J . Chrm. Tech. Biotechnol. 1992, 53, 33-38

Effect of Immobilisation on the Production of a-Amylase by an Industrial Strain of Bacillus amyloliquefaciens George Argirakos,* Kuganakathasan Thayanithyl & D. A. John Wase Biochemical Engineering Division, School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B 15 2TT, UK (Received 14 November 1990; revised version received 1 July 1991 ; accepted 29 July 1991)

Abstract: The effect of immobilisation of an industrial strain of Bacillus amyloliquefaciens in calcium alginate beads on production of a-amylase was investigated using lactose-based media in shake flasks and in a 0.3 dm3 glass fermenter. Although the microorganism was a good a-amylase producer in batch cultures of free cells, it was unable to produce the enzyme for extended periods either in repeated batch cultures, or in continuous cultivation. In each case, parallel tests with cells immobilised in calcium alginate beads gave still further reduced enzyme yields, and the free cells released into the broth from these beads probably contributed substantially to any amylase produced during these extended fermentations. After prolonged use, the core of the alginate beads accumulated hard insoluble material, with viable immobilised cells confined to a surface layer. Key words : Bacillus amyloliquefaciens, a-amylase, immobilised cells, continuous reactors, stability.

1 INTRODUCTION

made between fermentations using totally differing media. In the present work, we compared long-term (up to 696 h) a-amylase fermentation (using a strain of B. amyloliquefaciens immobilised in calcium alginate beads for batch, repeated batch and continuous cultivation), and making comparisons with further experimentally similar standard submerged fermentations. The results are reported in this paper.

The immobilisation of productive cells has long been advocated to improve productivity and decrease fermentation costs, on the assumption that the increased costs of immobilisation are more than offset by the extended productivity of the immobilised culture. It is therefore important to examine overall stability and productivity of immobilised cells over a length of time sufficient for this sort of information to be established. Unfortunately, current enthusiasms for immobilisation d o not appear to be matched by extended observations of culture productivity. For instance, production of a-amylase (EC 3.2.1.1) by immobilised live Bacillus amyloliquefaciens (or the closely related Bacillus suhtilis) has been investigated by a number of research groups.' Each of these reports claimed that immobilisation favoured enzyme production, but in each case the duration of the fermentation was relatively short, never exceeding 60 h at the most, and comparisons were even

2 MATERIALS AND METHODS 2.1 The Organism A high amylase-producing industrial strain of B. amyloliquefaciens (designated KTl)5-6was used throughout this work.

2.2

Media

2.2.1 Sources of constituents All materials were at least of general purpose or

* Present address: IBM Centre, Athens, Greece. $ To whom correspondence should be addressed. 33

J. Chem. Tech. Biotechnol. 0268-2575/92/$05.00 0 1992 SCI. Printed in Great Britain

G. Argirakos, K. Thayanithy, D. A . J . Wusc

34 bacteriological medium grade and were supplied by Fisons plc, Loughborough, U K unless otherwise stated. The soya flour was from Spillers Premium Products Ltd, Puckeridge, Ware, UK.

dilution rates, evaporation losses were assumed to be similar in both cases and no corrections were applied.

2.2.2 Inoculum medium This contained (g dm-'): malt extract 40.0, and yeast extract 20.0. The medium was also used for the production of biomass for immobilisation.

Sodium alginate powder ( 8 % (w/w); Protanal 1-F 10/20M, Protan A/S, Norway) was added to water and the mixture was sterilised. It was then stirred with a sterile glass rod to give a homogeneous solution, cooled to room temperature and mixed with an equal volume of water containing centrifuged and washed cells from batch cultures incubated for 24 h in inoculum medium. The resulting suspension was extruded through a hypodermic needle into 0.05 mol dm-3 CaCI, solution. The beads were cured at 4°C for about 1 h when they became firm. Beads of diameters from 2.0 to 3.0 mm were then transferred into production medium and cultivated for the required lengths of time. For batch and repeated batch incubations of immobilised bacteria, beads equivalent to 40 cm3 of gel were added to 60 cm3 of medium in 1.0 dm3 flasks whilst for continuous fermentations the gel to liquid ratio was set at 1 : 3.

