Gunnel Dalhammar

Degradation of polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-reducing conditions in enrichment cultures from northern soils.

ABSTRACT Thepotential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not wellunderstood, but such biodegradation would be very useful forremediation of polluted sites. Biodegradation of a mixture of 11different PAHs with two to five aromatic rings, each at a concentrationof 10 μg/ml, was studied in enrichment cultures inoculated withsamples of four northern soils. Under aerobic conditions, lowtemperature severely limited PAH biodegradation. After 90 days, aerobiccultures at 20°C removed 52 to 88% of the PAHs. The mostextensive PAH degradation under aerobic conditions at 7°C,53% removal, occurred in a culture from creosote-contaminatedsoil. Low temperature did not substantially limit PAH biodegradationunder nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene weredegraded. The most extensive PAH degradation under nitrate-reducingconditions at 7°C, 39% removal, occurred in a culturefrom fuel-contaminated Arctic soil. In separate transfer cultures fromthe above Arctic soil, incubated anaerobically at 7°C, removalof 2-methylnaphthalene and fluorene was stoichiometrically coupled tonitrate removal. Ribosomal intergenic spacer analysis suggested thatenrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, andVariovorax. Predominant populations from different soils oftenincluded phylotypes with nearly identical partial 16S rRNA genesequences (i.e., same genus) but never included phylotypes withidentical ribosomal intergenic spacers (i.e., different species orsubspecies). The composition of the enriched communities appeared to bemore affected by presence of oxygen, than by temperature or source oftheinoculum.

Development of nitrification inhibition assays using pure cultures of Nitrosomonas and Nitrobacter

Restricted requirements for nitrogen reduction at wastewater treatment plants have increased the need for assays determining the inhibition of nitrification. In this paper, two new assays studying ammonia oxidation and nitrite oxidation, respectively, are presented. As test organisms, pure cultures of Nitrosomonas and Nitrobacter isolated from activated sludge are used. The assays are performed in test tubes where the bacteria are incubated with the compound or wastewater to be tested. The nitrification rate is measured during 4 h and compared with reference samples. The test organisms were characterised with respect to temperature, pH and cell activity. Optimum temperature was 35 degrees C for Nitrosomonas and 38 degrees C for Nitrobacter; optimum pH was 8.1 for Nitrosomonas and 7.9 for Nitrobacter. There was a linear relationship between the nitrification rate and the cell concentration in the studied interval. The cell activity decreased slightly with storage time. A significant level of inhibition was calculated to 11% for the Nitrosomonas assay, and to 9% for the Nitrobacter assay. The assays are applicable to determination of nitrification inhibition in samples of industrial waste waters or influents of treatment plants, or chemical substances likely to be found in wastewater.

Biological degradation of selected hydrocarbons in an old PAH/creosote contaminated soil from a gas work site.

Abstract An old PAH/creosote contaminated soil (total ∼300 μg PAH/g soil) from a former gas work site in Stockholm, Sweden, has been treated at 20 °C with the addition of various nutrients and inoculated with bacteria (isolated from the soil) to enhance the degradation of selected hydrocarbons. Microcosm studies showed that the soil consisted of two contaminant fractions: one available, easily degraded fraction and a strongly sorbed, recalcitrant one. The bioavailable fraction, monitored by headspace solid phase microextraction, contained aromatics with up to three rings, and these were degraded within 20 days down to non-detectable levels (ng PAH/g soil) by both the indigenous bacteria and the externally inoculated samples. The nutrient additives were: a minimal medium (Bushnell-Haas), nitrate, nitrite, potting soil (Änglamark, Sweden), sterile water and aeration with Bushnell-Haas medium. After 30 days treatment most of the sorbed fractions were still present in the soil. Stirring or mechanical mixing of the soil slurries had the greatest effect on degradation, indicating that the substances were too strongly sorbed for the microorganisms. When stirring the choice of nutrient seemed less important. For the non-stirred samples the addition of nitrate with the bacterial inoculum showed the best degradation, compared to the other non-stirred samples. At the end of the experiments, accumulations of metabolites/degradation products, such as 9H-fluorenone, 4-hydroxy-9H-fluorenone, 9,10-phenanthrenedione and 4H-cyclopenta[def]phenanthrenone were detected. The metabolite 4-hydroxy-9H-fluorenone increased by several orders of magnitude during the biological treatments. Microbial activity in the soil was measured by oxygen consumption and carbon dioxide production.

