Design and Application of an Immobilized Loop Bioreactor for Continuous Beer Fermentation

672

R.H. Wijffels, R.M. Buitelaar, C. Bucke and J. Tramper i Eds) Immobilized Cells: Basics and Applications © 1996 Elsevier Science B.V. All rights reserved.

DESIGN AND APPLICATION OF AN IMMOBILIZED LOOP BIOREACTOR FOR CONTINUOUS BEER FERMENTATION. M. AndriesdXP.C. van Beveren (1), O. Goffin (1) and C.A. Masschelein (2) (1) Meura-Delta, Arenbergstraat 23, 2000 Antwerpen(Belgiiini) (2) CERIA-COOVI, Avenue Emile Gryzon 1, 1070 Brussels(Belgium)

Introduction Continuous production of beer in compact bioreactors can be done by immobilization of yeast. For application of such a system on large scale it is essential to use appropriate support materials. In literature adsorption of cells to solid surfaces such as porous glass and ceramic or attachment of cells to modified surfaces such as DEAE cellulose have been described as the most suitable means of immobihsing yeast for large industrial apphcations (1,2,4,5). Immobilized cells can only be appHed successfiilly for beer production if both productivity and quahty of the final product are high. Much progress has been made in recent years and several alternatives superior to the conventional batch technology exist today. Moreover, design engineering strategies have evolved to enhance the capabihties of immobilized ceU technology for the demanding requirements of large scale operation(3). In this study immobilization was carried out on preformed porous support, sihcon carbide matrices. These carriers allow optimisation of the pore size, pore size distribution and void volume as a fimction of biomass loading. Furthermore, external mass transfer Umitations are minimized by recirculating the bulk volume. By applying a multi-channel sintered sihcon carbide carrier in a loop reactor configuration beer with a similar composition and flavour profile as for a batch fermentation could be produced(3,7). Con^lete attenuation was achieved in a two stage bioreactor with a total residence time of two days. Stable production could be maintained over a period of six months. Materials and Methods Strain and medium A commercial lager yeast strain (C.007) from the CERIA laboratory culture collection was used for the immobilization for main lager fermentation experiments. The bioreactor was continuously fed with industrial hopped wort of 12 or 16 °P containing 40% non malt carbohydrates.

673 Wort oxygenation^) Oxygen in the wort feed stream is continuously supplied by diffiision through the MARPRENE connection tubing. The diffusion was detemuned in a bioreactor filled with water, de-aerated by CO2 sparging and measured with an INGOLD oxygen probe. The result is expressed as a fimction of time, recirculation rate and tube length, giving an oxygen supply of 9.7 |ig/renewal.m (renewal = volume/reckculation rate). Each reactor benig equipped with 2 m of tubing and operated at 0.5 renewals/min, 9.7 ^g of oxygen per mmute was transferred to the wort. Cleaning and sterilisation procedures(3,6J) Immobilized cells were removed by forward- and back- flushing the matrix with successively hot water (95°C), NaOH + detergent (pH 11, 60°C), hot water, alkahne detergent (0.2% v/v, 60 °C), hot water until pH 7, H2O2 (0.7% v/v, 60°C), hot water and steam The bioreactor wasfinallycon^letely sterihsed by either circulating a mixture of peracetic acid and hydrogen peroxide (1.5% v/v) for 20 minutes at ambient ten^erature or by steam sterihsation for 1 hour at 105°C. Analysis of wort and beer Wort density was measured using an Anton Paar DMA46 meter. Ethanol, higher alcohols and esters were determined by headspace gas chromatography with a FID detector. Fermentable sugars and amino acids were measured with HPLC. Results Applied carrier for immobilization(3) Iromobilization occurred on preformed matrices consisting of smtered Silicon Carbide and having the shape of a porous rod with 19 channels. Each matrix has a length of 90 cm, a diameter of 25 mm and each channel has a diameter of 2.5 mm. There is a void space of 180 ml per matrix available for the microorganisms to colonise and the pore sizes vary between 30 and 150 ^m. The advantages of using such a matrix are that the nutrients for the yeast cells, entrapped in the matrix, encounter no diffusion limitations due to the support material and are ideally distributed. There is no blocking of the support material so no special treatment of the wort is required. The material is inert, it has a high mechanical strength and chemical resistance allowing CLP. cleaning wdth known caustic and/or acid solutions. For sterilisation of the bioreactor steam or a mixture of peracetic acid and hydrogen peroxide was used. The characteristics of the carrier are summarised in figure 1.

