Microbial Dynamics of an Epilithic Mat Community in a High Alpine Stream

Studies were conducted to examine interrelationships between the heterotrophic and phototrophic populations within an epilithic community in the outlet stream of a high alpine lake. Levels of nitrates, phosphates, and total organic compounds in the lake were consistently near the lower limits of detectability. Microscopic examination of the community by phase-contrast light microscopy and scanning electron microscopy revealed diatoms, filamentous algae, and bacteria embedded within a dense gelatinous matrix. Chlorophyll a and primary productivity measurements had peak values in early August, with subsequent declines. Bacterial heterotrophic activity, as measured by V_max, turnover rate, and relative activity, increased significantly as the phototrophic community declined. This trend in heterotrophic activity was not accompanied by an increase in total bacterial numbers as determined by epi-illuminated fluorescence microscopy. These results suggest that the phototrophic community responded to changes in, or interactions among, various chemical and physical factors throughout the study period. The catabolic activity of the sessile bacteria appeared to be positively influenced by changes in the mat environment resulting from the decline of the phototrophic populations.     

Structure of planktonic microbial food web in a brackish stratified Siberian lake

The distribution of primary components of the microbial community (autotrophic pico- and nanoplankton, phototrophic bacteria, heterotrophic bacteria, microscopic fungi, heterotrophic flagellates, ciliates and heliozoa) in the water column of Lake Shira, a steppe brackish-water, stratified lake in Khakasia, Siberia (Russia), were assessed in midsummer. Bacterioplankton was the main component of the planktonic microbial community, accounting for 65.3 to 75.7% of the total microbial biomass. The maximum concentration of heterotrophic bacteria were recorded in the monimolimnion of the lake. Autotrophic microorganisms contributed more significantly to the total microbial biomass in the pelagic zone (20.2–26.5%) than in the littoral zone of the lake (8.7–14.9%). First of all, it is caused by development of phototrophic sulphur bacteria at the oxic-anoxic boundary. The concentrations of most aerobic phototrophic and heterotrophic microorganisms were maximal in the upper mixolimnion. Heterotrophic flagellates dominated the protozoan populations. Ciliates were minor component of the planktonic microbial community of the lake. Heterotrophic flagellates were the most diverse group of planktonic eucaryotes in the lake, which represented by 36 species. Facultative and obligate anaerobic flagellates were revealed in the monimolimnion. There were four species of Heliozoa and only three of ciliates in the lake.     

Technique for Enumeration of Heterotrophic and Phototrophic Nanoplankton, Using Epifluorescence Microscopy, and Comparison with Other Procedures

A new method is described that uses the fluorochrome primulin and epifluorescence microscopy for the enumeration of heterotrophic and phototrophic nanoplankton (2 to 20 μm). Phototrophic microorganisms are distinguished from heterotrophs by the red autofluorescence of chlorophyll a. Separate filter sets are used which allow visualization of the primulin-stained nanoplankton without masking chlorophyll a fluorescence, thus allowing easy recognition of phototrophic cells. Comparison with existing epifluorescence techniques for counting heterotrophic and phototrophic nanoplankton shows that primulin provides more accurate counts of these populations than the fluorescein isothiocyanate or proflavine techniques. Accuracy is comparable to that with the acridine orange technique, but this method requires only one filter preparation for the enumeration of both phototrophic and heterotrophic populations.     

Study of ectoparasitism of ultramicrobacteria of the genus Kaistia, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gram-negative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated, but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

Specimen Holder to Critical-Point Dry Microorganisms for Scanning Electron Microscopy

Critical-point drying of microorganisms for scanning electron microscopy can be rapidly and effectively accomplished by use of a newly described specimen holder. Up to eight different samples of spores or vegetative cells are placed between polycarbonate membrane filters in the holder and processed through solvent dehydration and critical-point drying using carbon dioxide without loss or cross contamination of microorganisms. Yeasts, molds, bacteria, and actinomycetes have been successfully processed.     

Microbial colonization of some duckweeds (Lemnaceae): Examination by scanning and transmission electron and light microscopy

Fresh specimens of the small floating aquatic macrophytes Lemna minor L. and Spirodela oligorhiza (Kurz) Hegelm., collected in Texas and Florida, were examined by scanning and transmission electron as well as light microscopy. Observations revealed that surfaces of the plants, including fronds and roots, were well colonized by bacteria and cyanobacteria as well as other microorganisms. Microorganisms were firmly attached and roots were shown to posses the mucilaginous layer commonly observed on other aquatic and terrestrial macrophytes. The rhizoplane of most plants was extensively colonized although some variability existed. Bacteria were also observed in the intercellular spaces of apparently healthy roots. The observations demonstrated some ultrastructural aspects of the association between some members of the Lemnaceae and attached epiphytic microorganisms.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Characterization of the Photosynthetic Apparatus and Proteome of Roseobacter denitrificans

The phototrophic capacity of aerobic anoxygenic phototrophic bacteria endows them with a selective advantage over other heterotrophic bacteria in the oligotrophic ocean. Here, we reported the phototrophic features and proteome of an aerobic phototrophic bacterium Roseobacter denitrificans under starvation stress. The fluorescence induction and relaxation measurements suggested that the photosynthetic capacity in R. denitrificans was preserved but was lower than in the photoautotrophic bacterium Rhodobacter sphaeroides. The existence of light-harvesting complexes (LH1 and LH2) and the reaction center (RC) in the native membrane were demonstrated through atomic force microscopy image analysis as direct evidence of their phototrophy. The homology-based LH1–RC complex structure was proposed in which RC was the Rb. sphaeroides homolog structure surrounded by the LH1. Moreover, the protein expression profiles of cells in the stationary phase under heterotrophic and mixotrophic conditions show that light enhanced or activated some proteins such as carbon monoxide dehydrogenase and NifU to cope with the low levels of amino acids and carbon sources under starvation conditions.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.     

