,o,App le Microbielogy and Biotechrology European

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European J. Appl. Microbiol, Biotechnol. 10, 2 1 1 - 2 2 1 (1980)

by Springer-V ~'rlag 9 1980

An Inoculum Technique for the Production of Fungal Pellets

J.C. van Suijdam, N.W.F. Kossen, and P.G. Paul Biotechnology Group, Department of Chemical Engineering, Delft University of Technology, Jaffalaan 9, NL-2628 BX Delft, The Netherlands

Summary. A simple standard inoculation procedure has been developed to obtain growth of fungi in the form of pellets. This technique made use of filamentous mycelium from a preculture as an inoculum, yielding many small pellets with a fairly homogeneous size distribution. At an early stage of growth the presence of a polymer (Carbopol-934) proved to be very important for the way spores germinate and lowered the agglomeration tendency. At a later stage of growth the influence of shearing forces becomes more predominant. Introduction

Pellets can be attractive as alternative growth form for the culture of fungi. The most important advantage of a pellet suspension is the significant decrease of the viscosity in comparison to a filamentous suspension. This enhances the desirable mixing and mass transfer properties of the suspension considerably. An additional advantage is the easier separation of the biomass from the broth. On the other side, however, a possible severe drawback may be introduced, i.e., decrease of growth and activity due to diffusional limitation of oxygen and/or other nutrients into the interior parts of the pellets. For the cultivation of mushrooms in submerged fermentations pellets are important for the flavour development, probably caused by autolysis in the center of the pellets. A secondary advantage thereby is the favourable texture of pellets for instance when used in soup production. Another application of mycelial pellets is the use as carrier material for immobilized enzymes where they can provide a better starting material for the production of immobilized mycelial particles in order to create a stable continuous process (Karube et al. 1977; Morikawa et at. 1979; Suzuki and Karube 1979). To our opinion it is also possible to use mycelial pellets as a support for whole cells. The large void fraction makes pellets especially suitable for this purpose. The inoculum quality is of prime importance for the rest of the course of a fermentation (Calam 1976; Meyrath and Suchanek 1972). Therefore the objective of this study was to develop a simple standard inoculation procedure to obtain pellets in fermentors, especially in a bubble column. This was a part of a more general study 0171-1741/80/0010/0211/8 02.20

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of the feasibility of the use of fungal pellets in industrial fermentations. The formation of pellets in submerged cultures may often have seemed to occur somewhat haphazardly. However, two extensive literature reviews have elucidated much of the influences playing a role upon the formation of pellets. The first one has been written by Whitaker and Long (Whitaker and Long 1973), the other one more recently by Metz and Kossen (1977). If one confines himself to a given chosen organism, strain and medium composition- factors mostly fixed in a process for reasons other than pellet formation -then three major variables remain that can be controlled: 1. inoculum concentration, 2. polymer additives, and 3. shearing forces in a fermentor. Generally it is stated that the criterion to be met for the development of pellets is an initial spore concentration smaller than 1011-1012 spores/m ~. Above this value filamentous growth occurs. The influence of surface-active agents and polymer additives upon the morphology of fungi, causing a more dispersed growth, has been well documented in literature. Elmayergi (Elmayergi 1975) obtained the most striking results out of a series of polymers with Carbopol-934. This anionic polymer had a strong tendency to change the morphology of Aspergillus niger from pellets into dispersed mycelium. Cationic polymers were found to have a reversed effect. The reason for the effect of agents such as Carbopol is that they prevent the agglomeration of spores into clusters, thus raising the effective spore concentration and hence stimulating pulpy growth. A similiar effect was observed by Cimerman et. al. (Cimerman et al. 1976) using native polymers like alginates and starch with the culture of A. niger. Addition of 0.1% alginate resulted in a 20-fold reduction of the spore/pellet ratio, causing the formation of more and smaller pellets. Concerning the influence of shearing forces, it is well known that mild agitation and shear stresses favours the development of pellets. Materials and Methods

Organisms Experiments were carried out with strains of Penicillium cbrysogenum strain A, obtained from Gist Brocades N.V. Delft; Sporotricbum pulverulentum Novobranova CBS 67171, obtained from Centraal Bureau voor Schimmelcultures and AspergiUus niger LMD 7352, obtained from the Laboratory of Microbiology, Delft University of Technology. The organisms were cultured on malt-agar slants (1% malt-agar, Oxoid) at 25~ and were transferred to fresh slants every 10 to 14 days. Every two months new spores from a dry and stock were used.

