Surface-enhanced Raman spectroscopy as a tool for probing specific biochemical components in bacteria.

Treatment of bacteria with silver yields intense and highly specific surface-enhanced Raman spectroscopy (SERS) spectra from various cellular chemical components located in the vicinity of the silver colloids. In particular, we demonstrate an extreme sensitivity to flavin components associated with the cell envelope and to their state of oxidation. Different spectra, possibly associated with DNA, carboxylates, and perhaps phosphates, are obtained from the soluble interior fraction of the cell.

Optimization of feeding profile for a fed-batch bioreactor by an evolutionary algorithm.

The optimal feeding profile of a fed batch process was designed by means of an evolutionary algorithm. The algorithm chromosomes include the real-valued parameters of a profile function, defined by previous knowledge. Each chromosome is composed of the parameters that define the feeding profile: the feed rates, the singular arc parameters and the switching times between the profile states. The feed profile design was tested on a fed-batch process simulation. The accepted profiles were smooth and similar to those derived analytically in other studies. Two selection functions, roulette wheel and geometric ranking, were compared. In order to overcome the problem of model mismatches, a novel optimization scheme was carried out. During its operation the process was sampled, the model was updated and the optimization procedure was applied. The on-line optimization showed improvement in the objective function for relatively low sample times. Choosing the sampling frequencies depends on the process dynamics and the time required for the measurements and optimization. Further study on experiments of fed-batch process demonstrated the use of complex, non-differentiable model and produced improved process performances using the optimal feeding profile.

Monitoring and control of pullulan production using vision sensor.

The production of the polysaccharide pullulan by the yeast-like fungi, Aureobasidium pullulans, is accompained by cellular morphogenetic changes. High productivity and yield of the process have been found to correlate with high concentration of yeast-like cells in the culture. The morphogenetic changes of A. pullulans cells depend on the culture conditions, e.g., dissolved oxygen, shear rate and medium composition. In order to improve the productivity of the process, a novel control law was formulated. A feeding strategy dependent on the culture cellular composition was designed and aimed to keep the yeast-like cell concentration high. The culture morphogenetic composition during the process was monitored by a recently developed vision sensor. Feeding was actuated when the yeast-like cell concentration decreased below a threshold. The proposed control strategy improved pullulan production by increasing both productivity and yield of the cells by 67% and 80%, correspondingly. The results point to the advantage and the potential of using the monitoring and control system and algorithm to increase productivity and yield in cellular bioprocesses.

Hybrid model building methodology using unsupervised fuzzy clustering and supervised neural networks.

This paper suggests a model building methodology for dealing with new processes. The methodology, called Hybrid Fuzzy Neural Networks (HFNN), combines unsupervised fuzzy clustering and supervised neural networks in order to create simple and flexible models. Fuzzy clustering was used to define relevant domains on the input space. Then, sets of multilayer perceptrons (MLP) were trained (one for each domain) to map input-output relations, creating, in the process, a set of specified sub-models. The estimated output of the model was obtained by fusing the different sub-model outputs weighted by their predicted possibilities. On-line reinforcement learning enabled improvement of the model. The determination of the optimal number of clusters is fundamental to the success of the HFNN approach. The effectiveness of several validity measures was compared to the generalization capability of the model and information criteria. The validity measures were tested with fermentation simulations and real fermentations of a yeast-like fungus, Aureobasidium pullulans. The results outline the criteria limitations. The learning capability of the HFNN was tested with the fermentation data. The results underline the advantages of HFNN over a single neural network.

A self-tuning vision system for monitoring biotechnological processes: I. Application to production of pullulan by Aureobasidium pullulans.

A prototype of a self-tuning vision system (STVS) has been developed to monitor cell population in fermentations. The STVS combines classical image processing techniques, neural networks and fuzzy logic technologies. By combining these technologies the STVS is able to analyze sampled images of the culture. The proposed system can be "tailored" with minimum effort by an expert who can "teach" the system to recognize cells by showing examples of different morphologies. After adaptation, the STVS is able to capture images, isolate the different cells, classify them according to the expert's criteria, and provide the profile of the cell's population. The system was applied to the classification and analysis of Aureobasidium pullulans. The importance of understanding the changes of population distribution during the fermentation and its effect in the production of pullulan are emphasized. The STVS can be used for monitoring and control of the cell population in small research fermentors or in large-scale production.

