Quantitation of microbial metabolism

Quantitation is a characteristic property of natural sciences and technologies and is the background for all kinetic and dynamic studies of microbial life. This presentation concentrates therefore on materials and methods as tools necessary to accomplish a sound, quantitative and mechanistic understanding of metabolism. Mathematical models are the software, bioreactors, actuators and analytical equipment are the hardware used. Experiments must be designed and performed in accordance with the relaxation times of the biosystem investigated; some of the respective consequences are discussed and commented in detail. Special emphasis is given to the required density, accuracy and reproducibility of data as well as their validation.     

Kinetics of microbial growth on pentachlorophenol

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Modelling fungal growth on surfaces

Fungal growth in time and space at the substrate surface was modelled for a simple system mimicking solid-state fermentation, using a polycarbonate Nucleopore membrane laid over a glucose solution. Biomass production depends on both tip density and the diffusion of glucose within the fungal hyphae. The model predicts early increases in both height and concentration, followed by a period in which the biomass profile moves with a constant wavefront. The rate of increase in height increases as tip diffusivity increases or as the Monod saturation constant for glucose decreases. © Rapid Science Ltd. 1998     

Use of ultrasonic energy in assessing microbial contamination on surfaces

Ultrasonic tanks were evaluated for their ability to remove viable microorganisms from various surfaces for subsequent enumeration. Test surfaces were polished stainless steel, smooth glass, frosted glass, and electronic components. The position of contaminated surfaces in relation to the ultrasonic energy source, distance of the ultrasonic source from the test surfaces, and temperature of the rinse fluid were some of the factors which influenced recovery. Experimental systems included both naturally occurring microbial contamination and artificial contamination with spores of Bacillus subtilis var. niger. The results showed that ultrasonic energy was more reliable and efficient than mechanical agitation for recovering surface contaminants. Conditions which increased the number and percentage of microorganisms recovered by ultrasonic energy were: using a cold rinse fluid, placing the sample bottle on the bottom of the ultrasonic tank, and facing the contaminated surfaces toward the energy source. It was also demonstrated that ultrasonic energy could be effectively used for eluting microorganisms from cotton swabs.     

Wipe-rinse technique for quantitating microbial contamination on large surfaces.

The Orca Basin is a hypersaline depression in the northern Gulf of Mexico with anoxic conditions observed in the lower 200 m of the water column. Measurements of adenosine 5′-triphosphate, heterotrophic potential, and uridine uptake made above and across the interface into the anoxic zone revealed the presence of an active microbial population approximately 100 m above the interface. Biomass and activity decreased at and just below the interface but increased near the bottom, consistent with similar observations made in the Cariaco Trench. The maximum adenosine 5′-triphosphate concentration above the interface of 5.9 ng/liter (2,173 m) is about eight times greater than the value found in oxygenated waters of corresponding depth in the absence of an anoxic zone. The maximum adenosine 5′-triphosphate concentration in the anoxic zone is approximately 15 times greater than that found in oxygenated water of similar depth, suggesting anoxia will support the development of a larger bacterial population. Our findings suggest that autotrophic bacteria may be the dominant physiological group in the region just above the interface.     

Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis.

By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.     

Mechanistic model for microbial growth on hydrocarbons

Based on available information describing the transport and consumption of insoluble alkanes, a mechanistic model is proposed for microbial growth on hydrocarbons. The model describes the atypical growth kinetics observed, and has implications in the design of large scale equipment for single cell protein (SCP) manufacture from hydrocarbons. The model presents a framework for comparison of the previously published experimental kinetic data.     

The retention of bacteria on hygienic surfaces presenting scratches of microbial dimensions

Aims: To produce surfaces of defined linear topographical features which reflect those found on worn and new stainless steel, to monitor the effect of feature dimensions on the retention of Listeria monocytogenes and Staphylococcus sciuri. Methods and Results: Surfaces were fabricated with parallel linear features of 30 microns or of microbial dimensions (1·02 and 0·59 μm width) and used in microbial retention assays with Staph. sciuri and L. monocytogenes. Retained cells were distributed uniformly across the smooth 30 micron featured surfaces but were retained in high numbers on microtopographies at the ‘peaks’ between the wide grooves. On smaller features, retention was attributed to the maximum area of contact between cells and substratum being attained, with cocci being embedded in 1·02‐μm‐width grooves, and rods aligned along (and across) the densely packed parallel 0·59‐μm grooves. Conclusions: The dimensions of surface features may enhance or impede cell retention. This phenomenon is also related to the size and shape of the microbial cell. Significance and Impact of Study: Findings may help describe and evaluate properties of hygienic and easily cleanable surfaces.     

