microorganisms vary substantially within an aquifer. In some samples, additions of inorganic nutrients resulted in a more rapid adaptation to the test substrate and a higher rate of metabolism, indicating that metabolism may have been limited by these nutrients. In other samples from the same aquifer layer, inorganic amendments had little or no influence on mineralization. In general, the addition of multiple inorganic nutrients resulted in a greater enhancement of degradation than did the addition of single substances. Additions of alternate carbon sources, such as glucose or amino acids, inhibited the mineralization of the xenobiotic substrates. This inhibition appears to be the result of the preferential utilization of the more easily degradable carbon amendments.     

Adaptation of aquatic microbial communities to hg stress

The mechanism of adaptation to Hg in four aquatic habitats was studied by correlating microbially mediated Hg volatilization with the adaptive state of the exposed communities. Community diversity, heterotrophic activity, and Hg resistance measurements indicated that adaptation of all four communities was stimulated by preexposure to Hg. In saline water communities, adaptation was associated with rapid volatilization after an initial lag period. This mechanism, however, did not promote adaptation in a freshwater sample, in which Hg was volatilized slowly, regardless of the resistance level of the microbial community. Distribution of the mer operon among representative colonies of the communities was not related to adaptation to Hg. Thus, although volatilization enabled some microbial communities to sustain their functions in Hg-stressed environments, it was not mediated by the genes that serve as a model system in molecular studies of bacterial resistance to mercurials.     

Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer.

The ability of subsurface microbial communities to adapt to the biodegradation of xenobiotic compounds was examined in aquifer solids samples from a pristine aquifer. An increase in the rates of mineralization of radiolabeled substrates with exposure was used as an indication of adaptation. For some compounds, such as chlorobenzene and 1,2,4-trichlorobenzene, slight mineralization was observed but no adaptation was apparent during incubations of over 8 months. Other compounds demonstrated three patterns of response. For m-cresol, m-aminophenol, and aniline intermediate rates of biodegradation and a linear increase in the percent mineralized with time were observed. Phenol, p-chlorophenol, and ethylene dibromide were rapidly metabolized initially, with a nonlinear increase in the percent mineralized with time, indicating that the community was already adapted to the biodegradation of these compounds. Only p-nitrophenol demonstrated a typical adaptation response. In different samples of soil from the same layer in the aquifer, the adaptation period to p-nitrophenol varied from a few days to as long as 6 weeks. In most cases the concentration of xenobiotic added, over the range from a few nanograms to micrograms per gram, made no difference in the response. Most-probable-number counts demonstrated that adaptation is accompanied by an increase in specific degrader numbers. This study has shown that diverse patterns of response occur in the subsurface microbial community.     

Influence of inorganic and organic nutrients on aerobic biodegradation and on the adaptation response of subsurface microbial communities

The influence of inorganic and organic amendments on the mineralization of ethylene dibromide, p-nitrophenol, phenol, and toluene was examined in subsurface soil samples from a pristine aquifer near Lula, Okla. The responses indicate that the metabolic abilities and nutrient requirements of groundwater microorganisms vary substantially within an aquifer. In some samples, additions of inorganic nutrients resulted in a more rapid adaptation to the test substrate and a higher rate of metabolism, indicating that metabolism may have been limited by these nutrients. In other samples from the same aquifer layer, inorganic amendments had little or no influence on mineralization. In general, the addition of multiple inorganic nutrients resulted in a greater enhancement of degradation than did the addition of single substances. Additions of alternate carbon sources, such as glucose or amino acids, inhibited the mineralization of the xenobiotic substrates. This inhibition appears to be the result of the preferential utilization of the more easily degradable carbon amendments.     

Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer

The ability of subsurface microbial communities to adapt to the biodegradation of xenobiotic compounds was examined in aquifer solids samples from a pristine aquifer. An increase in the rates of mineralization of radiolabeled substrates with exposure was used as an indication of adaptation. For some compounds, such as chlorobenzene and 1,2,4-trichlorobenzene, slight mineralization was observed but no adaptation was apparent during incubations of over 8 months. Other compounds demonstrated three patterns of response. For m-cresol, m-aminophenol, and aniline intermediate rates of biodegradation and a linear increase in the percent mineralized with time were observed. Phenol, p-chlorophenol, and ethylene dibromide were rapidly metabolized initially, with a nonlinear increase in the percent mineralized with time, indicating that the community was already adapted to the biodegradation of these compounds. Only p-nitrophenol demonstrated a typical adaptation response. In different samples of soil from the same layer in the aquifer, the adaptation period to p-nitrophenol varied from a few days to as long as 6 weeks. In most cases the concentration of xenobiotic added, over the range from a few nanograms to micrograms per gram, made no difference in the response. Most-probable-number counts demonstrated that adaptation is accompanied by an increase in specific degrader numbers. This study has shown that diverse patterns of response occur in the subsurface microbial community.     

