Surface roughness enhances upward migration of bacteria on polymer fibers above liquid cultures.

Monofilament polypropylene (PP) fibers, very similar to fibers that have been used as monofilament tailstrings of interuterine contraceptive devices, were suspended vertically in bacterial liquid monocultures so that a portion of a fiber extended above the liquid surface. In some cases these highly oriented, cold drawn fibers were abraded prior to insertion in the cultures in order to produce surface roughness characterized by axial channels and protruding microfibrils that partially peeled from the fiber surface thereby forming the channels. Extent of migration on a fiber was assessed by aseptically cutting it into small segments, followed by culturing each segment on agar containing growth medium. Such assessment of the PP fibers after 48 h of incubation in the cultures revealed upward migration of Eschericia coli, Pseudomonas aeruginosa, and Staphylococcus aureus over significantly longer distances on the pre-roughened fibers than on those not so pre-treated. Mean measured distances of migration during 48 h were: for E. coli 2.7+/-0.6 mm on roughened fibers (n = 16) and 0.4+/-0.7 mm on fibers not roughened (n = 17); for S. aureus 9.0+/-4.3 mm on roughened fibers (n = 13) and 0.2+/-0.3 mm on fibers not roughened (n = 14); for P. aeruginosa 8.5+/-3.7 mm on roughened fibers (n = 26) and 0.2+/-0.5 mm on fibers not roughened (n = 5). Although no statistically significant (95% confidence level) difference could be discerned between the migration distances of S. aureus and P. aeruginosa, each of these species migrated a greater distance on the PP than did E. coli. The migrations observed are attributed predominantly to wicking of the liquid cultures upward in the axial grooves developed on the surface of the PP by the eruption and peeling of microfibrils from the surface. Surface tension of the growth medium was significantly lower than that of water and its contact angle on PP was less than 90 deg, thereby indicating a tendency to wet the PP. Bacterial growth in the medium further reduced its contact angle on PP, thereby indicating an even greater tendency to wet PP after such growth.

Comparative behavior of E. coli and S. aureus regarding attachment to and removal from a polymeric surface.

Staphylococcus aureus was found to attach more readily on polypropylene fibers with greater resistance to subsequent washing off than Escherichia coli after immersion of the fibers up to 300 s in pure culture suspensions in mineral salts medium. Each of five different washing solutions were effective against E. coli, whereas only a solution that contained SDS was effective against S. aureus. Placement of inoculated fibers on nutrient agar for longer periods of time resulted in greater resistance to washing by both organisms, although S. aureus remained more resistant than E. coli.

Migration of bacteria along synthetic polymeric fibers.

E. coli, S. epidermidis, and B. distasonis were observed to migrate readily along polymer fibers impressed upon the surface of nutrient agar. E. coli was also observed to migrate readily along polymer fibers embedded in brain-heart infusion agar. Within periods of about 24 h, migration distances of about several centimeters were observed. No migration was observed in control experiments conducted on or in the same media, but without fibers. Migration speed was greatest for E. coli and slowest for B. distasonis. Cell population density was found to decrease rapidly with distance from a source culture. Swimming motility or natural convection in liquid between fiber and gel appears to be improbable based on the expected dimensions of capillary-condensed liquid between fiber and gel.

Investigation of production of dextran and dextransucrase by Leuconostoc mesenteroides immobilized within porous stainless steel.

Cells of Leuconostoc mesenteroides were immobilized within porus, stainless-steel (SS) supports and used for dextransucrase (DS) and dextran production. The pore size of the support significantly affected the dextran yields, which were greatest with average pore sizes of 2-5 microm. All immobilized-cell biocatalysts in porous stainless steel produced higher yields than free cells, with the exception of cells confined in submicrometer pores (0.5 microm). Coating supports of larger pore size (40 and 100 microm) with calcium alginate enhanced the cell-loading capacity of the supports and increased dextran and fructose yields in the cell-free broth. Controlled, fed-batch, DS production (activation), as a step preliminary to dextran production, significantly improved the subsequent dextran and fructose yields and shortened the time required to attain the maximum such yields. Scanning electron microscopy (SEM) of immobilized L. mesenteroides in stainless steel shows an irregular pattern of the microorganism inside the pores of the solid supports. Coating the porous solid supports with a cell-free calcium alginate layer led to an increase in the cell density inside the support. Cell growth inside the coated, porous stainless steel had no distinct growth form.

Polypropylene IUCD retrieval fibers: surface morphology, material properties, microbial attachment, and migration.

