Natural Microbial Populations Research Articles

Sulfentrazone sorption, desorption, and mineralization in soils from two tillage systems

Sulfentrazone sorption kinetics, desorption, and mineralization were evaluated in surface 7.5 cm of soils collected from long-term conventional-till (CT) and no-till (NT) plots. The soils used were Miami silt loam and Drummer silty clay loam from Illinois and Dundee silt loam from Mississippi. Sulfentrazone sorption kinetics in Dundee silt loam CT and NT soils were adequately described by a simple two-site equilibrium/kinetic model. Rapid initial sorption (within 1 h) was followed by a slower sorption and equilibrium, largely achieved by 72 h of shaking, with a negligible increase in sorption thereafter. The sorptionKfranged from 1.02 to 3.44 among the six CT and NT soils. TheKfvalues were greater for NT compared to their respective CT soils. Overall,Kfvalues were higher in Drummer silty clay loam followed by Dundee silt loam and Miami silt loam soil. TheNvalues were less than unity in all soils indicating nonlinear sorption. Sulfentrazone desorption was hysteretic with a very low rate of desorption. The total amount desorbed in four desorptions ranged from 58 to 72% of that sorbed. Less than 2.1% of applied14C-sulfentrazone was mineralized to14CO2 in Dundee silt loam CT and NT soils during a 77–d incubation. Relatively low mineralization of sulfentrazone suggests poor adaptability of native microbial populations that have not been exposed to this herbicide. Higher sorption and lower desorption of sulfentrazone in NT soils compared to CT soils suggest that NT systems (which tend to increase plant residues) may prolong sulfentrazone residence time in soil.

Enhanced degradation of lignosulfonated compounds by Streptomyces viridosporus

Degradation of lignosulfonated compounds by S. viridosporus strain T7A on nutritive or mineral culture media was quantified. Two lignosulfonated compounds, differing in their molecular mass distributions and chemical structures, were used: lignosulfonate (1–80 kDa) and vanisperse, a sulfonated oxylignin (1–20 kDa). The use of nutritive culture medium (containing Biosoyase) enhanced both the growth of S. viridosporus and production of lignin peroxidase (LiP) leading to enhanced lignocellulose degradation, but no lignosulfonated compound degradation was observed. A fraction of these compounds underwent a molecular transformation, producing non-biodegradable acid precipitable polymeric lignin (APPL). When a mineral culture medium (containing glycerol as additional organic carbon source) was used, approximately 21 and 35% of lignosulfonate and vanisperse were respectively degraded, with little APPL production. LiP activity was growth-associated and significant modifications of molecular mass distributions of both lignosulfonated compounds were observed. A mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent, was used to evaluate the biodegradability of lignosulfonate products from S. viridosporus cultures. This population was (i) unable to degrade lignosulfonate products, and (ii) decreased the lignosulfonate degradation capacity of S. viridosporus. Antagonistic effects of the mixed population on S. viridosporus were observed. In the light of this, bioaugmentation strategies involving addition of S. viridosporus are unlikely to be successful and alternative degradation strategies need to be developed.

A natural species concept for prokaryotes

Direct molecular analyses of natural microbial populations reveal patterns that should compel microbiologists to adopt a more natural species concept that has been known to biologists for decades. The species debate can be exploited to address a larger issue — microbiologists need, in general, to take a more natural view of the organisms they study.

Biodegradability of lignosulphonate by Streptomyces viridosporus strain T7A and a mixed natural microbial population antagonistic effects

AbstractThe ability of a mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent and Streptomyces viridosporus strain T7A, to degrade lignosulphonate was evaluated. S. viridosporus growing in a mineral medium containing glycerol (7 g/l) and lignosulphonate (1 g/l) allowed 20% of lignosulphonate to be degraded after 18 days of incubation. A culture of the mixed population on culture medium after S. viridosporus growth was unable to degrade lignosulphonate products. Moreover, antagonism between S. viridosporus and the mixed population or between S. viridosporus and the isolated strains from this population was observed. The enhancement of lignosulphonate biodegradation by naturally occurring microorganisms in association with S. viridosporus (bioaugmentation strategy) seems to be difficult.

