Growth of immobilised plant cells in reticulate polyurethane foam matrices

Summary An inoculum of initially freely suspended cell aggregates ofCapsicum frutescens was immobilised in porous polyurethane foam matrices. Subsequent growth and substrate consumption of these immobilised cells in batch culture were measured and compared with those of suspension cultures. The results showed that the maximum specific growth rate of freely suspended cells was slightly higher than that of immobilised cells but the overall growth patterns and final cell yields were similar.     

Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised in polyurethane foam support particles

Summary Oxidation of ferrous iron by Thiobacillus ferrooxidans cells passively immobilised in polyurethane foam particles, using both repeated batches and continuous operation, was studied in a laboratory-scale reactor. Repeated batches yielded complete oxidation at higher rates than single batches, providing resident inocula for subsequent batches. In continuous operation maximum ferric iron productivities were achieved at dilution rates well above theoretical washout values. At a dilution rate of 0.31 h^–1 [approximately three times the maximum specific growth rate (_max)], a productivity of 1.56 kg m^–3 h^–1, based on total ferric iron, or 1.0 kg m^–3 h^–1 based on dissolved ferric iron, was achieved. In addition, cells immobilised in the foam particles retained their oxidative ability for periods of up to 6 weeks when stored in the open air and could be reused immediately.     

Detoxification of hexavalent chromate by Amphibacillus sp. KSUCr3 cells immobilised in silica-coated magnetic alginate beads

Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe₃O₄) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe₃O₄ nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)₂, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca²⁺ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology.     

Glyptal as a support for enzyme immobilisation

Glyptal, a polyester obtained from phthalic anhydride and glycerol, was used as a support for protein immobilisation. Hydrazide groups were introduced in the polymer and then converted to azide groups, through which protein was covalently immobilised. Amyloglucosidase was used as a model and an insoluble water derivative was synthesised retaining 24 % of the specific activity of the native enzyme. Some properties of this immobilised enzyme were studied: Km (4.54 g.l^–1 using starch as substrate), optimal temperature (55°C) and half life (8 days). Furthermore, ferromagnetic-azide-glyptal derivative showed to be useful for the amyloglucosidase immobilisation.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Combined immobilized cultures producing fibrinolytic proteinases

The authors obtained combined immobilised systems composed of a culture producing fibrinolytic proteinases and a stimulating strain. The optimal ratio between the two cultures in gel was selected at a high starting cell density. Highly stable immobilised cultures were produced by growing the cells in gel particles. The interrelationship of the partners was studied in the binary immobilised culture. The biosynthetic activity of the system fell down to the level of a monoculture when the cells of the stimulating strain were eliminated from gel. The producing and stimulating strains are at equilibrium in associative immobilised cultures obtained by growing the cells in gel, and Arthrobacter is not eliminated. The mechanism of biosynthesis stimulation in a combined immobilised culture has been studied. Apparently, the procedure of immobilisation and the action of a stimulating compound exert the synergistic effect.     

Screening of porous and cellular materials for covalent immobilisation of Agaricus bisporus tyrosinase

We conducted a systematic study of covalent immobilisation of Agaricus bisporus tyrosinase onto typical enzyme carriers. Acrylic beads, two commercial silica gels with different pore structures and mesoporous silica foam (MCFs) beads functionalised using different organosilanes showed that only aminated MCFs offer active preparations with immobilisation efficiencies greater than 100% and a similar ratio of diphenolase (L-DOPA) to monophenolase (L-tyrosine) activities as the free enzyme. The native enzyme was entirely inactivated during incubation at 55°C for 30 min, whereas the enzyme immobilised on acrylic carrier or MCF retained 46 and 35%, respectively, of the initial activity after similar treatment. Susceptibility of native and immobilised tyrosinase to suicide inactivation in the presence of L-tyrosine and L-DOPA was tested in repeated batch tests. However, none of the preparations obtained in the L-DOPA solution was operationally stable enough to be used for practical applications.     