Highly drawn and oriented polypropylene fibers used for the retrieval thread of the Cu-7 intrauterine contraceptive device (IUCD) are compared as to surface morphology and crystallinity with polypropylene fibers prepared under different conditions. A series of experiments also demonstrates the colonization of the surface of polyolefin fibers by pathogenic bacteria that are often found in the human vagina. Moreover, it is demonstrated that the retrieval threads appear to encourage pathogenic bacteria to migrate across the surface of agar. The results also indicate that control of drawing and annealing can avoid the surface fibrillation and tendency to fail by separation into a bundle of multifilaments that are observed with the IUCD retrieval threads.

Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium-alginate beads: I. Batch and fed-batch fermentations.

In batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized-cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell-free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.

Production of dextransucrase and dextran by Leuconostoc mesenteroides immobilized in calcium-alginate beads: II. Semicontinuous fed-batch fermentations.

Cells of Leuconostoc mesenteroides immobilized in calcium alginate beads were used to produce dextransucrase (DS) in three sequential cycles of semicontinuous fed-batch fermentations. Each cycle consisted of a fed-batch DS production period of 24 h followed by a batch dextran production period for another 24 h. Free, suspended cells were used in only one cycle of fed-batch DS production followed by a dextran production period. It was impractically tedious to separate and reuse free cells. Increasing sucrose feed rate from 5 to 10 g/L h led to increases of the total enzymatic activity by about 88% with immobilized cells and by about 100% with free cells. In DS fed-batch semicontinuous fermentation, total enzymatic activity produced by immobilized cells was 1.35 and 1.56 times greater than that produced by free cells with respective sucrose feeding rates of 10 and 5 g/L h. These increases in enzyme productivity with immobilized cells, however, required total overall operating times three times longer (three cycles) than with free cells (one cycle). Growing the microorganism at optimum conditions for DS production also increased the dextran yield and shortened the time of conversion of sucrose to dextran, regardless of whether the cells were free or immobilized. Moreover, during three cycles of semicontinuous operation (144 h) immobilized cells produced more than three times as much dextran as free cells during one cycle (24 h).

Effect of beta-cyclodextrin on production of L-phenylacetyl carbinol by immobilized cells of Saccharomyces cerevisiae.

The rate and extent of microbial transformation of higher concentrations of benzaldehyde substrate to L-phenylacetyl carbinol (L-PAC) by immobilized cells of Saccharomyces cerevisiae ATCC 834 was markedly stimulated by addition of different concentrations of beta-cyclodextrin (BCD) to the fermentation medium. With 0.5, 1.0, and 1.5% BCD in the fermentation medium and cumulative doses of benzaldehyde of 12 and 14 g/L, significantly higher yields of L-PAC were obtained, about one- to twofold that of the yields of the control experiments. The favorable effects of BCD were evident in spite of its presence in stoichiometric concentrations significantly lower than those of benzaldehyde. The presence of BCD also appeared to stimulate microbial growth slightly. Enhanced cellular activity was reflected by faster D-glucose consumption and faster benzaldehyde utilization in the presence of BCD.

N-laurylbiotinamide as affinity surfactant.

N-laurylbiotinamide (NLB), which retains strong affinity for the protein avidin, was synthesized from biotin and N-laurylamine via the biotin ester of N-hydroxysuccinimide and characterized by NMR. When the synthesized NLB was used as a cosurfactant with AOT to form a reverse micellar system in isooctane, it was found to extend the pH range over which avidin can be transferred from a continuous aqueous solution to the reverse-micellar phase. This behavior is similar to that already reported for a different affinity surfactant, n-octyl beta-D-glucopyranoside.

Production of L-phenylacetyl carbinol by immobilized yeast cells: I. Batch fermentation.

Immobilization of Saccharomyces cerevisiae ATCC 834 within alginate beads enhances microbiological conversion of benzaldehyde to L-phenylacetyl carbinol (L-PAC), a precursor employed for synthesis of L-ephedrine. Yields of 90% L-PAC on benzaldehyde (initially 0.6% in medium) were obtained with immobilized cells, in contrast to about 10% with free cells which tend to form pellets in the presence of benzaldehyde. The predominant favorable action of immobilization appears to be a reduction in the toxic or inhibitory effects of benzaldehyde. With an initial benzaldehyde concentration of about 0.6% in the medium the optimum cell mass concentration was observed to be about 28 g cell mass (immobilized) per liter of medium.

Production of L-phenylacetyl carbinol by immobilized yeast cells: II. Semicontinuous fermentation.

