Relative quantitative PCR to assess bacterial community dynamics during biodegradation of diesel and biodiesel fuels under various aeration conditions

The degradation of diesel fuel, B20 blend and biodiesel in liquid cultures by a seven-member bacterial consortium was compared under conditions with full aeration or with limited aeration with nitrate added as main electron acceptor. Community dynamics was assessed employing real-time PCR and the ddCt method for relative quantification. Biodegradation rates increased with increasing biodiesel content, but were significantly reduced under conditions with nitrate. Despite large variations in biodegradation rates, magnitude changes in population numbers were typically observed only from zero to one order, regardless the type of fuel and electron acceptor. Only Comamonadaceae and Variovorax sp. distinctly preferred aerobic conditions, and during aerobic growth showed suppression as fuel contained more biodiesel. Thus, the consortium is relatively stable and most of the degraders can shift their metabolism from hydrocarbons to biodiesel. The stability of the consortium is of interest in the context of biodiesel-mediated biodegradation of petroleum hydrocarbons.     

DNA repair-deficient Chinese hamster ovary cells exhibiting differential sensitivity to gamma rays under aerobic and hypoxic conditions

The survival of the wild-type parent and two mutant lines of Chinese hamster cells, known to be defective in DNA repair, has been determined as a function of exposure to gamma rays under aerobic and hypoxic conditions. When compared to the wild-type line, one of the mutants selected for sensitivity to ethyl methyl sulfonate (EMS), and known to be defective in the repair of DNA strand breaks, exhibits a markedly enhanced sensitivity to aerobic irradiation but a reduced enhancement to hypoxic irradiation and thus an enhanced oxygen enhancement ratio (OER). In contrast, the other line, known to be defective in the incision step of excision repair, exhibits the reverse pattern of sensitivity and hence a reduced OER. The results are compared to findings in bacterial mutants and cells obtained from ataxia telangiectasia (AT) patients and heterozygotes.     

Production of Cold-Flow Quality Biodiesel from High-Acidity On-Edible Oils—Esterification and Transesterification of Macauba (<Emphasis Type="Italic">Acrocomia aculeata</Emphasis>) Oil Using Various Alcohols

Biodiesel is an alternative fuel that has been used for partial or total substitution of diesel to reduce its environmental impacts. Prior studies on this topic have focused on the quest for better synthesis process, new catalysts and low-cost non-food and raw materials to improve the economic and sustainable production as well as product quality. In this study, acidic oil from macauba, a palm tree native to South America that has no food uses, was converted into biodiesel. The esterification and transesterification reactions were performed with methanol, ethanol and isobutanol with the goal of improving the cold properties of the biodiesel. The isobutyl ester exhibited the lowest freezing point temperature but underperformed outside of international specifications for kinematic viscosity; it also exhibited a low ester content. The methyl and ethyl esters were within the specifications of the international standards for ester content, density, kinematic viscosity and sulphur content. The ethyl ester produced from macauba oil displayed better properties in cold conditions than methyl and isobutyl esters studied here, with a cold filter plugging point of 0 °C. Its onset crystallisation temperature was reduced from ?5.96 to ?13.41 °C when subjected to fractional crystallisation. The ethyl ester exhibited the best lubricity value among the other esters studied.     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

Influence of substrate surface loading on the kinetic behaviour of aerobic granules

In the aerobic granular sludge reactor, the substrate loading is related to the size of the aerobic granules cultivated. This study investigated the influence of substrate surface loading on the growth and substrate-utilization kinetics of aerobic granules. Results showed that microbial surface growth rate and surface biodegradation rate are fairly related to the substrate surface loading by the Monod-type equation. In this study, both the theoretical maximum growth yield and the Pirt maintenance coefficient were determined. It was found that the estimated theoretical maximum growth yield of aerobic granules was as low as 0.2 g biomass g^–1 chemical oxygen demand (COD) and 10–40% of input substrate-COD was consumed through the maintenance metabolism, while experimental results further showed that the unit oxygen uptake by aerobic granules was 0.68 g oxygen g^–1 COD, which was much higher than that reported in activated sludge processes. Based on the growth yield and unit oxygen uptake determined, an oxidative assimilation equation of acetate-fed aerobic granules was derived; and this was confirmed by respirometric tests. In aerobic granular culture, about 74% of the input substrate-carbon was converted to carbon dioxide. The growth yield of aerobic granules was three times lower than that of activated sludge. It is likely that high carbon dioxide production is the main cause of the low growth yield of aerobic granules, indicating a possible energy uncoupling in aerobic granular culture.     

