Natural Microbial Populations Research Articles

Occurrence of lysogenic bacteria in marine microbial communities as determined by prophage induction

Viruses are abundant and dynamic members of the marine microbial community, and it is important to understand the11 role in the ecology of natural microbial populations We have previously found lysogenic bacteria to be a significant proportion (43%) of the cultivable heterotrophic microbial population. As the majority of marine bacteria are not cultivable using standard plating methods, we measured the proportion of marine lysogenic bacteria in natural communities by prophage ~nduction. Mitomycin C, UV radiation, sunlight, temperature and pressure were used to induce prophage in lysogenlc bacteria from estuarine, coastal and oligotrophic offshore environments. To determine ~f hydrocarbon pollutants may cause the induction of marine lysogens, aromatic and aliphatic hydrocarbons (including Bunker C #6 fuel oil, phenanthrene, naphthalene, pyrene, and trichloroethylene) were also used as inducing agents. Induct~on was most often found in estuarine environments, where viral direct counts increased from 128.8 to 345% of the uninduced control, resulting in mortality of 10.5 to 67.3% (average 34%) of the bacterial population. Up to 38% of the bacterial population was lysogenized in estuanne environments, as calculated from an average burst size. Microbial populations from oligotrophic offshore environments were inducible at 3 of 11 stations sampled. Eight of the 11 samples (73 %) treated with polyaromatic hydrocarbons resulted in prophage induction in natural populations. Time series analysis was also conducted in 2 samples induced by mitomycin C from the Atlantic Ocean near the coast of North Carolina, USA. For both samples, significant decreases in bacterial numbers were detected in treated samples after 8 h of incubation. A significant increase of viruses was detected a t 8 h at one station and at 24 h at the other station after induction. This study indicates that natural lysogenic populations are sensitive to a variety of ~nduc ing agents, and induction occurs more frequently in coastal and estuanne environments than offshore environments.

Chlorimuron Adsorption, Desorption, and Degradation in Soils from Conventional Tillage and No‐Tillage Systems

AbstractNo‐tillage (NT) and other conservation management practices designed to enhance plant residue accumulation can affect the herbicides fate in soil. This study was designed to evaluate adsorption, desorption, and degradation of chlorimuron {ethyl 2‐[[[[(4‐chloro‐6‐methoxy‐2‐pyrimidinyl)amino]carbonyl]amino] sulfonyl]benzoic acid} in soils collected from long‐term conventional tillage (CT) and NT plots. Soils were Miami silt loam (fine‐silty, mixed, mesic, Typic Hapludalf) and Drummer silty clay loam (fine‐silty, mixed, mesic, Typic Endoaquoll) from Illinois, and Dundee silt loam (fine‐silty, mixed, thermic Aeric Ochraqualf) from Mississippi. Adsorption was determined on five concentrations of 14C‐chlorimuron (0.22–12.26 µmol kg−1) equilibrated for 72 h. Desorption in four cycles (1, 24, 24, and 24 h) was measured for samples initially sorbed at 2.60 µmol kg−1. Adsorption Kf values ranged from 0.56 to 6.53 and were higher in NT compared with CT soils. The N values were less than unity in all soils. Desorption was hysteretic and the total amount desorbed in four desorptions ranged from 18 to 51% of that sorbed. Desorption Kf values ranged from 0.60 to 2.74 and were higher in NT compared with CT soils. After 63 d incubation of 14C‐chlorimuron, 44 to 54% of applied remained as extractable chlorimuron and 10 to 16% of applied mineralized to 14CO2 among the soils. Half‐life of over 2 mo and relatively low mineralization suggests poor adaptability of native microbial populations to chiorimuron as substrate. Differences in the chlorimuron degradation pattern were minor between NT and CT soils, despite NT soils in general having greater organic C, microbial populations, and soil enzyme activity compared with CT soils.

Oxidation of polymetal sulfides by chemolithoautotrophic bacteria from deep‐sea hydrothermal vents