Direct and Rapid Analysis of the Adhesion of Bacteria to Solid Surfaces: Interaction of Fluorescently Labeled Rhodococcus Strain GIN-1 (NCIMB 40340) Cells with Titanium-Rich Particles.

A fluorimetric assay which enables direct and accurate analysis of the adhesion of bacteria to solid particles was developed. The assay is based on labeling of the bacteria with fluorescamine, which reacts with primary amino groups on the cell surface to yield a yellow fluorescence that is easily detectable by both fluorescence microscopy and spectrofluorimetry. As an example, fluorescent labeling of Rhodococcus strain GIN-1 (NCIMB 40340) cells enabled the detection and quantitative determination of their adsorption to TiO(inf2) and coal fly ash particles. Exposure of the cells to 10% acetone during the labeling reaction affected neither their viability nor their ability to adhere to these particles. Only a small fraction (;sim2%) of the total cell protein was labeled by fluorescamine upon staining of intact bacterial cells, which may indicate preferential labeling of certain proteins. Specificity studies carried out with the fluorescence assay confirmed previous findings that Rhodococcus strain GIN-1 cells possess high affinities for TiO(inf2), ZnO, and coal fly ash and low affinities for other metal oxides. In principle, the newly developed fluorimetric assay may be used for determination of cell adhesion to any solid matrix by either microscopic examination or epifluorescence measurements. In the present work, the adhesion of several other microorganisms to TiO(inf2) particles was tested as well, but their ability to adhere to these particles was significantly lower than that of Rhodococcus strain GIN-1 cells.

Adsorption of Rhodococcus Strain GIN-1 (NCIMB 40340) on Titanium Dioxide and Coal Fly Ash Particles.

Rhodococcus strain GIN-1 (NCIMB 40340) can be used to enrich and isolate a titanium-rich fraction from coal fly ash. The gram-positive bacterium was isolated by its ability to adhere strongly and rapidly to suspended particles of pure titanium dioxide or coal fly ash. Adsorption depends on the salt concentration and occurs in seawater. Lowering of the salt concentration or washing of particles with pure water did not, however, cause desorption of the bacteria from TiO(2) particles; this was achieved by strong alkaline treatment or combined treatment with sodium dodecyl sulfate and urea but not with dilute acids, alcohols, or cationic or nonionic detergents. The bacterium exhibits higher affinity towards oxides of Ti and Zn than to other oxides with similar distribution of particle size. Moreover, it adheres much faster to TiO(2) than to magnetite (Fe(3)O(4)) or Al(2)O(3). After about 1 min, more than 85% of the cells were adsorbed on TiO(2), compared with adsorption of only 10 and 8% to magnetite and Al(2)O(3), respectively. Adsorption of the bacteria on TiO(2) occurs over a pH range of 1.0 to 9.0 and at temperatures from 4 to over 80 degrees C. Scanning electron microscopy combined with X-ray analysis revealed preferential adherence of the bacterium to coal ash particles richer in Ti. Stronger adhesion to TiO(2) was also demonstrated in the translocation of bacteria, preadsorbed on magnetite, to TiO(2) particles. The temporary co-adhesion to magnetite and TiO(2) was exploited for the design of a prototype biomagnetic separation process in which bacterial cells serve as an adhesive mediator between magnetite and TiO(2) particles in a mixture of Al, Si, and Ti oxides that simulates their proportion in the ash.

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.

Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments.

An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. The enzyme retained its full activity at 20 to 55 degrees C, even after prolonged exposure (more than 30 days) to different concentrations of water-miscible organic solvents such as alcohols, glycols, pyridine, acetonitrile, dimethyl formamide, and dimethyl sulfoxide. The hydrolysis of 4-nitrophenyl laurate ester and of triglyceride emulsified in water was slightly accelerated with increasing concentrations of alcohols and glycols up to about 20% but was abolished with a further increase in alcohol concentration or in the presence of acetonitrile. In contrast, the rate of hydrolysis of these substrates in concentrated solutions of dimethyl formamide or dimethyl sulfoxide was markedly increased, by more than twofold and more than fivefold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous ethanol production by immobilized yeast reactor coupled with membrane pervaporation unit.

