Immobilization bacterial cells

De Rosa, M, Nicolaus, A., Gambacorta, B., Buonocore, V., and Poerio, E. (1980) Immobilized bacterial cells containing athermostable p-galactosidase, Biotechnol. Letters 2, 29 -34... 

Recent examples of process improvement have been reported by Davison and Thomson [11] and Kaufman et al. [12]. They studied the simultaneous fermentation and recovery of lactic acid in a biparticle fluidized-bed reactor using L. delbreuckii as the biocatalyst. The immobilized bacterial cells (on calcium alginate beads of 0.7-0.8 mm diameter) were fluidized in the liquid media in a column reactor (see Fig. 1). During fermentation, solid particles of lactic acid adsorbent (polyvinylpyridine resin) are added batchwise to the top of the reactor, and fall countercurrently through the biocatalyst. After the adsorbents have fallen through the reactor, they are recovered and the adsorbed lactic acid is recovered. The adsorbents not only remove acid produced but also effectively maintain the broth pH at optimal levels. The increase in lactic acid production is significant. The reported volumetric productivity of 4.6 g/l/h was a 12-fold increase over the reactor without the adsorbents. 

At present there are no totally satisfactory methods of commercially removing limonoid bitterness. Several new approaches are being developed which may lead to a more complete solution to the problem. These include specific adsorbents of limonoids, a biological reactor that uses immobilized bacterial cells, and the preharvest approaches which are presented in the text. 

Two types of immobilization are used for immobilizing glucose isomerase. The intracellular enzyme is either immobilized within the bacterial cells to produce a whole-ceU product, or the enzyme is released from the cells, recovered, and immobilized onto an inert carrier. An example of the whole-ceU process is one in which cells are dismpted by homogenization, cross-linked with glutaraldehyde, flocculated using a cationic flocculent, and extmded (42). 

Among the wide choice of reactor designs, the biofilm reactor is one of the best suited for azo-dye conversion as it meets two important process requisites. The first is related to the hindered growth feature of bacterial metabolism under anaerobic conditions. The second is related to the necessity to increase cell densities (see previous section) with respect to those commonly harvested in liquid broths [55, 56]. Except for bacteria that forms aggregates spontaneously, immobilization of cells on granular carriers and membrane reactor technology are the two common pathways to achieve high-density confined cell cultures in either discontinuous or flow reactors. 

The type of matrix used for immobilization of the recognition element for bacterial cell detection is crucial to achieve high sensitivity. Two important conditions should be considered specifically for bacterial detection (1) the accessibility of the recognition elements in the immobilization matrix for bacteria binding on the sensor surface and (2) to obtain the binding of the analytes within the most sensitive region of the evanescent field, immediately adjacent to the sensor surface. 

Work at the EPA Gulf Breeze Laboratory has demonstrated the potential usefulness of encapsulation in the bioremediation of PAHs. A model system has been developed in which a pure culture capable of degrading fluoranthene (strain EPA505) has been successfully encapsulated in polyurethane foam and polyvinyl alcohol (Baker et al., 1988). The capsules can be stored for several months at 4 °C with only minimal loss of viability. Upon addition of the capsules to moist soil, fluoranthene mineralization commenced in approximately the same way as observed when fresh bacterial cells were added to the soil. These results are shown in Figure 5.7a. Since the same inoculation size was used in all flasks during this experiment, the results suggest that the immobilization process does not significantly affect microbial activity. 

Subsurface environmental conditions are suboptimal with low temperatures and low concentrations of growth nutrients. The decline of bacterial inoculae by protozoan predation is of major concern in soil (Acea etal., 1988 Acea Alexander, 1988 Casida, 1989) but may not be a factor in saturated subsurface environments. Immobilization of cells to carrier material may enhance microbial survival in the environment through control of predation and supply of nutrients and moisture. Stormo Crawford (1992) developed a cell immobilization technique for production of small beads (2-50 /rm) consisting of agarose and cells of PCP-degrading Flavobacterium sp. Microencapsulated Flavobacteria efficiently degraded PCP and survived for two years in soil columns at environmental conditions (Stormo Crawford, 1994). These results show that microencapsulation may be a very useful tool in in situ bioremediation. 

Biofilm is commonly found within the labyrinthine waterways of cooling systems and is primarily composed of mixed populations of bacterial cells, immobilized within a fibrous matrix of ionic polymer (slime). 

Figure 1.15 The biocatalytic synthesis of acrylamide from acrylonitrile is performed in Japan on a scale of 10000 tons per year. The bacterial cells are immobilized in a poly(acrylamide) gel, and the process is run at pH 8.0-8.5 in semi-batch mode, keeping the substrate concentration below 3%.

The basic enzymatic procedure usually involves immobilization of bacterial cells containing aspartase activity or of semi-purified enzyme, in either a fed batch or a continuous, packed-column process. The starting material is maleic anhydride, an inexpensive, high volume, bulk chemical.56... 

Dipstick-based IgE testing allows rapid testing for positive IgE present in serum samples. In most cases these assays use total protein extracts (Straumann and Wuthrich 2000). However the proof of concept has also been performed with a recombinant protein, Bet v 1, the birch pollen allergen. Crystalline bacterial cell-surface layers were used as immobilization matrices. Covalently bound monoclonal anti-Bet v 1 antibody served as a monolayer onto the recombinant allergen was bound. Quantitative determination of Bet v 1-specific IgE from human serum samples was possible within 90 min (Breitwieser et al. 1996,1998). 

Breitwieser, A., C. Mader, I. Schocher, K. Hoffmann-Sommergruber, W. Aberer, O. Scheiner, U. B. Sleytr, and M. Sara. 1998. A novel dipstick developed for rapid Bet v 1-specific IgE detection recombinant allergen immobilized via a monoclonal antibody to crystalline bacterial cell-surface layers. Allergy 53 (8) 786-793. 

The rate constants and k refer to mineralization (m) and immobilization (i), respectively. Mineralization refers to production of NH3 and/or NH4 through microbial decomposition, whereas immobilization refers to incorporating N into bacterial cells (e.g., making up protein). 

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