The goal was to relate basic research to applications and to extract guidelines for characterization of immobilized cells in view of process design and application from the contributions. The manuscripts presented in these proceedings give an extensive and recent overview of the research and applications of immobilized-cell technology.
This publication contains full papers of both oral and poster presentations of the symposium "e;Immobilized Cells: Basics and Applications"e; that was held in Noordwijkerhout, The Netherlands, 26-29 November 1995. This volume covers recent developments in the field of immobilization e.g.: new support materials, characterization of support materials, kinetic characterizations, dynamic modelling, bioreactor types, scale up and applications are also given. Applications in the field of medicine, fermentation technology, food technology and environmental technology are described. Guidelines for research with immobilized cells. Based on the scientific sessions a strategy of research and methods for characterization of immobilized cells, especially in view of applications are given.The goal was to relate basic research to applications and to extract guidelines for characterization of immobilized cells in view of process design and application from the contributions. The manuscripts presented in these proceedings give an extensive and recent overview of the research and applications of immobilized-cell technology.
Cover 1
Contents 10
Preface 6
Acknowledgements 8
Keynote lecture 20
Why immobilize? 22
Basics 1: physics 36
Chapter 1. Alginate gels - Some structure-function correlations relevant to their use as immobilization matrix for cells 38
Chapter 2. Determination of biofilm diffusion coefficients using micro-electrodes 50
Chapter 3. Complexity and heterogeneity of microenvironments in immobilized systems 58
Chapter 4. Fundamentals of dispersion in encapsulation technology 66
Chapter 5. Stable support materials for immobilization of viable cells 74
Chapter 6. Evaluation of density function of pore size distribution of calcium pectate hydrogel 81
Chapter 7. Effects of formation conditions on size distribution of thermogel beads for cell immobilization 89
Chapter 8. Calcium pectate gel could be a better alternative to calcium alginate gel in multiple applications of immobilized cells 95
Chapter 9. Adhesion of Lactococcus lactis diacetylactis to surfaces 103
Chapter 10. Immobilization of Arthrobacter globiformis 193 cells into PVA cryogel. Dehydrogenation of steroid substrates 109
Chapter 11. Screening of immobilization materials for anaerobic wastewater treatment 117
Chapter 12. Ecologically pure process of acetate synthesis on diverse gaseous substrates by homoacetogenic bacteria, entrapped in poly(vinyl alcohol) cryogel 125
Chapter 13. Entrapment of Zymomonas mobilis cells into PVA-cryogel carrier in the presence of polyol cryoprotectants 131
Chapter 14. Some microorganisms during their entrapment in PAAG act as "biological accelerators" in how they affect the gel-formation rate 137
Chapter 15. Rhodococcus sp. immobilized by adsorption on chitin 145
Chapter 16. Immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam 151
Chapter 17. Effect of C/N ratio and cellulose type on the cellulolytic activity of free and immobilized Trichoderma reesei 155
Chapter 18. Analysis of fungal B-glucanase system produced under conditions of mycelial immobilization 159
Chapter 19. Immobilization as a contact stimulation of yeast cell wall alterations 167
Chapter 20. Modelling the immobilization of cells in a packed bed of porous carriers 173
Chapter 21. Porous silicone rubber as an immobilization matrix for microbial and mammalian cells: natural immobilization of a mass transfer limited culture environment 181
Chapter 22. Continuous fermentation by conventional and recombinant Saccharomyces cerevisiae immobilized in Ca-alginate beads hardened with trivalent ion 192
Chapter 23. Encapsulation by polyelectrolyte complex formation - a way to make hepatocyte cultures safe, efficient and on-line available 200
Chapter 24. Influence of the microenvironment on immobilized Gibberella fujikuroi 208
Chapter 25. Local mass transfer coefficients in bacterial biofilms using fluorescence recovery after photobleaching (FRAP) 215
Chapter 26. Characteristics and selection criteria of support materials for immobilization of nitrifying bacteria 224
Chapter 27. Stability of alginate gels applied for cell entrapment in open systems 232