A system comprised of an immobilized yeast reactor producing ethanol, with a membrane pervaporation module for continuously removing and concentrating the produced ethanol, was developed. The combined system consisted of two integrated circulation loops: In one the sugar-containing medium is circulated through the membrane pervaporation module. The two loops were interconnected in a way allowing for separate parameter optimization (e.g., flow rate, temperature, pH) for each loop.The fermentation unit was 2.0 L bioreactor with five equal segments, packed with 5-mm beads of immobilized yeasts. The bead matrix was a crosslinked polyacrylamide hydrazide gel coated with calcium alginate. The fast circulation loop of the bioreactor allowed for efficient liberation of CO(2) at the top of the immobilized yeast reactor. Continuous operation of the uncoupled reactor for over 50 days with inflowing defined medium or dilute molasses at a residence time of 1.25 h yielded ethanol at a rate of about 10 g/L h.The pervaporation unit was constructed from four 60-cm-long tubular membranes of silicone composite on a polysulfone support. The output from the fermentor was circulated through the inside of the tubes of a unit with a total surface area of 800 cm(2), having an average flux of 150 mL/h, and selectivities to ethanol vs. water up to 7. A vacuum of 30 mb was applied to the outside of the tubes, removing 20-30 g of ethanol per hour, which was collected in condensors. The continuous removal of ethanol, avoiding inhibition of the fermentation process, resulted in an improved productivity and allowed the use of high sugar concentrations (40% wt/vol) offering the potential of a compact system with reduced stillage.The combined system of ethanol production and removal enabled an operative steady state at which the liquid volume of the system, and the concentrations of ethanol within the reactor ( 4% wt/vol), as well as within the flux crossing the pervaporation membrane (17%-20% wt/vol) were kept constant. At the steady state, a 40% wt/vol sugar solution could be continuously added to the fermentor when 12%-20% wt/vol clear ethanol solution was continuously removed by the pervaporation unit. Membrane fouling was reversed by short washing steps, and continuous step operation was maintained by working with two different modules that were interchanged. In this manner, long term continuous operation (over 40 days) was achieved with a productivity of 20-30 g/L h, representing over a twofold increase relative to the continuously operated reactor uncoupled from the membrane and a fivefold increase in comparison with the value obtained fro a corresponding batch fermentation.

Isolation and characterization of a lipolytic bacterium capable of growing in a low-water-content oil-water emulsion.

A unique lipolytic bacterium was isolated in a selective growth system consisting of 99% triglycerides and a 1% water phase. The bacterium, termed Pseudomonas aeruginosa YS-7, was able to grow in an environment of low water content and could also survive amphipathic, osmotic, and matrical water stress in a triglyceride-rich culture. The isolated strain was identified as P. aeruginosa on the basis of standard physiological, biochemical, and serological assays. The strain is a gram-negative motile rod, aerobic, pigment forming, and capable of growing at 42 degrees C. It is highly tolerant of high concentrations of the cationic detergent cetyltrimethylammonium bromide and of the fatty acid salts derived from bacterial hydrolysis of the oil. Growth of the bacterium in a pure culture in a 99% triglyceride medium lasted until most of the water was evaporated or consumed. Growth was accompanied by triglyceride hydrolysis, which continued to occur even after growth saturation until the water was totally depleted. No loss of viability was observed when the culture was maintained under water-depleted conditions for an additional 40 h. A second cycle of bacterial growth and triglyceride hydrolysis was immediately initiated upon the addition of 1% (vol/vol) water to the culture. Lipase activity was stable regardless of changes in culture conditions. The isolated strain is uniquely resistant to severe water stress in a triglyceride-rich medium or under cold acetone precipitation compared with 12 other microbial strains, including bacteria and yeasts. Among these 12, only the lipolytic strains grew in the 99% triglyceride medium, but they reached a cell mass fourfold smaller than that of P. aeruginosa YS-7.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of emulsan in a fermentation process using soybean oil (SBO) in a carbon-nitrogen coordinated feed.

