Faculty Publications

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Open Access
Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in native, tilled, and successional soils
(Colorado State University. Libraries, 1999-07) Kowalchuk, George A., author; Bruns, Mary Ann, author; Stephen, John R., author; Paul, Eldor A., author; Prosser, James I., author; American Society for Microbiology, publisher
Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for Nitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospiracluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.
Open Access
Conversion of biovolume measurements of soil organisms, grown under various moisture tensions, to biomass and their nutrient content
(Colorado State University. Libraries, 1979-04) van Veen, Johannes A., author; Paul, Eldor A., author; American Society for Microbiology, publisher
Direct microscopic measurements of biomass in soil require conversion factors for calculation of the mass of microorganisms from the measured volumes. These factors were determined for two bacteria, five fungi, and a yeast isolated from soil. Moisture stress conditions occurring in nature were simulated by growth in two media using shake cultures, on agar plates, and on membranes held at 34, 330, and 1,390 kPa of suction. The observed conversion factors, i.e., the ratio between dry weight and wet volume, generally increased with increasing moisture stress. The ratios for fungi ranged from 0.11 to 0.41 g/cm3 with an average of 0.33 g/cm3. Correction of earlier data assuming 80% water and a wet-weight specific gravity of 1.1 would require a conversion factor of 1.44. The dry-weight specific gravity of bacteria and yeasts ranged from 0.38 to 1.4 g/cm3 with an average of 0.8 g/cm3. These high values can only occur at 10% ash if no more than 50% of the cell is water, and a specific conversion factor to correct past data for bacterial biomass has not yet been suggested. The high conversion factors for bacteria and yeast could not be explained by shrinkage of cells due to heat fixing, but shrinkage during preparation could not be completely discounted. Moisture stress affected the C, N, and P content of the various organisms, with the ash contents increasing with increasing moisture stress. Although further work is necessary to obtain accurate conversion factors between biovolume and biomass, especially for bacteria, this study clearly indicates that existing data on the specific gravity and the water and nutrient content of microorganisms grown in shake cultures cannot be applied when quantifying the soil microbial biomass.
Open Access
Effects of agronomic treatments on structure and function of ammonia-oxidizing communities
(Colorado State University. Libraries, 2000-12) Phillips, Carol J., author; Paul, Eldor A., author; Prosser, James I., author; Gross, Katherine L., author; Dollhopf, Sherry L., author; Harris, Dave, author; American Society for Microbiology, publisher
The aim of this study was to determine the effects of different agricultural treatments and plant communities on the diversity of ammonia oxidizer populations in soil. Denaturing gradient gel electrophoresis (DGGE), coupled with specific oligonucleotide probing, was used to analyze 16S rRNA genes of ammonia oxidizers belonging to the β subgroup of the division Proteobacteria by use of DNA extracted from cultivated, successional, and native deciduous forest soils. Community profiles of the different soil types were compared with nitrification rates and most-probable-number (MPN) counts. Despite significant variation in measured nitrification rates among communities, there were no differences in the DGGE banding profiles of DNAs extracted from these soils. DGGE profiles of DNA extracted from samples of MPN incubations, cultivated at a range of ammonia concentrations, showed the presence of bands not amplified from directly extracted DNA. Nitrosomonas-like bands were seen in the MPN DNA but were not detected in the DNA extracted directly from soils. These bands were detected in some samples taken from MPN incubations carried out with medium containing 1,000 μg of NH4+-N ml−1, to the exclusion of bands detected in the native DNA. Cell concentrations of ammonia oxidizers determined by MPN counts were between 10- and 100-fold lower than those determined by competitive PCR (cPCR). Although no differences were seen in ammonia oxidizer MPN counts from the different soil treatments, cPCR revealed higher numbers in fertilized soils. The use of a combination of traditional and molecular methods to investigate the activities and compositions of ammonia oxidizers in soil demonstrates differences in fine-scale compositions among treatments that may be associated with changes in population size and function.
Open Access
Effects of pH and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater
(Colorado State University. Libraries, 1998-10) Mahne, Ivan, author; Prinčič, Alenka, author; Tiedje, James M., author; Paul, Eldor A., author; Megušar, France, author; American Society for Microbiology, publisher
Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter−1), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant. The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined. Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis. Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level. The smaller changes in community structure brought about by the two pH extremes, however, were irreversible. Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative, Nitrosomonas eutropha C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the β subgroup of the class Proteobacteria. A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles.
Open Access
Isolation of saprophytic basidiomycetes from soil
(Colorado State University. Libraries, 1996-11) Harris, David, author; Thorn, R. Greg, author; Reddy, C. Adinarayana, author; Paul, Eldor A., author; American Society for Microbiology, publisher
A method with the combined advantages of soil particle washing, selective inhibitors, and an indicator substrate was developed to isolate saprophytic basidiomycetes from soil. Organic particles were washed from soil and plated on a medium containing lignin, guaiacol, and benomyl, which reduced mold growth and allowed detection of basidiomycetes producing laccase or peroxidase. The 64 soil samples yielded 67 basidiomycete isolates, representing 51 groups on the basis of morphology and physiology. This method should facilitate investigations into the biodiversity of soil basidiomycetes and yield organisms that are useful in bioremediation of soils contaminated with pesticides or other recalcitrant aromatic compounds.
Open Access
Microscopic counting and adenosine 5'-triphosphate measurement in determining microbial growth in soils
(Colorado State University. Libraries, 1977-09) Paul, E. A., author; Johnson, R. L., author; American Society for Microbiology, publisher
A microscopic technique utilizing dispersion of fungal hyphae in a Waring blender, filtration through membrane filters (Nucleopore Corp.), and counting on a fluorescence microscope was developed for counting fungal hyphal biomass. Nonfluorescent staining techniques of the soil-filter preparation did not give quantitative recoveries. Water-soluble aniline blue, which binds to the β-1,3-glucans of the fungal cell wall, made visualization of the hyphae by fluorescence possible. A range of fungi added to soil were quantitatively recovered. Adenosine 5'-triphosphate (ATP) was extracted from soil by lysis of the organisms with CHCl3 in NaHCO3, which prevented adsorption of the organic phosphorus to the soil colloids. Centrifugation and removal of CHCl3 was followed by dilution with pH 7.8 tris(hydroxymethyl)aminomethane buffer. ATP concentrations were measured by using the luciferase-luciferin light reaction. Since NaHCO3 interfered to some extent with this reaction, the standards were made up in equivalent mixtures of tris(hydroxymethyl)aminomethane buffer and NaHCO3. Recovery of ATP was rapid and quantitative in a range of soils. Measurement of the ATP and bacterial and fungal numbers in an incubated soil showed that fungal and bacterial population increases were delayed by phosphorus deficiency. Microbial populations were not affected at a later date. The ATP content of the soil system was reduced by phosphorus deficiency throughout the incubation period. This indicated that ATP could be altered without major changes in the microbial populations.
Open Access
Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities
(Colorado State University. Libraries, 2003-02) Blackwood, Christopher B., author; Marsh, Terry, author; Kim, Sang-Hoon, author; Paul, Eldor A., author; American Society for Microbiology, publisher
Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.

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