2.2.3 Batch production medium This contained (g dm-3): lactose. H,O 78.2; soya flour extract equivalent to 19 g of soya flour per dm3, yeast extract 15.6; casein hydrolysate 6.6, and MgS0,.7H20 0.4. This was used both for free submerged batch and repeated batch cultures, and immobilised cell batch and repeated batch cultures. For incubations of immobilised cells 2.94 g of CaC1,.2H20 was also added to improve gel ~tability,~ and the media were prepared with reduced water content so that the correct concentrations of the constituents were achieved upon the addition of the beads. 2.2.4 Continuous production medium This contained (g dm-3): lactose.H,O 20.0; casein yeast extract 2.0, KH2P0, 0.5, hydrolysate 5.0, MgS0,.7H20 0.2, CaCI2.2H,O 0.1 and 0.2 cm3 silicone anti-foam agent (Silcolapse 2000). For continuous cultivation the medium composition was adjusted in order to reduce its suspended solids content by replacing soya flour, the major contributor of suspended solids, by KH ,PO,.

2.4 Immobilisation

2.5

a-Amylase assay

The method used is a modification of the Wohlgemuth or S.K.B. method,* in which the carbon source (dextrin) was replaced by farina starch in phosphate buffer (pH = 6.0) to deal with the large quantities of amylase produced by the strain of B. amyloliguefaciens. Amylase activity was expressed in Wohlgemuth's ' X units cm-" of broth'. 2.6 Cell concentration

2.3 Cultivation procedures 2.3.1 Submerged cultures with ,free B. amyloliquefaciens organisms A loopful of an actively growing stock culture was transferred into 30 cm" of inoculum medium in a 500 cm3 conical flask. Following incubation (reciprocating shaker at 37f 1°C for 20 hj, 3.0 cm3of culture were transferred into 100 ern3 of production medium in a 1.0 dm3 conical flask which was then re-incubated as required. For repeated batch fermentations of free bacteria, washed, centrifuged organisms from a whole flask were transferred into a flask of fresh medium which was incubated as required. 2.3.2 Continuous cultivation Continuous cultures (in a 300 cm3 glass fermenter with a water jacket) were magnetically agitated and aerated through a sintered glass sparger at 1.0 v1v-l min-l. Broth temperature was maintained at 37-t 1°C and dilution rate was controlled at 0.3 h-l. As comparisons were made between immobilised cell cultures and free submerged cultures which both used similar air flow and

This was estimated by counting the number of microorganisms present by means of a haemocytometer of chamber dimensions 1.0 x 0.1 mm.

3 RESULTS 3.1 Comparison of batch fermentations with free cells and with immobilised cells Figure 1 shows a-amylase activities and numbers of bacilli present in the broths of flask cultures initiated either with free or with immobilised cells. Maximum enzyme titres for immobilised cell cultures were about 20% lower than those for free cell cultures ( 8 G 3 . 5 X units cm-3 is the usual norm for free cell cultivation). In contrast, there were more free cells in the cultures initiated with immobilised bacteria, and the occurrence of the peak in the numbers of free bacteria in the immobilised cultures was about 50 h later than that for non-immobilised cultures. This strongly suggests that the beads were acting merely as a source for continuous

a-Amylase production by B. amyloliquefaciens

35

I

m -

b

‘:i -

J 0

50

100

150 2 0 0 Time (h)

250

300

Fig. 1. Batch cultures in shale-flasks for free and immobilised cells. (a) Cell numbers in the broth: (0) cell numbers for the control batch culture (ern-", and (a)cell numbers released into the batch from the beads (cm-7. (b) Amylase titres in (0) the control batch culture, and ( 0 )the immobilised culture.

release of microorganisms into the medium. Macro- and microscopical inspection of the sectioned alginate beads sampled during the experiment revealed an uneven distribution of the biomass in the gel; live cells appeared to be restricted within a layer about 0.1 mm deep under the surface of the beads, while the core consisted of hard matter, apparently free from live organisms, indicating concentration gradients of nutrients and/or metabolic by-products within the alginate beads.

