Microscopy of biofilms

Identification, visualization and investigation of biofouling microbes are not possible without light, epifluorescence and electron microscopy. The first section of this chapter presents methods of quantification of microbes in biofilms and Catalyzed Reporter Deposition Fluorescent in situ hybridization (CARD-FISH). The second section provides an overview of Laser Scanning Confocal Microscopy (LSCM) imaging, which focuses mainly on the Fluorescent in situ Hybridization Technique (FISH) technique. This technique is very useful for visualization and quantification of different groups of microorganisms. The third section describes the principles of transmission (TEM) and scanning (SEM) electron microscopy. Traditional light and epifluorescent microscopy: Light microscopy is among the oldest methods used to investigate microorganisms. Several light microscopy techniques, such as bright field, dark field and phase contrast, enhance contrast between microorganisms and background. Epifluorescent microscopy is preferable over scanning electron microscopy (SEM) for bacterial size and abundance studies. Fluorescent in situ hybridization (FISH) allows quick phylogenetic identification (phylogenic staining) of microorganisms in environmental samples without the need to cultivate them or to amplify their genes using the polymerase chain reaction (PCR). This method is based on the identification of microorganisms using short (15-20 nucleotides) rRNA-complementary fluorescently labeled oligonucleotide probes (species, genes or group specific) that penetrate microbial cells, bind to RNA and emit visible light when illuminated with UV light. The chapter also lists the materials and equipment necessary for counting bacteria in biofilms using 4\u27,6-diamidino-2-phenylindole (DAPI) staining and catalyzed reporter deposition (CARD)-FISH. Confocal laser scanning microscopy: Laser scanning confocal microscopy (LSCM) imaging offers many advantages over conventional light and fluorescence microscopy, including the elimination of out-of-focus signal and the capability to collect images from serial sections of thick specimens. In situ hybridization with end-labeled oligonucleotide probes fluorescent in situ hybridization (FISH) and amplification of signal from those end-labeled probes catalyzed reporter deposition (CARD)-FISH are detailed in this chapter. LSCM systems are becoming increasingly common in multiuser equipment facilities in research institutions; the methods and sample preparation described are a summary of basic methods used in for determining the phylogenetic makeup of cells in fixed or live biofilms. There is an option to export the files into compatible file formats from image acquisition software, including jpg or tiff, which can then be annotated and saved in a separate location from the raw image files. Electron microscopy: This chapter describes the principal methods for biofilm preparation for Transmission electron microscopy (TEM) or Scanning electron microscopy (SEM) observations. TEM allows identification of the types of microorganisms present in biofilms because its high resolution enables the visualization of organelles specific to various types of microorganisms. Sample preparation for TEM is time consuming and requires highly skilled technicians or investigators experienced in thin sectioning. Investigators using core TEM facilities, however, are usually responsible for fixation of the sample. These investigators should also have an understanding of the processing steps following fixation, should troubleshooting become necessary. SEM and TEM are complementary in biofilm high-resolution characterization because TEM provides a two-dimensional image of intracellular organelles while SEM yields a three-dimensional rendering of the surface of the biofilm, thereby revealing the overall shape of the organisms composing the biofilm as well as their organization relative to each other and to the extracellular matrix

Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy

The common practice of counting bacteria using epifluorescence microscopy involves selecting 5–30 random fields of view on a glass slide to calculate the arithmetic mean which is then used to estimate the total bacterial abundance. However, not much is known about the accuracy of the arithmetic mean when it is calculated by selecting random fields of view and its effect on the overall abundance. The aim of this study is to evaluate the accuracy and reliability of the arithmetic mean by estimating total bacterial abundance and to calculate its variance using a bootstrapping technique. Three fixed suspensions obtained from a three-week-old marine biofilm were stained and dispersed on glass slides. Bacterial cells were counted from a total of 13,924 fields of view on each slide. Total bacterial count data obtained were used for calculating the arithmetic mean and associated variance and bias for sample field sizes of 5, 10, 15, 20, 25, 30, 35 and 40. The study revealed a non-uniform distribution of bacterial cells on the glass slide. A minimum of 20 random fields of view or a minimum of 350 bacterial cells need to be counted to obtain a reliable value of the arithmetic mean to estimate the total bacterial abundance for a marine biofilm sample dispersed on a glass slide

Antibacterial and Antifungal Activities of Cyanobacterial Strains Isolated from Hot Springs in Oman

