Comparison of the activity of immobilised and freely suspended Streptomyces coelicolor A3(2)

Streptomyces coelicolor was immobilised naturally in porous support materials and its growth, glucose uptake and actinorhodin production were compared with freely suspended culture using defined and complex media. When the defined medium was used, the most pronounced difference between the two cultures was the accumulation of actinorhodin extracellulary in freely suspended and intracellularly in immobilised cultures. In the complex medium, however, actinorhodin was excreted by both cultures. In addition, the complex medium yielded 50 times as much actinorhodin compared to the defined medium. Further increases in product concentration were obtained by repeated batches of immobilised culture, which showed stability for at least 3 months.     

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.     

Studies on the accumulation of heavy metal elements in biological systems

Summary Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl₂, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl₂ over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.     

Growth and nutrient uptake by immobilised tissue culture cells of celery (Apium graveolens)

Celery cell suspension immobilized in Ca-alginate and maintained in a medium containing 0.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,5-dichlorophenoxyacetic acid (3,5-D) as the auxin sources were compared with freely suspended cells in the same media. In a 2,4-D-containing medium the immobilized cells showed a reduced dry weight and a more uniform respiration rate, whereas the uptake of sucrose, phosphate and ammonia nitrogen was similar in both immobilized and freely suspended cells. In a 3,5-D medium, no increase in dry weight occurred in the immobilized cells, but a small increase in respiration rate showed that the cells were still viable. Uptake of sucrose, phosphate and ammonia nitrogen was reduced in the immobilized cells. The role of slow growing partially differentiated immobilised cells in the synthesis of plant secondary products is discussed.     

Use of polyurethane foam for the biodegradation of n-alkanes by immobilised cells of Pseudomonas

Pseudomonas strains C12B and A3 were separately immobilised in polyurethane foam and their ability to biodegrade n-decane or n-hexadecane tested and compared with that of freely suspended cells. In the presence of immobilised Pseudomonas C12B or A3 cells about 90% of n-alkane, originally present in liquid medium, was removed, 25%-40% of which was due to biodegradation.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production

Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH?<?6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5–7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale.     

Comparison of glucose fermentation by suspended and gel-entrapped yeast cells: An in vivo nuclear magnetic resonance study

Phosphorus-31 nuclear magnetic resonance ((31)P NMR) was used to compare the anaerobic metabolism of glucose by suspended and gel-entrapped Saccharomyces bayanus cells. The fermentation of glucose was carried out in a reaction system with continuous circulation through the NMR sample tube. The intracellular pH and the levels of some phosphorylated compounds were the levels of some phosphorylated compounds were noninvasively monitored by (31)P NMR while glucose, fermentation products, and biomass were determined by analytic techniques comparisons showed that no significant differences are observed in the relative concentrations in the spectra, but distinct profiles for the variation of both intracellular and extracellular pH are found. The internal pH of immobilized cells is maintained at a constant value throughout the fermentation as opposed to freely suspended cells for which a steady decrease in the internal pH occurs. A faster and stronger acidification is also observed in the external medium of the assays with suspended cells. Furthermore, higher yields for ethanol and biomass production and lower yields of fermentation by-products are obtained with immobilized cells. It is concluded that the higher intracellular pH achieved in the presence of the gel matrix had a regulatory effect on the metabolism which favored the ethanol production pathway. (c) 1993 John Wiley & Sons, Inc.     

Colonisation of polyurethane reticulated foam biomass support particle by methanogen species

Summary The ability of methanogen species to colonise reticulated polyurethane foam biomass support particles (BSP) in a continuous culture system using formate as carbon source was investigated. Scanning electron micrograph evidence and biomass measurements indicate that two methanogen species effectively colonised within the the matrix of the support particle.The freely suspended colonised BSP are resistant to washout, and a comparison of methane output of the immobilised culture and a liquid culture of the methanogens indicates the potential for process intensification of methane production.     

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques.Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.     

Biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp.

Freely suspended and Ca-alginate-immobilized cells of Pimelobacter sp. were used for degradation of pyridine. When the pyridine concentration was up to 2 g l^–1, freely suspended cells completely degraded pyridine regardless of the initial cell concentrations used. However, when the pyridine concentration increased to 4 g l^–1, the initial cell concentration in freely suspended cell culture should be higher than 1.5 g dry cell weight l^–1 for complete degradation of pyridine. In addition, a freely suspended cell culture with a high initial cell concentration resulted in a high volumetric pyridine-degradation rate, suggesting the potential use of immobilized cells for pyridine-degradation. When the immobilized cells were used for pyridine-degradation, neither specific pyridine-degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine-degradation rate in the range 0.082–0.129 g l^–1 hr^–1 could be achieved by the immobilized cells because of the high cell concentration. Furthermore, when the immobilized cells were reused in degrading pyridine at a concentration of 2–4 g l^–1 they did not lose their pyridine-degrading activity for 2 weeks. Taken together, the data obtained here showed the feasibility of using immobilized cells for pyridine-degradation.     

