Bjørn K. Jensen

Estimation of viable biomass in wastewater and activated sludge by determination of ATP, oxygen utilization rate and FDA hydrolysis

Abstract ATP content, oxygen utilization rate (OUR) and fluorescein diacetate (FDA) hydrolysis were tested for the ability to express the amount of viable biomass in wastewater and activated sludge. The relationship between biomass and these activity parameters was established in growth cultures made by inoculating a nutrient medium with either wastewater or activated sludge. Biomass was then determined directly by measurement of dry weight of growth culture (dw), and compared to data obtained by using the previously mentioned methods. In the exponential growth phase, ATP content showed the best correlation with biomass, while FDA hydrolysis in the sludge failed to show any such correlation. Conversion factors of 3 mg ATP/g dw, 300 mg O2/h g dw and 0.4 A/h (mg dw/ml) for ATP, OUR and FDA methods, respectively, were calculated. When the methods were applied for in situ determinations in four different wastewater plants, it was found that ATP content and respiration rate estimated viable biomass to range from 81 to 293 mg dw/g SS for raw wastewater and from 67 to 187 mg dw/g SS for activated sludge with a rather weak correlation between ATP and respiration measurements. The FDA hydrolysis estimated viable biomass to be higher than suspended solids, for which reason this parameter could not be recommended for determination of viable biomass in wastewater and activated sludge.

Microbial degradation of phenols and aromatic hydrocarbons in creosote-contaminated groundwater under nitrate-reducing conditions

Batch experiments were carried out to investigate the biodegradation of phenols and aromatic hydrocarbons under anaerobic, nitrate-reducing conditions in groundwater from a creosote-contaminated site at Fredensborg, Denmark. The bacteria in the creosote-contaminated groundwater degraded a mixture of toluene, phenol, the cresols (o-, m- and p-cresol) and the dimethylphenols 2,4-DMP and 3,4-DMP at both 10° and 20°C. Benzene, the xylenes, napthalene, 2,3-DMP, 2,5-DMP, 2,6-DMP and 3,5-DMP were resistant to biodegradation during 7–12 months of incubation. It was demonstrated that the degradation of toluene, 2,4-DMP, 3,4-DMP and p-cresol depended on nitrate or nitrite as electron acceptors. 40–80% of the nitrate consumed during degradation of the aromatic compounds was recovered as nitrite, and the consumption of nitrate was accompanied by a production of ATP. Stoichiometric calculations indicated that in addition to the phenols are toluene other carbon sources present in the groundwater contributed to the consumption of nitrate. If the groundwater was incubated under anaerobic conditions without nitrate, sulphate-reducing conditions evolved after ∼ 1 month at 20°C and ∼2 months at 10°C. In the sulphate-reducing batches disappearance of toluene, phenol, o-cresol and o-cresol was observed, whereas no removal of benzene, the xylenes, naphthalane, 2,3-DMP, 2,4-DMP, 2,5-DMP and 3,5-DMP was detected during 7 months of incubation.

Modelling TCE degradation by a mixed culture of methane-oxidizing bacteria

Abstract A model describing the growth of bacteria and the degradation of methane and trichloroethylene (TCE) based on the concept of competitive inhibition is proposed. The model has been applied to laboratory batch experiments representing different initial TCE concentrations (50–4300 μg/l) and initial methane concentrations (0.53–3.2 mg/l). The proposed model simulated successfully the data obtained for initial methane concentration (less than 1.8 mg/l), causing constant experimental growth conditions during the experiments. This indicates that the interactions between methane and TCE degradation can be explained as competitive inhibition. The model simulations of the results from the experiments with the highest initial methane concentration of 3.2 mg/l failed, supposedly because the growth conditions changed during the experiments. The proposed model is a useful engineering tool for design of treatment processes and in situ bioremediation schemes for degradation of TCE by methane-oxidizing bacteria.