Biocalorimetric and respirometric studies on biological treatment of tannery saline wastewater

This study is focused on the biodegradation of saline tannery effluent by identified halotolerant bacterial consortia in a bench-scale reaction calorimeter. A satisfactory agreement was observed between oxygen uptake rate profiles and heat flux–time curves confirming that, under strict aerobic conditions, calorimetry and respirometry provided the same information. Oxycalorific equivalent determined from our experiments was found to agree well to the theoretical value. A linear relationship was observed between chemical oxygen demand consumption and total metabolic heat production. The study confirmed that the heat release profiles could be used as an indirect parameter for online monitoring of the degradation process. The effect of salt (NaCl) inhibition on acetate and ammonia uptake by the halobacterial consortia is also reported in this paper.     

Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders: effect of the type of blend and the addition of biosurfactants

Biodegradation experiments for diesel/biodiesel blends in liquid cultures by-petroleum degrading microbial consortium showed that for low amendments of biodiesel (10%) the overall biodegradation efficiency of the mixture after seven days was lower than for petroleum diesel fuel. Preferential usage of methyl esters in the broad biodiesel concentration range and diminished biodegradation of petroleum hydrocarbons for 10% biodiesel blend was confirmed. Rhamnolipids improved biodegradation efficiency only for blends with low content of biodiesel. Emulsion formation experiments showed that biodiesel amendments significantly affected dispersion of fuel mixtures in water. The presence of rhamnolipids biosurfactant affected stability of such emulsions and altered cell surface properties of tested consortium.     

Lipase-catalyzed methanolysis of palm oil in presence and absence of organic solvent for production of biodiesel

Enzymatic production of methyl esters (biodiesel) by methanolysis of palm oil in presence and absence of organic solvent was investigated using Candida antarctica lipase immobilized on acrylic resin as a biocatalyst. Although, at least molar equivalent of methanol (methanol-palm oil ratio 3:1) is required for the complete conversion of palm oil to methyl esters, lipase catalyzed methanolysis of palm oil in absence of organic solvent was poisoned by adding more than 1/3 molar equivalent of methanol. The use of polar organic solvents prevented the lipase to be poisoned in methanolysis with a molar equivalent of methanol, and tetrahydrofuran (THF) was found to be the most effective. The presence of water in methanolysis of palm oil both in presence and absence of THF inhibited the reaction rate but this inhibition was considerably low in THF containing system. The palm oil-lipase (w/w) ratio significantly influenced the activity of lipase and the optimal ratio in presence and absence of THF was 100 and 50, respectively.     

Review of MTBE Biodegradation and Bioremediation

Conclusive evidence of methyl tert-butyl ether (MTBE) biotransformation and complete mineralization under aerobic conditions in environmental samples and enrichment cultures is reviewed, in addition to increasing evidence of MTBE biotransformation under anaerobic conditions. The metabolic pathway of MTBE appears to have two key intermediates, tert-butyl alcohol (TBA) and 2-hydroxy isobutyric acid (HIBA). The first enzyme in MTBE biodegradation has been identified as either a cytochrome P450 or a nonhemic monooxygenase in different isolates. Mixed and pure cultures of microorganisms have utilized MTBE as a sole carbon and energy source. Cometabolism of MTBE with n-alkanes at rates of 3.9 to 52 nmol/min/mg protein has been documented. The presence of co-contaminants such as BTEX has either not affected or seemed to limit MTBE biodegradation. Some studies of MTBE natural attenuation have attributed mass loss to biodegradation, while others have attributed mass loss to dilution and dispersion. Recent advances in the assessment of MTBE biodegradation have indicated the potential for natural anaerobic transformation of MTBE. In situ bioremediation of MTBE has been enhanced by adding air or oxygen, or by adding microorganisms and air or oxygen. Bioreactors have attained significant removal of MTBE from MTBE-contaminated influent. Despite historical concerns about the biodegradability of MTBE, several biological methods can now be used for MTBE remediation.     