In this study, cyanobacterial microbial mats from five hot springs in Oman, namely Al Kasfah Rustaq, Al Thwara Nakhl, Al–Ali Hammam, Gala and Bowsher, were characterized using direct microscopy. Nine monoclonal cyanobacterial cultures were obtained and their extracts in butanol, dichloromethane (DCM) and hexane were screened for antibacterial and antifungal activities. Direct microscopy revealed the presence of 12 different unicellular and filamentous morphotypes, with different distribution in the various mats. Temperature seems to be one of the most important parameters that accounts for the differences in cyanobacterial composition of the mats. Cells of the nine isolates and their aqueous supernatants were subsequently extracted with butanol, DCM and hexane. Dried extracts were tested against nine bacterial (i.e. gram +ve Staphylococcus aureus, Bacillus subtilis and gram –ve, Escherichia coli, Klebsiella pneumoniae, Salmonella choleraesuis, S. enterica, Psuedomonas aeruginosa, Providencia stuartii, and  Acinetobacter calcoaceticus) and two fungal pathogens (Rhizoctonia solani and Pythium sp.). All isolates exhibited antibacterial and antifungal activities, which depended mainly on the type of cyanobacterial culture, type of solvent used and the pathogen tested. The highest antibacterial activity was observed in Phormidium species, and butanol was found to be the most appropriate solvent to extract bioactivity from these cyanobacterial species. The results of this study suggest that thermal springs in Oman harbor diverse types of cyanobacteria, which may constitute an important source of antibacterial and antifungal compounds. Further investigation is needed to purify these compounds and find their chemical compositions and modes of action

The effect of surface colour on the formation of marine micro and macrofouling communities

<div><p>The effect of substratum colour on the formation of micro- and macro fouling communities was investigated. Acrylic tiles, painted either black or white were covered with transparent sheets in order to ensure similar surface properties. All substrata were exposed to biofouling at 1 m depth for 40 d in the Marina Bandar al Rowdha (Muscat, Sea of Oman). Studies were conducted in 2010 over a time course of 5, 10 and 20 d, and in 2012 samples were collected at 7, 14 and 21 d. The densities of bacteria on the black and white substrata were similar with the exception of day 10, when the black substrata had a higher abundance than white ones. Pyrosequencing <i>via</i> 454 of 16S rRNA genes of bacteria from white and black substrata revealed that Alphaproteobacteria and Firmicutes were the dominant groups. SIMPER analysis demonstrated that bacterial phylotypes (uncultured Gammaproteobacteria, <i>Actibacter</i>, <i>Gaetbulicola, Thalassobius</i> and <i>Silicibacter</i>) and the diatoms (<i>Navicula directa</i>, <i>Navicula</i> sp. and <i>Nitzschia</i> sp.) contributed to the dissimilarities between communities developed on white and black substrata. At day 20, the highest amount of chlorophyll <i>a</i> was recorded in biofilms developed on black substrata. SIMPER analysis showed that <i>Folliculina</i> sp., <i>Ulva</i> sp. and <i>Balanus amphitrite</i> were the major macro fouling species that contributed to the dissimilarities between the communities formed on white and black substrata. Higher densities of these species were observed on black tiles. The results emphasise the effect of substratum colour on the formation of micro and macro fouling communities; substratum colour should to be taken into account in future studies.</p> </div

Microbial diversity of eolian dust sources from saline lake sediments and biological soil crusts in arid Southern Australia

While microbial communities of aerosols have been examined, little is known about their sources. Nutrient composition and microbial communities of potential dust sources, saline lake sediments (SLS) and adjacent biological soil crusts (BSC), from Southern Australia were determined and compared with a previously analyzed dust sample. Multivariate analyses of fingerprinting profiles indicated that the bacterial communities of SLS and BSC were different, and these differences were mainly explained by salinity. Nutrient concentrations varied among the sites but could not explain the differences in microbial diversity patterns. Comparison of microbial communities with dust samples showed that deflation selects against filamentous cyanobacteria, such as the Nostocales group. This could be attributed to the firm attachment of cyanobacterial filaments to soil particles and/or because deflation occurs mainly in disturbed BSC, where cyanobacterial diversity is often low. Other bacterial groups, such as Actinobacteria and the spore-forming Firmicutes, were found in both dust and its sources. While Firmicutes-related sequences were mostly detected in the SLS bacterial communities (10% of total sequences), the actinobacterial sequences were retrieved from both (11–13%). In conclusion, the potential dust sources examined here show highly diverse bacterial communities and contain nutrients that can be transported with aerosols. The obtained fingerprinting and sequencing data may enable back tracking of dust plumes and their microorganisms

Impact of nitrate on bacterial structure and function in injection-water biofilms.