Chapter 28. Studies of cell viability of immobilized Chlamydomonas reinhardtii and glycerol photoproduction 240
Chapter 29. Effect of drying medium on the viability of dried Lactobacillus helveticus CNRZ 303 immobilized in calcium-alginate beads 248
Basics 2: physiology, mass transfer and dynamic modelling 256
Chapter 30. Comparison of retention and expression of recombinant plasmids between suspended and biofilm-bound bacteria degrading TCE 258
Chapter 31. Immobilized-cell growth: Diffusion limitation in expanding micro-colonies 268
Chapter 32. Competition and cooperation of microorganisms in a coimmobilized aerobic/anaerobic mixed culture 276
Chapter 33. Viability of immobilized cells: Use of specific ATP levels and oxygen uptake rates 283
Chapter 34. Gelatin immobilized growing yeast cells: Changes in the glycosylation level of external invertase and cell wall composition 291
Chapter 35. Nitrification activity of immobilized activated sludge evaluated by respiration rate 299
Chapter 36. Growth of immobilized cells: Results and predictions for membrane-attached biofilms using a novel in situ biofilm thickness measurement technique 309
Chapter 37. Cryo-electron microscopy of polymer particles in a high cell density synthetic biofilm 317
Chapter 38. Investigation of oxygen consumption by E. coli immobilized in a synthetic biofilm using a thin film plug reactor (TFPR) 323
Chapter 39. Investigation of gene expression in synthetic biofilms to extend the activity of immobilized whole cell catalysts 332
Chapter 40. Screening and design of immobilized biocatalysts through the kinetic characterization by flow microcalorimetry 339
Chapter 41. Pitfalls of initial reaction rate measurements 347
Chapter 42. Growth and eruption of gel-entrapped microcolonies 355
Chapter 43. Quantitative characterization of viability and growth dynamics of immobilized nitrifying cells 360
Chapter 44. Modelling of the biotransformation from geraniol to nerol by freely suspended and immobilized grape (Vitis vinifera) cells 368
Chapter 45. Modelling and experimental validation of cell and substrate evolution in an immobilized system 374
Chapter 46. Mass transfer limitations in a bioartificial pancreas 381
Chapter 47. Effect of dissolved oxygen concentration on pH-controlled fed-batch gluconate production by immobilized Aspergillus niger 389
Chapter 48. Alginate-immobilized thermotolerant yeast for conversion of cellulose to ethanol 398
Chapter 49. Further research on polysaccharide production by immobilized cells of the lichen Pseudevernia furfuracea (L.) Zopf. in polyacrylamide 403
Chapter 50. Study of parietin production by immobilized cells of Xanthoria parietina in calcium-alginate matrix 409
Chapter 51. Effects of immobilization on polyol production by Pichia farinosa 414
Chapter 52. Bioprocess engineering considerations in cyclosporin A fermentation by immobilized fungus Tolypocladium inflatum 421
Chapter 53. Nitrate uptake by immobilized growing Chlamydomonas reinhardtii 429
Chapter 54. Influence of the immobilization methodology in the stability and activity of P. putida UV4 immobilized whole cells 435
Chapter 55. Immobilization of the extremely thermophylic archaeon Pyrococcus furiosus in macro-porous carriers 443
Chapter 56. Immobilization of Mycobacterium sp. cells for sitosterol side chain cleavage in organic solvents 450
Chapter 57. Oxytetracycline production by free and immobilized cells of Streptomyces rimosus in batch and repeated batch cultures 456
Chapter 58. Bacterial conjugation within k-carrageenan gel beads: biotic and abiotic factors affecting plasmid transfer 463
Chapter 59. Plasmid stability in immobilized Bacillus subtilis continuous cultures 471
Chapter 60. Improved stability of a naturally occurring TOL plasmid in Pseudomonas putida by immobilization 477
Chapter 61. Strategies in the design of an enzymatic process for the synthesis of ampicillin: A whole cell E. coli recombinant penicillin amidase biocatalyst 483
Chapter 62. Immobilization of recombinant E. coli cells of with phenol-lyase activity 491
Chapter 63. Kinetics of a co-immobilized system 498
Chapter 64. Dynamic modelling of an integrated nitrogen removal system using co-immobilized microorganisms 505
Part 1: Immobilized-cell reactors 514
Chapter 65. Scale up aspects of immobilized cell reactors 516
Chapter 66. Problems in scale-up of immobilized cell cultures 524
Chapter 67. Performance of a liquid-impelled loop reactor with immobilized cells 530
Chapter 68. Effect of pulsation on morphology of Aspergillus niger and Phanerochaete chrysosporium in a fluidized-bed reactor 537
