Martin Hesselsøe

The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation of Nitrosomonas and Nitrosospira Strains

ABSTRACT Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. TwoNitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC50], 6 to 38 mg liter−1) was affected much less by LAS than the growth rate and viability (EC50, 3 to 14 mg liter−1) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter−1); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH4+ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge

Extensive physiological analyses of different microbial community members in many samples are difficult because of the restricted number of target populations that can be investigated in reasonable time by standard substrate-mediated isotope-labeling techniques. The diversity and ecophysiology of Rhodocyclales in activated sludge from a full-scale wastewater treatment plant were analyzed following a holistic strategy based on the isotope array approach, which allows for a parallel functional probing of different phylogenetic groups. Initial diagnostic microarray, comparative 16S rRNA gene sequence, and quantitative fluorescence in situ hybridization surveys indicated the presence of a diverse community, consisting of an estimated number of 27 operational taxonomic units that grouped in at least seven main Rhodocyclales lineages. Substrate utilization profiles of probe-defined populations were determined by radioactive isotope array analysis and microautoradiography-fluorescence in situ hybridization of activated sludge samples that were briefly exposed to different substrates under oxic and anoxic, nitrate-reducing conditions. Most detected Rhodocyclales groups were actively involved in nitrogen transformation, but varied in their consumption of propionate, butyrate, or toluene, and thus in their ability to use different carbon sources in activated sludge. This indicates that the functional redundancy of nitrate reduction and the functional versatility of substrate usage are important factors governing niche overlap and differentiation of diverse Rhodocyclales members in this activated sludge.

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

ABSTRACT Most heterotrophic bacteria assimilate CO2 in various carboxylation reactions during biosynthesis. In this study, assimilation of 14CO2 by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient 14CO2 during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of 14C-labeled organic substrates. Experiments with E. coli showed that 14CO2 was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO2-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [14C]oleic acid. However, the new HetCO2-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO2−, whereas the addition of O2 or NO3− initiated growth, as indicated by detectable 14CO2 assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO2-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO2-MAR results were supported by stable isotope analysis of 13C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of 13CO2. In conclusion, the novel HetCO2-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.

Effect of oxygen on survival of faecal pollution indicators in drinking water.

Aims:  The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water.

Degradation of organic pollutants by methane grown microbial consortia

AbstractMicrobial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge (“AAE-A2”), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 °C was 3.1 ± 1.2 mg l−1 and 8.7 ± 1.1 mg DBP (g protein × h)−1, respectively. AAE-A2 also showed fast degradation of BTX (230 ± 30 nmol benzene (mg protein × h)−1 at 20 °C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinustrichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83–92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas,Flavobacterium,Janthinobacterium and Rubivivax).

Development of nitrification hot-spots around degrading red clover (Trifolium pratense) leaves in soil

Abstract Litterbags with clover leaves in soil were buried in the field to study the influence of degrading clover leaves on the development of elevated nitrification activity (hot-spots) in space and time. Potential NH3 oxidation activity indicating the population size of ammonia-oxidizing bacteria was measured in soil samples taken in a zone a few millimetres thick around the leaves and in bulk soil. On 3 sampling days during leaf degradation, the potential NH4+ oxidation was significantly higher in leaf-associated soil than in bulk soil, the largest difference (factor of 2) occurring 24 days after burial. At all sampling occasions, NH4+ oxidation rates followed a normal distribution, except for a log-normal distribution in the leaf-associated soil sampled 24 days after burial when some very active samples (hot-spots) appeared. In a similar laboratory experiment we examined the effects of soil water on the development of nitrification hot-spots. We observed that the development of hot-spots did not take place in soil incubated at 60% of water-holding capacity (WHC), whereas they developed faster when the soil was incubated at approximately 100% WHC. It was concluded that soil water in combination with easily degradable organic N were essential parameters for the development of nitrification hot-spots in this soil.

Microbial Toxicity of Methyl tert-Butyl Ether (MTBE) determined with fluorescent and luminescent bioassays

The inhibitory effects of the fuel additive methyl tert-butyl ether (MTBE) and potential degradation products tert-butanol (TBA) and formaldehyde was examined using mixed microbial biomass, and six strains of bioluminescent bacteria and yeast. The purpose was to assess microbial toxicity with quantitative bioluminescent and fluorescent endpoints, and to identify sensitive proxies suitable for monitoring MTBE contamination. Bioluminescent Aliivibrio fischeri DSM 7151 (formerly Vibrio fischeri) appeared highly sensitive to MTBE exposure, and was a superior test organisms compared to lux-tagged Escherichia coli DH5α, Pseudomonas fluorescens DF57-40E7 and Saccharomyces cerevisiae BLYR. EC10 and EC50 for acute MTBE toxicity in A. fischeri were 1.1 and 10.9 mg L(-1), respectively. Long term (24h) MTBE exposure resulted in EC10 values of 0.01 mg L(-1). TBA was significantly less toxic with EC10 and EC50 for acute and chronic toxicity >1000 mg L(-1). Inhibition of bioluminescence was generally a more sensitive endpoint for MTBE toxicity than measuring intracellular ATP levels and heterotrophic CO2 assimilation. A weak estrogenic response was detected for MTBE at concentrations ⩾ 3.7 g L(-1) using an estrogen inducible bioluminescent yeast strain (S. cerevisiae BLYES). Microbial hydrolytic enzyme activity in groundwater was affected by MTBE with EC10 values of 0.5-787 mg L(-1), and EC50 values of 59-3073 for alkaline phosphatase, arylsulfatase, beta-1,4-glucanase, N-acetyl-beta-d-glucosaminidase, and leucine-aminopeptidase. Microbial alkaline phosphatase and beta-1,4-glucanase activity were most sensitive to MTBE exposure with EC50 ⩽ 64.8 mg L(-1). The study suggests that bioassays with luminescent A. fischeri, and fluorescent assays targeting hydrolytic enzyme activity are good candidates for monitoring microbial MTBE toxicity in contaminated water.

Quantification Of 14c-labeled Hydrophobic Organic Compounds In Soil Samples By A Scintillation Fluid Extraction Method

Hydrophobic organic chemicals labeled with 14C are often used as tracers to study chemical fate and transport in soil. Wet oxidation is the recognized but labor-demanding method used most often to measure the concentration of radioactive organic tracers in the soil. In this study, we test an alternative, simpler method for quantification of 14C-labeled hydrophobic organic chemicals in soil samples. The soil samples were extracted directly with scintillation fluid in glass scintillation vials (Scintillation Fluid Extraction, SFE method) and were subsequently analyzed by liquid scintillation counting. Application of internal standards showed no significant quenching or reduced counts of the 14C-labeled chemical caused by the presence of settled soil particles in the scintillation vials. Hence, the scintillation fluid could be used simultaneously as both scintillation and extraction media. Decreasing the amount of soil containing 14C-labeled chemicals added to the scintillation vial was shown to increase the extraction efficiency of the SFE method. Aging (contact time) and concentration were shown to affect the results of the SFE method. However, in a naphthalene diffusion experiment, where aging varied between almost no contact time and 20 days and concentration of naphthalene varied between ∼0 to 40 μg g−1, the estimated naphthalene diffusion coefficient was shown to be influenced only minimally when using the SFE method compared with the wet oxidation method. We emphasize that the SFE method is applicable only to sterile systems with no degradation or assimilation of 14C-labeled compounds. If this condition is met and there is appropriate consideration of the effects of chemical aging and concentration, the SFE method seems to be a useful and labor-saving alternative to the traditional wet oxidation method for determination of the concentration of 14C-labeled organic chemicals in soil samples.