3.2 Repeated batch fermentations Here, fermented broth was decanted, the beads were washed with sterile water and fresh medium was introduced at 48 h intervals over a period of 240 h. Similar flasks containing free organisms, permitted comparisons of a-amylase yields with those of immobilised bacteria fermentations. In this case, centrifuged and washed bacteria from a previous batch were introduced into the fresh medium. Figure 2 shows how a-amylase titres and the numbers of free organisms present in the broth varied as the fermentation progressed through 48 h cycles. For the free bacterial fermentations, ability to produce a-amylase decreased gradually, only about 50% of the initial activity remaining after five subsequent cultures, whilst culture density fluctuated between 2.6 x loy and 3.4 x lo9 organisms cm-3. For immobilised cultures, amylase assays were generally lower, and the rate of loss of productivity of a-amylase was much higher, so that

t

n

5

3.0

Z

O L

Time ( h )

Fig. 2. Repeated batch fermentations in shake-flasks. (a) The Progress of the control free cell cultures: at the end of each 48 h the amylase titre, and (0) the cell numbers ( ~ m - ~ ) period (0) were measured, and centrifuged, washed cells were then used to inoculate the next flask.(b) A similar test involving immobilised cells. At the end of each 48 h period ( 0 )the amylase titre achieved, and (m) the numbers of free cells released into the medium were measured before the beads were washed and the medium replaced.

after five cycles less than 1 YO of the initial activity remained. The numbers of bacteria released from the beads fell at first, then increased. In other words, at the end of the experiment, many more bacteria were being released than at the start, but they made hardly any amylase. A similar situation was also noted by Dixon,’ who was able to show that the culture was heterogeneous with respect to high-yielding cells, which declined in numbers as serial culture continued. It seemed likely that in the present case, steadily increasing numbers of lowyielding bacteria were being liberated. It was therefore assumed that this industrial strain, like most others, is unstable. Moreover, an inspection of various bead samples again showed clearly the uneven distribution of biomass in the gel previously described, together with the hardening and precipitation mentioned elsewhere in this report. Such conditions are not likely to be conducive to supporting a high-yielding strain over extended periods of time. 3.3 Continuous cultures Conventional continuous fermentations using free bacteria were continued for 696 h after initial batch

G. Argirakos, K. Thuyanithy, D. A . J . Wose

36

Time (h)

Fig. 3. Continuous cultivation of frcc cells and of immobilised cells in a 0.3 dm3 fermenter. (a) Amylase titres produced by (0) the control free cell continuous culture, and ( 0 )the immo-

bilised cell culture. (b) (0) Culture density of the free cell culture, and (H) cclls rclcascd into the medium from the immobilised culture.

operation for 24 h. Amylase activities and culture densities of broth samples, taken at intervals (Fig. 3), show that a-amylase activity reached a maximum value of only about 0.13 X units cm-3 at the end of the batch operation and fell sharply after the onset of continuous feeding. Although titres fluctuated, two 'plateau' phases can be observed on the graph; one at around 0.06 X units cm-3 between 48 h and 3 12 h, and a second at around a level of 0.03 X units cm-3 after 360 h. Microbial concentration remained relatively constant, roughly between 1 x lo9 and 3 x lo9 organisms cm-3 and no correlation between these two was evident. After about 120 h, considerable wall growth was observed, suggesting that subsequent biomass measurements on the broth did not truly reflect the average biomass content of the vessel. In spite of these variations, broth pH throughout the entire fermentation fluctuated only between 6.25 and 6.80, in contrast with the wide variations in pH values within batch cultivation on the same medium." Probably the buffering action of the incoming medium stabilises the continuous cultivation. Figure 3 also shows the results of continuous incubation of immobilised bacteria. Conditions were similar to those for free bacterial cultivation, except that the initial batch cultivation was omitted to control wall growth in the vessel at an early stage. However, although this was initially minimised, it became substantial by 3 14 h probably because the beads interfered with mixing. Clearly, this problem is likely to arise only in long-term immobilised bacterial fermentations. For the continuous immobilised culture, average aamylase activity, at around 0.016 X units cm-3, was

generally very much lower than that for free organisms over a similar period of time; nor was there any significant improvement in stability. Broth culture density varied between 2.6 x iOs and 6.0 x 10' organisms cm-S, around four times lower than the average for continuous fermentation with free bacteria. The pH value once again fluctuated between 6.20 and 6.80 (as for free organism cultivation). Clearly, comparisons between the two types of cultures are not complicated by pH differences. At the end of the test, a hard insoluble material had once again been formed in the cores of the beads. Qualitative analysis of this material showed that it consisted mainly of phosphates of calcium and magnesium. Microscopic examination confirmed that the material containing the live cells remained apparently intact as a gel in a layer only about 0.1 mm deep under the surface of the beads.