The significance of viruses to mortality in aquatic microbial communities

A variety of approaches including enumeration of visibly infected microbes, removal of viral particles, decay of viral infectivity, and measurements of viral production rates have been used to infer the impact of viruses on microbial mortality. The results are surprisingly consistent and suggest that, on average, about 20% of marine heterotrophic bacteria are infected by viruses and 10–20% of the bacterial community is lysed daily by viruses. The effect of viruses on phytoplankton is less certain, but ca. 3% of Synechococcus biomass may be lysed daily. The fraction of primary productivity this represents depends upon the relative biomass and growth rate of Synechococcus. Virus enrichment experiments suggest that the productivity of eukaryotic phytoplankton would be ca. 2% higher in the absence of viruses. Overall, probably about 2–3% of primary productivity is lost to viral lysis. There is considerable variation about these estimates; however, they represent a starting point for incorporating viral-mediated processes into aquatic ecosystem models.     

Metabolic efficiency and turnover of soil microbial communities in biodegradation tests.

Biodegradability screening tests of soil commonly measure 14CO2 evolution from radiolabeled test compounds, and glucose has often served as a positive control. When constant amounts of radiolabel were added to soil in combination with increasing amounts of unlabeled substrates, glucose and some related hexoses behaved in an anomalous manner. In contrast to that of formate, benzoate, n-hexadecane, or bis(2-ethylhexyl) phthalate, dilution of glucose radiocarbon with unlabeled glucose increased rather than decreased the rate and extent of 14CO2 evolution. [14C]glucose incorporation into biomass and Vmax values were consistent with the interpretation that application of relatively high concentrations of glucose to soil shifts the balance of the soil microbial community from the autochthonous (humus-degrading) to the zymogeneous (opportunistic) segment. The higher growth and turnover rates that define zymogeneous microorganisms, combined with a lower level of carbon incorporation into their biomass, result in the evolution of disproportionate percentages of 14CO2. When used as positive controls, glucose and related hexoses may raise the expectations for percent 14CO2 evolution to levels that are not realistic for other biodegradable compounds.     

Effects of endogenous substrates on adaptation of anaerobic microbial communities to 3-chlorobenzoate

Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.     

Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities.

Microbial communities in water from Baltimore Harbor and from the mainstem of Chesapeake Bay were examined for sensitivity to mercuric chloride, monomethyl mercury, stannic chloride, and tributyltin chloride. Acute toxicity was determined by measuring the effects of [3H]thymidine incorporation, [14C]glutamate incorporation and respiration, and viability as compared with those of controls. Minimum inhibitory concentrations were low for all metals (monomethyl mercury, less than 0.05 microgram liter-1; mercuric chloride, less than 1 microgram liter-1; tributyltin chloride, less than 5 micrograms liter-1) except stannic chloride (5 mg liter-1). In some cases, mercuric chloride and monomethyl mercury were equally toxic at comparable concentrations. The Chesapeake Bay community appeared to be slightly more sensitive to metal stress than the Baltimore Harbor community, but this was not true for all treatments or assays. For culturable bacteria the opposite result was found. Thymidine incorporation and glutamate metabolism were much more sensitive indicators of metal toxicity than was viability. To our knowledge, this is the first use of the thymidine incorporation method for ecotoxicology studies. We found it the easiest and fastest of the three methods; it is at least equal in sensitivity to metabolic measurements, and it likely measures the effects on greater portion of the natural community.     