Aerobic mesophilic sulfur‐oxidizing bacteria were tested for their ability to utilize a variety of natural and commercial polymetal sulfides as energy sources at near neutral pH. Substantial fixation of 14CO2 by natural microbial populations covering polymetal sulfide deposits was observed in in situ experiments conducted from DSV ALVIN at the Mid‐Atlantic Ridge hydrothermal vent sites at a depth of 3600 m. In shipboard experiments, similar rates were measured with natural populations on intact or pulverized sulfide minerals. In laboratory experiments with four vent isolates, the rate of 14CO2 incorporation depended on the particular strain and the type of polymetal sulfide substrate used. Vent sample material rich in chalcopyrite (CuFeS2) resulted in higher activities than on sphalerite (ZnS), galena (PbS), or chalcocite (CuS2) alone. Growth on polymetal sulfides, determined as protein, and I4CO2 fixation by strain MA‐3 (the most active of four isolates) were in the range of 5–10% of the corresponding values obtained on thiosulfate as the substrate. While the hydrothermal vent isolates lowered the pH to approximately 4.8 in thiosulfate medium, cultures grown on polymetal sulfides plateaued at higher pH values. Growth of isolates on polymetal sulfides was not enhanced by an addition of thiosulfate. Optimal growth occurred at near neutral pH on both the soluble and insoluble fractions of polymetal sulfides. The massive polymetal sulfide deposits at tectonic ocean spreading centers must be considered long‐term sources of electrons for the chemosynthetic production of biomass in the deep sea.

Existence and Development of Natural Microbial Populations in Wooden Storage Vats Used for Alcoholic Cider Maturation

Wooden maturation vats, traditionally used in the fruit-based alcoholic beverage industry, are gradually being superseded by vessels constructed from sterilizable materials, such as stainless steel...

Microbial degradation of paraquat sorbed to plant residues.

It was demonstrated that paraquat (1,1'-dimethyl-4,4'-bipyridynium dichloride) sorbed to plant residues was degraded by natural microbial populations associated with plants and/or soil under laboratory conditions. The microorganisms associated with plants showed a higher degradation rate than those from soil. The degradation rate was higher in rice straw (C/N ratio = 53) than in dropwort (C/N = 20) and Chinese milk vetch (C/N = 17). Urea, ammonium chloride, and sodium nitrate suppressed the paraquat degradation rate by microorganisms associated with rice straw, suggesting a relationship to nitrogen metabolism. The degradation rate was much higher under aerobic conditions than under anaerobic conditions. When rice straw containing sorbed paraquat was placed on the surface of soil or mixed with soil, the herbicide was degraded under the former condition but not under the latter condition. Monopyridone (1',2'-dihydro-1,1'-dimethyl-2'-oxo-4,4'-bipyridynium ion) and 14 CO 2 were detected as metabolites in rice straw spiked with [methyl- 14 C 2 ]-paraquat.

Electrical resistance tomography during in-situ trichloroethylene remediation at the Savannah River Site

Electrical resistance tomography was used to monitor in-situ remediation processes for removal of volatile organic compounds from subsurface water and soil at the Savannah River Site near Aiken, South Carolina. This work was designed to test the feasibility of injecting a weak mixture of methane in air as a metabolic carbon source for natural microbial populations which are capable of trichloroethylene degradation. Electrical resistance tomograms were constructed of the subsurface during the test to provide detailed images of the process. These images were made using an iterative reconstruction algorithm based on a finite element forward model and Newton-type least-squares minimization. Changes in the subsurface resistivity distribution were imaged by a pixel-by-pixel subtraction of images taken before and during the process. This differential tomography removed all static features of formation resistivity but clearly delineated dynamic features induced by remediation processes. The air-methane mixture was injected into the saturated zone and the intrained air migration paths were tomographically imaged by the increased resistivity of the path as air displaced formation water. We found the flow paths to be confined to a complex three-dimensional network of channels, some of which extended as far as 30 m from the injection well. These channels were not entirely stable over a period of months since new channels appeared to form with time. Also, the resistivity of the air injection paths increased with time. In another series of tests, resistivity images of water infiltration from the surface support similar conclusions about the preferential permeability paths in the vadose zone. In this case, the water infiltration front is confined to narrow channels which have a three-dimensional structure. Here, similar to air injection in the saturated zone, the water flow is controlled by local variations in formation permeability. However, temporal changes in these channels are minor, indicating that the permeable paths do not seem to be modified by continued infiltration.

Existence and Development of Natural Microbial Populations in Wooden Storage Vats Used for Alcoholic Cider Maturation

Wooden maturation vats, traditionally used in the fruit-based alcoholic beverage industry, are gradually being superseded by vessels constructed from sterilizable materials, such as stainless steel. However, it is shown that the surfaces of existing vats, even after cleaning, possess a unique, individual microbial profile. One 10,000-gallon oak vat was studied over a nine-week maturation period, and the changing microbial profile within the fermented cider was recorded. Microbial colonization of wood was also examined by suspending cubes of unused oak in the cider vat and visualized using scanning electron microscopy. A variety of microorganisms progressively colonized the porous structure of wood, which once established, would appear to persist throughout emptying, cleaning, and refilling cycles.