Comamonas denitrificans sp. nov., an efficient denitrifying bacterium isolated from activated sludge

To find a biomarker for denitrification in activated sludge, five denitrifying strains isolated from three wastewater treatment plants were studied. These strains were selected from among 1,500 isolates for their excellent denitrifying properties. They denitrify quickly and have no lag phase when switching from aerobic to anoxic conditions. All strains have the cd1-type of nitrite reductase. The strains are Gram-negative rods and they all grow as filamentous chains when cultivated in liquid solution. The strains differ in colony morphology when grown on nutrient agar. Almost full-length 16S rDNA sequences were determined and phylogenetic analysis revealed that these strains are positioned among members of the genus Comamonas in the beta-subclass of the Proteobacteria. Signature nucleotides and bootstrap percentages were also analysed to verify this position. Strains 110, 123T, 2.99g, 5.38g and P17 were < or = 96.7% similar to known strains, but > or = 99.7% similar to each other, as judged from their 16S rDNA sequences, and grouped tightly together in the phylogenetic tree. Sequence motifs in the 16S rRNA gene were also found, suggesting the monophyletic origin of these strains. Nevertheless, some strains differed from the others, for example strain 110 branches early from the other strains and 5.38g is phenotypically more inert. Therefore, it is proposed that strains 110, 123T, 2.99g and P17 are classified into a new species, Comamonas denitrificans sp. nov., while the taxonomic status of strain 5.38g will have to await the outcome of further studies. The type strain of Comamonas denitrificans is 123T (ATCC 700936T).

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.

Determination of hydrocarbons in old creosote contaminated soil using headspace solid phase microextraction and GC–MS

Headspace solid phase microextraction (HS-SPME) has been used together with GC-MS to analyze organic substances directly in a soil, heavily contaminated with PAHs/creosote (approximately 300 mg/kg soil), from an old gaswork site in Stockholm, Sweden. The HS-SPME results, both qualitative and quantitative, were compared with traditional liquid extraction using ethyl acetate/hexane (20:80). It was shown that the concentrations determined with HS-SPME at 60 degrees C correlated well, for compounds containing up to two and three aromatic rings (naphthalenes, acenaphthene, acenaphthylene and fluorenes, while a lower concentration was obtained for phenanthrene, anthracene, fluoranthene and pyrene. The total concentrations for each compound determined with HS-SPME ranged from 2 to 25 microg/g soil. Quantification was done using standard addition of compounds directly to the soil samples. The bioavailable fraction of the compounds in the contaminated soil at 20 degrees C was analyzed using external calibration by spiking sterile uncontaminated sand (same texture and particle size as the contaminated soil but without a heavily sorbed organic fraction) with hydrocarbon standards in different concentrations. Storage of exposed fibers at 20 degrees C showed that analysis should be done within two days to make qualitative measurements and earlier (as soon as possible) for quantitative determinations.

Comparison of inhibition assays using nitrogen removing bacteria: Application to industrial wastewater

Abstract Three pure culture assays for measurements of inhibition of the nitrogen removal process were tested and compared. The methods were performed using pure cultures of bacteria responsible for three reactions; ammonia oxidation, nitrite oxidation and nitrite reduction. The inhibition caused by 48 samples of industrial wastewater was determined and the results for the three inhibition assays were compared. The correlation coefficients were calculated to be between 0.63 and 0.73. The inhibition of the pure culture methods was also compared with results from another investigation, where the inhibition by the same wastewaters was determined by a screening method based on activated sludge. The best correlation was found with the ammonia oxidation assay. The heavy metal content was statistically analysed for relationships with inhibition using a linear model with stepwise linear regression. Although zinc was a significant inhibitor for all three methods, these responded differently in the second step, which indicate that the bacteria are not inhibited according to the same pattern. The relevance of the three pure culture assays as an inhibition test is discussed and the conclusion can be drawn that they are all needed to give a complete inhibition picture of wastewaters.