4

DISCUSSION AND CONCLUSIONS

Although the microorganism used in this work is a good a-amylase producer in batch fermentations, it gradually loses its activity in repeated batch or continuous culture^.^^'" Degradation of specially selected, re-selected, manipulated and/or mutated high-yielding strains is quite usual, especially exoenzyme-producing strains of Bacillus spp. ; the phenomenon has been attributed either to the loss of genetic information or to catabolite repression."-'* Since immobilisation is claimed to restrict growth rate, l 5 < l 6and therefore, presumably, reversion rate, it has been assumed that this method could help prolong production of a-amylase. Hence the current interest in performance of this microorganism when immobilised in alginate beads. However, as indicated earlier, the results of these experiments showed that immobilisation did not achieve the desired results. First, there was the growth of free bacteria. Such growth from beads containing immobilised Bacillus spp. has been reported by many authors:' '.17 the literature suggests that microbes release can be due either to abrasion, or to growth of organisms at the bead surface and release during long-term fermentations. This process of release appears unavoidable, since the support matrix will erode even in air-life fermenters.'* One objective of immobilisation was to reduce growth rate and prevent the strain degradation mentioned above : in this study, free growth was particularly prolific and circumvented this objective. Secondly, therefore, there was the problem of strain degradation. In this study, this appeared to be particularly severe and was exacerbated by extended periods of cultivation. The problem was clearly associated with organisms released from the beads as mentioned above. Thirdly, there was the decline of viable organisms in the cores of the beads, together with the precipitation of

37

a-Amylase production by B. amyloliquefaciens organic materials. Gradients within beads have already been observed for Bacillus spp.,2,3*17 and have been attributed to chemical degradation, or insufficiency of oxygen or other nutrients as discussed below. In this study, the chemical degradation was evidently time related, and in long-term continuous tests where actual precipitation occurred within the bead, the concentrations of inorganic salts were likely to have been increasingly inhibitory. Short-term immobilisation tests are not appropriate for examining this problem. Fourthly, a further affect of the decline of viability within the bead was that on continuous kinetics. At least 80 % of the gel volume was completely free from bacteria, and this, in turn, implies that most of the volume occupied by the gel was not part of the active culture, whereas the continuous tests assumed all the volume was involved. The actual dilution rate could therefore have been nearer 0.4 h-' than the calculated value of 0.3 h-l. Fifthly, there was the problem that mixing was severely hampered in the presence of large quantities of beads. This restriction of mixing gave rise to two further problems. Poorer mass transfer processes, particularly oxygen transfer, are suggested in Fig. 3 in which the amylase yields in these highly aerobic fermentationsS,10,19.20 are substantially higher in the free cell cultures than in the corresponding tests with immobilised cells. Poorer mixing also severely restricted broth velocity and hence turbulence close to the vessel walls, so that the bacteria which were released from the beads had more opportunity than their free-living counterparts to adhere to surfaces of the fermenter. These results do not agree with publications that report that the use of immobilised Bacillus spp. cells improved a-amylase In the latter case the free and immobilised cell cultures were compared under different conditions of growth such as different volumes of broth in flasks and different compositions of media. Indeed, in one case2the calculations which were made on the total amylase activity in flask cultures with immobilised cells are open to question. Finally, the authors believe that in many cases the reported relative advantage of the immobilised over the free cell cultures in, for example amylase production, is statistically insignificant. For instance, in conventional batch cultures of this particular strain of microorganism, it was found that the standard deviation of the highest amylase activity was about 13 % of the mean value." Overall, it would appear that methods of immobilisation of B. umyloliquefaciens involving calcium alginate beads for batch, repeated batch and continuous production of a-amylase lead to three major problems : low a-amylase yield, prolific growth of free cells in the broth, and progressively deteriorating conditions in the core of the beads. As a result, it is concluded that this method offers no significant advantage over conventional methods of fermentation in the amount or stability of amylase production.