Encapsulation of Pediococcus acidilactici cells in corn and olive oil microcapsules emulsified by peptides and stabilized with xanthan in oil-in-water emulsions: Studies on cell viability under gastro-intestinal simulating conditions

Three different encapsulation systems were developed in the form of oil-in-water acidic emulsions (pH 3.0) with the oil phase in the form of microdroplets in which Pediococcus acidilactici cells were enclosed. The first emulsion contained corn oil microdroplets (mean diameter 1.5 μm) emulsified with peptides and stabilized with SDS. The other two, were food grade systems with microdroplets of corn or olive oil (m.d. 2.1 and 2.2 μm, respectively) emulsified with peptides and stabilized with xanthan. In all systems, meat peptone, a rich source of peptides and amino acids, was provided in aqueous solution in which the cultures were suspended. Peptone derived peptides acted as emulsifiers and at the same time as nutrient substrates and osmoprotectants for cells. Emulsions were stored for 30 days at 4 °C. During this period, samples were examined for physical stability and viability of the encapsulated and freely suspended microorganisms present in the emulsions. Examinations were made through phase contrast microscopy and subsequent image capture and analysis with an automatic image analysis system. Viable and non-viable cells were discriminated on the basis of color differences produced through staining with trypan blue. The method permitted extraction of a large amount of information and produced large amounts of data through automated measurements on images. Emulsion characteristics and cell viabilities were also examined under conditions simulating the gastro-intestinal environment. Encapsulation proved to be critical since, following successive treatments of samples with simulated gastric juice (pH 2.0) and intestinal juice (pH 7.4), it ensured viability rates of encapsulated cells as high as 85% and delivered as much as 92% of the initially encapsulated cells to the target point. The formulated emulsion systems may have a large number of applications in the food sector provided further studies on engineering properties and improvements of stability over a wide pH range are carried out.     

Culture conditions for intracellular lipase production by Rhizopus chinensis and its immobilization within biomass support particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells.It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity.The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells.The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.     

Encapsulation of Pediococcus acidilactici cells in corn and olive oil microcapsules emulsified by peptides and stabilized with xanthan in oil-in-water emulsions: Studies on cell viability under gastro-intestinal simulating conditions

Three different encapsulation systems were developed in the form of oil-in-water acidic emulsions (pH 3.0) with the oil phase in the form of microdroplets in which Pediococcus acidilactici cells were enclosed. The first emulsion contained corn oil microdroplets (mean diameter 1.5 μm) emulsified with peptides and stabilized with SDS. The other two, were food grade systems with microdroplets of corn or olive oil (m.d. 2.1 and 2.2 μm, respectively) emulsified with peptides and stabilized with xanthan. In all systems, meat peptone, a rich source of peptides and amino acids, was provided in aqueous solution in which the cultures were suspended. Peptone derived peptides acted as emulsifiers and at the same time as nutrient substrates and osmoprotectants for cells. Emulsions were stored for 30 days at 4 °C. During this period, samples were examined for physical stability and viability of the encapsulated and freely suspended microorganisms present in the emulsions. Examinations were made through phase contrast microscopy and subsequent image capture and analysis with an automatic image analysis system. Viable and non-viable cells were discriminated on the basis of color differences produced through staining with trypan blue. The method permitted extraction of a large amount of information and produced large amounts of data through automated measurements on images. Emulsion characteristics and cell viabilities were also examined under conditions simulating the gastro-intestinal environment. Encapsulation proved to be critical since, following successive treatments of samples with simulated gastric juice (pH 2.0) and intestinal juice (pH 7.4), it ensured viability rates of encapsulated cells as high as 85% and delivered as much as 92% of the initially encapsulated cells to the target point. The formulated emulsion systems may have a large number of applications in the food sector provided further studies on engineering properties and improvements of stability over a wide pH range are carried out.     

Apparent free space and cell volume estimation: A non-destructive method for assessing the growth and membrane integrity/viability of immobilised plant cells

Pre-existing methods for measuring cell or organelle volume based on the selective permeability of biological membranes have been modified to make them suitable for determining the intracellular volume of immoblised cells. When a freely permeable substance (e.g. tritiated water) and an impermeable substance (¹⁴C labelled mannitol is often suitable) are mixed with an immobilised cell culture, the two substances are diluted to different degrees. The extent of the difference allows the total intracellular volume of intact cells to be calculated. This volume is shown to be a useful parameter for assessing cell growth. The application of the method to follow membrane integrity and cell viability is also discussed.Abbreviations 2,4D 2,4-dichlorophenoxyacetic acid - BSA Bovine serum albumen     

Glycolysis of red cells suspended in solutions of impermeable solutes. Intracellular pH and glycolysis.

The glycolytic rate human red cells suspended in a sucrose medium of low or physiological pH was higher than that of the cells suspended in Ringer's medium of the same. pH. The medium pHP-glycolytic rate curve of red cells suspended in soucrose media shifted to the acidic side by about one unit compared with that of cells suspended in Ringer's medium. Similarly, the pattern of glycolytic intermediates in red cells suspended in a sucrose medium resembled that in cells suspended in Ringer's solution of about one unit higher pH. These phenomena could be ascribed to the change of intracellular pH, which was measured by the 5,5'-dimethyl-oxazolidine-2,4-dione method. A similar stimulation of glycolysis was observed when sodium citrate was added to red cells suspended in Ringer's solution at constant pH. These observations indicate that membrane-impermeable non-electrolytes or anions stimulate glycolysis of red cells by elevation ofthe intracellular pH. Red cell glycolysis is influenced mainly by the intracellular pH rather than by the pH of the suspending medium.