Microbial Cell Concentration Research Articles

Fluorochrome in Monitoring Atmospheric Bioaerosols and Correlations with Meteorological Factors and Air Pollutants

The characteristics of bioaerosols in the ambient atmosphere were monitored by using epifluorescence microscopy with fluorochrome (EFM/FL) with five fluorescent dyes (AO, DAPI, SYTO-13, PI, and YOPRO-1). The correlations of bioaerosols with meteorological factors and air pollutants were simultaneously assessed. Results from EFM/FL were then compared with those using a commonly used culture-based method. The total microbial cell concentrations measured by the non-culture-based methods averaged about 8 × 105 cells/m3. However, culture-based method underestimated bioaerosol concentrations by the factor of 100 to 1000. The average viabilities were 0.66 by EFM/FL with PI staining, 0.35 with YOPRO-1 staining, and 0.0012 by the culture-based method. The viability by EFM/FL was much higher than the culturability. In summary, the total microbial cell concentration and viability were highly underestimated by the culture-based method. Moreover, based on culture-based and non-culture-based methods results, the total ...

Analysis of Bioaerosols from Chicken Houses by Culture and Non-Culture Method

In livestock breeding, high bioaerosol concentrations in environments such as chicken houses are an occupational health concern. Two sampling methods (impinger and filter) and three non-culture methods [epifluoresence microscopy with fluorochrome (EFM/FL), flow cytometry with the fluorochrome (FCM/FL), and fluorescent in situ hybridization (FISH)] were used to monitor the total concentration, viability, and culturability of bioaerosols in chicken houses. These results were compared to the commonly used culture method. Total microbial cell concentrations measured by the non-culture methods averaged about 5 × 10 7 cells/m 3 . However, culture method underestimated bioaerosol concentrations by a factor of 10. For sampler comparison, viability determined following impinger collection was higher than that following filter method. In conclusion, impinger was considered an appropriate sampler, and EFM/FL, FCM/FL, and FISH approaches could adequately assess total microbial cell concentration and viability of bioa...

Microbial growth and biofilm formation in geologic media is detected with complex conductivity measurements

Complex conductivity measurements (0.1–1000 Hz) were obtained from biostimulated sand‐packed columns to investigate the effect of microbial growth and biofilm formation on the electrical properties of porous media. Microbial growth was verified by direct microbial counts, pH measurements, and environmental scanning electron microscope imaging. Peaks in imaginary (interfacial) conductivity in the biostimulated columns were coincident with peaks in the microbial cell concentrations extracted from sands. However, the real conductivity component showed no discernible relationship to microbial cell concentration. We suggest that the observed dynamic changes in the imaginary conductivity (σ″) arise from the growth and attachment of microbial cells and biofilms to sand surfaces. We conclude that complex conductivity techniques, specifically imaginary conductivity measurements are a proxy indicator for microbial growth and biofilm formation in porous media. Our results have implications for microbial enhanced oil recovery, CO2 sequestration, bioremediation, and astrobiology studies.

Fluorochrome in Monitoring Indoor Bioaerosols

In this study total concentration, viability, culturability of indoor and outdoor bioaerosols in ten residential environments were studied by using a cultivating method and an epifluorescence microscopy method with fluorochrome (EFM/FL) using four different dyes (AO, DAPI, PI, and YOPRO-1). Results from the EFM/FL were then compared with those from the traditional cultivating method. The total indoor microbial cell concentrations measured by EFM/FL was 630 to 5200 times higher than that measured by the cultivating method; ranging from 1.38 × 106 to 4.48 × 106cells/m3 (microbial cells per cubic meter) with AO staining, and from 7.84 × 105 to 2.75 × 106 cells/m3 with DAPI staining, compared with the average concentration of 721 CFU/m3 for bacteria and 587 CFU/m3 for fungi by the cultivating methods. The viability of bioaerosols ranged from 0.63 to 0.92 by EFM/FL with PI staining, from 0.19 to 0.72 by EFM/FL with YOPRO-1 staining, and from 0.0002 to 0.0015 by the cultivating method. The viability determined ...