Dissimilatory Nitrate and Nitrite Reduction under Aerobic Conditions by an Aerobically and Anaerobically Grown Alcaligenes sp. and by Activated Sludge

The oxygen uptake of an Alcaligenes sp., isolated from activated sludge, was inhibited by small amounts of nitric oxide. The occurrence of this inhibition was dependent on the growth conditions and the pretreatment of the cells. Anaerobically grown cells, which had subsequently been aerated in a nitrogen-free medium, accumulated nitric oxide, after the addition of nitrate or nitrite. When the oxygen uptake was inhibited by nitric oxide, dissimilatory reduction of nitrate and nitrite proceeded under aerobic conditions at the same rate as in the absence of oxygen. Activated sludge removed nitric oxide actively under aerobic conditions and as a consequence the oxygen uptake of the sludge was not inhibited in the presence of nitrite. The rate of nitrate reduction under aerobic conditions was about 20% of that in the absence of oxygen.     

Influence of Low Oxygen Tensions and Sorption to Sediment Black Carbon on Biodegradation of Pyrene

Sorption to sediment black carbon (BC) may limit the aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in resuspension events and intact sediment beds. We examined this hypothesis experimentally under conditions that were realistic in terms of oxygen concentrations and BC content. A new method, based on synchronous fluorescence observations of ¹⁴C-pyrene, was developed for continuously measuring the uptake of dissolved pyrene by Mycobacterium gilvum VM552, a representative degrader of PAHs. The effect of oxygen and pyrene concentrations on pyrene uptake followed Michaelis-Menten kinetics, resulting in a dissolved oxygen half-saturation constant (K_om) of 14.1 μM and a dissolved pyrene half-saturation constant (K_pm) of 6 nM. The fluorescence of ¹⁴C-pyrene in air-saturated suspensions of sediments and induced cells followed time courses that reflected simultaneous desorption and biodegradation of pyrene, ultimately causing a quasi-steady-state concentration of dissolved pyrene balancing desorptive inputs and biodegradation removals. The increasing concentrations of ¹⁴CO₂ in these suspensions, as determined with liquid scintillation, evidenced the strong impact of sorption to BC-rich sediments on the biodegradation rate. Using the best-fit parameter values, we integrated oxygen and sorption effects and showed that oxygen tensions far below saturation levels in water are sufficient to enable significant decreases in the steady-state concentrations of aqueous-phase pyrene. These findings may be relevant for bioaccumulation scenarios that consider the effect of sediment resuspension events on exposure to water column and sediment pore water, as well as the direct uptake of PAHs from sediments.The aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) constitutes one of the main processes for dissipation of these toxic compounds from polluted soils and sediments. The oxygen dependence of this process has long sustained the belief that the anaerobic conditions usually found in environments such as sediments in estuaries and ports are the main cause of a long persistence of PAH pollution. However, recent findings have demonstrated that microorganisms can also use other electron acceptors, such as nitrate and sulfate, to oxidize PAHs in sediments (30, 33). Less attention has been given to aerobic biodegradation operating at low oxygen tensions. This process may also be important at the interface between anoxic sediments and the overlying waters. Resuspension of PAH-polluted anoxic sediments can result in the exposure of sediment particles to low concentrations of oxygen in the immediately overlying water column, thereby promoting the aerobic biodegradation of PAHs (22) under conditions in which they can be desorbed and taken up by competent microorganisms. The resulting decreases in the aqueous-phase concentration (and in the associated chemical activity) caused by oxygen-limited aerobic biodegradation may be relevant for bioaccumulation scenarios that consider exposure to the water column and sediment pore water, in addition to the direct uptake from sediments (23). Despite its significance, the capacity for prediction of aerobic biodegradation rates of PAHs at low oxygen tensions is still very limited. Whereas the oxygen dependence of fast biodegradation of PAHs in soils and sediments is a well-known phenomenon (5, 17), studies reporting precise measurements of the dissolved oxygen half-saturation constant (K_om) for biodegradation of PAHs—a key modeling parameter—are very scarce (7, 21). The only available estimation for a K_om value of a high-molecular-weight (HMW) PAH (5.9 μM) was provided for pyrene on the basis of growth rates on pyrene of a Mycobacterium strain in a fermenter (7). However, the pyrene concentration chosen (500 mg/liter)—well above the level of its aqueous solubility (0.13 mg/liter) known to support bacterial growth (34)—was not representative of those concentrations present in the environment.Besides the oxygen concentration, another factor that may control the biodegradation of sedimentary PAHs is their bioavailability. Due to their partitioning into sorbents, such chemicals exhibit only weak chemical activity gradients that promote their uptake and transformation by active microbial cells. Hence, the biodegradation rates are likely far below those corresponding to maximum rates, and they may reflect nonlinear biochemical dependencies. Also, these low rates may be due to the lower chemical activity of PAHs causing the microbial acquisition of the aqueous-phase chemicals to become a bottleneck for the biodegradation process (31). Examples of conflicts of bioavailability with biodegradation can be found when PAHs are predominantly sorbed onto solid aggregates (12) and dissolved in non-aqueous-phase liquids (28). Sorption is especially important in sediments. During recent years, the traditional, one-phase organic carbon (OC) partitioning model has been expanded for PAHs and other hydrophobic pollutants to include uptake both into OC and onto the ubiquitous, solid-phase products of incomplete combustion, collectively called black carbon (BC). Therefore, adsorption to BC and absorption to OC would occur in parallel during the sorption process (1, 2, 15). The new model has been useful in understanding field observations of the PAH solid-water distribution coefficient (K_d), which have evidenced a higher sorption capacity than would have been expected on the basis of OC content only (25, 26). Several studies have shown that strong sorption of PAHs to BC may also significantly limit biodegradation. For example, Ghosh et al. showed that 16 U.S. Environmental Protection Agency (USEPA) PAHs associated with carbonaceous coal-derived material present in harbor sediments exhibited negligible biodegradation rates in aerobic sediment slurries, whereas similar conditions led to significant losses (up to 75% after 2 months) of PAHs present in semisolid coal tar pitch (10). Little or no biodegradation was also observed for 3- to 6-ringed PAHs associated with BC-rich street dust added to soils to simulate diffuse pollution (18) and with naphthalene sorbed to granular activated carbon, a material similar to BC in its physicochemical characteristics, in suspensions of two different bacterial species with dissimilar modes of acquisition of the sorbed compound (14). Finally, Rhodes et al. examined the effect of BC on bioavailability of phenanthrene in soils (32). They found that the addition of BC to soils caused a significant decrease both in the total extent of mineralization and in extractability by the use of cyclodextrin solutions (32). Despite these advances in the field, it is still uncertain whether sorption to BC causes the sequestration of PAHs or whether their microbial assimilation is still possible, although at a very low rate. This gap in knowledge is a major limitation in predicting the fate of these chemicals in many contaminated sediments, making it difficult to achieve a proper perception of the risks posed by resuspensions in overlying waters and bioturbated sediment beds.We considered that sorption to sediment BC may limit the aerobic biodegradation of aqueous-phase PAHs such as pyrene and examined this hypothesis experimentally under conditions that were realistic in terms of concentrations of oxygen and suspended-solids typical for sediment resuspension events. For this aim, we developed a new method, based on synchronous fluorescence observations of ¹⁴C-pyrene, for both measuring the rates of uptake of dissolved pyrene at low oxygen concentrations by a representative PAH-degrading bacterial strain and simultaneously assessing the appearance of ¹⁴CO₂. The method also allowed us to characterize the evolution of aqueous-phase pyrene during biodegradation in initially equilibrated suspensions of sediment with a known content in black carbon. The information obtained experimentally was integrated in model calculations of the evolution of aqueous pyrene concentrations in sediment suspensions. To our knowledge, this is the first report connecting these two major factors—oxygen limitation and sorption to BC—in the biodegradation of HMW PAHs.     

