Continuous immobilized yeast reactor system for complete beer fermentation using spent grains and corncobs as carrier materials

Despite extensive research carried out in the last few decades, continuous beer fermentation has not yet managed to outperform the traditional batch technology. An industrial breakthrough in favour of continuous brewing using immobilized yeast could be expected only on achievement of the following process characteristics: simple design, low investment costs, flexible operation, effective process control and good product quality. The application of cheap carrier materials of by-product origin could significantly lower the investment costs of continuous fermentation systems. This work deals with a complete continuous beer fermentation system consisting of a main fermentation reactor (gas-lift) and a maturation reactor (packed-bed) containing yeast immobilized on spent grains and corncobs, respectively. The suitability of cheap carrier materials for long-term continuous brewing was proved. It was found that by fine tuning of process parameters (residence time, aeration) it was possible to adjust the flavour profile of the final product. Consumers considered the continuously fermented beer to be of a regular quality. Analytical and sensorial profiles of both continuously and batch fermented beers were compared.     

Growth model and metabolic activity of brewing yeast biofilm on the surface of spent grains: a biocatalyst for continuous beer fermentation

In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate (q(im)) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble-column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (X(im)act) of the total immobilized biomass (X(im)) was subsequently estimated at approximately X(im)act = 0.12 g(IB) g(C)(-1) (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell-to-carrier adhesion and cell-to-cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (X(im)act) and the maximum attainable biomass load (X(im)max) was included into the model. Since the average biofilm thickness was estimated at ca. 10 microm, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble-column reactor. Good agreement between model simulations and experimental data was achieved.     

Immobilization of yeast cells in PVA particles for beer fermentation

In this study, the potential of application of non-aggressive LentiKat^® technique for brewer's yeast immobilization on polyvinyl alcohol was assessed. High cell loads of about 10⁹ cells/ml were achieved by this procedure and immobilization procedure had no adverse effect on cell viability. The stability and activity of obtained immobilized biocatalyst was tested in the growth studies and fermentations. Immobilized cells exhibited high fermentation activity in both, laboratory and pilot-scale fermentations. In three successive gas-lift reactor fermentations the apparent attenuation of around 80% was reached after only 2 days, indicating good potential of immobilized cells for development of continuous primary beer fermentation. LentiKat^® particles showed high mechanical and fermentative stability, since they endured 30 days of operating time during 6-month period without significant change of cell activity, particle shape and particle size.     

Immobilized yeast cell systems for continuous fermentation applications

In several yeast-related industries, continuous fermentation systems offer important economical advantages in comparison with traditional systems. Fermentation rates are significantly improved, especially when continuous fermentation is combined with cell immobilization techniques to increase the yeast concentration in the fermentor. Hence the technique holds a great promise for the efficient production of fermented beverages, such as beer, wine and cider as well as bio-ethanol. However, there are some important pitfalls, and few industrial-scale continuous systems have been implemented. Here, we first review the various cell immobilization techniques and reactor setups. Then, the impact of immobilization on cell physiology and fermentation performance is discussed. In a last part, we focus on the practical use of continuous fermentation and cell immobilization systems for beer production.     

Detoxification of organophosphate nerve agents by immobilized dual functional biocatalysts in a cellulose hollow fiber bioreactor

A whole-cell technology for detoxification of organophosphates based on genetically engineered Escherichia coli cell expressing both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) onto cell surface was reported recently (Wang et al., 2002). This study reports the application of these biocatalysts when immobilized in a cellulose hollow fiber bioreactor (HFB) for the biodetoxification of a model organophosphate, paraoxon, in a continuous flow mode. In 24 h, 0.79 mg wet cell/cm2 fiber surface were immobilized onto cellulose fibers specifically and strongly through the cellulose binding domain, forming a monolayer demonstrated by Scanning Electronic Micrograph, and essentially no cell was washed away by washing buffer. The immobilized biocatalyst had a high performance of detoxifying paraoxon solution of 5,220 mumol/h x L reactor or 990 mumol/h x m2 reactor. The immobilized biocatalysts maintained a stable degradation capacity for 15 uses over a period of 48 days with only 10% decline in degradation efficiency under operating and storage conditions. In addition, the bioreactor was easily regenerated by washing with 1% sodium dodecyl sulfate (SDS), with 86.7% immobilization capacity and 93.9% degradation efficiency recovery. This is the first report using the HFB in a non-traditional way, immobilizing whole-cell biocatalysts by specific adhesion thus rendering the catalysis operation the advantages of low pressure drop, low shear force, and low energy requirement. The successful application of this genetically engineered dual functional E. coli strain in a model bioreactor shows its promise in large-scale detoxification of organophosphate nerve agents in bulk liquid phase.     