Cybernetic modeling of microbial growth on multiple substrates

The internal regulatory processes, which underlie a variety of behavior in microbial growth on multiple substrates, are viewed as a manifestation of an invariant strategy to optimize some goal of the cells. A goal-seeking or cybernetic model is proposed here, with the optimization obased on a short-term perspective of response to the environment. The model parameters are determined from the growth data on single substrates. The model predicts the entire range of microbial growth behavior on multiple substrates from simultaneous utilization of all sugars to sequential utilization with pronounced diauxic lags. It is shown to predict the many variations of the diauxic phenomenon in different growth conditions. The transients in continuous culture growth on mixed substrates caused by varying the feed strategies are easily simulated by this model. The framework of this model can be applied to batch or continuous culture growth of many bacteria on different combinations of substrates.     

The growth of saprophytic fungi on root surfaces

Summary Data are provided on the growth of non-pathogenic soil fungi on root surfaces. By growing roots from non-sterile soil into sterile vermiculite, and by regular isolation of fungi from the roots which had grown into the sterile vermiculite, it was shown that fungal growth down roots is slow. It is suggested that in the colonization of roots by non-pathogenic fungi successive lateral colonization from the soil is of greater importance than the growth of fungi down roots.     

Quantitation of intracellular polychlorobiphenyls during growth of Rhodotorula glutinis

A quantitative method to assay accumulated polychlorobiphenyls (PCB) in Rhodotorula glutinis is described which avoids any interference of extracellular adsorbed compounds. This method is based on a prior appropriate treatment of the cells before the extraction of derivatives by supercritical CO2 and GC analysis. The prior treatment consists in a double fixation of the samples with glutaraldehyde and osmium tetroxyde. After this fixation process the elimination of the extracellular PCBs can be achieved by washing the fixed yeast cells with organic solvents.     

Quantitation of fiber growth in transplanted central monoamine neurons

Summary Nerve fiber production by central noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) neurons was studied using immature brain tissue containing locus coeruleus, substantia nigra, or ventro-caudal medulla oblongata respectively, homologously grafted to the anterior chambers of rat eyes. A method was developed for quantitation of the fiber growth that occurs on the sympathetically denervated host irides as observed in whole mounts using Falck-Hillarp fluorescence histochemistry and by the uptake of ³H-metaraminol into the irides. Survival and growth in oculo of the three different areas were characterized by direct observations through the cornea in vivo for a number of pre- and postnatal stages of development of the donors, and the findings correlated to the degree of monoamine nerve fiber production on the host irides. The growth of fetal locus coeruleus transplants on irides was quantified using both fluorescence microscopical measurements of innervated areas and uptake of ³H-metaraminol. The uptake was well correlated to the histochemical measurements on individual irides, thus validating the fluorescence microscopical measurements of fiber production. The fiber growth of fetal locus coeruleus grafts on irides was followed for 20 weeks. The nerves increased in number and uptake capacity approximately linearly for 6 weeks whereafter the increase rapidly levelled off. On average, the final amount of nerve production by fetal locus grafts did not cover more than 1/3 of the host iris surface, and the average uptake of ³H-metaraminol by these nerves did not exceed 60% of that found in sympathetically intact control irides. The locus grafts produced a similar amount of fluorescent fibers in the host iris independent of the crown-rump length stage of the donor fetus and the final size of the transplants in oculo.The survival and growth of NA, DA and 5-HT neurons grafted from various postnatal donor rats was also followed by fluorescence microscopy. Locus coeruleus grafts produced markedly more fibers than the two other types of grafts when the donor was one week old or less, and DA grafts produced the least fibers of the three. Even from one month old donors some MA neurons survived grafting. Also, using prenatal donars, the locus coeruleus grafts produced many more fibers on the irides than did the DA grafts. It was concluded that the intraocular transplantation technique is very suitable for quantitative studies of nerve fiber production by immature monoamine neurons, and that it should be possible to study many other neuron systems in similar ways with this technique.Supported by the Swedish Medical Research Council (04X-03185), Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. The skilful technical assistance of Miss Ingrid Strömberg, Miss Maud Eriksson and Miss Gerd Boëtius is gratefully acknowledged. Thanks are due to Swedish Pfizer for the generous supply of Nialamid^®     

Improvement of biodesulfurization rate by assembling nanosorbents on the surfaces of microbial cells

To improve biodesulfurization rate is a key to industrialize biodesulfurization technology. The biodesulfurization rate is partially affected by transfer rate of substrates from organic phase to microbial cell. In this study, gamma-Al2O3 nanosorbents, which had the ability to selectively adsorb dibenzothiophene (DBT) from organic phase, were assembled on the surfaces of Pseudomonas delafieldii R-8 cell, a desulfurization strain. gamma-Al2O3 nanosorbents have the ability to adsorb DBT from oil phase, and the rate of adsorption was far higher than that of biodesulfurization. Thus, DBT can be quickly transferred to the biocatalyst surface where nanosorbents were located, which quickened DBT transfer from organic phase to biocatalyst surface and resulted in the increase of biodesulfurization rate. The desulfurization rate of the cells assembled with nanosorbents was approximately twofold higher than that of original cells. The cells assembled with nanosorbents were observed by a transmission electron microscope.     

New microbial growth factor.

A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a new microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight, and it has high specific activity. When added to the diets for a meadow vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.