Enhanced degradation of lignosulfonated compounds by Streptomyces viridosporus

Degradation of lignosulfonated compounds by S. viridosporus strain T7A on nutritive or mineral culture media was quantified. Two lignosulfonated compounds, differing in their molecular mass distributions and chemical structures, were used: lignosulfonate (1–80 kDa) and vanisperse, a sulfonated oxylignin (1–20 kDa). The use of nutritive culture medium (containing Biosoyase) enhanced both the growth of S. viridosporus and production of lignin peroxidase (LiP) leading to enhanced lignocellulose degradation, but no lignosulfonated compound degradation was observed. A fraction of these compounds underwent a molecular transformation, producing non-biodegradable acid precipitable polymeric lignin (APPL). When a mineral culture medium (containing glycerol as additional organic carbon source) was used, approximately 21 and 33% of lignosulfonate and vanisperse were respectively degraded, with little APPL production. LiP activity was growth-associated and significant modifications of molecular mass distributions of both lignosulfonated compounds were observed. A mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent, was used to evaluate the biodegradability of lignosulfonate products from S. viridosporus cultures. This population was (i) unable to degrade lignosulfonate products, and (ii) decreased the lignosulfonate degradation capacity of S. viridosporus. Antagonistic effects of the mixed population on S. viridosporus were observed. In the light of this, bioaugmentation strategies involving addition of S. viridosporus are unlikely to be successful and alternative degradation strategies need to be developed.

Amino acid variation in the GyrA subunit of bacteria potentially associated with natural resistance to fluoroquinolone antibiotics.

In studies of genetic diversity in natural microbial populations, we have analyzed nucleotide sequences in the quinolone resistance-determining region of the bacterial gyrA gene in ciprofloxacin-resistant and nonselected soil bacteria obtained from the environment. It is apparent that this sequence is highly variable, and resistance to fluoroquinolone antibiotics occurring in environmental populations of bacteria is due at least in part to natural sequence variation in this domain. We suggest that the development of new antimicrobial agents, including completely synthetic antimicrobials such as the fluoroquinolones, should incorporate the analysis of resistance mechanisms among microbes in natural environments; these studies could predict potential mechanisms of resistance to be encountered in subsequent clinical use of the agents and would guide chemical modification designed to evade resistance development.

Monitoring the Biological and Physical Reactivity of Dextran Carbohydrates in Seawater Incubations Using Flow Field-Flow Fractionation

In order to better understand the factors that modulate the size and reactivity of high molecular weight organic matter dissolved in seawater, fluorescently labeled dextrans were used as the model compounds whose molecular weight distributions were monitored by flow field-flow fractionation (flow FFF or F1FFF) during incubations in seawater matrices. Two fluorescein isothiocyanate (FITC) labeled dextrans (145k and 2M Da) were incubated in whole seawater (natural microbial population and natural dissolved organic matter (DOM) present), 0.02 μm filtered seawater (all microorganisms removed, but natural DOM largely unaltered), and UV-oxidized seawater (no microorganisms or natural DOM present). Flow FFF fractograms of the two FITC-dextrans incubated in UV-oxidized seawater showed no changes, signifying that the dextrans did not undergo any alteration or aggregation. The dextrans incubated in filtered seawater with natural DOM present resulted in fractograms that are shifted to higher retention times, consistent with aggregation of the dextran and natural DOM. In the whole seawater incubations, the complex changes in the fractograms over time indicated that the dextrans underwent both aggregation with natural DOM and degradation by heterotrophic microorganisms. The latter was confirmed by microscopic examination of the collected fractions after selectively staining the microorganisms with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI). The FITC dextrans were observed to be “attached” to the bacteria's outer cell membrane where they are subsequently hydrolyzed into small low molecular weight fragments by extracellular enzymes. Some of these degraded dextrans are assimilated by the bacteria and the rest (μ70%) are released into the seawater. Approximately half of the released degraded dextrans are of high enough molecular weight to be retained by flow FFF at the conditions employed in these studies. The data presented in this paper illustrate that, when used as both a qualitative and semipreparative tool, flow FFF can provide information on the relationships between natural and model DOM and microorganisms that would be difficult or impossible to obtain using other methods.

Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments

Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains. The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples. Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated. The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences relate to Nitrosomonas genes could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils. Such differences in community structure may affect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors.

Fenitrothion and 3‐methyl‐4‐nitrophenol degradation by two bacteria in natural waters under laboratory conditions

The ability of two bacterial strains, Flavobacterium sp. strain ATCC 27551 and Arthrobacter aurescens strain TW17, to degrade fenitrothion and the product of its hydrolysis 3‐methyl‐4‐nitrophenol (MNP) in natural water samples under laboratory conditions was studied. Fenitrothion was degraded by Flavobacterium sp whereas MNP was not degraded by this strain. In comparison, MNP was metabolized by A. aurescens and fenitrothion was not. The natural microbial populations did not decompose the fenitrothion and MNP, however Flavobacterium sp and R. aurescens degraded these compounds in the presence of the natural microbial population.