Fluorochrome and Fluorescent In Situ Hybridization to Monitor Bioaerosols in Swine Buildings

Total microbial cell concentration, viability, and culturability of bioaerosols in swine buildings were monitored by using epifluorescence microscopy with fluorochrome (EFM/FL) with four fluorescent dyes (AO, DAPI, PI, and YOPRO-1) and by using fluorescent in situ hybridization (FISH) with five oligonucleotide probes (fl-Univ, fl-EUB, cy-EUK, fl-PSMg, and fl-NotEUB) probes. Results from these two non-culture-based methods were then compared with those using a commonly used culture method. The total microbial cell concentration measured using the non-culture-based methods was 10 to 200 times higher than that using the culture method; from 5.48 × 106 to 2.18 × 107 cells/m3 with AO staining and from 5.03 × 106 to 2.13 × 107 cells/m3 with DAPI staining, compared with the average concentration of 1.02 × 105 CFU/m3 for bacteria and 1.27 × 103 CFU/m3 for fungi by the culture method. The viability ranged from 0.27 to 0.76 by EFM/FL with PI staining, from 0.02 to 0.60 with YOPRO-1 staining, from 0.53 to 0.79 by FISH, and from 0.002 to 0.033 by the culture method. The viability by EFM/FL and FISH were much higher than the culturability. In summary, the total microbial cell concentration and viability were highly underestimated by the culture method. Based on the FISH results, eubacteria and eukaryotes were the dominant components of the bioaerosols. In conclusion, EFM/FL and FISH methods can effectively assess the total microbial cell concentration and viability of bioaerosols in environmental samples.

Biosorption of heavy metals by Sphaerotilus natans: an equilibrium study at different pH and biomass concentrations

In the present work a lyophilised cell suspension of Spherotilus natans was studied as biosorbent material for cadmium and copper removal from aqueous solutions. The biomass was firstly characterised by potentiometric titration and evaluation of the major ionic content. The experimental data highlight that the biomass cell wall contains two main acidic groups with a total amount of 5 meq/g. Equilibrium biosorption trials of cadmium and copper were carried out to investigate the effects of two important experimental factors, pH and biomass concentration. As expected, both cadmium and copper biosorption extent was repressed by pH decrease. The effect of the biomass concentration changes both with the equilibrium pH value and the kind of metal adsorbed. In the case of copper biosorption at pH>5, the increase of biomass concentration causes a diminution of the maximum specific metal uptake due probably to cell aggregation phenomena; whereas at acidic pH values the previous trend is inverted perhaps because of the effect of partial hydrolysis of the bacterial cell wall constituents. A different behaviour was observed for cadmium biosorption in relation to the microbial cell concentration: the effect of biomass concentration is less evident and opposite with respect to copper at pH 6 and 3 and no definitive explanation was found for this case. Equilibrium modelling was performed for both metals by using the most used equations reported in the literature. A comparison of the biosorption characteristic of S. natans has been also performed with respect to some results reported by other researchers.

Modeling of sequencing batch reactors treating inhibitory and noninhibitory wastewaters

A mathematical model for sequencing batch reactors (SBR) that takes into account fill and react periods was developed and tested against experimental data from the literature. The Monod equation was used to describe microbial growth kinetics. The proposed model predicts the time dependent microbial cell and substrate concentrations during SBR treatment of wastes. A numerical method was used to solve the governing model equations which comprised nonlinear unsteady-state ordinary differential equations. Model simulations highlighted the importance of feed rate as a major operating parameter. For biodegradable wastes and low strength inhibitory wastes, an instantaneous fill is optimum whereas high strength inhibitory waste treatment is only possible with long fill times. The model predictions compared well with experimental results reported in the literature. This model is useful for further understanding of biological wastewater treatment kinetics by batch processes and may serve as a valuable design aid for practicing engineers.

Model for Effective Diffusivities in Aerobic Biofilms

Reliable estimates of effective diffusivities in biofilms are required to calculate the combined diffusion and biotransformation of soluble substrates. For aerobic biofilms, effective diffusivities have been previously correlated empirically to the biofilm dry densities. Biofilms consist primarily of microbial cells, insoluble extracellular polymeric substances (EPS), and interstitial water. Biofilms are therefore a natural, heterogeneous, multiphasic material. Effective diffusive properties of such materials, both biological and nonbiological, have historically been correlated to the phase-volume fractions and diffusive properties of the individual phases. This paper presents a conceptually similar model for effective diffusivities in aerobic biofilms. The model predicts separate effects for increasing concentrations of microbial cells and insoluble EPS. Predicted effective diffusivities are expressed as a fraction of the corresponding dilute aqueous values.

Effect of silver on the ferrous iron oxidizing ability of Thiobacillus ferrooxidans

The effect of silver on the iron oxidizing ability of Thiobacillus ferrooxidans was studied using electrochemical and other physicochemical techniques. Electrochemical investigation was conducted using a method based on change in redox potential. The rate of ferrous ion oxidation is influenced by pH (optimum pH range is 1.7–2.0), microbial cell concentration, and Fe(II) concentration. The mechanism of the bacterially mediated oxidation of ferrous iron is found to be influenced by temperature changes. In the vicinity of the optimum temperature (25°C), the reaction is likely to be controlled by the diffusion of Fe(II) ions, whereas below the temperature range 18–25°C, chemical reaction may be the rate-controlling factor. Ag(I) is detrimental to the microbial oxidation of Fe(II). In the presence of 10 mg/1 silver, the reaction may be chemically controlled over the temperature range 5.5–25°C. Inhibition of microbial Fe(II) oxidation in the presence of silver may take place via a mixed mechanism in which silver may bind with both the iron oxidizing enzyme(s) and the enzyme-Fe(II) complex.

