Immobilized Cell Technology Research Articles

Microbial Biocapsules as Generally Recognized-As-Safe Fungal-Based Immobilized Cell Technology for Precision Sequential Fermentations of Grape Must

This work focuses on the production of a white wine with a specific organoleptic profile by means of sequential fermentation using immobilized yeast in a system known as “microbial biocapsules”. Three fermentation conditions were created: sequential fermentation with immobilized yeast (SqFMB) employing a matrix composed by Aspergillus oryzae (pellet-forming fungus recognized as GRAS), sequential fermentations with non-immobilized yeast cells (SqF), and a control of spontaneous fermentation (SpF). To carry out these fermentations, Pedro Ximénez grape must was used and two non-Saccharomyces yeast strains, Debaryomyces hansenii LR1 and Metschnikowia pulcherimma Primaflora, and the Saccharomyces cerevisiae X5 strain were used. The wines produced were subjected to microbiological and chemical analyses in which metabolites that positively influence the wine profile, such as 1,1-diethoxyethane and decanal, are only produced in the SqFMB condition, and others, like nonanal, were detected in higher concentrations than in SqF and SpF. Microbiological analyses show that less non-Saccharomyces yeasts were isolated in the SqFMB condition than in SqF, which indicates an efficiency in the inoculation and removal method proposed. These results conclude that microbial biocapsules seem to be a good yeast carrier for wine elaboration; however, modulation of some variables like yeast concentration inocula, the employment of preadaptation methods or the use of yeast species with higher fermentative power need to be tested to improve the novel methodology.

Advanced Bioconversion of sugary wastewater: Integrated polyhydroxybutyrate and hydrogen productions in a continuous airlift two-chamber bioreactor

This study utilized a novel Continuous Airlift Two-chamber Bioreactor (CATBR) for the co-production of hydrogen and polyhydroxybutyrate (PHB) from mixed microbial cultures in sugary wastewater. Hydrogen generation was investigated in a defined bacterial consortium that contained mainly Clostridium species, and diverse microbial communities were studied in relation to PHB production, demonstrating the ability of diverse microbial communities to form stable microbial consortia. The impact of hydraulic retention time (HRT), pH, and air flow rate on PHB production was evaluated with special emphasis on volatile suspended solids (VSS).Optimal conditions for hydrogen and PHB production were established at a pH of 5.5, an aeration rate of 1500 mL/h, and an HRT of two days. Hydrogen yield was determined to be 3.14 ± 0.92 mL/g COD, and PHB yield was 31.32% (g PHB/g VSS). Production rates for hydrogen and PHB peaked at 0.23 ± 0.07 L/L.d and 0.64 g PHB/L.d, respectively, with a COD removal efficiency of 85 ± 0.67%. At an HRT of two days, the PHB generation rate was found to be 6–10 times higher than previously reported rates. The application of immobilized cell technology effectively prevented bacterial washout from the reactor, thereby ensuring stable operational conditions.

Use of yeast biocapsules as a fungal-based immobilized cell technology for Indian Pale Ale-type beer brewing.

Immobilized cell technologies (ICT) have been used in wort fermentation, beer maturation, or production of alcohol-free or low-alcohol beer. The purpose of ICT is to restrict intact cells to a specific location while allowing biological function. It improves cell stability, operational flexibility, and control in brewing, as well as ease in executing continuous operations. We investigated the use of yeast biocapsules for Indian Pale Ale (IPA) type beer wort fermentation, a novel ICT in brewing. Yeast biocapsules are a spherical yeast immobilization system in which yeast cells are encapsulated and connected to the hyphae of an inactivated hollow filamentous fungus pellet. Fermentations with yeast encapsulated in alginate beads, as the standard immobilization practice, and in free (non-immobilized) forms were carried out in parallel. We found that yeast biocapsules are a better option for cell reutilization than alginate beads, but worse for beer must clarity. Beer brewed with yeast biocapsules differed in concentration for five volatile compounds (acetaldehyde, diacetyl, ethyl acetate, 1,1-diethoxyethane, and isoamyl alcohol) and three sensory characters (persistency of the foam, malt, and yeast character). KEY POINTS: • Yeast biocapsules were investigated for beer wort fermentation • Biocapsules improve cell reutilization but are limited for beer clarification • Beer brewed with biocapsules is chemically different than conventional beer • Most sensory features did not differ between biocapsule and control beer.

