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Comparison of the fuel oil biodegradation potential of hydrocarbon-assimilating microorganisms isolated from a temperate agricultural soil.

Strains of hydrocarbon-degrading microorganisms (bacteria and fungi) were isolated from an agricultural soil in France. In a field, a portion was treated with oily cuttings resulting from the drilling of an onshore well. The cuttings which were spread at the rate of 600 g HC m-2 contained 10% of fuel oil hydrocarbons (HC). Another part of the field was left untreated. Three months after HC spreading, HC adapted bacteria and fungi were isolated at different soil depths in the two plots and identified. The biodegradation potential of the isolated strains was monitored by measuring the degradation rate of total HC, saturated hydrocarbons, aromatic hydrocarbons and resins of the fuel. Bacteria of the genera Pseudomonas, Brevundimonas Sphingomonas, Acinetobacter, Rhodococcus, Arthrobacter, Corynebacterium and fungi belonging to Aspergillus, Penicillium, Beauveria, Acremonium, Cladosporium, Fusarium, and Trichoderma were identified. The most active strains in the assimilation of saturates and aromatics were Arthrobacter sp., Sphingomonas spiritivorum, Acinetobacter baumanii, Beauveria alba and Penicillum simplicissimum. The biodegradation potential of the hydrocarbon utilizing microorganisms isolated from polluted or unpolluted soils were similar. In laboratory pure cultures, saturated HC were more degraded than aromatic HC, whereas resins were resistant to microbial attack. On an average, individual bacterial strains were more active than fungi in HC biodegradation.

Bacteria↗

Antibodies in normal and diseased pulps reactive with microorganisms isolated from deep caries.

Immunoglobulin molecules in the supernatant fluids (SF) from pulpal explant cultures have been observed to react with microorganisms implicated in infections of root canals. In this study, the reactivity of immunoglobulin molecules in the SF from normal and irreversible pulpitis pulps to six strains of predominant microorganisms isolated from the immediate layer of carious lesions above the pulps used for explant cultures was investigated using an enzyme-linked immunosorbent assay. Two ATCC strains of Eubacterium were also included in this assay. Specific antibodies to Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lactobacillus acidophilus (I), (II), Streptococcus mutans, Bacteroides intermedius, Eubacterium brachy, and Eubacterium alactolyticum in the SF from the normal and irreversible pulpitis tissues were observed with a large variation of antibody levels in both groups. Immunodiffusion assays of the SF revealed that IgG was the major class of immunoglobulin in the normal as well as the irreversible groups. The presence of natural antibodies in the normal pulps suggested a possible protective role of antibodies during the invasive process of caries.

Antibodies, Bacterial↗

Reactivity of IgG from explant cultures of periapical lesions with implicated microorganisms.

The presence of IgG in periapical inflammatory lesions suggests that immune responses participate in the disease process. The purpose of this investigation was to study the reactivity of IgG from the supernatant fluids of explant cultures of periapical lesions with microorganisms implicated in infections of endodontic origin. Ninety periapical lesions that had been contiguous with the apex of a root were removed and maintained in explant cultures. A dot-enzyme-linked immunosorbent assay (dot-ELISA) was used to demonstrate the presence of IgG in the supernatant fluids of the explant cultures reactive with a panel of microorganisms associated with infections of endodontic origin. The percentages of reactivity by dot-ELISA follow: Bacteroides intermedius (84%), B. buccae (12%), Porphyromonas (Bacteroides) gingivalis (50%), P. endodontalis (58%), P. asaccharolyticus (17%), Peptostreptococcus micros (44%), P. anaerobius (26%), Eubacterium alactolyticum (34%), Fusobacterium nucleatum (14%), and Actinomyces israelii (6%). At least one of the three species of B. intermedius, P. gingivalis, or P. endodontalis tested gave a positive dot-ELISA with 89% of the supernatant fluids from explant cultures of periapical lesions. A lack of cross reactivity of IgG in supernatant fluids from explants of periapical lesions was demonstrated for the four strains of black-pigmented Bacteroides/Porphyromonas by dot-ELISA.

Actinomyces↗

Influence of microorganisms on the environmental fate of radionuclides.