It is possible that the alternative technique of cell agglomeration might be more rewarding. Autoagglomerating cultures rely on the natural capabilities of the microorganisms involved to produce adhesive extracellular polymers which cause the microorganism to be retained in the interstices of a support medium. In such cases, this has the added advantage that subsequent movements of the support materials have a scouring action on the vessel, thus discouraging wall growth. In the present case, our strain of B. amyloliquefuciens has been shown to be very adhesive : for instance, this report describes adhesion to the walls of reactors, and future studies will address this possibility.

ACKNOWLEDGEMENTS The authors wish to express their gratitude to the late Mr G. Harding of ABM Chemicals Ltd for his interest and invaluable assistance during the early part of this research. The first author (G. A.) is also grateful to the State Scholarship Foundation of Athens for their financial support.

REFERENCES I . Kokubu, T., Karube, I. & Suzuki, S., a-Amylase production by immobilised whole cells of B. subtilis. Eur. J . Appl. Microbiol. Biotechnol., 5 (1978) 23340. 2. Shinmyo, A., Kimura, H. & Okada, H., Physiology of aamylase production by immobilized B. amyloliquefaciens. Eur. J . Appl. Microbiol. Biotechnol., 14 (1972) 7-12. 3. Chevalier, P. & de la Noue, J., Enhancement of a-amylase production by immobilized B. subtilis in an airlift fermenter. Enzyme Microb. Technol., 9 (1987) 53-6. 4. Chevalier, P. & de la Noue, J., Behavior of algae and bacteria co-immobilized in carrageenan, in a fluidized bed. Enzyme Microb. Technol. 10 (1988) 19-23. 5 . Thayanithy, K., Harding, G. & Wase, D. A. J., Rearrangement of lactose on sterilisation. Biotechnol. Lett. 4(7) (1982) 423. 6. Thayanithy, K., Wase, D. A. J. & Harding, G., Effect of steam sterilisation on a lactose medium. Process Biochem., 18(5) (1983) 17-19. 7. Baker, M . R., The development and application of a biological modelling fluid for mycelial fermentations. PhD thesis, University of Birmingham, UK, 1988. 8. Sandstedt, R. M., Kneen, E. & Blish, M. J., A standardized Wohlgemuth procedure for a-amylase activity. Cereal Chemistry, 16 (1939) 712-23. 9. Dixon, K., The production of alpha-amylase by Bacillus subtilis. PhD thesis, University of Birmingham, UK, 1975. 10. Thayanithy, K., A study on the production of a bacterial alpha-amylase. PhD thesis, University of Birmingham, UK, 1980. 11. Fencl, Z . , Iricica, J. & Kodesova J., The use of the multistage chemostat for microbial product formation. J. Appl. Chem. Biotechnol., 22 (1972) 405-16. 12. Heineken, F. G. & O'Connor, R. J., Continuous culture studies on the biosynthesis of alkaline protease, neutral protease and a-amylase by B. subtilis NRRL B341 I . J. Gen. Microbiol., 73 (1972) 3 5 4 4 .

38 13. Priest, F. G. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev., 41 (1977) 71 1-53. 14. Memmert, K. & Wandrey, C., Continuous production of Bacillus esoenzymes through redox-regulation. Ann. Rev. N Y Acad. Sci. 506 (1988) 63 1-6. 15. Tyagi, R. D. & Chose, T. K., Studies on immobilized S. cerevisiae. 1. Analysis of continuous rapid ethanol fermentation in immobilized cell reactor. Biotechnol. Bioengng, 24 (1982) 781-95. 16. Doran, P. M. & Bailey, J. E., Effect of immobilization on growth, fermentation properties, and macromolecular composition of S. cerevisiae attached to gelatin. Biotechnol. Bioengng, 28 (1986) 73-87.

G . Argirakos, K. Thayanithy, D. A . J. Wase 17. Baudet. C., Barbotin, J. N. & Guepsin-Michel, J., Growth and sporulation of entrapped B. subtilis cells. Appl. Environ. Microbiol., 45 (1983) 297-301. 18. Kloosterman, J. & Lilly, M. D., An airlift loop reactor for the transformation of steroids by immobilized cells. Biotechnol. Lett., 7(1) (1985) 25-30. 19. Martin, D., Production of alpha-amylase by bacterial fermentations. PhD thesis, University of Birmingham, UK, 1978. 20. Argirakos, G., Monoseptic immobilisation of Bacillus subtilis for the production of alpha-amylase. MPhil thesis, University of Birmingham, UK, 1989.