Kinetics of continuous production of β‐carotene in an airlift bioreactor

AbstractThe focus in the present work was on the continuous β‐carotene production from free fatty acids supplied by the soap stock as a sole carbon source for Rhodotorula rubra cells in an airlift bioreactor. The effects of dilution rate on the cell growth and β‐carotene production were studied by both freely suspended‐ and immobilized‐cell systems in the chemostat operations. The results showed that the immobilization of the cell on natural sponge of loofah significantly increased the optimum and maximum dilution rates from 0.0375 to 0.05 hr−1 and 0.055 to 0.150 hr−1, respectively. The immobilization process has practical role in balancing of cell proliferation in the matrix and wash out the grown cells containing β‐carotene from the bioreactor where β‐carotene productivity was enhanced from 5.4 to 23.4 mg L−1 hr−1 using the immobilized cell technology.

Immobilized mixed-culture reactor (IMcR) for hydrogen and methane production from glucose

Immobilized cell technology is a new technique to produce biogas. In the present study, an immobilized mixed-culture reactor (IMcR) in batch-mode operation was used for the production of hydrogen and methane simultaneously from glucose. Several factors, such as glucose concentration, temperature and fermentation time, were evaluated to determine the optimal conditions for hydrogen and methane production. Gas chromatography with a thermal conductivity detector (GC-TCD) and high-performance liquid chromatography (HPLC) were used to analyse the gas and effluent. The morphologies of the immobilized cells were characterized using scanning electron microscopy (SEM). The optimal conditions for hydrogen and methane production were obtained using a substrate with 5.0 g/L glucose at 60 °C for fermentation times of 48.0 h (hydrogen) and 72.0 h (methane). The maximum yields of hydrogen and methane at these optimal conditions were 37.0 ± 0.0 (×10−3) mol/mol glu and 39.0 ± 0.0 (×10−3) mol/mol glu, respectively. The chemical oxygen demand (COD) and pH gradually decreased with increasing fermentation time and temperature. However, the performance of the IMcR decreased over time due to cell damage and microorganism detachment from the cell. In conclusion, the IMcR system is a potential system for the simultaneous production of hydrogen and methane.

Consequence of phosphate solubilising microbes in sustainable agriculture as efficient microbial consortium: A review

Phosphorus is an essential nutrient for plant growth and yield. Currently, phosphorus fixation in soil is a major problem throughout the world as available phosphorus in soil is highly reactive with cationic elements to form complex compounds. Rock phosphate is one of the cheapest fertiliser and most abundant; however, its direct application in soils is not much effective for the availability of phosphorus to plant growth due to its low reactivity. Further, phosphorus is present as a fixed or complex form in soils, which is unavailable for plants. Phosphate solubilising microbes (PSMs) have potential to solubilise the complex form of phosphate in the available form of phosphorus to plants. PSMs have different mechanisms for solubilisation of phosphate by producing various acids and enzymes. Other mechanisms of phosphate solubilisation and mineralisation follow chelation and immobilised cell technology. All such phosphate solubilisation mechanisms by PSMs have been discussed in this article. The current need to develop genetically modified PSM as efficient PSMs strains for sustainable agricultural production is also discussed in the present review. The PSMs is known as effective biofertiliser for enhancing the plant growth, yield and nutrient content in crops as well as improve the soil fertility under sustainable agriculture. The main aim of this review was to elaborate the phosphate solubilising activities and their consequences for sustainable agriculture.

Aroma formation by immobilized yeast cells in fermentation processes.

Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes.