Microorganisms have a significant influence on the environmental fate of radionuclides in aquatic and terrestrial ecosystems with a multiplicity of physico-chemical and biological mechanisms effecting changes in mobility and speciation. Physico-chemical mechanisms of removal include association with extracellular materials, metabolites and cell walls which are features of living and dead organisms. In living cells, some physico-chemical processes are reversible, influenced by metabolism and changing environmental conditions. Metabolism-dependent mechanisms of radionuclide immobilization include sulphide precipitation, transport and intracellular compartmentation and/or sequestration by proteins and peptides. In addition, chemical reduction to less soluble forms can result in immobilization. Microbial processes involved in radionuclide solubilization include autotrophic and heterotrophic leaching, and complexation by siderophores and other metabolites. Such mechanisms are important components of biogeochemical cycles for radionuclides and should be considered in any analyses of environmental radionuclide contamination. In addition, several microorganism-based biotechnologies are receiving interest as potential treatment methods.

Absorption↗

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.

Bacteria, Aerobic↗

Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry.

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.

Bacteria↗

A rapid and specific method to screen environmental microorganisms for cephalosporin acylase activity.

Medically useful semisynthetic cephalosporin antibiotics are made from precursor 7-aminocephalosporanic acid (7-ACA). Cephalosporin acylase (CA), which catalyzes hydrolysis of both glutaryl-7-aminocephalosporanic acid (GL-7ACA) and cephalosporin C (CPC) to 7-ACA, is thus a very important enzyme for producing semisynthetic beta-lactam antibiotics. To facilitate the attempts of obtaining the microorganisms with higher CA activity from natural environments, a new and specific method for screening environmental microorganisms with cephalosporin acylase activity was developed. The core part of cephalosporin was replaced by 6-amino penicillinic acid (6-APA) to generate new substrates glutaryl-6-APA and adipoyl-6-APA for screening. Serratia marcescens that is sensitive to 6-APA and resistant to penicillin G, glutaryl-6-APA and adipoyl-6-APA was used as an indicator strain in an overlaid-agar screening system. A strain capable of producing cephalosporin acylase was selected from thousands of candidates by this method. Because of its specificity, simplicity and sensitivity, the method could be easily installed into a high-throughout system.

Bacteria↗

PCR-based detection of non-indigenous microorganisms in 'pristine' environments.

PCR-based technologies are widely employed for the detection of specific microorganisms, and may be applied to the identification of non-indigenous microorganisms in 'pristine' environments. For 'pristine' environments such as those found on the Antarctic continent, the application of these methods to the assessment of environmental contamination from human activities must be treated with caution. Issues such as the possibility of non-human dispersal of organisms, stability and survival of non-indigenous organisms in vivo, the sensitivity, reproducibility and specificity of the PCR process (and particularly primer design) and the sampling regime employed must all be considered in detail. We conclude that despite these limitations, PCR and related technologies offer enormous scope for assessment of both natural and non-indigenous microbial distributions.

Antarctic Regions↗

Modification of A-stat for the characterization of microorganisms.

Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms. Common for those cultivation methods is that one environmental parameter is programmed to change with constant change rate (change-stat) while the others are kept constant or in the range not affecting the growth characteristics. The environment response growth curves were obtained starting with chemostat (in A-stat and D-stat) or auxostat (in auxo-accelerostat) steady-state cultures followed by change of set-point value of the desired cultivation parameter. Physiological studies of Saccharomyces sp. and Lactococcus lactis were combined with validation of the different modifications of the A-stat method based on well-known cultivation techniques: chemostat, pH-auxostat, pO(2)-auxostat CO(2)-auxostat and fed-batch. The auxo-accelerostat was shown to be very efficient for cell characterization and dynamic studies in growth environments with excess of essential substrates. Choosing the rate of change of environmental parameters was shown to be critical in comparative physiological studies of microorganisms.

Carbon Dioxide↗

Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.

Biotechnology↗

In situ imaging of microorganisms in geologic material.