Conidia Sporulation was achieved in Kolle dishes on malt-agar with 1.25% calciumchloride. The spore suspensions were prepared by gentle washing the Kolle dishes with a 0. t%

An Inoculum Technique for the Production of Fungal Pellets

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Fig. 1. Photograph of spores of Penicillium cbrysogenum (magnification 550 x)

Triton X-100/distilled water solution and transferred to small serum bottles which were kept in the refrigerator. Care was taken not to use spores older than one month. The concentration of spores was counted under a microscope by means of a Thoma counting chamber. Chains and clusters of spores (see Fig. 1) were counted as one, assuming they give rise to the formation of only one nucleus growing into a pellet.

Growth Media The compositions of the synthetic growth media used for the culture of the organisms are given in Table 1_ Medium M10 and M33 were used for Penicitlium cbrysogenum in shake flasks and fermentors respectively. Temperature was kept at 25~ pH was controlled at 6.8 + 0.1. F o r Sporotricbum pulverulentum always medium M20 was used; temperature 40~ pH = 5.0. The conditions for Aspergillus niger were: medium M31 in shake flasks; M33 in fermentors; temperature 25~ pH = 5.0. Glucose, the limiting nutrient in these media, was added according to the required final biomass concentration. The media were sterilized at 121~ for 20 rain. with the exception of glucose and lactic acid which were sterilized separately at 110~ for 30 rain.

Additive As polymeric additive Carbopol-934 was used (B.F. Goodrich Chem. Comp.). Although Carbopol acts as a wetting agent with an affinity for water due to its hydrophilic nature, care was taken while dispersing the polymer to avoid clumps being formed. After dissolving the pH was adjusted.

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Table 1 Constituent

Glucose a Lactic acid Yeast extract (Oxoid) (NH4)2SO 4 KH2PO 4 KNO 3 MgSO 4 9 7 aq. FeSO 4 9 7 aq. CuSO 4 9 0 aq. ZnSO 4 9 7 aq. MnSO 4 9 4 aq. CaCI 2 9 0 aq. NaEDTA

Con centratio n kg/m 3 MIO

M20

M31 b

M33

10/20 10 7.5 -1.6 0.18 0.008 0.05 0.05 0.05 0.60

10/60 5 ----

10/20 3.5 -13.7 2.0 1.2 0.01 -0.002 0.01 -

10/60 10 1.5 0.5 0.18 0.008 0.05 0.05 0.05 1.3

a Glucose was added according to the required final biomass concentration b Medium M31 according to Tosoni and Glass (Tosoni and Glass 1963)

Apparatus T h e f e r m e n t a t i o n s were carried o u t in a 20-1 f e r m e n t o r (Biolafitte, t y p e S 2 0 L ) a n d a 20/3 0-1 w o r k i n g v o l u m e b u b b l e c o l u m n . P r e c u l t u r e s were carried o u t in shake flasks (0.5 or 2.0 1 E r l e n m e y e r flasks) or in a 2-1 Biolafitte f e r m e n t o r . T h e b u b b l e c o l u m n was especially designed and c o n s t r u c t e d for this p r o j e c t in t h e m e c h a n i c a l w o r k s h o p of o u r l a b o r a t o r y . A s c h e m a t i c diagram is given in Fig. 2. T h e f e r m e n t o r c o n s i s t e d of a glass c y l i n d e r e n c l o s e d b e t w e e n a stainless steel b o t t o m a n d top. A d o u b l e wall cooling j a c k e t was placed at t h e b o t t o m o f t h e c y l i n d e r to c o n t r o l t h e t e m p e r a t u r e . T h r o u g h this j a c k e t p r o b e s were i n s e r t e d for dissolved o x y g e n , pH a n d t e m p e r a t u r e c o n t r o l s y s t e m s as well as a s a m p l i n g p o r t w i t h an i n n e r d i a m e t e r of 10 m m . This design e l i m i n a t e d excessive g r o w t h of m y c e l i u m o n o b j e c t s sticking t h r o u g h t h e surface of t h e liquid, a n d e n s u r e d visual o b s e r v a t i o n o f t h e process. A special f e a t u r e o f t h e b u b b l e c o l u m n was t h e sparger (see Fig. 3.). It was m a d e o f e i g h t s i n t e r e d stainless steel pipes, w i t h a m e a n pore size o f 3 pan.