Microbial adaptation to degradation of hydrocarbons in polluted and unpolluted groundwater

The microbial adaptation to degradation of aromatic hydrocarbons in heavily polluted, slightly polluted and unpolluted groundwater was examined. In addition, adaptations of bacteria from aquifers polluted by fuel oil and gasoline were compared. Finally, it was tested whether addition of bacteria attached to subsurface soil, originating from the same wells as the groundwater had any effect on the degradation. The compounds used as substrate were: toluene, o-xylene, 1,3,5-trimethylbenzene, naphthalene, 1-methylnaphthalene, biphenyl, 2-ethylnaphthalene, and 1,4-dimethylnaphthalene. Initial concentrations of each component were in the range of 100 μg L−1 to 240 μg L−1. A simple techniques was used. The lengths of the lag periods were used to express the degree of adaptation to degradation of the hydrocarbons. In one experiment equal volumes of groundwater from the three sites were used as inoculum. In another experiment equal microbial numbers in the bottles were ensured by adding different volumes of groundwater. The experiments demonstrate that the bacterial community in the heavily polluted well has a higher degree of adaptation to hydrocarbon degradation than the community from the slightly polluted well. The bacteria from the unpolluted well are considered as being unadapted, because of long lag periods compared to the bacteria in the polluted wells (7 to 34 times longer). The sequence in which the compounds were degraded after inoculation with fuel oil polluted or gasoline polluted groundwater is related to the occurrence of these compounds in the water soluble fractions of the two petroleum products. Addition of soil reduced the lag periods in the bottles inoculated with heavily polluted groundwater, while no or minor effects were observed, when added to the bottles inoculated with slightly or unpolluted groundwater. During the experimental periods an increase in ATP content was observed.

Biodegradation of nitrogen- and oxygen-containing aromatic compounds in groundwater from an oil-contaminated aquifer

Abstract The aerobic biodegradation of oxygen and nitrogen heterocycles and o-cresol by subsurface bacteria in groundwater from an oil contaminated site at Zealand, Denmark, was compared to the biodegradation of these compounds in laboratory adapted suspended and fixed-film cultures. The aquifer at the abstraction site had a relatively high redox potential, since it contained nitrate. The groundwater (i.e. without the soil phase) had a high biodegradation potential for dibenzofuran, indole, quinoline, flourenone and o-cresol. All the compounds were degraded in groundwater within 5–15 days from an initial concentration of about 0.5 mg L−1 in both mixed substrate and single substrate experiments with an initial ATP concentration of 0.2 ng mL−1. Pyrrole, however, was not degraded in groundwater within 55 days in the mixed substrate experiment and very slowly, after a lag period of 20 days, in the single substrate experiment. The biodegadability picture found for groundwater in the mixed substrate experiment was similar to the results found with laboratory adapted suspended and fixed-film cultures. None of the compounds had any inhibitory effect on the biodegradation of naphthalene.

Stoichiometry and kinetics of microbial toluene degradation under denitrifying conditions

Batch experiments were carried out to investigate the stoichiometry and kinetics of microbial degradation of toluene under denitrifying conditions. The inoculum originated from a mixture of sludges from sewage treatment plants with alternating nitrification and denitrification. The culture was able to degrade toluene under anaerobic conditions in the presence of nitrate, nitrite, nitric oxide, or nitrous oxide. No degradation occurred in the absence of Noxides. The culture was also able to use oxygen, but ferric iron could not be used as an electron acceptor. In experiments with14C-labeled toluene, 34%±8% of the carbon was incorporated into the biomass, while 53%±10% was recovered as14CO2, and 6%±2% remained in the medium as nonvolatile water soluble products. The average consumption of nitrate in experiments, where all the reduced nitrate was recovered as nitrite, was 1.3±0.2 mg of nitrate-N per mg of toluene. This nitrate reduction accounted for 70% of the electrons donated during the oxidation of toluene. When nitrate was reduced to nitrogen gas, the consumption was 0.7±0.2 mg per mg of toluene, accounting for 97% of the donated electrons. Since the ammonia concentration decreased during degradation, dissimilatory reduction of nitrate to ammonia was not the reductive process. The degradation of toluene was modelled by classical Monod kinetics. The maximum specific rate of degradation, k, was estimated to be 0.71 mg toluene per mg of protein per hour, and the Monod saturation constant, Ks, to be 0.2 mg toluene/l. The maximum specific growth rate, μmax, was estimated to be 0.1 per hour, and the yield coefficient, Y, was 0.14 mg protein per mg toluene.