Nodularin concentrations in Lakes Alexandrina and Albert, South Australia, during a bloom of the cyanobacterium (blue-green alga)Nodularia spumigena and degradation of the toxin

Nodularin together with cell numbers of Nodularia spumigena were monitored during a toxic bloom of this cyanobacterium during the summer/autumn of 1994–1995 in Lakes Alexandrina and Albert, South Australia. There was a good correlation between cell numbers and toxin concentration over the course of the bloom at a number of locations. The correlation was poorer toward the end of the bloom, and may have reflected cell lysis and release of toxin to the surrounding water where it was rapidly degraded. The results suggested that the suitability of the lake water for drinking could be better determined by monitoring toxin content rather than cell numbers. Degradation of nodularin was studied and found to be more rapid in lake water that was supporting a bloom compared with water collected that had not experienced a bloom for several months. Half-lives were 24 and 54 h, respectively. There was no lag phase in water that was supporting a bloom, suggesting a relatively high population of microorganisms capable of degrading the toxin to be present. Toxin released to the lake water from lysing cells would therefore disappear very quickly. Toxin degradation was inhibited in the presence of copper ions. Copper sulphate treatment of blooms may therefore be doubly disadvantageous. Not only are cells lysed by copper sulphate addition, thereby releasing toxin to the surrounding water, but the natural microbial population capable of degrading the toxin may be destroyed, or the enzymes capable of toxin degradation inhibited. © 1997 John Wiley & Sons, Inc. Environ Toxicol Water Qual 12: 273–282, 1997

Towards a unified evolutionary genetics of microorganisms.

I propose here that evolutionary genetics, apart from improving our basic knowledge of the taxonomy and evolution of microbes (either eukaryotes or prokaryotes), can also greatly contribute to applied research in microbiology. Evolutionary genetics provides convenient guidelines for better interpreting genetic and molecular data dealing with microorganisms. The three main potential applications of evolutionary genetics in microbiology are (a) epidemiological follow-up (with the necessity of evaluating the stability of microbial genotypes over space and time); (b) taxonomy in the broad sense (better definition and sharper delimitation of presently described taxa, research of hidden genetic subdivisions); and (c) evaluation of the impact of the genetic diversity of microbes on their relevant properties (pathogenicity, resistance to drugs, etc). At present, two main kinds of population structure can be distinguished in natural microbial populations: (a) species that are not subdivided into discrete phylogenetic lineages (panmictic species or basically sexual species with occasional bouts of short-term clonality fall into this category); (b) species that are strongly subdivided by either cryptic speciation or clonal evolution. Improvements in available statistical methods are required to refine these distinctions and to better quantify the actual impact of gene exchange in natural microbial populations. Moreover, a codified selection of markers with appropriate molecular clocks (in other words: adapted levels of resolution) is sorely needed to answer distinct questions that address different scales of time and space: experimental, epidemic, and evolutionary. The problems raised by natural genetic diversity are very similar for all microbial species, in terms of both basic and applied science. Despite this fact, a regrettable compartmentalization among specialists has hampered progress in this field. I propose a synthetic approach, relying on the statistical improvements and technical standardizations called for above, to settle a unified evolutionary genetics of microorganisms, valid whatever the species studied, whether eukaryotic (parasitic protozoa and fungi) or prokaryotic (bacteria). Apart from benefits for basic evolutionary research, the anticipated payoff from this synthetic approach is to render routine and common-place the use of microbial evolutionary genetics in the fields of epidemiology, medicine, and agronomy.

The role of cyanobacterial exopolysaccharides in structuring desert microbial crusts

Microbial crusts are present on surfaces of soils throughout the world. A key feature of these crusts in arid zones is the abundance of filamentous sheath-forming and polysaccharide-excreting cyanobacteria. Several isolates of cyanobacteria were prepared from crust samples (Nizzana sand dunes, north-western Negev Desert, Israel). Optimal growth conditions for two such isolates of Microcoleus sp. were defined, and the role of the excreted polysaccharides in affecting the hydrological properties of crust-covered sand dunes was studied. Experiments with the native crust microbial population demonstrated the possibility of net primary productivity at both high relative air humidities and low moisture content.