Pellet Counting and Size Measuring Tecbnique Pellet c o n c e n t r a t i o n s a n d sizes were d e t e r m i n e d b y t a k i n g a large s a m p l e o u t of t h e f e r m e n t o r . This sample was p h o t o g r a p h e d as a w h o l e using a graphic film to o b t a i n p h o t o g r a p h s with g o o d c o n t r a s t . T h e p h o t o g r a p h s were t h e n analysed b y m e a n s o f a Q u a n f i m e t 7 2 0 S y s t e m (Image A n a l y s i n g C o m p u t e r , I M A N C O Ltd.). This Q u a n t i m e t 7 2 0 S y s t e m uses an a u t o m a t e d field s c a n n i n g t e c h n i q u e , p r o d u c i n g pellet c o u n t s a n d e n a b l i n g e v a l u a t i o n s o f t h e size d i s t r i b u t i o n . This m e t h o d is very c o n v e n i e n t to analyse large n u m b e r s o f particles. In this way very large a m o u n t s o f pellets can b e processed (in t h e o r d e r of h u n d r e d s per p h o t o g r a p h ) . T h e disadvantages are: t h e sampling techn i q u e b e c o m e s m o r e critical, t h e p h o t o g r a p h s have to be o f high q u a l i t y ( c o n t r a s t ) a n d all t h e pellets m u s t b e lying s e p a r a t e l y f r o m each o t h e r .

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Results and Discussion

Shake Flask Experiments In Table 2 the results of shake flask experiments with Sporotrichum pulverulentum are presented. Shake flasks were incubated at 40~ in an orbital incubator. Two types of flasks were used: without and with baffles. (Details are given by van Suijdam et al. 1978). The baffled flasks were shaken with an orbiting speed of 80 rpm, the others at 150 rpm. The inoculum concentration was 1010 spores/m3; measurements were performed after 42 h. As can be seen, the results confirm the influence of shearing forces and especially of Carbopol as an additive. A significant reduction in the spore/pellet ratio and the mean pellet diameter is observed. The most outspoken results are found when a combination of both baffles and Carbopol addition is applied: a decrease of more than 200 times in the spore/pellet ratio and a 80% decrease in the mean pellet diameter. Experiments with Penicillium chrysogenum and Aspergillus niger showed similar results. Adding Carbopol at a concentration of 3 kg/m 3 caused a decrease of approximately thirty times in the spore/pellet ratio. An additional effect of the use of Carbo-

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J.C.van Suijdam et al.

Table 2. Shake flask experiments with Sporotricbumpulverulentumshowing the influence of Carbopol and baffles Exp.

Carbopol kg/m3

Baffles

Spore/pellet ratio

Mean pellet diameter 10-3 m

$7/$8 s13/$14 $15/$16 $21/$26

3 3

+ +

8.8 103 0.25 103 1.9 103 40.0

2.6 1.3 1.7 0.3

pol was the absence of the mostly observed ring of wall growth in a shake flask. This probably improves the uniform germination of the spores. Finally, with all three organisms inoculum concentrations higher than 1011 spores in combination with the addition of Carbopol invariably produced dispersed mycelial growth. In previous work by Metz (Metz 1976) media were used containing CaCO 3 at the concentration of 3 kg/m 3 to cause pellet formation. The results of this study indicate that the presence of solid particles in the growth media is not essential for pellet formation.

Bubble Column Experiments In order to find the most suitable inoculation technique for a fermentation in a bubble column, three alternatives were investigated, i.e.: inoculation direct with spores into the fermentor, - from a preculture consisting of small pellets, and - inoculation from a preculture of pulpy mycelium. Inoculation with spores direct into the fermentor, was tested with all three organisms used. With Sporotricbum pulverulentum this resulted in a gradual development of the mycelium into pellets of fairly uniform size (see Fig. 4). This result hardly came as a surprise, since it is well known that Spor. pulverulentum forms pellets very easily. One disadvantage remains; it takes a very large amount of spores to obtain a desired pellet concentration. Experiments to study the behaviour of Aspergillus niger failed because of the very strong tendency of the spores to float on the surface of the broth. This flotation phenomenon is very common with spores of fungi. The low agitation intensity in a bubble column enhances this effect, making the results of an experiment like this, meaningless. With Penicillium chrysogenum this effect also occurred, although less pronounced. Agglomeration of spores with droplets of anti-foam (polypropylene glycol, P2000) caused an uneven germination of the spores and the formation of irregular shaped pellets (Fig. 5). Inoculation with a preculture consisting of small pellets cultured in shake flasks, resulted for all three organisms into the formation of a pellet type of suspension. In Fig. 6 the results of a pellet size distribution after 49 h of a fermentation with Aspergillus niger in the bubble column are shown. The relative frequency versus the pellet -

A n I n o c u l u m T e c h n i q u e for the P r o d u c t i o n o f Fungal Pellets

217

Fig. 4. Pellets of Sporotrichum pulverulentum, cultured in a bubble column. Initial spore concentration 8 1010 spores/m 3. Sample taken after 68 h; mean pellet diameter ~ 2 mm

Fig. 5. Growth of Penicillium chrysogenum in bubble column, after innoculation direct with spores

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J.C.van Suijdam et al.