Different abilities of eight mixed cultures of methane-oxidizing bacteria to degrade TCE

Abstract The ability of eight mixed cultures of methane-oxidizing bacteria to degrade trichloroethylene (TCE) was examined in laboratory batch experiments. This is one of the first reported works studying TCE degradation by mixed cultures of methane-oxidizing bacteria at 10°C, a common temperature for soils and groundwaters. Only three of the eight mixed cultures were able to degrade TCE, or to degrade TCE fast enough to result in a significant removal of TCE within the experimental time, when the cultures used methane as growth substrate. The same three mixed cultures were able to degrade TCE when they oxidized methanol, but only for a limited time period of about 5 days. Several explanations for the discontinued degradation of TCE are given. An experiment carried out to re-activate the methane-oxidizing bacteria after 8 days of growth on methanol by adding methane did not immediately result in degradation of methane and TCE. During the first 10–15 days after the addition of methane a significant degradation of methane and a minor degradation of TCE were observed. This experiment revealed that the ability of mixed cultures of methane-oxidizing bacteria to degrade TCE varied significantly even though the cultures were grown under the same conditions.

Transformation ofo-xylene too-methyl benzoic acid by a denitrifying enrichment culture using toluene as the primary substrate

A highly enriched denitrifying mixed culture transformedo-xylene cometabolically along with toluene by methyl group oxidation.o-Methyl benzaldehyde ando-methyl benzoic acid accumulated transiently as metabolic products ofo-xylene transformation. Transformation ofo-methyl benzyl alcohol ando-methyl benzaldehyde occurred independently of toluene degradation and resulted in the formation of a compound coeluting witho-methyl benzoic acid on a gas chromatograph. The cometabolic relationship between toluene ando-xylene could be attributed to a mechanism linked to the initial oxidation of the methyl group.

Microbial Properties Governing the Microbial Degradation of Polycyclic Aromatic Hydrocarbons

Twelve strains of PAH-degrading bacteria were isolated from soils sampled either at a coal-tar polluted site in Esbjerg Denmark, or at a fuel-oil polluted site in Tokol Hungary, by the spray-plate technique using phenanthrene as the substrate. The strains were characterized genetically by the Random Amplified Polymorphic DNA (RAPD) technique using the OPA15 and the OPA18 primers, and physiologically by the API 20NE test. When OPA15 was used as the primer, it was found that the strains isolated from the Esbjerg soils had almost identical DNA band patterns. When OPA18 was used, it was found that the strains had different DNA banding patterns showing that they were not reisolates. Physiologically the strains were quite similar when tested with the API 20NE test, but a cluster analysis divided the strains into two groups that corresponded to the sites where the soils were sampled. Four strains were characterized kinetically in fluid culture experiments with respect to growth rate and Monod half saturation constant. The growth rates were in the range between 0.02 per hour and 0.25 per hour, and the Monod half saturation constants were in the range between 5·10−6 g phenanthrene/L and 10−4 g phenanthrene/L. The genetic and physiological characterizations could not be correlated with the kinetic data, indicating that the RAPD analysis and the API 20NE test do not express microbial properties determining the rates of degradation. Two strains were used as inoculum in soil microcosm experiments. Inoculation had a profound effect on the degradation in the soil compared to uninoculated controls. In addition, the specific phenanthrene degradation rates were found similar to those obtained in the fluid culture experiments, indicating that kinetic properties of the bacteria were important for the degradation of phenanthrene in the soil microcosm experiments.

Changes in growth culture FDA activity under changing growth conditions

Abstract The FDA hydrolysis capacities and bacterial biomass concentrations (estimated by determination of ATP content) of growth cultures prepared from activated sludge and wastewater, were measured to find out whether the FDA activity would reflect bacterial biomass under different physiological states of the bacteria. The FDA activity/ATP ratio was calculated for different concentrations of autoclaved sludge. A faster decay rate of ATP relative to FDA hydrolysis activity was observed, thus causing changes in the ratio. Furthermore, comparison between values obtained from pure cultures and different soils revealed differences up to two orders of magnitude of the ratio. Based on these results it was concluded that the FDA activity should not be applied for measurements of viable biomass in environments in which different physiological conditions occur.