Review of MTBE Biodegradation and Bioremediation

Conclusive evidence of methyl tert-butyl ether (MTBE) biotransformation and complete mineralization under aerobic conditions in environmental samples and enrichment cultures is reviewed, in addition to increasing evidence of MTBE biotransformation under anaerobic conditions. The metabolic pathway of MTBE appears to have two key intermediates, tert-butyl alcohol (TBA) and 2-hydroxy isobutyric acid (HIBA). The first enzyme in MTBE biodegradation has been identified as either a cytochrome P450 or a nonhemic monooxygenase in different isolates. Mixed and pure cultures of microorganisms have utilized MTBE as a sole carbon and energy source. Cometabolism of MTBE with n-alkanes at rates of 3.9 to 52 nmol/min/mg protein has been documented. The presence of co-contaminants such as BTEX has either not affected or seemed to limit MTBE biodegradation. Some studies of MTBE natural attenuation have attributed mass loss to biodegradation, while others have attributed mass loss to dilution and dispersion. Recent advances in the assessment of MTBE biodegradation have indicated the potential for natural anaerobic transformation of MTBE. In situ bioremediation of MTBE has been enhanced by adding air or oxygen, or by adding microorganisms and air or oxygen. Bioreactors have attained significant removal of MTBE from MTBE-contaminated influent. Despite historical concerns about the biodegradability of MTBE, several biological methods can now be used for MTBE remediation.     