The role of cell immobilization in fermentation technology

The distinction between immobilized cell fermentation and immobilized cell biocatalysis is seldom made, though they are conceptually quite different. Unlike immobilized enzyme systems, immobilized viable cells can be used to carry out conventional fermentations. Microbial cells which would otherwise be freely dispersed (in almost colloidal suspension) within the fermentation environment can be encouraged to become attached in some way to a support (carrier), thus producing a discrete particulate solid phase. Such immobilization offers several potential advantages of a process engineering nature to the fermentation system. These include ease of handling and of cell separation, and lowering of bulk viscosity, as well as the obvious potential benefits of increased cell concentration.     

Food Bioconversions and Metabolite Production Using Immobilized Cell Technology

Abstract

This review explores recent advances in the use of immobilized cells for the production of metabolites used in the food industry, such as enzymes, amino acids, organic acids, alcohols, aroma compounds, polysaccharides, and pigments. Some food bioconversions such as fermentation of soy sauce and various hydrolysis are also considered. Special emphasis was placed on existing or potential industrial processes. This article also reports the effects of the reactor (configuration and working conditions), the immobilized cell physiological status (growing, nongrowing, or permeabilized), and of the carrier type, configuration, and size on the performance of immobilized cell systems. Compared with free cell fermentation, the main advantage of using immobilized cells is an increase in productivity, particularly in the case of continuous fermentation. For monoenzymatic reactions, nongrowing immobilized cells are often reported to exhibit a higher stability than free or immobilized enzymes.     

Continuous alcohol fermentation in an immobilized cell rotating disk reactor

The increasing interest in alcohol fermentation over these last years because of the energy crisis has been demonstrated by an increase in scientific research. After a brief analysis of the main results of the literature in the field of alcohol fermentation reactors, the use of a new type of immobilized cell reactor [the rotating biological surface (RBS) reactor] was studied. As is well known, the RBS reactor is a form of fixed-film reactor and can be described as a dynamic trickling filter. Our experimental apparatus employed a spongy material to trap the yeast cells on the disks. The results of fermentations carried out in the RBS reactor working in batch, in continuous with cell support, and in continuous without cell support have been presented in order to compare the different productivities and to assess the performance of the RBS immobilized cell reactor. An ethanol productivity of 7.1 g/L h was achieved in the RBS-ICR at a dilution rate of 0.3 h(-1), 2.5 times higher than the maximum productivity obtained in the RBS reactor without support at a lower dilution rate. The adoption of a spongy material as a cell immobilizer, combined with the use of the RBS reactor, enhances the particular advantages of both systems.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.     

Studies on immobilized Saccharomyces cerevisiae. III. Physiology of growth and metabolism on various supports

In earlier communications general analyses of rapid ethanol fermentation by Saccharomyces cerevisiae immobilized on inert supports were described. In this article physiology of growth and metabolism (parameters like rates of CO(2) evolution and O(2) uptake, respiratory quotient, and generation time) of Saccharomyces cerevisiae immobilized on different supports are reported. Values of the ratio of specific oxygen uptake rate for immobilized cells to free cells have been found to be 0.732, 0.781 and 0.785 for carrier A, carrier B, and covalently crosslinked controlled pore glass (CPG, specific surface area of 439 m(2) g(-1)), respectively. Rates of specific CO(2) evolution for immobilized cells to free cells for these supports are 0.784, 0.822, and 0.783, respectively. Marked reduction in generation time of Saccharomyces cerevisiae on all the supports has been observed. No change in size (4.8-5 mum) and specific growth rate (mu(m) = 0.275 h(-1)) of cells leaving the reactor has been observed.     