The effects of light intensity, inoculum size, and cell immobilisation on the treatment of sago effluent withRhodopseudomonas palustris strain B1

A study was carried out to determine a suitable light intensity and inoculum size for the growth ofRhodopseudomonas palustris strain B1. The pollution reduction of sago effluent using free and immobilisedR. palustris cells was also evaluated. The growth rate in glutamatemalate medium was highest at 4 klux compared to 2.5 and 3 klux. The optimal inoculum size was 10% (v/v). Both the COD and BOD of the sago effluent were reduced by 67% after three days of treatment. The difference in biomass production or BOD and COD removal with higher inoculum sizes of 15 and 20% was minimal. This could be attributed to limited nutrient availabillity in the substrate. The use of immobilised cells ofR. palustris reduced the pollution load 10% less compared to pollution reduction by free cells. Hence, there was no significant difference in using free or immobilised cells for the treatment of sago effluent.     

Immobilisation of the Thermostable l -aminoacylase from Thermococcus litoralis to Generate a Reusable Industrial Biocatalyst

A thermostable archaeal l -aminoacylase from Thermococcus litoralis has been used in immobilisation trials to optimise its application in industrial biotransformation reactions. Immobilisation techniques used included direct adsorption and crosslinking of the enzyme onto solid supports, bioencapsulation, and covalent bonding onto a variety of activated matrices. The most successful immobilisation methods were covalent binding of the enzyme onto glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per gram of support (wet weight for glyoxyl-Sepharose), respectively, with nearly 80% activity recovery in both cases. Enzyme immobilised onto glyoxyl-agarose was stabilised 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant l -aminoacylase immobilised onto Sepharose beads was constructed with the substrate, N -acetyl- dl -Trp, continuously flowing at 60°C for 10 days. No loss of activity was detected over five days, with 32% activity remaining after 40 days at 60°C. These results show the potential of the use of immobilised l -aminoacylase in biotransformation reactions for the production of fine chemicals.     

Immobilisation of the Thermostable l -aminoacylase from Thermococcus litoralis to Generate a Reusable Industrial Biocatalyst

A thermostable archaeal l -aminoacylase from Thermococcus litoralis has been used in immobilisation trials to optimise its application in industrial biotransformation reactions. Immobilisation techniques used included direct adsorption and crosslinking of the enzyme onto solid supports, bioencapsulation, and covalent bonding onto a variety of activated matrices. The most successful immobilisation methods were covalent binding of the enzyme onto glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per gram of support (wet weight for glyoxyl-Sepharose), respectively, with nearly 80% activity recovery in both cases. Enzyme immobilised onto glyoxyl-agarose was stabilised 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant l -aminoacylase immobilised onto Sepharose beads was constructed with the substrate, N -acetyl- dl -Trp, continuously flowing at 60°C for 10 days. No loss of activity was detected over five days, with 32% activity remaining after 40 days at 60°C. These results show the potential of the use of immobilised l -aminoacylase in biotransformation reactions for the production of fine chemicals.     

A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain

Abstract A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl₂, 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20–23 mg l^–1 and 0.5–2 mg l^–1, respectively). The maximum lysozyme yield achieved was about 23 mg l^–1 by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl₂). Response surface methodology was used to investigate the effect of pH and water activity (a_w). The best medium pH was 4.5–5.0, and bead a_w for optimum lysozyme yield was 0.94, regardless of polymer type.     

Specificity in the immobilisation of cell wall proteins in response to different elicitor molecules in suspension-cultured cells of French bean (Phaseolus vulgaris L.)

A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H₂O₂ and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post-translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed.     