674 Material : - Porous Rod with 19 Channels - Sintered Silicon Carbide Dimensions : - Length : 900 mm - Diameter : 25 mm - Diameter Channels : 2.5 mm - Void Volume : 180 ml (60%) - Pore Size : 30 - 150 ym. Advantages : - Ideal Shape : No Blocking of Support Material, Ideal Distribution of Fermentation Medium, No Diffusion Limitations - Inert Material, High Chemical Resistance and Mechanical Strength - Easy to Scale Up by Modular Assembly

Figure 1.

Characteristics of the silicon carbide immobilization matrix.

Figure 2 shows the asymmetrical pore size distribution, the pore sizes range between 30 ^m, near to the surface of the channels, to 150 ^m inside the silicon carbide structure resulting in a large surface available for colonisation of the yeast.

Figure 2.

The asymmetrical pore size distribution of the immobilization support structure.

Bioreactor configuration(3) The general bioreactor configuration is shown in figure 3. A continuous feed of nutrients is supplied to the bioreactor and therefore a continuous flow of product is obtained. The liquid phase is mixed completely, as a result there are no gradients in the concentration of nutrients, pH and temperature. Liquid is pumped through the channels and around the support units and recirculated as shown infigure3.

675 GAS EXHAUST NUTRIENTS IN

PRODUCT OUT

^Circulation Pioinp

Figure 3.

The general bioreactor configuration.

Primary fermentation with the immobilized loop bioreactor system Set-up for beer production Main lager fermentation was done in a two stage system in order to obtain an optimal balance between productivity and investment cost. The wort is continuously fed to the first bioreactor operating at 40 % apparent attenuation. The fermenting beer of the first stage is continuously transferred to the second bioreactor where end attenuation is reached. The beer, from the second stage is than stored in a beer tank before final treatment. (Seefigure4)

STAGE 1

STAGE 2

WORT VESSEL

WORT SUPPLY PUMP

Figure 4.

IMMOBILIZED YEAST REACTORS

Set-up for main lager fermentation.

HI^

676 Results obtained for primary fermentation using 12 and 16 °P wort As shown in table 1, steady state fermentation of 12 and 16 °P wort could be achieved with a flow rate of 1.3 ml/min in both cases. The enhanced productivity for fermenting 16°P wort could be explained by the increased specific sugar utiHsation in the first stage when using 16 °P wort, containing more easy fermentable sugars. Thus, a 38% increase in productivity was achieved only by using high gravity wort instead of 12°P wort. Operating tenq)erature was maintained at 15°C in both cases. Table 1.

Results obtained for main lager fermentation using industrial 12 and 16 °P wort. 12 °P wort

1 Apparent Attenuation (%) 1 Reactor Volimie (1) 1 Recirculation rate (renewals/hour) 1 Free yeast cone. (gDW/1) 1 Yeast immobilized (gDW) Total yeast mass (gDW) Temperature (°C) 1 Residual sugar cone, (g/1) Ethanol (%v/v) 1 Specific ethanol productivity (g/h.g

pw)

Specific sugar utilisation (g/h.g

pw) 1 Feed rate (ml/min)

Volumetric Productivity for beer with 4.9 %(v/v) Ethanol 1 (hi / year, matrix)

16 °P wort STAGE 1 STAGE 2 36 73 1.7 1.7 20 20 2.3 3.4 20.5 20.4 24.4 26.2 15 15 10 52 4.1 6.7 0.106 0.060

STAGE 1 40 1.7 20 1.6 20.5 23.2 15 42 2.7 0.068

STAGE 2 76 1.7 20 2.2 20.4 24.1 15 7 4.9 0.054

0.143

0.114

0.220

0.108

1.3 6.6

1.3 6.6

1.3 9.1

1,3 9,1

Table 2 shows the cort^arison between the flavour composition of batch and continuous produced beer. The batch fermentation was carried out in tall European Brewery Convention fermenting tubes, using the same operating conditions, the same yeast strain and wort as for the continuous fermentation. As is shown, the higher alcohol concentrations are sUghtly increased in the classical fermentation, but not significantly. However ester concentrations are significantly higher for the continuous fermented beer, giving it a more aromatic fiiU flavour. These ester concentration are easy controlable by adjusting the oxygenation, and in this way a similar profile as by a batch fermentation could be obtained.