A fermentation process for the microbial production of an amphipatic lipopolysaccharide, emulsan, has been established using a triglyceride carbon source in a coordinated carbon-nitrogen feed strategy. The polysaccharide was produced by the Acinetobacter strain at a productivity of about 0.5 g emulsan/L h while utilizing only the fatty acids (FA) portion of the triglycerides or free fatty acids that were fed into the medium.A useful correlation between the utilization of the fatty acids and the consumption of the nitrogen was found and employed for the practical feed strategy using an appropriate C--N ratio (7.7 g C/g N) of the soybeanoil (SBO) (carbon source) to the ammonium hydroxide base (nitrogen source). Either the pH control or the supervising computer system could accomplish the adequate balanced feed in to the fermentor. Lipolysis slowdown was overcome by switching from a triglyceride carbon source to a free fatty acids one. A yield of about 0.2 g emulsan/g fatty acids was obtained and a final concentration of over 20 g/L was reached. The polymeric product was found to be partially associated with the cell-oil complexes in the fermentation broth unless the oily carbon source was efficiently exhausted. A fedbatch fermentation that employed a shift of the carbon source feed from triglycerides to free fatty acids appeared to be an appropriate and feasible way of producing the polymer.

Production of exopolysaccharides by Acinetobacter strains in a controlled fed-batch fermentation process using soap stock oil (SSO) as carbon source.

The production of two extracellular capsular heteropolysaccharides by two different Acinetobacter strains has been studied in separate controlled fermentation processes with a view to their industrial applications as specific dispersing agents. The first, emulsan, is an extracellular polyanionic amphipathic heteropolysaccharide (MW 10(6) D) made by A. calcoaceticus RAG-1. It forms and stabilizes oil in water emulsions. The other, biodispersan (PS-A2), is another extracellular zwitterionic heteropolysaccharide (MW 51 kD) made by A. calcoaceticus A2. This polysaccharide disperses big solid limestone granules forming micron-size water suspension. Both polysaccharides are synthesized within the cells, exported to their outer surface to form an extracellular cell-associated capsule and released subsequently into the growth medium. The polymers were produced in a computer-controlled fed-batch intensively aerated fermentation process. A commercially available and cheap fatty acids mixture (soap stock oil) served as the carbon source, and was fed in coordination with the required nitrogen. The coordinated feed of carbon and nitrogen was operated on the basis of two metabolic correlations: The first correlation related the cell protein produced and the ammonium nitrogen consumed with the outcoming coeffients of 24 and 21 mM NH3/g protein for the emulsan and the biodispersan fermentations respectively. The second correlation linked the consumption of the fatty acids with that of the nitrogen source dictating the appropriate C/N ratio of the feed into the operating fermentor. These ratios were 7.7 g C/g N for the emulsan fermentation and 8.5 gC/g N in the case of the biodispersan production process.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency and amplitude effects during high-frequency vibration ventilation in dogs.

High-frequency external body vibration, combined with constant gas flow at the tracheal carina, was previously shown to be an effective method of ventilation in normal dogs. The effects of frequency (f) and amplitude of the vibration were investigated in the present study. Eleven anesthetized and paralyzed dogs were placed on a vibrating table (4-32 Hz). O2 was delivered near the tracheal carina at 0.51.kg-1.min-1, while mean airway pressure was kept at 2.4 +/- 0.9 cmH2O. Table vertical displacement (D) and acceleration (a), esophageal (Pes), and tracheal (Ptr) peak-to-peak pressures, and tidal volume (VT) were measured as estimates of the input amplitude applied to the animal. Steady-state arterial PCO2 (PaCO2) and arterial PO2 (PaO2) values were used to monitor overall gas exchange. Typically, eucapnia was achieved with f greater than 16 Hz, D = 1 mm, a = 1 G, Pes = Ptr = 4 +/- 2 cmH2O, and VT less than 2 ml. Inverse exponential relationships were found between PaCO2 and f, a, Pes, and Ptr (exponents: -0.69, -0.38, -0.48, and -0.54, respectively); PaCO2 decreased linearly with increased displacement or VT at a fixed frequency (17 +/- 1 Hz). PaO2 was independent of both f and D (393 +/- 78 Torr, mean +/- SD). These data demonstrate the very small VT, Ptr, and Pes associated with vibration ventilation. It is clear, however, that mechanisms other then those described for conventional ventilation and high-frequency ventilation must be evoked to explain our data. One such possible mechanism is forcing of flow oscillation between lung regions (i.e., forced pendelluft).