Comparison of anaerobic and aerobic biodegradation of mineralized skeletal structures in marine and estuarine conditions

The knowledge of the biodegradation rates is essential to studies of the biogeochemistry and ecology of aquatic systems. It helps us to quantify the production and uptake rates of chemical components and their recycling, and to understand the mechanisms and rates of organic matter accumulation in sediments. Experimental studies of biodegradation processes in six types of mineralized skeletons were performed in shallow-marine waters of Calvi Bay, Corsica and in estuarine waters of Roscoff, Brittany. Three types of mollusk shells, sea urchin skeletal plates, crab cuticle and fish vertebrae were exposed to oxic and anoxic conditions over periods of 15 days to 30 months. After recovery of the substrates, protein assays, bacterial counts and organic carbon analyses were performed.Quantitative protein assays and bacterial counts indicate that biodegradation of mineralized skeletal structures occurs at a slower rate in anoxic conditions than in oxic conditions. Bacterial analysis showed that in anoxic environment, less than 0.5% of the consumed organic matter is converted into bacterial biomass. The aerobic biodegradation rate was positively correlated with the organic content of the skeletons.Anoxic biodegradation of skeletons occurred at much slower rates in estuarine sediments than in shallow marine sediments. Preservation of skeletal structures in estuarine conditions appears to be correlated with the abundance of dissolved organic matter rather than with high sedimentation rates.     

A packed bed membrane reactor for production of biodiesel using activated carbon supported catalyst

In this study, a novel continuous reactor has been developed to produce high quality methyl esters (biodiesel) from palm oil. A microporous TiO₂/Al₂O₃ membrane was packed with potassium hydroxide catalyst supported on palm shell activated carbon. The central composite design (CCD) of response surface methodology (RSM) was employed to investigate the effects of reaction temperature, catalyst amount and cross flow circulation velocity on the production of biodiesel in the packed bed membrane reactor. The highest conversion of palm oil to biodiesel in the reactor was obtained at 70 °C employing 157.04 g catalyst per unit volume of the reactor and 0.21 cm/s cross flow circulation velocity. The physical and chemical properties of the produced biodiesel were determined and compared with the standard specifications. High quality palm oil biodiesel was produced by combination of heterogeneous alkali transesterification and separation processes in the packed bed membrane reactor.     

Lipase-catalysed production of biodiesel fuel from some Nigerian lauric oils

Fatty acids esters were produced from two Nigerian lauric oils, palm kernel oil and coconut oil, by transesterification of the oils with different alcohols using PS30 lipase as a catalyst. In the conversion of palm kernel oil to alkyl esters (biodiesel), ethanol gave the highest conversion of 72%, t-butanol 62%, 1-butanol 42%, n-propanol 42% and iso-propanol 24%, while only 15% methyl ester was observed with methanol. With coconut oil, 1-butanol and iso-butanol achieved 40% conversion, 1-propanol 16% and ethanol 35%, while only traces of methyl esters were observed using methanol. Studies on some fuel properties of palm kernel oil and its biodiesel showed that palm kernel oil had a viscosity of 32.40 mm2/s, a cloud point of 28 degrees C and a pour point of 22 degrees C, while its biodiesel fuel had a viscosity of 9.33 mm2/s, a cloud point of 12 degrees C and a pour point of 8 degrees C. Coconut oil had a viscosity of 28.58 mm(2)/s, a cloud point of 27 degrees C and a pour point of 20 degrees C, while its biodiesel fuel had a viscosity of 7.34 mm2/s, a cloud point of 5 degrees C and a pour point of -8 degrees C. Some of the fuel properties compared favourably with international biodiesel specifications.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Bioremediation strategies for diesel and biodiesel in oxisol from southern Brazil