Oligosaccharide degradation by anaerobic marine bacteria: Characterization of an experimental system to study polymer degradation in sediments

Enrichment of bacteria from anoxic marine sediments on specific carbohydrates yielded a reproducible model system with which to compare the degradation of a set of structurally related substrates. The goal was to approximate thc diversity of natural mixed microbial populations while reducing the complexity of studying the degradation of specific substrates in sediments. Structurally related substrates were used to compare the influence of substrate size, chemical linkage, and stereochemistry on carbohydrate degradation. Headspace gases (CO2, H2, CH4, H2S) were monitored by gas chromatography, and carbohydrate substrates were separated and quantified with high‐pressure liquid chromatography. The sequence of transformations observed in the cultures is consistent with the sequential substrate degradation expected in a consortium of anaerobic bacteria, and the transfer of carbon from substrates through to end products was followed quantitatively. A series of experiments showed that mixed cultures of marine bacteria distinguish between even small, closely related substrates that do not require extracellular hydrolysis prior to uptake. A galactose‐β(1,3)‐arabinose disaccharide (GLA) was degraded at half the rate of seven other similar disaccharides and three larger oligosaccharides. Results from transfer cultures indicate that the ability to degrade GLA probably is not widespread among bacteria. Although neither the component monomers of GLA nor its β(1,3) linkage is uncommon in marine environments, its specific combination of a hexose and a pentose may not fit standard carbohydrate transport systems in bacteria. Substrate structural features clearly can have a significant effect on degradation rates, even at molecular weights below the direct transport size limit.

Detection of soluble methane monooxygenase producing Methylosinus trichosporium OB3b by polymerase chain reaction.

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrophs cometabolizes haloaliphatic compounds such as trichloroethylene. Two 18-mer oligonucleotides as primary primers and a nested primer of the same length were selected to amplify specific DNA sequences of the sMMO gene cluster using polymerase chain reaction (PCR). Two DNA fragments of sizes 270 and 400 base pairs were obtained when purified DNA from the methanotroph Methylosinus trichosporium OB3b was used as template. The primers were specific for sMMO sequences of M. trichosporium, since none of the 13 bacterial isolates screened yielded the expected length of PCR-amplified DNA fragments. The detection limit of the PCR method was 5 x 10(2) cells of M. trichosporium. The sMMO sequences were successfully amplified in groundwater (containing native microbial population) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCGCTCATAAC-3'), RP1 antisense (5'-TCAGATGTCGGTCAGGGC-3'), FP2 sense nested (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGACATC-3').

Relative Role of Eukaryotic and Prokaryotic Microorganisms in Phenanthrene Transformation in Coastal Sediments

THE RELATIVE ROLE OF EUKARYOTIC VERSUS PROKARYOTIC MICROORGANISMS IN PHENANTHRENE TRANSFORMATION WAS MEASURED IN SLURRIES OF COASTAL SEDIMENT BY TWO DIFFERENT APPROACHES: detection of marker metabolites and use of selective inhibitors on phenanthrene biotransformation. Phenanthrene biotransformation was measured by polar metabolite formation and CO(2) evolution from [9-C]phenanthrene. Radiolabeled metabolites were tentatively identified by high-performance liquid chromatography (HPLC) separation combined with UV/visible spectral analysis of HPLC peaks and comparison to authentic standards. Both yeasts and bacteria transformed phenanthrene in slurries of coastal sediment. Two products of phenanthrene oxidation by fungi, phenanthrene trans-3,4-dihydrodiol and 3-phenanthrol, were produced in yeast-inoculated sterile sediment. However, only products of phenanthrene oxidation typical of bacterial transformation, 1-hydroxy-2-naphthoic acid and phenanthrene cis-3,4-dihydrodiol, were isolated from slurries of coastal sediment with natural microbial populations. Phenanthrene trans-dihydrodiols or other products of fungal oxidation of phenanthrene were not detected in the slurry containing a natural microbial population. A predominant role for bacterial transformation of phenanthrene was also suggested from selective inhibitor experiments. Addition of streptomycin to slurries, at a concentration which suppressed bacterial viable counts and rates of [methyl-H]thymidine uptake, completely inhibited phenanthrene transformation. Treatment with colchicine, at a concentration which suppressed yeast viable counts, depressed phenanthrene transformation by 40%, and this was likely due to nontarget inhibition of bacterial activity. The relative contribution of eukaryotic microorganisms to phenanthrene transformation in inoculated sterile sediment was estimated to be less than 3% of the total activity. We conclude that the predominant degraders of phenanthrene in muddy coastal sediments are bacteria and not eukaryotic microorganisms.