Response of water column microbial communities to sudden exposure to deltamethrin in aquatic mesocosms

Sudden exposure of an aquatic system to an insecticide can have significant effects on populations other than susceptible organisms. Although this is intuitively obvious, little is actually known about how such exposure might affect bacterial communities and their relative metabolic activity in ecosystems. Here, we assessed small sub-unit (ssu)-RNA levels in open and shaded 9 m(3) aquatic mesocosms (16 units - 2 x 2 factorial design in quadruplicate) to examine the effects of sudden addition of deltamethrin to the units. When deltamethrin was added, a cascade of bacterial then phytoplankton "blooms" occurred over time. The bacterial bloom, which most likely included organisms from the plastid/cyanobacterial phylogenetic guild, was almost immediate (within hours), whereas the phytoplankton (algal) bloom lagged by about 4 days. This sequential response can be explained by an apparent sudden release of nutrients consequent to arthropod death that triggered a series of responses in the microbial loop. Interestingly, bacterial blooms were noted in both open and shaded mesocosms, whereas the algal bloom was only seen in open units, suggesting that both deltamethrin addition (and presumptive nutrient release) and an adequate light supply was required for the phytoplankton response. Overall, this work shows that microbial activities as reflected by ssu-rRNA levels can respond dramatically via apparently indirect effects following insecticide application.     

Use of 16S-rRNA to investigate microbial population dynamics during biodegradation of toluene and phenol by a binary culture

Interspecies interactions and changes in the rate and extent of biodegradation in mixed culture-mixed substrate studies were investigated. A binary mixed culture of Pseudomonas putida F1 and Burkholderia sp. JS150 degraded toluene, phenol, and their mixture. Both toluene and phenol can serve as sole sources of carbon and energy for both P. putida F1 and strain JS150. To investigate the population dynamics of this system, a fluorescent in-situ hybridization method was chosen because of its ability to produce quantitative data, its low standard error, and the ease of use of this method. When the binary mixed culture was grown on toluene or phenol alone, significant interactions between the species were observed. These interactions could not be explained by a pure-and-simple competition model and were substrate dependent. Strain JS150 growth was slightly inhibited when grown with P. putida F1 on phenol, and P. putida F1 grew more rapidly than expected. Conversely, when the two species were grown together on toluene alone, P. putida F1 was inhibited while strain JS150 was unaffected. During growth of the mixed culture on a combination of toluene and phenol, the interactions were similar to that observed during growth on phenol alone; P. putida F1 growth was enhanced while strain JS150 was unaffected. Because of the observed interspecies interactions, monoculture kinetic parameters were not sufficient to describe the mixed culture kinetics in any experiment. This is one of the first reports of microbial population dynamics in which molecular microbial ecology and mathematical modeling have been combined. The use of the 16S-rRNA-based method allowed for observation and understanding of interspecies interactions that were not observable with standard culture-based methods. These results suggest the need for more investigations that account for both substrate and microbial interactions when predicting the fate of organic pollutants in real systems.     

Intensification of phenol biodegradation by humic substances

Phenol biodegradation was carried out in a batch system by the bacterial strain Cupriavidus metallidurans in the presence of potassium humate that was prepared by alkaline extraction from oxyhumolite. The experiments were focused on the assessment of the humate effect on biodegradation activity of the tested bacterial strain. The achieved results demonstrated that the humate has a positive influence on the biodegradation of phenol and reduces the incubation time necessary for phenol removal. Higher biodegradation rate and more intensive growth were observed during the cultivation in presence of humate in comparison to the cultivation without its addition. Adsorption of the humate on bacterial biomass was observed as well. Subsequently, a phenol biodegradation testing in a continuous-flow system using a biofilm reactor was also carried out. Although the reactor was inoculated by C. metallidurans only, the microbial composition under an aerobic non-aseptic condition during this long-term cultivation changed. The phenol removal efficiency obtained in the biofilm reactor was higher than 92% when phenol concentration in a treated medium was 1200 mg l⁻¹.     

Effects of glucose on phenol biodegradation by heterogeneous populations

The effect of the presence of more easily degradable alternative carbon sources on the biodegradation of toxic waste components is of great practical importance. In this work, a mixed phenol/glucose waste was fed to two heterogeneous populations acclimated to different conditions: one was acclimated to phenol as a sole source of carbon and one to a mixed phenol/glucose substrate. Batch substrate utilization experiments were performed under both growth and nonproliferating (no medium nitrogen source) conditions in order to assess substrate removal patterns at the levels of enzyme production and enzyme function. The results indicated that the substrate removal pattern exhibited by the cells was significantly influenced by the acclimation characteristics of the culture. The phenol acclimated cells showed an initial preference for phenol, but the presence of glucose hindered phenol removal rate under both growth and nonproliferating conditions. The cells acclimated to the mixed phenol/glucose waste demonstrated rapid initial glucose removal with a slower concomitant utilization of phenol; acclimation to the mixed waste evidently had a significant impact on the substrate removal pattern for this mixed substrate system.     