Chapter 69. The estimation of the optimum amount of solid support in an immobilized cell bioreactor 543
Chapter 70. Double-chambered bioreactors based on plane immobilized-cell membrane structures 551
Chapter 71. Continuous production and in situ extraction of isovaleraldehyde in a membrane bioreactor 557
Chapter 72. PEGASUS: Innovative biological nitrogen removal process using entrapped nitrifiers 565
Chapter 73. Treatment of nitrogenous wastewaters by immobilized cyanobacteria in an airlift-fluidized photo-bioreactor 575
Chapter 74. Photosynthetic bio-fuel cells using immobilized cyanobacterium Anabaena variabilis M-3 582
Chapter 75. Lactic acid fermentation using immobilized Lactobacillus casei cells 589
Chapter 76. Lay-out of fixed bed reactor systems for effective production of biologicals with immobilized animal cells 595
Part 2: Applications 602
Chapter 77. Industrial application of immobilized biocatalysts in Japan 604
Chapter 78. Enzyme production with immobilized filamentous fungi 611
Chapter 79. Microbiologial stability of an immobilized cell bioreactor with mixed lactic acid bacteria during continuous fermentation of milk 619
Chapter 80. Characterization of monoclonal IgA production and activity in hollow-fiber and fluidized-bed reactors 627
Chapter 81. Organoleptic profiles of different ciders after continuous fermentation (encapsulated living cells) versus batch fermentation (free cells) 634
Chapter 82. Application of nitrification by cells immobilized in polyethylene glycol 641
Chapter 83. Immobilized cell technology in food processing 652
Chapter 84. Bioencapsulation of carrot somatic embryos 660
Chapter 85. Alginate-polycation microcapsules for cell transplantation - Long time stability 668
Chapter 86. Fumaric acid production by Rhizopus arrhizus immobilized on polyurethane sponge 674
Chapter 87. Application of immobilized yeast cells in the brewing industry 680
Chapter 88. Design and application of an immobilized loop bioreactor for continuous beer fermentation 691
Chapter 89. Leuconostoc oenos entrapment: Application to continuous malo-lactic fermentation 698
Chapter 90. Practical use of an immobilized cell bioreactor for continuous prefermentation of milk 706
Chapter 91. Behavior of immobilized Nitrosomonas europaea and Paracoccus denitrificans in tubular gel for nitrogen removal in wastewater 714
Chapter 92. Treatment of high strength ammonia wastewaters using immobilized biomass 722
Chapter 93. Comparison of a system using immobilized microorganisms with a conventional activated sludge process for wastewater treatment 729
Chapter 94. Nitrification in PVAL beads: Influence of pH and temperature on nitrite oxidation 737
Chapter 95. The concept of combined phosphorus and nitrogen removal by immobilized biomass 744
Chapter 96. Anion-active surfactants degradation by immobilized cells 750
Chapter 97. Entrapped microbial cell process for treatment of chlorophenolic compounds 758
Chapter 98. Treatment of pentachlorophenol (PCP)-mineral salts medium by naturally immobilized cells of Arthrobacter strain ATCC 33790 764
Chapter 99. Aerobic degradation and dechlorination of low-chlorinated biphenyls by immobilized cells of a PCB degrading bacterial co-culture 769
Chapter 100. Biodegradation of phenol by a mixed culture entrapped in SiO2 films 776
Chapter 101. Propachlor and alachlor degradation by immobilized and suspended 781
Chapter 102. Morpholine degradation by strain Mycobacterium aurum MOI: Improvement of cells growth and morpholine degradation rate by cells immobilization 789
Chapter 103. Immobilized cells for applications in non-conventional systems 796
Chapter 104. Characterization of biofilm structure formed on nickel alloy fibre for ferrous sulphate oxidation by Thiobacillus ferrooxidans cells 804
Chapter 105. Biological sulphate reduction with synthesis gas: Microbiology and technology 812
Chapter 106. Development of mercury biotransformation process in fluidized bed reactor with immobilized microorganisms 819
Chapter 107. Cadmium removal in an airlift reactor 827
Chapter 108. Gastrointestinal protection of cellular component DNA within an artificial cell system for environmental carcinogen biomonitoring 833
Chapter 109. Immobilized transport mutants of bacterial cells in biosensor arrays. Improved selectivity for the simultaneous determination of glucose and lactose 840
Part 3: Guidelines 848
Chapter 110. Guidelines for the characterization of immobilized cells 850
Index of authors 854
Keyword index 858
Why immobilize?