The role of cyanobacterial exopolysaccharides in structuring desert microbial crusts

Microbial crusts are present on surfaces of soils throughout the world. A key feature of these crusts in arid zones is the abundance of filamentous sheath-forming and polysaccharide-excreting cyanobacteria. Several isolates of cyanobacteria were prepared from crust samples (Nizzana sand dunes, north-western Negev Desert, Israel). Optimal growth conditions for two such isolates of Microcoleus sp. were defined, and the role of the excreted polysaccharides in affecting the hydrological properties of crust-covered sand dunes was studied. Experiments with the native crust microbial population demonstrated the possibility of net primary productivity at both high relative air humidities and low moisture content.

Control of microbial souring by nitrate, nitrite or glutaraldehyde injection in a sandstone column

Microbial souring (production of hydrogen sulfide by sulfate-reducing bacteria, SRB) in crushed Berea sandstone columns with oil field-produced water consortia incubated at 60°C was inhibited by the addition of nitrate (NO3) or nitrite (NO 2 − ). Added nitrate (as nitrogen) at a concentration of 0.71 mM resulted in the production of 0.57–0.71 mM nitrite by the native microbial population present during souring and suppressed sulfate reduction to below detection limits. Nitrate added at 0.36 mM did not inhibit active souring but was enough to maintain inhibition if the column had been previously treated with 0.71 mM or greater. Continuous addition of 0.71–0.86 mM nitrite also completely inhibited souring in the column. Pulses of nitrite were more effective than the same amount of nitrite added continuously. Nitrite was more effective at inhibiting souring than was glutaraldehyde, and SRB recovery was delayed longer with nitrite than with glutaraldehyde. It was hypothesized that glutaraldehyde killed SRB while nitrite provided a long-term inhibition without cell death. Removal of nitrate after as long as 3 months of continuous addition allowed SRB in a biofilm to return to their previous level of activity. Inhibition was achieved with much lower levels of nitrate and nitrite, and at higher temperatures, than noted by other researchers.

Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements.

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An alignment of these sequences was used in a comparative analysis of RNase P RNA secondary and tertiary structure. The new sequences confirm the secondary structure model based on sequences from cultivated organisms (with minor alterations in helices P12 and P18), providing additional support for nearly every base pair. Analysis of sequence covariation using the entire RNase P RNA data set reveals elements of tertiary structure in the RNA; the third nucleotides (underlined) of the GNRA tetraloops L14 and L18 are seen to interact with adjacent Watson-Crick base pairs in helix P8, forming A:G/C or G:A/U base triples. These experiments demonstrate one way in which the enormous diversity of natural microbial populations can be used to elucidate molecular structure through comparative analysis.

Biodegradation of atrazine in surface soils and subsurface sediments collected from an agricultural research farm.

The purpose of the present study was to assess atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) mineralization by indigenous microbial communities and to investigate constraints associated with atrazine biodegradation in environmental samples collected from surface soil and subsurface zones at an agricultural site in Ohio. Atrazine mineralization in soil and sediment samples was monitored as 14CO2 evolution in biometers which were amended with 14C-labeled atrazine. Variables of interest were the position of the label ([U-14C-ring]-atrazine and [2-14C-ethyl]-atrazine), incubation temperature (25 degrees C and 10 degrees C), inoculation with a previously characterized atrazine-mineralizing bacterial isolate (M91-3), and the effect of sterilization prior to inoculation. In uninoculated biometers, mineralization rate constants declined with increasing sample depth. First-order mineralization rate constants were somewhat lower for [2-14C-ethyl]-atrazine when compared to those of [U-14C-ring]-atrazine. Moreover, the total amount of 14CO2 released was less with [2-14C-ethyl]-atrazine. Mineralization at 10 degrees C was slow and linear. In inoculated biometers, less 14CO2 was released in [2-14C-ethyl]-atrazine experiments as compared with [U-14C-ring]-atrazine probably as a result of assimilatory incorporation of 14C into biomass. The mineralization rate constants (k) and overall extents of mineralization (Pmax) were higher in biometers that were not sterilized prior to inoculation, suggesting that the native microbial populations in the sediments were contributing to the overall release of 14CO2 from [U-14C-ring]-atrazine and [2-14C-ethyl]-atrazine. A positive correlation between k and aqueous phase atrazine concentrations (Ceq) in the biometers was observed at 25 degrees C, suggesting that sorption of atrazine influenced mineralization rates. The sorption effect on atrazine mineralization was greatly diminished at 10 degrees C. It was concluded that sorption can limit biodegradation rates of weakly-sorbing solutes at high solid-to-solution ratios and at ambient surface temperatures if an active degrading population is present. Under vadose zone and subsurface aquifer conditions, however, low temperatures and the lack of degrading organisms are likely to be primary factors limiting the biodegradation of atrazine.