We studied the impact of NO3– on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, 15N and 35S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO3– was added to the seawater in an attempt to prevent bacterial H2S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO3– was mainly reduced to NO2–. In biofilms grown in the absence of NO3–, redox potentials of &lt;–450 mV at the metal surface suggested the release of Fe2+. NO3– addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO3– reduction occurred upon subsequent withdrawal of NO3–. Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO3– addition

Limitation of Microbial Processes at Saturation-Level Salinities in a Microbial Mat Covering a Coastal Salt Flat

Hypersaline microbial mats are dense microbial ecosystems capable of performing complete element cycling and are considered analogs of early Earth and hypothetical extraterrestrial ecosystems. We studied the functionality and limits of key biogeochemical processes, such as photosynthesis, aerobic respiration, and sulfur cycling, in salt crust-covered microbial mats from a tidal flat at the coast of Oman. We measured light, oxygen, and sulfide microprofiles as well as sulfate reduction rates at salt saturation and in flood conditions and determined fine-scale stratification of pigments, biomass, and microbial taxa in the resident microbial community. The salt crust did not protect the mats against irradiation or evaporation. Although some oxygen production was measurable at salinities of ≤30% (wt/vol) in situ, at saturation-level salinity (40%), oxygenic photosynthesis was completely inhibited and only resumed 2 days after reducing the porewater salinity to 12%. Aerobic respiration and active sulfur cycling occurred at low rates under salt saturation and increased strongly upon salinity reduction. Apart from high relative abundances of Chloroflexi, photoheterotrophic Alphaproteobacteria, Bacteroidetes, and Archaea, the mat contained a distinct layer harboring filamentous Cyanobacteria, which is unusual for such high salinities. Our results show that the diverse microbial community inhabiting this salt flat mat ultimately depends on periodic salt dilution to be self-sustaining and is rather adapted to merely survive salt saturation than to thrive under the salt crust. IMPORTANCE Due to their abilities to survive intense radiation and low water availability, hypersaline microbial mats are often suggested to be analogs of potential extraterrestrial life. However, even the limitations imposed on microbial processes by saturation-level salinity found on Earth have rarely been studied in situ. While abundance and diversity of microbial life in salt-saturated environments are well documented, most of our knowledge on process limitations stems from culture-based studies, few in situ studies, and theoretical calculations. In particular, oxygenic photosynthesis has barely been explored beyond 5 M NaCl (28% wt/vol). By applying a variety of biogeochemical and molecular methods, we show that despite abundance of photoautotrophic microorganisms, oxygenic photosynthesis is inhibited in salt-crust-covered microbial mats at saturation salinities, while rates of other energy generation processes are decreased several-fold. Hence, the complete element cycling required for self-sustaining microbial communities only occurs at lower salt concentrationsISSN:0099-2240ISSN:1098-533

Effect of organic solvents and acidic catalysts on biodiesel yields from primary sewage sludge, and characterization of fuel properties

<p>Biodiesel is considered the most convenient biofuel, due to its direct use in existing combustion engines; however, its production is not economically optimal due to processes that utilize costly substrates, require high energy expenditure or achieve low biodiesel yields. We used primary sewage sludge for the production of biodiesel and compared yields at different temperatures (40, 50 and 60 °C) and with different organic solvents (chloroform, toluene and hexane), acid catalysts (HCl and H₂SO₄) and catalyst concentrations (3, 5 and 7%). A maximum of 18 wt% biodiesel was obtained using chloroform with 3% H₂SO₄ at 40 °C. The conversion efficiency of the primary sludge to biodiesel under these conditions was 86.4%. Based on the fatty acid methyl ester composition of the obtained biodiesel and measured fuel properties, it was deduced that fuel from primary sludge is more suitable for areas with warmer climates than ones that reach temperatures close to 0 °C. However, the fuel properties of the produced biodiesel were within the limits of accepted international standards. We conclude that production of biodiesel from primary sewage sludge under optimized conditions reduces the impact of the feedstock on the environment, decreases overall costs and produces fuel with acceptable properties.</p

Geochemical and microbiological fingerprinting of airborne dust that fell in Canberra, Australia, in October 2002

During the night of 22-23 October 2002, a large amount of airborne dust fell with rain over Canberra, located some 200 km from Australia's east coast, and at an average altitude of 650 m. It is estimated that during that night about 6 g m-2 of aeolian dust fell. We have conducted a vast number of analyses to "fingerprint" some of the dust and used the following techniques: grain size analysis; scanning electron microscope imagery; major, trace, and rare earth elemental, plus Sr and Nd isotopic analyses; organic compound analyses with respective compound-specific isotope analyses; pollen extraction to identify the vegetation sources; and molecular cloning of 16S rRNA genes in order to identify dust bacterial composition. DNA analyses show that most obtained 16S rRNA sequences belong mainly to three groups: Proteobacteria (25%), Bacteriodetes (23%), and gram-positive bacteria (23%). In addition, we investigated the meteorological conditions that led to the dust mobilization and transport using model and satellite data. Grain sizes of the mineral dust show a bimodal distribution typical of proximal dust, rather than what is found over oceans, and the bimodal aspect of size distribution confirms wet deposition by rain droplets. The inorganic geochemistry points to a source along/near the Darling River in NW New South Wales, a region that is characteristically semiarid, and both the organic chemistry and palynoflora of the dust confirm the location of this source area. Meteorological reconstructions of the event again clearly identify the area near Bourke-Cobar as being the source of the dust. This study paves the way for determining the export of Australian airborne dust both in the oceans and other continents.No Full Tex