C. Wandrey Institute of Biotechnology, Research Centre Jülich, D-52425 Jülich, Germany
Introduction
Why immobilize? - The simplest answer may be: because nature does it! While scientists are requested to be mobile it is obviously advantageous for many biocatalytic systems to be immobile. A good example of this is Yellowstone Park (USA) where one can see thick mats of microorganisms and algae at the edge of hot springs. With a continuous supply of substrates, there is obviously selective pressure to become immobile. In nature different biocatalytic systems adapt remarkably well to reaction conditions, e.g. temperature, pH and substrate concentration in the effluent of the above mentioned hot springs.
Also in the laboratory one can see - sometimes unwanted - spontaneous immobilization. In chemostate cultures, which are operated close to washout conditions, microorganisms often develop a tendency to grow on the reactor wall. If nature shows such a clear tendency towards immobilization it is obvious that one also can exploit such phenomena in technical systems.
While the use of immobilized whole cells has already been known for a long time (e.g. production of acetic acid with microorganisms immobilized on pieces of wood) technical systems using immobilized isolated enzymes were applied much later. In the fifties, the first laboratory systems were described in the literature [1-2]. The first full-scale system using immobilized enzymes went into operation in 1969. The Tanabe Company, Japan, used immobilized acylase for the kinetic racemic resolution to obtain L-methionine [3].
The Japanese were also pioneers in the field of immobilized living and dead cells (production of ethanol from glucose, production of acrylamide from acrylnitrile) [4].
Full-scale systems using immobilized higher cells came into use in the eighties. A well known example is the plant of Bayer, USA, for the production of factor VIII. Here, the cells are not immobilized on a carrier but retained by a membrane in a continuously operated system [5].
In the following, some reasons, problems, and solutions are given which may occur, if enzymes, microorganisms, or higher cells are to be immobilized. The examples used are mostly from our own developments. It is not possible here to give a comprehensive overview, since there are now dozens of systems on an industrial scale - which are seldom described in detail - and thousands of systems in laboratory scale - which are better documented.
Immobilized enzymes
Enzymes should be understood here as enzymes in dead cells, partially purified enzymes, or purified enzymes. Immobilization can be achieved by carrier fixation, encapsulation, cross linking and/or the use of membranes for catalyst retention in a continuously operated system. In recent years there has been a trend towards using "macroporous" microcarriers. Such carriers can be produced from a mixture of glass powder and salt. After moulding and sintering the inert salt fraction is washed out [6]. The pore size and the porosity can be varied within a wide range by the appropriate selection of the particle size and the volume fraction of the salt so that even the centre of the particle can be reached by (reduced) enforced flow.
In our experience, homogeneous biocatalysts immobilized in membrane reactors often have advantages in comparison to heterogeneous biocatalysts. Catalytic systems become complex if a coenzyme is needed. Since a coenzyme is a transport metabolite it does not make sense to immobilize the coenzyme on a carrier. For readily water-soluble reactants one can exploit the fact that in the case of NAD very low coenzyme concentrations are needed to saturate the enzymes involved. The enzymes are retained in the membrane reactor, while the coenzyme is continuously dosed into the system.
For substrates with intermediate water solubility, the coenzyme can be covalently bound to soluble polymer in order to be retained by a membrane together with the enzymes.
If the water solubility of the reactants is very low, one can exploit this to deliver the substrate to the water phase from an organic solvent via a membrane. After the product is formed, it leaves the water phase and is obtained from the organic phase. In this case the entire catalytic system is "immobilized" in the water phase, while the organic solvent is a continuous phase [7].
Microorganisms
Microorganisms are immobilized (in a dead form), if no enzyme purification is required (e.g. production of acrylamide from acrylnitrile). In other cases living, non-reproducing cells, retained by a membrane or fixed to a carrier, are used. The microbial glucose oxidase catalyzes the production of gluconic acid from glucose while the simultaneously present catalase decomposes the hydrogen peroxide formed.