Analysis of the Virulence Characteristics of Bacteria Isolated from Bottled, Water Cooler, and Tap Water

A study was conducted to determine the numbers and types of bacteria found in three water sources—bottled water, water cooler water, and tap water—and to determine their virulence characteristics. A wide variety of water types were collected and each was analysed for Heterotrophic Plate Count (HPC) bacteria, Pseudomonas aeruginosa , and total coliforms. For each isolate, virulence characteristics were determined by enzyme analysis (10 associated with virulence), antibiotic susceptibility testing (natural and semi-synthetic antibiotics), acid lability (survival at pH 3-5), and cytotoxicity testing (HEp-2 cells). Results showed that all water sources had a normal bacterial content. Only 2 per cent of bottled water sources had P. aeruginosa . Total coliforms were isolated only from bottled water that used mixed (water alternating with milk) filling lines. Environmental bacteria did not produce significant enzymes associated with virulence, were not acid resistant, were susceptible to semi-synthetic antibiotics, and did not produce appreciable cytotoxicity. These natural aqueous bacteria were adopted to a water environment, did not grow well at conditions analogous to the human host, and did not have the characteristics associated with virulence. Future drinking water revisions and changes to the treatment processes should be directed towards the elimination of specific pathogens and to the prevention of exogenous sources of contamination rather than the elimination of natural water microbial populations. Keywords: virulence factors, drinking water, health risk, heterotrophic bacteria, water regulations.

Biodegradation of microcystin-LR by indigenous mixed bacterial populations

Microcystin-LR is a potent mammalian toxin which is known to have been responsible for the deaths of domesticated animals, and consequently there is concern as to its environmental fate. In laboratory experiments conducted using low levels (10 μg l −1) of microcystin-LR in reservoir water, primary biodegradation of the toxin was shown to occur in less than 1 week. An experiment designed to ascertain the degree of mineralisation of microcystin-LR, using natural microbial populations under aerobic conditions, showed that the peptide ring appears to be fairly resistant to biodegradation, as the degree of mineralisation was not high. However, as the toxicity of microcystin-LR is critically dependent on the orientation of the peptide ring with respect to the “Adda” (the β-amino acid which is unique to the blue-green algal toxins) side-chain, and it appears that this side chain is affected during biodegradation, it seems likely that a reduction in toxicity occurs.

Impact of a genetically engineered bacterium with enhanced alkaline phosphatase activity on marine phytoplankton communities

An indigenous marine Achromobacter sp. was isolated from coastal Georgia seawater and modified in the laboratory by introduction of a plasmid with a phoA hybrid gene that directed constitutive overproduction of alkaline phosphatase. The effects of this "indigenous" genetically engineered microorganism (GEM) on phosphorus cycling were determined in seawater microcosms following the addition of a model dissolved organic phosphorus compound, glycerol 3-phosphate, at a concentration of 1 or 10 (mu)M. Within 48 h, a 2- to 10-fold increase in the concentration of inorganic phosphate occurred in microcosms containing the GEM (added at an initial density equivalent to 8% of the total bacterial population) relative to controls containing only natural microbial populations, natural populations with the unmodified Achromobacter sp., or natural populations with the Achromobacter sp. containing the plasmid but not the phoA gene. Secondary effects of the GEM on the phytoplankton community were observed after several days, evident as sustained increases in phytoplankton biomass (up to 14-fold) over that in controls. Even in the absence of added glycerol 3-phosphate, a numerically stable GEM population (averaging 3 to 5% of culturable bacteria) was established within 2 to 3 weeks of introduction into seawater. Moreover, alkaline phosphatase activity in microcosms with the GEM was substantially higher than that in controls for up to 25 days, and microcosms containing the GEM maintained the potential for net phosphate accumulation above control levels for longer than 1 month.

Sequencing of (dA:dT) cloned mixed PCR products from microbial populations.

Because only between 1 and 10% of bacteria present in soil and aquatic environments are culturable using currently available methods, attempts to identify and quantify the nonculturable, majority population must circumvent the need for culture. Molecular biological techniques, particularly using 16S rRNA sequences, have substantial advantages over traditional culture-based methods for the characterization of natural microbial populations. One particularly interesting strategy involves the use of the polymerase chain reaction (PCR) to amplify target genes from DNA extracted directly from environmental samples. PCR-amplified target sequences can then be cloned to obtain a representative clone bank that reflects the diversity of a target sequence in the original population. After sequencing individual clones, comparative data analysis can be used to assess diversity among a natural population of microorganisms without the need to culture the organisms first.