Controls for rhizosphere microorganisms to study effects of vesicular-arbuscular mycorrhizae on Artemisia tridentata

Seven treatments were set up to test the effects of vesicular-arbuscular (VA) mycorrhizal fungi and other rhizosphere microorganisms on the growth of Artemisia tridentata ssp. tridentata. Soil sievings had no significant effect on root or shoot mass. Spores and surface-sterile spores were a poor inoculum source, but roots and fresh soil caused 45–75% mycorrhizal infection. Whereas root-inoculated plants still had low growth responses by the end of the experiment, fresh soil inoculum caused the greatest response, and partial fresh inoculum caused a lesser response. These results suggest that fresh soil is an appropriate inoculum for this plant-fungal-soil system, and that the major effect on plant growth of the fresh soil inoculum is from the mycorrhizal fungi and not from the other microorganisms, because the sievings had no effect on plant growth. In addition, soil dilution plating of saprophytic fungi showed 85% species similarity between sterile and fresh soil inoculum by the end of the experiment. Since the effects of non-VA microorganisms are complex and varied, we suggest that researchers work out the type of mycorrhizal controls that best suit their system.     

Effect of peat-bran inoculum ofTrichoderma species on biological control ofRhizoctonia solani in lettuce

A range of known biocontrol or plant growth-stimulating species ofTrichoderma orGliocladium were grown on peat-bran substrate to yield between 5×10⁷–3×10¹⁰ colony forming units (cfu's)g^–1 substrate after 14 days growth. Inocula were incorporated into peat:sand potting compost infested withRhizoctonia solani to give 7–8 × 10⁴ cfu's of antagonist g^–1 compost and assessed for biological control activity using lettuce seedlings. Six of the eight antagonists decreased daming-off and three of these consistently increased yield in comparison withR. solani treatment alone.Subsequently, peat-bran inoculum ofT. harzianum isolate TH1 was incorporated at 0.5% w/v intoR. solani infested potting compost. Both autoclaved and nonautoclaved inoculum ofT. harzianum TH1 decreased disease and increased yield. Incorporation of ethyl acetate-extracted autoclaved inoculum ofT. harzianum TH1 resulted in similar levels of biocontrol and improved plant growth as did incorporation of nonautoclaved and autoclavedT. harzianum TH1 inoculum. The need to standardize inocula and controls is emphasized.     

Kinetics of invertase immobilised on poly(phe-lys) coated polystyrene beads

Summary Surface of polystyrene beads was modified by poly(phe-lys) for invertase immobilisation. The optimum immobilisation conditions of invertase were; 0.01% (w/v) poly(phe-lys), 2% (v/v) glutaraldehyde at 25 °C and pH 4.5. The kinetics of sucrose hydrolysis by free and immobilised invertase in a batch reactor at pH 4.5 and 55 °C gave K_m and V_max values for sucrose with free and immobilised invertase of 81, 114 mM and 10.1, 9.2 mol glucose/min.mg enzyme, respectively. The deactivation rate constants of free and immobilised invertase were 0.0347 and 0.0098 min^–1, respectively.     

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.     

Growth promotion of Prunus rootstocks by root treatment with specific bacterial strains

A collection of bacterial strains obtained from a wide-range origin was screened for ability to promote growth in two types of Prunus rootstocks in a commercial nursery. Only few strains promoted growth significantly and consistently, and a strong specificity for the rootstock cultivar was observed. Irrigation of plants with Pseudomonas fluorescens EPS282 and Pantoea agglomerans EPS427 significantly increased plant height and root weight of the plum Marianna 2624 and the peach–almond hybrid GF-677, respectively. Plant height showed a higher rate of growth in early stages of development (2.6–3.5 times the non-treated controls), but the effect decreased with plant age. However, in aged plants growth promotion was more significant on root weight (1.9 times the non-treated controls) than on plant height. The efficacy of growth promotion and the persistence of strains in the root environment were dependent on the bacterial inoculum concentration applied. Increases in root development were maximum at inoculum concentrations of up to 8 log₁₀ CFU ml^–1 (ca 10 log₁₀ CFU L^–1 of potting mix). Population levels at the optimum inoculum concentration were around 7 log₁₀ CFU g f.w.^–1 root material at early stages of development and decreased to 4 log₁₀ CFU g f.w.^–1 after several months of development. The best plant growth-promoting strains were very diverse in secondary metabolite production and antagonistic ability against several plant pathogens.     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles

A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01–0.2 mg ml⁻¹). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.