Studies on ultrastructure and cultivation of microorganisms associated with zebra nematodes

Nematodes recovered from the hindgut of zebras were examined with scanning and transmission electron microscopy for microorganisms. Microorganisms were observed attached to the posterior extremities of two groups of nematodes, atractids and cyathostomes. Novel techniques were used to culture the microorganisms, and these included rinses to reduce contamination from hindgut flora and the design of the culture media. Electron microscopy revealed a flat bacterium not previously observed, as well as small rods and segmented filamentous bacteria. Culturing techniques resulted in isolation of a Propionibacterium species.Offprint requests to: R. C. Krecek.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Unique advantages of using low temperature scanning electron microscopy to observe bacteria

Summary Electron microscopy (EM) has greatly helped to elucidate our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be carried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for supplementary studies.Abbreviations EPS extracellular polysaccharide - EM electron microscopy - LTSEM low temperature scanning electron microscopy - SEM scanning electron microscopy - TEM transmission electron microscopy - TSA tryptic soy agar - TSB tryptic soy broth Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Study of ectoparasitism of ultramicrobacteria of the genus <Emphasis Type="Italic">Kaistia</Emphasis>, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

Structural and Geomicrobiological Characteristics of a Microbial Community from a Cold Sulfide Spring

The Ancaster sulfur spring is a cold (9°C) sulfur spring located near Ancaster, Ontario, Canada, which hosts an abundant and diverse microbial mat community. We conducted an extensive microscopical study of the microbial community of this spring using a number of techniques: phase light, confocal scanning laser microscopy, conventional scanning electron microscopy using both chemical/critical point drying and cryofixation preparative techniques, environmental scanning electron microscopy, and transmission electron microscopy. The latter two techniques were coupled with energy dispersive X-ray spectrometry for elemental analysis to complement wet geochemical data collected on bulk spring water and mat pore water. In the anoxic source of the spring, green and purple sulfur bacteria were found together with a sulfide-utilizing type of cyanobacteria that had the unusual characteristic of storing colloidal sulfur intracellularly. Deeper within the source, the mats were dominated by green sulfur bacteria and thick biofilms of cells that precipitated Fe and Zn sulfide minerals on their surfaces. Downstream from the source, thick, filamentous white mats lined the stream channel, formed by a diverse mass of nonphotosynthetic sulfur oxidizers, which were responsible for forming thick masses of spherical colloidal sulfur. These were distinguished by ESEM-EDS from cells by their simple elemental composition (only S was detected). Aqueous geochemistry analysis by ICP-MS showed that some elements (Fe, C, P, Zn, Mg, Ba) were present at higher levels in mat pore water than in bulk spring water. Our approach allowed us to gain an appreciation of the characteristics of this microbial community and allowed us to develop a good understanding of the types of microorganisms present and infer some of the relationships among the members of the community. In addition, we wish to convey the utility of a thorough microscopical approach in geomicrobiological and microbial ecology studies.     

Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Coexistence of mixotrophs, autotrophs, and heterotrophs in planktonic microbial communities

We examine what circumstances allow the coexistence of microorganisms following different nutritional strategies, using a mathematical model. This model incorporates four nutritional types commonly found in planktonic ecosystems: (1) heterotrophic bacteria that consume dissolved organic matter and are prey to some of the other organisms; (2) heterotrophic zooflagellates that depend entirely on bacterial prey; (3) phototrophic algae that depend only on light and inorganic nutrients, and (4) mixotrophs that photosynthesize, take up inorganic nutrients, and consume bacterial prey. Mixotrophs are characterized by a parameter representing proportional mixing of phototrophic and heterotrophic nutritional strategies. Varying this parameter, a range of mixotrophic strategies was examined in hypothetical habitats differing in supplies of light, dissolved organic carbon, and dissolved inorganic phosphorous. Mixotrophs expressing a wide range of mixotrophic strategies persisted in model habitats with low phosphorus supply, but only those with a strategy that is mostly autotrophic persisted with high nutrient supply, and then only when light supply was also high. Organisms representing all four nutritional strategies were predicted to coexist in habitats with high phosphorus and light supplies. Coexistence involves predation by zooflagellates and mixotrophs balancing the high competitive ability of bacteria for phosphorus, the partitioning of partially overlapping resources between all populations, and possibly nonequlibrium dynamics. In most habitats, the strategy predicted to maximize the abundance of mixotrophs is to be mostly photosynthetic and supplement nutritional needs by consuming bacteria.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Scanning transmission X-ray,laser scanning,and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.