Radiation Resistance of the Natural Microbial Population in Buffer Materials

AbstractThe radiation sensitivity of naturally occurring microorganisms in buffer materials vas investigated as veil as the sensitivity ofBacillus subtilisspores andAcinetobacter radioresistensin a buffer matrix. The D10values obtained in our radiation experiments varied from 0.34 to 1.68 kGy and it vas calculated that the surface of a nuclear fuel vaste container vould be sterilized in 9 to 33 d after emplacement, depending on the type of container, and the initial bioburden. This suggests that formation of biofilms and microbially influenced corrosion vould not be of concern for some time after emplacement. The results also indicated that sterilization throughout a 25 cm thick buffer layer is unlikely and that repopulation of the container surface after some time is a possibility, depending on the mobility of microbes in compacted buffer material.

Ribulose bisphosphate carboxylase gene expression in subtropical marine phytoplankton populations

Oceanic phytoplankton are known to fix CO2 primarily through the action of the enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase). The amino acid and nucleotide sequence of the large subunit of this enzyme have been conserved across the evolution of the chlorophytic plants (from cyanobacteria to higher plants via green algae) with approximately 80 and 70 % homology at the amino acid and nucleotide levels respectively. To understand the molecular regulation of this enzyme in phytoplankton, we have measured levels of the RuBPCase large subunit (rbcL) mRNA and DNA, in combination with rates of photosynthetic CO2 fixation, autofluorescent cell counts, and chlorophyll a in natural phytoplankton communities of Tampa Bay (Flonda, USA) and the southeastern Gulf of Mexico. We measured rbcL mRNA, rbcL DNA, and the ratio of rbcL mRNA to rbcL DNA by extracting RNA and DNA and probing the extracts with the Synechococcus PCC 6301 rbcL gene as a probe. Additionally, rbcL mRNA was amplified from certain samples using a reverse transcriptase-linked polymerase chain reaction procedure. In a transect from Tampa Bay seaward, levels of rbcL mRNA decreased 3to 8-fold from the estuarine environment to the offshore environment, and followed similar trends as photosynthetic CO2 fixation, picocyanobacterial counts, and chlorophyll a. In an offshore vertical profile, the subsurface maximum in the rbcL mRNA/DNA ratio coincided with the 60 m maximum in photosynthetic assimilation rates. In a die1 study using 150 1 of offshore water in a deck-top incubator, the rbcL mRNA.DNA ratio was nearly an order of magnitude greater (76 ng mRNA ng-' DNA) during the light period than in the dark (17 ng mRNA ng-' DNA). Likewise, the photosynthetic rate (PB) at constant illun~ination was highest during daylight hours (6.2 pg C pg-' chl a h-') and lowest at night (1.9 pg C pg-' chl a h-'). These results indicate that (1) carbon fixation in oceanic phytoplankton may be transcriptionally regulated at the level of the RuBPCase gene and (2) that the quantitatlon of particular target mRNAs and DNAs is an effective means to study the regulation of conserved gene functions in natural water column microbial populations.

Förderung von symbiose und Wachstum bei Luzerne durch kombinierte Beimpfung mit Rhizobium meliloti und Pseudomonas fluorescens

Summary Combined inoculation of the strains Rhizobium meliloti mel8 and Pseudomonas fluorescens PsIA12 to lucerne stimulated the development of young plants more than a single inoculation with Rhizobium . In field experiment about 2 years on Albic luvisol soil the mixed inoculants enhanced the yields of dry matter and raw protein and the uptake of phosphorus and potassium in shoots. As mechanisms of synergistic co-inoculation effects were observed increases in root surface area and stimulation of nodulation and nitrogenase activity in early stage of plant development. In addition there was a high significant increase in the activity of uptake hydrogenase in 10 7 mixed bacterial cells under ex planta conditions. The co-inoculation of both bacterial strains has effected the native microbial population in rhizosphere.

Light butter: natural microbial population and potential growth of Listeria monocytogenes and Yersinia enterocolitica

Yarrowia lipolytica, Bacillus polymyxa and Enterococcus faecium were the most frequent yeast and bacterial spoilage species associated with commercial light butter. Following inoculation and incubation at 4°C, strains of Y. enterocolitica and L. monocytogenes exhibited growth rates higher than those of the naturally occurring microflora. Listeria monocytogenes displayed a higher aptitude to proliferate in such food while the cell increment of Y. enterocolitica was limited.

Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss.