Effect of tracheal bias flow on gas exchange during high-frequency chest percussion.

High-frequency chest percussion (HFP) with constant fresh gas flow (VBF) at the tracheal carina is a variant of high-frequency ventilation (HFV) previously shown to be effective with extremely low tracheal oscillatory volumes (approximately 0.1 ml/kg). We studied the effects of VBF on gas exchange during HFP. In eight anesthetized and paralyzed dogs we measured arterial and alveolar partial pressures of CO2 (PaCO2) and O2 (PaO2) during total body vibration at a frequency of 30 Hz, amplitude of 0.17 +/- 0.019 cm, and tidal volume of 1.56 +/- 0.58 ml. VBF was incrementally varied from 0.1 to 1.2 l.kg-1.min-1. At low flows (0.1-0.4 l.kg-1.min-1), gas exchange was strongly dependent on flow rate but became essentially flow independent with higher VBF (i.e., hyperbolic pattern). At VBF greater than 0.4 l.kg-1.min-1, hyperventilatory blood gas levels were consistently sustained (i.e., PaCO2 less than 20 Torr, PaO2 greater than 90 Torr). The resistance to CO2 transport of the airways was 1.785 +/- 0.657 l-1.kg.min and was independent of VBF. The alveolar-arterial difference of O2 was also independent of the flow. In four of five additional dogs studied as a control group, where constant flow of O2 was used without oscillations, the pattern of PaCO2 vs. VBF was also hyperbolic but at substantially higher levels of PaCO2. It is concluded that, in the range of VBF used, intraairway gas exchange was limited by the 30-Hz vibration. The fresh gas flow was important only to maintain near atmospheric conditions at the tracheal carina.

Enhanced emulsan production in mutants of Acinetobacter calcoaceticus RAG-1 selected for resistance to cetyltrimethylammonium bromide.

Mutants of Acinetobacter calcoaceticus RAG-1 that produced elevated levels of the polymeric bioemulsifier emulsan were isolated on the basis of their resistance to the cationic surfactant cetyltrimethylammonium bromide (CTAB). Such mutants showed maximum enhancement in both overall yield and specific productivity of some two- to threefold over that of the wild type. In addition, the effect was also observed in a resting cell system in the presence of chloramphenicol, indicating that the mutation is not simply the result of faster growth. When CTAB-tolerant mutants were subjected together with the sensitive parent to the detergent under growing conditions, only the mutants were found to grow. The results suggest that the mutation for CTAB resistance leads to enhanced capsule production. This was confirmed quantitatively by a specific enzyme-linked immunosorbent assay for the cell-bound emulsan minicapsule.

Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus.

An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan free or associated with the bioemulsifier. The partially purified enzyme was found to interact specifically with the esterified fully active emulsan, but not with the deesterified polymer. A role for esterase in emulsan release from the cell surface was indicated when the enzyme was preferentially depleted from the cell surface under conditions in which emulsan was not released. Such cells lost the capacity to release the biopolymer.

Tolerance of Acinetobacter calcoaceticus RAG-1 to the cationic surfactant cetyltrimethylammonium bromide: role of the bioemulsifier emulsan.

Emulsan, the polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1, was found to enhance the tolerance of RAG-1 cells to the toxic effects of the cationic detergent cetyltrimethylammonium bromide (CTAB). Emulsan-mediated tolerance was obtained with the purified deproteinated apoemulsan; ca. 9 micrograms of apoemulsan neutralized 1 microgram of CTAB. Deesterified apoemulsan was only half as effective in protecting the cells from CTAB toxicity. Tolerance was also mediated by the cell-associated emulsan minicapsule. Mutants lacking this capsule were more sensitive to CTAB than the corresponding parent. The growth of mutants and parent cells in mixed-culture experiments demonstrated that the cell-associated polymer mediates CTAB tolerance in the early stages of growth. Once sufficient cell-free polymer has been released into the aqueous medium (ca. 0.5 micrograms/ml), this extracellular emulsan also plays a role in CTAB tolerance.