Maintaining yeast viability in continuous primary beer fermentation

Continuous fermentation is a long known and vastly studied process. The use of immobilized cell technology (ICT) is exploited in a significant number of studies owing to the associated high volumetric productivity, time savings and low capital demand. This work was aimed at solving one of the most relevant obstacles to implementing ICT on a large scale in beer fermentations, namely the control of biomass and the maintenance of cell viability in a gas-lift bioreactor. For this purpose, foam fractionation by skimming was proposed as a tool for control of continuous biomass concentration. The consequences of foaming on lignocellulosic yeast carrier losses were assessed and discussed. A steady consumption of sugars from wort, as well as consistent ethanol production, were achieved. The viability of the suspended cells in the reactor was compared with that of the cell population in the foam using flow cytometry. Results suggest that foam might be used as a promising tool to skim non-viable biomass out of the gas-lift reactor, thus ensuring the maintenance of a cell culture with optimum viability. Copyright © 2014 The Institute of Brewing & Distilling

Bioflavour production from orange peel hydrolysate using immobilized Saccharomyces cerevisiae

The rising trend of bioflavour synthesis by microorganisms is hindered by the high manufacturing costs, partially attributed to the cost of the starting material. To overcome this limitation, in the present study, dilute-acid hydrolysate of orange peel was employed as a low-cost, rich in fermentable sugars substrate for the production of flavour-active compounds by Saccharomyces cerevisiae. With this purpose, the use of immobilized cell technology to protect cells against the various inhibitory compounds present in the hydrolysate was evaluated with regard to yeast viability, carbon and nitrogen consumption and cell ability to produce flavour active compounds. For cell immobilization the encapsulation in Ca alginate beads was used. The results were compared with those obtained using free-cell system. Based on the data obtained immobilized cells showed better growth performance and increased ability for de novo synthesis of volatile esters of "fruity" aroma (phenylethyl acetate, ethyl hexanoate, octanoate, decanoate and dodecanoate) than those of free cells. The potential for in situ production of new formulations containing flavour-active compounds derive from yeast cells and also from essential oil of orange peel (limonene, α-terpineol) was demonstrated by the fact that bioflavour mixture was found to accumulate within the beads. Furthermore, the ability of the immobilized yeast to perform efficiently repeated batch fermentations of orange peel hydrolysate for bioflavour production was successfully maintained after six consecutive cycles of a total period of 240 h.

Microencapsulation of Probiotic Cells for Food Applications

The addition of microencapsulated probiotic cells to food products is a relatively new functional food concept. Most of the published scientific research in this field is not older than ten years. However, the technological background reaches back to the 1980s, where lactic acid bacteria were microencapsulated within the concept of the so-called immobilized cell technology (ICT). Target applications of ICT were continuous fermentation processes and improved biomass production. The methods adopted from immobilized cell technology were applied for the microencapsulation of probiotics, often optimized towards specific requirements associated with the protection of probiotic cells in food applications. However, there are still significant hurdles with respect to currently available methods for probiotic cell microencapsulation. This is mainly due to the fact that important characteristics of microcapsules based on ICT appear to be in conflict with the requirements arising from an application of probiotic microcapsules in food products, with particle size and inappropriate matrix characteristics being the most prominent ones. Based on this situation the aim of this review is to give a critical overview of the current approaches regarding the microencapsulation of probiotic cells for food applications and to report on emerging developments.

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures. The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.

Immobilization of Microbial Cells in Food Fermentation Processes

The purpose of this review is to emphasize existing trends and recent advances in the application of immobilized cell technology so as to implement some innovations in food industry associated with processing, preservation, and storage of the products based on the food safety issues. Attention is focused on the engineering aspects of the immobilized cell techniques with emphasis on the mass-balance-based mathematical modeling of the system. Some aspects of models for safety, quality, and competitiveness of the food processing sector are also presented. Ultimately, development of products with novel properties within the alcoholic beverages, meat processing/preservation, manufacture of cheese and bread, sweeteners and pigments, as well as nutraceuticals is also addressed.