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)

Methanotrophs for Clean-Up of Polluted Aquifers

Aquifers are vital reserves of drinking water which are under threat from pollution. Particular problems are posed by chlorinated compounds such as pesticides and solvents which native microbial populations are unable to degrade. Pump and treat regimes have proved unsuccessful since pollutants remain adsorbed to sediments but a possible solution is the use of introduced microorganisms to degrade pollutants in-situ. It is suggested that methanotrophs may be suitable candidates. Methanotrophs have an extraordinary range of degradative powers due to the non-specificity of their methane mono-oxygenase enzyme. We have shown that Methylosinus trichosporium OB3b is capable of degrading many common chlorinated pollutants co-metabolically when it is grown in a copper-depleted, oxygen-rich medium at neutral pH. In the subsurface however, such conditions do not exist and cultures grown in a medium made with untreated Cambridge aquifer water have a reduced range of degradative powers compared to similar cells grown in a medium made with distilled water. This means that to use methanotrophs for aquifer clean-up, the cells may need to be cultured above ground in ideal conditions and then introduced by some method of injection or infiltration. This may be possible because the degradative reactions are not coupled to growth and Methylosinus trichosporium OB3b cells maintain pollutant degrading ability up to 19 days after they have stopped growing. A suspension of these cells may thus be treated as a biocatalyst.

Bioremediation of groundwater pollution.

Significant progress has been made in the past year towards an understanding of the microbial processes in subsurface environments that may allow natural microbial populations to be employed for bioremediation of groundwater pollution. Among the highlights were: the discovery of several previously unknown xenobiotic-degrading abilities in groundwater microorganisms; progress in using the unique abilities of methanotrophs to oxidize halogenated solvents; and characterizations of microbial populations from subsurface soils.

Overview of a Large-Scale Bioremediation Soil Treatment Project

How long does it take to remediate 290,000 yd{sup 3} of impacted soil containing an average total petroleum hydrocarbon concentration of 3,000 ppm Approximately 15 months from start to end of treatment using bioremediation. Mittelhauser was retained by the seller of the property (a major oil company) as technical manager to supervise remediation of a 45-ac parcel in the Los Angeles basin. Mittelhauser completed site characterization, negotiated clean-up levels with the regulatory agencies, and prepared the remedial action plan (RAP) with which the treatment approach was approved and permitted. The RAP outlined the excavation, treatment, and recompaction procedures for the impacted soil resulting from leakage of bunker fuel oil from a large surface impoundment. The impacted soil was treated on site in unline Land Treatment Units (LTUs) in 18-in.-thick lifts. Due to space restraints, multiple lifts site. The native microbial population was cultivated using soil stabilization mixing equipment with the application of water and agricultural grade fertilizers. Costs on this multimillion dollar project are broken down as follows: general contractor cost (47%), bioremediation subcontractor cost (35%), site characterization (10%), technical management (7%), analytical services (3%), RAP preparation and permitting (1%), and civil engineering subcontractor cost (1%). Start-up of field workmore » could have been severely impacted by the existence of Red Fox habitation. The foxes were successfully relocated prior to start of field work.« less

Amino acid and ammonium utilization by heterotrophic marine bacteria grown in enriched seawater

The nitrogen metabolism of natural bacterial assemblages was studied in seawater cultures enriched with the dissolved materials released by the mussel Mytilus edulis. The mussel released dissolved organic C, NH4+, and dissolved organic N (DON), mostly in the form of combined amino acids (∼70–100%) that constituted 30% of the total N released. 15NH4+ techniques showed that the contribution of NH4+ to total microbial N demand during growth ranged from 50 to 88% even when large amounts of DON were utilized (∼70–260% of total bacterial N produced). An average of 80% of the DON used was converted to NH4+, showing simultaneous assimilation and regeneration of NH4+ by the natural microbial population and contradicting the idea that NH4+ assimilation occurs only when organic N is limiting.

Production of biologically refractory dissolved organic carbon by natural seawater microbial populations

Carbon from glucose and leucine added at natural concentrations to seawater was biologically transformed to higher molecular weight (mol. wt) dissolved materials that persisted through 6 months of incubation. At the end of incubation, the amount of carbon in high mol. wt dissolved fractions was approximately equal to the amount of carbon incorporated into particulate fractions. In tests of their resistance to biological utilization, only 1–17% of the high mol. wt materials were respired when re-incubated with seawater microbial populations, whereas 40–75% of the monomers were respired over the same time span. In situ transformations of biologically available carbon may be an important source of refractory dissolved organic carbon in the oceans.