In order to fully delineate the interactions of microorganisms with geological substrates, unequivocal identification of intact microbial cells within geologic samples is required without the disruption of either the rock texture or the relationship of the microorganisms to the mineral fabric. To achieve this objective we developed a protocol that enables the visualization of intact microbial cells in petrographic thin sections, avoids detaching the cells from their host mineral surfaces and avoids microbial contamination during the lapidary process. Propidium iodide and POPO-3, nucleic acid stains that specifically target double-stranded DNA and RNA were utilized for in situ visualization of cells in surface and subsurface basalts from northeastern Idaho. Additionally, examination of samples incubated with acetic acid-UL-14C via phosphor imagining facilitated the in situ visualization of 14C labeled biomass. Biomass observed was low (<10(7) cells/g). These observations indicate that the microbial distribution in these rocks exhibits a high degree of spatial heterogeneity at the sub-centimeter scale.

Bacteria↗

Industrial wastewater bioreactors: sources of novel microorganisms for biotechnology.

Microorganisms exist in nature as members of complex, mixed communities. The microbial communities in industrial wastewater bioreactors can be used as model systems to study the evolution of new metabolic pathways in natural ecosystems. The evolution of microbial metabolic capability in these bioreactors is presumably analogous to phenomena that occur in natural ecosystems. The microorganisms in these bioreactors compete for different carbon sources and constantly have to evolve new metabolic capabilities for survival. Thus, industrial bioreactors should be a rich source of novel biocatalysts.

Bacteria↗

Histamine and tyramine degradation by food fermenting microorganisms.

Microorganisms suitable for food fermentation were examined with regard to their potential to degrade histamine and tyramine. Out of 64 lactic acid bacteria evaluated in this study, 27 degraded histamine and one tyramine, respectively, with low activity. Among 32 strains of Brevibacterium linens and coryneform bacteria, 21 exhibited histamine and tyramine oxidase activity. None of 20 strains of Staphylococcus carnosus tested degraded histamine or tyramine. One strain out of nine strains of Geotrichum candidum degraded tyramine slightly. Among 44 strains of Micrococcus sp. examined, 17 degraded either one or two biogenic amines. In this study Micrococcus varians (M. varians) LTH 1540 exhibited the highest tyramine oxidase activity of all strains tested and was therefore investigated in detail. The enzyme was found to be located in the cytoplasm and was not membrane bound. The reaction end product p-hydroxyphenyl acetic acid was detected by HPLC analysis. An activity staining for the amine oxidase in a native polyacrylamide gel based on the formation of H2O2 during amine oxidation was developed. Resting cells of the strain exhibited optimal tyramine oxidase activity at a pH of 7 at 37-40 degrees C. The enzyme in the cell free extract had a pH optimum between 7-8. The enzyme activity was decreased by NaCl, glucose and hydralazine. Phenylethylamine and tryptamine were oxidized at lower concentrations than tyramine. The potential for amine degradation was not found to be associated with that of formation of biogenic amines, as 23 microorganisms with the ability to metabolise biogenic amines exhibited no decarboxylase activity toward histidine, tyrosine, phenylalanine, lysine or ornithine.

Fermentation↗

The evolution of social behavior in microorganisms.

Recent studies of microorganisms have revealed diverse complex social behaviors, including cooperation in foraging, building, reproducing, dispersing and communicating. These microorganisms should provide novel, tractable systems for the analysis of social evolution. The application of evolutionary and ecological theory to understanding their behavior will aid in developing better means to control the many pathogenic bacteria that use social interactions to affect humans.

Journal Article↗

Essential interactions between Thiobacillus ferrooxidans and heterotrophic microorganisms during a wastewater sludge bioleaching process.

The stimulating effect of heterotrophic microorganisms was investigated on the growth and on the ferrous iron oxidation of Thiobacillus ferrooxidans in synthetic media and in wastewater sludge. The addition of a sediment. Rhodotorula rubra isolate or a strain of T. acidophilus on two-layer agarose-gelled medium doubled the plating efficiency of T. ferrooxidans. In liquid cultures, R. rubra had a slight but significant effect on the growth rate of T. ferrooxidans. Moreover, the yeast allowed a faster initiation of the ferrous iron oxidation and acidification by T. ferrooxidans. In the bioleaching process, the co-culture of T. ferrooxidans with R. rubra or with the indigenous microbial assemblage from sludge was shown to be essential since the pure culture of T. ferrooxidans failed to oxidize ferrous iron and to acidify wastewater sludge. These results emphasize the importance of active heterotrophic microorganisms in the metal bioleaching activity of T. ferrooxidans in sludge.

Journal Article↗

The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments.