Leaks and spillages during the extraction, transport and storage of petroleum and its derivatives may result in environmental contamination. Biodiesel is an alternative energy source that can contribute to a reduction in environmental pollution. The aim of the present work was to evaluate biodegradation of diesel, biodiesel, and a 20% biodiesel-diesel mixture in oxisols from southern Brazil, using two bioremediation strategies: natural attenuation and bioaugmentation/biostimulation. Fuel biodegradation was monitored over 60 days by dehydrogenase activity, CO₂ evolution and gas chromatography. The bacterial inoculum employed for bioaugmentation/biostimulation consisted of Bacillus megaterium, Bacillus pumilus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia and PCR-DGGE using 16S RNAr primers showed that some members of this consortium survived in the soil after 60 days. The biodegradation of pure biodiesel was higher for bioaugmentation/biostimulation than for natural attenuation, suggesting that the addition of the microbial consortium, together with adjustment of the macronutrient ratio, increased biodiesel degradation. The results of dehydrogenase and respiratory activity, together with GC analysis, suggested that the presence of biodiesel may, by stimulating general microbial degradative metabolism, increase the biodegradation of petroleum diesel. The microbial community was altered by both treatments, with natural attenuation producing a lower diversity index than the amended soil. The bioaugmentation/biostimulation strategy was showed to have a high potential for cleaning up soils contaminated with diesel and biodiesel blends.     

Degradation of toluene by a mixed population of archetypal aerobes, microaerophiles, and denitrifiers: laboratory sand column experiment and multispecies biofilm model formulation

An experiment was conducted in a saturated sand column with three bacterial strains that have different growth characteristics on toluene, Pseudomonas putida F1 which degrades toluene only under aerobic conditions, Thauera aromatica T1 which degrades toluene only under denitrifying conditions, and Ralstonia pickettii PKO1 has a facultative nature and can perform nitrate-enhanced biodegradation of toluene under hypoxic conditions (DO <2 mg/L). Steady-state concentration profiles showed that oxygen and nitrate appeared to be utilized simultaneously, regardless of the dissolved oxygen concentration and the results from fluorescent in-situ hybridization (FISH) indicated that PKO1 maintained stable cells numbers throughout the column, even when the pore water oxygen concentration was high. Since PKO1's growth rate under aerobic condition is much lower than that of F1, except under hypoxic conditions, these observations were not anticipated. Therefore these observations require a mechanistic explanation that can account for localized low oxygen concentrations under aerobic conditions. To simulate the observed dynamics, a multispecies biofilm model was implemented. This model formulation assumes the formation of a thin biofilm that is composed of the three bacterial strains. The individual strains grow in response to the substrate and electron acceptor flux from bulk fluid into the biofilm. The model was implemented such that internal changes in bacterial composition and substrate concentration can be simulated over time and space. The model simulations from oxic to denitrifying conditions compared well to the experimental profiles of the chemical species and the bacterial strains, indicating the importance of accounting for the biological activity of individual strains in biofilms that span different redox conditions.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Methyl tert-butyl ether (MTBE) degradation by a microbial consortium

The widespread use of methyl tert-butyl ether (MTBE) as a gasoline additive has resulted in a large number of cases of groundwater contamination. Bioremediation is often proposed as the most promising alternative after treatment. However, MTBE biodegradation appears to be quite different from the biodegradation of usual gasoline contaminants such as benzene, toluene, ethyl benzene and xylene (BTEX). In the present paper, the characteristics of a consortium degrading MTBE in liquid cultures are presented and discussed. MTBE degradation rate was fast and followed zero order kinetics when added at 100 mg l(-1). The residual MTBE concentration in batch degradation experiments ranged from below the detection limit (1 microg l(-1)) to 50 microg l(-1). The specific activity of the consortium ranged from 7 to 52 mgMTBE g(dw)(-1) h(-1) (i.e. 19-141 mgCOD g(dw) (-1) h(-1)). Radioisotope experiments showed that 79% of the carbon-MTBE was converted to carbon-carbon dioxide. The consortium was also capable of degrading a variety of hydrocarbons, including tert-butyl alcohol (TBA), tert-amyl methyl ether (TAME) and gasoline constituents such as benzene, toluene, ethylbenzene and xylene (BTEX). The consortium was also characterized by a very slow growth rate (0.1 d(-1)), a low overall biomass yield (0.11 gdw g(-1)MTBE; i.e. 0.040 gdw gCOD(-1)), a high affinity for MTBE and a low affinity for oxygen, which may be a reason for the slow or absence of MTBE biodegradation in situ. Still, the results presented here show promising perspectives for engineering the in situ bioremediation of MTBE.