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PubMed1993

Detection of Escherichia coli in sewage and sludge by polymerase chain reaction.

A method in which the polymerase chain reaction (PCR) was used was developed to amplify either a uidA gene fragment or a 16S rRNA gene fragment from Escherichia coli in sewage and sludge. Because of interference caused by humic acidlike substances, crude DNA extracts were purified with a Sephadex G-200 spun column before the PCR was begun. A Southern analysis in which a nonradioactive chemiluminescent method was used was performed to confirm the presence of PCR products. The sensitivity of detection for PCR products when the chemiluminescent method was used was determined to be 30 ag of E. coli genomic DNA template. In seeded sludge, the PCR amplified the target DNA from 80 E. coli cells per g of sludge and 50 Shigella dysenteriae cells per g of sludge. Because only 0.05 aliquot of a sludge extract was used for the PCR, we deduced that the PCR detected target DNA equivalent to the DNA of 2.5 to 4 cells in the extract. The PCR amplified the uidA fragment from diluted sewage influents and effluents containing E. coli cells. Therefore, the PCR performed with a chemiluminescent gene probe can be used to detect the presence of potentially pathogenic microorganisms in sewage and sludge. This technique can be expanded to permit direct detection of pathogenic microorganisms in water samples, thus leading to enhanced public health protection.

Base Sequence↗
PubMed1993

Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Aprotinin↗
PubMed1995

Microbial degradation and humification of the lawn care pesticide 2,4-dichlorophenoxyacetic acid during the composting of yard trimmings.

The fate of the widely used lawn care herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) during the composting of yard trimmings consisting of primarily leaves and grass is an important unexplored question. In this study, we determined the extent of 2,4-D mineralization, incorporation into humic matter, volatilization, and sorption during the composting of yard trimmings. Yard trimmings (2:1 [wt/wt] leaves-grass) were amended with 14C-ring-labeled 2,4-D (17 mg/kg of dry weight) and composted in a temperature-controlled laboratory scale compost system. During composting, thermophilic microbes were numerically dominant, reaching a maximum of 2 x 10(11)/g. At the end of composting, 46% of the organic matter (OM) present in the yard trimmings was lost and the compost was stable, with an oxygen uptake rate of 0.09 mg of O2 per g of OM per h, and was well humified (humification index, 0.39). Mineralization of the OM temporally paralleled mineralization of 2,4-D. In the final compost, 47% of the added 2,4-D carbon was mineralized, about 23% was complexed with high-molecular-weight humic acids, and about 20% was not extractable (humin fraction). Less than 1% of the added 14C was present in water expressed from the finished compost, suggesting a low potential for leaching of 2,4-D. Very little volatilization of 2,4-D occurred during composting. It is of interest that our results indicate active mineralization of 2,4-D at composting temperatures of 60 degrees C because microbial 2,4-D degradation at thermophilic temperatures has not been previously documented.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay.

A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.

Bacteriological Techniques↗
PubMed1998

Humic acids as electron acceptors for anaerobic microbial oxidation of vinyl chloride and dichloroethene.

Anaerobic oxidation of [1,2-14C]vinyl chloride and [1, 2-14C]dichloroethene to 14CO2 under humic acid-reducing conditions was demonstrated. The results indicate that waterborne contaminants can be oxidized by using humic acid compounds as electron acceptors and suggest that natural aquatic systems have a much larger capacity for contaminant oxidation than previously thought.

Aerobiosis↗
PubMed1998

Effect of humic fractions and clay on biodegradation of phenanthrene by a Pseudomonas fluorescens strain isolated from soil.

The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 microg/ml. Humic acid at 10 microg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.

Aluminum Silicates↗
PubMed1998

Purification and characterization of laccase from Chaetomium thermophilium and its role in humification.

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45 degreesC both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70 degreesC and had half-lives of 24 and 12 h at 40 and 50 degreesC, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

Biodegradation, Environmental↗
PubMed1999

Inhibition of nitrifiers and methanotrophs from an agricultural humisol by allylsulfide and its implications for environmental studies.

Allylsulfide, an inhibitor of ammonia monooxygenase, was tested to determine its ability to inhibit nitrification and methane oxidation in pure cultures, in agricultural humisol enrichment cultures, and in humisol slurries. We confirmed that allylsulfide is a differential inhibitor of cultures of nitrifiers and methanotrophs at concentrations of 1 and 200 microM, respectively, which result in 50% inhibition. However, although a nitrifying enrichment culture added to sterilized humisol was inhibited 50% by 4 microM allylsulfide, 500 microM allylsulfide was necessary for 50% inhibition of the endogenous nitrifying activity in nonsterile humisol. We concluded that native nitrifiers were protected, possibly by being in colonial aggregates or sheltered microenvironments.