Another example is the use of immobilized yeast for coenzyme-dependent reductions. Glucose is used as the hydrogen source, taking advantage of the intracellular cofactor regeneration system. The microbial system is limited with respect to other nutrients so that the glucose is mainly used for cofactor regeneration and not for cell growth.
Anaerobic microorganisms are good examples of immobilization by means of membranes or carriers. One need not be afraid of substrate limitation with respect to oxygen. Furthermore, most of the carbon source is converted to the desired product (e.g. ethanol, acetic acid, biogas). Increasing space-time yield by increasing substrate concentration is limited by product inhibition or as in anaerobic waste water treatment, by the substrate concentrations available in practical waste waters. All these arguments strongly support the use of immobilization techniques. Nevertheless, one has to take into account the fact that in the mentioned systems cells reproduce and form carbon dioxide. Both of these "byproducts" must readily leave the reactor in order to avoid accumulation. For this purpose macroporous carriers or membranes can be used. With membranes a bleed technique has to be applied, where a minor part of the effluent leaves the reactor in an unfiltered state in order to avoid uncontrolled accumulation.
A first example of such systems is the production of ethanol from glucose by means of Zymomonas mobilis . Only 2.3 % (at most) of the glucose consumed by Zymomonas mobilis is needed for cell mass formation. That means that non-growth-coupled ethanol production has a negligible effect on ethanol selectivity. Growth (and thus ethanol formation) is strongly inhibited by ethanol. Cell growth becomes zero at ethanol concentrations above 70 g/l. With the given ethanol selectivity of ~0.5 g ethanol/g glucose it does not make much sense to use glucose concentrations higher than 140 g/l. One has to make a compromise between maximal product concentration and maximal space-time yield. Here, cell immobilization helps a lot, since for sensible ethanol concentrations the cell-specific ethanol production rate is comparatively low. One can nevertheless reach acceptable space-time yields by increasing the cell concentration. Both by carrier fixation as well as by the use of membranes, an at least 10-fold increase of catalyst concentration can be reached in comparison to chemostate conditions [8-9] (Figure 2).
In order to get rid of daughter cells and carbon dioxide and at least partially overcome product inhibition a two-stage fluidized-bed reactor using "macroporous" microcarriers was employed. Using an initial substrate concentration of 120 g glucose/l, an ethanol concentration of 50 g/l was reached (80 % conversion in the first stage, 99 % conversion in the second stage). Spacetime yield (at 99 % conversion) was 12 g/(lxh). In comparison to chemostate the space-time yield could be increased by the factor of 2.5. Since, the biomass concentration was increased 10-fold, the effectiveness factor was only about 0.25. An important additional advantage of the two-stage fludized-bed cascade in comparison to a chemostate was the fact that this system could be operated with a non-sterilized glucose feed stock, which is a byproduct of starch hydrolysis. When this feed stock was used in a chemostate, Lactobacilli became dominant after some time (shift from ethanol production to lactic acid production). In the fluidized-bed cascade contaminating Lactobacilli were continuously washed out, due to a monoseptic precolonization of the carrier with Zymomonas mobilis and the short residence time.
For a quite different purpose, the concentration of Zymomonas mobilis was increased in a membrane reactor. The reactor was placed into the core of the magnet of nuclear magnetic resonance machine for in-vivo NMR measurements of intracellular metabolites.
By decoupling the substrate and catalyst residence time in a chemostate with partial cell retention it was possible to measure intracellular metabolites...
| Erscheint lt. Verlag | 21.3.1996 |
|---|---|
| Sprache | englisch |
| Themenwelt | Medizin / Pharmazie ► Pflege |
| Medizin / Pharmazie ► Physiotherapie / Ergotherapie ► Orthopädie | |
| Naturwissenschaften ► Biologie ► Mikrobiologie / Immunologie | |
| Naturwissenschaften ► Biologie ► Ökologie / Naturschutz | |
| Naturwissenschaften ► Biologie ► Zellbiologie | |
| Technik ► Lebensmitteltechnologie | |
| Technik ► Medizintechnik | |
| Technik ► Umwelttechnik / Biotechnologie | |
| ISBN-10 | 0-08-053447-3 / 0080534473 |
| ISBN-13 | 978-0-08-053447-3 / 9780080534473 |
| Haben Sie eine Frage zum Produkt? |
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