Occurrence of nitrification inhibition in Swedish municipal wastewaters

The occurrence of substances inhibiting nitrification in Swedish municipal wastewaters was investigated using three methods: a screening method based on activated sludge and two pure culture methods based on Nitrosomonas and Nitrobacter. Influent samples from 109 wastewater treatment plants collected every day during one specific week were investigated. The three test methods were also compared. The results of the screening method showed that about 60% of the plants received wastewater containing inhibitory substances, although only 4% had considerable inhibition (>20%). With the two pure culture methods, inhibition was found at about 45% of the plants investigated, with considerable inhibition found at 13% and 20% of the plants with the Nitrosomonas and the Nitrobacter methods, respectively. The limit of detection was determined to be 5% inhibition for the screening method, 11% inhibition for the Nitrosomonas method and 13% inhibition for the Nitrobacter method. The pure culture methods found more samples strongly inhibitory or stimulating than the screening method. The highest correlation between the inhibition results from the three methods was found between the screening method and the Nitrosomonas method. It was also shown for sludge from several activated sludge treatment plants, that they were adapted to the toxic compounds present in the influent. At high inhibition this acclimatisation was less pronounced. No correlation was found for any of the methods between the inhibition and parameters such as the size of the plant, the geographic location, the content of ammonia, COD or conductivity in the influent, the presence of leachate or the percentage of industrial wastewater in the influent, or types of industries in the catchment area. However, the constantly highest inhibition was found at a plant with a large number of different industries connected. Any clear pattern for the variation of inhibition during the week was not found.

Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20 °C and 6 °C

Abstract A hydrocarbon mixture containing p-xylene, naphthalene, Br-naphthalene and straight aliphatic hydrocarbons (C14 to C17) was aerobically degraded without lag phase by a natural uncontaminated potting soil at 20 °C and 6 °C. Starting concentrations were approximately 46 ppm for the aromatic and 13 ppm for the aliphatic compounds. All aliphatic hydrocarbons were degraded within 5 days at 20 °C, to levels below detection (ppb levels) but only down to 10% of initial concentration at 6 °C. Naphthalene was degraded within 12 days at 20 °C and unaffected at 6 °C. At 20 °C p-xylene was degraded within 20 days, but no degradation occurred at 6 °C. Br-naphthalene was only removed down to 30% of initial concentration at 20 °C, with no significant effect at 6 °C. The biodegradation was monitored with head space solid-phase microextraction and gas chromatography–mass spectrometry.

Bacterial growth and biofilm production on pyrene

Enrichment cultures inoculated with Arctic soil yielded a biofilm that grew on pyrene and phenanthrene. In a 60-day period, the biofilm degraded 20 microg ml(-1) pyrene or 39 microg ml(-1) phenanthrene. Single colonized pyrene crystals (approximately 1.5x0.75x0.35 mm) yielded 10(11) culturable heterotrophs and 10(5) biofilm propagules. Analysis of ribosomal intergenic spacers identified six phylotypes in a clone library from the pyrene biofilm. Analysis of 16S rRNA gene sequences indicated that the phylotypes, in order of decreasing abundance, are most closely related to members of the genera Polaromonas, Sphingomonas, Alcaligenes, Caulobacter and Variovorax. Two isolates capable of growth on pyrene, both Pseudomonas spp., were obtained from the pyrene enrichment culture. Growth of microbial biofilms on polycyclic aromatic hydrocarbons has not been reported previously, and this mode of growth may have important effects on substrate uptake.