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

BackgroundOxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H2O2 from 0 to 130 ppm.ResultsAt the beginning of the culture, high cell density of 1013 CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H2O2 concentrations. However, after increasing the H2O2 concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H2O2 and to select two adapted colonies (HPR1 and HPR2) after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O2 than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells.ConclusionsOur study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H2O2 adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.

Population Yields and Vegetable Juice Fermentation of Leuconostoc mesenteroides Cultures Grown Under Free-Cell or Immobilized-Cell Technologies

Leuconostoc mesenteroides BLAC cultures were produced using classical free-cell fermentation technology and immobilized cell technology (ICT). The ICT process consisted of entrapping the culture into alginate or pectin beads, adding them to a growth medium, and incubating for 14 h to allow growth of the culture inside the beads. Cell populations, survival to freeze-drying and specific acidifying activity (SAA) following inoculation in vegetable juices were examined. In free cell fermentations, 1.3 × 1010 cells/mL were obtained while 1.6 to 2.0 × 1011 cells/g were found in beads. The total cell counts recovered per fermentor were similar in free-cell and ICT systems. In the ICT process, the free-cell level was only of 1% or less. No difference in population yields were noted between alginate and pectin gels. Survival to freeze-drying was between 65 and 80% and was not significantly affected by the prior fermentation process nor the alginate or pectin matrix. A methodology was developed to ascertain the free-cell content of the freeze-dried ICT cultures, and data showed that they contained 97.5% of gel-entrapped cells. Freshly prepared free-cell suspensions had four times higher SAA than comparative free-cell freeze-dried cultures. The dried ICT cultures had lower SAA than free-cell equivalents, but there was no significant effect of the ICT matrix (alginate or pectin) on SAA. The powders of the ICT culture were ground and sieved to obtain particles ranging from 38 μm to 1000 μm in diameter. The smaller the particle, the higher was its SAA. Fermentation of vegetable juices or pastes were carried out with the free and ICT cultures. In the ICT fermentation, the free-cell content of the inoculum represented 2.5% of the total population, but this increased to 88% after the 22 h incubation in vegetable juice. Data are discussed in relationship with potential benefits of ICT cultures in vegetable fermentations.

Continuous Beer Fermentation Using Immobilized Yeast Cell Bioreactor Systems

Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO(2) removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.

Primary beer fermentation by immobilised yeast—a review on flavour formation and control strategies

AbstractImmobilised cells are increasingly being used in bio‐industries and may also have benefits for the brewing industry. The major challenge to applying this technology successfully in breweries is focused on the main fermentation in combination with the secondary fermentation. In particular, the control and fine‐tuning of the flavour profile during the main fermentation require further investigation. In this review, the influence of immobilised cell technology on the production of the flavour‐active compounds (i.e. higher alcohols, esters and vicinal diketones) is discussed. Control strategies that are based on the manipulation of parameters during fermentation such as temperature, feed volume, wort gravity, wort composition and aeration are explained. Finally, bioreactor configurations that may facilitate immobilised cells in performing the primary fermentation are evaluated. Copyright © 2006 Society of Chemical Industry

Immobilized cell technology in beer brewing: Current experience and results

Immobilized cell technology (ICT) has been attracting continual attention in the brewing industry over the past 30 years. Some of the reasons are: faster fermentation rates and increased volumetric productivity, compared to those of traditional beer production based on freely suspended cells, as well as the possibility of continuous operation. Nowadays, ICT technology is well established in secondary fermentation and alcohol- free and low-alcohol beer production. In main fermentation, the situation is more complex and this process is still under scrutiny on both the lab and pilot levels. The paper outlines the most important ICT processes developed for beer brewing and provides an overview of carrier materials, bioreactor design and examples of their industrial applications, as well as some recent results obtained by our research group. We investigated the possible applications of polyvinyl alcohol in the form of LentiKats?, as a potential porous matrices carrier for beer fermentation. Given are the results of growth studies of immobilized brewer's yeast Saccharomyces uvarum and the kinetic parameters obtained by using alginate microbeads with immobilized yeast cells and suspension of yeast cells as controls. The results indicate that the immobilization procedure in LentiKat? carriers has a negligible effect on cell viability and growth. The apparent specific growth rate of cells released in medium was comparable to that of freely suspended cells, implying preserved cell vitality. A series of batch fermentations performed in shaken flasks and an air-lift bioreactor indicated that the immobilized cells retained high fermentation activity. The full attenuation in green beer was reached after 48 hours in shaken flasks and less than 24 hours of fermentation in gas-lift bioreactors.

Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M.

Exopolysaccharides (EPS) were produced by Lactobacillus rhamnosus RW-9595M during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid porous supports (ImmobaSil). Cultures were conducted in supplemented whey permeate (SWP) medium containing 5 or 8% (w/w) whey permeate. For free-cell batch cultures in 8% SWP medium, very high maximum cell counts (1.3 x 10(10) CFU ml(-1)) and EPS production (2350 mg l(-1)) were measured. A high EPS production (1750 mg l(-1)) was measured after four cycles for a short incubation period of only 7 h. Several methods for immobilized biomass determination based on analysis of biomass components (proteins, ATP and DNA) were tested. The DNA analysis method proved to be the most appropriate under these circumstances. This method revealed a high maximum immobilized biomass of 8.5 x 10(11) CFU ml(-1) support during repeated immobilized cell cultures in 5% SWP. The high immobilized biomass increased maximum EPS volumetric productivity (250 mg l(-1) h(-1) after 7 h culture) compared with free-cell batch cultures (110 mg l(-1) h(-1) after 18 h culture). High EPS productions were achieved during batch cultures of Lact. rhamnosus RW-9595M in SWP medium, exceeding 1.7 g EPS per litre. Repeated-batch cultures with immobilized cells resulted in increased EPS productivity compared with traditional free-cell cultures. The study clearly shows the high potential of the strain Lact. rhamnosus RW-9595M and immobilized cell technology for production of EPS as a functional food ingredient.

Growth and morphology of thermophilic dairy starters in alginate beads.

The aim of this research was to produce concentrated biomasses of thermophilic lactic starters using immobilized cell technology (ICT). Fermentations were carried out in milk using pH control with cells microentrapped in alginate beads. In the ICT fermentations, beads represented 17% of the weight. Some assays were carried out with free cells without pH control, in order to compare the ICT populations with those of classical starters. With Streptococcus thermophilus, overall populations in the fermentor were similar, but maximum bead population for (8.2 x 10(9) cfu/g beads) was 13 times higher than that obtained in a traditional starter (4.9 x 10(8) cfu/ml). For both Lactobacillus helveticus strains studied, immobilized-cell populations were about 3 x 10(9) cfu/g beads. Production of immobilized Lb. bulgaricus 210R strain was not possible, since no increases in viable counts occurred in beads. Therefore, production of concentrated cell suspension in alginate beads was more effective for S. thermophilus. Photomicrographs of cells in alginate beads demonstrated that, while the morphology of S. thermophilus remained unchanged during the ICT fermentation, immobilized cells of Lb. helveticus appeared wider. In addition, cells of Lb. bulgaricus were curved and elongated. These morphological changes would also impair the growth of immobilized lactobacilli.

Protecting probiotics by microencapsulation

Probiotics are live bacteria which transit the gastrointestinal tract and in doing so benefit the health of the consumer. An approach currently receiving considerable interest is the provision of a physical barrier against adverse environmental conditions for the living probiotic cells. In the past, microorganisms were immobilised or entrapped in polymer matrices for use in bio-technological applications. The physical retention of the cells in the matrix facilitated the separation of the cells from their metabolites. As the technique of immobilisation or entrapment improved, the immobilised cell technology has evolved into encapsulation of live bacterial cells.