Estimation of microbial cell concentration in suspension culture by the osmotic pressure measurement of culture broth

A method for estimating microbial cell concentration in suspension cultures even under heterogeneous conditions was developed on the basis of changes in osmotic pressure of the medium. During batch cultivation of Saccharomyces cerevisiae and Candida brassicae, there was a linear relationship between increase in cell concentrations (ΔX) and the difference between osmotic pressure change in the broth (ΔPc) and the osmotic pressure increase due to product accumulation (ΔPp) regardless of the product (ethanol) concentration in the broth. A linear relationship between (ΔX) and (ΔPc — ΔPp) was also observed when medium containing solid substrate (wheat germ) was used. An enzymatic method for separating cells from the solids was developed and cell concentrations in broths containing solid substrates could be measured accurately. During the batch production of bialaphos (a herbicide) by Streptomyces hygroscopicus using a medium containing solid substrates, the cell concentration could also be estimated by the developed methods.

Detection and removal of Escherichia coli using fluorescein isothiocyanate conjugated monoclonal antibody immobilized on bacterial magnetic particles.

A novel fluoroimmunoassay method using bacterial magnetic particles for the highly sensitive detection of bacteria has been developed. Fluorescein isothiocyanate (FITC) conjugated monoclonal anti-Escherichia coli antibody was immobilized onto bacterial magnetic particles (BMPs) using a heterobifunctional reagent, N-succinimdyl 3-(2-pyridyldithio)propionate (SPDP). E. coli cells were reacted with FITC-antibody-BMP conjugates for 15 min in an inhomogeneous magnetic field which enhanced aggregation. The cell/BMP complexes sedimented, causing relative fluorescence intensity of the solution to decrease with increasing microbial cell concentration. A linear relationship was obtained between the relative fluorescence intensity and cell concentration in the range of 10(2)-10(6) cells/mL. Selectivity of this detection system was satisfactory. Monoclonal antibody immobilized on BMPs was also applied to the specific removal of E. coli from the bacterial suspension.

Electrochemical sensor for viable microbial cell concentration based on a functional polymer that captures microorganisms alive

We have prepared an electrochemical sensor that rapidly determines the concentration of viable cells of aerobic and facultatively anaerobic microorganisms in aqueous suspension. It consists of a flow cell containing an oxygen electrode and non-woven cloth coated with a pyridinium-type polymer. When a sample suspension of microorganism cells is passed through the electrochemical sensor at a constant flow rate, the microbial cells are captured and accumulated on the surface of the non-woven cloth in the living state. The consumption of oxygen by the captured living cells appears as a decrease in the electric current. The rate of decrease of the electric current is porportional to the concentration of viable microbial cells. Each measurement requires about 10 min. The minimum detectable concentration of living microbial cells expressed as turbidity at 610 nm is 0.005–0.023. The minimum detectable concentration in colony count is 3.7×10 5 cells ml −1 in the case of Escherichia coli.

Electrochemical sensor for viable microbial cell concentration based on a functional polymer that captures microorganisms alive

We prepared an electrochemical sensor that rapidly determines the concentration of viable cells of aerobic and facultatively anaerobic microorganisms in aqueous suspension. It consists of a flow cell containing an oxygen electrode and non-woven cloth coated with 10 mg·g −1 of a copolymer of N-benzyl-4-vinylpyridinium chloride and styrene, a pyridinium-type polymer. When a sample suspension of microorganisms cells is passed through the electrochemical sensor at a constant flow rate, the microbial cells are captured and accumulated on the surface of the non-woven cloth in the living state. The consumption of oxygen by the captured living cells appears as a decrease in the electric current. Rate of decrease of the electric current is proportional to the concentration of viable microbial cells. A control suspension of bacterial cells killed by autoclaving gave a negligible decrease in the electric current. Each measurement required about 10 min. The minimum detectable concentration of living microbial cells expressed as turbidity at 610 nm was 0.005 to 0.023. The minimum detectable concentration in colony count was 3.7 × 10 5 cells·ml −1 in the case of Escherichia coli.