固定化米根霉发酵制L-乳酸

采用海藻酸钙包埋法固定化米根霉（Ｒｈｉｚｏｐｕｓｏｒｙｚａｅ）,菌体在颗粒表面形成一层菌丝膜,有利于氧气和其它营养物质的传递;三相流化床生物反应器结构简单、动力消耗低、反应器内物质混合均匀、氧传递量大于固定化米根霉的需氧量,非常适合好氧的固定化米根霉发酵。利用它进行重复使用固定化米根霉的间歇发酵或连续发酵制备Ｌ 乳酸,整个过程一般可持续两周以上。固定化米根霉的产酸速率达１６～１８ｇ／Ｌ ｂｅａｄ．ｈｒ,得率为７０～８０％,反应器生产能力约为传统搅拌罐的３倍。采用海藻酸钙包埋法固定化米根霉在三相流化床生物反应器中进行发酵可以有效地提高Ｌ 乳酸的生产效率,具有良好的工业应用前景。     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

发酵生物制氢研究进展

综述了近年来发酵生物制氢领域的研究进展?在菌种方面,除了对现有产氢菌种的深入研究外,还采用生物学,分子生物学及生物信息学手段建立产氢菌种库;在氢酶的研究方面,已逐步从基因确定、功能研究拓展到基因工程构建高效产氢菌研究：而在与废弃生物质处理相结合的反应过程方面,研究主要集中在利用不同种类的废弃物的产氢和高效产氢反应器上。此外,还初步总结了目前对发酵制氢可行性和经济性的评价,并对其发展方向提出了新的看法。     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Assessment of Nitrate Removal Coupled with Fe (II) Oxidation and Analysis of Microbial Communities in a Moving-Bed Biofilm Reactor

A moving-bed biofilm reactor (MBBR) was used with an immobilized Pseudomonas sp. SZF15 strain in a Yu long filter (a novel bioactive carrier), which has the ability to remove nitrate and Fe (II) simultaneously. Optimum operating conditions were achieved with a nitrate removal ratio of 95.62% and an Fe (II) oxidation ratio of 88.09%. Meanwhile, high-throughput sequencing results confirmed that the immobilized Pseudomonas sp. SZF15 was not lost during the operation. This study lays the foundation for practical application of groundwater treatment.     

Distribution of Bacterial Contaminants in a South African Lager Brewery

Bacteria isolated from contaminated pitching yeast, fermenting wort and beer samples from a South African lager brewery over a one-year period were tentatively identified by an improved, rapid diagnostic procedure as pediococci (41%), homofermentative lactobacilli (5%), heterofermentative lactobacilli (9%), Acetobacter spp. (7%), Gluconobacter spp. (13%) and Hafnia protea (25%). Pediococci and lactobacilli dominated samples taken from fermentation, storage and 'bright' beer tanks but were absent from pitched wort samples, from collection vessels and the single pitching yeast sample investigated. Acetic acid bacteria and H. protea were widely distributed in collection vessel, fermentation and storage tank samples, and H. protea was isolated from recycled pitching yeast.     

Immobilized pepsin: Properties and use to prevent haze formation in beer and wine

Summary The characteristics of a new immobilized pepsin preparation are shown as a function of pH-value and temperature. Optimal working conditions for beer and wine treatment are derived from the stability and activity data. It was found that good protein stability of the beverage results when at least 50 g immobilized enzyme per hl beer per h (alternatively 20 g per hl wine per hour) are applied in a special type of packed bed reactor. Since the foam stability of beer is decreased by the enzyme treatment, better perspectives for industrial application are seen in wineries than in breweries.     

Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk

The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 ± 1.86 g/l, an optimal ethanol concentration of 87.91 ± 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h.     

聚乙烯醇固定化酵母细胞制备CTP研究

以聚乙烯醇为固定化载体,固定化冷冻处理过的啤酒酵母细胞从CMP制备CTP;分别从胶的型号、浓度和固定化方法的优化等方面摸索了最适的固定化条件,固定化细胞在试验条件下连续发酵8次,转化率维持在85%～95%。同时,还对固定化细胞的稳定性进行试验研究,并用HPLC对产品进行了分离鉴定。