Agriculture↗
PubMed2003

Members of a readily enriched beta-proteobacterial clade are common in surface waters of a humic lake.

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. beta-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments. In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment. Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.

Betaproteobacteria↗
PubMed2003

Disinfection of water containing natural organic matter by using ozone-initiated radical reactions.

Ozone is widely used to disinfect drinking water and wastewater due to its strong biocidal oxidizing properties. Recently, it was reported that hydroxyl radicals ((.)OH), resulting from ozone decomposition, play a significant role in microbial inactivation when Bacillus subtilis endospores were used as the test microorganisms in pH controlled distilled water. However, it is not yet known how natural organic matter (NOM), which is ubiquitous in sources of drinking water, affects this process of disinfection by ozone-initiated radical reactions. Two types of water matrix were considered for this study. One is water containing humic acid, which is commercially available. The other is water from the Han River. This study reported that hydroxyl radicals, initiated by the ozone chain reaction, were significantly effective at B. subtilis endospore inactivation in water containing NOM, as well as in pH-controlled distilled water. The type of NOM and the pH have a considerable effect on the percentage of disinfection by hydroxyl radicals, which ranged from 20 to 50%. In addition, the theoretical T value of hydroxyl radicals for 2-log B. subtilis removal was estimated to be about 2.4 x 10(4) times smaller than that of ozone, assuming that there is no synergistic activity between ozone and hydroxyl radicals.

Bacillus subtilis↗
PubMed2005

Isolation of soil bacteria adapted to degrade humic acid-sorbed phenanthrene.

The goal of these studies was to determine how sorption by humic acids affected the bioavailability of polynuclear aromatic hydrocarbons (PAHs) to PAH-degrading microbes. Micellar solutions of humic acid were used as sorbents, and phenanthrene was used as a model PAH. Enrichments from PAH-contaminated soils established with nonsorbed phenanthrene yielded a total of 25 different isolates representing a diversity of bacterial phylotypes. In contrast, only three strains of Burkholderia spp. and one strain each of Delftia sp. and Sphingomonas sp. were isolated from enrichments with humic acid-sorbed phenanthrene (HASP). Using [14C]phenanthrene as a radiotracer, we verified that only HASP isolates were capable of mineralizing HASP, a phenotype hence termed "competence." Competence was an all-or-nothing phenotype: noncompetent strains showed no detectable phenanthrene mineralization in HASP cultures, but levels of phenanthrene mineralization effected by competent strains in HASP and NSP cultures were not significantly different. Levels and rates of phenanthrene mineralization exceeded those predicted to be supported solely by the metabolism of phenanthrene in the aqueous phase of HASP cultures. Thus, competent strains were able to directly access phenanthrene sorbed by the humic acids and did not rely on desorption for substrate uptake. To the best of our knowledge, this is the first report of (i) a selective interaction between aerobic bacteria and humic acid molecules and (ii) differential bioavailability to bacteria of PAHs sorbed to a natural biogeopolymer.

Adaptation, Physiological↗
PubMed1972

Effect of several clay minerals and humic acid on the survival of Klebsiella aerogenes exposed to ultraviolet irradiation.

The effect of various clay minerals and humic acid on the survival of Klebsiella aerogenes exposed to ultraviolet (UV) irradiation was investigated. A protective effect was observed and found to depend on the specific light absorption and light scattering properties of the clay minerals and the humic acid used. The higher the specific absorption, the better was the survival of K. aerogenes after UV irradiation. Bacterial survival was lower in clays saturated with divalent cations (Ca, Zn) than in those homoionic to monovalent cations (K).

Absorption↗
PubMed1976

Respiration rates for determining the effects of urea on the soil-surface organic horizon of a black spruce stand.

The respiration rates of microflora of layers of soil-surface organic horizon of a black spruce (Picea mariana (Mill.) B.S.P.) stand have been studied manometrically under controlled conditions of moisture, temperature, and aeration in the presence of urea and other nitrogen and mineral amendments. L.F., and F2 samples from field plots fertilized with 448 kg N/ha as urea in 1961 had still in 1971 greater respiration rates than similar samples from unfertilized field plots. In lab tests, addition of urea (112, 280, and 448 kg N/ha or 875, 2187, and 3500 ppm N) stimulated the endogenous respiration of each layer. The stimulation was greater when 2187 ppm N was applied and when moisture and temperature of the layers were maintained at 60% water-holding capacity and 20 degrees C during the 42-day incubation period. Addition of Mg, Ca, and K to urea-fertilized layers increased respiration while P and S decreased it. Addition of NH4NO3 and (NH4)2SO4 impaired the endogenous respiration. The endogenous respiration and moisture, temperature, and fertilizer effects decreased in the order L,F1, and F2 layers.

Bacteria↗