Effects of invasive aquatic carrion on soil chemistry and terrestrial microbial communities

Carrion plays a crucial role in the recycling of nutrients and organic matter in ecosystems. Yet, despite their ecological importance, studies addressing the relevance of carrion originated from invasive alien species (IAS) in the interface between aquatic and terrestrial ecosystems are uncommon, especially those assessing belowground effects. In this study, we carried out a manipulative experiment to assess the impact of massive mortalities of the Asian clam Corbicula fluminea (Müller, 1774) as a carrion subsidy evaluating possible effects on the terrestrial soil chemistry and the structure of a microbial (bacteria and fungi) community. We placed five levels of C. fluminea density (0, 100, 500, 1000 and 2000 ind. m^?2) and samples were collected 7, 30 and 90 days after clams’ addition. The results revealed that C. fluminea carrion have a significant effect belowground, especially on nutrients content (mainly NH₄ ⁺, NO₂ ^?, NO₃ ^? and PO₄ ^3?), fungal biomass and fungal and bacterial diversity. Given the predicted increase and intensification of extreme climatic events and the widespread distribution of several aquatic IAS (including bivalve species such as C. fluminea) the ecological importance of these massive mortalities (and resulting carrion) cannot be ignored because they may affect microbial communities with significant impacts on nutrient cycling, even in adjacent terrestrial habitats.     

Influence of gaseous VOC concentration on the diversity and biodegradation performance of microbial communities

In this work, the influence of toluene gas concentration on the isolation of toluene degrading microbial communities from activated sludge was studied. Toluene biodegradation at gas phase concentration of 10?g?m^?3 (R1) resulted in process instability with removal efficiencies (RE) lesser than 33?%, while operation at toluene gas phase concentrations of 300?mg?m^?3 (R2) and 11?mg?m^?3 (R3) was stable with RE ranging from 74 to 94?%. The consortium isolated in R1 exhibited the highest tolerance toward toluene but the lowest biodegradation performance at trace level VOC concentrations. Despite R2 and R3 showed a similar sensitivity toward toluene toxicity, the microbial community from R2 supported the most efficient toluene biodegradation at trace level VOC concentrations. The Shannon-Wiener index showed an initial biodiversity decrease from 3.2 to 2.0, 1.9 and 2.7 in R1, R2 and R3, respectively. However, while R2 and R3 were able to recover their initial diversity levels by day 48, this loss in diversity was permanent in R1. These results showed that traditional inoculum isolation/acclimation techniques based on the exposure of the inoculum to high VOC concentrations, where toxicity tolerance plays a key role, may result in a poor abatement performance when the off-gas stream is diluted.     

Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities

Microbial communities in water from Baltimore Harbor and from the mainstem of Chesapeake Bay were examined for sensitivity to mercuric chloride, monomethyl mercury, stannic chloride, and tributyltin chloride. Acute toxicity was determined by measuring the effects of [3H]thymidine incorporation, [14C]glutamate incorporation and respiration, and viability as compared with those of controls. Minimum inhibitory concentrations were low for all metals (monomethyl mercury, less than 0.05 microgram liter-1; mercuric chloride, less than 1 microgram liter-1; tributyltin chloride, less than 5 micrograms liter-1) except stannic chloride (5 mg liter-1). In some cases, mercuric chloride and monomethyl mercury were equally toxic at comparable concentrations. The Chesapeake Bay community appeared to be slightly more sensitive to metal stress than the Baltimore Harbor community, but this was not true for all treatments or assays. For culturable bacteria the opposite result was found. Thymidine incorporation and glutamate metabolism were much more sensitive indicators of metal toxicity than was viability. To our knowledge, this is the first use of the thymidine incorporation method for ecotoxicology studies. We found it the easiest and fastest of the three methods; it is at least equal in sensitivity to metabolic measurements, and it likely measures the effects on greater portion of the natural community.