677 Table 2.

Comparison between the flavour profile of a continuous and a batch fermented beer using the same wort and yeast stram.

1 N-Propanol (mg/1) 1 Isobutanol (mg/1) 1 Isoamylalcohol (mg/1) Ethylacetate (mg/1) 1 Isoamylacetate (mg/1)

CLASSIC 18 14 67 18 1.1

IMMOBILIZED 12 13 54 31 L3

Utilization of wort sugars and amino acids during continuous fermentation through the immobilized two-stage bioreactor Continuous fermentation experiments were performed at 15 °C and the response of the unmobilized yeast population to the indhddual wort sugars and amino acids on each stage of the bioreactor is shown in table 3. In terms of utilisation of glucose, maltose and maltotriose, sequential uptake patterns as measured in the outflow streams of stage 1 and 2 were similar to those of a discontinuous fi'ee cell system. Interestingly, amino acids were taken up very rapidly and the utilisation of group I and 11 was almost complete in the outflow of stage 1. These findings confirm the excellent performance of both the matrix structure and loop reactor design with respect to the supply of nutrients to the immobilized yeast population. Table 3.

Individual wort sugar and amino acid utilisation in the two stage immobilized system for primary fermentation.

1 % Utilisation 1 Glucose 1 Maltose 1 Maltotriose

Stage 1 100 39 11

Stage 2 100 95 90

97 100 88 99

97 100 100 100

88 93 99 100 80

100 99 100 100 100

95 65 70 68 67 59

100 97 99 95 94

GROUP I 1 Serine Threonine Asparagine 1 Arginine GROUP n 1 Isoleucine 1 Leucine Lysine 1 Methionine 1 Valine GROUP m 1 Glycine 1 Alanine 1 Phenylalanine 1 Tyrosine Tryptophane Histidine

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678 Conclusion A fully continuous immobilized yeast cell system was developed for the production of lager beer. A sintered silicon carbide carrier has been chosen for its high mechanical strength, loading capacity and hydrodynamic properties in combination with the multi-channel loop reactor design where the solid liquid contact area was maximised. For optimal volumetric productivity, a two stage configuration in continuous mode was adopted to achieve complete attenuation. The system was stable and produced beer of excellent quality with a total residence time of 2 days and with a composition and flavour profile similar to that of beer produced by batch fermentation. Amino acids and wort sugars were taken up sequential and Free Amino Nitrogen levels in the final beer were low. References 1. Aivasidis, A., Wandrey, C.H., Eils, H.G. and Katzke, M., proceedings of the European Brewery Convention Congress, Oslo, 1993, 569-576 (pages) 2. Linko, M. and Kronlof, J., proceedings of the European Brewery Convention Congress, Oslo, 1993, 353-360 (pages) 3. Krikilion, Ph., Andries, M., Gofifin, O., van Beveren, P.C. and Masschelein, C.A., Proceedings European Brewery Convention, 25th Congress Brussels 1995, p 419 4. Masschelein, C.A., Critical reviews in biotechnology 14(2), 1994, 155-177 (pages) 5. Pajunen, E., Gronqvist, A. and Ranta, B., proceedmgs of the European Brewery Convention Congress, Lisbon, 1991, 361-368 (pages) 6. van de Winkel, L., van Beveren, P.C. and Masschelein, C.A., proceedings of the European Brewery Convention Congress, Lisbon, 1991, 577-584 (pages) 7. van de Winkel, L., van Beveren, P.C, Borremans, E., Goossens, E. and Masschelein, C.A., proceedings of the European Brewery Convention Congress, Oslo, 1993, 307-314 (pages)