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of Aspergi!lus niger after 49 h in a bubble column

diameter intervals are given in this figure. The total number of observations was 118. As can be seen, a very wide size distribution is obtained. The third inoculation technique examined made use of a preculture of a filamentous type of mycelium. This mycelium was either cultured in a shake flask or in a small 2 1 working volume stirred fermentor. It was astounding to find that a true filamentous type of mycelium in the bubble column always gradually developed into more dense hyphal flocs until finally becoming clearly distinguishable pellets. In Figs. 7 - 1 0 an impression is given of this phenomenon taking place with a culture of Penicillium cbrysogenum. Figure 7 shows the loose flocs of the pulpy mycelium form the preculture; following transfer to the bubble column after a few hours we already see the individual hyphae and loose flocs aggregate (see Fig. 8). After 2 0 - 3 0 h pellets are formed, at first as chain-like nuclei within floccose filamentous mycelium, later on more and more as individual pellets (Figs. 9 and 10). This phenomenon can very likely be attributed to the mild shearing forces present in a bubble column, highly stimulating the formation of pellets. In Fig. 1 l a - c the pellet size distributions are given for a culture of Aspergillus niger as a function of time. Clearly discernable is the increase of the 'mean' pellet diameter with time. A comparison of the size distributions given in Figs. 6 and 1 lc - both samples taken at a similar growth phase - illustrates the observation that the method using a pulpy preeulture produces smaller pellets of a more homogeneous size. This is of course an advantage from an engineering point of view. Taken into account the number of spores used in the preculture, the spore/pellet ratio decreased in this way from 2 - 2 0 to 0 . 1 - 0 . 2 for Penicillium chrysogenum. This means that from one spore finally 5 to 10 pellets were formed. To make sure the preculture consisted solely of filamentous mycelium the following procedure was held: in a shake flask 3 kg/m 3 Carbopol was added to the medium, this together with a high inoculum concentration of spores (>1011 spores/m 3) always produced pulpy growth. Later on in this project it was found that lowering the Carbopol concentration first to 1 kg/m 3 and even to 0.5 kg/m 3 did not change this effect.

An Inoculum Technique for the Production of Fungal Pellets

219

Fig. 7. Pulpy mycelium of Penicillium chrysogenum from preculture; 44 h after inoculation Fig. 8. Developing pellets; 10 h after transfer to Tower Fermentor Fig. 9. Pellets, 17 h after transfer Fig. I0. Pellets, 27 h after transfer

In a stirred fermentor, adding Carbopol in combination with a relatively high stirrer speed had the same results. It was found, however, that one restriction is imposed upon this inoculation technique: the inoculum should not be too heavy, i.e. initial concentrations exceeding about 1 kg/m 3 resulted in pulpy growth.

Conclusions An inoculation technique using filamentous mycelium to produce pellets in a bubble column proved to be a convenient method, yielding many small pellets with a fairly homogeneous size distribution. In the early phase of the germinanon of fungal spores the presence of polymers like Carbopol is very important, in that this strongly effects the electrostatic forces among spores. In a later phase of the growth of mycelium the influence of shearing forces become more predominant.

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J.C.van Suijdam et at,

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Acknowledgement. The autors acknowledge the help of the Department of Civil Engineering of the Delft University of Technology for putting to our disposal the Quantimet 720 System.

References

Calam CT (1976) Process biochemistry, April 7--12 Cimerman A, Johanides V, Skafar S (1976) Paper presented at the Fifth International Fermentation Symposium, Berlin Elmayergi H (1975) J Ferment Technol 5 3 : 7 2 2 - 7 2 9 Karube I, Hirano KI, Suzuki S (1977) Biotechnol Bioeng 1 9 : 1 2 3 3 - 1 2 3 8 Metz B (1976) Ph D Thesis, Delft University of Technology Metz B, Kossen NWF (1977) Biotechnol Bioeng 19:781--799 Meyrath J, Suchanek J (1972) Methods in microbiology. 7B: 159--209 Morikawa Y, Karube I, Suzuki S (1979) Biotechnol Bioeng 21:261 --270 Suijdam JC van, Kossen NWF, Joha AC (1978) Biotechnol Bioeng 20:1695--1709

An Inoculum Technique for the Production of Fungal Pellets

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Suzuki S, Karube I (1979) Production of antibiotics and enzymes by immobilized whole cells. ACS Symposium Series 106:59--72 Tosoni AL, Glass DG (1963) Canadian patent 671.647 Whitaker A, Long PA (1973) Process biochemistry, November 27-31 Received March 27, 1980