Enumeration of Microorganisms in Metalworking Fluids Using Photometric Methods

This paper will discuss two approaches to assaying concentrations of microorganisms in metalworking fluids using light. One approach employs fluorescent dye to stain the DNA within microbial cells suspended in the fluid. The intensity of light emitted by the bound dye is measured to provide an estimate of microbial concentrations. The second approach is based upon a light-producing reaction of ATP, a measure of the energetic state that is essentially constant for all living cells. The rate of light production is proportional to the concentration of ATP and, therefore. to the concentration of microbial cells; the light emitted is then a measure of the number of cells in a sample of fluid. Both approaches provide reproducible and reliable results. These methods have the potential of providing rapid, on-site measurement of microbial loads and are being explored as alternatives to standard plate count and dip slide methods that require an incubation period of up to two days to achieve results.

Spectrophotometric method for high biomass concentration measurements

Continuous monitoring of concentration is important in the optimal control of bioreactors. Spectrophotometry provides a simple, accurate, and rapid way for measuring cell concentration of unicellular microorganisms, based on either Beer's law or calibration curves prepared using standard solutions. However, this method is limited to a low range of microbial concentrations, because Beer's law deviates significantly at high concentrations. In the present investigation, based on experimental work, a new technique is posed to monitor the concentration of microbial cells as high as 100 g DW/L. this is achieved by using a mixture of known concentration as reference, rather than the "ideal blank" with zero concentration of analyte. As a result, a new equation is developed that, although applied here only to microbial concentration, in principle can be used for monitoring the concentration of any optically sensitive material.

Effect of longitudinal mixing on microbial growth in a multi‐stage column bioreactor

AbstractThe performance of a multi‐stage column bioreactor for nonlimited microbial growth was studied. The back‐flow model was employed to describe the flow pattern of the microbial suspension. Analytical solutions for calculating the microbial cell concentrations under steady‐state and unsteady‐state operations, with and without cells in feed, were obtained and the cell growth behavior was examined. Only when microbial cells were present in the feed and the reaction number was less than a critical number was a stable steady state achieved: the microbial cell concentration was then significantly affected by the extent of longitudinal mixing. In the absence of microorganisms in the feed, the steady state was not stable with either washout or nonlimited growth resulting.

Continuous turbidometric measurements of microbial cell density in bioreactors using a light-emitting diode and a photodiode

A simple device for optimal measurements of the concentration of microbial cells in a bioreactor is described. It consists of a green or red light-emitting diode as light source and a photodiode as light detector. The device can be used for continuous measurements on cell suspensions circulated through a measuring chamber comprised of a tube or viral. The diodes can also be mounted in glass tubes inside a bioreactor for continuous in situ measurements. The use of a biexponential calibration function allows the diode signal to be used to measure cell concentration over a range of more than a factor of 100.

Microorganisms of Groundwater and Decomposing Refuse as Viewed by the Transmission Electron Microscope

The chief problem in obtaining an objective view of native microbial communities is lack of suitable experimental techniques. In our laboratory a simple preparative method was used in order to make electron microscopical investigations of indigenous microorganisms from ground-water and decomposing municipal refuse possible. This method includes (i) suspension of the complex microflora from the refuse sample in a buffered water solution or a direct use of groundwater samples; (ii) embedding of microorganisms from the suspension on the surface of collodion filmed and carbon coated electron microscopical grids by direct centrifugation in the Beckman Airfuge; (iii) a one step staining with 1,5% phosphotungstic acid at pH 6.8 in centrifuge rotor chambers. The main advantage of our method is based on a supporting effect of the centrifugal force towards the concentration and adhesion of microbial cells at the surface of electron microscopical grids. Electron microphotographs of microorganisms from ground-water, and decomposing municipal refuse are presented.

Selective determination of microbial cells by graphite electrode modified with adsorbed 4,4′-bipyridine

The concentrations of microbial cells in suspensions can be measured with a graphite electrode modifed with adsorbed 4,4′-bipyridine, by using cyclic volummetry or differential pulse volummetry. The relationship between the peak current and the cell concentration for Saccharomyces cerevisiae is linear over the range 0.03 × 10 8—2.0 × 10 8 cells ml −1. Yeast, gram-positive bacteria and gram-negative bacteria gave different peak potentials, which may be useful for classification purposes. Electron transfer between the microbial cells and the 4,4′-bipyridine-modified electrode is mediated by coenzyme A in the cell wall.

Factors affecting microbial growth and polysaccharide production during the fermentation of Xanthomonas campestris cultures

Fermentations of Xanthomonas campestris have been carried out on laboratory and pilot plant scales using various organic nitrogen sources in order to test their effectiveness in polysaccharide (xanthan) production. It was discovered that high nitrogen concentrations give highest yields of crude product and result in a need for only short fermentation times to achieve maximum product formation. These products, however, have inferior solution rheology to those produced from low-nitrogen media due partly to their high concentrations of co-precipitated microbial cells and partly to differences in tertiary molecular structure.