Indigenous mixed populations of anaerobic microorganisms from an irrigation tailwater drain and submerged agricultural chemical waste pit readily biodegraded the major isomer of endosulfan (endosulfan I). Endosulfan I was biodegraded to endosulfan diol, a low toxicity degradation product, in the presence of organic carbon sources under anaerobic, methanogenic conditions. While there was extensive degradation (>85%) over the 30 days, there was no significant enhancement of degradation from enriched inocula. This study demonstrates that endosulfan I has the potential to be biodegraded in sediments, in the absence of enriched microorganisms. This is of particular importance since such sediments are prevalent in cotton-growing areas and are typically contaminated with endosulfan residues. The importance of minimizing non-biological losses has also been highlighted as a critical issue in determining anaerobic biodegradation potential. Seals for such incubation vessels must be both oxygen and pollutant impermeable. Teflon-lined butyl rubber provides such a seal because of its resistance to the absorption of volatiles and in preventing volatilization. Moreover, including a 100 mM phosphate buffer in the anaerobic media has reduced non-biological losses from chemical hydrolysis, allowing biodegradation to be assessed.

Journal Article↗

Heat-killed microorganisms induce PAI-1 expression in human peritoneal mesothelial cells: role of interleukin-1alpha.

Human peritoneal mesothelial cells (HMCs) have a critical role in maintaining the intraperitoneal balance between fibrinolysis and coagulation by expressing the fibrinolytic enzyme, tissue-type plasminogen activator (tPA), as well as a specific plasminogen activator inhibitor (type 1; PAI-1). During bacterial peritonitis, the balance between intraperitoneal generation and degradation of fibrin is disturbed. As a consequence, severe peritoneal damage occurs, which is one of the leading causes of patient dropout from continuous ambulatory peritoneal dialysis (CAPD) therapy. Cultured HMCs isolated from omental biopsy specimens were used to study the effect of heat-killed strains (2 x 10(8)/mL) of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli on the synthesis of tPA and PAI-1. Conditioned media were obtained by incubating cells with the different bacterial strains. tPA and PAI-1 antigen concentrations were measured in the cell supernatants by enzyme-linked immunosorbent assay. Each of the three heat-killed microorganisms induced a time-dependent increase in PAI-1 synthesis. After a 48-hour incubation period, the strongest effect was seen in the presence of S aureus (3.5-fold versus control), followed by S epidermidis (2.5-fold versus control) and E coli (1.5-fold versus control). Under the same conditions, tPA antigen levels did not change after exposure to S aureus or E coli, whereas the addition of S epidermidis resulted in enhanced tPA antigen production (2-fold versus control). The increase in PAI-1 synthesis in the presence of the heat-killed microorganisms was preceded by similar changes in interleukin-1alpha (IL-1alpha) levels. Inhibiting the activity of IL-1alpha with a neutralizing antibody significantly reduced bacterial-induced PAI-1 production. Our results indicate that the fibrinolytic imbalance during bacterial peritonitis depends on the bacterial species. The increase in PAI-1 synthesis, not the decrease in the production of tPA, alters mesothelial fibrinolytic activity. Because the increase in PAI-1 expression is significantly quenched by blocking the activity of IL-1alpha, the mesothelial release of this cytokine is involved in bacterial-induced changes in the fibrinolytic system.

Bacterial Vaccines↗

Functional, regulatory and indicator features of microorganisms in man-made ecosystems.

Functional, regulatory and indicator features of microorganisms in development and functioning of the systems and sustaining stability of three man-made ecosystem types has been studied. 1) The functional (metabolic) feature was studied in aquatic ecosystems of biological treatment of sewage waters for the reducer component. 2) The regulatory feature of bacteria for plants (producer component) was studied in simple terrestrial systems "wheat plants-rhizospheric microorganisms-artificial soil" where the behavior of the system varied with activity of the microbial component. For example with atmospheric carbon dioxide content elevated microbes promote intensification of photosynthesis processes, without binding the carbon in the plant biomass. 3) The indicator feature for the humans (consumer component) was studied in Life Support Systems (LSS). High sensitivity of human microflora to system conditions allowed its use as an indicator of the state of both system components and the entire ecosystem. Grant numbers: N99-04-96017, N15.

Biomass↗