The cyclic, semicontinuous production of L-phenylacetyl carbinol (L-PAC) from a benzaldehyde substrate by Saccharomyces cerevisiae ATCC 834 immobilized in calcium alginate beads was substantially enhanced to about 4.5 g/L in a second cycle by reactivation in fresh medium for 24 h, following an earlier 24-h period of production from substrate. Intermittent feeding of benzaldehyde was employed (four doses in 3 h). In subsequent similar cycles, however, the production returned to that produced in the first cycle, viz. L-PAC concentration of 2-3 g/L in the medium. Production of L-PAC was also increased by adaptation of the cells over 200 h of exposure to the benzaldehyde substrate (compared to wild-type cells) and by continuous (as compared to intermittent) feeding of the substrate. A liter as great as 10 g/L was obtained with wild-type cells by continuous feeding of benzaldehyde over 6 h. Immobilization not only protected the cells from toxic effects of substrate but also permitted them to be used during 7 cycles of semicontinuous operation over more than 200 h.

Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads.

In fed-batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 microm and pore size of 0.14 microm) produced the highest total enzymatic activity, followed by Celite R633, alginate-coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate-coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme-enriched, immobilized-cell cultures was higher than that obtained from free-cell culture under similar conditions.

Amperometric assay of coenzyme-dependent oxidoreductase enzymes in a flow-through cell.

We have demonstrated that a simple electrochemical cell can serve as a detector of NADH concentration in a flow system thereby providing an assay technique for NADH dependent enzymes. When this is applied to NADH produced by enzymatic reaction, then a reproducible measure of enzyme activity is obtained. This method of enzyme activity assay is applicable to a number of oxidoreductase enzymes which employ NAD+ or NADP+ as coenzymes to achieve substrate modification. The presence of electroactive species in samples of human serum has proved a serious problem in the electrochemical analysis of serum activity. These species produce a large background anode current at the anode voltage appropriate for NADH oxidation. The presence of this high current limits the usefulness of amplification of the current output to detect small changes in NADH concentration.

Electrochemical characterization of immobilized NAD+.

Nicotinamide adenine dinucleotide (NAD+) has been covalently attached to alginic acid using carbodiimide coupling, thereby producing a macromolecular adduct of NAD, which can be rendered either soluble or insoluble by adjustment of pH. It was found that this NAD+-alginic acid complex was enzymatically active, and also that the oxidized form could be electrochemically reduced without loss in enzymatic activity. This NAD+ adduct has now also been polarographically characterized as to its two-step reduction waves, which are slightly shifted toward more cathodic potential as compared to free NAD+. When controlled electrolysis was conducted to reduce the bound NAD+ at the cathode, the NADH so formed by electrochemical action was found to be again oxidizable either enzymatically or electrochemically without loss in co-enzymatic function. The NADH adduct produced by electrochemical reduction of the NAD+ adduct has also been characterized by voltammetry.

Preparation and properties of soluble-insoluble nicotinamide coenzymes.

A soluble-insoluble form of nicotinamide adenine dinucleotide (NAD+), which can be rendered either soluble or insoluble by simply adjusting the pH, has been prepared by covalently coupling NAD to alginic acid using 1,2,7,8-diepoxyoctane. The NAD bound to the alginic acid showed the coenzymic function in the soluble state and could be collected for further use as precipitate by lowering the pH to below 3. Coupling soluble-insoluble coenzymes with insolubilized apoenzymes is possible in fluidized and fixed-bed reactors.

Electrolytic regeneration of the reduced from the oxidized form of immobilized NAD.

A covalently bound adduct of nicotinamide adenine dinucleotide (NAD) with alginic acid has been found to be enzymatically active and to undergo electrochemical oxidation or reduction without significant loss of its enzymatic activity. The preparation of the adduct itself (from NAD+, alginic acid, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate) is also accomplished with substantially complete retention of enzymatic activity. This adduct has been converted from the oxidized to the reduced form by controlled potential electrolysis using mercury and stainless-steel electrodes. This electrolytically produced NADH complex could be oxidized again to the enzymatically active NAD+ complex by enzymatic reaction with the proton acceptor, 2,6-dichlorophenol indophenol, as catalyzed by diaphorase. Using this electrolytic method with immobilized NAD, it is now possible to carry out redox reactions in which NADH is enzymatically oxidized to NAD+, with the simultaneous electrolytic regeneration of the reduced form, NADH, from the oxidized form, NAD+, produced in the enzymatic reaction.

Electrochemical regeneration of nicotinamide adenine dinucleotide.

Reduced nicotinamide adenine dinucleotide (NADH) has been characterized electrochemically by solid electrode voltammetry and controlled potential electrolysis. Photometric and enzymatic assay showed that enzymatically active nicotinamide adenine dinucleotide (NAD-+) could be regenerated electrolytically from its reduced form without the use of so-called electron mediators. Complete regeneration of enzymatically active NAD can be expected in pyrophosphate buffers and phosphate buffers during the electrolysis. Advantages of electrochemical regeneration of coenzymes are discussed, especially with regard to immobilization of enzymes.